The Role of Cell Interactions in Early Neurogenesis Cargese 1983

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The Role of Cell Interactions in Early Neurogenesis Cargese 1 983

Edited by

A.-M. Duprat Laboratory of General Biology ERA-CNRS 327 Paul Sabatier University Toulouse, France

A. C. Kato University Medical Center Geneva, Switzerland

and

M. Weber Laboratory of Fundamental Pharmacology and Toxicology LP-CNRS 8221 Toulouse, France

Plenum Press New York and London Published in cooperation with NATO Scientific Affairs Division

Proceedings of a workshop on The Role of Cell Interactions in Early Neurogenesis, held May 29-June 3, 1983, in Cargese, Corsica, France

Library of Congress Cataloging in Publication Data

Main entry under title:

The Role of cell interactions in early neurogenesis.

(NATO Advanced science institutes series. Series A, Life sciences) Proceedings of a workshop sponsored by CNRS, NATO, and EMBO, held at the

Institut d'etudes scientifiques in Cargese, Corsica, May 29-June 3, 1983. "Published in cooperation with NATO Scientific Affairs Division."

Includes bibliographical references and index. 1. Developmental neurology-Congresses. 2. Cell interaction-Congresses. I.

Duprat, A.-M. 1939- .11. Kato, A. C., 1944- .111. Weber, M., 1948-IV. Centre national de la recherche scientifique (France) V. North Atlantic Treaty Organization. VI. European Molecular Biology Organization. VII. North Atlantic Treaty Organization. Scientific Affairs Division. VIII. Series. DNLM: 1. Nervous System-embryology-congresses. 2. Cells-physiology-congresses. 3. Cell Differentiation-congresses. WL 101 W926r 1983 QP363.5. R64 1984 591.3'32 84-9877 ISBN 978-1-4684-1205-5 ISBN 978-1-4684-1203-1 (eBook) DOl 10.1007/978-1-4684-1203-1

©1984 Plenum Press, New York Softcover reprint of the hardcover 15t edition 1 984 A Division of Plenum Publishing Corporation 233 Spring Street, New York, N.Y. 10013

All rights reserved. No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise, without written permission from the Publisher

PREFACE

The workshop entitled "The Role of Cell Interactions in Early Neurogenesis" was held at the Institut d'Etudes Scientifiques in Cargese, Corsica on May 29th to June 3rd, 1983. The setting was ideal for a small meeting whose purpose was to generate as much discussion as possible between the various participants.

One of the major topics of this conference was neural induction, that is, the first step in neurogenesis. Neural induction remains poorly understood at the molecular level as compared to the sub­sequent steps of neuronal migration and differentiation. It thus appeared important to unite different researchers working on this fundamental process of neural induction with scientists studying later steps of neurogenesis in order to exchange ideas and suggestions.

We would like to acknowledge the invaluable assistance of Marie­France Hanseler, the administrative assistant of CNRS who is respon­sible for the Institute. The workshop was primarily sponsored by CNRS (Centre National de la Recherche Scientifique, France), NATO and EMBO (European Molecular Biology Organization). Additional financial support was generously provided by the International Society of Developmental Biologists, the European Developmental Biology Organiz­ation, the European Council, the Universite Paul Sabatier (Toulouse), la Societe Francaise de Biologie du Developpement, les Etablisse­ments Fabre (Fr~nce) et les Etablissements Sarget (France).

The proceedings from this workshop comprise the present volume and there are five general chapters. In addition to the principal lectures given at the Institute, many participants presented short reports and all of these are included in this volume.

v

A.-M. Duprat A. C. Kato M. Weber

CONTENTS

1 NEURAL INDUCTION

Early Events in the Neurogenesis of Amphibians A. M. Duprat, L. Gualandris, P. Kan, and

F. Foulquier

Early Embryonic Induction: The Ectodermal Target Cells

H. Grunz

Clonal Restrictions During Early Development of the Frog Embryo

M. Jacobson

The Mechanism of the Amphibian Primary Induction at the Cellular Level of Organization

S. L,svtrup

Sequential Induction of the Central Nervous System L. Saxen

Neural-inducing Activity of Newly-mesodermalized Cells and Cellular Alterations of Induced Neurodermal Cells

A. S. Suzuki, T. Kaneda and T. Ueno

A Molecular Aspect of Neural Induction in CYNOPS Presumptive Ectoderm Treated with Lectins

K. Takata, K. Y. Yamamoto and N. Takahashi

Neural Embryonic Induction H. Tiedemann

vii

3

21

39

55

67

75

83

89

viii CONTENTS

2 CELL MIGRATION AND EARLY STAGES OF DIFFERENTIATION

Adrenergic Differentiation in the Automatic Nervous System

P. Cochard

Search for Stem Cells and Their Characteristics in the Mouse Hypothalamus

F. de Vitry

Post-transcriptional Regulation of Ontogenetically Modulated Proteins in the Nervous System

H. Soreq

Role of the Extracellular Matrix in Neural Crest Cell Migration

J. P. Thiery, R. Rovasio, J. L. Duband, A. Delouvee, M. Vincent and H. Aoyama

Neurodifferentiation in Cultures of F9 Teratocarcinoma Cells

J. Wartiovaara, P. Liesi, H. Hervonen and L. Rechardt

3 NEURONAL AND GLIAL MARKERS

Intermediate Filaments as Markers of Neuronal Differentiation

F. Alfonsi, M. Darmon, N. Forest and D. Paulin

Immunochemical Studies on the D2-Glycoprotein E. Bock and K. M611g!rd

Molecular Heterogeneity in Peripheral Glia R. Mirsky and K. R. Jessen

Plasma Proteins and Fetal Brain Development N. R. Saunders

Stage Specific Antigens on Oligodendrocyte Cell Surfaces

I. Sommer and M. Schachner

Cell-type-specific Molecules: Identification of Glycolipid Binding Sites for Soybean Agglutinin and Differences in the Surface Glycolipids of Cultured Adrenergic and Cholinergic Sympathetic Neurons

A. D. Zurn

109

123

131

139

145

157

177

181

191

201

207

CONTENTS ix

4 ELECTROPHYSIOLOGICAL APPROACH TO NEUROGENESIS

Membrane Excitability in Ciliary Ganglion Neurons and in Mesencephalic Neural Crest Cells

C. R. Bader. D. Bertrand. E. Dupin and A. C. Kato

Regulation of Endplate Channel Gating H. R. Brenner

Electrical Excitability. Regional Differentiation and the Ionic Control of Early Development

P. Guerrier. M. Moreau and L. Meijer

The Early Differentiation of Neuronal Membrane Properties

N. C. Spitzer

The Control of Neuronal Differentiation by Intra­cellular Sodium

A. E. Warner

5 FACTORS INVOLVED IN NEURONAL SURVIVAL. DEVELOPMENT AND DIFFERENTIATION

Purification of a Neurotrophic Protein from Mammalian Brain

Y.-A. Barde and H. Thoenen

Studies of the Development of Central Noradrenergic Neurons in vitro

U. di Porzio and M. Estenoz

Nerve Growth Promoters in the Embryonic Chick T. Ebendal

The Roles and Limitations of Growth Factors in Neuronal Development

D. Edgar

Cell Interactions During Formation of the Neuro­muscular Junction. The Search for Muscle­derived Motoneuron Growth Factors

C. E. Henderson

A Spinal Cord Derived Neurotrophic Growth Factor for Spinal Nerve Sensory Neurons

R. M. Lindsay and C. Peters

215

225

229

239

251

263

271

279

287

291

299

x

Inhibition of Proteolytic Activity as Modulation of Neurite Outgrowth

D. Monard and J. Gunther

Surface-bound and Released Neuronal Glycoproteins and Glycolipids

P. H. Patterson

In Vitro Studies on the Maturation of the Ascending Mesencephalic Dopaminergic Neurons

A. Prochiantz, S. Denis-Donini, M.-C. Daguet-de Montety, M. Mallat, A. Herbert and J. Glowinski

Brain Factors Supporting Proliferation of Neuronal Cells in Culture

M. Sensenbrenner, I. Barakat and G. Labourdette

Plasticity in the Neurotransmitter Phenotype of Rat Sympathetic Neurons in Primary Culture

J.-P. Swerts, M.-C. Giess, C. Mathieu, E. Sauron, A. Le Van Thai and M. Weber

INDEX

CONTENTS

307

311

319

331

335

345

NEURAL INDUCTION

EARLY EVENTS IN THE NEUROGENESIS OF AMPHIBIANS

(PLEURODELES WALTL, AMBYSTOMA MEXICANUM)

A. M. Duprat, L. Gualandris, P. Kan, and F. Foulquier

Laboratoire de Biologie generale 118, Route de Narbonne, 31062 Toulouse, France

NEURAL DETERMINATION AND TARGET CELL SURFACE

Neural induction is one of the oldest and yet still unsolved problems of Embryology. This key phenomenon is the first step in many of the complex processes occurring in the differentiation of target cells to mature neural cells and is the subject of intense and sometimes contradictory research.

The nature of the signal and the molecular mechanism of the transmission of this signal are still unclear (L6vtrup, 1975, 1983; Tiedemann, 1976; Landstrom and L6vtrup, 1977, 1979; Grunz et al., 1975, 1979; Saxen et al., 1977a, 1977b; Toivonen, 1979; Takata et al., 1981; Yamamoto et al., 1981; Gualandris and Duprat, 1981; Duprat et al., 1982; Jacobson, 1982). For Jacobson, "the basic morpho­logical plan of the central nervous system (CNS) can be identified in mid-blastula stages and the early origin of the basic morphological pattern of CNS development is under control of the egg cytoplasm". However in general embryologists agree that gastrulation is an important step in the ontogeny of the nervous system (inductive or permissive step?).

We feel, along with other authors, that a new conceptual frame­work is needed for studying the process of neural determination, based more on the essential role played by the target cells than on the inducer.

Using lectin probes under carefully controlled conditions (Gualandris et al., 1983) we have previously reported the important role played by cell surface glycoconjugates and their topography in this process. Experiments were performed to elucidate the nature and

3

4

No treatment : control

or Ilectin Treatment sugar

lectin + sugar

Fluorescence Observ.

A. M. DEPRAT ET AL.

Presumptive fctoderm ,

~~:l'Bla"toporal o Waiting in

Lip

~ Holtfreter medium

~) ~ Association

Lt1.J Ectod ./B1. lip

! "" 0RemOval of B1. lip

E:::!

"" of Ectod. /

"'- Dissociation

In toto explant Dissociated Ectod. cells

Fig. 1. Experimental Procedure.

turnover of the glycoconjugate molecules present on target cell surfaces of Pleurodeles waltl, using fluorescent labelled lectins specific for N-Ac-a-D-galactosamine, a-D-galactose (SBA); a-D-mannose and a-D-glucose (PSA, LCA and Con A).

- According to the methodology described Figure 1, when ecto­derm, which had been previously treated with lectins (SBA, PSA, LCA), was associated with natural inducing tissue (4 h), neural induction was strongly inhibited. During this period, ectoderm surface confor­mation is modified (Figure 2-5). The treated target cells after culturing in vitro for several weeks, differentiated into normal epidermal cells (Figure 6). Treated ectoderm observed ultrastruc­turally, immediately, 4 and 24 hours after treatment, showed a normal morphology.

EARLY EVENTS IN NEUROGENESIS

Fig. 2. At the beginning of the treatment, only the cell perip is fluorescent. SBA-FITC treated ectoderm.

Fig. 3. External side of ectoderm after 10 ron treatment: fluor pattern shows caps. SBA-FITC treated ectoderm.

- When the association between previously treated ectodern blastoporal lip was maintained for longer (24 h), neural induct occurred (glycoconjugate turnover repaired normal organization cell surface; Table 2).

The structural integrity of the plasma membrane at the gas stage seems necessary for neural determination to occur.

6 A. M. DEPRAT ET AL.

Fig. 4. Internal side of ectoderm after 10 mn treatment: fluorescent fine network on each cell. SBA-FITC treated ectoderm.

Fig. 5. Extracellular matrix on the inner surface of ectoderm. (N-Ac a D galactosamine, a-D galactose, N-Ac glucosamine, mannose, fucose, a-D mannose, a-D glucose glycoconjugate molecules and fibronectin), SBA-FITC treated ectoderm.

Several hypotheses were proposed:

1. The "neural signal" requires specific membrane receptors and a-D-galactose, a-D mannose and glucose containing complexes could be directly concerned.

2. Normal organization of the target cell membrane is necessary for the transmission of the "neural signal". The lectins involved in reorganization of the membrane structure (glycoconjugate reorganiz­ation), inhibit the neural process.

EARLY EVENTS IN NEUROGENESIS 7

if

m

i.f.

gl y

Fig. 6. (a) Ciliated epithelial cell (m: melanin, i.f.: intermediate filaments, 1: lipid droplet, y: yolk platelet, gl: glycogen particles). (b) Secretory cell and epidermal cell (ECM: extracellular matrix, mv: microvilli, j: cell junctions, N: nucleus, i.f.: intermediate filaments.

3. Lectin molecules bound to membrane sites, crowd the cell surface and prevent the passage of the inductive signal.

We have attempted to elucidate the nature and turnover of the glycoconjugate molecules which are present on target cell surfaces and could be involved directly or indirectly in the neural process.

Ectoderm explants (double cell layers) were treated in vitro with lectin probes (SBA, PSA, LCA, Con A) conjugated with fluoro­chromes (FITC and TRITC), in order to check the binding of the lectins at the cell surface, to investigate the resulting modifica­tions, to compare the behavior of the outer and the inner surfaces of the target tissue and to observe the kinetics of changes in the explants in vitro.

8 A. M. DEPRAT ET AL.

Our experiments indicate that:

1. The molecular reorganization with respect to lectin binding are different for the external and the internal surfaces of the ectoderm.

- External surface: fluorescence pattern shows caps (Figure 3). The capping phenomenon described in isolated cells, can also

occur when the cells are aggregated in a tissue.

- Internal surface: a) fluorescence develops into a fine network on each cell with a spoke-like structure which converges towards a central point (Figure 4).

The surface glycoconjugates include N Ac-a-D galactosamine, a-D galactose, a-D mannose, a-D-glucose receptors. Moreover, the embryo­nic cells react with pea lectin indicating the presence on the cells of N-glycan complex: N-Ac glucosamine, mannose, fucose receptors, which until now had only been detected in adult tissue (Kornfeld et al., 1981).

2. The intensity of fluorescence is: Con A < LCA < PSA < SBA. The ratio of external side/internal side fluorescence is constant ~~) -Table 1.

Table l. Comparative Study of the Fluorescence Intensity (in Arbitrary Units), with Different Lectins, of the Internal and the External Surfaces of Treated Ectoblast.

Lectins Fluorescence Fluorescence Fluorescence of lectins in of the external of the internal solution N surface N surface N (50 /Jg ml- 1)

SBA 100.52 ± 0.398 20 46.92 ± 1.921:l 15 96.84 ± 3.090 15 *100 *45.76 ± 1. 91 *96.33 ± 3.07

PSA 20.99 ± 0.436 11 6.54 ± 0.295 33 11.83 ± 1.485 39 *100 *31.15 ± 1.40 *56.36 ± 7.07

LCA 52.02 ± 0.89 10 6.74 ± 0.053 20 11.44 ± 0.161 30 *100 *12.95 ± 0.10 *21.99 ± 0.31

CON A 54.64 ± 0.81 10 2.26 ± 0.010 34 4.215 ± 0.495 55 *100 * 4.13 * 7.71 ± 0.90

N Numbers of measurements. * The actual values observed are in small print; bold print

corresponds to values expressed as a "100" of the fluorescence of the lectin solution used.

EARLY EVENTS IN NEUROGENESIS 9

3. The kinetics of changes in the explants in vitro, over 24 hand longer are shown in Table 2, using labelled SBA (idem for labelled PSA and LeA). These experiments reflect the disappearance of lectin-binding receptors (experiment 4) and the formation of new receptors (experiment 5).

The results should be compared with our previous data showing that in vitro neural determination does not occur when the ectoderm is previously treated with lectins, (50 ~g ml- 1 for 30 min) perhaps because of plasma membrane disorganization and/or saturation of the eventual receptors for "neural signal". This phenomenon is revers­ible after a 20 h resting period probably because the renewal of glycoconjugates partially reorganizes the normal plasma membrane structure and/or forms new free receptors.

Thus, the molecular organization of the plasma membrane of neural target cells at the gastrula stage, appears to be important in the mechanism of neural determination.

Table 2. Quantitative Analysis of the Fluorescence Intensity Against Time, on Treated Ectoblast. - FITe-SBA Treatment

Treatment

a) Lectin solution (50 /-lg ml- 1 )

Immediate observation • Observ. after 24 h

b) Experiment 3 3 mn treatment

c) Experiment 4 3 mn treatment + 24 h normal medium

d) Experiment 5 3 mn treatment + 24 h normal medium + 3 mn treatment

e) Experiment 6 24 h treatment

Fluorescence values

(mean ± s.d.)

23.99 ± 0.102 25.29 ± 0.293

5.01 ± 0.143

2.89 ± 0.052

9.30 ± 0.143

19.22 ± 0.533

N (Number of

measurements)

19 44

69

70

72

73

The Kolmogorov-Smirnov test pointed out significant differences between all these experiments at P < 0.01 (see Results). Similar results were obtained with PSA and LeA.

10 A. M. DUPRAT ET AL.

During gastrulation it is the inner surface of the ectoderm which undergoes the "neural signal". We observed a network of extra­cellular matrix (ECM) (Figure 5), on this inner surface (containing N Ac-a-D galactosamine, a-D-galactose, N-Ac glucosamine, mannose, fucose, a-D-mannose, a-D-glucose glycoconjugate molecules and fibro­nectin). Could this ECm playa role in this neural process? No such network of ECM was ever observed on the outer surface of the ecto­derm. According to the methodology described (Figure 1), the blast­oporal lip was associated in vitro on the outer surface of isolated ectoderm: neural determination occurred (45/45 positive cases) as for a normal blastoporal lip association on the inner surface of the explant.

The ECM on the inner surface of the ectoderm does not seem to play a role in neural determination. In agreement with Johnson (1977), Nakatsuji et al., (1982), Boucaut et al., (1983a,b), this matrix seems involved in cell migration and morphogenetic movements of gastrulation.

EARLY NEURAL DIFFERENTIATION

What are the acquired neural differentiating potentialities of the newly determined neural cells (at the gastrula stage)?

Our interest is focused upon this question. What will be the behavior of these neural cell precursors isolated from the normal embryonic environment (chiefly ulterior chordamesodermal influence)?

We adapted microsurgical and culture methods to study behav­ioral, morphological, neurochemical and biochemical characteristics of these neural cell precursors which were excised from late gastrula or early neurula embryos and isolated in primary cultures in uncon­ditioned medium.

Behavioral and Morphological Events

Microsurgery performed in appropriate ionic solutions allowed the separation of neural plate (central nervous system precursor) and of neural fold (peripheral nervous system precursor) from underlying chordamesoderm (Figure 7). These three components, then dissociated (Barth dissociating medium, EDTA, pH 8.6, 10 min.) were cultured (Barth medium) on a dried collagen substrate either in isolation or in combination.

- When neural plate + neural fold + chordamesoderm cells were cultured together (cocultures) (Figure 8-11) some neurons reaggre­gated in large clusters and their neurites formed thick fascicules. Other neurons remained in isolation and gave rise to a sparse network

EARLY EVENTS IN NEUROGENESIS

Neural «primordium»

Isolated

Neural plate

----------~------------Transverse sections

Isola ted

Neural fold

Fig. 7. Schema of experimental procedure.

11

Fig. 8. Coculture, 12 days culture: * neuron with neurite, • muscle cells, ...... melanocytes, .... aggregated epidermal cells.

12 A. M. DUPRAT ET AL.

of fine neurites. Besides glial cells, other cell types differen­tiated (myoblasts, chordal cells, melanoblasts, several epidermal cell types, fibroblasts •••• ).

- In cultures from neural plate, neurons reaggregated strongly and only some large clusters of cells with thick fascicules of neur­ites were observed (Figure 9).

- In cultures from neural fold, dispersed neurons or small aggregates were observed plus glial, melanoblast, fibroblast, epider­mal cell types (Figure 10).

This suggests that at a very early stage, the neuronal precur­sors have acquired distinctive adhesive properties.

An ultrastructural study (3, 5, 7, 10, 12, 15, 21 day cultures) showed the following normal neuronal differentiation in cocultures (Figure 11): typical neurons with characteristic neurotubules, neuro­filaments, dense core vesicles and clear vesicles, numerous syn­apses, ••• In cultures from neural plate and from neural fold, a normal ultrastructure differentiation was also observed.

The morphological events taking place in neuronal precursors isolated in vitro seem normal and identical to those observed in vivo.

Neurochemical and Immunological Aspects

a) Acetylcholinesterase activity. - The method of Karnovsky and Roots was used in the presence of iso-OMPA (10- SM). Control experi­ments were carried out with BW 284 CSI (substrates employed: acetyl­thiocholine or butyrylthiocholine).

In cocultures, in isolated neural plate cells and in isolated, neural fold cells, the presence of AChE was detected in neuronal cells as well as in muscle cells (Figure 12).

b) Catecholamine fluorescence: was observed in cocultures, in neural plate and neural fold cultures using the glyoxylic acid method. Some cells are GIF positive (Figure 13-14).

c) Immunofluorescence staining. - In 12 day old cocultures, release of fibronectin and collagen was demonstrated from fibroblast or epithelial cells: a strongly fluoresceing network can be seen with immunofluorescence using antibodies against fibronectin (from Dr. ,T. P. Thiery - chick antibodies and Dr. J. C. Boucaut - Axolotl anti­bodies). Staining with anti-FN antibodies gave negative results in 2 or 3 day cultures.

EARLY EVENTS IN NEUROGENESIS 13

Fig. 9. Culture from isolated neural plate cells (12 days culture): neurons reaggregated strongly, thick fascicules of neurites.

Fig. 10. Culture from isolated neural fold cells (12 days culture): dispersed neurons with thin neurites.

Presence of collagen was also demonstrated between spreading cells cultured on glass or plastic coverslips (without dried collagen).

Neurofilament proteins: Fluorescent staining with antibodies against 200 K and 68 K neurofilament antigens (from Dr. D. Paulin) showed a positive result. Similar experiments will be performed on isolated neural plate cells and isolated neural fold cells (Figure 15).

14 A. M. DUPRAT ET AL.

Fig. 11. Ultrastructural micrographies: (a) Neuron with neuronal process. (b) Transverse section through neurites with neurotubules and neurofilaments. (c) Dense core vesicles in neuronal processes.

Tetanus toxin (T.T.) binding molecules: fluorescent staining with anti-T.T. antibodies (from Dr. Bizzini) demonstrated the appear­ance and the location of T.T. binding markers on neuronal plasmalemma (Figure 16).

d) 3H-dopamine uptake: was observed radio-autographically in cocultures, in isolated neural plate cells and in isolated neural fold cells, using HPLC purified 3H-DA (5.10-7M, spec. activo 18 Ci/mM) in the presence of pargylin (10-4M) and with or without desmethylimipramine (Figure 17).

EARLY EVENTS IN NEUROGENESIS IS

Fig. 12. Coculture, 12 days culture: acetylcholinesterase staining.

Fig. 13. Catecholamine fluorescence observed using glyoxylic acid method, 6 day culture.

Neurotransmitter Synthesis

(This work was performed in collaboration with Dr. M. Weber, Lab. Pharmacol. Toxicol. Fond., CNRS, Toulouse).

Experiments carried out, using the method of Mains and Patterson (1973) to test the ability of cells to synthesize and accumulate acetylcholine (ACh) when provided with ~H-choline (Figure 18-19), indicated:

16 A. M. DUPRAT ET AL.

Fig. 14. Idem Figure 13, 14 days culture.

Fig. 15(a,b) Fluorescent staining observed with antibodies against 200 K neurofilament antigens.

1. - No ACh synthesis at early neurula stage; 2. - Isolated neural plate cells or isolated neural fold cells

primary culture synthesized a small quantity of ACh (after 1, 2, 3 weeks);

3. - ACh synthesis was increased in cocultures; 4. - Isolated chordamesodermal cells did not synthesize any ACh; 5. - ACh synthesis was increased in isolated neural plate +

underlying chordamesoderm cells, whereas a small quantity of ACh was detected in isolated neural fold + underlying mesoderm cells;

6. - No catecholamine (3H-tyrosine) synthesis was detected at early neurula stage.

EARLY EVENTS IN NEUROGENESIS

Fig. 16. Appearance of tetanus toxin (T.T.) binding molecules on neuronal processes. (a) phase contrast micrograph. (b) fluorescent staining with anti-TT antibodies.

v

/ \ . f ...... •

" ..

Fig. 17. 3H-dopamine uptake observed radioautographically (v: varicosities).

) 7

18

~ 1\1 v

"'0 '"' ., 10 ~ ! o E a.

<Il .5 o 5 .J:: o >. ... <Il o CO

A. M. DUPRAT ET AL.

-1 c. ,It".,

/1-------j--------! NP // ~NF

O~~~==~,===-~I 2 3

weeks

Fig. 18. Ability of cells to synthesize and store acetylcholine. NP, Isolated neural plate cells: NF, Isolated neural fold cells.

'" 1\1 v ... l2 "' ~

15

~10 <Il "0 E a. <Il c o .J:: .2 5 >. ... <Il o CO

I

I

,1---J • -- •

1 I

Co ultures

O~----------~----------+-~

2 weeks

Fig. 19. Ability of cells to synthesize and accumulate acetyl­choline. NP + eM (neural plate cells co-cultured with underlying chordamesodermal cells); NF + M (neural fold cells co-cultured with underlying mesodermal cells).

EARLY EVENTS IN NEUROGENESIS 19

Experiments are now being carried out to complete these results.

In conclusion, neural precursors isolated from the embryo at an early stage after neural determination and cultured in an uncon­ditioned medium, have acquired the ability to express the following neuronal differentiating properties:

- presence of neuronal phenotype and normal ultrastructural pattern;

- acetylcholinesterase and some GIF positive neurons (catecholaminergic neurons);

- differentiation of neuronal markers (neurofilament proteins, tetanus toxin binding molecules);

- catecholamine uptake (3H-dopamine); - synthesis and storage of acetylcholine.

At the gastrula stage, neuroblasts have acquired the ability to phenotypically differentiate, to synthesize, store and take up neuro­transmitters. Our results point out a stimulating effect of non­neuronal cells (chordamesoderm) in neurotransmitter synthesis.

REFERENCES

Boucaut, J. C., and Darribere, T., 1983a, Cell Differ., 12:77-83. Boucaut, J. C., and Darribere, T., 1983b, Cell Tissue Res.,

234: l35-145. Duprat, A. M., Gualandris, L., and Rouge, P., 1982, J.Embryol.Exp.

Morphol. 70:171-187. Grunz, H., Multier-Lajous, A. M., Herbst, R., and Arkenberg, G.,

1975, W.Roux's Archiv.Entwicklungsmech Org., 178:277-284. Grunz, H., and Staubach, J., 1979, W. Roux's Entwicklungsmech Org.,

186:77-80. Gualandris, L., and Duprat, A. M., 1981, Differentiation, 20:270-273. Gualandris, L., Rouge, P., and Duprat, A. M., 1983, J.Embryol.Exp.

Morph., 77:183-200. Jacobson, M., 1982, in: "Neuronal Development," N. C. Spitzer, ed.,

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256:6633-6640 Landstrom, U., and L6vtrup, S., 1977, Acta Embryol.Exp., 171-178. Landstrom, U., and L6vtrup, S., 1979, J.Embryol.Exp.Morphol.,

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20 A. H. DUPRAT ET AL.

Saxen L., 1977, in: "Cell and Tissue Interactions", J. W. Lash, M. M. Burger, ed~, Raven Press, N.Y., 1-9.

Saxen, L., Karkinen-Jaaskelainen, M., Lehtonen, E., Nordling, S., and Wartiovaara, J., 1977, in: "Cell Surface Interactions in Embryogenesis", G. Poste:- and G. L. Nicolson, eds., Nicolson, North-Holl. Div. ASP Biol.Med.Press.,Amst., 331-407

Takata, K., Yamamoto, Y., and Ozawa, R., 1981, W.Roux's Arch.Devel.Biol., 190:92-96.

Tiedemann, J., 1976, J.Embryol.Exp.Morphol., 35:437-444. Toivonen, S., 1979, Differentiation, 15:177-181. Yamamoto, K. Y., Ozawa, R., Takata, K., and Kitoh, J., 1981, W.Roux's

Arch.Devel.Biol., 190:313-319.

EARLY EMBRYONIC INDUCTION:

THE ECTODERMAL TARGET CELLS

Horst Grunz

Universitat Essen (FB 9 - Zoophysiologie) Universitatsstr. 5 4300 Essen 1, FRG

Spemann's and Hilde Mangold's famous transplantation experi­ment[1] showed that in amphibians competent ectoderm (presumptive epidermis) could be triggered to differentiate into neural tissues by the upper blastopore lip of early amphibian gastrulae. Spemann entitled this area with inducing activity as the organisator (organ­izer), because it organizes the formation of the central nervous system. In the following decades the interest of embryologists has been focused on the question, which factors located in the upper blastopore lip are responsible for the process of primary embryonic induction. Morphogenetic factors, which induce in competent ectoderm the formation of endodermal, mesodermal and neural derivatives, could be isolated from different sources[2-13]. A vegetalizing factor could be isolated in highly purified form from chicken embryos[14,15]. It is now generally accepted that these factors, which in contrast to growth factors can be entitled as determination factors, are protein in nature.

When talking about primary embryonic induction special interest must be focused not only on the inducing system (chordamesoderm), but also on the responding tissue (the ectodermal target cells). The interaction between inducing and reacting tissue is reciprocal (=wechselseitig)[16] and it is not possible to discuss phenomena and processes of the target cells without mentioning the inducing tissue. Vogt's vital dye staining experiments in 1923[17] showed that the chordamesoderm invaginates and forms a layer of tissue, also called the archenteron roof, in the very vicinity of the overlaying neuro­ectoderm. The precise molecular interactions between the inducing chordamesoderm and the ectodermal target cells leading to the formation of the central nervous system are still a matter of specu­lation. In contrast to other systems (for example the insulin-model)

21

22 H. GRUNZ

the inducer(s) (neuralizing factor(s)), responsible for primary embryonic induction is not available in highly purified form, a fact which has so far prevented the study of the primary molecular events during induction between the 'natural' inductor and the receptors of the target cells. However, I shall report below about recent prom­ising approaches of several authors, which can be considered as a first weak illumination of the 'black box', as Paul Weiss[18] des­cribed the ectoderm.

it is well-known from various experiments that the ectoderm is a totipotent tissue [see 19,20 for review]. The segregation into mesoderm, endoderm and ectoderm takes place very early in the devel-0pment[21-23]. The competent ectodermal target cells are able to differentiate into almost all embryonic structures under the influ­ence of appropriate inducers. Isolated untreated ectoderm develops into so called atypical epidermis. However, with exception of the linear alignment, which in normogenesis depends on the underlaying mesenchyme[24], this 'irregular' epidermis is ultrastructurally very similar to 'normal' epidermis[25,26J. The ectoderm develops into a tissue containing a considerable amount of cells covered with well differentiated cilia. Therefore, ectoderm cannot be described as tissue without any self-differentiation tendencies. The reaction of the target cells (competent ectoderm) to inducing stimuli can be tested by three main methods:

1. the implantation method[27], 2. the sandwich-techniqueL28] , 3. the hanging-drop-method or filter-platelet-method[29-31].

In the two last techniques, isolated ectoderm (the target cells) can be exposed to inducers under in vitro conditions eliminating the influence of those tissues, which in normogenesis are located in the vicinity of the ectoderm.

COMPETENCE OF THE TARGET CELLS

Holtfreter and Hamburgerl32] described competence as "the physi­ological state of a tissue which permits it to react in a morpho­genetically specific way to determinative stimuli". The problem of competence is extensively reviewed in Saxen and Toivonen[19J and Nakamura and ToivonenL20]. It could be shown by in vivo and in vitro experiments that the competence of the ectodermal target cells to react to neural and mesodermal inducing stimuli decreases gradually during gastrulation and is finally lost in the early neurula[33-36]. In earlier experiments we showed that the period of mesodermal com­petence could be prolonged by the inhibition of protein synthesis[37]. However, these results cannot answer the question as to which factors at the molecular level are responsible for the loss of competence. It could be argued that the treatment with cycloheximid also inhibits

EARLY EMBRYONIC INDUCTION 23

the synthesis of restriction factors, proteins in nature, which will cause a gradual limitation of the expression potentials of the genome during gastrulation. The loss of competence as a result of a gradual decrease of the permeability of the plasmamembrane for inducing factors during gastrulation is less probable. So far a decrease or loss of receptors for inducers (neuralizing factor) cannot be absol­utely excluded.

INFLUENCE OF IONS AND CYCLIC AMP AND THE PHENOMENA OF AUTONEURALIZATION, SPECIES SPECIFICITY, INITIAL CELL MASS, PATTERN FORMATION

Autoneuralization effects, i.e. the differentiation of ectoderm into neuroid or archencephalic (forebrain) structures, can be observed after the treatment of competent ectoderm under non­physiological conditions (high pH, Ca-free solution, hypotonic medium, urea etc.). Especially the change in the ionic environment of the cultured ectoderm can cause an autoneuralization[35,38,39]. Such effects are species specific in such that autoneuralization can be brought about in certain spawns of Ambystoma mexicanum under culture conditions, which never cause neural differentiations in isolated ectoderm of Triturus pyrrhogaster[40J or Triturus alpestris. Barth's experiments were carried out with Ambystoma mexicanum ectoderm. This species cannot be considered as a suitable test material for studies of neural inductions. For this reason also experiments of L6vtrup et al., and Wahn and coworkers must be dis­cussed with great care and precaution[41,42,43J. They reported that cyclic AMP should be able to stimulate ectoderm of Ambystoma to differentiate into neural tissues. The authors also used very small pieces of ectoderm, a -condition, which can (because of an unphysio­logical leakage of the cells) significantly influence the results of differentiation[44-47]. Using very small pieces of ectoderm Niu demonstrated contradictory to nearly all the results of other authors (showing that inducers are protein in nature) that RNA should be responsible for neural induction.

We have shown that in Triturus alpestris ectoderm cyclic AMP or their mono- or dibutyrylic derivatives cannot evoke neural induc­tions[48]. Triturus alpestris can be considered as a species, which is highly resistent to autoneuralization effects. Using Triturus alpestris ectoderm as test system we have shown that there are no differences in the sodium, potassium and calcium content of induced and non induced cells within the first 24 h after the beginning of the incubation with vegetalizing factor. Treatment of Trit¥ru~ alpestris ect~erm with ouabain, causing a change of the Na /K ratio, the Ca ionop¥ore+A 23187~nd Holtfreter solution with reduced amounts of Na , K and Ca could not evoke inductions[49].

24 H. GRUNZ

Concerning the neuralization effects by changes of ionic concen­trations or/and the pH of the culture medium it could be argued that at least in Ambystoma and also in a few other species certain steps in the chain of events leading to neuralization can easily be started by unspecific means, which in normogenesis is triggered in a specific way by the 'natural' inducer. That means that neuralization can more or less be easily evoked in competent ectoderm of all species by different unspecific factors, high or low pH, Ca-free medium, urea, CO~-shock or mechanical damage of the tissue or treatment with meth­yleneblue[50]. On the other hand with exception of lithium chloride no other unspecific substance or unphysiological treatment is able to cause mesodermalization or endodermalization of competent ectoderm[36,51,52]. The autoneuralization experiments and the results after LiCI-treatment indicate that the target cells appar­ently contain neuralizing and mesodermalizing activity, which is located in the living ectodermal cell in a masked form. By killing of the ectoderm with different treatments (heating or treatment with ethanol) the ectoderm acquires neuralizing inducing activity. After the treatment of ectoderm with phenol the ectoderm develops weak mesodermal inducing activity[53,~4].

Very important factors for the formation of specific differen­tiation patterns are the exposure time of ectoderm to the inducer and the species used. Johnen[55,56] has shown that Ambystoma ectoderm will form archencephalic brain structures after 5 min contact to archenteron roof, while the realization of similar structures in Triturus vulgaris needs at least 4 h interaction. Increasing expo­sure periods in both species resulted in the formation of deuter­encephalic and spinocaudal tissues. In contrast to Triturus we could observe in Ambystoma that chordamesoderm adheres to competent ectoderm in a much shorter period after the combination of both tissues [Grunz, unpublished results]. This could explain the faster reactivity of Ambystoma in Johnen's experiments. Using vegetalizing factor as an inducer a prolongation of the exposure time and an increase of the inducer concentration will result in a typical change of the pattern of induced endodermal and mesodermal structures[57j, Results of different experimental approaches indicate that the veg­etalizing factor causes a labile vegetalization of the ectoderm. The formation of endoderm and/or mesoderm derived tissues depends on secondary inducing interactions and cell communication[47,58,59].

For this information transfer between cells within the same layer and pattern formation gap junctions must be taken into con­sideration[60].

CHANGE OF CELL AFFINITY AND CELL SURFACE ARCHITECTURE OF THE ECTODERMAL TARGET CELLS AFTER INDUCTION

For several decades the reaction of the target cells to inducing stimuli could only be identified after several days culture (up to 14

EARLY EMBRYONIC INDUCTION 25

days, endoderm even much longer) after the beginning of the treatment with inducers. Macroscopic observation and histological analysis were the only methods used to find out the final result of the induc­tion process. During this period many secondary processes have taken place in the target cells after the primary signal of the inducer. Therefore the primary events during the very beginning of the induc­tion are of great interest (see next section). Since these processes are very complex so far only few but promising informations about this period are available. In the last years we acquired some knowl­edge about the processes, which in contrast to histological analysis can be identified several hours after the beginning of induction. It has been shown that cell affinity changes within 24 h after induction with vegetalizing factor. The induced target cells acquire in con­trast to untreated ectodermal cells an affinity to endoderm. This phenomena can be observed by changes of morphogenetic movements of the target cells in whole embryos[61] or by comparison of the sorting out behavior of induced and non induced ectodermal cells[31]. These processes become visible macroscopically within 24 h of the beginning of the treatment with inducer. During a period of 24 h a change of the surface architecture of the target cells can also be observed by scanning electron microscopy[26]. Furthermore the ectodermal target cells after induction show a reduced binding capacity for cat ionized ferritin[62]. Already about 6 h after the treatment with highly purified vegetalizing factor induced cells can be distinguished from untreated cells in their contact behavior to plastic culture dishes[63]. While untreated ectodermal cells (dissociated single cells) stay free on tissue culture dishes in spheric form, induced ectoderm takes up contact with the substratum within 6 h and the cells show fibroblast-like morphology. This demonstrates that ap­parently the structure of the plasma membrane changes very rapidly after the induction, which is responsible for the adhesion of the cells to the substratum.

POSSIBLE MODE OF INTERACTION BETWEEN THE TARGET CELLS AND INDUCING FACTORS

It is still unclear how in normogenesis the inducing signals are transferred from the inducing tissue (chordamesoderm) to the over­laying target cells (neuroectoderm). By in vitro transfilter experi­ments it can be concluded that a freely diffusible neuralizing factor is migrating to the reacting tissue[64-66]. Our electron microscopic observations have shown that cell contacts exist between the inducing and reacting tissue during the period of gastrulation[67]. Toivonen and Saxen[66] argued that for primary neuralization (archencephalic induction) no cell contacts are needed. On the other hand close cell-to-cell contacts are postulated for the formation of the more caudal neural structures (Nieuwkoop's term transformation).

26 H. GRUNZ

However, the in vitro transfilter experiments cannot absolutely exclude the possibility that in normogenesis short distance diffusion of a neuralizing factor takes place in zones of close cell contacts between the inducing chordamesoderm and the reacting ectodermal target cells. In any case the mode of interaction of the target cells with different inducers is quite different. Recent experiments with inducing factors immobilized on BrCN-Sepharose or BrCN-Sephadex show that the vegetalizing factor apparently must be internalized to become biologically active, while the neuralizing factor (causing archencephalic brain structures) seems to interact with the plasma membrane of the target cells[68,69]. Autoradiographic studies with 3H-acetylated vegetalizing factor have shown that the inducer distri­butes quickly (within 4 h) in the intercellular spaces of the target tissue followed by a gradual internalization. Six to twenty hours after the beginning of the treatment with vegetalizing factor silver grains can also be found over the cytoplasma and the nuclei [Grunz, unpublished results]. However, the precise mode of the action at the molecular level is still obscure. In the case of the interaction of the neuralizing factor with the target cells some promising infor­mation exists. In the laboratory of Dr. Tiedemann fractions with neuralizing activity have been isolated from Xenopus laevis embryos. This activity is thought to be stored in ribonucleoprotein particles, which in association with the cytoskeleton could leave the inducing cells (chordamesoderm) via exocytosis (coated vesicles, coated pits). However, the factor does not necessarily remain bound to particles but could leave the chordamesoderm cells in the form of free dif­fusible molecules. Apparently the inducing factor is not an inte­grated protein of the plasma membrane (cited in Tiedemann[70]). Coated pits and coated vesicles were observed by Grunz and Staubach[67] in chordamesodermal cells. Such structures also play an important role in other systems, for example for the removal of the insulin receptor[71,72], and also in exocytosis[73]. After exocy­tosis the neuralizing factor could migrate from the inducing tissue to the target cells. Extracellular matrix (consisting of proteogly­cans) could aid this transport. This view is supported by the fact

'that from this extracellular material a neuralizing factor (protein or glycoprotein) has been extracted[70]. How after its migration the neuralizing factor interacts with receptors on the plasma membrane of the target cells is still a matter of speculation. Also no experi­mental data are available about the information transfer from the plasma membrane to the genome. The view that neuralization is initi­ated by the interaction of neuralizing factors with specific recep­tors on the plasma membrane is also supported by recent results with Concanavalin A. Takata and coworkers[74] have shown that ectoderm treated with certain lectins (Concanavalin A or Ulex europeus agglutinin) differentiates into archencephalic brain structures. Con A reacts with a-D-mannose or a-D-glucose, UEA with fucose residues of glycoproteins on the plasma membrane. It could be argued that these lectins interact with the same receptors on the plasma membrane, which are specific for the 'natural' inducer. On the other

EARLY EMBRYONIC INDUCTION 27

hand Duprat[75] has shown that in ectoderm pretreated with SBA, PSA, LCA followed by the exposure to the inducing stimulus of upper blastopore lip, neural inductions are strongly inhibited. We obtained similar results two years ago by the pretreatment of ectoderm with wheat germ agglutinin (WGA) [Grunz, unpublished results]. Takata and coworkersl74] have shown, excluding the possi­bility of toxic effects of the lectin by preventing its internaliz­ation by coupling to Sepharose, that immobilized Con A is still able to induce neural structures in competent ectoderm. The contradictory results of Duprat's group and Takata's group are explained by Takata on the basis of binding studies with DBA and Con A. He pointed out that a tremendous amount of ferritin-conjugated DBA, which shows no significant inducing activities, binds to the surface of ectodermal target cells, while ferritin-conjugated Con A molecules are much less frequent. His explanation for the different results is that Con A and UEA in contrast to other lectins, which could cause an unspecific perturbation of the plasma membrane organization, could interact with glycoproteins of the plasma membrane resulting in a specific modu­lation of receptor positions, which activate further steps in the reaction chain leading to neural induction. Using Xenopus laevis ectoderm we also observed a neuralizing effect after treatment with 100 ~g/ml Con A for 1 h (Figure 1). However, the percentage of cases forming more voluminous archencephalic brain structures is increased by the incubation in cytochalasin B containing culture medium (10 ~g/ml, for 5 min), which is an inhibitor of microfilament organiz­ation, prior to the Con A-treatment (Figure 2). On the other hand the reaction of the target cells to the vegetalizing factor is de­creased after cytochalasin treatment. Toxic effects of cytochalasin B could be excluded by control series. The results can be explained by the different mode of action of the vegetalizing and neuralizing factor. That the influence of the vegetalizing factor is inhibited could mean that the processes of endocytosis necessary for the internalization and biological activity of the vegetalizing factor are inhibited. On the other hand apparently the steric arrangement of the receptors within the plasma membrane needed for the inter­action with the neuralizing factor, is not disturbed by the distor­tion of the microfilaments, which are considered to be correlated with (glyco) proteins of the plasma membrane. The effect of the cytochalasin B can also be explained in another way. After treatment with this drug the cells temporarily round up (during the period of competence: about 5 h at 20°C in Xenopus), which results in an increase of free cell surface (including the former intercellular contact areas) accessible to Con A. Takata also pointed out that a certain amount of Con A must be kept bound to the cell surface for a certain period of time for neural stimulation of the target cells. The weaker stimulation of .ectoderm by Con A omitting the cytochalasin B treatment can be explained in our experiments with Xenopus laevis ectoderm. In contrast to urodelean ectoderm (Takata's group used Triturus pyrrhogaster) the anuran ectoderm consists of two distinct cell layers[76]. In isolated ectoderm only the inner ectoderm layer

28 H. GRUNZ

Fig. 1. Xenopus ectoderm (both layers, see also Figure 3 C) has been treated with Con A (100 ~g/ml for 1 h). A small brain structure (neu) has been formed by the ectoderm. su = succer.

Fig. 2. Xenopus ectoderm (both layers) has been treated with cyto­chalasin B (10 ~g/ml for 5 min) prior to the incubation with Con A (100 ~g/ml, 1 h). In contrast to the series without cytochalasin B treatment (see Figure 1) the ectoderm has formed relatively large brain structures (neu).

cell layers[76]. In isolated ectoderm only the inner ectoderm layer is exposed to the lectin, while the inner side of the outer layer is protected by the neighboring inner ectoderm layer (Figure 3). The former outer side of the egg (outer surface of the superficial ectoderm layer) binds much less Con A [74]. This means that in

EARLY EMBRYONIC INDUCTION 29

Xenopus laevis only about half of the ectodermal cells of the explant are exposed to the Con A. Apparently in some explants this is below the critical value needed for a neural stimulation of the ectodermal target cells. Furthermore it must be mentioned that in several cases the explants lose considerable parts of the inner ectodermal cells, which can be easily recognized by their low amount of pigment. Such a loss of inner ectodermal cells (segregation of the inner from the outer ectoderm) could also be observed when sandwiches were cultured in modified Leibovitz-Medium L-15 [76].

Several authors (cited in Asashima and Grunz[76]) concluded from vital dye staining and microsurgical experiments that the central nervous system of anurans develops mainly from the inner ectodermal layer. In normogenesis only the inner layer comes into contact with the inducing underlaying chordamesoderm during gastrulation. As­suming that the neural induction takes place by close cell-to-cell contacts and/or short distance diffusion of a neuralizing factor, it would be reasonable to assume that only the inner ectoderm layer is stimulated to form neural tissues. On the other hand the outer ectoderm layer should also be able to form neural structures, when the inner layer, which could act as a barrier, is removed and the outer layer alone is exposed to the inducing stimulus. The inner and outer ectoderm layer can be separated mechanically by fine glass needles[76]. Recent preliminary results indicate that only the inner ectoderm layer reacts to Con A treatment (100 ~g/ml for 1 h) with the formation of neural (archencephalic) structures.

However, we have shown using upper blastopore lip as inducer that the outer ectoderm layer is also able to form well developed brain structures[76]. Our results from homoplastic transplantations were, of course, of limited significance, because we could not dis­criminate neural inductions as a result of self-differentiation of the living inducer (blastopore lip) on the one hand, and neural tissue which has been formed from the target cells on the other hand. However, very recent xenoplastic experiments with Triturus vulgaris blastopore lip as inducer and outer ectoderm of Xenopus laevis clearly show that the outer ectoderm is able to form well differ­entiated brain structures (Figure 4, compare with Figure 5 inner ectoderm as reacting tissue). The inducing and reacting tissue and their derivatives can easily be distinguished, because Xenopus laevis nuclei with two nucleoli as markers are much smaller than Triturus vulgaris nuclei. The lower reactivity of the outer ectoderm to Con A can be explained as follows. Apparently the inner ectoderm layer binds a much larger amount of Con A than the outer ectoderm. Since only the inner ectoderm forms neural structures under our experi­mental conditions (100 ~g/ml Con A for 2 h) it could be argued that the amount of Con A bound to the outer ectoderm is not sufficient for a neuralizing effect. This concept is corroborrated by the fact that a small piece of ectoderm (both layers) treated with 100 ~g/ml Con A for 2 h is not able to differentiate or cause neural inductions, when

30 H. GRUNZ

Xenopus [aevis Triturus vulgaris

t r-"-'" wilh Concol'lCllldill A - - - - \ - - - . - . r- . -----.. -. ....."'''''M I

I I " , ' ~comt:iMl.otion ~ ~~~~

I I' I inner; outef or both ... ectodermol layers

.... . __ _ ~ ___ __ _ rinSing inlO<.-D-monnose ---- 1 ---- · ~ ~

<:::> <:::> <:> e I I I ,. ___ ! _ ___ _ cUlture. for 5 days at 18" - +- - - - - -+ --- -_.

ciliated epidermis neural ino...c:t ion neural indu.ction neural derivatives formed

A B c

by all ectodefmol loyers of Xenopu5

o

Fig. 3 . Schematic representation of the experiments performed with Xenopus laevis and Triturus vulgaris. A,B,C. After the treatment of the outer (A), inner (B) or both (C) ectodermal layers with Concanavalin A (100 ~g/ml for 1 h) sandwiches were formed from two caps of ectoderm. Only series Band C formed neural derivatives under our experimental conditions, which apparently depends on the amount of Con A bound to the single cell and the explant (small arrows ~) . Treatment of ectoderm with cytochalasin B (10 ~g/ml for 5 min) prior to the incubation with Con A results in an increase of the per­centage and the size of neural structures. D. Upper blastopore lip of Triturus vulgaris has been isolated in the early gastrula and kept for 12 h in culture prior to the combination with either outer, inner or outer + inner ecto­derm layers of Xenopus laevis (xenoplastic combination). The Xenopus ectoderm of all three series has been induced by the Triturus blastopore lip and has formed well differen­tiated brain structures.

EARLY EMBRYONIC INDUCTION 31

Fig. 4. Outer ectoderm of Xenopus laevis and blastopore lip of Triturus vulgaris 5 days after combination (see also Figure 3 D). Brain structures (neu) and epidermis (ep) have developed from Xenopus ectoderm. The inducing tissue (blastopore lip = bl) can clearly be distinguished from the reacting tissue (ectoderm). Xenopus nuclei (small arrow), containing two nucleolei, are much smaller than the nuclei of Triturus cells (large arrow).

Fig. 5. Inner layer of Xenopus ectoderm and blastopore lip of Triturus 5 days atter combination. The Xenopus ectoderm has differentiated into epidermis (ep) and archencephalic brain structures. In this section an optic vesicle (opt) with tapetuum (ta) and lens (Ie) is shown. bl = Still histo­logically unorganized, yolk-rich derivatives of the blasto­pore lip of Triturus. In contrast to Xenopus the develop­ment of Triturus is much slower. Histologically well dif­ferentiated structures can be observed after about 12 days culture at 18°C (Xenopus: about 5 days).

32 H. GRUNZ

wrapped by two caps of untreated competent ectoderm (both layers) in the form of a sandwich (Figure 6). Apparently the amount of Con A bound to a certain number of cells (initial cell mass, exposure time of Con A) plays an important role for the realization of well differ­entiated neural structures. Furthermore there could exist a close relationship between exposure time and reactivity of the target cells during a specific period within the cell cycle. Taken together we can conclude that in normogenesis the neural induction takes place by the interaction of chordamesoderm and ectodermal target cells in zones of close cell-to-cell contact by short distance diffusion of neuralizing factor.

It should be mentioned that Harris and Zalik[77] have isolated an endogeneous lectin with a-D-galactoside binding specificity in Xenopus laevis, which has been implicated to play a ro~e in cell­to-cell and cell-to-substrate adhesiveness. The activ~y of this lectin protein in nature depends on the presence of Ca • ++his+fact could explain why amphibian cells can be dissociated by Ca /Mg -free media and the reaggregation is prevented by inhibitors of de novo protein synthesis[78,79]. Whether this lectin or other endogen­ous lectins possess neuralizing inducing activity has not yet been tested. It could be that endogenously synthesized proteins struc­turally similar to lectins play a role in the inducing process by interaction with glycoprotein receptors on the target cells.

Fig. 6. A piece of Xenopus ectoderm (both layers = imp), treated with Con A (100 ~g/ml for 2 hours), was wrapped by untreated ectoderm (both layers) in the sandwich method. The ectoderm has differentiated into ciliated epidermis only. Similar results were obtained after implantation of untreated ecto­derm. This means that the possibility of self-differen­tiation of Xenopus ectoderm into mesodermal, endodermal and neural derivatives without induction processes can be ex­cluded by this experiment and the results shown in Figures 4 and 5 (experiments with inducer).

EARLY EMBRYONIC INDUCTION 33

The information transfer of inducing signals from the plasma membrane to cell constitutents and the nucleus resulting in a repro­gramming of the target cells is completely obscure. Experiments with 125J labelled vegeta1izing factor have shown that the inducer has a high affinity to single and double stranded DNA[80].

Results in other systems could be helpful as working concepts for further experimental work. Insulin binds to specific receptors on the plasma membrane of the target cells, which causes the regu­lation of a variety of metabolic processes (cited in Roth and Casse11[81]. Recent results suggest that the insulin receptor is itself a protein kinase[81]. Protein kinases, which selectively phosphorylate tyrosine residues, are thought to be correlated with the regulation of normal and neoplastic growth. Such kinases are associated with viral transformation[82]. Tyrosine residue kinase activity is also associated with growth factor receptors. The epidermal growth factor with associated kinase activity is thought to phosphorylate cellular proteins on tyrosine residues (cited in Rubin and Earp[83]). The transforming gene products of Rous sarcoma virus and other tumor viruses are protein kinases, which are associated with the N-termina1 end to the cytoplasmic side of the plasma membrane and which phosphorylate tyrosine residues in cellular fractions and in cultured cells (cited in Rubin and Earp[83]). These results, obtained in other systems, should be taken into consider­ation for future concepts and studies of the processes taking place on or in the ectodermal target cells during primary embryonic induc­tion.

Summarizing we can postulate the following chain of events during neural induction. An inducing factor is synthesized in the inducing chordamesoderm, possibly a constituent of ribonucleoprotein particles. The factor could leave the inducing tissue by exocytotic processes in cooperation with the cytoskeleton system. The extracel­lular matrix (ECM) between the inducing and reacting tissue could support the transport of the inducer to the target cells. Specific glycoprotein receptors, components of the plasma membrane of the ectodermal target cells, will interact with inducer molecules fol­lowed by a cascade of reactions within the target cells leading to neural differentiation.

REFERENCES

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2. S. Toivonen and T. Kuusi, Imp1antationsversuche mit in verschiedener Weise vorbehandelten abnormen Induktoren bei Triton, Ann.Soc.Zool.-bot.fenn.Vanamo. 13:1-19 (1948).

34 H. GRUNZ

3. T. Yamada, Regional differentiation of the isolated ectoderm of the Triturus gastrula induced through a protein extract, Embryologia, 1:1-20 (1950).

4. T. Yamada, Induction of specific differentiation by samples of proteins and nucleoproteins in the isolated ectoderm of Triturus gastrulae, Experientia, 14:81-87 (1958).

5. T. Kuusi, Uber die chemische Natur der Induktionsstoffe im Implantationsversuch bei Triton, Experientia, 7:299-300 (1951a).

6. T. Kuusi, Uber die chemische Natur der Induktionsstoffe mit besonderer Berucksichtigung der Rolle der Proteine und der Nucleinsauren. Diss., Helsinki, Ann.Soz.Zool.-bot.fenn.Vanamo 14:1-98, (1951b).

7. H. Tiedemann and H. Tiedemann, Versuche zur chemischen Kennzeichnung von embryonalen Induktionsstoffen, Hoppe-Seyler's Z.Physiol.Chem., 306:7-32 (1956).

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9. H. -H. Chuang, Effects of alcohol and heat treatment on inductive ability, Acta.Biol.Exp.Sinica, 8:3-4 (1963).

10. I. Faulhaber, Anreicherung des vegetalisierenden Induktions­faktors aus der Gastrula des Krallenfrosches (Xenopus laevis) und Abgrenzung des Molekulargewichtbereiches durch Gradien­tenzentrifugation, Hoppe-Seyler's Z.Physiol.Chem., 351: 588-594 (1970).

11. I. Faulhaber and L. Lyra, Ein Vergleich der Induktionsfahigkeit von Hullenmaterial der Dotterplattchen- und Microsomen­fraktion aus Furchungs- sowie Gastrula- und Neurulastadien des Krallenfrosches Xenopus laevis, Wilhelm Roux' Archives, 176:151-157 (1974).

12. J. Kawakami, S. Noda, K. Kurihara, and K. Okuma, Vegetalizing factor extracted from the fish swimbladder and tested on pre­sumptive ectoderm of Triturus embryos, Wilhelm Roux' Archives 182:1-7 (1977).

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EARLY EMBRYONIC INDUCTION 35

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30. T. Yamada and K. Takata, A technique for testing macromolecular samples in solution for morphogenetic effects on the isolated ectoderm of the amphibian gastrula, Develop.Biol., 3:411-423 (1961) •

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36 H. GRUNZ

34. P. D. Nieuwkoop, Neural competence of the gastrula ectoderm in Ambystoma mexicanum. An attempt at quantitative analysis of morphogenesis, Acta Embryol.Morph.Exp., 2:13-53 (1958).

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36. H. Grunz, Experimentelle Untersuchungen fiber die Kompetenzver­haltnisse frfiher Entwicklungsstadien des Amphibein-Ektoderms, Wilhelm Roux' Archives, 160:344-347 (1968).

37. H. Grunz, Abhangigkeit der Kompetenz des Amphibien-Ektoderms von der Proteinsynthese, Wilhelm Roux' Archives, ~65:91-102 (1970).

38. L. G. Barth, Neural differentiation without organizer, J.Exp. Zool., 87:371-383 (1941).

39. L. G. Barth and L. J. Barth, The sodium dependence of embryonic induction, Develop.Biol., 20:236-262 (1969).

40. S. Karasaki, On the mechanism of the dorsalization in the ecto­derm of Triturus gastrulae caused by precytolytic treatments. I. Cytolytical and morphogenetic effects of various injurious agents, Embryologia, 3:317-334 (1957).

41. S. L~vtrup, U. Landstrom, and H. L~vtrup-Rein, Polarities, cell differentiation and primary induction in the amphibian embryo, Biological Reviews, 53:1 (1978).

42~ S. L~vtrup, Epigenetic mechanisms in the early amphibian embryo. Cell differentiation and morphogenetic elements, Biological Reviews, 58:91-130 (1983).

43. H. L. Wahn, L. E. Lightbody, and T. T. Tchen, Induction of neural differentiation in cultures of amphibian undetermined presumptive epidermis by cyclic AMP derivatives, Science, 188:366-369 (1975).

44. G. V. Lopashov, Die Entwicklungsleistungen des Gastrulaektoderms in Abhangigkeit von Veranderungen der Masse, Biol.Zbl., 55: 606-615 (1935).

45. W. B. Muchmore, Differentiation of the trunk mesoderm in Amblystoma maculatum. II. Relation of the size of presumptive somite explants to subsequent differentiation, J.Exp.Zool.; 134:293-310 (1957).

46. E. M. Deuchar. Effect of the cell number on the type and stability of differentiation in Amphibian ectoderm, Exp.Cell Res., 59:341-343 (1969).

47. H. Grunz, Change of the differentiation pattern of amphibian ectoderm after the increase of the initial cell mass, Wilhelm Roux' Archives, 187:49-57 (1979).

48. H. Grunz and H. Tiedemann, Influence of cyclic nucleotides on amphibian ectoderm, Wilhelm Roux' Archives, 181:261-265 (1977).

49. G. Siegel, H. Grunz, and H. Tiedemann, (in preparation). 50. Waddington, Needham, Brachet, Studies on the nature of the

amphibian organization centre. Ill. The activation of the evocator, Proc.Roy.Soc •• London, 120:173-198 (1936).

51. Y. Masui, Alteration of the differentiation of gastrula ectoderm

EARLY EMBRYONIC INDUCTION 37

under influence of lithium chloride, Mem.Konan Univ., Sci.Ser., 4:79-102 (1960).

52. D. O. E. Gebhardt and P. D. Nieuwkoop, The influence of lithium on the competence of the ectoderm in Ambystoma mexicanum, J.Embryol.Exp.Morph., 12:317-331 (1964).

53. J. Holtfreter, Der EinfluS thermischer, mechanischer und chemischer Eingriffe auf die Induzierfahigkeit von Triton­Keimteilen, Wilhelm Roux' Archives, 132:225-306 (1934).

54. H. Tiedemann, U. Becker, and H. Tiedemann, Uber die primaren Schritte bei der embryonalen Induktion, Embryologia, 6:204-218 (1961).

55. A. G. Johnen, Experimental studies about the temporal relation­ships in the induction process. I. Experiments on Amblystoma mexicanum, Proc.Acad.Sci.Amst.Ser.C., 59:554-561 (1956).

56. A. G. Johnen, Experimental studies about the temporal relation­ships in the inducing process. II. Experiments on Triturus vulgaris, Proc.Acad.Sci.Amst.Ser.C., 59:652-660 (1956).

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68. Hildegard Tiedemann and J. Born, Biological activity of vegetalizing and neuralizing inducing factors after binding to BAC-Cellulose and CNBr-Sepharose, Wilhelm Roux' Archives, 184:285-299.

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71. J. Y. Fan, J. -L. Carpentier, P. Gorden, E. van Obberghen, N. M. Grunfeld, and L. Orci, Receptor-mediated endocytosis of insulin: Role of microvilli, coated pits, and coated vesicles, Proc.Natl.Acad.Sci. USA, 79:7788-7791 (1982).

72. J. L. Goldstein, R. G. W. Anderson, and M. S. Brown, Coated pits, coated vesicles, and receptor mediated endocytosis, Nature, 279:679-682 (1979).

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CLONAL RESTRICTIONS DURING EARLY DEVELOPMENT

OF THE FROG EMBRYO

Marcus Jacobson

Department of Anatomy, University of Utah School of Medicine, Salt Lake City UT 84132

INTRODUCTION

I shall define clonal restriction as the restriction of develop­mental activities of cells determined by their origin from specified progenitors, and shared by all cells descended from the same pro­genitors. Three kinds of clonal restriction will be dealt with in this article: restriction of mingling in the embryo (Jacobson, 1982, 1983); restriction of mingling of embryonic cells in tissue culture (Jacobson and Klein, in preparation); restriction of formation of connections between neurons and their target cells (Moody and Jacobson, 1983).

Previous work has shown that there are clonal restrictions of cell dispersal and mingling during normal development in Xenopus (Jacobson, 1983). Clonal analysis, using intracellularly injected horseradish peroxidase (HRP) as a heritable intracellular tracer, has shown that restriction of cell mingling originates at the midblastula stage when four ancestral groups of balstomeres can be identified on each side called anterior-median (AM), anterior-lateral (AL), posterior-medial (PM) and posterior-lateral (PL) (Figure 1; Jacobson 1983). The A}1 groups fuse in the midline so that there are finally a total of seven groups. Each group gives rise to descendants which exclusively populate a morphological domain, called a compartment, at later stages of development (Jacobson, 1982, 1983). Descendants of each ancestral cell group mingle with one another in the same com­partment but do not cross compartment boundaries. No mingling occurs between cells derived from different ancestral cell groups.

In other words, there are clonal restrictions of cell mingling based on the ancestry of cells from different blastomere groups. It

39

40

VENTRAL

M. JACOBSON

512-CELL 8T AGE

e­m ." -t

Fig. 1. Fate map (anterior oblique case: The star is at the animal pole of the embryo which becomes the head end, the arrow points to the vegetal pole, which becomes the rear end) or blastomeres at the S12-cell stage. Blastomeres are shown on the right side of the embryo and the regions of the central nervous system populated by their descend­ants is shown by symbols on the left side of the embryo. Blastomeres belong to groups that populate compartments: AM, anterior-median group populates ventral telencephalon, diencephalon, mesencephalon and retina on both sides; AL, anterior-lateral group, populates the dorsal telencepha­lon, diencephalon, mesencephalon and retina on the same side as well as the cranial neural crest; PM, posterior­medial group populates the ventral rhombencephalon and spinal cord on the same side; PL, posterior-lateral group populates the dorsal rhombencephalon and spinal cord on the same side as well as the trunk neural crest.

CLONAL RESTRICTIONS DURING DEVELOPMENT 41

is important to emphasize that these clonal restrictions of cell mingling start before gastrulation and thus must play a role in segregating cells of different clonal origin during gastrulation movements. The restrictions of cell mingling start operating before the formation of primary germ layers and before the appearance of different cellular phenotypes. Many types of cells belonging to all the primary germ layers differentiate in each compartment. The compartment is, therefore, a morphological unit, not related to histological patterns.

I do not regard the compartment hypothesis as finally proven. Much evidence of clonal restriction of cell mingling has been obtained (Jacobson, 1983) as well as evidence of compartmental re­strictions of neuronal connectivity (Moody and Jacobson, 1983). However, more work remains to be done to discover the initial con­ditions in the egg and early cleavage stages which result in the foundation of blastomere groups from which the compartments orig­inate. Much work remains to discover the nature of the compartment­specific cellular properties and the ways in which the are expressed as restrictions of cell mingling.

METHODS

These observations were made on embryos of the clawed frog, Xenopus laevis. This species has many advantages: fertilized eggs are obtained easily, they are relatively large (about 1.2mm dia­meter), they develop rapidly, reaching tailbud stages about 24 hours after fertilization. Embryos are selected which have a regular pattern of pigmentation which indicates the dorso-ventral and anterior-posterior (animal-vegetal) axes of the fertilized egg and blastula. After cleavage starts, further selection occurs of those embryos that have regular and symmetrical patterns of cleavage so that individual blastomeres can be identified in a series of embryos (Jacobson, 1981).

To trace the clone of cells that descends from any single blastomere a tracer enzyme, horseradish peroxidase, was injected into a single blastomere in a large series of embryos at 2- to 512-cell stages. In control experiments, the tracer did not diffuse out of the injected cell, did not interfere with normal development, was transmitted to all the descendants of the injected cell, and could be detected in those descendants even after they had differentiated (Jacobson and Hirose, 1978, 1981; Hirose and Jacobson, 1979). The tracer enzyme was detectable in the outgrowing axons and dendrites of neurons that had inherited the tracer from the initially injected ancestral cell (Jacobson, 1983). Therefore, we were able to see how the labeled axons were related to labeled target cells (Moody and Jacobson, 1983).

42 M. JACOBSON

Embryos were fixed at various times after the initial injection and processed histologically to show the presence of HRP. We rec­orded the positions. numbers and types of labeled cells in the serially sectioned embryos. The number of labeled cells was counted in embryos injected at the 2- to 512-cell stages and fixed before the beginning of gastrulation (stage 10 of the normal table ot Nieuwkoop and Faber. 1956).

RESULTS: RESTRICTION OF CELL DISPERSAL AND MINGLING IN THE INTACT EMBRYO

The cells were found to divide almost synchronously. every 33 minutes at 20°C. and reached the 12th cell generation at the begin­ning of gastrulation (Newport and Kirschner, 1982). After that stage the cell cycle time increased to about 2 hours and another 2 or 3 mitotic divisions occurred before the end of gastrulation (stage 12~ - 13). The cells of the early neural plate were. therefore. at the 15th and 16th cell generation (Jacobson. 1984). The observed number of labeled cells was close to the expected number (2n where n is the number of cell generations produced between the time of injection of the label and fixing the embryo). This shows that little or no cell death occurred during the cleavage stages and during gastrulation.

There was a one-to-one relationship between the position of the initially labeled ancestral cell and the position of its labeled clone when the embryo was fixed before the onset of gastrulation. This was because coherent clonal growth occurred during the first twelve cleavages and cell migration started only after the 12th cell generation. Cell migration occurred very rapidly after the 12th cell generation so that. by the 13th or 14th cell generation. the labeled cells had dispersed and mingled with unlabeled cells. There were two regions: one containing labeled cells mingled with unlabeled cells and another containing no labeled cells. The region in which the labeled cells were dispersed is called the clonal domain.

When all the results were combined and compared it was found that. with rare exception every blastomere labeled at the 512-cell stage distributed all its descendants into one of seven clonal domains in the embryo. There was a one-to-one relationship between each of those seven clonal domains and seven groups of blastomeres in the 512-cell embryo. These are called ancestral cell groups. Each ancestral cell group consists of about 14 to 26 blastomeres but the exact number in each embryo cannot be determined from these results because only one ancestral cell was labeled in each case. The number of ancestral cells was estimated from the composite map of blasto­meres injected at the 512-cell stage (Figure 1) and from the percent­age of labeled cells found in the clonal domain after injecting a single cell at the 512-cell stage. The descendants of each ancestral cell injected at the 512-cell stage were mingled with unlabeled cells

CLONAL RESTRICTIONS DURING DEVELOPMENT 43

which were descendants of other ancestral cells belonging to the same group, but no mingling occurred between descendants of different ancestral cell groups (Jacobson, 1983). Clonal restriction of cell mingling and cell dispersal resulted in development of morphological domains, which are called compartments, populated exclusively by the descendants of a single ancestral cell group. Between compartments, lines of clonal restriction developed in the embryo which cells did not cross (Figures 2 and 3).

The question of whether further lines of clonal restriction develop later remains to be investigated. There appears to be no reason why further compartmentation should not occur at later stages. However, the evidence shows that clonal restriction of cell dispersal to a single compartment does not occur in many cases after injection of single cells at the 256-cell or earlier stages. Cells injected at the 256-cell stage frequently distributed their descendants into two of the seven compartments, while cells injected at the 128-cell stage distributed their descendants into more than two compartments in many cases. These results show an abrupt change in the behavior of the clones descended from single ancestral cells of the 512-cell stage when compared with clones descended from single cells of the 256-cell and earlier stages.

The descendants of a single ancestral cell group mingled in a single compartment and abrupt transitions occurred between neighbor­ing compartments. Examples of abrupt transitions are illustrated in Figures 2 and 3 which show the distribution of CNS descendants ·of blastomeres that were neighbors but were in different ancestral cell groups at the 512-cell stage. In these cases the neighboring blasto­meres contributed all their descendants to completely different compartments. However, in 8 of 91 cases injected at the 512-cell stage some labeled cells were located outside the compartment in which all the other labeled cells were confined (Jacobson, 1983). There are several possible causes of these "aberrant" cases (Jacobson, 1983): the injected blastomere may not have completed cleavage at the time of injection or some blastomeres may not have become "committed" to a single ancestral cell group at the time of injection or the "commitment" may have been labile or reversible in some cases. More work is required to resolve this problem.

The relationship between cell lineage and cell phenotype is not fixed at the stages that we have examined, that is, there is no clonal restriction of phenotype at the 512-cell stage. The same types of cells differentiated in different compartments and different cell types appeared in the same compartment. For example the same types of cells were found on both sides of the compartmental boundary between dorsal and ventral parts of the retina. Many different types of cells, belonging to all three classical germ layers, different­iated in all the clones which also contribute to the nervous system. Every blastomere that contributed descendants to the central nervous

44 N. JACOBSON

Fig. 2. Reconstructions of every section in a complete series in the coronal plane through the retinae and central nervous system of two specimens after injecting HRP into indivi­dual blastomeres (13 and 24 in Figure 1) at the 512-cell stage. These blastomeres were neighbors but belonged to different groups (AM and AL, respectively) and they dis­tributed their descendants into different compartments.

CLONAL RESTRICTIONS DURING DEVELOPMENT 45

Fig. 3. Reconstructions of every section in a complete series in the coronal place through the CNS in two specimens injected at the 512-cell stage and killed at tailbud stages. Upper specimen: after injection of HRP into blastomere 66 (in the posterior-lateral blastomere group). Lower specimen: after injection into blastomere 47 (in the anterior-lateral blastomere group). Blastomeres 47 and 66 were neighbors at the 512-cell stage.

46 M. JACOBSON

system gave rise to several types of neurons and also probably to glial cells. The identification of nerve cell types that were labeled was relatively unambiguous in the case of well characterized neurons such as Rohon-Beard neurons, primary motoneurons, retinal ganglion cells, Mauthner's neuron, commissural neurons of the spinal cord, neurons of cranial motor nuclei and neurons of cranial and spinal sensory ganglia. However, identification of glial cells was difficult or impossible. The use of cell markers specific for neurons and for glial cells is planned as part of the next phase of research. By injecting single ancestral cells at progressively later cell generations, it should be possible to determine when phenotypic restrictions may be initiated. Injection of individual blastomeres becomes increasingly more difficult as the cells decrease in size at later generations. An alternative method is easier at the 10th generation and later, namely transplantation of single labeled cells to the same position in an unlabeled embryo.

Coventionally, the definition of cell commitment requires trans­planting or explanting cells to see whether they continue different­iating according to their original program or whether they change their program in response to the new conditions. Therefore, experi­ments are planned in which cells labeled with HRP are transplanted, in a series of embryos at progressively later cell generations, from one compartment of the donor to a different compartment of an un­labeled host at the same stage. In other experiments the host and donor will be at different stages.

CLONAL RESTRICTION OF CELL MINGLING IN VITRO

A small group of cells from one ancestral cell group or from one compartment can be explanted in vitro. We have observed that many types of cells differentiated in such explants within 2 or 3 days. However, identification of types of nerve cells in those explants cannot be made by conventional histological staining methods and cell-specific markers will be required. While investigation of "commitment" or of clonal restriction of phenotype requires un­ambiguous methods of identifying various types of cells, investi­gation of clonal restriction of cell mingling merely requires an unambiguous method of identifying all cells belonging to the same clone and of distinguishing them from cells belonging to other clones. This can be accomplished by labeling one clone with HRP and seeing how the labeled cells behave when placed in contact with unlabeled cells of different clonal origin.

We have completed the first of a series of experiments of this kind. A small group of cells was excised from a single compartment of a completely labeled embryo at the 512 to 1024-cell stage and placed in vitro in direct contact with a group of cells from a dif­ferent compartment excised from an unlabeled embryo at the 1024-cell

CLONAL RESTRICTIONS DURING DEVELOPMENT 47

stage. The combinations were fixed after 16 to 44 hours in vitro, sectioned and reacted histochemically to show the labeled cells. The results are shown in Figure 4. The cells continued to express their clonal restriction of mingling: little or no mingling of cells occurred when the combinations originated from different compartments whereas complete or considerable mingling occurred in the majority of cases when the combinations originated from the same compartment.

THE POSSIBLE MECHANISMS OF RESTRICTION OF CELL MINGLING AT COMPARTMENT BOUNDARIES

It is now evident that some properties or functions of the blastomere groups result in restriction of mingling of descendants of different blastomere groups. In turn, these restrictions of cell mixing during morphogenetic movements result in the formation of morphological domains, called compartments. Our experiments show that mingling occurs between cells in each compartment but not bet­ween compartments (Jacobson, 1982, 1983). Why the cells are permit­ted to move inside the compartment but are restricted from leaving the compartment is the problem addressed here.

There are three likely mechanisms of restriction which may result in the observed compartmentation. Firstly, a differential adhesion mechanism by means of which cells of the same compartment selectively adhere to one another. Secondly, a mechanism controlling cell motility so that cells are motile when in the company of other cells of the same compartment but mutual inhibition of cell movement

25

,-U) 20 w U)

« u ~ 15 0:: w OJ ::E 10 ::::l 2:

5

-

I

• SAME o DIFFERENT

I .~ h h ~ --, o 234567

Not Mixed

MINGLI NG INDEX

Totolly Mixed

Fig. 4. Mingling index in combinations of two blastomere groups, either the same (black) or different (white). O=complete separation of two groups; 7=complete mingling of cells of two groups, after 12 to 45 hours in vitro.

48 N. JACOBSON

occurs when cells of different compartments come into contact. A number of possible types of contact inhibition of movement have been reported (reviewed by Martz and Steinberg, 1973; Heaysman, 1978). Thirdly, a compartment-specific control of cell division such that cell division is inhibited when cells of different compartments come into contact and especially when a cell belonging to one compartment becomes entirely displaced into another compartment. This could result in some slowing of the rate of mitosis at the compartmental boundaries and could result in complete inhibition of mitosis in a cell that had become totally surrounded by cells belonging to a different compartment.

A number of possible mechanisms might result in contact mediated inhibition of cell division (Abercrombie, 1962; Eagle and Levine, 1967; Martz, 1969, 1973; Martz and Steinberg, 1972) and there is considerable discussion about the mechanisms in different systems (see Glaser 1980 for discussion). One can also conceive of a mechan­ism whereby cell division is controlled by differential cell adhesion so that when a cell belonging to one compartment is surrounded by cells belonging to another compartment the former rounds up because it does not adhere to the surrounding cells. Folkman and Tucker (1980) have shown that inhibition of cell division occurs when cells round up when they are unable to adhere to their substrate whereas cell division is stimulated in cells which adhere strongly to the substrate or to other cells.

There is considerable evidence, in several different systems, for all three mechanisms. The aim of our experiments will be to determine whether these mechanisms play a role in the development of compartments during early embryonic development in Xenopus.

There is an immense literature on differential cell adhesiveness and on cell adhesion molecules, which have been reviewed (Steinberg, 1964, 1970, 1981; Moscona, 1974; Lilien et al., 1979). There is no doubt that cell recognition and adhesion are very important mechan­isms of morphogenesis and their molecular mechanisms are rapidly becoming known (Kemp, 1973; Hausman and Moscona, 1975; Thiery et al., 1977, 1982; Yamada and Olden, 1978; Edelman, 1983). However, it is essential to point out that experimental analysis of cell adhesion has been done almost entirely on tissues derived from embryos which have completed gastrulation (the major morphogenetic process of early embryonic development) and in most cases the analysis has been done on differentiated tissues.

The studies of Steinberg on 4-8 day chick embryo tissues (heart, liver, neural retina) showed that dissociated cells can sort out after reaggregation (Steinberg, 1963, 1970, 1981; reviews). Similar studies of cell recognition and adhesion have also been done on cells that had already completed most of their morphogenetic movements and had reached their definitive places in the embryo, for example neural

CLONAL RESTRICTIONS DURING DEVELOPMENT 49

retina (Hausman and Moscona, 1975, 1976; Grunwald et al., 1981; Magnani et al., 1981; Moscona, 1972a,b; Rutishauser et al., 1976; Cheng-Ming Chuong et al., 1982), neural crest cells (Rovaisio et al., 1983), to give only some of the pertinent references. This is to emphasize that few studies have been done on embryos during the period of most active morphogenetic cell movements before the begin­ning of cell differentiation. The studies of Holtfreter (1939), Townes and Holtfreter (1955), Curtis (1961) and Nosek (1978) were on selective cell reaggregation of cells derived from embryos at the end of gastrulation and during neurula stages. The sorting-out behavior of blastula and early gastrula cells has not previously been invest­igated. In the light of the compartment hypothesis (Jacobson, 1982, 1983) it becomes necessary to investigate the interactions between blastula and early gastrula cells that result in compartmentation.

Cell locomotion (Nakatsuji and Johnson, 1982) and cell contact (Johnson, 1976) during amphibian gastrulation had received consider­able attention before the discovery of clonal restrictions of cell dispersal and mingling (Jacobson, 1983). Those findings may be related to the development of compartments in the amphibian embryo. Cells start moving individually after the 12th generation in Xenopus. This has been termed the "midblastula transition" (Signoret and Lefresne, 1971; Newport and Kirschner, 1982) although it really occurs at the end of the blastula stage in Xenopus (Jacobson, 1984). Before the 12th generation the cells divide very rapidly and almost synchronously, every 33 minutes at 20°C, but they do not move inde­pendently. The cells pass through the 13th to 15th cell cycle during gastrulation in Xenopus (Jacobson, 1984) and they migrate as indivi­dual cells during that time (Nakatsuji and Johnson, 1982).

Migration of Xenopus cells during gastrulation has been studied in vitro as well as in vivo. Using time-lapse cinematography. Keller (1978) showed that the surface cells of Xenopus "exchange neighbors by sliding past one another" during gastrulation. The motility of Xenopus cells during gastrulation resembles that of living teleost embryos in that cells mainly migrate as individuals or as small groups and not as large coherent sheets (Wourms, 1972a,b; Trinkaus, 1973). Similar motility of individual cells dissociated from Xenopus gastrulae has been observed in vitro (Nakatsuji and Johnson, 1982). Those Xenopus cells migrated at mean rates of more than 300 ~m/hour (Nakatsuji and Johnson, 1982). The ultrastructure of Xenopus cells during gastrulation movements has been studied by transmission elec­tron microscopy (Nakatsuji, 1976) and by scanning electron microscopy (Keller and Schoenwolf, 1977; Nakatsuji et al., 1982). Pertinent findings were that the cells had a leading process 0.5 to 1 ~m wide that was filled with microfilaments and that had focal membrane specializations at the tip, apparently for adhering to components of the substrate or to neighboring cells. It is obvious that consider­able work remains to be done to characterize the ultrastructure of those cells at different phases of locomotion, at different positions in the embryo, and at different stages of development.

50 M. JACOBSON

The conditions in the extracellular environment as well as the glycoproteins on the cell surface are important in initiating and directing movements of cells during gastrulation in amphibians. Fibronectin appears between cells of the blastula (Boucaut and Darribere, 1983) and gastrulation movements are completely inhibited by injecting an antibody to a cell-binding fragment of fibronectin into the blastocoel of Pleurodeles (J. P. Thiery, personal communi­cation). The importance of cell surface glycoproteins in amphibian gastrulation movements is also shown by findings that gastrulation is arrested after inhibition of glycoprotein synthesis (Romanovsky and Nosek, 1980) or after treatment with lectins (O'Dell et al., 1974; Moran, 1974; Boucaut et al., 1979). A possible role of fibronectin in promoting entry of cells into the division cycle (Couchman et al., 1982) should also be kept in mind.

CLONAL RESTRICTIONS OF NEURONAL CONNECTIVITY

We have studied the clonal relationships between primary moto­neurons and the muscle cells that they innervate (Moody and Jacobson, 1983) and between Rohon-Beard neurons and the epidermal cells that they innervate (Jacobson and Huang, in preparation). There are about 100 primary motoneurons on each side of the spinal cord. Their peripheral neurites (axons) contact epidermal cells and their central neurites connect with primary motoneurons via spinal interneurons. All the links in this primary reflex: epidermis - Rohon-Beard neuron - interneuron - primary motoneuron - muscle, originate at the end of gastrulation in Xenopus (Lamborghini, 1980) and start functioning soon after closure of the neural tube.

Our clonal analysis, using HRP as a tracer, shows that Rohon­Beard neurons and the epidermis of the dorsal trunk originate in the posterior-lateral blastomere group (i.e. they are in the posterior­dorsal compartment). The primary motoneurons and the ventral so~itic muscle which they innervate, both originate from the posterior-medial blastomere group (i.e. they are in the posterior-ventral compartment).

Those neurons inherited the HRP from their ancestral cells and the tracer passed into their outgrowing neurites which allowed us to trace the time of initial axonal outgrowth, direction, pathway and termination of the outgrowing axons. The Rohon-Beard axons started growing at stage 19-20 of the Nieuwkoop and Faber (1956), form 2 or 3 branches shortly after exiting from the spinal cord, and arrived at their epidermal target cells 2 or 3 hours later. In almost all cases in which the axons could be traced to the epidermis, the labeled axons targeted on labeled (therefore clonally related) epidermal cells.

CLONAL RESTRICTIONS DURING DEVELOPMENT 51

The primary motoneurons which inherited the label from their ancestral cells also showed a highly significant preferred associa­tion with clonally related (i.e. labeled) myotubes in the ventral half of the myotome. The axons were first seen emerging from primary motoneurons at stage 21 to 22 of Nieuwkoop and Faber (1956). The majority of branches supplied clonally related myotubes (Figure 5). These observations with the light microscope do not show when syn­aptic connections are made with the target cells.

The preferential association of those neurons with clonally related target cells suggests that a clonal restriction of neuronal connectivity operates. It is noteworthy that the Rohon-Beard neurons and primary motoneurons are the first neurons to originate. The hypothesis is that they inherit compartment-specific cellular proper­ties which allow them to associate preferentially with other cells belonging to the same compartment. We have found no regular compartment-specific associations between neurons and their targets that originate later in development (Jacobson and Huang, in preparation).

The Rohon-Beard axons are the pioneer sensory axons and the primary motoneuron axons are the pioneer motor axons. They are

Fig. 5. Camera lucida drawing of coronal section at the level of the spinal cord, showing a labeled primary motoneuron projecting a labeled axon which branches to supply labeled myotubes. A single blastomere was injected with HRP at the 64-cell stage and the embryo was fixed at late tailbud stage 34. Bar equals 50 ~m.

52 M. JACOBSON

destined to die before the end of the larval period. but before doing so they serve as guides for the secondary. definitive. sensory and motor axons. The regular spacing of Rohon-Beard neurons and primary motoneurons along the length of the spinal cord may be the basis for subsequent spacing of sensory and motor spinal roots.

We have distinguished between primary and secondary nervous circuitry. The primary circuitry develops very early and very rapidly and forms the basis of the elementary reflex activity. The secondary. definitive nervous circuitry develops later to replace the primary system. The primary nervous circuitry is formed by a rela­tively small number of neurons that originate during and shortly after gastrulation. They form a scaffolding or framework on which the secondary circuits are later laid down. Because the primary neurons are less than a few hundred microns from their targets. and because the primary axons grow at rates of 50-75 pm/hour in the frog embryo. the primary circuits are formed very rapidly (Jacobson and Huang. in preparation). Chemospecificity. namely a preferential association between axons and targets. appears to be required for development of the primary circuits - neither chemotaxis nor pathway specificity seems to be required. During growth of the embryo the distances between neurons and targets increase, and it may appear to be difficult for axons to reach the appropriate targets without some form of guidance. The problem of pathway selection and target selec­tion by outgrowing secondary axons would be greatly simplified if the secondary axons grow to their targets by simply following the pioneer axons.

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Glaser, L., 1980, Mediator of Developmental Processes, in: "The Cell Surface:" pp.79-97. S. Subtelny and N. K. Wessels:-eds., Aca­demic Press, New York.

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73:3594-3598. Hirose, G., and Jacobson, M., 1979, Dev.Biol. 71:191-202. Jacobson, M., 1980, Trends Neurosci. 1:3-5. Jacobson, M., 1981, J.Neurosci. 1:918-922. Jacobson, M., 1982, Vol. 5, Neuronal Development in: "Current Topics

In Neurobiology," pp.45-99. N. Spitzer, ed.:-Plenum, New York. Jacobson, M., 1983, J. Neurosci. 3:1019-1038. Jacobson, M., 1984, J. Neurosci. In press. Jacobson, M., and Hirose, G., 1978, Science, 202:637-639. Jacobson, M., and Moody, S. M., 1984, J. Neurosci, In Press. Jacobson, M., and Hirose, G. 1981, J. Neurosci. 1:271-284. Johnson, K. E., 1976, J.Cell Sci., 22:575-583. Keller, R. E., 1975, Dev.Biol. 42:222-241. Keller, R. E., and Schoenwolf, G. C., 1977, Roux Arch.Dev.Biol.

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Rutz, J., 1979, in: "Surfaces of Normal and Malignant Cells", R. O. Hynes, ed.:-Wiley, New York.

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Martz, E., 1973, J.Cell Physiol. 81:39-48. Martz, E., and Steinberg, M. S., 1973,'J.Cell Physiol. 81:25-38. Moody, S. M., and Jacobson, M., 1983, J.Neurosci. Moran, D., 1974, J.Exp.Zool., 188:361-365. Moscona, A. A., 1974, pp.67-99. in: "The Cell Surface In

Development", A. A. Moscona:-ed, Wiley, New York. Moscona, M. L., Degenstein, L., Byun, D. Y., and Moscona, A. A.,

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THE MECHANISM OF THE AMPHIBIAN PRIMARY INDUCTION AT THE

CELLULAR LEVEL OF ORGANIZATION

Soren Lovtrup

Department of Zoophysiology University of Ume! o S-901 87 UMEA, Sweden

The main presumptive fates of the early amphibian ectoderm are twofold: it may become epidermis or it may differentiate into the notochord - neural plate - neural crest complex. From the innumer­able observations inspired by the "organizer" experiments by Spemann and Mangold (1924) it is also known that the latter course requires contact being established between the ectoderm and the invagitating endoderm. It is usually asserted that this phenomenon of "primary induction" involves that the endoderm imposes upon the ectoderm the faculty to form the several patterns of cell differentiation just mentioned.

This terminology unquestionably is meant to imply that the contact between endoderm and ectoderm involves a change in the pre­sumptive fate of the latter, partly with respect to the patterns of differentiation which the cells assume, and partly also with respect to the morphogenetic events in which they become engaged.

Ignoring the latter point in the present context I shall pro­pose, in complete agreement with current notions, that cell differ­entiation implies differential transcription of the genome. If this is true, and if the mechanism of "induction" indicated here is cor­rect, then it follows that the endoderm somehow is capable of chang­ing the part of the genome in the ectodermal cells which is available for transcription. In a more convenient terminology we may say that the endoderm has changed the program encoded in the ectodermal cells.

Before going on I shall briefly touch an epistemological con­cept, the causal postulate, which asserts that if two entities are different at t = tl' then they were also different at t = to, or else they have been exposed to different environmental influenc;s. In the

55

56 S. L0VTRUP

case discussed the second alternative clearly prevails, one part of the ectodermal cells has been subjected to a stimulation which has changed its presumptive fate, the other part has preserved the orig­inal program.

In order to distinguish betwe~n these different environmental influences, one might use the words "activation" and "induction", respectively, the latter representing the situation where a change of program takes. However, previous experience has shown that this terminology may lead to a discussion of words rather than facts, and in an attempt to avoid this outcome, I propose here to distinguish between "instructive" and "permissive" events, the former word obvi­ously referring to the situation where the program is changed.

On these premises it is possible to formulate rather unambigu­ously the question I plan to consider in the present paper, viz: Is the "inductive" interaction between endoderm and ectoderm in the amphibian embryo an instructive or a permissive event? Since we may assume that there is only one program represented in the fertilized egg, we may also ask: which of the two paths of ectodermal cell differentiation prevails in the genome at the outset of ontogenetic development?

Most previous studies of "primary induction" have been made either on whole embryos or on explants large enough to allow for some morphogenetic events taking place. If the primary effect of the "induction" is to control cell differentiation, morphogenesis being only the causal consequence of the existence of cells representing different patterns of differentiation, then it seems that these previous studies have been unnecessarily complex, and therefore also unnecessarily difficult to interpret.

It is my firm belief that the manner a problem is posed may often imply an essential short cut to its solution. Sometimes inter­pretative simplifications may be obtained by studying a problem on a lower level of organization. I shall submit that "primary induction" may be studied at the cellular level, and that this approach has the advantage to exclude the occurrence of morphogenetic events.

In the following text are outlined the results and the con­clusions which have been obtained from experiments designed to answer the question suggested here.

EXPERIMENTAL APPROACH

We know that in the normal embryo the process of cell differen­tiation is ruled by interactions between cells from different parts (germ layers) of the embryo. Therefore, if we want to know the nature of the pattern of differentiation originally encoded in the

MECHANISM OF PRIMARY INDUCTION 57

genome, it is necessary to work under conditions where such inter­actions do not take place: that is, it is necessary to study the differentiation of cells explanted in vitro.

In order to obtain an easily interpretable answer to our question there are two further provisos which must be fulfilled. First, the explants must be taken from the embryo before the onset of cell differentiation in vivo. And since morphological and chemical observations agree in demonstrating that this happens in association with the process of gastrulation, the explants should be taken from a middle to late blastula. Second, in order to ensure that no morpho­genetic events take place, the explants must be very small.

In the work to be reported here (L6vtrup, Landstrom and L6vtrup-Rein, 1978; L6vtrup, 1983), small explants (~ 25 cells) were isolated from the axolotl blastula and cultured in vitro. As shown in Figure I, the explants were taken from ten different regions, corresponding to the ectoderm: (2), (4), (5), (6) and (8), the meso­derm: (3) and (7) and the endoderm: (1), (9) and (10).

Spontaneous Differentiation

It turned out that after some days of culture three different cell types appear which, in fact, approximately represent the three germ layers. Thus, the ectodermal cells differentiate into epi­dermis, usually prevailing in the form of spherical vesicles consist­ing of ciliated and non-ciliated vesicles, covered at inside by a basal lamina of reticulate collagen (Figure 2a).

a

veg

Fig. 1. Diagram outlining the various regions of the axolotl blastula from which explants were taken: (1) and (9), endoderm; (2) and (8), equatorial ectoderm; (4), (5) and (6), animal ectoderm; (3) and (7), mesoderm; (10) circum­polar vegetal cells.

58 S. L¢VTRUP

Fig. 2. (a), Aggregate consisting of ciliated and non-ciliated ectodermal (epidermal) cells (75X). (b), Aggregate consist­ing of mesodermal cells (75X). (c), Fibroblast-like endo­dermal cell (Ruffini cell), belonging to the class of s010-filocytes or sf-cells (750X). (d), Ectodermal sf-cell (750X). (e), Ectodermal sf-cell (225X). (f), Mesodermal sf-cell (225X). (g), Endodermal sf-cell (225X).

MECHANISM OF PRIMARY INDUCTION 59

The mesoderm cells form small aggregates, spherical or irregu­lar, as the case may be, and covered by a distinct surface coat (Figure 2b). Various lines of evidence strongly indicate that this pattern represents undifferentiated embryonic cells; we hope to support this interpretation by further empirical evidence at a very early occasion.

The endodermal cells do not form aggregates, but migrate on the substrate away from the explants (Figure 2c). These cells, which are morphologically similar to some of the variety of cells which are called "fibroblasts", have elsewhere been suggested to represent a particular class of cells (solo-filocytes or sf-cells), which in­cludes several distinct patterns of differentiation (L6vtrup, 1974; 1983). It should be pointed out that these cells originate in explants from regions (1) and (9), but not from region (10). It appears that the latter cells cannot differentiate; in the normal embryo their fate is to end up in the archenteron to be used for food.

On the given premises the heading of this section is slightly misleading. The three observed patterns of differentiation are spontaneous in the sense that they arise in the absence of any par­ticular efforts on our behalf. However, even if we ignore the undif­ferentiated cells, two patterns of differentiation remain, and on the basis of our premises we may clearly infer that one of these repre­sent a spontaneous or permissive event, whereas the origination of the second must be an instructive event. We have thus narrowed down our problem to a choice between two alternatives, and we shall give the answer to this issue in the next section.

STIMULATED DIFFERENTIATION

The wealth of prevailing knowledge bears out that the endoderm affects the differentiation of the ectoderm, not vice versa. And thus it is close at hand to infer that the pattern represented by the sf-cell is the one encoded in the genome of the fertilized egg.

We shall try to substantiate this conclusion in a stepwise fashion. As a first approach one may consider to make mixed cul­tures, containing both endodermal and ectodermal cells. Under these conditions some epidermis may be observed, but in many explants an outgrowth of cells is observed, which are similar to the endodermal sf-cells, although smaller and containing melanin granules rather than yolk platelets (Figure 2d, 2e and 2g).

We have interpreted this experiment to represent a model of amphibian primary induction, as it appears on the cellular level of organization. Some of the puzzle associated with the notion that the presumptive gut induces the presumptive skin to become nervous tissue

60 S. L0VTRUP

is surely relieved by this observation. For if the endoderm is indeed an inductor, it is not a heterotypic inductor of the kind suggested by the preceding clause, but a homotypic inductor which imposes upon the ectodermal cells its own pattern of differentiation.

But we should not forget the other possibility, namely, that the ectoderm actually is programmed to become sf-cells, but that this program cannot be realized unless the cells at an early time are stimulated (activated) through a permissive rather than an instruc­tive reaction.

There is a lot of data in the literature which support this notion. Thus, the classical experimental work on primary induction showed that a great number of completely unspecific substances may change the differentiation of the ectodermal cells (cf Needham. 1942). Indeed. rough treatment leading to "sublethal cytolysis" was in some instances enough to ensure the formation of nerve cells from the ectoderm (Barth. 1941, Ho1tfreter. 1947).

Under these circumstances it must evidently be concluded that the specificity of the response resides in the ectoderm. not in whatever treatment evokes the response. Indeed. the obvious conclu­sion to be derived from these many experimental observations was reached by Woerdeman (1936). who suggested that the ectoderm is programmed to form nerve cells. but that this will occur only when they are properly stimulated. Woerdeman thus regarded amphibian primary induction as a permissive rather than an instructive process.

In our work we have collected a large amount of evidence sup­porting this hypothesis. First of all. when properly stimulated. even the mesodermal cells form sf-cells. which morphologically are intermediate between ectodermal and endoderma1 sf-cells (Figure 2f).

Above it was asserted that ectodermal sf-cells arise in mixed cultures. In point of fact. we have made very few of this kind of experiments, for it very soon turned out that the same effect may pe obtained much more easily by adding various chemicals to the medium.

Our work a1~ng these lines may be subdivided into three parts. First we have Li • known to promote vegeta1ization in the sea urchin embryo. This phenomenon implies the furtherance of the formation of mesenchyme cells (sf-cells) at the cost of endoderma1 and/or ecto­dermal cells. This effect is completely analogous to the one ob­served in the amphibian embryo. when LiC1 is applied in 10 mM sol­ution.

Second. it has been found that cultured fibroblasts (sf-cells?) produce and secrete. indeed. may be covered by a layer of heparan sulphate (Kraemer, 1971). We also know that this substance is syn­thesized in substantial amounts in the early amphibian embryo. in

MECHANISM OF PRIMARY INDUCTION 61

time with the ongoing cell differentiation (Kosher and Searls, 1973; Hoglund and L6vtrup, 1976). As there is some indication that this synthesis may take place in the endodermal sf-cells, we conjectured that heparan sulphate might be the "inductor".

Experiments seemed to corroborate this inference, in concentra­tions of 0.1-1 ~g/ml heparan sulphate provokes the formation of ectodermal sf-cells in vitro (Landstrom and L6vtrup, 1977). However, in agreement with the previous students of "primary induction" we soon found that the search for the amphibian "inductor" is an elusive undertaking. Thus we found that dextran sulphate, a completely unnatural product in the present context, being active at 1 ~g/ml; chondroitin sulphate is also active, although only in higher concen­trations (10 ~g/ml).

The observations discussed so far roundly support Woerdeman's idea that the primary induction in amphibia is not an induction in the sense of being an instructive event; rather, everything suggests that it is a permissive phenomenon, an activation.

This notion forms the starting point of the third group of experiments. We have investigated the effect of a number of sub­stances known to be directly or indirectly inv~lved in the activation of cells. It is thought that the effect of Li may be associated with the fact that it accumulates in the cells, because it cannot be eliminated by the sodium pump. If this idea is correct one might expect ouabaine, an i~hibitor of the sodium pump, to have an effect similar to that of Li. So it has, and so has A23187, the calcium ionophore, and valinomycin, the potassium ionophore. The cyclic mononucleotides (cyclic AMP and cyclic GMP) were found to be active in exceedingly low concentrations. The protein hormones prosta­glandin El and glucagon promote the formation of ectodermal sf-cells, but it is inhibited by the steroid hormone testosterone, in the presence of 10 mM LiCI (Table 1). Most of the substances listed in this table are supposed to exert their function through receptors in the cell surface. In complete agreements with our results it has been found that if the surface receptors are blocked with lectins neural induction is suppressed (Duprat, Gualandris and Rouge, 1982).

All these results thus corroborate Woerdeman's proposition, dating back almost half a century. Hence, I shall conclude that the program available for transcription in the fertilized egg - and also in the unfertilized egg in those cases where haploid embryos develop normally - is the one specifying the basic cell type, which is called "the solo-filocyte".

AUTODIFFERENTIATION

It may be objected that I have not here presented a cellular model corresponding to the primary induction in the amphibian embryo,

62 S. L0VTRUP

Table 1. Activation by various agents of the differen­tiation of animal ectodermal cells isolated from the axolotl blastula. The data in this table generally represents the results obtained on 60 explants in two separate experiments (2x30). In several cases more explants were studied.

Concentration Differentiated aggregates (%)

Ouabain 10 j.JM

1 j.JM 100 oM

10 oM 1 oM

100 pM A 23187

1 j.JM 100 oM

10 oM Valinomycin

10 j.JM 1 j.JM

100 oM Adenosine-3'-5'-cyclic monophosphate

10 j.JM 1 j.JM

100 oM 10 nl1

1 oM 1 pM 1 fM

Guanosine-3'-5'-cyclic monophosphate 10 j.JM

1 j.JM 100 oM

10 oM 1 oM 1 pM 1 fM

Prostaglandin El 100 j.JM

10 j.JM Glucagon

2.5 '10-5 lU Controls (Li+ or heparan sulphate) Testosterone

1 ng/ml Controls (no addition)

o o o

17 40 45

60 50 43

o 38 38

80 74 64 60 30 30 17

65 67 74 70 44 38 37

54 37

40 60-90

o o

MECHANISM OF PRIMARY INDUCTION 63

for that process results in the formation of nerve cells, and so far has only been accounted for the formation of sf-cells, ectodermal and mesodermal, as the case may be. But all that is now required is patience, for once the sf-cells have appeared in the culture dishes -usually after four days - they continue to undergo differentiation.

At this stage a further distinction is necessary, namely, be­tween different regions in the ectoderm: the differentiation of the cells from the regions (4), (5) and (6), "the animal ectoderm", deviates from that of the cells from regions (2) and (8), "the equa­torial ectoderm". Thus, cells from the former regions differentiate in a particular temporal sequence such that mesenchyme cells are formed on the fifth (Figure 3a), nerve cells on the sixth (Figure 3b) and melanophores on the eighth or ninth day (Figure 3c).

The characterization of the mesenchyme cells is based only on their form, but we believe that these cells like their counterparts in vivo synthesize hyaluronate. We intend to test this supposition in the nearest future. Originally we discriminated the nerve cells only on the basis of the formation ofaxons, but lately we have demonstrated the presence of cholinergic and adrenergic cells by cytochemical methods. The melanophores are characterized partly by their content of melanin, partly by their radially symmetrical shape. Synthesis of melanin may occur also in other cell types, particularly under experimental conditions (L~vtrup, 1983).

Cells from regions (2) and (8), the equatorial ectoderm, differ­entiate into notochordal cells, distinguished by their extensive swelling (Figure 3d) and into elongated fibroblasts (Figure 3e). Mesodermal cells differentiate into muscle cells (Figure 3f) and fibroblasts.

We have no specific ideas about the nature of the agents which direct the differentiation of ectodermal and mesodermal cells along these various lines, but we surmise that they are cytoplasmic fac­tors.

In studies similar to those reported here, Epperlein (1978) has made a distinction between cholinergic neural plate (brain) nerve cells and adrenergic neural crest nerve cells. If this notion is accepted, it appears that in our cultures we have nerve cells rep­resenting partly the neural plate (cholinergic cells), and partly the trunk neural crest (mesenchyme cells, adrenergic and cholinergic nerve cells and melanophores).

This state of affairs vindicates the assertion that the differ­entiation patterns which we by various experimental means can provoke in explants of animal ectoderm represent a model of amphibian primary induction on the cellular level.

64 S. L0VTRUP

-.- -'" .. --......-, ... , • 1. '. ott .

:'.

Fig. 3. (a), Mesenchyme cell (7S0X). (b), Axons extending from nerve cells located in the central aggregate (lSOX). (c), Melanophore (7S0X). (d), Swollen chordocytes (2S0X). (e), Elongated fibroblasts (lSOX). (f), Muscle cells (37SX).

MECHANISM OF PRIMARY INDUCTION 65

CONCLUSION

I shall venture to submit that on the cellular level the riddle behind amphibian primary induction has been solved. And the solution spells that if by induction we mean an instructive event. then there is no induction whatsoever. for the cells are from the outset pro­grammed to form the differentiation patterns characterizing the neural ectoderm. However. the ectodermal cells cannot realize this program unless they are subject to a permissive activation.

In the absence of this stimulation the cells normally differen­tiate into ciliated epidermis. According to the causal postulate this differentiation must involve an instructive event. We do not know anything about the nature of the inductor involved. but it may be presumed that the tonicity of the medium plays a decisive role. since the differentiation is suppressed at high tonicities (Landstrom. 1977. L6vtrup. 1983).

The classical experiments on amphibian primary induction has led to the distinction between at least two inductors. essentially respon­sible for induction in the head and in the trunk. respectively (cf Saxen and Toivonen. 1962). However. this distinction is possible only on the basis of morphological criteria. and this implies that the interpretation of the results has been complicated by the fact that both cell differentiation and morphogenetic events take place in the explants. ---

Returning once more to the causal postulate it is quite obvious that the difference in form between head and trunk must have a cause. which we with some justification may refer to one or more morpho­genetic factors (cells or macromolecules). And since these morpho­genetic elements regularly originate as the results of cell differen­tiation processes. it may be expected that morphogenetic events often follow as the direct consequence of cell differentiations occurring in the embryo.

It might indeed be possible to apply the concept of "induction" in such contexts. However. considering the fundamental differences between the processes of cell differentiation and morphogenesis. this terminology is hardly recommendable. In point of fact. the failure to distinguish between cell differentiation and morphogenesis may for years have delayed the proper understanding of the nature of amphib­ian primary induction.

Now that we understand this phenomenon as far as cell differen­tiation is concerned. we may take a fresh look at the problem of morphogenesis. Here many problems remain to be solved. but I think that the answer to one of the most challenging ones, the difference between head and trunk morphogenesis is within reach. In fact, the extensive swelling which occur in head, as compared to the trunk

66 S. L0vTRUP

(except in the frog tadpole) probably may be referred to a greater synthesis of hyaluronate in the former than in the latter location.

REFERENCES

Barth, L. C., 1941, Neural differentiation without organizer, J.Exp. Zool., 87:371-383.

Duprat,-X:-M., Gualandris, L., and Rouge, P., 1982, Neural induction and the structure of the target cell surface, J.Embryol.Exp. Morphol., 70:171-187.

Epperlein, H. H., 1978, Differentiation of neural derivatives in the ectomesenchymal-endodermal interaction-system (EElS) of Triturus alpestris in tissue culture, in: "Form-shaping Move­ments in Neurogenesis", C. -0. Jacobson and T. Ebendal, eds., pp.123-127, Almqvist and Wiksell International, Stockholm.

Hoglund, L. R., and L~vtrup, S., 1976, Changes in acid mucopolysac­charides during the development of the frog Rana temporaria, Acta Embryol.Exp., 1976:63-79.

Holtfreter, J., 1947, Neural induction in explants which have passed through a sublethal cytolysis, J.Exp.Zool., 106:197-222.

Kosher, R., and Searls, R., 1973, Sulfated mucopolysaccharide syn­thesis during the development of Rana pipiens, Devel.Biol., 32:50-68.

Kraemer, P. M., 1971, Heparan sulphates of cultured cells. II. Acid soluble and precipitable species of different cell lines, Biochemistry, 10:1445-1451.

Landstrom, U., 1977, On the differentiation of prospective ectoderm to a ciliated cell pattern in embryos of Ambystoma mexicanum, J.Embryol.Exp.Morphol., 41:23-32.

Landstrom, U., and L~vtrup, S., 1977, Is heparan sulphate the agent of the amphibian inductor? Acta Embryol.Exp., 1977:171-178.

L~vtrup, S., 1974, Epigenetics - A Treatise on Theoretical Biology, John Wiley, London.

L6vtrup, S., 1983, Epigenetic mechanisms in the early amphibian embryo Cell differentiation and morphogenetic elements, BioI. Rev., 58:91-130. -­

L~vtrup~., Landstrom, U., and L~vtrup-Rein, H., 1978, Polarities, cell differentiation and primary induction in the amphibian embryo, Biol.Rev., 53:1-42.

Needham, J., 1942, "Biochemistry and Morphogenesis," The University Press, Cambridge.

Saxen, L., and Toivonen, S., 1962, "Primary Embryonic Induction," Logos, London.

Spemann, H., and Mangold, H., 1924, Uber Induktionen von Embryonalan­lagen durch Implantation von artfremder Organisatoren, Arch. Mikros.Anat.Entw.mech., 100:599-638. ---­

Woerdeman, C., 1936, Embryonic "induction" by chemical substances, Konink.Akad.van Wetensch. Amsterdam, Proc.Sect.Sci., 39:306-314.

SEQUENTIAL INDUCTION OF THE CENTRAL NERVOUS SYSTEM

Lauri Saxen

Department of Pathology University of Helsinki 00290 Helsinki 29, Finland

INTRODUCTION

Primary determination and early segregation of the central nervous system (CNS) form a chain of events in which the beginning can be traced back to oogenesis. Already then the Amphibian oocyte develops an animal/vegetal polarity which, in fact, can be visualized in many species as an uneven distribution of the yolk and the pigmen­tation. Following the first four or five cleavages, cells of the two halves become determined toward ectodermal and endodermal directions, respectively. The prospective inductor of the CNS, the mesoderm, is subsequently formed. Then the actual neural induction takes place during gastrulation and is followed by the segregation of the main regions of the CNS.

In this overview, the biology of this sequential determination will be outlined to create a proper framework for the recent approaches at the molecular level of these events.

Determination of the Neural Inductor

The prospective inductor of the CNS, the mesoderm, develops at the dorsal marginal zone between the animal (ectodermal) and the vegetal (endodermal) halves. The self-differentiation capacities of this zone can already be demonstrated at an early morula stage. During mid-blastula stage the mesoderm acquires its inductive proper­ties when tested against competent gastrula ectoderm (cf Nakamura, 1978). Separation and recombination of different parts of an early blastula have suggested that 'at least most of the mesoderm is derived from the ectodermal half, and that this event is determined by an

67

68 L. SAXEN

inductive interaction between the two dissimilar tissues of the animal and vegetal poles (Nieuwkoop, 1973). The mechanism of this event is completely unknown, but it is tempting to speculate that the "vegetalizing factor" characterized by Tiedemann (1982, this volume) acts upon the ectoderm at this stage of development.

Determination of the Presumptive Neuroectoderm

During early gastrulation, the prospective neural plate area of the ectoderm has still retained its multipotency and can experiment­ally be converted into practically all derivatives of the three germ layers. During gastrulation it becomes irreversibly determined toward neural direction, and this is a consequence of its contact with the invaginating mesoderm, the inductor or "organizer" (Spemann and Mangold, 1924). During invagination the inductive properties of the mesoderm become further modified: the mesoderm to invaginate first, the prospective precordal plate, will induce head structures while the caudal part, the future axial mesoderm, develops into an

Fig. 1. Demonstration of the effect of a "head" and a "tail" inductor, respectively, in implantation experiments on Triturus gastrulae (From Saxen and Toivonen, 1962).

SEQUENTIAL INDUCTION OF THE CNS

inductor of the trunk and tail formations (cf Saxen and Toivonen, 1962) (Figure 1).

69

The process of neuralization of the ectoderm and its regional­ization to the different levels of the CNS can be mimicked in experi­ments by using as inductors various heterologous tissues or their fractions with varying inductive properties. Many of these exert a typical "head"-inductor effect leading to the differentiation of cranial neural structures only. Others produce neural structures belonging to the hindbrain and tail regions always accompanied by various amounts of mesodermal derivatives. Furthermore, tissues and purified preparations showing an exclusively mesodermalizing action have been found (Toivonen, 1953, Tiedemann, 1981).

Based on the hypothesis that the ultimate fate of the multi­potent ectoderm is determined by two inductive principles, a neural­izing and a mesodermalizing one, we performed the following experi­ment (Toivonen and Saxen, 1955): competent gastrula ectoderm was exposed, either in vivo or in vitro, to a neuralizing and a mesoder­malizing inductor, respectively. In addition, the combined effect of these inductors was examined by exposing the target cells to both of them simultaneously. The results showed that the combined action of the two inductors was not merely a summation of their separate effects, but, in addition, neural structures of the more caudal regions were obtained. Subsequent experiments indicated that we were dealing with a quantitative action of the two inductors; when the neuralizing and the mesodermalizing inductors were mixed in different ratios, an increasing proportion of the mesodermalizing component brought about a gradual caudalization of the CNS structures (Saxen and Toivonen, 1961). These observations, therefore, were compatible with our "two gradient hypothesis", postulating two active principles distributed in the inductor tissue in a gradient fashion and produc­ing various ratios of the Nand M factors (Toivonen and Saxen, 1955).

Sequentiality of the Determination of the CNS

Following the two-step activation-transformation theory of Nieuwkoop et al. (1952), we then analyzed the time course of the induction of the CNS assuming a primary and a secondary action upon the ectodermal cells (Saxen et al., 1964) (Figure 2). Again the ectoderm was exposed separately to the neuralizing and the "caudal­izing" inductors for 24 h - a period known to be sufficient to com­plete the primary action (Toivonen, 1958) and also to abolish the responsiveness (competence) of the ectoderm to respond to such stim­uli (Nieuwkoop, 1958; Toivonen, 1958; Leikola, 1963). Then the inductor was removed from the ectodermal explants, the ectoderm was disaggregated and reaggregated to be followed up for an extended period in vitro. The third type of explants consisted of combined reaggregates of the two above types, i.e. neuralized and caudalized cells disaggregated and mixed after 24 h.

70 L. SAXEN

FOREBRAIN HINDBRAIN SPINAL CORD

Fig. 2. Schema of the disaggregation-reaggregation experiment of Saxen et al., 1964, demonstrating the two-step induction of the CNS (From Saxen, 1983).

Analysis of the reaggregates showed that the single inductors had led to the expected formations in the reaggregates: the neural­izing inductor produced exclusively cranial neural structures, while the "caudal" inductor triggered differentiation of most caudal neural structures (spinal cord) accompanied by mesodermal derivatives. In the combined reaggregates, both the most cranial and the caudal structures of the CNS were reduced in size and number and the midd1e region, the hindbrain derivatives, dominated . The apparent con­clusion was that these were determined by a secondary interaction of the neuralized vs. mesodermalized cells after the initial 24 h induc­tion period. The interpretation would be compatible with the results obtained with mixed inductors, as there we initially induced varying amounts of mesodermal cells which then interacted with the neuralized component.

Ultimately the experimentation was brought closer to normal in in vivo situation by repeating the above experiment with cells ob­tained from normal embryos at an early neurula stage (Toivonen and Saxen, 1968). Pieces were dissected from (1) the cranial plate region, and (2) the caudal part of the axial mesoderm. In both

SEQUENTIAL INDUCTION OF THE CNS 7I

FOREBRAIN L L--.I.--..J ~ E~ ~ HINDBRAIN

SPINAL CORD

PER CENT 50 100 50 100 50 100 50 100 50 100 50 100 ABC D E F

Fig. 3. Results of the disaggregation experiment with tissue fragments of a Triturus neurula to demonstrate the action of the axial mesoderm to the regionalization of the anterior neural plate (From Saxen, 1983, after Toivonen and Saxen, 1968).

cases, the cells were predetermined and developed after disaggrega­tion according to their prospective fate, i.e. to cranial neural or mesodermal structures. When combined and analyzed after in vitro cultivation, the neural structures had been converted into more caudal ones, hindbrain and spinal cord. An increase of the relative amount of mesodermal cells in these aggregates gradually shifterd all CNS structures toward the caudal type (Figure 3). The final result thus closely resembled that obtained with combined inductors as described above.

CONCLUSIONS

Experiments carried out on Amphibian embryos and described here suggest that determination and early segregation of the CNS include at least three distinguishable steps controlled by inductive tissue interactions: the prospective inductor becomes determined at an early blastula stage probably by an ectodermal/endodermal interaction. Following invagination it exerts a determinative influence on the competent prospective neuroectoderm which thus becomes uniformly neuralized. The ultimate segregation of the neural plate to the main regions of the CNS is subsequently induced by the axial mesoderm acting upon neuralized cells.

The hypothesis of the two gradients in the inductor tissue seems compatible with all the above findings as far as an effect upon the ectoderm is concerned. It does, indeed, imply two active compounds, "signal substances" and there is additional evidence of the existence of different compounds with the two different modes of action. Chemical fractination investigations of Tiedemann (1981, 1982, this volume) have repeatedly resulted in two fractions with a different inductive action. When combined to Sepharose beans, the neuralizing fraction is not inactivated, while the vegetalizing one loses its

72 L. SAXEN

inductive capacity (Born et al., 1980). Experiments where the induc­tor tissue is separated from the gastrula ectoderm distinguish like­wise between the two types of inductors: an interposed filter pre­venting actual contacts of the interacting cells does not permit the transmission of a mesodermalizing action which apparently requires direct cell-to-cell contacts, while the neuralizing message is carried by diffusable substances (Saxen, 1961; Toivonen, 1979). However, in addition to two types of signal substances, other factors possibly affecting the destiny of the ectodermal cells should be considered. These include changes in the responsiveness of the target cells, temporal changes in the ectodermal/mesodermal inter­relationships (contact vs. no contact), and single physical factors possibly resulting from the movements of the interacting components.

REFERENCES

Born, J., Grunz, H., Tiedemann, H., and Tiedemann, H., 1980, Biological activity of the vegetalizing factor: decrease after coupling to polysaccharide matrix and enzymatic recovery of active factor. W. Roux's Arch., 189:47-56.

Leikola, A., 1963, The mesodermal and neural competence of isolated gastrula ectoderm studied by heterogeneous inductors, Ann. Zool.Soc.Vanamo, 25:2-50.

Nakamura, 0., 1978, Epigenetic formation of the organizer, pp. 179-220, in: "The Organizer - A Milestone of a Half-Century from Spemann," O. Nakamura and S. Toivonen eds., Elsevier/ North Holland Biomedical Press, Amsterdam.

Nieuwkoop, P. D., 1958, Neural competence of the gastrula ectoderm in Ambystoma mexicanum. An attempt at quantitative analysis of morphogenesis. Acta Embryol.Morphol., 2:13-53.

Nieuwkoop, P. D., 1973, The "organization center" of the amphibian embryo: its origin, spatial organization, and morphogenetic action. Adv.Morphog. 10:1-39.

Nieuwkoop, P. D., et al., 1952, Activation and organization of the central nervous system. I. Induction and activation. II. , Synthesis of a new working hypothesis. J.Exp.Zool., 120:1-108.

Saxen, L., 1961, Transfilter neural induction of Amphibian ectoderm. Devel.Biol., 3:140-152.

Saxen, L., (in press). Neural induction. Int.J.Neurol. Saxen, L., and Toivonen, S., 1961, The two-gradient hypothesis in

primary induction. The combined effect of two types of induc­tors mixed in different ratios. J.Embryol.Exp.Morphol., 9:514-533.

Saxen, L., and Toivonen, S., 1962, Primary Embryonic Induction. Academic Press, London.

Saxen, L., and Toivonen, S., and Vainio, T., 1964, Initial stimulus and subsequent interactions in embryonic induction. J.Embryol.Exp.Morphol., 12:333-338.

SEQUENTIAL INDUCTION OF THE CNS 73

Spemann, H., and Mangold, H., 1924, Uber induktion von Embryonalanlagen durch Implantation artfremder Organisatoren. w. Roux's Arch., 100:599-638.

Tiedemann, H., 1981, Pattern formation and induction in amphibian embryos. Fortschr.Zool., 26:121-131.

Tiedemann, H., 1982. Signals of cell determination in embryogenesis, pp. 275-287. in: "Biochemistry of Differentiation and Morpho­genesis, 33. Colloquim - Mosbach." Springer Verlag, Heidelberg.

Toivonen, S., 1953, Bone-marrow of the guinea-pig as a mesodermal inductor in implantation experiments with embryos of Triturus. J.Embryol.Exp.Morphol., 1:97-104.

Toivonen, S., 1958, The dependence of the cellular transformation of the competent ectoderm on temporal relationships in the induc­tion process. J.Embryol.Exp.Morphol., 6:479-485.

Toivonen, S., 1979, Transmission problem in primary induction. Differentiation, 15:177-181.

Toivonen, S., and Saxen, L., 1955, The simultaneous inducing action of liver and bone-marrow of the guinea-pig in implantation and explantation experiments with embryos of Triturus. Exp.Cell Res., ~.3:346-357.

Toivonen, S., and Saxen, L., 1968, Morphogenetic interaction of presumptive neural and mesodermal cells mixed in different ratios. Science, 159:539-540.

NEURAL-INDUCING ACTIVITY OF NEWLY-MESODERMALIZED CELLS

AND CELLULAR ALTERATIONS OF INDUCED NEURODERMAL CELLS

Akio S. Suzuki, Teruo Kaneda and Tetsuro Ueno

Department of Biology Kumamoto University Kumamoto-Shi, Japan

In primary embryonic induction of amphibia, there are two main problems: neural-inducing activity of the organizer (chorda-mesoderm) and transformation of the presumptive ectoderm into neurodermal tissues. Many attempts to analyze the problems have been made from various points of view. It is still quite important to investigate the appearance of neural-inducing activity and cellular alterations of induced neurodermal cells. In the present paper, several results on the temporal relationship between mesodermalization and neural­inducing activity of actor cells, and on cellular alterations of induced neurodermal cells are presented.

APPEARANCE OF NEURAL-INDUCING ACTIVITY

The presumptive mesoderm exhibits two unique properties: it forms the primary mesodermal axis by its capacity to differentiate and induces neurodermal structures in the ectoderm. As demonstrated by Nieuwkoop (1969) and Nakamura et al. (1971a), the formation of mesoderm in the future marginal zone occurs from the blastula stage onward. On the other hand, recent studies on the appearance of the neural-inducing activity of the dorsal marginal zone show that this activity is accumulated progressively in the cells of the future marginal zone (Nakamura et al., 1971b; Malacinski et al., 1980; Asashima, 1980). However, it is not clear whether or not the mesodermal-differentiating capacity is directly coupled with neural­inducing activity.

In the present study, newly-mesodermalized ectoderm was used to analyze the temporal relationship between mesodermalization and neural-inducing activity. Mesodermalized ectoderms were obtained by

75

76 A. S. SUZUKI ET AL.

using two methods: (a) the presumptive ectoderm of the Nile-blue stained embryo (stage 11. beginning of the gastrula) was transplanted into the upper portion of the dorsal marginal zone of the host. a non-stained stage 11 embryo (Kaneda 1981). After 12 hrs the trans­planted ectoderm was removed from the host embryo. (b) the presump­tive ectoderm of stage 11 embryo was mesodermalized by contact with the swimbladder of Carassius for three hours (Kawakami. 1976). In order to investigate the neural-inducing activity. both mesodermal­ized ectoderms were combined with competent ectoderm of stage 12b gastrula and the combinants were cultured for one week.

The results are shown in Table 1. Both mesodermalized ectoderms acquire the high ability to induce neurodermal structures in the reactor ectoderm by its neural-inducing activity. The neurodermal structures are obtained in 15 cases out of 18 combinants and 22 cases out of 28 combinants in the series of transplantation and swimbladder experiments. respectively. Most of them are deutero-spinocaudal. This fact suggests that the neural-inducing activity of the actor cells (organizer) appears in coupling with their mesodermalization, because it is clear that the differentiated neurodermal structures originated from the combined competent ectoderm (data not shown).

CELLULAR ALTERATIONS OF INDUCED NEURODERMAL CELLS

The analysis of cellular alterations of induced neurodermal cells would be instructive for understanding of mechanism in neural induction. For this purpose, many attempts to analyze the cellular changes of neurodermal cells immediately after the inducing stimulus have been performed (Tarin 1971; Grunz and Staubach 1979; Keller 1980, Barbieri et al., 1980). In the present studies; in order to clarify cellular identification of neuralization after an inductive action, measurements of several indices of shape, volume, and

Table 1. Frequency and Pattern of Neural Differentiation in the Ectoderm Explants which are combined with the Newly­Mesodermalized Ectoderm.

Newly- Total Frequency of Brain Spinal Neural mesodermalized No. of neural differ- cord cells ectoderm by explants entiation No.(%)

Transplantation 18 15(83) 2(11) 15(83) 3(17)

Swimbladder 28 22(79) 3(11) 20(71) 2( 7)

Control* 18 17(94) 1( 6) 17(94) 3(17)

*Control; Dorsal lip of late gastrula is used as the actor. Histo-logical examination is performed one week after in vitro culture.

NEURAL-INDUCING ACTIVITY 77

adhesivity of neurodermal cells were made during gastrulation and early neurulation. The changes of cell volume and cell adhesivity will be reported.

Progressive Changes in Cell Volume of Ectoderm

As shown in Figure 1, the cell volume of induced neuroderm still continues to decrease during late gastrula and early neurula. Aver­age cell volume at early neurula (stage 18) becomes 2.0 x 104 ~m3, one-half of that at late gastrula (state 13b). The data show that cell size of the neuroderm abruptly decreases until mid-gastrula and after late gastrula, the decrease becomes slower. It is, further­more, shown that the deviation becomes smaller in accordance with the developmental process. The volume distribution histogram at each developmental stage is shown in Figure 2.

'"'E ~

~ 0

x

.. E :J

0 >

.. u

40

30 :

20

10

o 9 (-8)

Origin of No. of neuro-ectoderm

cells embryos Cell volume (Xlo"pm3)

MeantSD

Blastula (st.9) 9 Gastrula I (11) 5

II (12a) 8 1II(12b) 7 IV(13b) 7

Neurula I (15) 6 II (18) 5

:

11 12a 12b 13b 15 (0) (6) (12) (1 8) (24)

Developmental stage (Time)

26·6 t4·1 10.312.1 6.5 tl·7 5.411·0 3·5 to·5 2·8!0.3 1·9 t O·2

18 (36)

Fig. 1. Progressive changes in cell volume of the dorsal ectoderm during early development. The isolated ectoderm was dissociated by treatment with CMF-Steinberg's solution and the diameter of single cells was measured. The volume of each cell was calculated assuming that it was a sphere of a cylinder. At each developmental stage, 5 to 9 embryos were used and the volume of about 100 cells per embryo was determined.

78 A. S. SUZUKI ET AL.

2oot .... _....... St.ll

: _~._~I-I"""'P"I"""I -.....-.... St.12a

St.12b

~

;f!. St.13b

>-u C CII ::J cr CII ...

LL

St.15

St.18

20

Cell volume <Xl04 jJm3)

Fig. 2. Volume distributions of the dorsal ectoderm cells at each developmental stage.

There are marked differences in cell volume distribution between before mid-gastrula and after late gastrula. The neuroderms before mid-gastrula (stage 12b) contain heterogeneous cells with respect to their size and the heterogeneity decreases abruptly after late gas­trula (stage 13b): there is only one peak (3.5 x 104 ~m3) on the size distribution histogram (Figure 2). Judging from the fact that neur­alization of the ectoderm is realized just before the end of gastru­lation (Suzuki et al., 1975), it is reasonable to conclude that the homogeneity of cell size is closely related with the process of morphological events in neural induction. The conclusion is also supported by the fact that the neuroderm of early neurula (stage 18) consists completely of homogeneous cells in cell volume (Figure 2).

NEURAL-INDUCING ACTIVITY 79

Cell Reaggregation of Ectoderm

During the morphogenic process of amphibian gastrulation, cell­ular adhesiveness is markedly modified as are intercellular contact and cell morphology (Baker, 1965; Monroy et al., 1976; Smith et al., 1976). Recently, Bellairs et al. (1978) and Matsuda (1980) pointed out that the cell surface properties might be related to cytodiffer­entiation in early embryogenesis. The cell adhesivity is analyzed by reagglutinability of dissociated single cells. The neurectoderm isolated from vari~s sta~s of development was dissociated into single cells in Ca - Mg free Steinberg's solution and then the dissociated cells were reaggregated by rotating the culture flask on a gyratory shaker for 1 hr, 5 hr, and 24 hr.

As shown in Figure 3, there are remarkable differences in the reaggregation of neurectodermal cells between the gastrula and the neurula. The neurectodermal cells of the gastrula form a reaggregate of cluster type, but the induced neurodermal cells of the neurula form a reaggregate of the rosary type. The rosary type reaggregates of neurula are observed immediately after rotating the culture flask within one hour, but the neurectodermal cells of the gastrula gradu­ally form the cluster type reaggregate. Both reaggregates finally form one large aggregate after 24 hrs of rotation. It is also clear that there is a gradual decrease in capacity of reaggregation during gastrulation. Accordingly, it is difficult for newly-induced neuro­dermal cells of late gastrula (stage 13b) to form the reaggregates within a short period (Table 2).

Table 2. Reaggregating Capacity and Pattern of Dissociated Single Cells of Cynops Ectoderm. The degree of Cell Reaggregation is Represented by an Index of 1 - Nt/No, where No is the Total Particle Number Prior to the Initiation of Reaggregation and Nt is the Total Number of Particles at Rotation Period t(hr).

Rotation Cell Cell Source Isolated From Period Source (hr) Stage Stage Stage Stage Stage Stage

11 12a 12c l3b 15 17b

1 pNE pEE + +--+ +--+

5 pNE ++ ++ + +++ +++ pEE ++ ++ ++ +++ +++ +++

pNE: presumptive neurectoderm or neural plate. pEE: presumptive epidermal ectoderm or ventral epidermis. + and - indicate the degree of cell reaggregation of the cluster type and the rosary type, respectively.

80 A. S. SUZUKI ET AL.

A

~t.12c St . 1 )b

~~ . St.l.5 • ": ~.

:fj. St . 17b 1 mm .."' .•

, St.17b 1 mm'

Fig. 3. Light micrographs showing reaggregation in vitro of dissociated single cells of presumptive neurectoderm and neural plate from gastrulae and neurulae. The isolated tissues were dissociated into single cells in CMF-Steinberg's solution for 60 min. The dissociated single cells were transfered to normal Steinberg's solution containing 5% fetal calf serum. The cell suspension was placed in 2 ml culture flask coated with 2% agar and rotated on a gyratory shaker at a constant speed of 100 rpm at 23°C for 1 hr(A) and 5 hr(B).

On the contrary, the epidermal ectoderm does not show any stage­specificity in a reaggregation pattern but a stronger reagglutin­ability after gastrulation. The reaggregates of the epidermal cells are of the cluster type during gastrulation and early neurulation. Therefore, the tissue-specific adhesiveness is caused differently between neuroderm and epidermis after neural induction. Scanning electron microscope studies also shows that there are differences in cell contact between both ectoderms. These facts demonstrate the important role played by cell surface structures in cellular differ­entiation.

REFERENCES

Asashima, M., 1980, Wilhelm Roux's Arch., 188:123-126. Baker, P. C., 1965, Cell BioI., 24:95-116. Barbieri, F. D., Sanchez, S. S., and Del Pino, E. J., 1980,

J.Embryol.Exp.Morph., 57:95-106.

NEURAL-INDUCING ACTIVITY 81

Bellairs, R., Curtis, A. S. G., and Sanders, E. J., 1978, J.Embryo.Exp.Morph., 46:207-213.

Grunz, H., and Staubach, J., 1979, Wilhelm Roux's Arch., 186:77-80. Kaneda, T., 1981, Develop.Growth and Differ., 23:553-564. Kawakami, I., 1976, J.Embryol.Exp.Morph., 36:315-320. Keller, R. E., 1980, J.Embryol.Exp.Morph., 60:201-234. Malacinski, G. M., Chung, H-M., and Asashima, M., 1980,

Develop.Biol., 77:449-462. Matsuda, M., 1980, J.Embryol.Exp.Morph., 60:163-177. Monroy, A., Baccetti, B., and Denis-Donini, S., 1976, Devel.Biol.,

49:250-259. Nakamura, 0., Takasaki, H., and Ishihara, M., 1971a, Proc.Japan

Acad., 47:313-318. Nakamura~, Takasaki, H., Okumoto, T., and Iida, H., 1971b,

Proc.Japan Acad., 47:203-208. Nieuwkoop, P. D., 1969, Wilhelm Roux's Arch., 162:341-373. Smith J. L., Osborn, J. C., and Stanisstreet, M., 1976,

J.Embryol.Exp.Morph., 36:513-522. Suzuki, A., Kuwabara, K., and Kuwabara, Y. 1975, Develop.Growth and

Differ., 17:343-353. Suzuki A., and Ikeda, K., 1979, Develop.Growth and Differ.,

21:175-188. Tarin, D., 1971, J.Embryol.Exp.Morph., 26:543-570.

A MOLECULAR ASPECT OF NEURAL INDUCTION IN CYNOPS

PRESUMPTIVE ECTODERM TREATED WITH LECTINS

Kenzo Takata, Kiyoko Yamazaki Yamamoto and *Noriko Takahashi

Radioisotope Center, Nagoya University, Chikusa Nagoya 464 and *Laboratory of Biochemistry Nagoya City University Medical School, Showa Nagoya 467, Japan

Little has been reported on what happens when the competent ectoderm first receives the inductive stimulus, or the location of the cellular site on which the inductive stimulus exerts its effect. Recently, the primary involvement of cell surface in the inducing mechanism of neural tissues has been stressed (Tiedemann and Born, 1978; Grunz and Staubach, 1979; Takata et al., 1981, 1984; Yamamoto et al., 1981; Duprat et al., 1982). Experiments using Sepharose beads, on which the neural-inducing factor (vegetalizing factor: Tiedemann and Born, 1978; Con A: Takata et al., 1981) was immobi­lized, indicated that even if the neural-inducing factor acts on the cell surface alone, it can evoke the competent ectoderm to differen­tiate into neural tissues, and does not necessarily have to enter into the cytoplasm to exert its effect. This possible mode of inducing action of Con A seems to agree with the evidence that retention of a certain amount of mitogen (Con A) for a certain length of time on the cell surface is a prerequisite for stimulating lympho­cytes to blasto formation, but that mitogen taken up into cytoplasm is not involved in the mechanism of stimulation of cell division (Pauli et al., 1973).

Considering the above-mentioned evidence on the basis of the well-known fact that lectins specifically bind to sugar residues of glycoproteins existing on the cell surface (plasma membrane), it is quite plausible that the neural receptors primarily consist of glyco­proteins (Takata et al., 1981).

To proceed with our idea, we have to 1) examine the mode of binding of Con A to the competent ectoderm 2) examine the role of

83

84 K. TAKATA ET AL.

sugar residues on the cell surface in the inducing machinery, 3) isolate the sugar moiety from the cell surface glycoprotein, and 4) identify its chemical nature. For these purposes the following experiments were conducted.

1) The presumptive ectoderm was excised from St. 11 gastrulae and the thus prepared explants were incubated in a Steinberg balanced salt solution (SBSS) containing 3H-Con A (20 ~Ci/ml, specific activity: 45.5 Ci/mM) and the radioactivity of explants was measured at certain intervals. The amount of Con A bound to explants reached the saturation level 2 hr after incubation (Figure 1). The number of bound Con A molecules per cell was calculated according to the radio­activity of the explant, which consists of about 3,800 cells on the average and was found to be 3.3 x 10 7• This value seems smaller than that reported on human lymphocytes or cultured mouse cells. The difference may be partly due to the fact that the cell surface bet­ween neighboring cells could not be fully exposed to the labelling medium, even though Cynops presumptive ectoderm consists of a mono­layer (Yamamoto et al., 1981). It should be noted here that at any time during the course of labelling, more than 50% of the total amount of 3H-Con A bound to the explant could be released by chasing with a-D-mannose and that such partial removal of bound Con A resulted in a failure of neural induction. When the chasing was started, however, 3 h after Con A-treatment, the induction frequency remained high.

These results imply that to induce the presumptive ectoderm, a certain amount of Con A must be kept bound to the cell surface for a certain period of time. On the other hand, Con A molecules which were not removed by a-D-mannose should be those taken up into the cytoplasm and might not be involved in the induction mechanism. This assumption may be supported by the facts that although immobilized Con A on Sepharose beads could not be taken up into cytoplasm (cf. Takata et al., 1981), it was as active as soluble Con A, and that the

3

III • ..., ~ • 'ci.2 x ril

M

"-:;: 1 P< 0

... 0 rl

0 0.5 1.0 2.0 3.0 hr

Incubation Time in 3H- Con A (20°C)

Fig. 1. Time course changes in amount of bound 3H-Con A.

LECTIN TREATMENT OF PRESUMPTIVE ECTODERM 85

continuous presence of Con A on the cell surface but not in the cytoplasm is required to stimulate lymphocytes (Pauli et al., 1973).

We reported previously in scanning electron microscopic studies considerable changes of the ectoderm cell surface by the treatment of Dolichos biflorus agglutinin (DBA) which does not have any signifi­cant inducing activity (Yamamoto et al., 1981). In transmission electron microscopic observation we confirmed using ferritin­conjugated DBA that a tremendous amount of DBA molecules is bound on the cell surface, whereas ferritin-conjugated Con A molecules are much less frequent than DBA. These observations indicate that the mere perturbation of molecular organization in the plasma membrane caused by binding of lectins is not a trigger for setting the induc­ing machinery into operation but that there should be a certain modification in the plasma membrane, leading to commencement of induction.

2) To examine whether the sugar residues in the plasma membrane play a primary role as the receptor responding to the neural stimu­lus, the following experiments were conducted. The ectoderm explants were first treated with neuraminidase at pH 6.1 (Nakarai Chemicals, Ltd.) to remove sialic acid which, if present, disturbed the enzyme activity in the next step treatment. Subsequently, explants were treated with almond glycopeptidase, which specifically cleaves e­aspartylglucosylamine linkage in glycoproteins and thus could be an excellent tool for analyzing asparagine-linked (mannose-containing) oligosaccharides (Takahashi, 1977; Takahashi and Nishibe, 1981). The control explants were incubated without enzymes in the same buffer solution (pH 6.1). After the treatment both the experimental and control explants were induced by Con A (300 ~g/ml for 3 hr) and cultured in SBSS for two weeks. The neural induction frequency of the experimental and the control was 13% and 71% respectively (Figure 2). These results indicate that the mannose-containing oligo­saccharides of glycoproteins on the cell surface of Cynops presump­tive ectoderm play a role in the commencement of neural induction.

3) and 4) Isolation and characterization of oligosaccharides on the cell surface. Early blastulae were raised up to the early gas­trula stage in SBSS containing 3H-mannose (50 ~Ci/ml) for labelling oligosaccharides of cell surface glycoproteins. The presumptive ectoderm thus labelled was excised, fixed in glutaraldehyde fluid and subjected to enzymatic treatments (Figure 3). To compare cell sur­face oligosaccharides with intracellular ones, the latter were iso­lated from the homogenate of explants from which surface oligo­saccharides had been removed. Several kinds of oligo saccharides labelled with 3H-mannose were obtained through filtration on a Bio­Gel ·p-4 column. It could be ascertained from the fractionation profiles that extracellular fractions were different from intra­cellular ones. This also implies that the fractions we obtained from the intact explants were not contaminants from the intracellular ones, but were of extracellular origin (Takata et al., 1984).

86 K. TAKATA ET AL.

a ' ..

b

Fig. 2. (a) Neural tissue induced by Con A - treatment without enzymes, x 230. (b) Explant treated with neuraminidase and almond glycopeptidase showing no neural tissue, x 230.

There has been no crucial evidence, at present, concerning the molecular mechanism participating at the confronted surfaces between organizer and competent ectoderm during embryonic induction, never­theless a great deal of effort has been done to elucidate events or factors involved in the induction mechanism (Yamada, 1958; Saxen, 1961; Tarin et al., 1973; Tiedemann and Born; 1978; cf. Nakamura and Toivonen, 1978). We use lectins as probes for identifying the cell­ular sites, on which neural-inducing stimuli act, and for character­izing the nature of molecules ~onstituting the sites (receptors).

LECTIN TREATMENT OF PRESUMPTIVE ECTODERM

Blastulae labelled with 3H-mannose

I Presumptive ectoderm excised at St.ll gastrulae

I fixed with 2.5 % glutaraldehyde

I Sialic acid removed by N/IOO HCI (90°C, I hr)

I Sugar chains released by glycopeptidase

I

Residual tis~ue fragments I

Supernatant

I I ground by a glass rod applied to gel-filtration

I (Bio-Gel P-4) digested with pepsin 500 pI fractioris collected

I I digested with glycopeptidase R~dioactivity counted

I I

Solid fraction I

Soluble fraction (CELL SURFACE FRACTIONS)

I applied to gel-filtration

(Bio-Gel P-4) 500 pI fiactions collected

I Radioactivity counted

(INTRACELLULAR FRACTIONS)

87

Fig. 3. Schematic diagram representing the isolation procedure of sugar chains from Cynops presumptive ectoderm.

Experimental results we obtained so far seem substantial enough to indicate that at least one of the cell surface molecules which are responsive to the neural-inducing stimuli is an asparagine-linked oligosaccharide in glycoproteins. On the other hand, the organizer factor acting on the target cells during gastrulation is still ob­scure. In the experiments using the transfilter induction system (Saxen, 1961, Tarin et al., 1973), it was shown that certain com­ponents are transmitted from organizer to the presumptive ectoderm during gastrulation. At present, it is not known whether those components are lectin-like substances. Related to this, however, it is of interest that an endogenous lectin has been found in Xenopus embryos (Harris and Zalik, 1982) as well as in chick embryos (Cook et al., 1979). It may be possible that our experimental systems with lectins shed light on the molecular basis of neural induction in the target cells (competent ectoderm cells).

REFERENCES

Cook, G. M. W., Zalik, S. E., Milos, N., and Scott, V., 1979, J.Cell Sci., 38:293-304.

Duprat, A. M., Gualandris, L., and Rouge, P., 1982, J.Embryol.Exp. Morphol., 70:171-187.

88 K. TAKATA ET AL.

Grunz, H., and Staubach, J., 1979, Roux's Arch.Devel.Biol., 186: 77-80.

Harris, H. L., and Zalik, S. E., 1982, Roux's Arch.Devel.Biol., 191: 208-210.

Nakamura, 0., and Toivonen, S., eds., 1978. "Organizer - A mile-stone of a half-century from Spemann". Elsevier/North-Holland Biomedical Press, Amsterdam.

Pauli, R. M., DeSalle, L., Higgins, P., Henderson, E., Norin, A., and Strauss, B., 1973, J.lmmunol., 111:424-432.

Saxen, L., 1961, Devel.Biol., 3:140-152. Takahashi, N., 1977, Biochem. Biophys.Res. Commun. 76:1194-1201. Takahashi, N., and Nishibe, H., 1981, Biochim.Biophys.Acta,

657:457-467. Takata, K., Yamamoto, K. Y., and Ozawa, R., 1981, Roux's Arch.Devel.

BioI., 190:92-96. Takata,~ Yamamoto, K. Y., Ishii, I., and Takahashi, N., 1984, Cell

Differen., in press. Tarin, D., Toivonen. S •• and Saxen, L •• 1973. J.Anat.,115:147-148. Tiedemann, H., and Born, J., 1978, Roux's Arch.Devel.Biol., 184:

285-299. Yamada, T., 1958, in: "A Symposium on the Chemical Basis of

DevelopmentTliW. D. McElroy and B. Glass eds •• Johns Hopkins Press.

Yamamoto, K. Y •• Ozawa, R., Takata, K., and Kitoh, J., 1981, Roux's Arch.Devel.Biol., 190:313-319.

NEURAL EMBRYONIC INDUCTION

H. Tiedemann

Institut fUr Molekularbiologie und Biochemie Freie Universitat Berlin

In 1923 it was discovered by Hilde Mangold and Hans Spemann that the blastoporal lip, the presumptive mesodermal anlage, induces in ventral ectoderm a second neural anlage. In normal development ventral ectoderm does not form any nervous tissue but epidermis. The so-called "organizer experiment" was confirmed by many other investi­gators and with other experimental techniques. It was a very import­ant discovery which finally led to a better understanding of cell determination and differentiation, but it was also an untimely one. In the 1920's many facts on the biochemistry and the subcellular organization of cells had not been discovered, which later proved to be relevant to the investigation of induction and differentiation and many experimental tools were not yet developed.

I will first describe experiments on the isolation, chemical nature and subcellular distribution of neural inducing factors and then try to correlate these factors with their possible mechanism of action.

For the test of the biological action of inducing factors a test system is needed. Because early gastrula ectoderm is not yet irre­versibly determined to ectoderm and can be channelled into other pathways of differentiation, ectoderm of early gastrula stages is very suitable for testing inducing factors. Different test methods have been developed (Figure 1). The most convenient is the implan­tation method whereby whole gastrulae are used. A pellet of the material to be tested is implanted into the blastocoel so that by the gastrulation movements it is brought into contact with the ventral ectoderm which can be induced (Mangold, 1923). Forehead inductions induced by neuralizing factor are shown in Figures 2a and b. The substances can be tested in different concentrations; before the

89

90 II. TIEDEMANN

A

Fig. 1. Test methods for inducing factors. Ectoderm of an early gastrula (Et) is brought into contact with the inducer (Ind) either by the implantation method or by the sandwich method. In this method the inducer is wrapped into two pieces of isolated gastrula ectoderm (Et). In the third method ecto­derm is explanted in salt solution (Sol) and covered with silk to prevent curling off.

preparation of the pellet an inert non-inducing protein is added in different proportions. Another method adopted from conventional tissue culture methods gave the same results. In this method a piece of isolated ectoderm is covered with silk to prevent its curling off and then cultured in salt solution to which inducing factors were added (Becker et al., 1959). The third test method is the so-called sandwich method which was developed by Holtfreter. The pellet of inducing factor is wrapped in two pieces of ectoderm like a sandwich (Holtfreter, 1933a). In both methods which use isolated ectoderm possible host influences are excluded.

We have isolated inducing factors from 9 to 11 day old chicken embryos which have a high inducing activity. A vegetalizing factor Mr ~ 13,000) has been purified to homogeneity which induces primarily

NEURAL EMBRYONIC INDUCTION 91

. . a

b

Fig. 2. a) and b) Medium size forehead inductions with an eye on the belly of Triturus alpestris larvae. Fractions contain­ing inducing factor were implanted into the blastocoel of the early gastrula stage.

endoderm and by secondary interactions mesoderm-derived organs (noto­chord, myomeres, renal tissue, blood cells; Geithe et al., 1981; Asahi et al., 1979; Tiedemann, 1982). A neuralizing factor which induces foreheads with eyes has been enriched (Tiedemann et al., 1963) but could not be highly purified because the factor is tightly bound to subcellular structures. Both factors are inactivated by proteolytic enzymes (trypsin, pepsin) and are therefore protein in nature (Tiedemann et al., 1960).

More recently we have isolated neuralizing factors from eggs and early stages of amphibian embryos, where these factors act in normal development. This will make it easier to investigate the mechanism of action of these factors in normogenesis. The isolation of in­ducing factors from amphibian embryos is very difficult because they have to be separated from the large amount of yolk platelets which have no inducing activity. It has previously been shown that in­ducing factors can be extracted from amphibian embryos (Faulhaber, 1970). We have isolated subcellular fractions from the homogenate of Xenopus embryos and tested these for their inducing activity. The

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NEURAL EMBRYONIC INDUCTION 93

scheme of fractionation is shown in Figure 3. The 6,000 (9,000) rpm sediment contains all the yolk, pigment granula and chromatin. It is mechanically contaminated with vesicles and particles from the super­natant. The inducing activity of the sediment is very much reduced after several extractions with sucrose (1.5 M)-polyvinylpyrrolidon (5%) buffer or with deoxycholate containing buffers. The residual yolk has a very low inducing activity (Table 1).

When the material which was extracted from the 6,000 (9,000) rpm sediment was repeatedly separated on stepwise sucrose gradients (Janeczek et al., 1983) the material in the 50% sucrose layer has a low inducing activity. This fraction consists mostly of plasma membranes as was shown by their labelling with diazotized 35S-sulfan­ilic acid. The small inducing activity of plasma membranes suggests that inducing factors are obviously not integral proteins of the plasma membranes. Plasma membranes contain binding sites for neural inducing factors (Born et al., 1980) to which a small amount of factor could be bound. This or a contamination with other subcel­lular structures could explain the small inducing activity. Factor which is loosely attached to the membrane could on the other hand be separated from the plasma membrane by the fractionation procedure.

The supernatant of the 6,000 (respectively 9,000) rpm sediment was centrifuged for 2 h at 100,000 x g. The supernatant shows neural inducing activity (Table 1). A histological section of an archen­cephalic induction is shown in Figure 4. The inducing activity of the supernatant did not change when protease inhibitors as phenyl­methylsulfonylfluoride, leupeptin, pepstatin and a-2 macroglobulin were added before homogenization. This shows that the factor in the supernatant is not released from particulate material by proteolytic cleavage. Such a possibility has to be excluded because proteolytic enzymes are activated by homogenization. Size exclusion chromato­graphy on Sephacryl S 200 has shown that the factor is present in a wide range of different size classes. The size classes from about 30,000 to 200,000 Daltons are probably not due to the formation of complexes as HPLC chromatography under denaturing conditions has shown. This suggests an in vivo processing of the factor by limited proteolytic cleavage. The factor bands by isoelectric focusing under non-denaturing, as well as denaturing conditions in 6 M urea, in the slightly acidic range at pH 5-6.

The highest neural inducing activity is found in two fractions which are derived from the 100,000 x g microsomal sediment after repeated gradient centrifugation (Tiedemann, 1982). One of these fractions contains a heterogeneous population of small vesicles (Table 2). Some are filled with more or less electron dense material, as the electron micrograph shows. When this vesicle frac­tion was treated with the non-ionic detergent Triton X 100 and cen­trifuged, all the activity was found in the sediment. RNP particles which are present in the fraction could be removed by treatment with

Tab

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NEURAL EMBRYONIC INDUCTION 95

Impl.

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Fig. 4. Induction of an eye by a high speed supernatant from Xenopus gastrulae. L = lens; T = tapetum; Impl. = implant. Brain is present in other serial sections of this induction. Magnification: x 100.

EDTA. These particles have no inducing activity. The active factor in this vesicle fraction can be largely extracted with slightly alkaline buffer at pH 8.5-9.0.

The second fraction with a very high neural-archencephalic inducing activity contains ribonucleoprotein particles (Table 2; Figure 5) which were separated from ribosomal subunits on a stepwise sucrose (25-65%) gradient in the presence of EDTA. The ribosomal subunits do not have inducing activity (Tiedemann, 1982; Janeczek et al., 1983). The RNP particles were further fractionated on linear sucrose gradients. The highest inducing activity show particles in the 18 - 27 S region (fractions 7 - 16 in Figure 6). The RNP par­ticles are contaminated with degradation products of ribosomal sub­units. The preparation of the particles was therefore carried out in the presence of ribonuclease inhibitor from human placenta. The inducing activity is then partially shifted to larger particles. This suggests that the RNP particles are in part derived from larger ribonuclease and EDTA sensitive ribonucleoprotein complexes by limited degradation with ribonuclease. A histological section of an archencephalic induction induced by diluted RNP particles is shown in Figure 7.

'D

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NEURAL EMBRYONIC INDUCTION 97

Fig. 5. Electronmicrograph of negatively stained RNP particles. The particles were prepared in the presence of ribonuclease in­hibitor and isolated from fraction 13 of the linear sucrose gradient (Figure 6).

'"' 3 E c::

0 <0

~ 2 Ql u c:: m

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Fractions

Fig. 6. Centrifugation of gastrula RNP particles on a linear (10-30%) sucrose gradient (16 h at 35,000 rpm; Spinco rotor SW 41). The gradient was fractionated and the absorbance monitored at 260 nm.

Whether the RNP particles with high neural inducing activity are all of cytoplasmic origin or whether they are also derived from heterogeneous nuclear ribonucleoprotein particles released from broken nuclei is not yet known.

The neural archencephalic inducing activity of the RNP particles could depend on the protein or the RNA moiety of the particles.

98 H. TIEDEMANN

Fig. 7. Induction of eye, nose and brain by RNP particles diluted with y-globulin. Impl. = implant; E = eye surrounded by dark pigmented tapetum; N = nose; B = brain. The lumen in the induced nose and larger masses of nerve cells and fibers are present in other serial sections of this induction. Magnification: x 100.

Incubation with ribonuclease did not change the inducing activity, whereas incubation with trypsin (or with proteinase K) largely abol­ishes the inducing activity of the particles (Table 3; Janeczek et al., 1983). This suggests that the protein moiety is responsible for the inducing activity of particles derived from gastrula stages as well of particles from eggs. Obviously maternal messenger RNA has no inducing activity.

The protein moiety of gastrula RNP particles was extracted with phenol and the proteins isolated from the phenol layer. The isolated protein has a lower inducing activity than the RNP particles {Table

Tab

le

3.

Ind

uci

ng

acti

vit

y o

f RN

P p

art

icle

s aft

er

trea

tmen

t w

ith

pro

teo

lyti

c e

nzym

es o

r w

ith

ri

bo

nu

clea

se A

Siz

e o

f in

du

ctio

ns

(%)

Ind

uce

d r

egio

ns

(%)

Tre

atm

ent

of

No

of

Po

siti

ve

Fo

re-

Hin

d-

un

spec

. n

ot

fracti

on

ca

ses

%

Lar

ge

Med

ium

S

mal

l h

ead

h

ead

n

eu

ral

specif

ied

Try

psi

n

(eg

g)*

28

43

0

0 43

3

11

0 29

C

on

tro

l (e

gg

)*

28

71

0 25

46

43

0

7 21

R

ibo

nu

clea

se A

(e

gg)*

* 30

43

0

4 39

9

0 7

27

Rib

on

ucl

ease

A

(gast

rula

)**

27

22

0

4 18

8

0 8

6 C

on

tro

l (g

ast

rula

)**

30

23

0

3 20

6

0 9

8

*D

ilu

ted

wit

h o

ne p

art

y

-glo

bu

lin

befo

re p

rep

ara

tio

n f

or

the

bio

log

ical

test.

*

*D

ilu

ted

wit

h t

hre

e p

art

s y

-glo

bu

lin

befo

re p

rep

ara

tio

n f

or

the

bio

log

ical

test.

§:i

c:! ~ tzj ~ ~ §i:l

H

tJ

H S c:!

tJ

H

H o Z

\0

\0

100 H. TIEDEMANN

2). This could depend on a chemical modification, especiallyoxi­dation of the protein, but it is also possible that the intact par­ticles are more easily taken up by the cells than the isolated pro­tein. Ribonucleic acid isolated from the aqueous phase has no in­ducing activity (Table 2).

The inducing capacity of the protein was somewhat enhanced when the protein was reduced with 2-mercaptoethanol. The protein is soluble in 75% formic acid but does not dissolve in more dilute formic acid. This suggests that at least some of the proteins are hydrophobic. The protein was then fractionated by high pressure size exclusion liquid chromatography in 75% formic acid on a derivatized silica gel. The main inducing activity was found in a fraction which contains molecules from 10,000 to 16,000 Dalton (Table 2). Whether the inducing activity which was found in fractions with higher mol­ecular weight is due to an aggregation of the factor or to the pres­ence of precursor molecules from which the factor with lower mol­ecular weight arises in the cells by limited proteolytic cleavage, is not yet known.

How can the distribution of neural inducing factors in different subcellular fractions be correlated with the mechanism ot induction and tissue differentiation? The neuralizing factors are present in eggs, early embryonic stages and in gastrula ectoderm in a biological inactive masked form. Very little soluble neuralizing factor is found in the high speed supernatant from eggs and early embryonic stages as well as the high speed supernatant from uninduced ectoderm. Neuralizing factor is however found in high speed supernatants from the homogenate of gastrula stages and in the high speed supernatant from neural plate homogenates (John et al., 1983a). In the gastrula stage the factor is exported from the mesoderm which induces the neural plate. The induced neural plate itself then acquires neural inducing activity (Mangold, 1933; Tiedemann-Waechter, 1960), probably due to an export of the factor. It is therefore not unlikely that the soluble factor in the high speed supernatant included molecules which are to be exported from the tissues with neural inducing act~v­ity (mesoderm, neural plate). This hypothesis is further supported by the finding that neural inducing factor could be isolated from the extracellular space between the mesoderm and the overlying dorsal ectoderm which is induced (John et al., 1983b). The small vesicles with high neural inducing activity could be involved in the export of neuralizing factor by exocytosis. The excreted factor is probably bound to receptor sites on the plasma membranes of competent cells and neural differentiation is then mediated by a signal which is generated on the plasma membrane of these cells. The nature of this signal and the chain of reactions which actuate and conduct the signal are not known. An activation of the factor in the RNP par­ticles could be involved in the process of signal transfer to the target site within the cells. To prove this hypothesis further investigations, especially with immunological methods are however

NEURAL EMBRYONIC INDUCTION

needed. A precursor product relationship between the factors in different subcellular fractions is also not excluded.

101

In the experimental test system (Figure 1) the activated RNP particles could on the other hand be taken up by the ectoderm cells as many other proteins are taken up (Vainio et al., 1962). Thereby the membrane mediated steps could be circumvented.

It has been shown that the masked factor in the ectoderm can partially be activated by homogenization and freezing of the ectoderm and fully activated by treatment with ethanol (Figure 8). These are treatments which denature complex protein or ribonucleoprotein struc­tures, but do not inactivate the neuralizing factor(s). Neuralizing factor is found in the high speed supernatant when the ectoderm was treated before homogenization with Cytochalasin Band Colcemid to disaggregate cytoskeletal elements or when the homogenate was incu­bated for 5 h, so that a partial autolysis could take place. These treatments do however not lead to full activation (John et al.,

Fig. 8. Induction of eye, nose and brain by activated factor from gastrula ectoderm. L = lens; T = tapetum; N = nose; B = brain. The lumen of the induced nose and larger masses of nerve cells with fibers are present in other serial sections of this induction. Only a small piece of the total implant (Impl.) is seen in this serial section. Magnification: x 100.

102 H. TIEDEMANN

1983a). It is not unlikely that in normal development the factor is unmasked by a modification or partial degradation of molecules which form complexes with the factor.

There is no indication that cyclic nucleotides or Ca++ - ions are rate limiting signals in induction. Cyclic nucleotides, their mono- and dibutyryl derivatives (Grunz et al., 1977) or treatment of ectoderm with the calcium ionophore A 23187 do not evocate inductions as shown by the careful experiments by Grunz and Hildegard Tiedemann (unpublished) with ectoderm of Triturus alpestris. Changes in the sodium postassium ratio after treatment with Quabain (10-4 M) did lead to the formation of a few mesenchyme and perhaps blood cells besides large masses of ectoderm.

It has been reported that cyclic nucleotides and the Caionophore A 23187 can elicit neural differentiation in the ectoderm of Amby­stoma mexicanum (Wahn et al., 1975; L~vtrup et al., 1983). But in the experiments of Grunz and Hildegard Tiedemann a high rate of autoneuralization occurred in Ambystoma ectoderm explants under the conditions of the test without any additions to the culture medium. The percentage of explanted ectoderm which formed neural tissues in the control series (up to 85%) differed from batch to batch of Amby­stoma eggs. The ectoderm of Triturus alpestris shows under the same conditions no autoneuralization.

The total inducing activity of the ectoderm does not increase after neural induction. This is a further argument for the acti­vation hypothesis which is probably also true for the so-called autoneuralisation process. The first event in autoneuralisation which takes place, as was already mentioned, very easily in Ambystoma ectoderm but not in Triturus ectoderm, could be due to an export of neuralizing factor by exocytosis. The exported factor could in turn induce other ectodermal cells by the membrane mediated mechanism of neural induction. Alternatively a direct activation of masked intra­cellular factors is not excluded.

Since the discovery of the inducing activity of the presumptive dorsal mesodermal area ("Urdarmdach") and the totipotency of ecto­dermal cells, other theories have been proposed. Goerttler (1925) came to the conclusion that the morphogenetic movements which are associated with gastrulation and neurulation are the essential fac­tors which determine embryonic differentiation. Similar ideas were proposed by other authors. The induction of well organized organs by highly purified factors disproves this theory. Changes in the cell shape and coordinated movements are of course necessary for the formation of typical organ structures. The histiotypic different­iation of cells is however not prevented when typical morphogenetic movements can not take place under certain conditions in tissue culture (ectoderm covered with silk to prevent its curling up; Becker et al., 1959). Changes of cell affinities {which besides changes in the cytoskeletal architecture are important for the movement of cells

NEURAL EMBRYONIC INDUCTION 103

and cell complexes) occur after the cells are determined for a cer­tain pathway of differentiation (Kocher-Becker et al., 1965).

Other authors have argued that gastrula ectoderm is a mosaic of already determined cells. It has been supposed (Ave et al., 1968) that inducing factors select cell species with "inhomogeneous devel­opmental potencies" (i.e. neural or mesodermal potencies) because a larger part of ectodermal cells was killed by mesodermalizing or neuralizing fractions used by the authors. It has however been shown that with the appropriate inducers almost the total cell population of isolated ectoderm can be induced either to endodermal-mesodermal or neural-epidermal tissues.

It can not be deduced from fate maps which cells of a certain developmental stage are already determined to their later fate in development. Fate maps have been constructed by marking certain regions of eggs and early embryos by vital staining (Vogt, 1929). Jacobson (1982) has injected lactoperoxydase into cells of early Xenopus embryos to mark certain regions. Such experiments give information on cell lineage and spatial arrangements of cell clusters within certain boundary lines (provided that the label is not trans­ferred to neighboring cells). There is no doubt that certain ances­tral cells give rise to distinct groups of differentiated cells. But these experiments do not give information on the means and the time at which cells become determined. Jacobson (1982) believes in his compartmentation theory that "the primary morphological pattern of the CNS (of Xenopus) arises in the midblastula stage". But this lacks any experimental evidence. It is very unlikely that the basic mechanism of the determination of the CNS in Xenopus is different from Triturus. It has recently been shown (Asashima et al., 1983) that also ectoderm isolated from early gastrulae of Xenopus does not develop into nervous tissue, but can be induced to neural tissue or tissues which are in normogenesis derived from endoderm or mesoderm depending on the inducer which is used. Ectoderm of early gastrulae of Xenopus or Triturus is however not "undetermined". After isol­ation, ectoderm develops into epidermis like cells (Grunz, 1977). This means that the ectoderm is already determined, but the state of determination is labile and can be changed (s. also Tiedemann, 1982).

Jacobson (1982) explains the formation of a second neural system in the ventral ectoderm in the experiment of Spemann and Hilde Mangold by migration of neural ancestor cells from the dorsal to the ventral side. Similar ideas on an inherent polarity of the ectoderm had already been proposed by Goettler in 1927 and proven to be wrong by Holtfreter in 1933 (b+c). Holtfreter has shown that heteroplas­tically transplanted dorsal ectoderm (presumptive neural tissue) as well as ventral ectoderm can be induced to neural tissue. Further­more the total amount of neural tissue in embryos with the normal head and a large forehead or hindhead induction on the ventral side can be much larger than in normal embryos. The experiments on the

104 H. TIEDEMANN

differentiation capacities of parts of the gastrula were extensively reviewed by Holtfreter and Hamburger (1955).

In conclusion the experiments show that there are very complex interactions at three different levels. Inducing factors are already present in different amounts in all regions of eggs and early embryos in a masked, inactive form. They become activated in different regions of the embryo at different stages of development. The acti­vation may depend on quite different mechanisms. The acquisition of neural inducing activity of the presumptive mesodermal area in the morula to blastula stage is correlated with an export of the factor from this tissue. The third level is the ability to react to an inducing factor (the so-called competence of a tissue). This ability could depend on the formation of ,plasma membrane binding sites for certain factors, but on the other hand also on the state of chromatin which may include tissue specific protein-DNA interactions modifying the accessibility of genes. In experiments with W. and M. Minuth (Minuth et al., (1981) it was shown that in the ectoderm an intrinsic program for the synthesis of nuclear protein is running down. But this program is changed when the ectoderm is induced to develop into other tissues. Hybridization experiments with cloned DNA revealed a stage specific expression of genes at the level of transcription and RNA-processing (Dworkin and Dawid, 1980; Knochel and John, 1982). Which genes are regulated in neural differentiation is however not yet known. In general. events which are intrinsic to cells of cer­tain regions are modified by extrinsic signals which are generated in neighboring tissues.

Acknowledgements

This investigation was supported by the Deutsche Forschungs­gemeinschaft (Sonderforschungsbereich Embryonale Entwicklung und Differenzierung) und Fonds der Chemischen Industrie.

REFERENCES

Asahi. K. -i •• Born. J., Tiedemann. H., Tiedemann. H •• 1979, Roux's Arch.Dev.Biol., 187:231-244.

Asashima, M., Grunz, H., 1983, Differentiation, 23:206-212. Ave, K., Kawakami, I., Sameshima, M., 1968, Dev.Biology, 17:611-626. Becker, U., Tiedemann, H., Tiedemann, H., 1959, Z.Naturf., 14b:

608-609. Born, J., Grunz, H., Tiedemann, H., Tiedemann, H., 1980, Roux's Arch.

Dev.Biol., 189:47-56. Dworkin, M. B., Dawid, I. B., 1980, Dev.Biol., 76:449-464. Faulhaber, I., 1970, Hoppe-Seyler's Z.Physiol.Chem., 351:588-594. Geithe, H. -P., Asashima, M., Asahi, K. -i., Born, J., Tiedemann. H ••

Tiedemann, H •• 1981. Biochim.Biophys.Acta. 676:350-356.

NEURAL EMBRYONIC INDUCTION

Goerttler, K., 1925, Roux's Arch.Dev.Biol., 106:503-541. Goerttler, K., 1927, Roux's Arch.Dev.Biol., 112:517-576. Grunz, H., 1977, Roux's Arch.Dev.Biol., 181:267-277.

105

Grunz, H., Tiedemann, H., 1977, Roux's Arch.Dev.Biol., 181:261-265. Holtfreter, J., 1933a, Roux's Arch.Dev.Biol., 128:584-633. Holtfreter, J., 1933b, Roux's Arch.Dev.Biol., 127:591-618. Holtfreter, J., 1933c, Roux's Arch.Dev.Biol., 127:620-775. Holtfreter, J., Hamburger, V., 1955, in: "Analysis of Development",

B. H. Willier, P. A. Weiss, V.1ffamburger, eds., W. B. Saunders, Philadelphia a. London, pp. 230-296.

Jacobson, M., 1982, in: "Current Topics in Neurobiology", N. C. Spitzer, ed.,lPlenum Press, New York, pp.45-99.

Janeczek, J., John, M., Born, J., Tiedemann, H., Tiedemann, H., 1983, in preparation.

John, M., Born, J., Tiedemann, H., Tiedemann, H., 1983a, Roux's Arch. Dev.Biol., in press.

John, M., Janeczek, J., Hoppe, P., Tiedemann, H., Tiedemann, H., 1983b, Roux's Arch.Dev.Biol., 192:45-47.

Knochel, W., John, M. E., 1982, Europ.J.Biochem., 122:11-16. Kocher-Becker, U., Tiedemann, H., Tiedemann, H., 1965, Science, 147:

167-169. L6vtrup, S., Perris, R., 1983, Cell Diff., 12:171-176. Mangold, 0., 1923, Arch.Entw.Mech.Org., 100:198-301. Mangold, 0., 1933, Naturwiss., 21:761-766. Minuth, W., Minuth, M., Tiedemann, H., 1981, IRCS Medical Science,

9:36 Spemann, H., Mangold, H., 1924, Arch.Entw.Mech.Org., 100:599-638. Tiedemann-Waechter, H., 1960, Roux's Arch.Dev.Biol., 152:303-338. Tiedemann, H., Tiedemann, H., Kesselring, K., 1960, Z.Naturforschg.,

15b:312-319. Tiedemann, H., Becker, U., Tiedemann, H., 1963, Biochim.Biophys.Acta,

74:557-560. Tiedemann, H., 1982, in: 23. Colloquium Ges. BioI. Chem.: "Bio­

chemistry of Differentiation and Morphogenesis", L. Jaenicke, ed., Springer Verlag, Berlin-Heidelberg, pp.275-287.

Tiedemann, H., 1982, in: "Biochemical Development of the Fetus and Neonate", C. T-:-Jones, ed., Elsevier Biomedical Press, pp. 65-99.

Vainio, T., Saxen, L., Toivonen, S., Rapola, S., 1962, Expt.Cell Res. 27:.'>27-538.

Vogt, W. -C., 1929, Roux's Arch.Dev.BioI." 120:385-706. Wahn, H. L., Lightbody, L. E., Tchen, T. T., 1975, Science, 188:

366-369.

2

CELL MIGRATION AND EARLY STAGES OF DIFFERENTIATION

ADRENERGIC DIFFERENTIATION IN THE AUTONOMIC

NERVOUS SYSTEM

Philippe Cochard

Institut d' Emhryologie 49, bis avenue de la Belle Gabrielle Nogent-sur-Marne 94130, France

INTRODUCTION

The problem of the mechanisms generating neuronal diversity in terms of neurotransmitter expression has been extensively studied in the autonomic nervous system. Autonomic neurons utilize a number of well characterized neurotransmitters including catecholamines (CA) and acetylcholine (ACh). These neurons derive from a transient embryonic structure, the neural crest. From a dorsal midline pos­ition on the neural primordium, neural crest cells migrate in the embryo, following precise pathways and settle in various localiz­ations; some of them aggregate lateral to the aorta to form adren­ergic sympathetic chain primordia, while others migrate more ventrally in the gut mesenchyme and eventually give rise to cholin­ergic and peptidergic enteric neurons (see Le Douarin, 1982, for references).

It is now well established that, as a population, neural crest cells are not predetermined to express the catecholaminergic or the cholinergic phenotypes (Le Douarin and Teillet, 1974; Le Douarin et al., 1975). Moreover, in certain experimental conditions, a high degree of plasticity in neurotransmitter expression has been demon­strated in the developing autonomic neuron, which, perhaps, persists into adulthood (see Patterson, 1978; Le Douarin, 1982). These exper­iments have revealed the predominant role played by the environment encountered by autonomic neurons and their precursor cells during ontogeny, on the promotion and regulation of transmitter choice.

To further define the role of the environment on crest cell development and elucidate its nature, we focused on the study of the mechanisms regulating the expression of the adrenergic phenotype.

109

110 P. COCHARD

Sensitive techniques are available to demonstrate in situ the pres­ence of individual adrenergic phenotypic characters during ontogeny: the CA-biosynthetic enzymes and the CA themselves. Using these markers in normal development as well as in a variety of experimental conditions, we have addressed specific questions: when and where are individual noradrenergic characters initially expressed? Is this early expression irreversible in vivo? Under which factors is it stimulated or inhibited?

INITIAL EXPRESSION OF THE ADRENERGIC PHENOTYPE DURING NORMAL DEVELOPMENT (see Figure 1 and Table 1)

Using immunocytochemical and CA-histofluorescence techniques, we have shown in the rat embryo that neither CA nor the enzymes respon­sible for their biosynthesis are detectable in neural crest cells during their initial dorso-ventral migration. Tyrosine hydroxylase (TOH), dopamine-~-hydroxylase (DBH) and CA first appear simul­taneously at 11 days of gestation, soon after crest cells have coalesced at the dorso-lateral aspect of the aorta to form the primordia of paravertebral ganglia (Figure la) (Cochard et al., 1978, 1979). This indicates that as soon as the enzymes are present in detectable quantities they are metabolically active. In addition, the remarkable degree of synchrony in the appearance of two major enzymes of the CA biosynthetic pathway suggests that their expression is regulated by the same mechanism. Similarly, in the chick embryo, CA cannot be detected before presumptive sympathetic neurons have reached their destination and are found first in sympathetic ganglion primordia at 3.5 days of incubation (Enemar et al., 1965; Allan and Newgreen, 1977).

However, the environment of the aorta is not the only site in which the initial expression of the adrenergic phenotype normally occurs. In the rat embryo, we have described a population of cells in ectopic position within the gut mesenchyme, that, at 11 days of gestation, display immunoreactivities for both TOH and DBH. (Coch,ard et al., 1978, 1979). Their morphological and biochemical features are identical to those of sympathoblasts developing at the same stage along the aorta (Figure Ib). Moreover, their time of appearance, location and the recent evidence that at least some of them elaborate neurofilament proteins (Cochard and Paulin, unpublished results) indicate that they are of neural crest origin, and represent precur­sor cells of enteric ganglia. At 12 days of gestation, they become more numerous and can be found almost throughout the gut.

However, the adrenergic phenotype is only transiently expressed in the gut by these cells, since it is not detectable after the 14th gestational day (Table I), (Cochard et al., 1978, 1979; Teitelman et al., 1979). In addition to CA synthetic abilities, these transient catecholaminergic gut cells also exhibit a high affinity uptake

ADRENERGIC DIFFERENTIATION I I I

• . -, -.. ' . • M ... . "

~

~ - -En \..

• .. . :.

, .. e •

,- ... -

Fig. 1. Demonstration of tyrosine hydroxylase (TOR) by immunofluor­escence in rat embryos at 12 days of gestation. a - TOR­immunoreactivity is detectable in the cytoplasm and cyto­plasmic processes of sympathoblasts aggregated at the dorso-lateral aspect of the aorta (Ao). b- At the same stage, TOR-immunoreactive cells (arrows), morphologically similar to sympathoblasts, are also found in the gut mesen­chyme (M). Note the presence of blood cells displaying non-specific fluorescence. En denotes the endodermal layer of the gut. Bars represent 20 ~m.

Tab

le

1.

Dev

elop

men

t o

f in

div

idu

al

adre

ner

gic

ch

ara

cte

rs i

n t

he

rat

embr

yo.

CA a

nd t

heir

sy

nth

esiz

ing

enz

ymes

ty

rosi

ne h

yd

rox

yla

se

(TO

R)

and

do

pam

ine-

S-h

yd

rox

yla

se

(DB

R)

app

ear

sim

ult

aneo

usl

y a

t 11

da

ys o

f g

est

ati

on

(E

ll),

aft

er

cre

st cell

mig

rati

on

, in

sym

path

o­b

last

s an

d n

euro

bla

sts

loca

ted

in

th

e g

ut

mes

ench

yme.

T

he h

igh

aff

init

y u

pta

ke

syst

em

for

no

rad

ren

alin

e (C

A u

pta

ke)

is

dem

on

stra

ble

in

bo

th p

op

ula

tio

ns

at

E12

. A

ll t

hes

e in

div

idu

al

adre

ner

gic

ch

ara

cte

rs p

ers

ist

in s

ym

pat

ho

bla

sts

wh

erea

s at

E14

, TO

R,

DBR

and

CA

, b

ut

no

t th

e u

pta

ke

syst

em,

have

dis

app

eare

d i

n g

ut

neu

rob

last

s (c

on

tro

l).

Row

ever

, th

e ad

ren

erg

ic p

hen

oty

pe

of

thes

e cell

s ca

n b

e "r

escu

ed"

by t

reati

ng

th

e m

oth

er w

ith

g

luco

co

rtic

oid

s o

r by

in

trae

mb

ryo

nic

in

jecti

on

s o

f n

erv

e gr

owth

facto

r (N

GF)

.

Day

s o

f g

est

ati

on

10

11

12

13

14

17

Dev

elop

men

tal

even

ts

Cre

st cell

G

ut

co

lon

izati

on

m

igra

tio

n

Gan

gli

on

ag

gre

gat

ion

CA s

yn

thesi

s +

+

+

+

+

(T

OR

, D

BR)

Sym

path

o-b

last

s

CA u

pta

ke

? +

+

+

+

~+

+/-

+

CA s

yn

thesi

s +

G

ut

neu

ro-

(TO

R,D

BR

) Gluc~icoids

+

+

+

+

bla

sts

NGF

CA u

pta

ke

? +

+

+

+

••

• ?

N

>-.;

(')

0 (') ~ t:1

ADRENERGIC DIFFERENTIATION 113

system specific for noradrenaline (Jonakait et al., 1979). However, this last noradrenergic character does not disappear together with transmitter synthesizing ability and can still be demonstrated at later stages, at least up to the 17th day of gestation (Table 1). This and the fact that the catecholaminergic cells are able to pro­liferate (Teitelman et al., 1981) strongly suggest that their dis­appearance at 14 days of gestation is not due to cell death but rather to the loss of some adrenergic traits, namely the capacity to synthesize CA, while the ability to take up noradrenaline is retained for at least several additional days.

The fate of these cells is still an open question. A very likely hypothesis is that they would acquire other transmitter properties. Such a shift in neurotransmitter phenotypic expression has been well documented in rat sympathetic neurons which, under specific culture conditions, lose the ability to synthesize CA while developing cholinergic properties (see Patterson, 1978 and Swerts et al., this volume). It is interesting to note that in both systems not all adrenergic characters are lost, since the sympathetic cholin­ergic cells also retain the high affinity CA uptake system. Thus it is possible that the transient catecholaminergic gut cells ultimately become cholinergic enteric neurons. Alternatively, they might con­vert to one of the numerous putative transmitters expressed by enteric neurons.

Transient adrenergic cells with a similar developmental time course have also been found in the gut of the mouse embryo (Teitelman et al., 1981 and our own observations) but so far we have been unable to detect such cells in the avian digestive tract (Cochard and Le Douarin, unpublished observations).

In any case, these observations indicate that the expression of the adrenergic phenotype is not always a stable and irreversible process. Furthermore, environmental factors appear to playa criti­cal role even during normal development, since adrenergic different­iation is maintained in sympathoblasts within the sclerotome whereas it disappears in neuroblasts located in the gut mesenchyme.

TISSULAR AND HORMONAL INFLUENCES ON THE EXPRESSION OF THE ADRENERGIC PHENOTYPE

Respective Roles of the Notochord and of Vascular Environments (see Figures 2, 3, 4 and Table 2)

From the above, it appears that adrenergic differentiation occurs only when presumptive sympathetic neurons have reached their destination at the dorsolateral aspect of the aorta, within the sclerotomal part of the somite. Then the question arises as to whether an inductive process, responsible for the expression of the

114 P. COCHARD

adrenergic phenotype, acts while crest cells are migrating dorso­ventrally or when they are in their final location. In vivo and in vitro culture experiments by Cohen (1972) and Norr (1973) indicated that crest cells receive differentiating signals on their route. More specifically the development of the catecholaminergic phenotype was found to depend upon cellular contacts between neural crest cells and the somitic mesenchyme. However, this last structure acquired its inductive capacity only after being previously conditioned by the neural tube and the notochord.

These results, and the fact that sympathoblasts first develop in the vicinity of the notochord, prompted us to investigate whether this structure could, by itself, promote adrenergic cell different­iation. We used, as substratum for crest cell development, the hind-gut mesenchyme, removed from the embryo at 5 days of incubation, before it had been colonized by enteric neuron precursor cells. In various series of experiments, the aneural colorectum was associated with the quail neural primordium, in the presence or absence of the notochord and cultured on the chick chorio-allantoic membrane (CAM) for 2 to 10 days.

In all cases, crest cells migrated into the gut and different­iated into neurons that were shown to express cholinergic traits (Smith et al., 1977). In the absence of the notochord the adrenergic phenotype was never expressed in the cells which had seeded the gut. In some cases, however, CA-containing cells sometimes associated in ganglion-like structures developed along blood vessels of the CAM from some erratic neural crest cells which migrated out of the ex­plant at some distance of the gut wall (Figure 2) (Teillet et al., 1978).

In contrast, when the notochord was included in the explant. groups of cells expressing TOH and CA developed in the site of the myenteric plexus and occasionally also in the submucosal plexus (Figure 3) (Teillet et al., 1978; Co chard et al., unpublished results). Thus the fundamental role of the notochord is demonstrated by its ability to promote the appearance of the adrenergic phenotype in an ectopic environment such as the gut mesenchyme, where, in the avian embryo, it is not normally expressed (Table 2). Moreover, recent data suggest that the notochord may elicit adrenergic traits in vitro in ciliary ganglion neurons which normally would not have expressed them (Teitelman et al., 1982).

Our experiments also demonstrate that adrenergic differentiation can occur independently of the notochord in the environment of the CAM blood vessels. Other ectopic vascular sites have been recently shown to provide excellent conditions for adrenergic cell different­iation: when grafted in the umbilical cord (Lamers et al., un­published results) and in the liver parenchyma (Figure 4) (Cochard and Le Douarin, unpublished results) neural crest cells express

ADRENERGIC DIFFERENTIATION 115

Fig. 2. Association of the quail neural primordium (neural tube plus neural crest) with the aneural colorectum of a 5-day chick embryo. Culture of explant for 7 days on the chick chorio-allantoic membrane (CAM). a - Cells which have migrated out of the explant and aggregated along blood vessels (BV) of the CAM in ganglion-like structures display intense catecholamine (CA)-histofluorescence. b - The same section, stained by the Feulgen-Rossenbeck technique, shows that all the catecholaminergic cells are of crest origin since they express the quail nuclear marker. Bar represents 50 )Jm.

catecholaminergic traits. However, in the absence of the notochord, adrenergic differentiation is delayed by several days. This suggests that factors similar to that produced by the notochord are also present in highly vascularized environments, but are produced later in development (Table 2).

From these results, it is conceivable that adrenergic different­iation is elicited by the notochord in neural crest cells as they migrate dorso-ventrally. Thac the notochord in this process acts directly on the presumptive sympathoblasts or indirectly through the sclerotomal cells which constitute the crest cell environment at this stage is still largely unknown. Later in development, the environ­ment of blood vessels, in the vicinity of which most adrenergic

116 P. COCHARD

Fig. 3. Same experiment as in figure 2, except that a quail notochord has been explanted together with the neural primordium. a - In the presence of this axial structure, a group of cells localized between the endoderm (En) and the smooth muscle (SM) of the gut exhibit intense CA-histo­fluorescence. In the absence of the notochord, catechola­minergic cells were never found in the gut wall. b - The same section, stained by the Feulgen-Rossenbeck technique, shows that the fluorescent cells come from the neural crest, as evidenced by the quail nuclear marker. Bar represents 50 ~m.

ganglia develop, might provide additional cues to reinforce or stabilize a neurotransmitter phenotype that at early stages is very labile (see above, and also Patterson, 1978 and Le Douarin, 1982, for references).

In turn, the fact that the adrenergic phenotype is never expressed in the avian digestive tract or only transiently in that of rodents indicates that such cues are lacking in the gut environment. Alternatively, the gut could release factors promoting the expression of other neurotransmitter characters and excluding that of the adren­ergic metabolism.

ADRENERGIC DIFFERENTIATION 117

Table 2. Influence of the notochord on the differentiation of catecholaminergic properties in crest cells associated with the aneural colorectum on the chorio-allantoic membrane (CAM). Numbers indicate the number of cases in which CA-containing cells were found (in the CAM or along the gut wall) over the total number of grafts observed. In control grafts, some crest cells develop catecholaminergic traits in the CAM, but significant numbers of positive cases are only found after 4 days of grafting. In no case were CA-containing cells found along the gut wall.

In contrast, the presence of the notochord results in the development of adrenergic cells soon after the beginning of the graft and both in the CAM and along the gut.

Duration of the graft (days)

Localization of CA-containing cells

Controls

Notochords

CAM

1/9

3/10

2 - 4

Gut

0/9

3/10

Role of Glucocorticoids (see Figure 5)

CAM

5/5

11/11

5 - 7

Gut

0/5

8/11

The nature of the factors involved in the regulation of the expression of catecholaminergic traits is still a question mark. Nevertheless, several lines of evidence suggest that corticoid hor­mones might playa role in their stimulation and/or stabilization (see for instance McLennan et al., 1980; Fukada, 1980). Moreover, it has been shown that raising maternal glucocorticoid hormones enhances and prolongs expression of noradrenergic phenotypic characters in the transient catecholaminergic gut cells of the rat embryo (Jonakait et al., 1980, 1981).

We have recently tested the effect of glucocorticoids in our experimental system described above. A ring"of silicone plastic polymer (Silastic, Daw Corning) containing 1 to 20 pg of hydrocorti­sone or corticosterone was implanted on the CAM, around the associ­ation of neural crest and hind-gut. In most cases, we observed a dramatic stimulation of adrenergic differentiation: numerous groups of cells with a high CA content developed around and within the gut wall (Figure 5) (Cochard and Le Douarin, unpublished results). The extent of catecholaminergic differentiation was much larger than that previously observed under the influence of the notochord.

118 P. COCHARD

Fig. 4. Association of the quail neural crest with the liver rudiment of a 5-day chick embryo. Although in normal development crest cells never encounter the liver paren­chyma, in these experimental conditions, they migrate between hepatic cords and nearly all of them differentiate into CA-producing cells grouped in small clusters. Bar represents 20~m.

Preliminary experiments have shown that the same result can be obtained in similar explants isolated in vitro, thus ruling out the possibility of an indirect effect of the hormone through the circu­lation of the host embryo. Consequently, glucocorticoids act either directly on neural crest cells or, alternatively, on the gut mesen­chyme, possibly to inhibit the release of factors that would normally antagonize adrenergic differentiation.

A comparable effect of corticoid hormones has been obtained in our laboratory on dissociated crest cells co-cultivated with a var­iety of mesenchymal cells including those of the intestine (Fauquet et al., in preparation).

Whatever the mechanisms involved, these data suggest that ster­oid hormones might be a major component in the regulation of the biochemical differentiation of autonomic neurons, by specifically stimulating adrenergic traits in neuroblasts with an unstable trans­mitter phenotype.

ADRENERGIC DIFFERENTIATION 119

Fig. 5. Influence of glucocorticoids on the differentiation of crest cells. The same type of explant as described in figure 2 (i.e. neural primordium plus aneural gut, without the notochord) has been treated for 6 days by hydro­cortisone. This low power view of a section of the explant shows that under the influence of the corticoid hormone, numerous strands of cells found in the CAM and also around and within the smooth muscle layer of the gut (SM) display intense CA-histofluorescence. The dashed line indicates the separation between the gut proper and the mesenchyme of the CAM. Bar represents 50~m.

CONCLUS IONS

The experimental data gathered over the last decade has abun­dantly documented the initial pluripotentiality of the crest cell population with respect to neurotransmitter differentiation and the critical role played by the environment in this process (see Le Douarin, 1982, for a general review). In vivo experiments, in rat or avian embryos, have indicated that the acquisition of adrenergic transmitter characters occurs at least in two stages (see also Black, 1982). Initial expression of the adrenergic phenotype only takes place after crest cells have completed their dorso-ventral migration and very likely depends upon specific interactions between them and the somitic-notochordal environment. However, this first phase of development is not stable and irreversible. Further maintenance appears necessary for ongoing expression of an initially labile phenotype. This may be ensured by the continuous exposure to the same "initiation factor(s)" and/or other factors. Molecules such as nerve growth factor (Kessler et al., 1979, 1981) and glucocorticoids have been shown to maintain and enhance transmitter phenotypic

120 P. COCHARD

expression in immature autonomic neurons. Thus, it is conceivable that local concentrations in specific areas of these, or similar factors, are responsible for the differentiation pattern observed in the definitive state.

The elegant work of Patterson and coworkers (see Patterson, 1978) on newborn rat sympathetic neurons cultivated in vitro has revealed the existence of a soluble "cholinergizing" factor, i.e. mediating the conversion from an adrenergic to a cholinergic pheno­type. Its purification and biochemical characterization are cur­rently in progress (Weber, 1981; see also Swerts et al., this volume). Obviously, the ultimate isolation and characterization of the putative factors involved in adrenergic differentiation, and particularly that of the initial signal(s), can only be achieved by an in vitro approach. Experiments in our laboratory have shown that molecules present in fetal calf serum and in 9-day chick embryo extract stimulate adrenergic phenotypic expression in cephalic or trunk neural crest cells (Ziller et al., 1979; Fauquet et al., 1981). These studies indicate that direct intercellular contacts between crest cells and the non-neuronal cells constituting their immediate environment might not be necessarily required to elicit adrenergic differentiation.

Acknowledgements

The author wishes to express his gratitude to Professor N. Le Douarin for constant help throughout this study and very stimulating discussions. This work was supported by the Centre National de la Recherche Scientifique, by a grant from the Delegation Generale a la Recherche Scientifique et Technique, and by a grant from the NIH, ROI DEO 4257 03 CBY.

REFERENCES

Allan, I. J., and Newgreen, D. F., 1977, Am.J.Anat., 149:413-421. Black, I. B., (1982), Science, 215:1198-1204. Cochard, P., Goldstein, M., and Black, I. B., 1978, Proc.Natl.Acad.

Sci. USA, 75:2986-2990. Cochard, P., Goldstein, M., and Black., I. B., 1979, Dev.Biol.,

71: 100-114. Cohen, A. M., 1972, J.Exp.Zool., 179, 167-182. Enemar, A., Falck, B., and Hakanson, R., 1965, Dev.Biol., 11:268-283. Fauquet, M., Smith, J., Ziller, C., and Le Douarin, N.M., 1981,

J.Neurosci., 1:478-492. Fukada, K., 1980, Nature, 287:553-555. Jonakait, G. M., Bohn, M. C., and Black, I. B., 1980, Science,

210:551-553. Jonakait, G. M., Bohn, M. C., Markey, K., Goldstein, M., and Black,

I. B., 1981, Dev.Biol. 88:288-296.

ADRENERGIC DIFFERENTIATION 121

Jonakait, G. M., Wolf, J., Co chard , P., Goldstein, M., and Black., I. B., 1979, Proc.Natl.Acad.Sci.USA 76:4683-4686.

Kessler, J. A., Cochard. P., and Black., I. B., 1979, Nature 280:141-142.

Kessler, J. A., Cochard, P., and Black., I. B., 1981, Adv.Neurol., Vol.29: pp. 115-123, Neurofibromatosis. Raven Press, N.Y.

Le Douarin, N. M., 1982, The neural crest. Cambridge University Press, Cambridge.

Le Douarin, N. M., Renaud, D., Teillet, M. A., and Le Douarin. G. H •• 1975. Proc.Natl.Acad.Sci.USA. 72:728-732.

Le Douarin. N. M •• and Teillet. M. A •• 1974. Dev.Biol. 41. 162-184. McLennan, I. S., Hill. C. E •• and Hendry. I. A •• 1980. Nature.

283:206-207. Norr. S. C •• 1973, Dev.Biol. 34:16-38. Patterson. P. H., 1978. Ann. Rev.Neurosci. 1:1-17. Smith. J •• Co chard , P •• and Le Douarin. N. M., 1977. Cell Different,

6:199-216. Swerts. J. P •• Le Van Thai. A •• and Weber. M., 1983. The role of cell

interactions in early neurogenesis (This volume, pp. ). Teillet. M. A •• Cochard. P •• and Le Douarin, N. M •• 1978. Zoon.

6:115-122. Teitelman. G •• Baker. H •• Joh. T. H •• and Reis. D. J •• 1979.

Proc.Natl.Acad.Sci.USA. 76:509-513. Teitelman, G •• Gershon, M. D •• Rothman. T. P •• Joh. T. H •• and Reis,

D. J., 1981. Dev.Biol •• 86:348-355. Teitelman, G •• Iacovitti. L •• Grayson, L •• Joh, T. H., and Reis, D.

J •• 1982, Soc.Neurosci. 12th Annual Meeting. Abstracts vol. 8:70.7

Weber. M. J., 1981, J.Biol.Chem., 256:3447-3453. Ziller. C •• Smith, J., Fauquet, M., and Le Douarin, N. M., 1979,

Prog.Brain Res., 51:59-74.

SEARCH FOR STEM CELLS AND THEIR CHARACTERISTICS

IN THE MOUSE HYPOTHALAMUS

F. de Vitry

College de France Neuroendocrinologie Cellulaire 75231 Paris, Cedex 05

Generation of cell diversity is one of the major problems under­lying brain development. Extensive histological and autoradiographic studies have led to some cells of the neural tube being considered as the ultimate progenitors of all neurons and glia in the adult central nervous system. If neuronal and glial cell lineages derive from the same cell lining the ventricle, one should be able to localize it in the mouse embryonic brain. If they derive from two different ven­tricular cells, stem cells should be generated at an even earlier stage than brain ventricular formation, the 9th day of gestation in the mouse.

Let us recall that in vivo the hypothalamus consists of the lateral and ventral walls of the third ventricle. The mouse fetus hypothalamus begins its development on the 10th day of gestation, soon after the closure of the neural tube. Cells of the ventricular zone divide rapidly and then migrate to their final position. Al­though the majority of the final divisions of the hypothalamic neurons occurs between day 12~ and 14~, some neurons are still div­iding up to embryonic day 16 (or even later) as indicated by auto­radiographic studies[l]. Genesis of ependymal cells start at em­bryonic day 15[2] and we could visualize glial ependymal tanicytes, which line the ventral zone of the ventricle, at embryonic day 19 using an anti-GFA serum. Thus neuronal and glial cell lineages seem to coexist during fetal life, while the brain develops.

Two main axes of research were undertaken in order to explore at which stage of development nerve stem cells are generated, their characteristics and potentialities, and whether they are localized in situ at the ventricular lumen.

123

124 F. DE VITRY

IN VIVO STUDY: A PRESUMPTIVE COMMON PRECURSOR FOR NEURONAL AND GLIAL CELL LINEAGES AMONG VENTRICULAR CELLS

In order to trace cell lineages. we have followed the cellular localization of a neuronal specific and a glial specific protein (y-y enolase and S-100 respectively) from the adult to embryonic day 17 in the ependymal zone of the third ventric1e[3]. This proved to be successful in finding cells containing both antigens. and lining the ventral part of the third ventricle. These cells were observed only between embryonic day 17 and day 3 postnatally by immunocytochemical methods. A transient detachment of some of these ventricular cells (often double cells) could be visualized before their migration into the hypothalamus tissue. as in the migration of post-mitotic neurons. They remained as bifunctional cells up to day 10 postnatally. Later in the development, they differentiated into separate cells, one type containing y-y enolase and the other S-100. like neurons and glial cells. If these" bifunctional ventricular cells represent common precursors of neuronal and glial cell lineages. one should conclude that stem cells are still generated around birth in a precise area of the mouse central nervous system. This brain region fulfills its complete maturation around day 15 postnatally.

ESTABLISHMENT OF PRIMITIVE NERVE CELL LINE F7, REFLECTING PROPERTIES OF NERVE STEM CELLS

In order to understand mechanisms underlying cell diversifi­cation from a common precursor. one should dispose of the in vitro system. For that purpose we isolated a hypothalamic primitive nerve cell line as a tool to characterize nerve stem cells and study their potentialities.

Isolation of F7 Clonal Cell Line

Clonal cell line C7 isolated from primary cultures of 14 day old mouse hypothalamus embryo (A/J) after SV 40 transformation, initially retained the capacity to synthetise neurophysin and vasopressin, a function unique to some hypothalamic neurons in vivo[4]. Subclones of the original C7 line were isolated and morphologically, histo­chemically and immunochemica1ly characterized. The primitive nature of one of these clones, F7, derived from such neurosecretory cells, has been established on the basis of its ultrastructural features: it resembled a primitive neuroepithelial cell. It gave a negative response to all of the tests that we used to identify neurosecretory cells.

Characteristics of F7 Cell Line

Up to now F7 is a stable line. It has been shown to synthetize

STEM CELLS IN THE HYPOTHALAMUS

F7 STEM CELLS

{

GFA 14- 3- 2

SOMATOSTATIN MET-ENKEPHALIN

NEUROSECRETORY CELL NEUROPHYSIN

OLIGODENDROCYTE CELL J CARBONIC ANHYDRASE II lMYELIN BASIC PROTEIN

Fig. 1. Schematic representation of F7 cell properties and potentialities.

125

2 neuropeptides: somatostatin and met-enkephalin[5]. It is recog­nized by an antibody against 14-3-2 (y-y enolase). using the peroxy­dase method. It contains GFA as shown by immunocytochemistry and this staining is specific since it is not present if the antibody is preadsorbed by GFA (unpublished results). Thus F7 line synthetizes low levels of a neuronal specific and a glial specific protein. as well as 2 neuropeptides (cf. Figure 1).

Comparison with in vivo Properties of Nerve Cells

As previously mentioned. we found a simultaneous localization of a neuronal specific and a glial specific protein (y-y enolase and S-100) in the same ventricular cells between embryonic day 17 and postnatal day 3. Concerning peptides. met-enkephalin has been found by day 16 of gestation in the rat fetus[6] and its distribution in the embryo is unlike that of the adult. Other peptides appear tran­siently during development[7]. The role of neuropeptides in early development remains to be elucidated. Thus the properties of F7 cells reflect those of embryonic hypothalamic cells in vivo.

Demonstration of the Bipotentiality of F7 Cells

F7 cell line provides a unique model to analyse how the choice and expression of a particular brain phenotype is controlled. under the effect of exogeneous inducers. .

NEURONAL DIFFERENTIATION

The primitive nervous cell line F7 when injected subcutaneously into syngeneic mice. gives rise to tumors which after subculture

126 F. DE VITRY

STEM CELLS IN THE HYPOTHALAMUS 127

Fig. 2. F7 stem cells: phase contrast. (a) Control cells grown under • routine conditions in serum containing medium (x 240). (b)

Neurosecretory neuronal cells obtained after transplantation of F7 cells into syngeneic mice (x 320). (c) Oligo­dendrocyte-like cells obtained after culture of F7 cells in defined cultured conditions without serum (x 240).

b

Fig. 3. (a) Immunocytochemical staining of oligodendrocyte-like F7 cells using carbonic anhydrase (CA II) antiserum. (b) Control with CA II antiserum previously adsorbed by purified antigen.

128 F. DE VITRY

contain not only primitive cells but also a new type of differen­tiated cells (Figure 2). These newly differentiated cells can be distinguished from primitive cells on the basis of their morpho­logical and growth rate alteration, their neurophysin content and their positive reaction towards S glucuronidase activity, a neuro­secretory marker in vitro. F7 cells appear thus as progenitor of some secretory neurons[8].

GLIAL DIFFERENTIATION

We have devised recently a serum-free medium able to support the survival and the proliferation of the primitive nervous cell line F7, and characterized a few agents which, added to this basal medium, induce oligodendrocyte-like differentiation[9]. Under the combined action of cholesterol, a retinal factor (EDGF) and brain extract, the cells acquire the capacity to express carbonic anhydrase II and myelin basic protein, 2 proteins exclusively localized in oligo­dendrocytes in vivo (cf. Figure 3). F7 cells can thus be induced to express the oligodendrocyte phenotype under defined conditions in vitro.

CONCLUSION

As indicated in Figure 1, the capacity of F7 cells to switch from an immature nervous cell either to a neurosecretory neuronal cell type or to an oligodendrocyte-like cell, depending on environ­mental conditions, demonstrate that they are bipotential stem cells of the central nervous system. Other binary choices have already been observed in vitro[10,11] or in vivo[12]. Malignant cells such as teratocarcinomas have been shown to be capable of reversal to normalcy when grown in normal environment[13]. Our model appears suitable for analysis, at the molecular level, of the mechanisms underlying the expression of a particular phenotype in the brain and the exact nature of the interacting signals.

REFERENCES

1. K. Niimi, I. Harada, Y. Kusaka, and S. Kishi, The ontogenic development of the diencephalon of the mouse, Tokushima J.Exp.Med., 8:203-238 (1962).

2. G. Das, Gliogenesis and ependymogenesis during embryonic devel­opment of the rat. An autoradiographic study, J.Neurol.Sci., 43:193-204 (1979).

3. F. De Vitry, R. Picart, C. Jacque, L. Legault, P. Dupouey, and A. Tixier-Vidal, Presumptive common precursor for neuronal and glial cell lineages in the mouse hypothalamus, Proc.Natl. Acad.Sci.USA, 77:4165-4169 (1980).

STEM CELLS IN THE HYPOTHALAMUS 129

4. F. De Vitry. M. Camier. P. Czernichow. P. Benda. P. Cohen. and A. Tixier-Vidal. Establishment of a clone of mouse hypothalamic neurosecretory cells synthesizing neurophysin and vasopressin. Proc.Natl.Acad.Sci.USA. 71:3575-3579 (1974).

5. F. Cesselin. M. Hamon. S. Bourgoin. N. Buisson. and F. De Vitry. Presence of met-enkephalin in a somatostatin-synthesizing cell line derived from the fetal mouse hypothalamus. Neuropeptides. 2:351-369 (1982).

5. E. L. Knodel and E. Richelson. Methionine-enkephalin immuno­reactivity in fetal rat brain cells in aggregating culture and in mouse neuroblastoma cells. Brain Res •• 197:565-570 (1980) •

7. L. W. Haynes. D. G. Smyth. and S. Zakarian. Immunocytochemical localization of 3-endorphin (lipotropin c-fragment)> in the developing rat spinal cord and hypothalamus. Brain Res •• 232:115-128 (1982).

8. F. De Vitry. Growth and differentiation of a primitive nervous cell line after in vivo transplantation into syngeneic mice. Nature. 267:48-50 (1977).

9. F. De Vitry. J. P. Delaunoy. J. Thibault. N. Buisson. N. Lamande. L. Legault. A. Delasalle. and P. Dupouey. Induction of oligodendrocyte-like properties in a primitive hypothalamic cell line by cholesterol. a retinal factor and brain extract, EMBO J ••• 2:199-203 (1983).

10. S. Landis and P. Patterson. Neural crest cell lineages. TINS. 4: 172-175 (1981). --

II. M. Darmon. M. Buc-Caron. D. Paulin. and F. Jacob. Control by the extracellular environment of differentiation pathways in 1003 embryonal carcinoma cells: study at the level of specific intermediate filaments. EMBO J •• 1:901-906 (1982).

12. N. Le Douarin. The ontogeny of the neural crest in avian embryo chimaeras. Nature. 286:663-669 (1980).

13. B. Mintz and K. Illmensee. Normal genetically mosaic mice produced from malignant teratocarcinoma cells. Proc.Natl. Acad.Sci.USA. 72:3585-3589 (1975).

POST-TRANSCRIPTIONAL REGULATION OF ONTOGENETICALLY

MODULATED PROTEINS IN THE NERVOUS SYSTEM

Hermona Soreq

Department of Neurobiology Weizmann Institure of Science Rehovot 76100, Israel

INTRODUCTION

The highly complex functions of the numerous cell types in the nervous system require the expression of many more genes than the number expressed in other tissues[l). This high complexity is re­flected at the post-transcriptional levels of mRNA and protein. Consequently, over 70% of the different mRNAs and proteins in the nervous system are scarce molecules, and their average concentration in the tissue is much lower than one molecule per cell[2). Part of the proteins produced by these mRNAs specify the functions respons­ible for the specialized properties of particular cell types. It is, therefore, of major importance to study the post-transcriptional regulation of their expression and to localize them to specific cell types and periods in nervous system development.

To select developmentally regulated proteins, and to assign these particular proteins to specific cell types, we conduct simul­taneous analysis of in vivo protein composition and of the trans­lation products directed by mRNA in vitro (in reticulocyte lysate) or in ovo (in microinjected Xenopus oocytes). In the following, this molecular biology approach to Development Neurosciences will be briefly described.

SELECTION OF CELL-TYPE SPECIFIC MONOCLONAL ANTIBODIES INTERACTING WITH NASCENT POLYPEPTIDE CHAINS SYNTHESIZED IN VITRO BY CEREBELLAR mRNA

Utilizing in vitro translation of cerebellar mRNA, we have found that the production of a number of abundant cerebellar polypeptides

131

132 H. SOREQ

is regulated differently during ontogenesis of the cerebellum of normal rats[3], as compared to the irradiation-agranulated rat cere­bellum[4] and to the cerebellum of staggerer mice[5]. Monoclonal antibodies (mAb) to such polypeptides should enable the assignment of the corresponding proteins to defined cell types and periods in cerebellar development. In order to study the post-transcriptional regulation of these proteins, the mAb should recognize the polyamino sequence of the proteins. To select for such antibodies, we im­munized mice with cerebellar proteins eluted from regions in poly­acrylamide gels, where differences in synthesis were observed. Hybridoma cell lines secreting mAb were obtained by fusion with NSO myeloma cells. The mAh were screened in two different ways:

1) against 125 I -Iabeled cerebellar proteins, representing the total proteins in the cerebellum, including the contribution of incoming fibers;

2) against JOS-Iabeled polypeptides, translated in vitro from cerebellar mRNA, representing the biosynthetic potential of endogenous cerebellar cell bodies alone.

mAb which were found to be positive by both selection methods were examined for their binding to frozen cerebellar sections by indirect immunofluorescence~ In addition to mAb which stained most of the cell types, we identified mAb which selectively stained specific subcellular regions in various cell types. These include Bergmann fibers of Golgi type II epithelial cells, cell bodies of neurons in both the molecular and the granular layer, and meningeal cells[6]. Such characterized mAb will serve as probes to follow the post-transcriptional regulation of the corresponding cerebellar antigenic markers.

USE OF IN OVO BIOASSAYS IN DEVELOPMENTAL NEUROSCIENCES

Labeling techniques or in vitro translation assays are not sensitive enough to detect scarce mRNA sequences or their translation products. To study the post-transciptional regulation of non­abundant proteins with known functions, we employ "in ovo" bioassays. In these, we use microinjected Xenopus oocytes as a translation system, where the examined mRNA sequences are expressed into bio­logically active proteins.

Xenopus oocytes have been extensively used as an expression system for a variety of microinjected mRNAs[7-9J. They perform efficiently the translation, processing and various post-trans­lational modifications[10]. In the case of mRNA species directing the synthesis of secretory proteins, they secrete the correct trans­lational productsL11,12]. Furthermore, microinjection of a synthetic leader peptide modulates the rate of secretion from the oocytes[13].

POST-TRANSCRIPTIONAL REGULATION 133

In the oocytes. injected mRNAs are being translated into bio­logically active and correctly sequestered protein products[14-16]. An impressive example is the synthesis of a functional acetylcholine receptor. The biosynthesis of such receptors from microinjected mRNA involves glycosylation. assembly of 4 different subunits and inser­tion. in the correct orientation. into the oocyte membrane[17]. In our hands. translation in vitro of mRNA from embryonic rat brain results in AChR polypeptides which are still devoid of the receptor native conformation. Such polypeptides could only be precipitated by a mAb specific for non-conformational determinants of Torpedo AChR. but not by mAb specific for the receptor cholinergic site or by a-Bungarotoxin. On the other hand. in injected oocytes. AChE was not only translated but also the correct post-translational modifications and maturation of the newly synthesized polypeptides took place. resulting in a native. membrane bound molecule. which probably pos­sesses the receptor binding site[18]. The quantitative in ovo bio­assays for scarce mRNAs coding for nervous system enzymes. growth factors such as EGF[19]. and receptors. promise to be valuable both for biological and molecular studies. in addition to being a neces­sary requirement for the preparation and amplification of cDNA probes.

POST-TRANSCRIPTIONAL MODULATION OF NERVOUS SYSTEM PLASMINOGEN ACTIVATOR

Plasminogen activators (PAs) are highly specific serine pro­teases which convert the inactive zymogen plasminogen~ abundant in body fluids. into the active protease plasmin[20]. Minute amounts of PA induce a strong. highly localized and short-termed proteolysis. The synthesis and secretion of PA in various tissues have been as­sociated with migration of normal and malignant cells[21]. The widespread distribution of plasminogen-dependent proteolytic pro­cesses in various tissues and cell lines. the continued presence of plasminogen in the cerebrospinal fluid and the generality of the proteolysis induced by PA led us to explore the possibility that post-transcriptional modulation of PA is actively involved in pro­teolytic mechanisms required for neuronal migration and functioning.

Using an autoradiographic technique to detect PA activity in situ. we found PA in the brain to be localized in neuronal-rich-Cell layers such as the cerebellar granular layer and the hippocampal dentate gyrus[22]. PA was also found in growing processes and growth cones of differentiating neuroblastoma cells[23]. Indeed. in hom­ogenates of mature bovine brain we found PA to be associated with synaptosomal membranes[24]. Low activities of electrophoretically separated PA species. such as those observed in glioma cells in culture. were detected and characterized by a sensitive radiometric blotting technique[25].

134 H. SOREQ

To monitor the expression of PAmRNA into the biologically active enzyme, we used an in ovo bioassay[26). This bioassay has been combined with the in situ PA autoradiography, to show that cerebellar granular neurons produce and express PA activity throughout develop­ment and particularly during migration[27).

It is not clear yet by our observations whether PA is equally abundant in different types of neurons. The mechanisms regulating the production and subcellular distribution of the enzyme during neuronal development and under different biological conditions remain to be revealed. Determination of the structural organization of PA gene(s) in brain DNA may yield insights into the various modes of expression of this enzyme in different brain cell types. These issues can now be approached using the techniques described above.

BIOSYNTHESIS OF BRAIN CHOLINESTERASES

The development of cholinergic synapses is accompanied by ac­cumulation of the membrane acetylcholine receptor (AChR) and of the acetylcholine hydrolyzing enzyme, acetylcholinesterase (AChE).

Cholinesterases (ChEs) display multiple molecular forms. These differ in their cell type[28) and subcellular localization[29), immunological properties[30), substrate specificity[31), regulation and, presumably, physiological function. The regulation of ChEs production may involve presynaptic and post-synaptic control at different post-transcriptional steps through the pathway of gene expression. To examine this issue, we employ an in ovo bioassay to monitor the expression of ChEmRNA into catalytically active ChEs[32). To detect minute activities of ChE, we developed a highly sensitive microfluorometric method capable of measuring few picomoles of re­leased thiocholine[33). Microinjection of mRNA from the embryonic human brain, size-fractionated by sucrose gradient centrifugation, revealed that the biosynthesis of brain ChEs is directed by multiple mRNAs of various lengths[34).

To investigate the post-transcriptional regulation of ChEs in particular brain cell types, we employ frozen primary human gli­ogenous tumors and meningiomas. In the neuroectoderm-originated gliomas, we detect up to 40% of ~ChE in two ChE forms, of sediment­ation coefficients ca. 4.5 Sand 10 S. In the mesenchyme-originated meningioma the lighter (4.5 S) form of ChE appears exclusively, and AChE activity predominates (>90%). mRNA from both sources produces ChE in microinjected oocytes. Thus, both dedifferentiated gliogenous cells and meningeal cells within the human nervous system appear to synthesize ChEs, which differ in their substrate specificities and migration properties[35).

POST-TRANSCRIPTIONAL REGULATION l~

The regulation of ChEs expression was examined in the developing cerebellum, where assignment of cholinergic and cholinoceptive func­tions to specific cell types and development periods is still a matter of controversy. We found the level of AChEmRNA to be maximal in the neonate cerebellum and to decrease with postnatal development, when granular neurons proliferate. Furthermore, we found that agran­ulation does not alter the content, substrate specificity or sedi­mentation characteristics of ChEs, nor does it affect ontogenetic changes in these parameters. The specific activity of ChE increases with cerebellar development, and is even higher in the irradiation­agranulated cerebellum. However, unfractionated mRNA from normal cerebellum, from the agranular cerebellum of staggerer mutants and from the cerebellum of irradiated rats all induced the production of apparently similar levels of ChE in microinjected oocytes[36]. This indicates that in spite of the fact that AChE activity resides pri­marily in the granular layer of the mature rat cerebellum[37], most of the activity is not produced by granular neurons but is contri­buted by incoming fibers. Parallel conclusions have been reached regarding the cerebellar muscarinic acetylcholine receptors[38].

Are different genes responsible for the production of the various ChE forms in different cell types? If so, are these genes related to each other in their evolutionary origin, nucleotide se­quence, orientation and regulation of expression? To answer these questions, and others, we now carry out (using the AChEmRNA bioassay) molecular cloning experiments, aimed at obtaining DNA probes for cholinesterase gene(s).

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1. J. P. Bantle and W. E. Hahn, Complexity and characterization of polyadenylated RNA in the mouse brain, Cell., 8:139-150 (1976).

2. B. B. Kaplan, B. S. Schachter, H. H. Osterburg, J. S. de Villis, and C. E. Finch, Sequence complexity of polyadenylated RNA obtained from brain regions and cultured rat cells of neural origin, Biochem., 17:5516-5524 (1978).

3. H. Soreq, A. Safran, and R. Zisling, Ontogenetic variations in cerebellar gene expression, Dev.Brain Res., 3:65-79 (1982).

4. D. Eliyahu and H. Soreq, Degranulation of rat cerebellum induces selective variations in gene expression, J.Neurochem., 38: 313-321 (1982).

5. H. Soreq, A. Safran, and D. Eliyahu, Modified composition of major ontogenetically regulated mRNAs and proteins in the cerebellum of old mice and of staggerer mutants, Dev.Brain Res., Vol. 10:73-82 (1983).

6. A. Safran, Z. Eshhar, D. M. Phillips, and H. Soreq, Selection of cell-type specific monoclonal antibodies interacting with nascent polypeptide chains synthesized in vitro by cerebellar mRNA. Neurosci.Soc.Abstrs., 9:263.13 (1983).

136 H. SOREQ

7. J. B. Gurdon, C. D. Lane, H. R. Woodland, and G. Marbaix, Use of frog eggs and oocytes for the study of messenger RNA and its translation in living cells, Nature, 233:177-182 (1971).

8. C. Lane and J. Knowland, The injection of RNA into living cells: The use of frog oocytes for the assay of mRNA and the study of the control of gene expression, in: "The Biochemistry of Animal Development", R. Weber, ed. ,Vol. III, Acad. Press, pp. 145-181 (1975).

9. G. Marbaix and G. Huez, Expression of messenger RNA injected into Xenopus oocytes, in: "Transfer of Cell Constituents into Eukaryotic cells", J. E. Celis, A. Graessmann, and A. Loyter, eds., Plenum Press, N.Y., pp.347-381 (1980).

10. C. Lane, The fate of foreign proteins introduced into Xenopus oocytes, Cell, 24:281-282 (1981).

11. A. Colman and J. Morser, Export of proteins from oocytes of Xenopus laevis, Cell, 17:517-526 (1979).

12. G. Valle, H. Besley, and A. Colman, Synthesis and secretion of mouse immunoglobulin chains from Xenopus oocytes, Nature, 291:338-340 (1981).

13. R. Koren, Y. Burstein, and H. Soreq, Synthetic leader peptide modulates secretion of proteins from microinjected Xenopus oocytes, Proc.Natl.Acad.Sci.USA, 80:7205-7210 (1983).

14. C. Labarca and K. Paigen, mRNA directed synthesis of catalytic­ally active mouse B-glucuronidase in Xenopus oocytes, Proc. Natl.Acad.Sci.USA, 74:4462-4465 (1977).

15. E. O. Long, N. Grass, C. T. Wake, H. P. Mach, S. Carrel, R. Accalla, and B. Mach, Translation and assembly of HLA-DR antigens in Xenopus oocytes injected with mRNA from a human B-cell line, The EMBO Journal, 1:649-654 (1982).

16. J. D. Richter and D. L. Smith, Differential capacity for trans­lation and lack of competition, between mRNAs that segregate to free and membrane-bound polysomes, Cell, 27:183-191 (1981). --

17. E. A. Barnard, R. Miledi, and K. Sumikawa, Translation of exo­genous mRNA coding for nicotinic acetylcholine receptors pro­duces functional receptors in Xenopus oocytes, Proc.R.Soc. Lond.B, 215:241-246 (1982).

18. D. Mochly-Rosen, H. Soreq, and S. Fuchs, In vitro and in ovo translation of the putative rat brain nicotinic acetylcholine receptor, I.J.Med.Sci., (1983).

19. M. Burmeister, J. Schlessinger, and H. Soreq, Biosynthesis of EGF in mRNA-microinjected Xenopus oocytes, 15th FEBS meeting, in press, (1983).

20. E. Reich, Activation of plasminogen: a widespread mechanism for generating localized extracellular proteolysis, in: "Bio­logical Markers of Neoplasia: Basic and applied aspects", Elsevier, pp.491-500 (1978).

21. R. K. Marotti, D. Belin, and S. Strickland, The production of distinct forms of plasminogen activator by mouse embryonic cells, Dev.Biol •• 90:154-159 (1982).

POST-TRANSCRIPTIONAL REGULATION

22. H. Soreq and R. Miskin, Plasminogen activator in the rodent brain, Brain Res., 216:361-374 (1981).

137

23. H. Soreq, R. Miskin, A. Zutra, and U.Z. Littauer, Modulation in the levels and localization of plasminogen activator in dif­ferentiating neuroblastoma cells, Dev.Brain Res., 7:257-270 (1983).

24. N. Zisapel, R. Miskin, M. Laudon, and H. Soreq, Plasminogen activator is enriched in the synaptosomal plasma membrane, Brain Res., 248:129-139 (1982).

25. R. Miskinand H. Soreq, Sensitive autoradiographic quantifi­cation of electrophoretically separated proteases, Anal. Biochem., 118:252-258 (1982).

26. R. Miskin and H. Soreq, Microinjected Xenopus oocytes synthesize active human plasminogen activator, Nuc.A.Res., 9:3355-3363 (1981).

27. H. Soreq and R. Miskin, Plasminogen activator in the developing rat cerebellum: Biosynthesis and localization in granular neurons, Dev.Brain Res., in press (1984).

28. J. A. Edwards and S. Brimijoin, Divergent regulation of acetyl­cholinesterase and butyrylcholinesterase in tissues of the rat, J.Neurochem., 38:1393-1402 (1982).

29. M. Lazar and M. Vigny, Modulation of the distribution of acetyl­cholinesterase molecular forms in a murine neuroblastoma x sympathetic ganglion cell hybrid cell line, J.Neurochem., 35:1067-1079 (1980).

30. Z. Rakonczay, G. Vincendon, and J. P. Zanetta, Heterogeneity of rat brain acetylcholinesterase: a study by gel filtration and gradient centrifugation, J.Neurochem., 37:662~669 (1981).

31. A. M. Graybiel and C. W. Ragsdale, Pseudocholinesterase staining in the primary visual pathway of the macaque monkey, Nature, 299:439-441 (1982).

32. H. Soreq, R. Parvari, and I. Silman, Biosynthesis and secretion of catalytically active acetylcholinesterase in Xenopus oocytes microinjected with mRNA from rat brain and from Torpedo electric organ, Proc.Natl.Acad.Sci.USA, 79:830-835 (1982).

33. R. Parvari, I. Pecht, and H. Soreq, A microfluorometric assay for cholinesterases, suitable for multiple kinetic determin­ations of picomoles of released thiocholine, Anal.Biochem., Vol. 133:450-456 (1983).

34. H. Soreg, D. Zevin-Sonkin and N. Razon, Expression of cholinesterase gene(s) in human brain tissues: translational evidence for multiple mRNA species, Submitted.

35. N. Razon, D. Zevin-Sonkin, I. Silman, and H. Soreq, mRNA from Human brain gliomas and meningiomas directs the synthesis of cholinesterase in microinjected Xenopus oocytes, Int.J.Dev. Neurosci., in press, (1984).

36. H. Soreq, R. Parvari, and I. Silman, Ontogenic changes in acetylcholinesterase and its translatable mRNA in normal and irradiation-agranulated rat cerebellum, Abst.Soc.for Neurosci., p.416 (1982).

138 H. SOREQ

37. J. A. Olscholwka and V. K. Vijayan, Postnatal development of cholinergic neurotransmitter enzymes in the mouse cerebellum. Biochemical, light microscopic and electron microscopic cyto­chemical investigations, J.Comp.Neurol •• 191:77-101 (1980).

38. H. Soreq, D. Gurwitz, D. Eliyahu, and M. Sokolovsky, Altered ontogenesis of muscarinic receptors in agranular cerebellar cortex, J.Neurochem., 39:756-763 (1982).

ROLE OF THE EXTRACELLULAR MATRIX IN NEURAL

CREST CELL MIGRATION

Jean Paul Thiery, Roberto Rovasio, Jean Loup Duband, Annie Delouvee, Michel Vincent and Hirohiko Aoyama

Institut d' Embryologie du C.N.R.S. et du College de France, 49, bis Avenue de la Belle Gabrielle 94130 Nogent-sur-Marne, France

INTRODUCTION

Embryogenesis involves a series of mechanisms that control rapid cell proliferation, cell-cell recognition and cell differentiation. A variety of theories have been formulated to account for the remark­able precision with which the different parts of embryos develop.

It is generally accepted that the control and the coordination of cell behavior should be highly specific and require many inter­active molecules. However, induction, synchrony in development, recognition, and pattern formation have not yet been shown to involve a vast repertoire of cognins and informative molecules.

In an attempt to address the question of how much specifity is required in morphogenetic processes, we have chosen the neural crest as a "simple" model system. Indeed, not only can mechanisms of cell migration and cell differentiation be best approached during neural crest development but also certain aspects of neurogenesis can be studied in a much less complex environment than in the central ner­vous system.

THE NEURAL CREST

Definition

In vertebrates, neural crest cells separate progressively from the dorsal border of the neural epithelium and migrate as individual mesenchymal cells. During most of their displacement, neural crest

139

140 J. P. THIERY ET AL.

cells are interacting with a three dimensional fibrillar network of glycoproteins and glycosaminoglycans. At their site of arrest, neural crest cells differentiate, partly under the control of the local environment. into many different tissues. The peripheral nervous system, with the exception of a few cranial sensory ganglia, der~ves, as well as melanocytes, and some endocrine and paraendocrine cells are derived from the neural crest. The massive contribution of crest cells to cranio-facial structures has also been well documented[1].

Migration Pathways

1. The extracellular matrix. Very early in embryogenesis, high molecular weight glycoproteins and glycosaminoglycans accumulate in extracellular spaces. The chemical nature of the extracellular matrix (ECM) is under current investigation but it is already estab­lished that laminin, collagens, fibronectin, hyaluronic acid and chondroitin sulfate are abundant and their precise localizations were recently established by histochemical and immunocytological tech­niques[2-S]. As shown in Figure 1 it appears that collagens 1 and III, fibronectin and glycosaminoglycans form the fibrillar structures of the ECM, whereas laminin, collagen IV and possibly glycosamino­glycans and fibronectin are found in the basal lamina. Structural studies on fibronectin have revealed that this glycoprotein can interact through specific domains with several other ECM components and most importantly with the cell surface[9]. Fibronectin has shown to favor cell substrate adhesion and in some instances also to promote cell motility.

Due to its high hydration properties it was proposed that hya­luronic acid could participate in the formation of the space utilized by migrating cells[10]; whereas collagens which readily polymerize to form fibers, might serve as a backbone for ECM assembly[11]. However little is known about the spatially and temporally regulated synthesis, assembly and remodelling of the ECM[12].

2. Routes of migration. Using anti-fibronectin antibodies, we have described the distribution of fibronectin throughout the time of neural crest cell migration. We found that neural crest cells move along fibronectin-rich basement membranes of adjacent tissues rather than penetrating within tissues. Most crest cells migrate as a confluent sheet in narrow spaces that appear transiently ahead of the front of migration. Neural crest cells utilize the available extra­cellular spaces, and accumulate locally where physical obstacles such as fused basement membranes or tissues are present[13,14]. In the trunk, crest cells facing the intersomitic spaces can reach the most ventral structures of the embryo where they differentiate into adren­ergic autonomic ganglia and plexuses; whereas crest cells that en­counter the somites are prevented from migrating very far ventrally and condense along the neural tube to form the dorsal root ganglia.

NEURAL CREST MIGRATION 141

~~'~~~.f+ ~H + +++ +++ BL

G.J.

T.G ..

Fig. 1. Interactions between ECM components, mesenchymal and epithelial cells, BL: basal lamina; BM: basement membrane; C.I, III: Collagen I, III; D: desmosome; FN: tibronectin; GJ: gap junction; HA: hyaluronic acid; TG: tight junction; 2: reticulation site; 2: collagen binding domain; 3: cell binding domain; 4: glycosaminoglycan binding domain.

Role of Fibronectin in Crest Cell Adhesion and Migration

In in vitro binding assays, it was shown that most crest cells adhere and flatten rapidly to fibronectin-coated substrata; whereas only a few crest cells attach to collagen, and these remain round. Crest cells also adhere efficiently to fibronectin-rich ECM deposited by fibroblasts. This binding was prevented by specific anti­fibronectin antibodies.

Most embryonic cells cultured in vitro are able to synthetize and deposit a fibronectin-rich fibrillar matrix, however neural crest

142 J. P. THIERY ET AL.

f. N.

CAM.

Fig. 2. Proposed mechanism for neural crest cell migration and homing. Crest cells are released (1) from neural epithelial cells and migrate in transiant narrow pathways(2). At defined sites, crest cells round up (3) and adhere together to form ganglion rudiments (4,5) changes in cell-cell and cell-ECM adhesive properties are shown to correlate with the presence of N CAM and FN respectively.

cells, in their great majority, do not secrete fibronectin[15]. These data suggested that in vivo neural crest cells adhere to fibro­nectin present in ECM deposited by the surrounding tissues.

In vitro, migration pathways made by creating alternating zones of fibronectin, glass or other ECM components were used to demon­strate that crest cells migrated specifically on the fibronectin regions as a dense population of cells. Time-lapse video analyses revealed unique motility properties of crest cells; persistence in their direction of movement can only be obtained when confluency is maintained[16]. Finally both in vitro and in vivo, neural crest ' cells are prevented from migrating in the fibronectin-rich ECM when monovalent antibodies directed against FN are introduced in the pathways.

Our studies indicate that the mechanisms allowing crest cells to reach their final destination involve a) narrow and transient path­ways b) rapid cell proliferation c) fibronectin as a substrate for migration. However there is evidence that fibronectin is a necessary but not sufficient component for migration.

This unique response of crest cells to fibronectin-rich fibrillar matrices may be directly linked to their inability to produce fibronectin. Recent reports provide additional support to this hypothesis[17-19].

NEURAL CREST CELL MIGRATION 143

Gangliogenesis

When reaching sites of differentiation into ganglia. neural crest cells acquire new adhesive properties. In particular. the neural cell adhesion molecule (NCAM) is expressed at the crest cell surface at the time of aggregation into ganglion rudiments. Con­comittant with the appearance of NCAM. it was found that fibronectin disappears locally at the site of aggregation of crest cellsL20].

The developmentally regulated synthesis and degradation of ECM components such as fibronectin as well as the emergence of new cell surface properties are likely to be involved directly in gangliogen­esis. Nonetheless the exact time sequence is not yet established.

Concluding Remarks

The mechanisms that ensure proper directionality and precise localization of crest cells may require only a few molecules that are otherwise not restricted to the immediate environment of these cells. Our data suggest that it is not necessary to invoke a series of complex events mediated by highly specific molecules. rather vari­ation in the relative concentration of several ECM molecules can modulate the behavior of crest cells and induce new modes of inter­actions (Figure 2).

REFERENCES

1. N.

2. A.

3. M. 4. C.

5. H.

6. D.

7. J. 8. J. 9. R.

10. B.

11. J.

12. E.

M. Douarin. The Neural Crest", University Press, Cambridge (1982). Furthmayer "Immunochemistry of the Extracellular Matrix", CRC Press, Boca Ranton, Florida (1982). A. Derby, Dev.Biol. 66:321-336 (1978). Vanroelen, L. Vakaet., and L. Andries, J.Embryol.Exp.Morphol. 56:178 (1980). Von Den Mark, K. Von Der Mark, and S. Gay, Develop.Biol. 48:237-249 (1976). R. Critchley, M. A. England, J. Wakely, and R. O. Hynes. Nature, 280:498-500 (1979). L. Dub and , and J. P. Thiery, Dev.Biol. 94:337-350 (1982). L. Duband, and J. P. Thiery, "In Preparation" (1983). O. Hynes, and K. M. Yamada, J.Cell BioI. 95:369-377 (1982). P. Toole, Neuronal Recognition Barondes, S.H. in "Current Topics in Neurobiology", Plenum Press, New York, pp 275-329 (1976). A. McDonald, D. G. Kelly, and T. J. Brockelmann, J.Cell BioI. 92:485-492 (1982). D. Hay, "Cell Biology of Extracellular Matrix", Plenum Press, New York (1981).

144 J. P. THIERY ET AL.

13. J. L. Dub and , and J. P. Thiery, Dev.Biol. 93:308-323 (1982). 14. J. P. Thiery, J. L. Duband and A. Delouvee Dev.Biol. 93:324-343

(1982). 15. D. F. Newgreen, and J. P. Thiery, Cell and Tiss.Res. 211:269-291

(1980). 16. R. A. Rovasio, A. Delouvee, K. M. Yamada, R. Timpl, and J. P.

Thiery, J.Cell BioI. 96:462-473 (1983). 17. C. S. Le Lievre, G. G. Schweizer, C. M. Ziller, and N. M. Le

Douarin Dev.Biol. 77:362-378 (1980). 18. C. A. Erickson, K. W. Tosney, and J. A. Weston, Dev.Biol. 77:

142-156 (1980). 19. J. R. Couchman, D. A. Rees, M. R. Green and C. G. Smith, J.Cell

BioI. 93:402-410, (1982). 20. J. P. Thiery, J. L. Dub and , U. Rutishauser and G. M. Edelman

Proc.Natl.Acad.Sci.USA. 79:6737-6741 (1982).

NEURODIFFERENTIATION IN CULTURES OF F9

TERATOCARCINOMA CELLS

Jorma Wartiovaara*,**, Paivi Liesi*** Heikki Hervonen**** and Leena Rechardt****

Departments of *Medical Biology, **Electron Microscopy ***Virology and ****Anatomy, University of Helsinki Siltavuorenpenger 20 A, 00170 Helsinki 17, Finland

Mouse teratocarcinoma derived cell lines have widely been used as a model to study early developmental phenomena. Such studies utilize the fact that cells of a number of embryonal carcinoma (EC) lines can differentiate in vitro mimicking early developmental events (cf Graham, 1977; Martin, 1980). The direction and extent of differ­entiation can be influenced by monitoring the culture conditions and using external factors including retinoic acid and cyclic AMP. Such studies have e.g. enabled the comparison of endodermal type of dif­ferentiation of teratocarcinoma cells with endoderm formation in the early mouse embryo (cf Wartiovaara et al., 1980; Wartiovaara and Leivo, 1982).

NEURODIFFERENTIATION OF EC CELLS

It has become evident that neural differentiation can also be induced or promoted in some EC cell lines (Adamson et al., 1977; Kuff and Fewell, 1980; Darmon et al., 1981; Pfeiffer et al., 1981; Paulin et al., 1982; Paulin, 1983). When F9 EC-cells are cultured in the presence of retinoic acid, in aggregate form or by adding cyclic AMP, they show a differentiation to endoderm-like cells (Strickland and Mahdavi, 1978; Hogan et al., 1981). Kuff and Fewell (1980) have shown that cultivation of F9 cells in the presence of both retinoic acid and dibutyryl cAMP leads also to formation of neural like cells and to a large increase in biochemically demonstrable specific acetylcholinesterase activity in such cultures. They could not, however, obtain conclusive evidence for neuronal differentiation of F9 cells in their 3-4 day old cultures.

145

146 J. WARTIOVAARA ET AL.

CHOICE OF DIFFERENTIATION PARAMETERS IN F9 CELL CULTURES

We have recently studied with F9 cells to what extent neural characteristics are expressed during differentiation under various culture conditions and with different agents. We have also tried to characterize how such neuronal differentiation effects expression of matrix molecules and cell-substrate relations. In these experiments long term, up to 3 week-cultivation in low serum concentration (3% fetal calf serum) was used. The cells were grown in the continuous presence of retinoic acid (10- 7 M) and in some cultures also dibutyryl cAMP (10- 3 M) was included in the medium after an initial 2 day culture in only retinoic acid. The cultures were analyzed for the following markers:

1) cytochemically demonstrable specific acetylcholinesterase (AChE) activity,

2) uptake of I-DOPA (10- 5 M) demonstrated by glyoxylic acid induced fluorescence,

3) neurofilaments demonstrated by indirect immunofluorescence with antibodies against neurofilament triplet proteins or 200 k dalton neurofilament protein,

4) glial fibrillar acidic protein, myelin basic protein or galactocerebroside as seen by immunofluorescence,

5) extra- and intracellular laminin and fibronectin as seen in immunofluorescence.

The results of the above experiments are summarized in Tables 1 and 2 indicating the various parameters at 6, 12 and 18 days of culture or at 14 days for the matrix components.

ENDODERMAL-LIKE DIFFERENTIATION

In F9 cultures treated only with retinoic acid, cells with flattened endoderm-like morphology appear in one to two days as described earlier (Strickland and Mahdavi, 1978). In older cultur~s also spindle-like cells and cells with short extensions can be found. These cells show I-DOPA uptake independent of culture time but lack AChE activity (Table 1). Also no neural filament or glial fibrillar acidic protein positive cells are to be found.

NEURAL-LIKE DIFFERENTIATION

In F9 cultures treated with retinoic acid and cAMP, the results include the appearance of bipolar cells which show histochemically detectable AChE activity in the perikarya and the processes (Table 1). In prolonged cultures also multipolar neural-like cells with long connecting processes are formed in numbers. Such cells show Nissl-substance in kresyl violet staining and presence of AChE ac-

NEURODIFFERENTIATION OF TERATOCARCINOMA

Table 1. Differentiation Parameters in Induced F9 Cell Cultures

Parameters

AChE

I-DOPA uptake

NF

200 kD

GFA MB GC

Abbreviations:

Treatment

RA RA+ db cAMP

RA RA+ db cAMP

RA RA+ db cAMP

RA RA+ db cAMP

RA RA+ db cAMP

AChE, acetylcholinesterase NF, neurofilament triplet proteins 200 kD, 200 k dalton neurofilament protein GFA, glial fibrillar acidic protein MB, myelin basic protein GC, galactocerebroside RA, retinoic acid

Days of Culture

6

+

+ +

12

+

+ +

18

+t-

+ +

+t-+

db cAMP, dibutyryl cyclic adenosine monophosphate

Treatments:

147

RA, 3% fetal calf serum containing medium, supplemented with 10-7 M RA, was changed every 3-4 days on F9 cells grown on gelatin coated glass coverslips.

RA + db cAMP, as above but after initial use of only RA for 2 days, the medium was supplemented with both RA (10-7 M) and db cAMP (10- 3 M).

tivity (Figure 1). After I-DOPA treatment such cells exhibit blue­green amine-fluorescence which is intensified in the presence of 10-5 M nialamide (Wartiovaara and Rechardt, 1982).

In 18 day old cultures treated with retinoic acid and cAMP strongly fluorescing cells can be detected by immunofluorescence staining with antibodies against neurofilament triplet proteins

148 J. WARTIOVAARA ET AL.

Table 2. Laminin and Fibronectin Expression in F9 Cell Cultures

Supplements Laminin In Medium Intrac. Extrac.

+ + RA ++ +

RA+ db cAMP +++ +

Abbreviations: RA, retinoic acid db cAMP, dibutyryl cyclic AMP Intrac., intracellular fluorescence Extrac., extracellular fluorescence

Fibronectin Intrac. Extrac.

++ ++ +

Culture method as in Table 1, indirect immunofluorescence staining of paraformaldehyde fixed cells after 14 days of cultivation, post-treated with 0.05% NP 40 for intra­cellular staining but omitted in staining for only extra­cellular material.

(Figure 2; Liesi et al., 1983a). These cells have ramifying inter­connecting processes often forming large networks. In 12 day or younger cultures positive cells were not found. Staining with anti­bodies against only 200 k neurofilament antigen did not show any positive cells even in 18 day cultures.

In the F9 cultures treated with retinoic acid and cAMP also astrocyte-looking cells can be found. However, no expression of glial fibrillar acidic protein, myelin basic protein or galactocere­broside could be detected in immunofluorescence even after prolonged cultivation times (Table 1; Liesi et al., 1983a).

LAMININ AND FIBRONECTIN EXPRESSION

With respect to the expression of the matrix glycoproteins laminin and fibronectin distinct changes occur during induction of F9 cells that are dependent on the direction of the differentiation (Table 2). Retinoic acid alone induces fibronectin expression and increases the laminin content of the endoderm-type cells.

Treatment with both retinoic acid and cAMP has a potentiating effect on the laminin content of the induced neural-type cells (Figures 3 and 4). However, the cells lose their fibronectin content and only few extracellular strands are seen to remain in the cultures (Figure 5). The neural-type cells are still often connected with the processes to the fibronectin containing strands on the culture sub-

NEURODIFFERENTIATION OF TERATOCARCINOMA 149

Fig. 1. F9 teratocarcinoma cells cultured for 12 days in the presence of 3% fetal calf serum, retinoic acid (10- 7 M) and dibutyryl cAMP (10- 3 M). Staining for specific acetyl­cholinesterase activity (Karnovsky-Roots thiocholine method; 10-0 M iso-OMPA as an inhibitor for non-specific esterases). Note enzyme activity in bi- and multipolar cell bodies (arrows) and processes (arrowheads). x 950.

stratum. There seems to be a progressive loss of fibronectin fluor­escence with ageing of the culture. Also the few remaining endo­dermal type of cells become fibronectin negative (Figure 5). It is therefore conceivable that the diminishing fibronectin strands are remnants of a protease sensitive matrix produced by the endodermal type of cell at the onset of the culture. The tendency of the neural-type cells to associate with the fibronectin strands is inter­esting. Migrating chick neural crest cells seem to follow fibro­nectin rich pathways and focal disappearance of fibronectin leads to cessation of crest cell migration (Thiery et al., 1982). The re­lationship of neurogenesis to extracellular matrix needs, however, still further study.

CONCLUSIONS

Adamson et al., (1977) first described in detail the induction of acetylcholinesterase with the neural type of differentiation in cultures of certain teratocarcinoma lines in vitro. First, in recent

150 J. HARTIOVAARA ET AL.

Fig. 2. F9 cells treated with both retinoic acid and dibutyryl cAMP for 18 days and stained in indirect immunofluorescence with antibodies against neurofilament triplet proteins. Neuron­like cells exhibit intense fluorescence both in cell bodies and processes. x 950.

Fig. 3. Scanning electron micrograph of F9 cells cultured for 14 days in retinoic acid and dibutyryl cAMP. Note extensive interconnecting processes in cells with rounded bodies. x 800.

NEURQDIFFERENTIATION OF TERATOCARCINOMA 151

Fig. 4. Cells as in Figure 3 stained for extra- and intracellular laminin. Strong fluorescence is visible both in cell bodies and processes. x 700.

years more interest has been focused on this question in several cell lines including F9 cells (Kuff and Fewell, 1980; Darmon et al., 1982; Paulin et al., 1982; Wartiovaara and Rechardt, 1982). The endodermal differentiation of F9 cells has been quite extensively characterized (cf Hogan et al., 1983) but conclusive evidence for their neural differentiation has been lacking. The present results suggest that F9 line teratocarcinoma cells can also differentiate into definite neurons in vitro but need prolonged culture times under the con­ditions used. Further studies are now in progress to characterize to what extent different neurotransmitters and -peptides can be expres­sed in the F9 cells. Preliminary experiments demonstrate that leu­enkephalin and serotonin-like immunoreactivity can be induced in F9 cell cultures (Liesi et al., 1983b). Also the association of neural like cells with fibronectin containing extracellular strands, as described in the present paper, is a phenomenon that, like fibro­nectin association with migrating chick neural crest cells (Thiery et al., 1983), suggests a role for the extracellular matrix in early neurogenesis.

Acknowledgements

The antibodies used in the present study were kind gifts from Dr. D. Dahl (anti-neurofilament proteins, anti-GFA protein, anti­myelin basic protein and anti-galactocerebroside), Dr. R. Timpl (anti-Iaminin) and Dr. A. Vaheri (anti-fibronectin). The skillful technical assistance of Ms. Elina Waris is greatly appreciated.

152 J. \~ARTIOVAARA ET AL.

Fig. 5. Cells as in Figure 3 stained for extra- and intracellular fibronectin. SA phase, 5B fluorescence micrograph. Note absence of intracellular staining both in neural-type cells and in endodermal-type flat cell (on left). Extracellular fluorescent strands traverse the substratum and associate with endoderm and neural type of cells. x 800.

NEURODIFFERENTIATION OF TERATOCARCINOMA 153

REFERENCES

Adamson, E. D., Evans, M. J., and Magrane, G. G., 1977, Biochemical markers of the progress of differentiation in cloned terato­carcinoma cell lines, Eur.J.Biochem., 79:607-615.

Darmon, M., Buc-Caron, M.-H., Paulin, D., and Jacob, F., 1982, Control by the extracellular environment of differentiation pathways in 1003 embryonal carcinoma cells: study at the level of specific intermediate filaments, EMBO J., 1:901-906.

Graham, C. F., 1977, Teratocarcinoma cells and normal mouse embryo­genesis, in: "Concepts in Mammalian Embryogenesis", ed., M. I. Sherman, MIT Press, Cambridge, Mass., pp.315-394.

Hogan, B. L. M., Taylor, A., and Adamson, E., 1983, Cell interactions modulate embryonal carcinoma cell differentiation into parietal or visceral endoderm, Nature (London), 291:235-237.

Hogan, B. L. M., Barlow, D. P., and Tilly, R., 1983, F9 teratocarcin­oma cells as a model for the differentiation of parietal and visceral endoderm in the mouse embryo, Cancer Surveys, 2:115-140.

Kuff, E. L., and Fewell, J. W., 1980, Induction of neural-like cells and acetylcholinesterase activity in cultures of F9 terato­carcinoma treated with retinoic acid and dibutyryl cyclic adenosine monophosphate, Devel.Biol., 77:103-115.

Liesi, P., Hervonen, H., Rechardt, L., and Wartiovaara, J., 1983a, Induction of neural filament antigens in F9 teratocarcinoma cells, The Histochemical Society, 34th Annual Meeting, Charleston, USA.

Liesi, P., Rechardt", L., and Wartiovaara, J., 1983b, NGF has an en­hancing effect on neuronal differentiation of F9 teratocar­cinoma cells in vitro, 7th European Neuroscience Congress, Hamburg.

Martin, G. R., 1980, Teratocarcinomas and mammalian embryogenesis, Science, 209:768-776.

Paulin, D., Jakob, H., Jacob, F., Weber, K., and Osborn, M., 1982, In vitro differentiation of mouse teratocarcinoma cells monitored by intermediate filament expression, Differentiation, 22:90-99.

Paulin, D., 1983, Intermediate filaments during neuronal morpho­genesis, in: "NATO Advanced Study Institut Series", eds., A. M. Duprat, A. Kato, and M. Weber, Plenum Press, (in press)

Pfeiffer, S. E., Jakob, H., Mikoshiba, K., Dubois, P., Guenet, J. L., Nicolas, J. -F., Gaillard, J., Chevance, G., and Jacob, F., 1981, Differentiation of a teratocarcinoma line: Preferential development of cholinergic neurons, J.Cell Biol., 88:57-66.

Strickland, S., and Mahdavi, V., 1978, The induction of different­iation in teratocarcinoma cells by retinoic acid, Cell, 15: 393-403. --

Thiery, J. P., Duband, J. L., and Delouvee, A., 1982, Pathways and mechanisms of avian trunk neural crest cell migration and localization, Devel.Biol., 93:324-343.

154 J. WARTIOVAARA ET AL.

Thiery, J. P., Rovasio, R., Duband, J. L., Delouvee, A., Vincent, M., and Aoyama, H., 1983, Role of the extracellular matrix in neural crest cell migration, in: "NATO Advanced Study Institut Series", eds., A. M.lDuprat, A. Kato, and M. Weber, Plenum Press, (in press)

Wartiovaara, J., Leivo, I., and Vaheri, A., 1980, Matrix glyco­proteins in early mouse development and in differentiation in teratocarcinoma cells, in: "The Cell Surface: Mediator in Developmental Processes~ eds., S. Subtelny and N. K. Wessells, Academic Press, New York, pp.30S-324.

Wartiovaara, J., and Leivo, I., 1982, Basement membrane matrices and early mouse development, in: "New Trends in Basement Membrane Research", eds., K. Kiihn,Ii'. Schone and R. Timpl, Raven Press, New York, pp.239-246.

Wartiovaara, J., and Rechardt, L., 1982, Neuronal differentiation in induced F9 teratocarcinoma cell, Joint Meeting of the American - Japanese Histochemical Societies, Vancouver, Canada.

3

NEURONAL AND GLIAL MARKERS

INTERMEDIATE FILAMENTS AS MARKERS

OF NEURONAL DIFFERENTIATION

Fabienne Alfonsi*, Michele Darmon*,** Nadine Forest*, and Denise Paulin*,**

*Universite Paris 7, and **Institut Pasteur Department de Biologie moleculaire 25 rue du Dr. Roux, 75015 Paris, France

Various polypeptides organized in fibrous networks forming microtubules, microfilaments and intermediate-filaments are impli­cated in axonal transport[l]. In the cytoplasm of all differentiated cells, each reseau is built with filaments constituted by a repeat of theosame type of unit: Sand y actins form microfilaments (diamet~r 70 A), as tubulin dimers polymerize in microtubules (diameter 250 A) and one of the five polypeptide types autoas~embles to give inter­mediate sized filaments (diameter 80 to 140 A). A variety of as­sociated proteins are bound to the filaments with different affini­ties. Associated proteins could differ with the cell species confer­ring specific cell type properties.

Different variants of these major constituants are expressed according to species, developmental stage, cell type and pathological situation. In the mammalian genome, at least 20 actin genes or pseudogenes are detected[2] but high resolution electrophoresis gel analysis and aminoacid sequence determination show six different actin sequences[3].

As in other tissues Sand y variants are synthesized in brain. The various a forms are only found in muscle tissues where they represent one of the major constituants. In contrast, in the mammal brain, one observes a large diversity of different tubulins; charge heterogeneity is increased by post-translational modifications. Multiple tubulin variants in the nervous system are synthesized during embryonic development[4].

The third category of polypeptides able to form filaments (10 nmeters diameter) is more heterogenous than the two others and is

157

Fil

amen

t T

ype

a. K

erat

ins

Des

min

V

imen

tin*

Gli

al F

ilam

ent

Pro

tein

N

euro

fila

men

t P

rote

in

Tab

le 1

. In

term

edia

te

(10

nm)

Fil

amen

ts P

rop

erti

es

Tis

sues

o

r C

ells

M

Wx1

0-;"

Ep

ith

eliu

m

68-6

3-60

58-5

4-52

M

uscl

e 50

-55

Mes

ench

yme

52-5

7-54

58

Gli

al*

*

51

Neu

rone

s 68

-160

-210

65

-140

-200

A,A

, N

°

52-6

0

101-

109

37-4

5 18

-33

70

k 18

-35

Com

mon

Seq

uenc

e

ARG

-TY

R-G

LU-G

LU-G

LU-V

AL_

ALA

_LEU

_ARG

ILE

LE

U-T

YR

-GLU

-GLU

-GLU

-ILE-

AR

G-V

AL-

LEU

A

RG

-LEU

-GLU

-GLU

-GLU

-ILE-

AR

G-H

IS-L

EU

ASP

-ALA

-LEU

-AR

G-G

LN-A

LA-L

YS-

GLN

-GLU

--

--

SER

-ASN

-GLU

-TY

R-A

RG

-AR

G-G

LY

ASP

-ALA

-VA

L-A

RG

-ALA

-ALA

-LY

S-A

SP-G

LU­

VA

L-SE

R-G

LU-S

ER-A

RG

-AR

G-L

EU

*Vim

enti

n o

rig

inall

y d

escr

ibed

as

the

inte

rmed

iate

fil

amen

t o

f m

esen

chym

al c

ell

s is

fou

nd

in:

Mel

anoc

ytes

, le

ns

ep

ith

eli

al

cell

s, ir

is e

pit

hel

ium

, Sc

hwan

n cell

s,

epen

dym

al c

ell

s, ast

ro­

cyte

s,

dev

elo

pin

g n

euro

nes

. **

No

inte

rmed

iate

fil

amen

ts in

oli

go

den

dro

cyte

s o

r m

icro

gli

a.

\J1

00

I'%j ~ I'%j ~ til

H

t%j

>-3 ~ .

INTERMEDIATE FILAMENTS 159

subdivided into five classes according to their immunological proper­ties[6]. The 10 nmeters filaments share common properties: resist­ance to extraction in low and high salt buffers as well as in non­denaturing detergents; common partial aminoacid sequences and immunological determinants[s,9]. All of the different types of intermediate filament polypeptides appear in phosphorylated and non-phosphorylated and non-phosphorylated forms and different vari­ants could be expressed.

The first table shows the five types of polypeptides which constitute intermediate filaments: alpha keratin in epithelium, desmin in muscle, vimentin in mesenchymal derivatives, glial acidic fibral protein in some glial cells and neurofilaments in neurones. The observation that different cells use different gene products for the construction of intermediate filaments probably reflects func­tional differences and for a given tissue, variant expression could be related to the regulation of cell expression.

As shown in Tabie 2, neurofilaments are composed of three poly­peptides with low (70 k), medium (160 k) and high (210 k) molecular weights: for each polypeptide, a large heterogeneity is seen due to post-translational modifications such as phosphorylation and partial proteolysis[8]. A relative simplification has been developed during evolution for mammals and birds as compared to the trout, perhaps reflecting an increase in constraint involved in the protein func­tion.

Table 2 • Neurofilament Subunits

Species High MW Medium MW Low MW

Bovine 210 160 70 Human 200 ISS 68.5 Rat 200 147 68 Mouse 200 145 68 Chicken 180 ISS 70 Frog 180 170,160 70 Trout 210 140-195 70-77

THE THREE POLYPEPTIDES ARE ORGANIZED TO FORM THE NEUROFILAMENTS

Studies on in vitro reconstitution lead to information concern­ing the way that the triplet polypeptides are arranged within the individual neurofilament. First, only the 70 k polypeptide is able to reassemble into an intermediate filament in the absence of the other two polypeptides[lb,ll]. No filament formation was observed when one hundred and sixty k and two hundred k protein alone or mixture of both was subjected to the same procedure. The 160 k and

160 F. ALFONSI ET AL.

the 210 k polypeptides copolymerized into an intermediate filament only if 70 k was present. When the 70 k filaments are examined by electron microscopy they resemble the 10 nmeters filaments obtained after reconstitution of desmin filaments of muscle. In the presence of 210 k polypeptides, conspicuous surface alterations and whisker­like extensions are seen. There projections are absent on filaments obtained from 70 k alone. These results (Figure 1) suggest that the 70 k polypeptides form the core of the filament and the 200 k poly­peptides are tightly associated accessory proteins.

Second, antibodies specific for 70 k produce continuous decor­ation of each neurofilament whereas the antibody for 210 k gave discontinu04s labelling with a periodic arrangement[12]. The 210 k could function to maintain order and spacing among intracellular elements or could serve to mediate mechano-chemical coupling of organelles with filaments. Decoration with antibodies against 150 k gives a periodic arrangement like the picture of microtubules period­ically arranged due to associated proteins MAP2 •

Third, are all the neurones composed of the three polypeptides? Immunofluorescence studies, in vivo, show no segregation of the triplet proteins in normal adult neurones but some negative reaction has been described in embryos and in pathological situations as we shall see later. However, 210 k proteins could be found primarily in axones whereas dendrites and cell bodies are stained more densely with 160 k and 70 k. In other cases, similar labelling of axonal structures with the three antibodies are found[13]. Association of neurofilament proteins with tubulin is seen in electron microscopy on

A B

!~~~~ ~~>--~~J;-~,,~ ~~-~~--, t ~~,~--~~~==~~----~~~~-------

; 200K : ~

c .

I I

'------ 1000 A ---___ ....

Fig. 1. Neurofilament decoration with antibodies (a) against 200 k and (b) against 70 k detected by electron microscopy. (c) Schematic representation of neurofilament organization [derived from reference 10-13]

INTERMEDIATE FILAMENTS 161

invertebrate axones and reveals an extensive network of cross bridges, suggesting that the two structures are linked in vivo to form part of an axoplasmic filamentous network.

Other evidence of interfilament links comes from the finding that they are transported together as the major elements of the slow component of the axonal transport[15]. However, interruption of slow axonal transport by certain drugs is accompanied by an accumulation of the neurofilaments at the most proximal part of the axon whereas microtubules are not affected. Biochemical data point to an associ­ation between microtubules and neurofilaments. Neurofilament protein was a contaminant of the microtubule preparation while associated microtubule proteins and tubulin are contaminants of the neurofila­ment preparation[16,17,18,Table 3].

During nerve growth, the cell body and neurite remain stationary while the growth cone crawls forward over the substratum. The neur­ites do no grow from the base but instead new material appears to be added at the growing tip. Membrane, proteins and other materials synthesized in the cell body are transported along the neurite to the growth cone via axoplasmic transport[14]. In radiotracer experi­ments, Lazek and Hoffman have demonstrated that five proteins make up the majority of the slow component. Two of these appear to be tubulins while the remaining three[68,145,200] are the neurofila­ments.

STRUCTURAL HOMOLOGIES BETWEEN THE THREE PROTEINS OF THE TRIPLET NEUROFILAMENT, OR ACROSS SPECIES ARE REVEALED WITH PEPTIDE MAP ANALYSES AND IMMUNOLOGICAL REACTIVITY

Peptide map analyses show that the 210 k neurofilament proteins from different origins have more similarities between them than when one compares these for 160 k or 70 k in the same organismsLI9,20]. Genetic polymorphism for the 210 k polypeptide has been described for the rabbit nervous system[21].

At least three different messengers code for the three proteins as demonstrated by translation of RNA from mouse, rabbit and rat brain[32-34]. Sequence homologies were demonstrated using monoclonal antibodies[22-24]. By screening one hundred hybridoma supernatants obtained in the same fusion experiment, we found eleven of them directed against neurofilament proteins but each one with a different specificity (Table 4). Five are directed against one polypeptide; one identified 70 k, three identified 160 k, one identified 200 k (Figure 2). Six others detected two or three of the NF polypeptides; two react with 70 + 160 k, two with 160 + 210 k, two with the three polypeptides 70 + 160 + 210 k. For each clone, two successive sub­clonings produced supernatants with exactly the same reactivity, eliminating the possibility of a mixture of antibodies.

162

•• ",,160 K

~ 70 K

~«"TU ~C;O K ~

B c

F. ALFONSI ET AL.

o

I

.""",, 210 K

~ 70 K .

Fig. 2. Reactivity of monoclonal antibodies against neurofilament polypeptides. After transfer on nitrocellulose sheets, incubation with different antibodies are performed and revealed with peroxidase activity in the presence of DAB. (a) coomassie blue staining. Cytoskeleton polypeptides isolated from bovine brain as described in [18]. Extracts are analysed after migration on SDS gel electrophoresis. 210 k, 160 k, 70 k polypeptides represent the triplet of neuro filaments; tubulin 55 k and GFA 50 k are also present. Peroxidase revealed antibody directed against: (b) 70 k, (c) 160 k, (d) 200 k, (e) the triplet and associate polypep­tides.

Evidence for differences in the reactive site for the monoclonal antibodies directed against the same polypeptides is obtained by using native neurofilaments (see Figure 3a and Table 4). One mono~ clonal antibody against the 70 k + 160 k couple does not react with the total filament whereas the other does, indicating in the first case that the reactive site is masked and in the second case that the site is exposed. The same kind of differences are obtained with the monoclonal antibodies having the triple activity. For one monoclonal antibody, the reactivity is very high on the native filament and is lost after SDS gel electrophoresis, indicating an antibody directed against a conformational determinant.

We could conclude that at least four classes of antigenic deter­minants are present: (1) one class of determinant is shared by all types of intermediate filaments (2) one class is present on two or three polypeptides of the triplet (3) one class is subunit specific (4) one class is subunit specific and species specific.

Tab

le 3

. P

oly

pep

tid

es a

sso

cia

ted

or

co

nst

itu

tin

g f

ilam

ents

in

rat

bra

in a

nd s

pin

al

cord

Mo

lecu

lar

wei

gh

t x

10

-3

Syno

nym

pHi

235

230

Fo

dri

n

5.7

5

.7

210

150

70

neu

rofi

lam

ent

trip

let

5.5

5

.4

5.3

68

61

58

54

ass

ocia

ted

p

rote

ins

vim

enti

n

S-t

ub

uli

n

MAP

N

FAP

5.6

5

.6

5.5

5

.4

Tab

le 4

. M

onoc

lona

l A

nti

bo

dy

Reacti

vit

ies

(a)

(b)

(b)

Rad

io-I

mm

uno

Ass

ay o

n Im

mu

no

det

ecti

on

aft

er

Imm

un

od

etec

tio

n a

fter

Tra

nsf

er

from

SD

S T

ran

sfer

from

SD

S A

nti

-B

ovin

e P

oly

pep

tid

es

Nat

ive

Ele

ctr

op

ho

resi

s E

lectr

op

ho

resi

s b

od

ies

70

160

210

k F

ilam

ent

70

160

210

68

145

200

Bov

ine

Mou

se

1 44

00

+

2 34

00

1100

+

+

+

+

3

5700

+

4

5300

18

00

300

+

+

+

+

5 19

00

4500

31

00

1600

SM

EAR

+

+

+

6 54

00

200

+

7 11

00

700

+

+

+

+

8 18

00

SMEA

R SM

EAR

9 90

0 25

00

1500

+

+

+

+

10

26

00

3300

10

00

+

+

+

+

+

+

(a)

rad

ioacti

vit

y i

s e

xp

ress

ed in

cpm

fo

r ea

ch a

ssay

(b

) +

po

siti

ve re

sult

mea

ns

fix

ati

on

of

mo

no

clo

nal

an

tib

od

y d

ete

cte

d a

s d

escr

ibed

in

ref.

5

; sm

ear

mea

ns

a d

iffu

se r

eacti

on

.

H z H

tt:I

50

~ t:::I

H ~ tt:I

GFA

'-r

:l H

t-<

5.6

~ z H

(J

)

0'\

w

164 F. ALFONS! ET AL.

INTE~ffiDIATE FILAMENTS

NEUROFILAMENT EXPRESSION IN MOUSE BRAIN IS DETECTED AT DAY 9 OF EMBRYOGENESIS

165

During mouse embryogenesis in the first three days, no inter­mediate filaments are detected in the blastomere cells. At day 4, when the cells differentiate to give the first epithelium, keratins are synthesized in the trophectoderm, then in the endoderm[25-271. The remaining undifferentiated stem cells do not express any kind of intermediate filaments.

The vimentin expression is detected at day 8 in mesodermal derivatives[281. In transverse sections of a 9 day embryo at the cephalic level, vimentin filaments are present in neural crest and some neural tube cells. At trunk levels, vimentin reactivity appears later in the neural tube as shown in Figure 4c. It is also found in the lateral plates and intermediate cell mass but not in the somites, except in its dermomyotomal moiety after dissociation of the selero­tome.

70 k neurofilament reactivity is first found at this stage in a few cells of the ventral region of the midbrain and hindbrain; no positive cells could be found in the forebrain or in the trunk neural tube at this stage. AT 11.5 day of embryogenesis, 70 k is present in a variety of central and peripheral neuronal structures such as the intermediate layer, dorsal root ganglia, sympathetic ganglia, and enteric neurone precursors. In addition, 70 k sera decorated nerves coming from various origins: ventral root fibers and vagal fibers (see Figure 3g). At this stage, few cells show a transitory coexist­ence of vimentin and 70 k neurofilaments (see Figure 4).

Are the three proteins 70 k, 160 k and 145 k, expressed with the same timing? In our experiments using mouse embryos, we were able to detect 145 k and 200 k at the same stage as 70 k. Discrepancies are observed in results about 200 k. In the rat, Shaw and Weber only

Fig. 3. Microphotograph of immunofluorescent structures decorated • with specific antibodies directed against NF or vimentin.

(a) Primary culture from mouse mesencephalic brain region reacts with monoclonal antibodies directed against the triplet polypeptides. (b) Same area in phase microscopy. (c) Axone and growth cone of chicken cells cultured during 3 days. Note absence of staining of the growth cone. (d) Same area in phase microscopy. (e) Double labelling with monoclonal human antivimentin and rabbit polyclonal 70 k antibodies. Chicken cells from spinal cord of 4 day old embryo cultured during 1-3 days. (g) Mouse embryo sections incubated with 70 k antibodies. Note the strong staining of fibers coming from ganglia at 11.5 days of embryogenesis. in (h); sympathetic ganglia in (g).

166 F. ALFONS! ET AL.

Fig. 4. Vimentin detection in mouse embryos 7 to 11 days old. (a) Frozen section of 7 day embryo incubated with antivimentin. (b) Same area in phase microscopy. (c) Fixed section embedded in PEG are incubated with IgM monoclonal anti­vimentin followed by fluorescein coupled anti~. Cells of neural tube of 11 day old embryo show a positive reaction.

found the 200 k expression at approximately the time of birth[29] using one monoclonal antibody[30].

In the brain of a 5 day prenatal rat, 68 k and 145 k anti­bodies stained the neuronal cells. The two antigens appear to have an identical distribution. A section from a 1 day postnatal rat cerebral cortex is stained strongly with a 70 k antibody but not with a 200 k antibody. At the same stage, nerve fibers in rat tongue are positive for 70 and 200 k. These results support the idea that the expression of the three proteins could be different in different parts of the nervous rat system, the 200 k being the last to be synthesized.

COEXISTENCE OF VIMENTIN AND NEUROFILAJIENT POLYPEPTIDES IN DEVELOPING NEURONES

Double labelling with antibody to neurofilaments and vimentin show that both coexist, at least transiently in the course of matur­ation of the neuroblasts during neurogenesis in chicken, mouse and

INTERMEDIATE FILAMENTS 167

rat. However, further neurone maturation leads to a loss of vimentin and only neurofilaments are expressed. In adult neurones, vimentin has not been detected except in the axonless horizontal cell of the retinal layer. These cells have unusual features and could be inter­mediate between neuronal and glial cells [see references in Tables 5 and 6].

The switching off of the vimentin coding genes, which normally occurs during development, takes place in vitro; information avail­able on primary cultures or in established cell lines suggest that it happens, in response to environmental signals of differentiation.

IN VITRO EXPRESSION OF INTERMEDIATE FILAMENTS HAS BEEN STUDIED IN PERMANENT CELL LINES

Mouse neuroblastoma and rat pheochromocytoma when maintained in exponential growth in serum containing medium express primarily vimentin and a very small quantity of neurofilament polypep­tidesl55,56]. When induced to differentiate with NGF or CCA, out­growth of neurite-like processes can be observed and are character­ized with anti 70 k, 145 k and 200 k antibodies. These cell lines are determined to give neuronal cells and glial; other derivatives have not been formed so far.

Table 5. Intermediate Filament Composition in Neural Cells

Neuroectoderm derived: • Melanocytes[35J • Lens epithelial cells[36] • Iris epithelium[37]

Schwann cells[38,39,49] Ependymal cells[41] Brain astrocytes[40-42,48,58] Immature glia[43,44] Mature astroglia[40,47J Oligodendrocytes[48] Microglia[48] Retina[54]

(horizontal cell axonless of adult mouse)

Developing neurones[44-47, 51,52,55,56,65]

Mature neurones[50]

Vimentin

+ + + + + + + +

+

+

GFA

+

+

Triplet NF

+

+ +

Tab

le 6

. C

oex

iste

nce

of

Vim

enti

n a

nd N

euro

fila

men

t P

oly

pep

tid

es in

Neu

ron

al C

ell

s 0

' 0

0

Co

exis

ten

ce

Sp

ecie

s T

issu

es o

r C

ell

s E

mbr

yo

stag

es

Tre

atm

ent

Vim

enti

n

Vi

+ N

F N

F R

ef.

chic

ken

ch

ick

+

n

eu

ral

tub

e 2

-3

day

s in

sit

u

+

0.7

5

stag

e

18

+

45

Sta

ge

l3

po

st m

ito

tic

neu

rob

last

q

uail

n

eu

ral

cre

st

6-1

0

som

ites

in

vit

ro c

ult

ure

+

+

+

47

ch

ick

en

spin

al

cord

4

-5

day

s in

vit

ro c

ult

ure

+

+

+

31

sp

inal

gan

gli

a 9

-12

d

ays

+

+

+

46

mou

se

neu

ral

cre

st

9-1

4 d

ays

in s

itu

9

day

s ll

.5

day

s 14

d

64

neu

ral

tub

e g

ang

lio

n

mou

se

mes

ence

ph

alo

n

l3

day

s in

vit

ro cu

ltu

re

no

no

70

k 50

d

uri

ng

8

day

s 14

5 k

200

k ra

t ce

ph

alic

reg

ion

15

d

ays

in v

ivo

+

+

+

44

a l3

d

ays

in v

itro

cu

ltu

re

+

3 d

ays

+

mou

se

neu

rob

last

om

a in

vit

ro n

eu

rite

+

+

56

in

du

ctio

n w

ith

CCA

66

ra

t ph

eoch

rom

ocyt

oma

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mou

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65

~ 10

09

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+

7

0,1

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52

"=j

0

200

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00 k

51

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+

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I m

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~

INTERMEDIATE FILA}lliNTS 169

By contrast, another type of stem cell line leads to different derivatives according to their origin and environmental condition. From the inner cell mass of the blastocyst, permanent embryonal cell lines have been established, directly (EK) or after transfer in ectopic sites (EC) (see Figure 5).

EC and EK cells are pluripotent, tumoral and able to different­iate in vivo or in vitro. In vivo, cells injected into the blasto­cyst join the cells of the inner cell mass and participate in the formation of many of the tissues of the resulting chimeric mouse. When injected into ectopic sites, they give tumors with formation of different tissues. In vitro, under certain conditions, some lineages lead to various different phenotypes[51,52,65].

When treated with retinoic acid in the presence of dibutyryl­cAMP, the 1009, 1003 or F9 cells gave rise to several types of dif-

BLASTOCYST EGG CYLINDER

CYlokel81L

EK No

Cyl0 e tal "--"IIIIIIIIIIi~tfI,..

TERATOCARCINOMA

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EjC DIFfERENTIATED CELLS

I009-1\L Nourolll .monr • GrA

EMBRYOID BODY AGGREGATE f9 ~ KerlllllLn

PCC3..RA...- Desm.n V .men l Ln

Fig. 5. Intermediate filament expression in teratocarcinoma cells and corresponding tissues in mouse embryo from blastocyst to 8 day stage. Schematic representation of relationship between embryo and tumoral counterpart.

Tab

le

7.

Path

olo

gic

al

Sit

uati

on

s[6

2]

Wal

leri

an d

egen

erat

ion

[59

] d

isap

pea

ran

ce o

f n

euro

fila

men

ts

Neu

rop

ath

ies

[62]

ac

cum

ula

tio

n o

f n

euro

fila

men

ts

• in

du

ced

by

neu

roto

xin

s rD

PN

alum

iniu

m

m-h

exan

e ac

ryla

mid

e m

eth

ylb

uty

lket

on

e

• p

eri

ph

era

l n

euro

nal

sy

stem

in

fan

tile

neu

roax

on

al d

istr

op

hy

am

yo

tro

pic

la

tera

l sc

lero

sis

Alz

hei

mer

's d

isease

(p

rese

nil

e d

emen

tia)

[61

]

Tum

ors

neu

rob

last

om

a[6

0]

pheo

chro

moc

ytom

a g

ang

lio

neu

rob

last

om

a

accu

mu

lati

on

of

fila

men

ts

app

ear

neg

ativ

e fo

r n

euro

fila

men

ts i

n

som

e ca

ses

pre

sen

ce o

f n

euro

fila

men

ts

neu

rofi

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dete

cti

on

in

axo

ns

-.J

o "':I ~ "':I ~

en

H

tI:1

t-3 ~

INTERMEDIATE FILAMENTS 171

ferentiated cells. After 6 days of treatment, one class of cells reacts with anti-neurofilament antibodies. Aggregation of cells before retinoic acid treatment promotes differentiation. Conditioned medium was also used. Neurofilament proteins appear in a sequential way:[51] at the beginning, preneurones contain vimentin in their small extensions concomittant with morphological differentiation and the 70 k protein is synthesized and coexists with vimentin. Reactiv­ity with anti 200 k develops later and in a discontinuous fashion. Fourteen days after plating, most neurones containing 70 k were not found to contain vimentin. The different situations found in the derivatives of the three cell lines probably correspond to different maturation stages. With the 1003 line, the neurones are different from that found in adult tissues because only two proteins are detec­ted (70 k and 200 k). These neurones have veratridine-sensitive .sodium channels. With the 1009 line, cholinergic neurotransmitters and choline acetyltransferase activities appear and the majority of the neurones after 6 days of treatment display only neurofilament proteins and vimentin is not detected[52].

Interestingly, tumor cells retain the intermediate filament type of their cell of origin; positive staining was shown with antibodies specific for 68 k and for 200 k in the case of human ganglioneuro­blastoma and pheochromocytoma. Not all neuroblastoma tumors have positive staining for neurofilaments supporting the idea that they might originate from a cell type which lacks intermediate filaments; furthermore in these tumors, the cells negative for neurofilaments are also negative for vimentin[60] (Table 7).

Tetanus-toxin binding (found also with one type of astro­cyte[48]) and neurotransmitter molecules (such as acetylcholine and catecholamines which are also detected in neural crest cells[47]), should not be considered as markers of neuronal differentiation exclusively. The presence of the three neurofilament polypetides characterize mature neurones; so far, the expression of even one neurofilament protein has not been demonstrated in non-neuronal cells.

Acknowledgements

Part of this work was supported by a grant from INSERM 811018. We thank P. Cochard, C. Henderson, A. Prochiantz and J. -Po Thierry for results summarized in Figure 3. We are indebted to Gabriel Merle for help in preparation of the manuscript and to A. Delacourte for results in Table 2 and Figure 2.

REFERENCES

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172 F. ALFONSI ET AL.

2. P. Soriano, P. Szabo, and G. Bernardi, The scattered distri­bution of actin genes in the mouse and human genomes, The Embo Journal, 1: (1983).

3. J. Vandekerckhove, K. Weber, The complete amino acid sequence of actins from bovine aorta, bovine heart, bovine fast skeletal muscle and rabbit slow skeletal muscle: a protein­chemical analysis of muscle actin differentiation, Differentiation, 14:123-133 (1979).

4. P. Denoulet,'B. Edde, C. Jeantet, and F. Gros, Evolution of tubulin heterogeneity during mouse brain development, Biochimie, 64:165-172 (1982).

5. K. Dellagi, J. C. Brouet, J. Perreau, and D. Paulin, Human mono­clonal IgM with autoantibody activity against intermediate filaments, Proc.Natl.Acad.Sci.USA, 79:446-450 (1982).

6. B. H. Anderton, Intermediate filaments: a family of homologous structures, J.of Muscle Research and Cell Motility, 2:141-166 (1981).

7. M. Osborn. N. Geisler. G. Shaw. G. Sharp, and K. Weber, Inter­mediate filaments. Cold Spring Harbor Symposia on Quantitat­ive Biology, vol. XLVI. Organization of the Cytoplasm, Cold Spring Harbor Laboratory, (1982).

8. R. A. Nixon, B. A. Brown, and C. A. Marotta, Posttranslational modification of a neurofilament protein during axoplasmic transport: implications for regional specialization of CNS axons, J.of Cell BioI., 94:150-158 (1982).

9. R. M. Pruss, R. Mirsky, M. C. Raff, B. Anderton, and R. Thorpe. A monoclonal antibody demonstrates that intermediate fila­ments share a common antigen, J.Cell BioI., 87:178A (1980).

10. N. Geisler. and K. Weber, Self-assembly in vitro of the 68,000 molecular weight component of the mammalian neurofilament triplet proteins into intermediate-sized filaments, J.Mol. Bio!., 151:565-571 (1981). --

II. R. K. H. Liem, and S. B. Hutchinson. Purification of individual components of the neurofilament triplet: filament assembly from the 70,000-dalton subunit, Biochemistry USA. 21(13):3221 (1982) •

12. M. Willard, and C. Simon, Antibody decoration of neurofilaments, J.of Cell BioI., 89:198-205 (1981).

13. G. A. Sharp. G. Shaw, and K. Weber, Immunoelectronmicroscopical localization of the three neurofilament triplet proteins along neurofilaments of cultured dorsal root ganglion neurones, Exp.Cell Res., 137:403-413 (1982).

14. E. R. Kuczmarski, and J. L. Rosembaum, Studies on ~he organ­ization and localization of actin and myosin in neurons, J.Cell BioI., 80:356-371 (1979).

15. R. G. Nagele, and F. J. Roisen, Ultrastructure of a new micro­tubule-neurofilament coupler in nerves, Brain Research, 253: 31-37 (1982).

16. M. L. Shelanski, J. F. Leterrier, and R. K. H. Liem, Evidence for interactions between neurofilaments and microtubules, Neurosci. Res. Program. Bull, 19(1):32 (1981).

INTERMEDIATE FILAMENTS 173

17. A. Matus, R. Bernhardt, and T. Hugh-Jones, High molecular weight microtubule-associated proteins are preferentially associated with dentritic micro tubules in brain, Proc.Natl.Acad.Sci.USA, 78(5):3010-3014 (1981).

18. R. Thorpe, A. Delacourte, M. Ayers, C. Bullock, and B. H. Anderton, The polypeptides of isolated brain 10 nm filaments and their association with polymerized tubulin, Biochem.J., 181:275-284, (1979).

19. P. T. Davison, and R. N. Jones, Neurofilament proteins of mammals compared by peptide mapping, Brain Research, 182:470-473 (1980).

20. B. A. Brown, R. A. Nixon, P. Strocchi, and C. A. Marotta, Characterization and comparison of neurofilament proteins from rat and mouse eNS, J.of Neurochem., 36(1):143-153 (1981).

21. H. Czonek, D. Solfer, K. Mack, and H. M. Wisnfewski, Similarity of neurofilament proteins from different parts of the rabbit nervous system, Brain Research, 216(2):387 (1981).

22. V. Lee, H. L. Wu, and W. W. Schlaepfer, Monoclonal antibodies recognize individual neurofilament triplet proteins, Proc. Natl.Acad.Sci.USA, 79:6089-6092 (1982). -----

23. J. Wood, and B. Anderton, Monoclonal antibodies to mammalian neurofilaments, Biosci.Rep., 1:263-8 (1981).

24. F. Alfonsi, N. Forest, F. Gosselin, and D. Paulin, Structural homologies between the three proteins of the triplet neuro­filament or across species are revealed with monoclonal anti­bodies, (in preparation).

25. D. Paulin, C. Babinet, K. Weber, and M. Osborn, Antibodies as probes of cellular differentiation and cytoskeletal organiz­ation in the mouse blastocyst, Exp.Cell Res., 130:297-304 (1980).

26. P. Brulet, C. Babinet, R. Kemler, and F. Jacob, Monoclonal anti­bodies against trophectoderm specific markers during mouse blastocyst formation, Proc.Natl.Acad.Sci.USA, 77:1113, (1980).

27. B. W. Jackson, C. Grund, S. Winter, W. W. Franke, and K. Illmensee, Formation of cytoskeletal elements during mouse embryogenesis II. Epithelial differentiation and inter­mediate-sized filaments in early postimplantation embryos, Differentiation, 20:203-216 (1981).

28. w. W. Franke, C. Grund, C. Kuhn, B. W. Jackson, and K. Illmensee, Formation of cytoskeletal elements during mouse embryogenesis. III. Primary mesenchymal cells and the first appearance of vimentin filaments, Differentiation, 23(1):43 (1981).

29. G. Shaw, and K. Weber, Differential expression of neurofilament triplet proteins in brain development, Nature, 298:277-279 (1982)

30. E. Debus, G. Fluegge, K. Weber, and M. Osborn, A monoclonal antibody specific for the 200 k polypeptide of the neuro­filament triplet, The Embo Journal, 1:41-45 (1982).

) 74 F. ALFONSl Kl' AL.

31. C. E. Henderson, M. Huchet, and J. P. Changeux, Two distinct activities affecting neurite outgrowth from embryonic chicken spinal neurons, Devel.Biol., (in press) (1983).

32. H. Czosnek, D. Soifer, and H. M. Wisniewski, Studies on the bio­synthesis of neurofilament proteins, J.Cell BioI., 85:726-734 (1980) •

33. P. Strocchi, D. Dahl, and J. M. Gilbert, Studies on the bio­synthesis of intermediate filament proteins in the rat CNS, J.Neurochem., 39:1132-1141 (1982).

34. C. Hall, and L. Lim, Developmental changes in the composition of polyadenylated RNA isolated from free and membrane-bound polyribosomes of the rat forebrain, analysed by translation in vitro, Biochem.J., 196:327-336 (1981).

35. w. W. Franke, E. Schmid, M. Osborn, K. Weber, Different inter­mediate filaments distinguished by immunofluorescence micro­scopy, Proc.Natl.Acad.Sci.USA, 75:5034-5038 (1978).

36. F. C. S. Ramaekers, M. Osborn, E. Schmid, K. Weber, H. Bloemendal, and W. W. Franke, Identification of the cyto­skeletal proteins in lens-forming cells, a special epitheli­oid cell type, Exp.Cell Res., 127:309-327 (1980).

37. D. Barritault, Y. Courtois, and D. Paulin, Biochemical evidence that vimentin is the only in vivo constituent of the inter­mediate-sized filaments in adult bovine epithelial lens cells, Biol.Cellulaire, 39(3):335-338 (1980) •.

38. T. Raju, A. Bignami, and D. Dahl, In vivo and in vitro differen­tiation of neurons and astrocytes in the rat embryo. Immuno­fluorescence study with neurofilament and glial filament anti~era, Devel.Biol., 85:344-357 (1981).

39. E. R. Abney, P. B. Barlett, and M. C. Raff, Astrocytes, epen­dymal cells and oligodendrocytes develop on schedule in dis­sociated cell cultures of embryonic rat brain, Devel.Biol., 83:301-310 (1981).

40. D. Dahl, A. Bignami, K. Weber, and M. Osborn, Filament proteins in rat optic nerves undergoing Wallerian degeneration. Localization of vimentin, the fibroblastic 100 A filament protein, in normal and reactive astrocytes, Exptl.Neurol., 73:496 (1981).

41. J. Schnitzer, W. W. Franke, and M. Schachner, Immunocytochemical demonstration of vimentin in astrocytes and ependymal cells of developing and adult mouse nervous system, J.Cell BioI., 90:435 (1981).

42. s. H. Yen, and K. L. Fields, Antibodies to neurofilament, glial filament and fibroblast intermediate filament proteins bind to different cell types of the nervous system, J.Cell BioI., 88:115 (1981).

43. G. Shaw, M. Osborn, and K. Weber, An immunofluorescence micro­scopical study of the neurofilament triplet proteins, vimentin and glial fibrillary acidic protein within the adult rat brain, Europ.J.Cell BioI., 26:68-82 (1981).

INTERMEDIATE FILAMENTS 175

44a. A. Bignami. T. Raju. and D. Dahl. Localization of vimentin, the non-specific intermediate filament protein. in embryonal glia and in early differentiating neurons. In vivo and in vitro immunofluorescence study of the rat embryo with vimentin and neurofilament antisera, Develop.Biol., 91(2):286 (1982).

44b. D. Dahl. D. C. Rueger, A. Bignami. K. Weber. and M. Osborn. Vimentin, the 57,000 dalton protein of fibroblast filaments. is the major cytoskeletal component in immature glia, Eur.J. Cell Bio!.. 24: 191 (1981). --

45a. S. J. Tapscott. G. S. Bennett, Y. Toyoma. F. Kleinbart. and H. Holtzer. Intermediate filament proteins in the developing chick spinal cord. Develop.Biol •• 85:40 (1981).

45b. S. J. Tapscott. G. S. Bennett, and H. Holtzer, Neuronal pre­cursor cells in the chick neural tube express neurofilament proteins, Nature, 292(5826):836 (1981).

46. M. Jacobs, Q. Lim Choo, and C. Thomas, Vimentin and 70 k neuro­filament protein co-exist in embryonic neurones from spinal ganglia, J.of Neurochem •• 38(4):969-977 (1982).

47. C. Ziller, E. Dupin, P. Brazeau, D. Paulin, and N. M. Le Douarin. Early segregation of a neuronal precursor cell line in the neural crest as revealed by culture in a chemically defined medium, Cell, 32:627-638 (1983).

48. M. Raff, The role of cell interactions in early embryogenesis. This symposium.

49. R. Mirsky, The role of cell interactions in early embryogenesis. This symposium.

50. Prochiantz, A. Delacourte. M. C. Daguet, and D. Paulin, Inter­mediate filament proteins in mouse brain cells cultured in the presence or absence of fetal calf serum, Exp.Cell Res •• 139 (1982).

51. M. Darmon, M. L. Buc-Caron, D. Paulin, and F. Jacob. Control by the extracellular environment of differentiation pathways in 1003 embryonal carcinoma cells: study at the level of specific intermediate filaments, The Embo Journal, 1(8):901-906 (1982).

52. D. Paulin. H. Jakob, F. Jacob. K. Weber, and M. Osborn, In vitro differentiation of mouse teratocarcinoma cells monitored by intermediate filament expression. Differentiation. 22:90-99 (1982) •

53. B. A. Brown, R. A. Nixon, P. Strocchi, and C. A. Marotta, Characterization and comparison of neurofilament proteins from rat and mouse CNS, J.Neurochem., 36:143 (1981).

54. U. C. Drager, Coexistence of neurofilaments and vimentin in a neurone of adult mouse retina. Nature, 303:169-172 (1983).

55. I. Virtanen, V. P. Lehto, E. Lehtonen, T. Vartio, S. Stenman, P. Kurki, o. Wager, J. V. Small, D. Dahl, and R. A. Badley, Expression of intermediate filaments in cultured cells, J.Cell Sci., 50:45-63 (1981).

56. A. o. Jorgensen, L. Subrahmanyan, C. Turnbull, and V. Kalnins, Localization of the neurofilament protein in neuroblastoma

176 F. ALFONSI ET AL.

cells by immunofluorescent staining, Proc.Natl.Acad.Sci.USA, 79(9):3192-3196 (1976).

57. H. Jakob, and Forecht, (unpublished results). 58. A. Bignami, and D. Dahl, Differentiation of astrocytes in the

cerebellar cortex and the pyramidal tracts of the newborn rat. An immunofluorescence study with antibodies to a protein specific to astrocytes, Brain Research, 49:393 (1973).

59. D. Dahl, P. Strocchi, and A. Bignami, Vimentin in the central nervous system. A study of the mesenchymal-type intermediate filament-protein in wallerian degeneration and in postnatal rat development by two-dimensional gel electrophoresis, Differentiation, 22:185-190 (1982).

60. M. Osborn, M. Altmannsberger, G. Shaw, A. Schauer, and K. Weber, Various sympathetic derived human tumors differ in neuro­filament expression, Virchows Arch. (Cell Pathol.), 299:1-16

61. R.

62. E.

63. E.

64. P. 65. J.

66. M.

(1982). M. Marchbanks, Biochemistry of alzheimer's dementia, J.Neurochem., 39(1):9-15 (1982). Rungger-Brandle, G. Gabbiani, The role of cytoskeletal and cytocontractile elements in pathologic processes, Cyto­skeleton in Pathology, 110(3):361-392 (1983). Lazarides, Intermediate filaments chemical heterogeneity in differentiation, Cell, 23:649 (1981). Co chard , and D. Paulin, (manuscrit in preparation). Wartiowara, P. Liesi, H. Hervonen, and L. Recherdt, Neural differentiation in F9 teratocarcinoma cultures. This symposium. M. Portier, B. Croizat, and F. Gros, Sequence changes in cytoskeleton components during neuroblastoma differentiation, Febs Letter, 86(2):283-288 (1982).

IMMUNOCHEMICAL STUDIES ON THE D2-GLYCOPROTEIN

Elisabeth Bock* and Kjeld M6llg~rd**

*The Protein Laboratory. University of Copenhagen 34. Sigurdsgade. DK-2200 Copenhagen N. Denmark **Institute of Medical Anatomy. Dept. A. University of Copenhagen. The Panum Institute 3. Blegdamsvej, DK-2200, Copenhagen N. Denmark

The use of marker molecules identifiable by immunochemical methods is an important tool for cellular identification and in defining subtypes of cells. problems most acute in cell cultures. developing tissues and in tissues undergoing pathological changes. The D2-glycoprotein was originally introduced as a marker of synaptic membranes (Bock & J~rgensen. 1975). By immunocytochemical procedures D2 was later demonstrated on the entire surface of neurons in primary cultures of fetal rat brain (Bock et. al •• 1980b) and the protein has since been used as a neuronal cell marker for distinguishing neurons from glial cells in culture (Bock et al •• 1982b). In a recent study antibodies against D2 were found to inhibit fasciculation of neurites from cultured rat sympathetic ganglia and D2-glycoprotein was shown to be immunochemically related to the chick neural cell adhesion molecule (N-CAM) (J6rgensen et al •• 1980). It was therefore suggested that D2 is involved in adhesion phenomena between neurons.

Quantification of D2 has been performed by means of polyspecific antibodies raised against synaptic membranes (Bock and Braestrup. 1978). A fractionation procedure for human brain. rat brain and chick brain has recently been described and specific antibodies against these D2-glycoproteins have now been produced (Ibsen et al •• 1983a; Rasmussen et al., 1982; Bock et al., 1983b) thus enabling the set-up of more sensitive quantification assays such as enzyme linked immunosorbent assay (Ibsen et al •• 1983a) and the possibility of performing immunocytochemical studies on well fixed paraffin embedded material from many species.

177

178 E, BOCK AND K, M0LLGARD

Polypeptide analysis of purified D2 by means of sodium-dodecyl­sulphate polyacrylamide-gel electrophoresis (SDS-PAGE) has shown that adult rat brain and human brain D2 is composed of two polypeptide bands of apparent MW 150,000 and 125,000, respectively, and fetal D2 is composed of one polypeptide band in the MW range 150,000-200,000 (Rasmussen et aI" 1982; Ibsen et al., 1983a). D2 binds to several lectins including concanavalin A and wheat germ lectin (Bock et aI" 1983a), Analysis of lectin interactions has indicated a difference in carbohydrate composition between fetal and adult D2, Furthermore, lectin positive and lectin negative forms of D2 seem to be present in varying proportions during development, indicating a heterogeneity of the carbohydrate moiety of D2 at any stage of development,

An amphiphilic nature of D2 has been demonstrated (J6rgensen, 1977; Albeck and Bock, 1982), However, also a soluble form of D2 has been demonstrated in cerebrospinal fluid (J6rgensen and Bock, 1975), in amniotic fluid and in serum (Ibsen et aI" 1983a,b), It therefore seems as if the epitopes of the D2-glycoprotein exist on both a membrane-bound amphiphilic molecule and on a hydrophilic molecule, The relationship between these two forms is unclear,

The membrane topography of D2 has been studied by immunoabsorp­tion, lactoperoxidase catalysed 125I-Iabelling and treatment with proteolytic enzyme of intact and lysed synaptosomes, By all pro­cedures D2 was found to be localized on the external surface of the synaptic plasma membrane (Bock et aI" 1980a),

D2 has been demonstrated in all investigated areas of the central nervous system (Bock and Braestrup, 1978), Biochemical studies of D2 in the developing mouse (Jacque et aI" 1976) and chick brain (Bock et aI" 1983b) have shown that the molecule appears very early in gestation. Thus, D2 could already be demonstrated in the brain of a five day chick embryo, Maximum concentration was found at the days before hatching and thereafter the concentration decreased slightly, The glycoprotein has also been demonstrated in various human adult and fetal organ extracts, although in much lower amo~nts than in the nervous system (Bock et aI" 1983a), The concentration was higher at early stages of development than in adult tissues,

The regional distribution and cellular localization of the D2-glycoprotein in human embryonic nervous system has been invest­igated by immunocytochemistry at the light microscope level in sections of human embryonic brain, spinal cord and some neural crest derivatives, A positive staining reaction for D2 was observed in intermediate and marginal zones of fore-, mid- and hindbrain already in the earliest (15 mm crown-rump length) embryo examined, whereas the ventricular zone as well as nerves and ganglia were largely negative at this stage. By 20 mm crown-rump length the majority of the neuronal membranes in the CNS and peripheral nerves exhibited positive staining for D2, A similar stainability was found in sen-

D2-GLYCOPROTEIN

•

---

, . 7!":. .-.-'. " ," " . (

f"- "

179

Fig. 1. Sagittal section through the brain stem and head of a seven week human embryo (22mm crown-rump length) stained for D2-glycoprotein. Note the strong stainability of the efferent fibers in cranial nerves III, IX and X and the weaker stain­ability of the sensory ganglia (e.g. V). The ventricular zone (VZ) is very weakly stained compared to the intermedi­ate and marginal zones. Developing neurons in the neural layer of the retina are positive in contrast to the cells of the pigment layer which appear dark due to the presence of densely packed small pigment granulas. x 50.

sory cranial nerve ganglia and dorsal root ganglia as well as in sympathetic and parasympathetic ganglia. A typical staining reaction for D2 is shown in Figure 1.

D2 was originally described as a neuronal membrane marker. The relationship to the Chick N-CAM and the inhibition of fascicle form­ation between neurites by antibodies against D2, indicates that the protein may be involved in neuronal adhesion phenomena. If indeed the D2-glycoprotein is a cell adhesion molecule, the function may be explained on the assumption that the glycoprotein on one type cell surface is recognized by the surface of a complementary cell in­volved. Two basically different mechanisms can be considered: 1) The

180 E. BOCK AND K. M0LLGARD

order and number of adhesion molecules organized with other membrane components at the cell surface is complementary to the same order and number of adhesion molecules on the counter part cell surface (Steinberg's self-self interaction model) (Steinberg, 1963). 2) The molecules that can recognize or interact with the cell adhesion molecule may be lectins or enzymes, a so-called "lock and key" inter­action. Recent studies indicate a self-self interaction (Rutishauser et al., 1982). Our recent demonstration of D2-immunoreactivity in other organs than the nervous system indicates that D2 may be a more general cell adhesion molecule, or a member of a family of cell adhesion molecules present on plasma membranes of parenchymal cells of many organs at various stages of development.

REFERENCES

Albeck, M. J., and Bock, E., 1982, "Prot ides of the Biological Fluids", vol. 29, H. Peeters, ed., Pergamon Press, Oxford, pp 151-154.

Bock, E., Berezin, V., and Rasmussen, S., 1983a, "Protides of the Biological Fluids", vol. 30, H. Peeters, ed., Pergamon Press, Oxford, pp. 75-78.

Bock, E., Bjerrum, O. J., Gombos, G., J~rgensen, o. S., Reeber, A., Vincendon, G., Wechsler, W., Yavin, E., and Yavin, Z., 1980a, in: "Synaptic Constituents in Health and Disease", M. Brzin, ~ Sket, and H. Bachelard, eds., Mladinska knjiga, Ljubljana, Pergamon Press, London, pp 210-223.

Bock, E., and Braestrup, C., 1978, J.Neurochem. 30:1603. Bock, E., and J~rgensen, O. S., 1975, FEBS Lett 52:37. Bock, E., Rasmussen, S., Albeck, M., Sensenbrenner, M., Pettmann, B.,

and Louis, J. C., 1983b, "Electroimmunochemical Analysis of Membrane Proteins", O. J. Bjerrum, ed., Elsevier, Amsterdam, pp. 275-286.

Bock, E., Yavin, Z., J~rgensen, O. S. and Yavin, E., 1980, J.Neurochem., 35:1297.

Ibsen, S., Berezin, V., N~rgaard-Pedersen. B., and Bock, E., 1983~, J.Neurochem., 356-362.

Ibsen, S., Berezin, V., N~rgaard-Pedersen, B., and Bock, E., 1983b, J.Neurochem., 363-366.

Jacque, C. M., J~rgensen, o. S., Baumann, N. A., and Bock, E., 1976, J.Neurochem. 27:905.

J~rgensen, o. S., 1977, FEBS Lett. 79:42. J~rgensen, o. S., and Bock, E., 1975, Scand.J.lmmunol. 4 suppl.2:25. J~rgensen, o. S., Delouvee, A., Thiery, J.-P., and Edelman, G. M.,

1980, FEBS Lett. 111:39. Rasmussen, S., Ramlau, J., Axelsen, N. H., and Bock, E., 1982,

Scand.J.Immunol. 15:179. Rutishauser, U., Hoffmann, S. and Edelman, G. M., 1982, Proc.Natl.

Acad.Sci.USA., 79:685. Steinberg, M. S., 1963, Science, 141:401.

MOLECULAR HETEROGENEITY IN PERIPHERAL GLIA

Rhona Mirsky and Krist jan R. Jessen

Department of Anatomy University College London

Immunohistochemical studies have transformed our picture of the chemical complexity of both the CNS and PNS. The chemical specific­ity of antibodies combined with the visual resolution obtained by microscopic examination of tissue sections or cell cultures has allowed chemical analysis of the component parts of the nervous system at a cellular level, and provides a means of detecting mole­cular heterogeneity in both morphologically similar and different types of cells. This is well illustrated in the case of neuropep­tides. The use of antibodies recognizing 30-40 different peptides or peptide fragments in immunohistochemical studies has revealed complex and often partly overlapping patterns of distribution in central and peripheral neurons, which generally cut across, rather than coincide with, traditional population categories based on morphology or trans­mitter type (e.g. Schultzberg et al., 1980).

Another type of neuronal heterogeneity has been uncovered by the use of monoclonal antibodies. These have usually been selected on the basis of their ability to recognize antigens, often of unknown chemical identity, which distinguish between sUbpopulations of neur­ons, on the basis of either developmental or morphological/anatomical variation. For example, PNS derived rat neurons can be distinguished from CNS derived neurons because they express a protein recognized by an antibody 38/D7 which is not present on the surface of CNS derived neurons (Vulliamy et al., 1981). Many other examples of this type could be given, and it is evident from the work of several groups that the neuronal subcategories revealed by the use of this method are generally different from those detected by morphological, trans­mitter or neuropeptide analysis (McKay et al., 1981).

181

182 R. MIRSKY AND K. R. JESSEN

These immunohistochemical findings have been given a functional interpretation in a few instances, especially in the case of the peptides. Nontheless, it is clear that the biological significance of the great majority of antigens and molecules so far detected immunohistochemically in neurons, remains to be explained. Already, however, the demonstration of this bewildering spectrum of molecular heterogeneity among neurons has had an important influence in many areas of neurobiology, including developmental biology, and stimu­lated a great deal of new research.

In turning from studies on neurons, to those on glial cells, it seems that questions about diversity and heterogeneity have received much less attention. This is especially evident in the current approach to peripheral glia. In sharp contrast to the growing recog­nition of diversity among peripheral neurons, the current trend has tended to emphasize basic similarities between peripheral glia. The increasing use of the term "Schwann cell", originally referring to one phenotype only, to cover all peripheral glia, reflects this view, which has been reinforced by experiments on myelination showing that two of the peripheral glial phenotypes can revert from one to the other, depending on environmental stimuli (Aguayo et al., 1976; Weinberg and Spencer, 1976; Mirsky et al., 1980). It may therefore be worth pointing out that in fact peripheral glia exhibit marked differences in structure and morphological relationship with neurons. On the basis of these two criteria alone they can be unambiguously divided into the three broad classes of satellite cells, associated with neuronal cell bodies in the sensory and sympathetic ganglia, Schwann cells, associated with axons, either non-myelinating or myelinating, and enteric glial cells, found in the enteric nervous system, in addition to smaller groups such as the glia of the olfact­ory nerve. The degree of phenotypic difference between these groups of cells is quite comparable to that seen in central glia between the structure and neuro-glia relationships of atrocytes, oligodendrocytes and microglia. On the other hand, studies on myelination suggest that in PNS glia, cell-cell interactions playa greater role in dictating the glial phenotype than such interactions do in the eNS. In the case of myelination this results in individual neurons being able to signal the glial phenotype most appropriate for the normal functioning of that neuron. It is important to know whether this type of interaction extends to other aspects of neuro-glial relation­ships or whether it is restricted to the myelination event. Thus, at one extreme glia might be cells which are fine tuned by the neurons they surround to provide the type of microenvironment or other ser­vices needed by that particular neuron, the control of myelination being only on example of many, or, on the other hand glia may serve only general support functions of the type demanded by all neurons from all glia. Myelination would then be the only example where specific requirements of a neuronal subpopulation are met. Func­tional requirements are generally reflected in chemical composition. Therefore, in the first case, peripheral glia would be expected to

PERIPHERAL GLIA CELLS 183

express chemical heterogeneity, perhaps comparable to that found in neurons. In the second case, if these cells serve a uniform, general role, this should be reflected in a high degree of molecular homogen­eity. In this paper, we will review recent studies from our own laboratory which show that satellite and Schwann cells and, to a lesser extent enteric glia, do indeed express complex distribution patterns of both the cell membrane and intracellular antigens that we have so far investigated. Some of the antigens examined are highly restricted in distribution to glia, while others are less so. The antibodies used are listed in Table 1. The results are presented separately for each main cell type before more general considerations are discussed.

SCHWANN CELLS

The distribution of antigens can be most clearly understood by dividing the Schwann cells into two functional categories, non­myelinating and myelinating. Only two of the antibodies used, those recognizing the cell surface protein Ran-I and the intermediate filament protein vimentin, were present on all Schwann cells exam­ined. Interestingly Ran-I was present at much higher levels in cultured Schwann cells than on Schwann cells in freshly teased pre­parations of sciatic nerve. It is present early in development, being clearly seen on the surface of Schwann cells in 1 day old cultures from 13 day rat embryo DRG. In contrast, the surface anti­gens Ran-2 and ASE3 antigen were present on non-myelinating Schwann cells only, in situ, but their cell type and developmental distri­butions were quite different from each other. ASE3 antigen, like Ran-I, was present on Schwann cells in cultures from embryo DRG (15 days old embryo was the earliest examined) and was present on all the Schwann cells in a partially teased preparation of sciatic nerve from new born rat, a time when myelination has not begun. It is also present on a majority, if not all, the Schwann cells in partially teased preparations from the pre-and post-ganglionic sympathetic trunks of adult rats, where there are few myelinated fibres, and it is on unmyelinated fibres in both the dorsal and ventral roots of adult rats. In contrast, Ran-2 appears for the first time on Schwann cells relatively late in development (14-21 days postnatal) and is seen on non-myelinating cells in all nerves surveyed. These include sciatic nerve, pre- and post-ganglionic sympathetic trunks and dorsal and ventral roots. A5E3 antigen is expressed by Schwann cells in culture, whereas Ran-2 is not.

Previous studies show that the myelin related molecules, galac­tocerebroside and sulfatide, and the proteins Pu and P1 (Basic pro­tein) are present on all myelinating Schwann cells in situ (Brockes et al., 1980; Winter et al., 1982). However, the myelin protein P~ is present in only a subpopulation of myelinating Schwann cells (Trapp et al., 1979). This uneven distribution is particularly

Ant

ibod

y

An

ti-R

an-l

Ant

i-R

an-2

A5E

3

An

ti-v

imen

tin

Ant

i-G

FAP

Ant

i-G

FAP

3

An

ti-g

luta

min

e sy

nth

etas

e

Ant

i-fi

b ro

nec

tin

An

ti-g

lact

oce

re­

bro

s id

e

An

ti-P

o

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ti-P

1 (b

asic

p

rote

in)

An

ti-P

2

An

tig

en

Pro

tein

Pro

tein

Pro

tein

, 14

5kd

Pro

tein

, 54

kd

Pro

tein

, 49

kd

Pro

tein

, 49

kd

Pro

tein

Pro

tein

, 23

0kd

Gly

coli

pid

Pro

tein

, 29

kd

Tab

le

1

Abs

orbe

d m

ouse

p

oly

clo

nal

Mou

se m

onoc

lona

l,

IgG

2a

Mou

se m

onoc

lona

l,

IgG

2a

Rab

bit

po

lycl

on

al

Rab

bit

po

lycl

on

al

Mou

se m

onoc

lona

l,

IgM

Rab

bit

po

lycl

on

al

Rab

bit

po

ly c

lon

al

Mou

se m

onoc

lona

l,

IgM

Rab

bit

po

lycl

on

al

Pro

tein

, 1

4,

17,

Rab

bit

po

lycl

on

al

18

.5 a

nd 2

1. 5

kd

Pro

tein

, 14

-14k

d R

abb

it p

oly

clo

nal

Don

or a

nd

/or

Ref

.

Fie

lds

et

al.

, 19

75

Bro

ckes

et

al.

, 19

79;

Raf

f et

al.

, 19

79

Bart

lett

et

al.

, 19

81

J.

Gav

rilo

vic

, J.

W

inte

r an

d R

. M

irsk

y

Ram

aeke

rs e

t al.

, 19

82

R.

Pru

ss;

Jess

en a

nd M

irsk

y,

1980

Alb

rech

tsen

et

al.

, 19

83

Dr.

O

.-S

. Jo

rgen

sen

; M

arti

nez-

Her

nand

ez e

t al.

, 19

77

War

tio

vaa

ra e

t al.

, 19

74

Ran

scht

et

al.

, 19

82

Bro

ckes

et

al.

, 19

80

Dr.

M

. K

adlu

bow

ski

and

Dr.

R

.A.C

. H

ughe

s

Kad

lubo

wsk

i an

d H

ughe

s,

1980

00

-i

'- i::d

g:j

i::d ~ ~ ~

i::d

t....

t:rJ

til

til ~

PERIPHERAL GLIA CELLS 185

evident in dorsal roots, where many of the Schwann cells surrounding the smaller fibres appear not to express the protein. Galactocere­broside appears on the surface of Schwann cells in the sciatic nerve more than one day before the onset of meylination, whereas PO, Pi and Pz are not immunohistochemically detectable until the onset of mye­lination (Winter et al., 1982). Myelin associated moleculas are not found associated with Schwann cells not destined to make myelin and are rapidly lost in culture from Schwann cells which are removed from contact with the appropriate axonal signal (Mirsky et al., 1980).

Whereas the intermediate filament protein vimentin is detectable inside all Schwann cells in situ (Yen and Fields, 1981), GFAP-like immunoreactivity is found associated only with non-myelinating Schwann cells not only in sciatic nerves (Yen and Fields, 1981) but also in a wide variety of peripheral nerves. Among nerves surveyed most if not all fibres which are not myelinated, are ensheat¥ed by Schwann cells which express GFAP. In nerves which have GFAP Schwann cells, all the Schwann cells associated with a particular nerve appear to be positive, suggesting that a neuronal signal may be important in the induction of GFAP-like proteins in Schwann cells (K.R. Jessen, R. Thorpe, R. Mirsky, J.Neurocytol., in press).

In contrast, glutamine synthetase can be detected in subpopu­lations of both non-myelinating and myelinating Schwann cells.

In development vimentin is expressed more strongly by Schwann cells from new born rats than adult rats whereas the converse in true for GFAP, which is not detectable in the sciatic nerve of newborn rats, barely detectable in sciatic nerve of 5 days old rats, and which attains adult levels of expression between 14 and 21 days. When Schwann cells from older animals are cultured they appear to retain their GFAP-like reactivity, a clear difference from the expression of myelin related molecules.

ENTERIC GLIAL CELLS

When visualized in freshly dissected myenteric plexus prep­arations, Ran-1 is absent from the surface of enteric glia in situ, but is clearly seen on a majority if not all enteric glia in culture. In contrast, Ran-2 which is present on some but not all astrocytes in situ and in culture, is present on enteric glia in situ, both in -­freshly dissected preparations and in sections of colon, but is absent from enteric glial cells in culture. As in Schwann cells, the antigen appears relatively late in development. It is not detectable on the surface of enteric glia in myenteric plexus from 7 day old rats, is barely detectable at 2 weeks of age, but can be clearly seen in rats 3 weeks or older.

186 R. MIRSKY AND K. R. JESSEN

A characteristic which enteric glia share with non-myelinating Schwann cells is the expression of A5E3 antigen, which is present both in situ and in cuture. This antigen is also expressed by smooth muscle cells and perineurial cells but not by astrocytes in situ.

Enteric glia, like astrocytes, express the intracellular pro­teins, vimentin, GFAP and glutamine synthetase both in situ and in culture. Neither the myelin associated molecule galactocerebroside, nor fibronectin are expressed by enteric glia in culture in any significant quantity. In whole mount preparations of the colon, from which either longitudinal muscle or mucosa have been stripped to allow visualization of the myenteric or submucous plexuses, respect­ively, it can be seen that the GFAP-reactivity is very strong in the enteric glia within the ganglia of both the submucous and myenteric plexuses, whilst the glial cells in the interconnecting strands between the ganglia show little or no reactivity. On the other hand, vimentin seems to be present in approximately equal amounts in the glial cells within the ganglia and those in the interconnecting strands. Glutamine synthetase reactivity, like GFAP reactivity, appears to be stronger in the glial cells within the ganglia than in the cells of the interconnecting strands.

In development, vimentin is more strongly expressed by the enteric glial cells of newborn rats than at later ages. The levels appeared to fall slowly over the first three postnatal weeks and adult levels of expression are seen in enteric glial cells of three week old rats. In contrast, GFAP reactivity, though clearly detect­able in enteric glia of newborn rats, did not rise to adult levels until between two and three weeks of age (K.R. Jessen and R. Mirsky, 1983).

SATELLITE CELLS

Ran-1 appears to be present on all satellite cells. This can be seen in situ using a preparation of DRG preincubated with collagen~se and hyaluronidase and trypsin inhibitors which is gently teased before incubation and antibody. To visualize the neuronal soma and satellite cells round them the preparation is lightly fixed and then squashed under a coverslip to give a monolayer of cells. Many satellite cells are Ran-2 negative in situ.

The A5E3 antigen can be seen associated with all satellite cells in frozen sections of DRG from adult rats.

Many satellite cells, especially those surrounding the large neurons in the DRG, express very strong vimentin-like reactivity. Some satellite cells surrounding small neurons in the DRG, and some neurons in the SCG express little vimentin reactivity. In the satellite cells of the SCG and the DRG at cervical thoracic and

PERIPHERAL GLIA CELLS 187

lumbar levels, a small minority of neurons were surrounded by satellite cells expressing GFAP-like reactivity, although the number was considerably smaller than the vimentin positive population, especially in the DRG (K.R. Jessen, R. Thorpe, R. Mirsky, J.Neurocytol., in press).

Developmentally, strongly vimentin positive satellite cells are seen in DRG of new born rats, but the reactivity does not appear to decline with age. In contrast, the size of the GFAP positive satel­lite cell population increased markedly with age. Highest numbers were seen in rats 4 months or older in age, and GFAP positive satel­lite cells which were often hard to find in young animals, were never detected in animals younger than 3 weeks old. Glutamine synthetase reactivity has not so far been examined in satellite cells.

HETEROGENEITY IN GFAP FILAMENTS

Combined use of polyclonal anti-GFAP sera and a monoclonal antibody to human GFAP (anti-GFAP 3) prepared and characterised by Albrechtsen, Von Gerstenberg and Bock (1983) allowed us to compare GFAP-like immunoreactivity in the rat CNS and PNS. Polyclonal anti­sera used both immunohistochemically and in immunoblotting experi­ments showed that the widespread GFAP-like reactivity found through­out the peripheral nervous system resides in a protein of molecular weight apparently identical (49kd) to that of astrocyte GFAP. None­theless, the GFAP of most peripheral glia appears to differ from GFAP of astrocytes by lacking an antigenic determinant defined by the monoclonal antibody, anti-GFAP 3. This was shown in double label immunohistochemical experiments with poly clonal and monoclonal anti­body. 'Both antibodies labelled astrocytes and Bergmann glia in frozen sections of cerebellum, while in the sciatic nerve and DRG. Schwann and satellite cells which were clearly labelled with the polyclonal antibody showed no staining with the monoclonal antibody. In enteric glia. only a small subpopulation of the glia within the ganglia labelled with the monoclonal antibody. Furthermore, SDS and PAGE followed by immunoblotting of rat brain and sciatic nerve extracts with anti-GFAP 3 revealed an immunoreactive band in the GFAP position in brain, but no detectable activity in sciatic nerve. The simplest interpretation of these results is that the polyclonal GFAP sera recognize two or more closely related filament proteins of identical molecular weight, one of which is not recognized by the monoclonal antibody. While the protein recognized by the monoclonal antibody is abundant in astrocytes in the CNS it is apparently not present in sciatic nerve or satellite cells. On the other hand, it is present in low but variable amounts in enteric glial cells (K.R. Jessen R. Thorpe, R. Mirsky, J.Neurocytol •• in press).

Schw

ann

cell

s (a

) N

on-m

yeli

n fo

rmin

g

(b)

My

elin

to

rmin

g

Sate

llit

e c

ell

s

En

teri

c g

lia

Ast

rocy

tes

Tab

le

2 P

rop

ert

ies

of

Peri

ph

era

l G

lial

Cell

s In

Sit

u

Ran

-l

Ran

-2

ASE

3

+

+

+

+

+

+/-

+

+

+

+/-

Vim

enti

n

+ +

+ +

+

GF

AP

-lik

e G

luta

min

e F

ibro

necti

n

reacti

vit

y s

yn

theta

se

+*

+/-

+/-

+/-

*

+/-

*

+/-

+

+

+/-

Ind

icate

s th

at

the

an

tig

en

is

foun

d on

a

sub

po

pu

lati

on

of

cell

s w

ith

in

* F

or

dis

cu

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n o

f h

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ty a

mon

g G

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P-l

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men

ts,

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ory

ex>

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Z

PERIPHERAL GLIA CELLS 189

DISCUSSION

A comparison of the antigenic properties of the three main types of peripheral glial cells in situ is presented in Table 2. Several conclusions emerge. The first, and most important is that it is possible to find considerable heterogeneity of antigenic expression not only between the different categories of peripheral glia but also within the three main types, indicating that it is not difficult to find molecular heterogeneity within the peripheral glia, even with the limited range of antibodies used in this study. Of the antigens examined. something is known about the functions of glutamine synthe­tase, fibronectin and the myelin associated molecules. much less is known about the role of the intermediate filament proteins, and the significance of the Ran-i, Ran-2 and A5E3 antigens is at present obscure.

Of the three main cell types examined the enteric glia show least heterogeneity and a marked similarity to astrocytes. although there does seem to be a small difference between cells within the ganglion and those in the interconnecting strands with respect to expression of both GFAP-like reactivity and glutamine synthetase. Unsurprisingly. considering the enormous specialization of the mem­brane that occurs when myelin is formed, the biggest differences are seen between non-myelinating and myelin forming Schwann cells, though even this case. glutamine synthetase is found in subpopulations of both non-myelinating and non-myelinating cells and Ran-i and vimentin are expressed by both types. The satellite cell population appears to show highly variable expression of the intermediate filament proteins. vimentin and GFAP, with GFAP-like expression being seen only in the satellite cells round the largest neurons in older animals.

The differences observed in expression of GFAP proteins in the CNS and PNS show for the first time that there is heterogeneity among glial filaments. Although enteric glia express small but variable amounts of the GFAP recognized by the monoclonal anti-GFAP 3. the majority of the glial filaments present in the enteric glia. and all those present in subpopulations of Schwann and satellite cells are different from. but clearly closely related to astrocyte GFAP. This is shown both by the immunohistochemical and by the immunoblotting results with polyclonal GFAP antibodies. It is possible that the difference between the GFAP found abundantly in astrocytes and that found in most peripheral glia is related to a post-translational modification, but it seems more likely that the difference may derive from small differences in amino acid sequences between GFAP subunits, as has been shown for another family of intermediate filaments, the cytokeratins.

Clearly. it is now important to establish whether chemical heterogeneity of the type described above is controlled by the neur­ons. If this turns out to be the case, as seems possible from

190 R. MIRSKY AND K. R. JESSEN

studies on myelination, it is tempting to speculate that peripheral glia in general may functionally meet needs specific to the neurons they contact in addition to providing general support functions.

REFERENCES

Aguayo, A. J., Charron, L., and Bray, G. M., 1976 J.Neurocytol. 5:565-573.

Albrechtsen, M., von Gerstenberg, A. C., and Bock, E., J.Neurochem. (in press).

Bartlett, P. F., Noble, M. D., Pruss, R. M., Raff, M. C., Rattray, S., and Williams, C. A., 1981. Brain Res. 204:339-352.

Brockes, J. P., Fields, K. L., and Raff, M. C., 1979, Brain Res., 165:105-118.

Brockes, J. P., Raff, M. C., Nishiguchi, D. J., and Winter, J., 1980, J.Neurocytol., 9:67-77.

Fields, K. L., Gosling, C., Megson, M., and Stern, P. L., 1975, Proc.Nat.Acad.Sci.USA., 72:1286-1300.

Jessen, K. R., and Mirsky, R., 1980, Nature, 286:736-737. Jessen, K. R., and Mirsky, R., 1983, J.Neurosci., 3:2206-2218. Kadlubowski, M., and Hughes, R. A. C., 1980, J.Neuro.Sci., 48:171-8. Martinez-Hernandez, A., Bell, K. P., and Norenberg, M. D., 1977,

Science, 195:1356-1358. McKay, R., Raff, M. C., and Reichardt, eds., Monoclonal Antibodies to

Neural Antigens, Cold Spring Harbor Press, Cold Spring Harbor, 1981.

Mirsky, R., Winter, J., Abney, R. R., Pruss, R. M., Gavrilovic, J., and Raff, M. C., (1980). J.Cell BioI., 84:483-494.

Raff, M. C., Fields, K. L., Hakomori, S-I., Mirsky, R., Pruss, R. M., and Winter, J., 1979, Brain Res., 174:283-308.

Ranscht, B., Clapshaw. P. A., Price, J., Noble, M., and Seifert, W., 1982, Proc.Nat.Acad.Sci., 79:2709-2713.

Schultzberg, M., Hokfelt, T., Nilson, G., Terenius, L., Rehfeld, J., Brown, M., Elde, R., Goldstein, M., and Said, S., 1980, Neurosci., 5:689-744.

Trapp, B. D., McIntyre, L. J., Quarles, R. H., Sternberger, N. H.,' and Webster, H. De F., 1979, Proc.Nat.Acad.Sci., 76:3552-6.

Vulliamy, T., Rattray, S., and Mirsky, R., 1981, Nature 291:418-419. Wartiovaara, J., Linder, E., Ruoslahti, E., and Vaheri, A., 1974,

J.Exp.Med., 140:1522-1533. Weinberg, H., and Spencer, P. S., 1976, Brain Res., 113:363-378. Winter, J., Mirsky, R. and Kadlubowski, M., 1982, J.Neurocytol.,

11:351-362. Yen, S.-H., and Fields, K. L., 1981, J.Cell BioI., 88:115-126.

PLASMA PROTEINS AND FETAL BRAIN DEVELOPMENT

N. R. Saunders

Department of Physiology and Centre for Neuroscience University College London

Several studies have demonstrated the presence of a number of plasma proteins (a-fetoprotein, fetuin, transferrin and albumin) in the developing brain of many different species including rat[l], mouse[2], sheep[3], pig[4] and human[5]. These proteins and some other plasma proteins have been found in high concentrations in cerebrospinal fluid in early stages of brain development[3,4,6-9]. The work described in this paper is concerned with two main ques­tions. 1) Where do the plasma proteins in fetal csf and brain orig­inate from? 2) What is their functional significance?

PLASMA PROTEINS IN FETAL BRAIN AND CHOROID PLEXUS

The presence of several plasma proteins in developing brain has been most clearly illustrated by immunocytochemistry. The first plasma protein to appear in the developing brain in the sheep fetus is fetuin, at about the time of neural tube closure. In the early stages of neural tube development, shortly after its closure, several plasma proteins have been identified and appear to have both an extracellular and intracellular distribution. By 35 days gestation (20 mm CRL) a-fetoprotein. fetuin and albumin appear to be intra­cellular. especially within the cytoplasm of presumed developing neurons; subsequently these proteins are also found within nerve processes. In contrast, in early stages of brain development trans­ferrin appears to be predominantly associated with nerve processes although later in development in some brain regions it is also found within the cell bodies of some neurons (e.g. after 40 days in some diencephalic nuclei). Transferrin associated with nerve processes appears to be mainly on their surface and may well be largely extra­cellular in contrast to the intracellular distribution of AFP, fetuin

191

192 N. R. SAUNDERS

and albumin. Using a double staining technique it has been shown in the pig fetus that developing neurons may contain both fetuin and AFP[lO]. In both the sheep and the pig fetus fetuin appears to have the most widespread distribution and prolonged presence of all the proteins so far investigated.

Detailed studies of the distribution of AFP, fetuin, albumin and transferrin in brains of sheep, pig and rat fetuses are in progress. In the forebrain, the developing cells are often stained in their characteristic layers but not necessarily for each protein at the same stage of development, although some cells do contain more than one plasma protein. The ventricular and subventricular layers in the sheep fetal brain contain cells positive for fetuin, AFP and/or albumin from 20 days gestation until about 60 days gestation (term is 150 days). The cortical plate especially in the early stages of its formation (35-40 days) contains many cells that are positive for fetuin; some AFP and albumin positive cells are usually also present. The most striking observation so far is that whereas the cell bodies of a high proportion of neurons in the cortical plate at 35 days gestation contain fetuin, the regions on either side of the cortical plate in which mainly cell processes are found stain for transferrin. It is in this region that synaptogenesis is occurring[ll]. This is particularly interesting because of the finding that transferrin is an essential component in defined media for culturing neurons (and some other cell types) in vitro[12].

Studies o,f the distribution of plasma proteins in human and sheep fetal choroid plexus at different gestational ages have been published[13-l5]. In the telencephalic choroid plexus of both species 40% or more of the epithelial cells were positive for albumin, AFP and fetuin (sheep) in very early stages of development, e.g. up to 40 days gestation in sheep fetuses. Subsequently only 5-1070 of cells were positive for each protein. There was a species difference for the myelencephalic choroid plexus. In the human fetus it showed similar high proportions of cells positive for each protein early in development and this high proportion persisted for much longer than in the telencephalic choroid plexus. In contrast in tile sheep the myelencephalic choroid plexus did not have more than about 5% of cells positive for anyone plasma protein at any stage of development.

PROTEIN IN FETAL CSF

Fetal csf is characterized by a high concentration of protein, the peak occurring at different ages in different species (Fig. 1). For example in the sheep and pig fetus there is a peak of 1000 mg/IOO ml or more total protein at 30 days gestation. In the rat the peak is much lower at about 320 mg/IOO ml and this occurs around the time of birth. In the sheep and probably also the pig this high concen-

PLASMA PROTE INS 193

mg/100ml

600

200

20 40 60 80 100 120

fetal age (days)

Fig. 1. Total protein concentration (Lowry method, mg/100 ml) in csf of various species at different gestational and post-natal ages. Arrows indicate time of birth. Term in the sheep is 150 days and in the pig is 120 days.

tration of protein can be accounted for by only a small number of plasma proteins. Table 1 summarizes the data so far available on individual protein concentrations in csf at the time of the peak total protein concentration, in 4 species. In the sheep fetus the decline in csf protein concentration occurred first in csf within the lateral ventricles (Fig. 2). Although the concentrations decline with increasing gestational age in both lateral ventricular and cisternal csf, that in the latter at any age was always higher than in the former. Such a difference is of course well known in the adult.

ORIGIN OF PLASMA PROTEINS IN FETAL CSF AND BRAIN

The most likely origin of the plasma proteins in fetal csf and brain is from the plasma itself either by crossing the cerebral vessels or the choroid plexus in the immature brain. Extensive

194 N. R. SAUNDERS

Table 1. Proteins in Fetal csf (mg/100 ml). Concentrations of Individual Proteins at the Peak Total Protein Concentration in Different Species.

Sheep (31 days)

Albumin 121 a-Fetoprotein 532 Fetuin 229 aI-Antitrypsin 427 Transferrin 51 Total protein (Lowry) 1143

mg/loomg 160 30

1200

800

400

L IV L

40

Pig Rat Human (31 days) (22 days) (19 weeks)

85 143 406 76 72 113 + +

++++ ++ ++ 195 62 6 961 317 375

60days

IV L IV

Fig. 2. Total· protein concentration in lateral ventricular (L) and IVth ventricular (IV) csf of fetal sheep at 30, 40 and 60 days gestation.

studies of the ultrastructure of cerebral endothelial cells and of choroid plexus epithelial cells in the developing brain have been carried out[11,16]. These showed that, contrary to the view usually expressed in textbooks of·Physiology, the blood-brain and blood-csf. barriers (Le. the tight junctions between cerebral endothelial cells and between the choroid plexus epithelial cells) are well formed at the earliest stages investigated, e.g. 20 days gestation in the sheep embryo. Studies of the transfer of plasma proteins from blood to csf and brain and from csf to brain have also been carried out. These studies produced a number of unexpected observations. At 60 days gestation marker plasma proteins (either human plasma proteins esti­mated by immunoassay or 125I-sheep albumin) injected intravenously, reached the natural steady state level in cisternal csf for several proteins (AFP, albumin, transferrin) by 3 to 6 hours after injec­tion[6]. However, at earlier stages of development (30-40 days gestation) in similar experiments marker proteins reached only about one third of the natural steady state (csf/plasma) concentration in cisternal csf and scarcely penetrated at all into lateral ventricular csf (Table 2). In all these experiments the physiological state of

PLASMA PROTEINS 195

the fetus (arterial blood pressure, heart rate and blood gases) was monitored carefully. The fetuses were maintained without much change in their state for up to 6 hours. In these same experiments no penetration of marker protein into fetal brain could be detected (not even on autoradiographic examination of brain sections from fetuses injected with 125I-Iabelled proteins). Furthermore, when artificial csf containing either 125 I - a lbumin or horseradish peroxidase was perfused through the ventricular system of 60 day fetal sheep for 4 to 5 hours no penetration of protein into the brain could be de­tected[17]. However, at 125 days gestation horseradish peroxidase penetrated several mm into the brain after similar periods of ven­triculocisternal perfusion as previously shown by Brightman and Reese[*J in adult mice. This lack of penetration of protein from csf into brain in 60 days fetal sheep has been shown to be due to the presence of an unusual type of intercellular junction that restricts the intercellular space in the neuroependyma to about 2 nm[17J.

The lack of penetration of proteins into brain either from blood or from csf suggested that the plasma proteins in brain might orig­inate from local synthesis. Also since marker proteins failed to reach the natural steady state level in 30 to 40 day fetuses it seemed possible that some of the plasma proteins in fetal csf might originate from secretion by either choroid plexus or brain following synthesis in situ.

The possibility of synthesis of plasma proteins by newborn rat brain was suggested by the experiments of Ali et al., (1983). This has been further investigated in fetal brain and choroid plexus of rats, sheep and pigs by incubation of tissues (brain, choroid plexus and liver) in vitro in Krebs solution or MEM containing 3H-Ieucine. After incubation at 37°C for 1 to 3 h the tissue was extracted over­night at 4°C in 2% Triton X200 and Trasylol[18]. Labelled plasma

Table 2. Penetration of 125I-labelled Protein from Plasma into csf in Forebrain (Lateral Ventricle) and Hindbrain (IVth Ventricle) at 40 Days Gestation in Fetal Sheep.

Age (days)

40 (38-43)

Protein

125I - a lbumin

Rat 1.. os C/100 sec/g csf are C/100 sec/g plasma

csf/plasma x 100

Forebrain Hindbrain

0.35 ± 0.03 14.2 ± 1.1

Paired samples obtained 3-6 h after i.v. injection of labelled protein[19].

196 N. R. SAUNDERS

proteins in both the incubation medium and tissue extract were sep­arated by immunoprecipitation in two dimensional electrophoresis. It is clear from these studies that in all 3 species 3H-Ieucine was incorporated into several plasma proteins (AFP, albumin, transferrin and fetuin) by both choroid plexus and brain during a restricted period of fetal brain development. Table 3 shows results from exper­iments in sheep fetuses of 30, 40 and 60 days gestation. The highest levels of incorporation of 3H-Ieucine into albumin, fetuin and AFP in forebrain occurred at 30 days, with a marked decline subsequently. The highest level of incorporation into transferrin in brain was at 40 days although the scatter of results was very large. In the IVth ventricular choroid plexus, the level of incorporation into albumin, fetuin and AFP did not change much between 30 and 40 days but in­creased in the case of transferrin. Subsequently incorporation into albumin and AFP declined considerably but was little changed for fetuin and transferrin. Liver is shown for comparison and shows a generally higher level of incorporation into each protein than in either brain or choroid plexus.

Cycloheximide (0.1 roM) added to the incubation medium blocked incorporation of 3H-Ieucine into plasma proteins. The high level of incorporation of 3H-Ieucine into several plasma proteins in brain at 30 days correlates well with the peak concentrations of these pro­teins in csf at this age[19]; also the apparent increased incorpor­ation into transferrin in brain between 30 and 40 days correlates with the increase in csf transferrin concentration which occurs between these ages[19]. In contrast, in plasma although the trans­ferrin concentration increases between 30 and 40 days, there is no significant change in AFP concentration. This reinforces the con­clusion that at 30 and 40 days gestation the csf concentrations of plasma proteins are probably more dependent upon the level of syn­thesis and release of these proteins by brain and choroid plexus than on transfer from plasma.

FETUIN

It has been of particular interest to study fetuin in fetal brain and csf, partly because in the pig and sheep fetus it appears to have an even more widespread distribution and earlier appearance than AFP. Also bovine fetuin has been shown to have a number of interesting effects in tissue culture including effects on cells from species not known to possess fetuin[20.21]. Until recently fetuin was thought to be confined to species of the order Artiodactyla (cattle, sheep, goats, pigs). Recently its species distribution has been investigated using 4 different anti-fetuin antisera (two anti­cattle, anti-sheep and anti-pig) and the peroxidase-antiperoxidase method applied to plasma samples dried on to agarose gels[22]. This study has shown that fetuin has a much wider species distribution than previously thought. It has been identified in at least 5 Orders

Tab

le 3

. In

corp

ora

tio

n o

f 3

H-l

euci

ne

into

sh

eep

feta

l b

rain

tis

sues

in v

itro

. T

issu

e sa

mpl

es

wer

e in

cub

ated

in

Kre

bs

solu

tio

n c

on

tain

ing

~H-leuc~ne at

37°C

fo

r 1

h.

Tis

sues

wer

e ex

tracte

d i

n T

rito

n X

I00

and

Tra

sylo

12

. In

div

idu

al p

lasm

a p

rote

ins

wer

e se

par

ated

by

imm

un

op

reci

pit

atio

n w

ith

sp

ecif

ic a

nti

sera

in

cro

ssed

im

mu

no

elec

tro

ph

ore

tic

pla

tes.

R

esu

lts

are

ex

pre

ssed

as

mea

n CP

M x

1

0-2

/mg

wet

wei

gh

t ti

ssu

e ±

S

.E.M

. A

ge

in d

ays

from

co

nce

pti

on

(t

erm

in

she

ep is

15

0 d

ays)

.

Feta

l A

ge

N

Alb

umin

F

etu

in

Fo

reb

rain

30d

7 1

0.0

8 ±

2

.13

8

.67

±

2.6

0

40d

8 3

.81

±

2.4

1

3.1

2

± 0

.59

60

d 8

0.5

8

± 0

.42

1

.98

±

0.8

5

Liv

er

30d

10

26

.04

±

6.3

9

33

'4 ±

7

.04

40

d 6

50

.61

±

11

.50

2

6.4

8

± 6

.16

60

d 8

27.1

7 ±

5.3

7

8.2

2 ±

1

.65

IV V

en

tric

ula

r C

horo

id P

lex

us

30d

2-3

1

6.6

3 ±

5

.29

3

.76

±

2.7

0

40d

3 1

7.8

9 ±

6

.08

6

.97

±

3.4

0

60d

3 (2

.16

±

2.1

6)

5.6

2 ±

4

.09

a-F

eto

pro

tein

6.3

5

± 1

.44

8

.02

±

3.7

1

1.0

9 ±

0

.29

60

.69

±

8.9

4

56

.54

±

16

.50

15

.45

± 3

.73

18

.30

±

3.1

4

17

.23

±

7.0

1

(2.7

8 ±

2

.78

)

Tra

nsf

err

in

5.9

9 ±

1.

5S

12

.49

±

8.0

2

2.4

4 ±

0

.94

22.2

2 ±

4.1

2

18.4

7 ±

3.4

8

6.6

2

± 0

.98

8.7

9

15

.96

±

3.9

8

14

.24

±

11

.26

'"d ~ en ~ '"d ~

\;3

H Z

en

1.0 ......

198 N. R. SAUNDERS

of Eutherian Mammals (Artiodactyla, Primates, Rodentia, Carnivora and Perissodactyla) and in Metatherian (marsupial) Mammals. The ident­ification of fetuin in human fetal plasma has been confirmed by using crossed immunoelectrophoresis.

FUNCTIONAL SIGNIFICANCE OF PLASMA PROTEINS IN FETAL CSF AND BRAIN

It is possible that the high concentration of plasma protein in fetal csf is principally a consequence of protein loss from cells disintegrating during the normal growth and development of the brain. It is not yet possible to estimate the relative contribution of brain and choroid plexus to the csf concentration of individual proteins and it may well change with age. The actual level is probably a balance between secretion (or loss accompanying cell breakdown) from brain and from choroid plexus together with some penetration from blood to csf especially later in gestation. Onset of flow of csf through the ventricular system and return of csf to the circulating blood via the subarachnoid space probably contributes to the marked decline in protein concentration in fetal csf, but this has not been well investigated so far. The different distribution of some of the plasma proteins in fetal brain that has been described above suggests that these proteins are involved in particular stages of the develop­ment of at least some neurons in brain development. It is not clear whether the functional importance of these plasma proteins relates to their known binding properties in adult animals or to some quite new properties. The observation that proteins such as AFP, fetuin and albumin have a predominantly intracellular distribution in neuronal cell bodies whereas transferrin may be largely extracellular and associated with nerve processes is intriguing when compared with recent observations on the effects of changing protein composition on neuronal growth in vitro. Thus extracellular transferrin appears to be an essential component of defined media for the in vitro culture of neurons[12]. In contrast neurons appear to grow well in low concentrations (0.5%) of serum proteins but are inhibited by the higher concentrations (5% or more) usually used in culture media[23].

Acknowledgement

I should like to thank the Wellcome Trust and the Agricultural Research Council, UK for their support of my research over many years. I should also like to thank my colleagues at University College London and in the University of Copenhagen for all their patient hard work.

PLASMA PROTEINS 199

REFERENCES

1. J. 2. C. 3. K. 4. M. 5. K. 6. K. 7. K. 8. K. 9. K.

10. M. 11. K. 12. J. 13. M. 14. M. 15. M. 16. K. 17. N. 18. E. 19. M. 20. T. 21. S. 22. K.

23. H.

24. M. 25. K.

26. R.

27. M.

* M.

Trojan and J. Uriel. C.R.Acad.Sci •• 289:1157-1160 (1979). D. Toran-Allerand. Nature. 286:733-735 (1980). M. Dziegielewska. et al •• J.Physiol •• 318:239-250 (1981). E. Cavanagh. et al •• Dev.Neurosci •• 5:492-502 (1982). M~llg!rd. et al •• Neurosci.Lett •• 14:85-90 (1979). M. Dziegielewska. et al •• J.Physiol •• 300:441-445 (1980a). M. Dziegielewska. et al •• J.Physiol •• 300:457-465 (1980b). M. Dziegielewska. et al •• Dev.Biol •• 83:193-200 (1981). M. Dziegielewska and N. R. Saunders. Comp.Biochem.Physiol •• 66B:307-311 (1981). E. Cavanagh. et al •• In preparation (1983). M~llg!rd and N. R. Saunders. J.Neurocytol •• 4:453-468 (1975). E. Bottenstein. et al •• Exper.Cell Res •• 125:183-190 (1980). Jacobsen. et al •• Develop.Brain Res •• 3:239-250 (1982a). Jacobsen. et al •• Develop.Brain Res •• 3:251-262 (1982b). Jacobsen. et al •• Develop.Brain Res •• In press. (1983). M~llg!rd. et al •• Nature. 264:5583. 293-294 (1976). R. Saunders and K. M~ll!rd. Trends.Neurosci •• 4:56-60 (1981). Bock. J.Neurochem •• 19:1731-1736 (1972). E. Cavanagh. et al •• Submitted to Dev.Brain Res •• (1983). T. Puck. et al •• Proc.Nat.Acad.Sci •• 59:192-199 (1968). Yachnin. J.Exp.Med •• 141:242-256 (1975). M. Dziegielewska. et al •• Comp.Biochem.Physiol •• 76A:2.241-245 (1983). G. Coon and C. N. Sinback. in "Growth of Cells in Hormonally Defined Media". D.A. Sirbasku et al.. ed •• Cold Spring Harbor. NY. pp 1009-1016 (1982). Ali. et al •• Dev.Brain Res •• 6:47-55 (1983). M. Dziegielewska and N. R. Saunders. Comp.Biochem.Physiol.. 73A:2. 327-329 (1982). G. Ham. in "Growth of Cells in Hormonally Defined Media". D.A.Sirbasku et al •• ed •• Cold Spring Harbour. NY. pp 39-60 (1982) • E. Molliver. et al •• Brain Res •• 50:403-407 (1973). w. Brightman and T. S. Reese. J.Cell BioI •• 40:648-677 (1969) •

STAGE SPECIFIC ANTIGENS ON OLIGODENDROCYTE CELL SURFACES

I. Sommer and M. Schachner

Institute of Neurobiology University of Heidelberg, FRG

The goal of our research is to investigate the differentiation of the myelin forming cells in the central nervous system, the oli­godendroglia. Differentiation of a given cell type often depends on interactions with other cell types, a phenomenon that has been shown to be relevant also for oligodendrocytes in the case of Wallerian Degeneration. As interactions among cells are most likely mediated by the cell surface, we have concentrated on the investigation of cell surface components.

We have used the hybridoma technology to define antigenic markers that are cell type, cell surface and stage specific. Mono­clonal antibodies that recognize such marker molecules are ideally suited for identification and separation of living cells at a given differentiative stage. Biochemical analysis of the respective anti­gens gives insight into the cell's molecular requirements to exert its specific functions. An ultimate goal is to define the function of these antigens with the help of in vitro myelinating systems.

This article will concentrate on eleven monoclonal antibodies designated 01-011 that react with oligodendrocyte cell surfaces. The corresponding antigens have been characterized with respect to their developmental appearance in vivo in normal mice and in the hypomye­linating mutant jimpy. The distribution of the 0 antigens in vitro on oligodendrocyte subclasses and the biochemical properties of the antigens have been investigated.

The first hybridomas secreting 0 antibodies were obtained from fusions of myelomas with splenocytes of mice that had been immunized with homogenates of bovine corpus callosum. Later all monoclonal antibodies reacting with oligodendrocyte cell surfaces which arose

201

202 I. SOMMER AND M. SCHACHNER

from different immunizations in Dr. Schachner's laboratory were included in a comparative analysis.

Four antibodies designated 01 to 04 have been studied most extensively[1-4]. Cell surface specificity of the antibodies was established by indirect immunofluorescence using live brain cell cultures in which intracellular components are not accessible to antibodies. Cell type specificity was assessed in early postnatal cerebellar cultures which contain all of the major neural cell classes. In double labeling experiments using toxins and rabbit antisera against established cell type specific markers (Tetanus Toxin and antibodies to Tetanus Toxin to identify neurons, glial fibrillary acidic protein for astrocytes, fibronectin for fibroblasts and galactocerebroside to identify oligodendrocytes) it was shown that the 0 antibodies reacted with oligodendrocytes but not with neurons, astrocytes or fibroblasts. Antigens 01 and 02 were found only on galactocerebroside posi~ive+cells, whereas 03 and 04 stained the same cells as 01 and 02 (04 /01 ) and in addition a class of cel*s t~at could not be identified with the markers available (04 /01 ).

In cultures from embryonic brain 04 and 03 positive cell~ weEe detectable before 01 and 02 positive c$,ls+suggesting that 04 /01 cells were less differentiated+tha~ 04 01 cells[2]. In orde+ to+ confirm the hypothesis that 04 /01 cells are precursors to 04 /01 cells ~he tormer were labeled in vitro with 04 TRITC antibody after all 04 /01 cells had been eliminated by complement dependent immuno­cytolysis. After two days in vitro the majority of these 04 TRITC labeled cells were sho~ to_express 01 antigen on their surface in~ica~ing that most 04 /01 cells had indeed differentiated into 04 /01 cells[4]. An ultrastructural analysis confirmed this identi­fication [1] •

Unfortunately the specificity of the antibodies 01 to 04 to oligodendrocytes was lost when intracellular structures were access­ible to the antibodies. In fixed cultures or in brain sections, P1 to 04 stained filamentous structures in astrocytes and neuronal elements.

During our search for additional stage specific antigens we detected 6 new antibodies reacting with oligodendrocyte cell surfaces which we called 05 to 011 (5) and the list is still expanding. These antibodies arose from the same immunfzations as 01 to 04 except for 07 and 011 which originated from mice injected with chicken synap­tosomes and rats immunized with mouse cerebellar membranes solubil­ized in DOC and prepurified on glycerol gradients respectively. All of the antigens proved to be oligodendrocyte specific according to the criterion that they were exclusively found on the surface of cells that belonged to the 04 positive cell population. In addition to their specificity for oligodendrocyte cell surfaces in vitro,

STAGE SPECIFIC ANTIGENS 203

antigen 05 to 011 were confined to white matter and adjacent areas in situ.

The developmental expression of the 0 antigens in tissue sec­tions of brain and spinal cord has been investigated only with anti­bodies 05 to 011 because of the difficulty to distinguish astrocytic staining in white matter from oligodendrocyte specific staining in the case of 01 to 04. In sections of spinal cord antigens OS, 06 and 07 are detectable by indirect immunoperoxidase methods at birth followed by 08 and 09 at day 2, 010 at day 4 and 011 at day 7. In the spinal cord of the mutant jimpy (which is defective in CNS­myelination due to the inability of oligodendrocytes to differentiate properly) the appearance of the 0 antigens in development is delayed. At the earliest age tested (day 3) antigens 05 and 06 are detectable in much lower amounts as compared to the normal male control litter­mates. Antigen 07 was detected at day 5, followed by 08 and 09 at day 13. Antigen 011 was detected at minimal amounts at day 21; 010 was never detected in spinal cord or any other brain region at the ages tested (up to day 32). In other areas of normal or mutant brain like in the cerebellum, the appearance of the antigens follows the same developmental pattern.

The 0 antigens are not restricted to the CNS but are also found in peripheral nerves with one exception: 010.

In monolayer cultures of early postnatal mouse cerebellum the population of cells stained by different 0 antibodies was always included in the 04 positive cell population however the degree of overlap with 04 positive cells varied between 98% to 5% depending upon the antibody used. The highest overlap with 04 was found for 03,05 and 06, decreasing for 02,01 and 07 being even lower for 08>09> 010>011. Antigen OIl was found on about 5% of all 04 positive cells in cultures of seven day old mouse cerebellum maintained in vitro for three days.

The developmental expression of the ° antigens at various times in culture and the developmental pattern of antigen expression in tissue sections (mentioned above) suggest that one ° antigen is acquired after another on the oligodendrocyte cell surface in the following order: 04,03,05,06 - 02,01,07, - 08 - 09 - 010 - OIl. Detection of later appearing antigens on a given cell would therefore indicate a further advanced stage of differentiation.

Like the in vivo antigen, 010 could not be demonstrated in cultures of jimpy mutants obtained from different brain regions although antigen OIl which appears after 010 during normal develop­ment was present on few cells. The idea that loss of 010 antigen may be related to the inability of jimpy oligodendrocytes to make myelin is at this point mere speculation. However, this idea is consistent with the absence of 010 in normal PNS and the fact that in jimpy mice only the central nervous system is affected.

204 I. SOMMER AND M. SCHACHNER

For the biochemical characterization of antigens 01 to 09 we chose a thin layer chromatography immune-overlay assay similar to a western blot because all of the antigens except for 010 and maybe 011 are heat stable and soluble in chloroform/methanol. Using commer­cially available glyco10pid standards we have been able to identify antigens 03.04.05.06 as su1fatide and antigens 01.02.07 as ga1ac­tocerebroside. Both of these components are known to be highly enriched in myelin and oligodendrocyte membranes. Antigens 08 and 09 are not identified yet. they are not identical with ga1actocerebro­side or su1fatide nor with each other according to their different mobilities in a 70/30/5 (chloroform/methanol/water) solvent system. Antigen 010 could not be detected in this kind of assay however we know that it is trypsin-sensitive and heat-labile indicating that it is a protein.

Analysis of the effects of antibody application to mye1inating or myelinated cultures will give us some clues as to the function of the antigens in oligodendrocyte-axon interaction and in the inter­action of oligodendrocyte membranes with each other.

Recent experiments have shown that we have to be extremely careful with the definition of markers. The classical neuronal marker Tetanus Toxin has been found on astrocytes and possibly also on precursor cells that give rise to oligodendrocytes and astro­cytes[6]. In addition vimentin. thought to be specific for astro­cytes in the nervous system. has been found on 04 positive cells (Meier et a1 •• unp.). In the embryonic chick peripheral nervous system 04 positive cells (most likely Schwann cells) have been shown to have a high affinity uptake for NGF and noradrenalin. both proper­ties so far thought to be neuron specific[7]. These findings emphasize that in each experimental system the "markers" have to be recharacterized in order to remain the valuable tool they can be if used properly.

REFERENCES

1. G. Berg and M. Schachner. Immune e1ectronmicroscopic identi­fication of 0 antigen bearing oligodendrocytes. Cell and Tissue Res •• 219:313-325 (1981).

2. M. Schachner and S. U. Kim and R. Zehn1e. Developmental express­ion in central and peripheral nervous system of oligodendro­cyte cell surface antigens (0 antigens) recognized by mono­clonal antibodies. Dev.Bio1. 83:328-338 (1981).

3. I. Sommer and M. Schachner. Monoclonal antibodies to oligodendro­cyte cell surfaces: an immunocyto10gica1 study in the central nervous system. Dev.Bio1. 83:311-327 (1981).

4. I. Sommer and M. Schachner. Cells that are 04 antigen-positive and 01 antigen-negative differentiate into 01 antigen-positive oligodendrocytes. Neurosc.Lett. 29:183-188 (1982).

STAGE SPECIFIC ANTIGENS 205

5. I. Sommer, C. Lagenaur and M. Schachner, Stage specific antigens 05 to 011 on oligodendrocyte cell surfaces detected by mono­clonal antibodies. Soc.Neurosci.Abstr. 8:246 (1982).

6. M. C. Raff, R. H. Miller and M. Noble, A glial progenitor cell that develops in vitro into an astrocyte or an oligodendrocyte depending on the culture medium, Nature, in press (1983).

7. H. Rohrer and I. Sommer, Simultaneous expression of glial and neuronal properties by chick ciliary ganglion cells during development, J. Neurosci., in press (1983).

CELL-TYPE-SPECIFIC MOLECULES: IDENTIFICATION OF GLYCOLIPID BINDING SITES FOR SOYBEAN AGGLUTININ AND DIFFERENCES IN THE SURFACE GLYCO­LIPIDS OF CULTURED ADRENERGIC AND CHOLINERGIC SYMPATHETIC NEURONS

Anne D. Zurn

Department of Biochemistry University of Geneva, Sciences II 30, q. Ernest Ansermet CH-1211 Geneva 4, Switzerland

The formation of precise neural connections during development of the brain is a prerequisite for the normal function of the nervous system. Establishment of these specific synapses is regulated both by a genetic program intrinsic to either the neurons and/or their target cells and by epigenetic (environmental) factors.

Epigenetic factors can consist of soluble substances (trophic factors) released by either neural or non-neural tissues and directed to neurons[l]. Nerve growth factor is the best characterized such trophic molecule[2]. Other factors can be present on the surface of cells and allow axons to reach their target cells (contact guidance). Finally, unique components on the surface of neurons and their target cells (cell-type-specific markers) can be involved in specific cell recognition and allow the establishment of precise neural connections[3,4].

In the last twenty years numerous reports from studies both in vivo and in vitro have appeared which support Sperry's chemoaffinity hypothesis that macromolecules on the surface of cells mediate speci­fic cell-cell recognition and association (for reviews see 5,6,7). But although many experiments bring evidence supporting the chemo­affinity hypothesis, very little is still known about the chemical nature of the surface molecules involved in cell-cell recognition. Edelman and his colleagues have isolated a neural cell adhesion molecule (N-CAM) from retinal tissues in culture which is responsible for the aggregation of dissociated neural retinal cells[lO]. N-CAM seems to be involved in adhesive interactions between surfaces of neuronal cells in various species and in neuron-muscle interactions in vitro[11,12,13,14]. Lander and colleagues have purified a heparan

207

208 A. D. ZURN

sulfate proteoglycan from corneal endothelial cells which promotes neurite outgrowth of autonomic neurons in culture. This proteoglycan may function by increasing adhesion between the neuronal plasmalemma and the substratum[15].

In 1970 Roseman had suggested that cell recognition and adhesion might be mediated via the carbohydrates on cell surfaces[16]. Sev­eral reports have appeared since then which indicate that surface carbohydrates (glycoproteins, glycolipids and glycosaminoglycans) might be involved in cell adhesion, neuronal recognition and synapto­genesis: the adhesion of ventral retina to dorsal tecta seems to depend on a galactosyltransferase located on ventral retina and on terminal S-N-galactosamine residues on dorsal tecta[8]; Sanes and Cheney[17] found a synapse-specific carbohydrate in skeletal muscle by using Dolichos biflorus lectin. This carbohydrate, which is present in the basal lamina of the synaptic cleft, might playa role in cell recognition during formation of the neuro-muscular junction. Carbohydrate moieties of gangliosides (acidic glycosphingolipids containing sialic acid present in larger amounts in the brain than in most other tissues[18]) might also playa role in synaptogenesis since they seem to be enriched in synapses (as shown by increased binding of choleratoxin and tetanus toxin in the synaptic cleft[19,20]). Neutral glycosphingolipids (GSL), another class of glycolipids having no sialic acid, are also major components of the nervous system. Ganglioside composition is different in various regions of the brain, and glycolipid patterns change, becoming more complex during brain development[22]. It is not clear, however, whether these differences are due to changes in neuronal or glial GSL composition or both. Furthermore, nothing is known yet about the function of these glycolipids in the nervous system[21).

Cultures of adrenergic and cholinergic sympathetic neurons provide a system where it is possible to correlate GSL differences with neuronal function and development. Neurons from the newborn rat superior cervical ganglion can be maintained in culture in the vir­tual absence of nonneuronal cells[23]. When these neurons are grown in the absence of nonneuronal cells they are adrenergic, but in the presence of certain nonneuronal cells, or medium conditioned by those cells, they become cholinergic[24,25]. Thus the phenotype of these neurons, including the type of transmitter they synthesize and the type of synapses they form, can be experimentally controlled. The adrenergic and cholinergic sympathetic neurons also differ in the proteins they spontaneously secrete into the medium[26] and in cer­tain of their surface glycoproteins[27]. Differences have also been observed in the binding of bacterial toxins and plant lectins using morphological techniques[28]. Of particular interest was the finding that axonal membranes of the adrenergic neurons bind soybean agglutin­in(SBA) at a fivefold higher density than those of the cholinergic neurons. Ricinus communis agglutinin-60 (RCA), however, binds equally well to both types of neurons. SBA and RCA have the same

CELL-TYPE-SPECIFIC MOLECULES 209

monosaccharide specificity although they have different affinities for galactose (Gal) and N-acetyl-galactosamine (GalNac)[29].

To identify possible glycoproteins recognized by these two lectins, sodium dodecylsulfate extracts of neurons were electro­phoresed on polyacrylamide gradient gels[30]. The gel was then fixed and incubated in the presence of 125 I _SBA or 125_RCA[3l]. The auto­radiographs of these gels reveal that RCA binds to several glycopro­teins whereas SBA does not. However, both lectins specifically label material migrating ahead of the dye front. Since this is the region where glycolipids migrate and since several glycolipids contain terminal Gal and GalNac residues, the possibility that the lectins are binding to glycolipids was further tested. Cultured neurons were extracted in chloroform-methanol[32]. The lower phase containing neutral glycolipids, lipids and phospholipids, and the upper phase containing the gangliosides, were separated by thin layer chroma­tography (TLC) and incubated in the presence of 125I -labeled lectins[33]. The autoradiographs show that SBA and RCA bind to two neutral glycolipids extracted from these neurons as well as to sev­eral glycolipid standards. One of these glycolipids co-migrates with globoside (GL-4). The other one migrates slightly behind asialo­GMI(GAI) and is termed GL-X[30].

The polyacrylamide gel and the TLC method assess binding of lectins to solubilized glycolipids. In the intact plasma membrane, however, such glycolipids might have their carbohydrate chains masked by bulky carbohydrate chains of glycoproteins or other glycolipids and thus may not be accessible to the lectins. Therefore a different approach was employed to determine whether glycolipids bind SBA or RCA on living sympathetic neurons in culture. Neurons were surface labeled by means of galactose oxidase (GAO) treatment in the presence or absence of SBA or RCA, followed by reduction with [3H]borohy­dride[30,34]. If one of these lectins binds to an accessible glyco­lipid on the neuronal membrane, it may prevent or decrease subsequent labeling of the glycolipid by GAO and (3H)borohydride. TLC analysis of the lower phase material after surface labeling in the presence and the absence of SBA reveals that the two glycolipids labeled with GAO and (3H)borohydride have the same chromatographic behavior as the glycolipids which bind 125 I _SBA and 125I-RCA. Their labeling is decreased by about 50% in the presence of 100 ~g/ml SBA, but not with RCA. Increasing concentrations of SBA produce a maximal inhibition of labeling of 50-60% for both glycolipids and this inhibition is obtained with 4-5 x 10-7M SBA. RCA, however, increases the labeling of the two glycolipids at low concentrations (10-7_10-bM). This increase in labeling could be due to binding of RCA to glycoproteins and redistribution of glycoconjugates on the surface, exposing them to GAO. The 20-30% inhibition obtained with higher concentrations of RCA probably does not reflect specific binding to the glycolipids because high concentrations were necessary (lO-OM) and no plateau value was reached. In other experiments. neither SBA nor RCA in-

210 A. D. ZURN

hibited the labeling of any of the gangliosides labeled by the GAO­[3H]borohydride method. Thus SBA and RCA bind to different glycocon­jugates on the intact neuronal plasma membrane. SBA binds to two neutral glycolipids, one co-migrating with GL-4 and the other one with an uncharacterized glycolipid, GL-X. It shows only very faint labeling of glycoproteins extracted from these neurons. RCA, on the other hand, binds to several neutral glycolipids and gangliosides after extraction, but no evidence was obtained for binding to glycolipids in the intact membranes. Since RCA shows specific binding to a number of extracted glycoproteins, some of these are most probably the neuronal RCA binding sites.

Since adrenergic axons have a fivefold higher density of SBA binding sites than cholinergic axons and since SBA binds to two neutral glycolipids on these neurons, the question arose as to whether cholinergic neurons in fact contain a smaller amount of these glycolipids on their surfaces than adrenergic neurons. To test this possibility, neurons grown under adrenergic or cholinergic conditions for three weeks were surface-labeled with the GAO-(3H)-borohydride method and their neutral glycolipid content analyzed by TLC[30]. Both GL-4 and GL-X are labeled less on cholinergic than on adrenergic neurons. The amount of label was 60-70% and 40-60% lower, respect­ively. The major gangliosides of the adrenergic and cholinergic neurons were also analyzed either after surface labeling with GAO and (~H)-borohydride or after metabolic labeling with N-acetyl-D-(3H)­mannosamine, a specific precursor of sialic acid. Both labeling procedures reveal quantitative differences in the ganglioside pattern of the two types of neurons.

Thus quantitative differences in both the neutral glycolipid and the ganglioside composition of sympathetic neurons grown under adrenergic or cholinergic conditions have been detected. This supports the hypothesis that neurons might have unique components on their surfaces responsible for specific cell recognition.

REFERENCES

l. S. 2. L. 3. R. 4. M. 5. A.

6. W. 7. D. 8. R. 9. D.

10. J.

S. Varon and R. P. Bunge, Ann.Rev.Neurosci., 1:327 (1978). A. Greene and E. M. Shooter, Ann.Rev.Neurosci., 3:353 (1980). W. Sperry, Proc.Natl.Acad.Sci., 50:703 (1963). Schachner, J.Neurochem., 39:1 (1982). A. Moscona, in "Neuronal Recognition", S. H. Barondes, ed., Chapman and Hall, p.205 (1976). Frazier, and L. Glaser, Ann. Rev. Biochem., 48:491 (1979). I. Gottlieb and L. Glaser, Ann.Rev.Neurosci., 3:303 (1980). B. Marchase, J.Cell.Biol., 75:237 (1977). Purves, W. Thompson and J. W. Yip, J.Physiol., 313:49 (1981). P.Thiery, R. Brackenbury, U. Rutishauser, G. Edelman, J.Biol. Chem., 252:6841 (1977).

CELL-TYPE-SPECIFIC MOLECULES 211

11. S. Hoffman, B. Sorkin, P. White, R. Brackenbury, R. Mailhammer, U. Rutishauser, B. Cunningham, and G. M. Edelman, J.Biol.Chem., 257:7720 (1982).

12. J. B. Rothbard, R. Brackenbury, B. A. Cunningham, and G. M. Edelman, J.Biol.Chem., 257:11064 (1982).

13. C. M. Chuong, D. McClain, P. Streit, and G. M. Edelman, Proc.Natl.Acad.Sci., 79:4234 (1982).

14. M. Grumet, U. Rutishauser, and G. M. Edelman, Nature, 295:693 (1982) •

15. A. D. Lander, D. K. Fujii, D. Gospodarowicz and L. F. Reichardt, J.Cell.Biol., 94:574 (1982).

16. S. Roseman, Chem.Phys.Lipids, 5:270 (1970). 17. J. R. Sanes, and J. M. Cheney Nature, 300:646 (1982). 18. R. W. Ledeen, in "Complex Carbohydrates of Nervous Tissue." R.

U. Margolis andlR. K. Margolis, eds., Plenum, New York pp.1-23 (1979).

19. H. A. Hansson, J. Holmgren and L. Svennerholm, Proc.Nat.Acad.Sci.USA, 74:3782-3786 (1977).

20. R. B. Rogers, and S. H. Snyder, J.Biol.Chem., 256:2402-2407 (1981) •

21. S. Hakomori, Ann.Rev.Biochem., 50:733 (1981). 22. L. M. Irwin, and C. C. Irwin, Dev.Neurosci., 2:129 (1979). 23. R. E. Mains, and P. H. Patterson, J.Cell.Biol., 59:329 (1973). 24. P. H. Patterson, and L. L. Y. C. Chun, Dev.Biol., 60:473 (1977). 25. M. J. Weber, J.Biol.Chem., 256:3447 (1981). 26. K. J. Sweadner, J.Biol.Chem., 256:4063 (1981). 27. s. J. Braun, K. J. Sweadner and P. H. Patterson, J.Neurosci.,

1:1397 (1981). 28. M. Schwab, and S. L. Landis, Dev.Biol., 84:67 (1981). 29. H. Debray, D. Decout, G. Strecker, G. Spik and Montreuil,

Eur.J.Biochem., 117:41-44 (1981). 30. A. D. Zurn, Dev.Biol., 94:483 (1982). 31. K. Burridge, Proc.Natl.Acad.Sci., 73:4457 (1976). 32. G. Dawson, R. McLawhon, and R. J. Miller, Proc.Natl.Acad.Sci.,

76:605 (1979). 33. J. L. Magnani, D. F. Smith and V. Ginsburg, Anal.Biochem.,

109:399 (1980). 34. G. Gahmberg, and S. Hakomori, J.Biol.Chem., 250:2447 (1975).

4

ELECTROPHYSIOLOGICAL APPROACH TO NEUROGENESIS

MEMBRANE EXCITABILITY IN CILIARY GANGLION NEURONS

AND IN MESENCEPHALIC NEURAL CREST CELLS

C. R. Bader. D. Bertrand. E. Dupin and A. C. Kato*

Department of Physiology and *Department of Pharmacology Centre Medical Universitaire. 1211 Geneve 4 Switzerland

INTRODUCTION

One intriguing question in neurobiology concerns the origin of neuronal cells. What causes neuronal differentiation and can this process be delayed or modified? A prerequisite to answer these questions is to be able to determine when differentiation begins in a given developing neuronal system. One characteristic of well devel­oped neurons is the presence of several specialized membrane struc­tures called channels. Channels are generally selectively permeable to certain ions and this permeability can be modulated by voltage. neurotransmitters and sometimes by both. Our purpose was to follow a developing system using well-identified membrane channels as markers for the development of neurons. A model that seemed suitable for this type of study is the monolayer culture of ciliary ganglion and mesencephalic neural crest cells of the avian embryo. The mesen­cephalic neural crest is a transient structure in the neural primor­dium of vertebrate embryos that forms (apart from a series of other structures) the cellular elements of the ciliary ganglion (Le Douarin, 1982).

MATERIALS AND METHODS

The methods used for the culture of chick ciliary ganglion neurons (Kato and Rey, 1982) and quail mesencephalic neural crest (Dupin. 1982; Ziller et al., 1981 and 1983) have been previously described. The techniques for superfusion and electrophysiological recording from neurons in culture have also been described (Bader et al •• 1982). In small neurons such as those of the ciliary ganglion

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218 C. R. BADER ET AL.

from 4 day-old chick embryos or from freshly plated quail neural crest, the whole cell recording technique was used (Hamill et al., 1981).

RESULTS AND DISCUSSION

Mature ciliary ganglion neurons in culture

The strategy used in the present study was to characterize first the membrane properties of a 'mature neuronal system' and then to proceed with a study of earlier stages of its development. In ciliary ganglion neurons from 11 day-old chick embryos, four membrane channels could be identified in addition to the channel modulated by acetylcholine (Bader et al., 1982). The identification of these channels was done by using a combination of pharmacological blocking agents. The result of a typical experiment is illustrated in Figure 1. Injection of a depolarizing pulse of current in a neuron trig­gered an overshooting action potential (Figure lA, control). When an agent known to block voltage-dependent potassium channels (tetra­ethylammonium, TEA) was added to the medium, the response to the pulse of current changed to a longer lasting action potential com­posed of a brief peak followed by a less depolarized but longer lasting plateau (Figure lA, TEA). The response in the presence of TEA was also characterized by a long lasting hyperpolarization at the end of the action potential. This suggested that the block of voltage-dependent potassium channels by TEA had revealed the presence of other channels.

The presence of other channels was confirmed by substituting cobalt for calcium in the superfusion medium which also contained TEA (Figure lB; TEA, Co); in the presence of cobalt the action potential had a shorter duration, although longer than the control, and the afterhyperpolarization was suppressed. Finally, when tetrodotoxin (TTX) was added to TEA and cobalt in the medium, the cell became a passive R-C system in the physiological range of voltage.

These results suggest the existence of voltage-dependent sodium channels (TTX block), potassium channels (TEA block) and calcium channels (cobalt block). None of these channels, however, can ac­count for the long lasting afterhyperpolarization seen in the pre­sence of TEA alone (Figure lA). A possibility for this hyperpolar­ization was the existence of a channel activated by the accumulation of intracellular calcium during the long lasting action potential. This possibility was tested by injecting the calcium chelator EGTA into a cell superfused with TEA. As seen in Figure lC, the action potential became extremely long and the hyperpolarization at the end of the action potential was suppressed. The results in Figures lA and lC were consistent with the existence of a calcium-activated potassium current that could be blocked by preventing an increase in

MEMBRANE EXCITABILITY IN NEURONS 219

intracellular calcium concentration. A further confirmation for the presence of such a channel was provided by an experiment in which cesium was injected intracellularly to block the calcium-activated potassium current (Tillotson and Horn, 1978); intracellular cesium also caused a marked increase in the duration of the action potential recorded in the presence of extracellular TEA (Figure ID), and the afterhyperpolarization was suppressed.

Although future experiments may demonstrate the existence of other channels in ciliary ganglion neurons, we chose the voltage­dependent sodium, potassium and calcium currents and the calcium­activated potassium current as markers in our study on membrane development.

Early stage ciliary ganglion neurons in culture

The next step was to investigate the membrane properties of ciliary ganglion neurons at the earliest stage when a structure resembling a ganglion could be recognized (fourth day of embryonic life). At this stage, the neurons are small (10 ~m diameter), but their spherical shape and small processes immediately after plating permit the use of voltage clamp with the whole cell recording tech­nique. Combining the voltage clamp technique and the pharmacological blocking agents described above, we found that even at these early stages of development, the currents described in more mature neurons are already present (Bader, Bertrand and Kato, 1983).

The results of these experiments are illustrated in Figure 2. Our reference to evaluate voltage-dependent currents in Figures 2A and 2B will be the situation where TTX, TEA and cobalt were present. It can be seen in this particular condition that when a cell is stepped in voltage clamp from a holding voltage of - 80 mV to + 5 mV, the feedback current provided by the clamping apparatus is essen­tially rectangular (label TEA, TTX, Co; the initial transient peak is the current required to bring the membrane capacity to + 5 mV); thus, the cell behaves as a passive R-C system. Figure 2A shows the cur­rent recorded during a same voltage step in the absence (control) and in the presence of the three blocking agents. Under normal con­ditions there is an early inward current (downward trace), immedi­ately followed by an outward current (upward trace). In the presence of TTX and TEA (Figure 2B), the early inward peak is suppressed, as well as the outward current that immediately follows. Instead there is a delayed slow inward current which is followed by an outward current. The results in Figure 2 can be explained in the following way. The early inward current is a sodium current (blocked by TTX) , and the outward current that immediately follows is a potassium current (blocked by TEA). The block of these two currents unmasks another inward current, which can be blocked by cobalt; we conclude that this is a voltage-dependent calcium current. Finally the block

220

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-1~: [ __ --'f pA

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C. R. BADER ET AL.

A

outward

inward

B

outward I inward I

Fig. 2. Voltage clamp recordings in ciliary ganglion neurons from a 4 day-old chick embryo. Whole cell recording was made in a neuron 4 hr after dissociation and plating. A. Two super­imposed recordings from the same cell; upper part, current traces (we adopt the convention that inward current is n~ga­tive and outward current is positive) and lower part, volt­age traces as recorded intracellularly. Control trace: superfusion with control medium (Bader et al., 1982). TEA, TTX, Co trace: superfusion with a medium containing TTX (5 x 10-6M), TEA (10 mM) and cobalt (3 mM) instead of cal­cium. B. Two superimposed recordings from the same cell as in A. TEA, TTX trace: superfusion with a medium containing TTX (5 x 10-0M) and TEA (10 mM). Co + TEA, TTX trace: superfusion in a medium containing TTX and TEA with the ad­dition of cobalt (3 mM) instead of calcium. The effects of the drugs were fully reversible.

MEMB~~ EXCITABILITY IN NEURONS 221

with cobalt also suppresses the late outward current observed in the presence of TTX and TEA. This would be expected if there were a calcium-activated potassium current like that described above.

Therefore, at the earliest embryonic stage when the ciliary ganglion could be recognized, there were already the same four mem­brane currents as observed in the mature neurons. These currents may not have reached their final maturation, in the sense that with further development, their current density per unit membrane surface may differ. Nevertheless the neuronal differentiation is already quite advanced at that stage with respect to electrophysiological properties.

Mesencephalic neural crest cells in culture

It was necessary to examine even earlier stages of the neuronal development. In this system it is possible to study the mesen­cephalic neural crest as a source of precursor cells for the ciliary ganglion. At the beginning of their migration, mesencephalic neural crest cells can be excised from 36 hr. old quail embryos and cultured as explants (Fauquet et al., 1981; Dupin, 1982; Ziller et al., 1981 and 1983). Some of the cells in defined medium begin to grow pro­cesses and after 4 to 5 days in culture have morphological character­istics of neurons as revealed by immunocytochemical demonstration of tetanus binding sites and of neurofilament proteins (Ziller et al., 1983). Although these cells from the migrating mesencephalic neural crest were considered as differentiated neurons on several criteria (Dupin, 1982; Ziller et al., 1981 and 1983), it remained to be demonstrated that they were electrically excitable.

In Figure 3 we show that mesencephalic neural crest cells in culture for 5 days are able to generate action potentials. Thus, the mesencephalic neural crest explants in culture give rise to cells that can be characterized as neurons according to electrophysiologi­cal criteria. Using the voltage clamp technique and pharmacological agents, we investigated the development of membrane excitability in mesencephalic neural crest cells. After one day in culture, two membrane currents were present, a voltage- and time- dependent potas­sium current and a leakage current. After 48 hr, a sodium current sensitive to TTX was found in all cells studied and a calcium current was found in some of the cells. Later, these currents were recorded in all cells, they increased in magnitude with time in culture and were present for up to at least 7 days in culture (Bader, Bertrand, Dupin and Kato, 1983). Thus the sequence of development of membrane currents in quail mesencephalic neural crest neurons differs from that described in amphibian neurons in vitro (Spitzer and Lamborghini, 1976) and in vivo (Baccaglini and Spitzer, 1977; Baccaglini, 1978). In amphibian neurons, firm evidence exists for a calcium current appearing before a sodium current.

222 C. R. BADER ET AL.

o

mV

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Fig. 3. Action potential in a mesencephalic neural crest cell cul­tured for 5 days in a chemically defined medium. A cell was recorded with a patch pipette filled with KCl (95 mM), Tris­HCl (45 mM) and NaCI (5 mM). Depolarizing with a 30 pA pulse of current (applied at time zero, bar in the figure) was sufficient to bring the cell to threshold.

Acknowledgements

This work was supported by grants from the Swiss National Science Foundation (3.625.0.80 and 3.625.0.82 to C. R. Bader and D. Bertrand; 3.675.0.80 and 3.230.0.82 to A. C. Kato). We are grateful to Mrs N. Collet for typing the manuscript and Mr. F. Pillonel for preparing the graphs.

REFERENCES

Baccaglini, P. I., and Spitzer, N. C., 1977, Developmental changes in the inward current of the action potential of Rohon-Beard neurones, J.Physiol., 271:93-117.

Baccaglini, P. I., 1978, Action potentials of embryonic dorsal root ganglion neurones in Xenopus tadpoles, J.Physiol., 283:585-604.

Bader, C. R., Bertrand, D., and Kato, A. C., 1982, Chick ciliary ganglion in dissociated cell culture. II. Electrophysiological properties, Develop.Biol., 94:131-141.

MEMBRANE EXCITABILITY IN NEURONS 223

Bader, C. R., Bertrand, D. Dupin E., and Kato, A. C., 1983, Development of electrical membrane properties in cultured avian neural crest, Nature, (London), 305:808-810.

Bader, C. R., Bertrand, D., and Kato, A. C., 1983, Membrane currents in a developing parasympathetic ganglion, Develop.Biol., 98:515-519.

Dupin, E., 1982, Differenciation et proliferation cellulaires au cours de l'ontogenese du systeme nerveux autonome chez l'oiseau; etudes in vivo et in vitro. These de 3e cycle, Paris, France.

Fauquet, M., Smith, J., Ziller, C., and Le Douarin, N. M., 1981, Differentiation of autonomic neuron precursors in vitro: cholinergic and adrenergic traits in cultured neural crest cells, J.Neurosci., 1:478-492.

Hamill, O. P., Marty, A., Neher, E., Sakmann, B., and Sigworth, F. J., 1981, Improved patch clamp techniques tor high resolution current recording from cells and cell-free membrane patches, Pflugers Arch., 391:85-100.

Kato, A. C., and Rey, M. -J., 1982, Chick ciliary ganglion in dis­sociated cell culture. I. Cholinergic properties, Develop. BioI., 94:121-130.

Le Douarin, N. M., 1982, The Neural Crest, Cambridge University Press.

Spitzer, N. C., and Lamborghini, J. E., 1976, The development of the action potential mechanism of amphibian neurons isolated in culture, Proc.Natl.Acad.Sci.USA, 73:1641-1645.

Tillotson, D., and Horn, R., 1978, Inactivation without facilitation of calcium conductance in caesium-loaded neurones of Aplysia, Nature (London), 273:312-314.

Ziller, C., Le Douarin, N. M., and Brazeau, P., 1981, Differenciation neuronale de cellules de la crete neurale cultivee dans un milieu defini, C.R.Acad.Sci., Paris, 292:111,1215-1219.

Ziller, C., Dupin, E., Brazeau, P., Paulin, D., and Le Douarin, N. M., 1983, Early segregation of a neuronal precursor cell line in the neural crest as revealed by culture in a chemically de­fined medium, Cell, 32:627-638.

REGULATION OF ENDPLATE CHANNEL GATING

H. R. Brenner

Department of Physiology University of Basel, Vesalgasse 1 CH-4051 Basel, Switzerland

During the formation of a motor endplate, the subsynaptic mem­brane undergoes a complex series of structural and functional changes which seem to be induced by the motor neuron. The embryonic acetyl­choline receptors (AChR.s.) in mammalian and amphibian myotubes differ in a number of properties from those at the mature end­plate[1]. They are free for lateral diffusion in the cell membrane, their metabolic stability is low (tl ~ 24 hrs) and they can be acti­vated by curare. When activated by2ACh their ionic channels remain open for about 4 ms. Before the myotubes become innervated, they are sensitive to acetylcholine (ACh) along their entire length. Shortly after the myotube is contacted by motor neurone, ACLR clusters appear in the sarcolemma underneath the nerve terminal. This clustering involves a redistribution of pre-existing extrajunctional ACLR.s. Within the next 2-3 weeks, the AChR.s at the developing endplate increase in number and density, while extrasynaptic ACh sensitivity gradually declines as a result of beginning muscle activity. During this period, the turnover of the junctional receptors slows to reach a half life t! of about 10 days, activation by curare is lost and an immunologically distinct form of AChR appears. Finally, over the last 10 days of this period, the apparent mean open time of the junctional channels is reduced from 4 ms to about 1 ms and their conductance is increased[2]. At about the same time, junctional folds are formed in the subsynaptic membrane. Little is known about how these developmental changes are controlled by the neuron and how they might be related.

In recent years, muscle activity has been recognized to be of crucial importance for postsynaptic receptor metabolism. In adult animals the development of extrajunctional ACh sensitivity after denervation can be suppressed by chronic stimulation of the muscle

225

226 H. R. BRENNER

whereas junctional ACh sensitivity remains unaffected[3]. The role of muscle activity for sub synaptic differentiation has been invest­igated during the surgically induced development of ectopic endplates in the soleus muscle of adult rats. For this purpose, the fibular nerve is cut, transplanted onto the proximal endplate-free zone of the soleus muscle and allowed to grown in among the superficial muscle fibres for about 3 weeks. Functioning ectopic endplates will begin to form in the extrajunctional membranes 2~-3 days after the soleus nerve is cut. They then develop along a pattern similar to that observed during ontogenic development: receptors are accumu­lated subsynaptically[4] and metabolically stabilized[5], junctional AChesterase (AChE) appears[6], channel gating becomes fast[7] and folds are formed in the sub synaptic membrane. In this system, the muscle can be chronically stimulated via implanted electrodes. It therefore allows to distinguish between nerve dependent and activity dependent developmental changes.

The formation of ectopic endplates can be prevented if extra­junctional denervation supersensitivity is suppressed by direct stimulation of the muscle[3]. However, muscle activity seems to promote the differentiation of the subsynaptic membrane, once a positionally stable receptor accumulation has been induced by the nerve terminal. When the foreign nerve is cut 2 days after soleus nerve section[3] and the muscle is stimulated directly, the initially multiple clusters on each fibre will be reduced in number. The remaining ones increase in size and reach similar appearance and distribution as in preparations with intact foreign innervation after multiple innervation has been reduced[8]. Conversely, nonstimulated fibres retain a high number of immature clusters.

After a brief neuromuscular contact, the development of junc­tional folds and of fast channel gating becomes independent of the continued presence of a nerve terminal. 2~-5 days after soleus nerve section, the ectopic endplates are morphologically and functionally immature: sub synaptic folds are absent and channel gating is still of the slow extrajunctional type. If the muscle is then kept mechan­ically active, either by direct stimulation or by allowing the soleus nerve to reinnervate the soleus muscle at the original endplate sites, the properties of mature endplates will develop, even when further neural influence is excluded by denervation at this time[9]. The neural signals inducing these changes act therefore at an early stage of junctional development. Channel conversion is completed sooner at the denervated endplates of chronically stimulated muscles than during normal ontogenetic development. This and the observation that during ontogenesis, fast gating channels appear earlier in white than in red muscle fibres suggest a crucial role of muscle activity for channel conversion.

At mature endplates, the metabolic stability of the AChR.s remains dependent on muscle activity and/or the presence of the

ENDPLATE CHANNEL GATING 227

terminal, respectively. Shortly after denervation, the metabolic stability of the junctional AChR.s is greatly reduced, whereas fast channel gating and junctional folds are maintained for weeks.

AChR accumulate only at contacts made by cholinergic but not by other neurons[lO]. Thus, preganglionic autonomic neurons will read­ily form functional neuromuscular synapses with sub synaptic special­izations such as fast channel gating and junctional folds[ll]. Therefore, the neural signals responsible for the induction of the endplate membrane appear to be common to different types of cholinergic neurons. Their nature, however, remains unknown. Several factors have been isolated from various sources of nervous tissue which promote AChR synthesis and clustering in the absence of innervation. Endplate formation may therefore be initiated by a diffusible substance released from the neuron. However, the rel­evance of these factors remains to be substantiated.

Measurements of endplate current noise under various experi­mental conditions as described and data obtained with the patch clamp technique[12] which resolves single channel events with high temporal resolution have shed some light on the possible mechanisms of channel conversion during endplate development. Thus, fast channel gating per se is not dependent on innervation as the junctional type of channels can be found - though at a small fraction - both in the extrasynaptic membrane of chronically denervated muscle and in embry­onic muscle cells which have never been under neural influence[12]. This finding and the fact that fast channel kinetics are maintained inspite of receptor replacement at denervated endplates exclude the possibility of a direct e.g. covalent chemical modification of AChR.s themselves by a neurally released substance. The mechanism of channel conversion could be explained by the following hypothesis which is consistent with all experimental data available[7]. Follow­ing their insertion into the sarcolemma, the AChR.s can adopt two conformations which determine the junctional and the extrajunctional type of gating. The conformations are at equilibrium. In the non­innervated sarcolemma, the 'nonjunctional' conformation is favored. Upon contact with the muscle, the nerve leaves a 'footprint' in the sub synaptic membrane which at this site may then be modified by an activity dependent muscle factor in such a way that the equilibrium is shifted towards the 'junctional' conformation. Alternatively, the neural 'footprint' may be left in the basal lamina which in regenera­ting muscle fibres directs new receptor accumulations and junctional fold formation to where the original endplates had been[13]. Channel conversion itself may be related to the appearance of junctional folds, as the two changes develop simultaneously during the differ­entiation of the endplate membrane[7].

228 H. R. BRENNER

REFERENCES

1. D. 2. B. 3. T. 4. T. 5. C. 6. T. 7. H. 8. T. 9. H.

10. M. 11. P.

12. o. 13. S.

M. Fambrough, Physiol.Rev., 59:165-227 (1979). Sakmann and H. R. Brenner, Nature, 276:401-402 (1978). L~mo and C. R. Slater, J.Physiol., 275:391-402 (1978). L~mo and C. R. Slater, J.Physiol., 303:173-189 (1980a). G. Reiness and C. B. Weinberg, Dev.Biol., 84:247-254 (1981). L~mo and C. R. Slater, J.Physiol., 303:191-202 (1980b). R. Brenner, and B. Sakmann, J.Physiol., 337:159-171 (1983). L~mo, Biol.Cell, 45:381 (1982). R. Brenner, T. Meier, and B. Widmer, Nature, submitted (1983). W. Cohen, and R. R. Weldon, J.Cell BioI., 86:388-401 (1980). Breitschmid and H. R. Brenner, J.Physiol., 312:237-252 (1981). P. Hamill, and B. Sakmann, Nature, 294:462-464 (1981). J. Burden, P. B. Sargent and U. J. McMahan, J.Cell BioI., 82:412-425 (1979).

ELECTRICAL EXCITABILITY, REGIONAL DIFFERENTIATION

AND THE IONIC CONTROL OF EARLY DEVELOPMENT

P. Guerrier, M. Moreau and L. Meijer

Station Biologique 29211 Roscoff

GENERAL OBJECTIVE

Cell differentiation may result either from the distribution between cells of regionally organized territories (mosaic), or from the emergence of new properties into equivalent territories submitted to different extracellular stimuli reSUlting in different intracel­lular activation processes.

A good model for such a mechanism is given by the study of meiosis reinitiation in the starfish where the hormone I-Methyl­adenine (I-MeAde) is not required to enter the cell but only to stimulate membrane receptors which may act by liberating an intra­cellular second messenger[I,2J. Cell contact may eventually act to produce such a similar cell differentiation as demonstrated to be the case in the embryo of PatellaL31.

Our fundamental hypothesis is that cell differentiation results from the recognition of external signals which are necessarily selec­ted and transformed at the level of the plasma membrane. It is also clear that this structure acts as a transducer for many other stimuli which playa crucial role in cell life, e.g., controlling the rein­itiation of nuclear divisions, protein synthesis, changes in func­tion, all processes which, at the onset of development, may be regu­lated at the post-transcriptional and even at a post-translational level. Our studies focus on different aspects of this membrane control.

229

230 P. GUERRIER ET AL.

IONIC CONTROL OF OOCYTE MATURATION

Oocytes of most animals are blocked during prophase (diplotene) of the first maturation division, at the germinal vesicle (GV) stage. GV appears as a huge nucleus containing the DNA equivalent for 4 N chromosomes to be dispatched into the ootid and the potential three polar bodies. Meiosis reinitiation may be triggered by quite dif­ferent stimuli and it may proceed in more than one step, possibly showing a second arrest either in metaphase I (some invertebrates) or in metaphase 2 (most vertebrates). These last blocks are released upon further fertilization or activation.

Meiosis Reinitiation as Triggered by Hormones

We are studying this process in both the amphibian and the starfish oocyte. The responsible hormones, progesterone and I-MeAde, are released by the follicle cells following the action of neuro­hormones and these relay hormones act directly at the cell plasma membrane level. In the starfish, resumption of the maturation di­visions is independent of transcription and translation, whereas some protein synthesis seems to be involved in amphibians[I]. This indi­cates that starfish meiosis reinitiation involves the transformation of pre-existing elements and it has been recognized that protein phosphorylation greatly increases following hormone stimulation. In both the starfish and the amphibian oocyte, the maturation process results in the formation of an intracellular maturation promoting factor or MPF, which appears in the cytoplasm before germinal vesicle breakdown (GVBD). This factor is able to promote maturation fol­lowing its injection into recipient non-stimulated GV intact oocytes and it retains its activity in vitro if protected against dephos­phorylation. Serial transfers of cytoplasm demonstrate that this factor is autocatalytically amplifiable, whereas heterologous trans­fers show that it is devoid of specificity, governing the division of somatic as well as germ cells. Since various mimetics have been described that do not share any structural analogy to the natural, agonists but can also trigger maturation, it is clear that the hor­mones are not involved intracellularly and that an efficient common second messenger is required.

Potential second messengers such as cyclic nucleotidesL4J or pH [2] have been discarded in the case of the starfish, whereas a reduc­tion in the level of cyclic nucleotides seems to be an important factor in the amphibian oocyte, where both a reduction in c-AMP­dependent protein kinase activity and an increase in c-AMP-indepen­dent phosphorylation appear to be required[5]. In bot~ cases, the most likely e~ficient second messenger seems to be Ca2 since a transient Ca2 surge+ as evidenced via intracellular injection of photoproteins or Ca2 sensitive electrodes, has been shown to occur both in the starfish[6] and the amphibian[7] following stimulation of

ELECTRICAL EXCITABILITY IN EARLY DEVELOPMENT 231

the+oocytes by the responsible hormones and mimeti~s. In addition, Ca2 iontophoresis or even increasing external Ca2 proved to be efficient in t+iggering maturation[l]. Finally, it has also been shown that Ca2 and the biological responses were affected in the same way by various modulators, either inhibitors or even activators such as, for example, dbc-AMP or the A subunit of cholera toxin in the case of the starfish oocyte.

+ Such a Ca2 response may have various intracellular effects as well as reflexive effects on various ionic permeabilities. In this particular case, it appeared, however, that electrophysiological techniques could only tell us something ab~ut these secondary effects but were unable to reveal the decisive Ca2 event which, in the starfish, began less than 2 seconds after hormone addition and was achieved within 30 seconds. The overall inte.rated process of matur­ation is, however, rather complex and the Ca2 response, while neces­sary, did not appear sufficient to bring about GVBD. Additional events are required which must dev~lop throughout the hormone depen­dent period, which exceeds the Ca2 releasing period by 4 min and may involve enzyme or substrate availability.

The way in which Ca2+ could exert its effect has also been investigated and we now possess strong evidence suggesting the in­volvement of calmodulin and polyamines. Specifically, calmodulin antagonists, applied in the external medium and during the hormone dependent period, will inhibit the biological response, unless the oocytes had been first subjected to a brief treatment with trypsin.+ The response is also suppressed by 8 specific inhibitors of the Ca2

dependent enzyme transglutaminase which is similarly affected by other inhibitors of the biological response. such as the amines procaine. nicotine and NH~. and the anticalmodulin drugs[2].

Finally. we recently demonstrated that a simple mechanical breakdown of the GV was sufficient to promote maturation and render the oocyte fertilizable and able to develop normally. in the absence of any hormone treatment. It thus appears that the hormone induced MPF can not be taken as responsible for these ultimate features but. possibly. only for disrupting the nuclear envelope[8].

Meiosis Reinitiation as Triggered by Sperm

This particular situation is encountered in the bivalve molluscs where various mimetics are also effective such as KC1. the ionophore A23l87 and NHq• Changing the ionic composition of the ex~ernal medium. performing q5Ca tracer flux analysis. recording H efflux and using ionic channel bl~ckers showed that this process required sim­ultaneously both a Ca2 influx and a change in the internal p¥. Only ammonia proved to be efficient in the absence of external Ca2 but the response was also blocked. in this case. by D-600 which has been

232 P. GUERRIER ET AL.

+ previously found to inhibit intracellular Ca2 release in the star-fish[9].

We also recently observe$ that ConA could produce GVBD and that this process was not only Ca2 dependent but also energy dependent, in contrast with KCl-induced maturation. ConA induced GVBD is also blocked by various inhibitors susceptible to act on the cytoskeleton such as TFP, CB and low pH[lO].

The relatively small size of the bivalve oocyte is not really favorable to classical electrophysiology and microinjection. How­ever, it may represent a good relatively transparent material fo+ using various membrane fluorescent probes (pH, potential and Ca2

indicators).

FERTILIZATION AND THE ELECTRICAL FAST BLOCK TO POLYSPERMY

Fertilization seems to be accompanied by a change in the elec­trical membrane properties which may preclude the penetration of supernumerary spermatozoon. Since the work of Jaffe[ll] on the sea urchin egg, it is agreed that this blockade is electrical in nature, resulting from an abrupt inversion of the resting potential which becomes positive for some minutes. Artificially raising the poten­tial (voltage clamp) precludes fertilization. This response of the plasma membrane is an early response preceding the rising of the fertilization membrane and so~e evidence suggests that at this stage only sperm is able to open Na channels[12]. Biophysical methods are required to provide further information on sperm receptors and ionic channels gating. Useful methods will include patch-clamp and photo­bleaching. Work is currently in progress using three selected favor­able materials: the amphibian where one can compare the situations found in the monospermic anurans and in the physiologically poly­spermic urodeles[13], the bivalves[14] and the scaphopod Dentalium where there is no trace of cortical granule exocytosis and no ferti­lization membrane elevation.

ELECTRICAl. EXCITABILITY AND CELL POLARITY

One used to consider that polarity which governs early morpho­genesis is settled into the egg structure from the beginning. We have shown, however, that it develops in relation to asymmetrically oriented processes and that this also holds for the so-called 'mosaic embryos'. Thus, in Limax, the animal-vegetal polarity which will define the future cephalo-caudal axis is only definitively fixed after the extrusion of the second polar body[15]. Dorsoventral polarity emerges even later, in relation to epigenetic processes linked to the mode of cleavage and which involve cell interactions or intracellular activation processes[16].

ELECTRICAL EXCITABILITY IN EARLY DEVELOPMENT 233

The independence of morphogenesis upon an abnormal distribution of cytoplasmic stuffs, resulting after centrifuging the egg, suggests that a cortical or membranal organization may account for such a stability, even though it may express itself via the cytoskeleton framework.

Recently, we described that some regional differences existed between the electrical properties of the plasma membrane of the mosaic egg of Dentalium during first cleavage, when a vegetative polar lobe is transiently isolated from the animal zone where the furrow cuts between two equivalent sister blastomeres. These will only get different morphogenetic properties when the polar lobe further fuses with one of them. When half a lobe is made to fuse with each cell, a double embryo is produced[17]. On the other hand, lobeless embryos are known to give rise to radialized larvae without symmetry, which lack mesoderm, the apical tuft and such organs as eyes, foot, velum and shell. At this stage of the first cleavage, isolated polar lobe and individual isolated blastomeres which form at the animal pole (Figure 1) differ in that excitability (the capacity to give action potentials) is restricted to the lobe[18].

Such an observation appeared to us quite stimulating since it suggested the existence of a large regional mosaicism in the plasma membrane organization, which was related to the animal-vegetal polar­ity and could potentially account for some morphogenetic effect. The fact that a cluster of bacteria was specifically attached to the vegetal pole of the Dentalium egg seemed to confirm such a view[19] .

v

INTACT TREFOIL

~"}.-­

-0-

POLAR LOBE

• "

QQ CELL

f--l

100p.m

t 100 msec

\20 nA 100 msec

Fig. 1. Voltage vs time responses to applied current obtained from Dentalium embryos at the trefoil stage - 100 millisecond current pulses applied at 2 second intervals[18].

234 P. GUERRIER ET AL.

Similar indications pointing to a regional organization of the plasma membrane are also found in the literature. Thus, sperm receptors seem to be restricted to the animal pole in the anuran amphibian oocyte. Moreover, it had been incidentally described that a I-MeAde dependent action potential could only be evoked in a nucleated, but not in an anucleated, fragment prepared from the oocyte of the star­fish Astropecten aurantiacus[20]. Even though these authors specifi­cally stressed the presence of the nucleus, such an observation could be alternatively taken as an indication for the existence of a regional organization of the cell membrane related to the animal­vegetal axis. It has been shown, via centrifugation experiments, that the portion of the oocyte surface in close contact with the nucleus always gave rise to the animal pole of the larva, even in those conditions where polar bodies had been forced to be emitted in a different 10cation[21].

Unfortunately, this assumption has not been confirmed by further studies. Both in Astropecten aurantiacus from Banyuls and ~ irregularis from Roscoff, we found that isolated animal nucleated and vegetal anucleated halves did not differ at all in their electrical membrane properties (Figure 2).

On the other hand, we also found that the egg of the mud snail Ilyanassa. which presents a polar lobe sharing the same morphogenetic capacities as the Dentalium one. did not exhibit regional differences in its electrical membrane properties at first cle~vage[22]. Only the unfertilized egg was found able to produce Ca2 -dependent pulse evoked action potentials. even though its re~ting potential is actually very low (-17 ± 8 mv. n = 18) and K independent at this stage (Figure 3A and B). -

In our opinion. it is worthwhile pursuing this line of study. using more refined techniques such as patch-clamp. Moreover. in Dentalium. it would be useful to determine whether the electrical localizations reported are present from the origin or are only set up in relation to such polarized events as those which occur during polar body extrusion. We know th~t the unfertilized oocyte of Dentalium has a highly negative K -dependent resting potential and is actually excitable (Figure 3C). Clear cut landmarks for the polarity of this oocyte do exist and it is quite easy to cut it in different pieces at any stage of the maturation process. which is usually triggered following sperm penetration in the metaphase 1 blocked oocytes.

Studying further evolution of the electrical membrane properties of the animal micromeres in this or other mosaic embryos and charac­terizing the differentiation of new ionic channels at this level are worthy of further investigation.

ELECTRICAL EXCITABILITY IN EARLY DEVELOPMENT

. • rL __ _

~~L.., __ _

-~--~---.--0.55

o~mv MeAde

:::f 11 1111111--rT"T1T1111 1 1 1~111 111~""--1 ------'rnrm c 10 20 30 40 50min

I II i ! 1111111 1 ! i III i ! 1111 111111

235

·1

.1

Fig. 2. Behavior of bissected oocytes of the starfish Astropecten irregularis. A - halves prepared from a whole oocyte fixed in a Falcon Petri dish with protamine sulfate and cut with a glass needle. The animal nucleated and vegetal anucleated halves thus obtained were further isolated in different vessels. B - typical response to depolarizing current pulse observed in the vegetal anucleated half. The same potential shift from the polarized to the depolarized state is observed as with the intact oocyte or the animal nucleated half . C - continuous recording of the electri cal response to I-Methyladenine as observed in a vegetal anucleated frag­ment. Applied current for monitoring changes in resistance was 0.75 nA.

EARLY NEUROGENESIS

As cited above, the mosaic embryo of annelids, molluscs and ascidians constitutes an interesting model for the study of early neurogenesis since it is established that the cerebral ganglia rapidly arise from progeny of the first animal quartet of micromere. Microsurgical experiments which would tell us much about an eventual role of cell interaction in this process, are easy to design.

To date, the only significant work which has been performed concerning the origin of excitability remains the work of Hagiwara and Miyazaki[23] concerning the so called 'differentiation without cleavage' as observed in the egg of the annelid Chaetopterus. How­ever some of the features observed may not correspond to those occur-

236 P. GUERRIER ET AL.

Fig. 3. Action potentials recorded from unfertilized oocytes of Ilyanassa (A, normal response; B, in presence of 10 nM cobalt) and Dentalium (C). Resting potentials were -30mV for Ilyanassa and -55 mV for Dentalium. Pulses duration were 2 sec in A and B; 0.2 sec in C.

ring during normal development. Moreover it has not been shown whether some ionic channels represent more than the introduction of specialized membrane patches related to the differentiation of cilia as has been observed in Paramecium[24]. The use of eggs of a larger size than the Chaetopterus egg may give important results. A com­parative analysis of the in situ cell excitability at various stages and of that of cells isolated at each generation would be particu­larly relevant. It could reveal the importance of the pattern of cell interaction previously found to play such an important role during differentiation of the mesoderm in Patella[3].

CONCLUSION

We think that the very early activation processes we are studying may stand out as pertinent models for understanding those

ELECTRICAL EXCITABILITY IN EARLY DEVELOPMENT 237

which occur later during development of the vertebrates. It seems significant that an external signal, recognized by the plasma mem­brane, can completely modify cell properties by changing intracel­lular ionic activities, thus activating some relevant enzymes. Similar processes, which do not necessarily involve any internaliz­ation or transfer of material, may well play an important role in introducing significant differences between cells, by which further gene expression may be controlled.

In the early mollusc embryo, it is also quite significant that one can easily force any vegetal blastomere to become the mesoderm mother cell, only by changing cell-cell relationships, provided that the operation is performed at the right stage. It is worth stressing that such a 'regulation' uses the same ways as those found in normal development.

More specific investigations about the nature of those inform­ative signaling processes which control embryogenesis and cell dif­ferentiation are needed, without such uninformative self explanatory notions as potencies, gradients, regulation, induction and maps, which add nothing to our purely descriptive analysis and only mask our ignorance.

REFERENCES

1. P. Guerrier, M. Moreau, L. Meijer, G. Mazzei, J. P. Vilain, and F. Dube, The role of calcium in meiosis reinitiation, Progress Clin.Biol.Res., 91:139-155 (1982).

2. L. Meijer and P. Guerrier, Maturation and fertilization in star­fish oocytes, Internat.Rev.Cytol., (in press) (1983).

3. J. A. M. Van Den Biggelaar and P. Guerrier, Dorsoventral polarity and mesentoblast determination as concomitant results of cellular interactions in the mollusc Patella vulgata, Develop.Biol., 68:462-471 (1979).

4. G. Mazzei, L. Meijer, M. Moreau, and P. Guerrier, Role of calcium and cyclic nucleotides during meiosis reinitiation in starfish oocytes, Cell Diff., 10:139-145 (1981).

5. J. L. Maller and E. G. Krebs, Regulation of oocyte maturation, in: "Current Topics in Cellular Regulation", Academic Press, New York, pp.271-311 (1980).

6. M. Moreau, P. Guerrier, M. Doree, and C. C. Ashley, 1-methyl­adenine-induced release of intracellular calcium triggers meiosis in starfish oocytes, Nature, 272:252-253 (1978).

7. M. Moreau, J. P. Vilain, and P. Guerrier, Free calcium changes associated with hormone action in amphibian oocytes, Develop. BioI., 78:201-214 (1980).

8. P. Guerrier, L. Meijer, M. Moreau, and F. J. Longo, Hormone independent GVBD induces cytoplasmic maturity in the starfish oocyte, J.Exp.Zool., 226:303-309 (1983).

238

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10.

11.

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P. GUERRIER ET AL.

F. Dube and P. Guerrier, Activation of Barnea candida (Mollusca, Pelecypoda) oo~ytes by sperm or KC1, but not by NH4D1, requires a Ca2 influx, Develop.Biol., 92:408-417 (1982).

L. Dufresne-Dube, C. Metivier, F. Dube, and P. Guerrier, Acti­vation of Barnea candida oocytes by concanavalin A, Exp.Cell Res., (submitted).

L. ~Jaffe, Fast block to polyspermy in sea urchin eggs in electrically mediated, Nature, 261:68-71+(1976).

M. Gould-Somero, Localized gating of egg Na channels by sperm, Nature, 291:254-256 (1981).

M. Charbonneau, M. Moreau, B. Picheral, J. P. Vilain, and P. Guerrier, Fertilization of amphibian eggs: comparison of electrical responses between anurans and urodeles, Develop. BioI., 98:304-318 (1983).

L. Dufresne-Dube, F. Dube, P. Guerrier, and P. Couillard, Absence of a complete block to polyspermy after fertilization of Mytilus galloprovincialis (Mollusca, Pelecypoda) oocytes, Develop.Biol., 97:27-33 (1983).

P. Guerrier, Origine et stabilite de la polarite animale vegetative chez quelques Spiralia, Annls.Embryol.Morph., 1: 119-139 (1968).

J. A. M. Van Den Biggelaar, and P. Guerrier, Origin of spatial organization, in: "Biology of Mollusca", N. H. Verdonk and J. A. M. Van den Biggelaar, eds •• Academic Press, (1983).

P. Guerrier, J. A. M. Van den Biggelaar, K. Van Dongen, and N. Verdonk, Significance of the polar lobe for the determination of dorsoventral polarity in Dentalium vulgare (da Costa), Develop.Biol., 63:233-242 (1978).

L. A. Jaffe, and P. Guerrier, Localization of electrical excita­bility in the early embryo of Dentalium, Develop.Biol., 83: 370-373 (1981).

L. Dufresne-Dube, P. Guerrier, and B. Picheral, An ultrastruc­tural analysis of Dentalium vulgare gametes with special reference to fertilization, J.Ultrastr.Res., 83:242-257 (1983) •

B. Dale, A. de Santis, and M. Hoshi, Membrane response to 1-, methyladenine requires the presence of the nucleus, Nature, 282:89-90 (1979).

H. Shirai, and H. Kanatani, Effect of local application of 1-methyladenine on the site of polar body formation in starfish oocyte, Develop.Growth Differ., 22:555-560 (1980).

M. Moreau, and P. Guerrier, Absence of regional differences in the membrane properties from the embryo of the mud snail Ilyanassa obsoleta, Biol.Bull., 161:320 (1981).

S. Hagiwara, and S. Miyazaki, Changes in excitability of the cell membrane during 'differentiation without cleavage' in the egg of the annelid Chaetopterus pergamentaceus. J.Physiol.(London), 272:197-216 (1977).

K. Dunlap, Localization of Ca channels in Paramecium caudatum, J.Physiol.(London), 271:119-133 (1977).

THE EARLY DIFFERENTIATION OF NEURONAL

MEMBRANE PROPERTIES

Nicholas C. Spitzer

Department of Biology, B-022 University of California, San Diego La Jolla, CA 92093

Studies of neural induction involve assessments of neuronal differentiation following experimental manipulations. These have in the past relied principally on neuroanatomical descriptions of neurite outgrowth, which sample an important neuronal phenotype. In recent years it has become possible to analyze the differentiation of key cytoplasmic specializations of neurons, such as their neurotrans­mitter synthetic capacity, and the differentiation of their charac­teristic membrane properties. Some of the recent progress in under­standing the development of neuronal membrane properties will be reviewed. These assays are likely to be useful in future studies of neural induction for several reasons. First, the increasing ease of application of the techniques involved invites their general use. Second, acquisition of neuronal membrane properties occurs very early in normal embryonic development, recommending them for rapid evalu­ation of neuronal induction. Third, different neurons exhibit dif­ferent constellations of properties appearing in particular sequences (Spitzer and Lamborghini, 1981). A broader characterization of neuronal development affords the opportunity to distinguish the induction of different neuronal types. Fourth, the expression of neurotransmitter synthetic enzymes of membrane proteins are likely to be the results of activities of single genes, in contrast to the more complex phenomenon of neurite extension which involves mUltiple gene products. The analysis of the molecular events involved in neuronal induction may be correspondingly facilitated.

Neurons possess a number of types of ion channels, which account for some of the functions of these cells. Voltage-dependent channels open in response to changes in membrane potential and allow cells to make action potentials. Chemically sensitive channels open in re­sponse to neurotransmitters and mediate chemical synapses. Other

239

240 N. C. SPITZER

channels involved in the production of electrical synapses permit currents to flow directly between cell interiors. Precursor cells often lack the capacity to make action potentials or to respond to neurotransmitters, although there are some exceptions, most notably among egg cells (Hagiwara and Jaffe, 1979; see also Baud, Kato and Marcher, 1982). Precursor cells are often electrically coupled, however.

A general conclusion from the studies of the differentiation of nerve and muscle membranes is that their characteristic properties first appear in an immature form and change during the course of further development. When cells first become electrically excitable they often make Ca-dependent action potentials that are of long duration; these are subsequently converted to brief Na-dependent impulses. Cells that are coupled become uncoupled. When cells begin to respond to neurotransmitters, the localization of receptors is often diffuse; however the ionic basis of these responses is constant during development, in spite of changes in the distribution of recep­tors.

THE DEVELOPMENT OF THE ACTION POTENTIAL

Studies of the development of neuronal membrane properties have been expedited by choice of favorable preparations. The Rohon-Beard neurons of the amphibian spinal cord have been very useful for this work. There are about two hundred of these cells, which are primary sensory neurons (Robers and Clarke, 1982), located on the dorsal aspect of the spinal cord where they are accessible for electro­physiological examination. They have their birthdate during the gastrula stage of development, along with five other populations of neurons, which are the first in the embryo to exhibit this step in neural differentiation (Lamborghini, 1980). The birthdate is remark­ably synchronous in the population, with 90% of cells completing their final round of DNA synthesis in a period of six hours. This event occurs close to the time of primary induction (Tarin, 1971).

The precursor cells from which Rohon-Beard neurons arise have been identified at the sixteen-cell stage of the embryo (Jacobson, 1981). Cells of the neural plate do not appear to be electrically excitable (Palmer and Slack, 1970; Slack and Warner, 1975). When Rohon-Beard cells are first impaled with microelectrodes at the neural tube stage, about six hours after their birthdate, they are unable to generate action potentials. Within a few hours they are capable of making impulses when depolarized. These impulses are hundreds of milliseconds in duration, and depend chiefly on an influx of Ca, as shown by experiments involving ion substitutions and the application of pharmacological blocking agents (Baccaglini and Spitzer, 1977). At the tailbud stage, roughly one day of develop­ment, the action potential acquires a Na component, and gradually

NEURONAL MEMBRANE PROPERTIES 241

shortens to an average duration of tens of msec. The same tests show that the initial peak is Na-dependent, while the later plateau is Ca-dependent. As early as three days of development, upon attainment of the larval stage, the impulse, which is now about one msec in duration, in principally Na-dependent.

The electrical excitability of amphibian dorsal root ganglion neurons has also been investigated. These cells arise later than the Rohon-Beard neurons, over a prolonged period of time, and take over the role of primary sensory neurons as the Rohon-Beard cells die. Ca-dependent impulses are observed in the smallest and presumably youngest neurons, and the frequency with which they are observed declines with increasing embryonic age, while that of Na-dependent impulses rises. The results suggest that these neurons follow the same developmental sequence seen in Rohon-Beard neurons (Baccaglini, 1978). The maturation of amphibian olfactory neurons follows the same pattern of development (Strichartz et al., 1980). The out­growing axons produce action potentials which are initially Ca­dependent, and later depend on Na.

There are an increasing number of instances in which the devel­opment of electrical excitability of vertebrate neurons has been observed to occur in vitro. Neurons from the presumptive spinal cord of the amphibian neural plate can be grown in dissociated cell cul­ture. The action potentials elicited from the cell bodies of these neurons exhibit the same change in the ionic dependence of the inward current, from Ca to Na, along the same time course as the Rohon-Beard neurons in vivo (Spitzer and Lamborghini, 1976). Since these cul­tures contain neurons which innervate skeletal muscle and are presum­ably motor neurons, this aspect of differentiation is not unique to sensory cells. Furthermore, the excitability of the neurites of these cells has been studied in culture, where they can be clearly visualized at early stages, and the ionic dependence of their action potentials also shifts from Ca to Na (Willard, 1980). Dorsal root ganglion cells of the mouse generate action potentials that first depend on Ca and Na, and later on Na alone (Matsuda et al., 1978). Murine neuroblastoma cells can make Ca-dependent action potentials, and acquire Na-dependent impulses during subsequent differentiation (Miyake, 1978). Cerebral cortical cells from the chick embryo make action potentials that are Ca- and Na-dependent; the Ca component of the inward current apparently disappears during subsequent develop­ment (Mori et al., 19~2). However, there are a number of cases in which the ionic basis of the action potential has not been observed to change with increasing age in culture. In some cases, recordings could have been made sufficiently late that changes would have al­ready occurred, or the culture medium could have been inadequate to support normal development. In others the possibility remains that development involves no change in ionic dependence of the action potential.

242 N. C. SPITZER

Toxin-binding assays are a useful approach to identifying the time of appearance of ion channels and quantitating their changing levels during development. Although probes for the voltage-sensitive Na channels have been used successfully (Berwald-Netter et al •• 1981). the application of comparable ligands to the study of Ca channels has not been described.

Among the invertebrates. ganglionic neurons in the grasshopper embryo are accessible to microelectrode impalements at very early stages (Goodman and Spitzer. 1979; Heathcote, 1981). The action potential elicited from the cell bodies of dorsal unpaired median (DUM) interneurons, as well as those derived from a different pre­cursor neuroblast, depend on an influx of both Ca and Na when the cells first become excitable, and either is sufficient to support an impulse. At later stages both ions are needed to produce an action potential. This change in the ionic mechanism of the action poten­tial in the cell body stands in contrast to the absence of change in the Na-dependent action potentials arising in the axons (Goodman and Spitzer. 1981).

During the course of regeneration neurons synthesize new mem­brane that becomes electrically excitable. This process seems to recapitulate the pattern of differentiation seen during development. The regenerating neurites of embryonic guinea pig dorsal root gang­lion cells in vitro initially contain voltage-dependent Ca channels which later disappear (Fukuda and Kameyama, 1979). The giant axons of the adult cockroach initially produce Ca-dependent action poten­tials in the proximal stump close to the site of transection; later the membrane generates normal Na-dependent impulses (Meiri, Spira and Parnas, 1981).

The shift in ionic dependence of the impulse from Ca to Na during embryogenesis has also been seen in some striated and cardiac muscle cells (see Spitzer. 1982, for review). However, tunicate skeletal muscle cells lose the Na component of their impulse while retaining the Ca component (Takahashi et al., 1971; Takahashi and Yoshii, 1981). Thus a change in ionic dependence, rather than its direction. may be the most general phenomenon in the development of action potentials.

THE DEVELOPMENT OF ELECTRICAL UNCOUPLING

Cells that will arrive at different states of terminal differen­tiation are frequently coupled by low resistance junctions at an early stage and become uncoupled at a particular time during their development. This phenomenon was initially observed in a variety of electrically inexcitable cells (e.g. Potter et al., 1966; Sheridan, 1968; Spitzer, 1970; Lo and Gilula, 1979). More recently it has been described in the differentiation of nerve and muscle. Rohon-Beard

NEURONAL MEMBRANE PROPERTIES 243

neurons are electrically coupled at the early neural tube stage, before they are able to make action potentials. Current injected into one cell spreads to others, presumably via gap junctions (Spitzer, 1982). This coupling is voltage dependent, in that the degree of coupling depends on the voltage difference between the cells. A shift in the membrane potential of one cell, away from that of its neighbors, markedly reduces the strength of coupling for as long as the potential difference is maintained. Other cells in the spinal cords of the same embryos exhibit electrical coupling that is not voltage-dependent. This appears to be the same process first described for isolated pairs of amphibian blastomeres (Spray, Harris and Bennett, 1979).

Rohon-Beard neurons are still electrically coupled when Ca action potentials can first be elicited from these cells, and an impulse in one cell can be sufficient to trigger an impulse in a nearby coupled cell. More commonly the Ca action potential in one cell causes a subthreshold depolarization of the second which de­creases during its course, perhaps reflecting voltage-dependent uncoupling. These cells become completely uncoupled from one another around the time of appearance of the Na component of the action potential although other cells are still coupled. The Rohon-Beard cells remain uncoupled during their further development. The coup­ling and uncoupling of amphibian spinal neurons in cell culture has not yet been investigated.

The uncoupling of DUM neurons from their progenitor neuroblast, and the uncoupling of other tissues from one another, have been described for the grasshopper embryo (Goodman and Spitzer, 1979, 1981). The disappea~ance of gap junctions between ganglion cells of the amphibian retina has been reported (Dixon and Cronly-Dillon, 1972). Mesoderm cells of amphibian embryos and striated muscle cells in rat embryos become uncoupled at specific times (Blackshaw and Warner, 1976b; Dennis et al., 1981). Thus, stage-specific uncoupling appears to be a general process.

THE DEVELOPMENT OF NEUROTRANSMITTER SENSITIVITY

In contrast to the developmental changes in features of ion channels mediating action potentials and electrical coupling, the channels involved in responses to neurotransmitters have properties that appear to be constant from the time of their first appearance. The application of a variety of neurotransmitters to Rohon-Beard neurons at neural tube stages is without effect on their membrane potential or conductance. However, these cells begin to respond at the early tailbud stage, and are depolarized by gamma-aminobutyric acid (GABA); roughly half of the cells are also depolarized by glycine (Bixby and Spitzer, 1982a). The responses to both of these neurotransmitters seem to appear at the same time, and the cells

244 N. C. SPITZER

remain insensitive to a host of other compounds. Examination of the ionic basis of the response to iontophoretic application of GABA in matur~ cells+reveals that it is the result of a conductance increase to Na and K , with a reversal potential of -30 mV. The same re­versal potential is observed in newly sensitive cells, strongly suggesting that the ionic dependence of the response is constant. At all stages of development this response is blocked by picrotoxin or curare and exhibits desensitization like that seen for many other neurotransmitter receptors.

Some features of this chemosensitivity do change, however. The sensitivity of cells to GABA increases about tenfold during their maturation, probably as a result of an increase in the number of receptors or in some properties of single channels, or both. Furthermore, the glycine response is transient, and can no longer be elicited by 3~ days of development. Since the number of Rohon-Beard cells is stable during this time, the response seems to have been lost from the existing population.

The development of neurotransmitter sensitivity of amphibian spinal neurons in culture parallels development in vivo in most respects. The time of first appearance, initial sensitivity, re­versal potential, pharmacology and desensitization of the response to GABA are the same for one class of cultured neurons as they are for the Rohon-Beard neurons in the spinal cord. Other neurons, which are hyperpolarized by GABA and glycine and depolarized by glutamate, and thus likely to be motor neurons, first begin to yield these responses at the same age in culture (Spitzer and Bixby, 1982). The reversal potential of the response to GABA in these cells is -60 mV, which is the value reported for mature motor neurons in vivo.

Ganglionic DUM neurons of the grasshopper become sensitive to both acetylcholine (ACh) and GABA at an early stage of embryogenesis. The appearance of responses to both neurotransmitters seems to occur at the same time. The reversal potentials are constant during devel­opment, implying that the ionic dependences of the responses are unchanging (Goodman and Spitzer, 1970, 1980). The pharmacology of the responses, as well as the lack of desensitization, also appear invariant with development.

Vertebrate skeletal muscle cells are sensitive to ACh. The reversal potential of this response is constant from the time of its first appearance early in the differentiation of these cells (Fambrough and Rash, 1971; Fischbach, 1972; Cohen and Kullberg, 1974; Steinbach, 1975; Blackshaw and Warner, 1976a, Ohmori and Sasaki, 1977), although there are dramatic changes in the distribution of ACh receptors. Again, constancy of the ionic selectivity of the channels seems indicated. There may be an exception of this widespread phe­nomenon, however, in the response of the chick atrium to ACh, which has been reported to change (Pappano, 1972).

NEURONAL MEMBRANE PROPERTIES 245

THE DEVELOPMENTAL SIGNIFICANCE OF CHANGING MEMBRANE PROPERTIES

The functions of Ca-dependent action potentials at early stages of development remain to be identified. These impulses of long duration allow the influx of large amounts of Ca which could have profound effects on cellular metabolism. Alternatively, the action of these impulses could be electrical rather than ionic. The re­versal of the sign of the membrane potential for substantial periods of time could be essential for the insertion of membrane components. Agents that selectively block Ca channels may allow resolution of this issue (Dunlap and Fischbach, 1978; Bixby and Spitzer, 1982b). It has recently been shown that conditions which promote Ca entry into the growth cones of cultured neuroblastoma cells lead to morpho­logical changes that are probably associated with neurite elongation (Anglister et al., 1982). Na-dependent impulses cannot playa role in early development in those cases in which they appear after con­siderable differentiation has occurred (e.g. Goodman and Spitzer, 1979) or when their blockage by tetrodotoxin (TTX) has no effect on some aspects of later development (Obata, 1977, Harris, 1980, 1981). The chronic application of TTX is toxic to some cells with Na­dependent action potentials, however, perhaps as a consequence of the blockage of trophic interactions (Bergey et al., 1980), and may alter normal synapse elimination (Van Essen, 1982).

The precise role of electrical coupling and subsequent uncoup­ling between embryonic cells is unknown. Metabolic cooperativity has been demonstrated between coupled cells in culture (Gilula, Reeves and Steinbach, 1972). Induction could involve cellular interactions via these specialized junctions in vivo, which would not be needed at later stages. Although Rohon-Beard cells develop their action poten­tial mechanisms and become sensitive to neurotransmitters while they are becoming uncoupled, the relationship is not obligatory (Bixby and Spitzer, 1982a). The development of amphibian spinal neurons in dissociated cell culture indicates that if coupling is necessary for early development, it must be required prior to the neural plate stage at which the cultures are prepared. The significance of the voltage-dependent feature of electrical coupling is still obscure. The Ca-dependent action potentials could cause transient uncoupling, which might be important for independent development of the cells or as a prelude to permanent uncoupling. Neurons can also become func­tionally uncoupled although the electrical synapse persists (Rayport and Kandel, 1980).

The early appearance of neurotransmitter sensitivity in embry­onic cells could reflect the specialization of the membrane required for the formation of chemical synapses. However there is no evidence for synapses on the cell bodies of the neurons whose development has been ~tudied, like a number of mature neurons whose somal sensitivity has been examined. The increase in GABA sensitivity of Rohon-Beard cells is unusual, since embryonic muscle fibers have been seen to

246 N. C. SPITZER

accumulate ACh receptors at the neuromuscular junction and lose them elsewhere (e.g. Blackshaw and Warner, 1976a; Ohmori and Sasaki, 1977). The existence of the early, developmentally transient glycine sensitivity of Rohon-Beard neurons, like the transient neurotrans­mitter sensitivity of rat Purkinje cells (Crepel, Dupont and Gardette, 1982), raises the possibility that these ion channels may have some role in events other than synaptogenesis.

THE ROLES OF RNA AND PROTEIN SYNTHESIS IN DIFFERENTIATION OF NEURONAL MEMBRANE PROPERTIES

There is presently little information about the molecular basis for the appearance of different membrane properties during embryo­genesis and the subsequent changes in these phenotypes during matur­ation. These processes may involve the synthesis and insertion of new channel proteins, or modification of existing membrane compon­ents. This issue has been addressed by examining the effects of specific metabolic inhibitors applied to cells during a restricted period of their development, to determine the timing of RNA or pro­tein synthesis required for the expression of different properties. There have been several studies of the temporal dependence of the effects of RNA synthesis inhibitors on the cellular differentiation of amphibian nerve and muscle (Duprat, Zalta and Beetschen, 1966; Stocker and Bride, 1980). This approach has demonstrated that the RNA and protein synthesis needed for the expression of neurotrans­mitter synthesis follows that required for neurite outgrowth in the development of the fruit fly and the mouse (Seecof, 1977; Bloom and Black, 1979).

Dissociated cell cultures containing amphibian spinal neurons are attractive for such studies, since neuronal differentiation in vitro parallels that in vivo, in several respects discussed abov~ The timing of the macromolecular syntheses necessary for the develop­ment of the Na-dependent impulse in these cultured neurons has been investigated. The early application of actinomycin D, to block RNA synthesis, and cycloheximide or puromycin, to suppress protein syn~ thesis, prevents the appearance of the mature impulse (O'Dowd, 1981; Blair, 1981). The neurons continue to make Ca-dependent action potentials of long duration, and the basal state of the membrane (resting potential, input resistance) is unaffected, as is the devel­opment of other cellular properties (neurite extension, voltage­dependent K channels). This effect is further limited, in that application of these inhibitors at later times does not prevent the appearance and maturation of the Na-dependent action potentials. There is similar evidence for transcriptional control of the develop­ment of this phenotype in cultured chick muscle cells (Kano and Suzuki, 1982). If biochemical excision of single phenotypes can be achieved by the appropriately timed application of reversible inhib­itors, the removal of the block should permit determination of the effects on subsequent development.

NEURONAL MEMBRANE PROPERTIES 247

More specific probes are needed to distinguish between the hypotheses of new synthesis and modification of existing molecules. Antibodies to these membrane proteins and techniques for identifying their messenger RNAs should reveal the times of onset of transcrip­tion and translation. One may hope to obtain developmental time­tables for the appearance of RNAs and proteins that give rise to various membrane properties. Advances in molecular biology and immunology suggest that such a detailed understanding of the early program of neuronal development is within the foreseeable future.

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THE CONTROL OF NEURONAL DIFFERENTIATION BY

INTRACELLULAR SODIUM

Anne E Warner

Department of Anatomy and Embryology University College London Gower Street, London

It has long been held that the first event in the development of the nervous system in the amphibian embryo is an interaction between mesoderm cells, brought into position by gastrulation movements, and the overlying dorsal ectoderm cells. This event has been called primary induction and is discussed elsewhere in this volume. Recently this view has been challenged by Marcus Jacobson (1982 and this volume), who argues that the nervous system is derived from 7 com­partments, whose founder cells are established at the early-mid blastula stage. Following gastrulation the neural tube is con­structed as a result of an extensive series of morphogenetic move­ments as the dorsal surface of the ectoderm develops first the neural groove and then the neural folds. The area encompassed by the neural folds, the neural plate, will form the central nervous system, both neurones and supporting cells, while the edges of the neural folds are destined to give rise to the neural crest. As the neural groove deepens and the neural folds lift, the folds from either side are brought together in the mid-line, where the ectoderm fuses to form a continuous sheet and the neural tube becomes a separate entity. Neural crest cells then begin their characteristic migration away from the dorsal surface of the neural tube to form the sympathetic and sensory ganglia, Schwann cells and a variety of other deriva­tives. Work in a number of laboratories has shown that the fate of neural crest cells is dependent upon the environment in which they eventually find themselves.

The first differentiated neurones, capable of generating action potentials and releasing and responding to transmitter substances, appear in the neural tube very shortly after it closes. These neur­ones are Rohan-Beard cells, primitive sensory neurones and motoneur­ones. The electrical properties of Rohan-Beard cells have been

251

252 A. E. HARNER

investigated by Nick Spitzer and his colleagues (see Spitzer, this volume). The motoneurones form the first functional neuromuscular junctions with developing myotome cells, again very shortly after the neural tube closes (Blackshaw and Warner, 1967a; Kullberg, Lentz and Cohen, 1977). The elements of the first reflex arc are thus con­structed at an early stage in the developing Xenopus tadpole.

Autonomous differentiation of small explants of presumptive neural tissue from the neural plate region occurs reprod~cibly from about the time of the appearance of the neural groove in both axolotl and Xenopus embryos (Duprat et al., this volume; Messenger and Warner, unpublished). Neurones which appear in such cultures from the axolotl synthesize catecholamines and cholinesterase (Duprat et al., this volume); those from Xenopus similarly synthesize catechol­amines and cholinesterase and are electrically excitable (Messenger and Warner, unpublished). Neural crest derivatives, such as pigment cells which respond to melatonin, also appear in such cultures (see Messenger and Warner, 1977). The number of neurones that different­iate from early neural plate stages is very small, but as neurulation proceeds the density of neurones produced in dispersed cultures increases and cultures made from the late neural fold stages onwards (Nieuwkoop and Faber, stage 18) contain large numbers of different­iated neurones (Messenger and Warner, 1979 and unpublished).

These findings suggest that the events which commit neuronal precursor cells to their eventual fate take place between gastru­lation and the late neural fold stage. A similar conclusion with regard to the anatomical organization of the nervous system can be derived from experiments carried out by Carl-Olaf Jacobson (1964). Using the classical grafting techniques of experimental embryology, Jacobson showed that rotation of portions of the neural plate led to rotation of anatomical structures if the grafts were done at the late neural fold sta3e, whereas grafts performed at the beginning of neurulation lead to regulation, so that anatomical structure was not altered.

The properties of cells within the neural plate over this period of development would seem well worth examining, since understanding the processes which lead to commitment of neuronal precursors to fulful their developmental fate may give clues as to how they are set in motion and also how they might lead to the switching on of genes which express the phenotypic properties of neuronal cells. For the remainder of this article I shall consider evidence which suggests that the intracellular concentration ot sodium ions plays an import­ant role in determining whether presumptive neurones in the neural plate are able to complete their differentiation and express their developmental fate.

The starting point for this work lay in the observation that the cells of early embryos are in direct communication with each other

INTRACELLULAR SODIUM 253

via a pathway which is probably mediated by the intercellular mem­brane structure, the gap junction (cf. Potter, Furshpan and Lennox, 1966 and review by Wolpert, 1978). Although the time of appearance of this channel varies from species to species it seems .that at times when classical grafting experiments suggest that embryonic cells are able to interact and influence each other, all cells in the embryo are in direct communication with each other through this pathway. Since the original observations by Potter ~t a1., (1966) there has been considerable speculation about the role that this pathway might. play in the passage of developmentally important information from one cell to the next. One argument in support of such a hypothesis would be if the commitment of a cell, or group of cells, to follow a par­ticular developmental fate were accompanied by the disappearance of this channel between them and their neighbors. For the nervous system it turned out (Warner, 1973) that the disappearance of elect­rical coupling, and therefore presumably the gap junctional channel, between cells destined to form the nervous system and cells destined to form other ectodermal derivatives, does riot occur until the neural tube closes. However the degree bf electrical coupling within the neural plate does begin to fa1i off as closure of the neural tube approaches (Blackshaw and Warner, 1976b). These experiments then neither support nor oppose the theory that gap junctions may be involved in specification of the nervous system, since it is possible that the correlative event ~s the cessation of synthesis of gap junction proteins with gap junctions disappearing with a time course dictated by the turn-over time.

On this occasion I do not wish to return to the complex and controversial issue of the role of gap junctions during development except to comment that one proposed method of signal transfer during primary induction has been through the gap junctional channel. The substitution of the compartment theory of Marcus Jacobson is unlikely to bring about any great resolution of this issue since the proper­ties of gap junctions within and between compartments may be differ­ent (cf. Warner and Lawrence, 1982).

In the course of experiments to examine current spread within the ectoderm during neurulation it became apparent that despite the presence of a low electrical resistance channel between ectoderm and presumptive neural cells the electrical properties of the two groups )f cells were not the same. Thus by the late neural fold stage cells lying in the region of the neural plate have resting potentials ~lmost 20mV more negative than,their companions residing within the presumptive ectoderm (Warner, 1973). This difference in resting ?otentia1 appears during neurulation. Before the neural folds begin to lift membrane potentials within the ectoderm are uniform at about - 40mV, regardless of developmental fate (Blackshaw and Warner, 1967b). As neurulation proceeds.ce11s in the neural plate gradually ~cquire more negative resting potentials, reaching between -60 and -70mV by the late neural fold stage (Blackshaw and Warner, 1967b).

254 A. E. WARNER

This alteration in resting membrane potential occurs over the inter­val suggested by both tissue culture and grafting experiments to cover the commitment of presumptive neural cells to express the neuronal phenotype. Cells in the ectoderm retain relatively low resting potentials of about -40mV throughout neurulation. These findings raise two interesting questions. Firstly what is the mec­hanism underlying this increase in resting membrane potential? Secondly is it causally or casually related to the commitment of presumptive nerve cells?

The first question can be addressed by examining the membrane properties of neural plate cells more closely. An increase in rest­ing potential could be generated by an increase in the potassium­permeability of the cell membrane relative to that of other cations in neural plate cells. The problem with this mechanism is that it is difficult to envisage how an alteration in passive properties could sustain an increase in resting potential in some cells of an elect­rically inter-connected network. Alternatively the increase in negativity could be both generated and sustained by activation of a pump such as the sodium pump, which normally exports three sodium ions in exchange for two potassium ions. By examining the effect of increasing the extracellular concentration of potassium (in prepar­ations wounded in the belly ectoderm to ensure penetration of the solution into the intercellular spaces) Blackshaw and Warner (1976b) showed that there was no substantial alteration in the membrane permeability to potassium in neural plate cells between early and late neural fold stages. They further found that raising extracellu­lar potassium to 20 mM at early neural fold stages, about 1 hour before the natural increase in resting potential, hyperpolarized neural plate cells. This hyperpolarization, which could be as much as 30 mV, was blocked by the cardiotonic steroids ouabain or stro­phanthidin (10-6M) suggesting that it reflected activation of the sodium pump (see Glynn, 1957). Since the natural increase in resting potential was also blocked by these two specific inhibitors of the sodium pump it seemed likely that this too reflected sodium pump activation in neural plate cells, but not ectoderm cells, during neurulation.

The question whether this increase in activity of the sodium pump is related to neuronal differentiation was addressed by Messenger and Warner (1979). Xenopus embryos were exposed to the cardiotonic steriod strophanthidin while they were neurulating, a hole being made in the belly wall to ensure penetration of the sodium pump inhibitor into the intercellular spaces. At the end of neur­ulation the drug was washed away from the intercellular spaces and the embryos left to develop for a further two days in the absence of the pump inhibitor. Histological examination of treated embryos and their controls revealed that sodium pump inhibition during neur­ulation, when the membrane potential increase takes place, had per­manent consequences for the subsequent development of the nervous

INTRACELLULAR SODIUM 255

system. The brains and eyes of the treated tadpoles contained many cells, suggesting that drug treatment had not lead to cell death, but there was little sign of axon outgrowth and the normal, orderly arrangement of neuroblasts from ependymal to marginal layers of the developing nervous system was absent. Thus it seems that if acti­vation of the sodium pump during the neural plate stages is prevented then neurones derived from the neural plate fail to fulfil their developmental fate. The phenotype of the many cells present within the neural tube after Na pump inhibition is not known; they may represent cells which have followed a pathway leading to glial cell, rather than neuronal cell, differentiation.

Although such experiments show clearly that sodium pump inhi­bition during neurulation has longlasting effects on the development of neurones it is difficult to obtain quantitative information be­cause of the inherent variability in the response of embryos to teratogens. To overcome this problem it is necessary to have a system which allows the number of neurones which differentiate after treatment to be measured. This was achieved by developing a tissue culture system where the degree of neuronal differentiation could be defined by cell counting (Messenger and Warner, 1979). The neural tube, notochord and somites are dissected out from embryos at the time of closure of the neural tube. They are dispersed into single cells and then dispersed into petri dishes, each dish containing material from 3 embryos. Differentiation takes place in Ringer solution with 10% foetal calf serum and penicillin/streptomycin to reduce bacterial contamination. After 18-24 hrs each petri dish contains a monolayer of differentiated cells. The cultures contain neurones, muscle cells, pigment cells and a variety of mesenchymal cells and fibroblasts which cannot be identified unequivocally. The time course of differentiation is the same as that seen in the whole embryo. All the evidence so far obtained suggests that the differ­entiated cells have similar properties to those that differentiate in the whole embryo. Thus muscle cells contain striations and will contract either if contacted by a nerve or if stimulated electric­ally. Endplate potentials can be recorded in response to spontaneous activity in contracting neurones and muscle cells in close proximity to each other are electrically coupled, as in the whole embryo at this stage of development (Blackshaw and Warner, 1976c). Cells that have the morphological characteristics of neurones, a phase bright cell body, neurites and growth cones, can be stained specifically by tetanus toxin (Vulliamy and Messenger, 1981) or by antibodies to neurofilament proteins (Breckenridge and Warner, unpublished). They synthesize catecholamines and cholinesterase and are electrically excitable. Pigment cells respond to melatonin (Messenger and Warner, 1977). The cultures remain viable for 5 to 7 days, when death of some of the neurones first begins. By this time the proportion of fibroblasts and mesenchymal cells has greatly increased by cell division. After 18 to 24 hrs in culture each petri dish contains 60,000 to 70,000 differentiated cells.

256 A. E. HARNER

Differentiation is assessed by counting the cells in between 20 to 40 microscope fields taken at random from 3 petri dishes. The number of-"nerve cells and the number of muscle cells are counted in each fiEUd- and expressed as a pro~ortion of the total number pf cells for that'fie1d. Frequency h1stogr~ms can then be constructed showing the percentage of neurones and muscles cells that differentiate in cultiires'made from control embryos and those treated during'neur­ulation. 'Three important features of this method deserve 'comment. Firstly the cells are always' co~nted at between 18 and 24 hrs, before the mesenchymal and fibroblast population begins to divide,. Secondly the use-of a number of embryos greatly reduces variability in the cultures 'so' that results may b~ oirect1y compared from one day to the next. Thirdly, regardless of treatment, differentiation always takes place in' the absence of drugs so that both control and treated embryos undergo differentiation under the same culture conditions. Further details of these techniques can be found in Messenger and Warner (1979) 'and Breckenridge and Warner (1982). . '; ,

Using this method it has been possible to demonstrate (Messenger and Wa~er, 1979; Breckenridge and Warner, 1982):

" " .1._" •

a)

b)

c)

d)

e)

f)

That the dose response relation for the effect of the sodium pump inhibitor strophanthidin on neural differentiation lies between 10-7M and 10-SM, much the same as that found when exam­i~ing the effects of glycoside on Na and K fluxes in red cells (Glynn, 1957). That glycoside treatment reduces the overall neuronal population by 70%. _The remaining 30%, of neurones are probably derived from the neural crest since they can be largely abolished by an anti-body to Nerve Growth Factor. This implies that inhibition of the sodium pump during neurulation only affects the neural plate derived neurones. Whether neural crest derived neurones are sensitive to Na pump inhibition at some other stage of development is not known. The total number of cells that differentiate and the number of muscle cells that differentiate are not affected by inhibition of the Na pump; the effect is restricted to neurones. ' Raising the concentration of potassium in strophanthidin con­taining solution reduces the effectiveness of glycoside treat­ment, as expected from the known comp~tition between glycosides and potassium at the external sttes of the sodium pump. Analogues which are much less effective at inhibiting the sodium pump in red cells are similarly less able to inhibit neuronal differentiation. Lowering the concentration of potassium in the intercellular spaces to below 1 mM during neurulation also prevents neuronal differentiation. This finding further supports the view that the Na pump is important since the degree of activation of the pump is critically dependent upon the level of extracellular potassium.

INTRACELLULAR SODIUM 257

These findings make it unlikely that the effects of strophanthi­din on neural differentiation are related to some non-specific action of the cardiotonic steriod and greatly strengthen the view that it is inhibition of the sodium pump that is relevant. Messenger and Warner (1979) further showed that the sensitivity of differentiating neur­ones to sodium pump inhibition is restricted to the time when the membrane potential is increasing. Treatment before the neural folds lift, or from Nieuwkoop and Faber stage 17 (late neural fold) to the time of closure of the neural tube, are both without effect on the number of neurones that subsequently differentiate.

Why does inhibiting the sodium pump at this time during neur­ulation have this effect? An answer to this question could give clues to the events which occur during normal development. There are three obvious possibilities.

(i) The permeability of gap junctions is reduced. This may occur because the concentration of intracellular calcium rises when the sodium pump is inhibited (cf. Blaustein, 1974; Bers and Ellis, 1982) and it has been shown that this can lead to un­coupling of heart muscle cells (Weingart, 1977). This possibil­ity can be tested by examining whether an increase in extra­cellular calcium potentiates the inhibitory effect of strop han­thidin on differentiating neurones. Breckenridge and Warner (1982) showed that when Cao was increased at the same time as inhibiting the sodium pump, differentiating neurones were pro­tected. This finding makes it very unlikely that the effect of strophanthidin is brought about by a reduction in intercellular communication within the neural plate. However it does riot necessarily mean that the low resistance electrical pathway has no role to play at this stage of development of the nervous system.

(ii)The abolition of the increase in resting potential produced by blocking the sodium pump is responsible for the reduction in subsequent differentiation of neurones. This can be tested by measuring the resting potential in embryos which have been rescued from the consequences of sodium pump inhibition by an increase in extracellular calcium. In such circumstances the resting potential in neural plate cells remains at the level found before Na pump activation (close to -40mV) throughout neurulation (Breckenridge and Warner, 1982). It is therefore unlikely that the increase in resting membrane potential norm­ally generated by activation of the sodium pump is essential for the subsequent differentiation ot neurones. This finding is in keeping with the demonstration by Messenger and Warner (1979) that neural differentiation proceeds normally in embryos treated with 100mM potassium during neurulation.

(iii)The intracellular concentration of sodium during neurulation is the factor controlling ability of neural plate neurones to express their developmental fate. When the sodium pump is inhibited the intracellular concentration of sodium rises (and that of potassium falls). If the internal Na concentration is

258 A. E. WARNER

crucial then any manipulation which leads to a fall in intra­cellular sodium should be able to protect neurones against the inhibitory effect of cardiotonic steriods. The protection afforded by an increase in extracellular calcium would fit this notion since it is known that when Cao is raised Na falls, even if the Na. pump is blocked (Deitmer and Ellis, 1978). On this hypothesi~ strontium ions should also protect differentiating neurones, magnesium and manganese ions should not and manganese ions should oppose the protective effect of raising extracellu­lar calcium (cf. Deitmer and Ellis, 1978). Similarly lowering extracellular sodium, so reducing the passive leak of sodium ions into neural plate cells, should also oppose the consequen­ces of blocking the sodium pump (Ellis, 1977; Deitmer and Ellis, 1978; Ellis and Deitmer, 1978). In a series of experiments designed to test these predictions of the hypothesis that intra­cellular sodium is the controlling factor, Breckenridge and Warner (1982) demonstrated that all the predictions were met, both when examined in tissue culture and in the whole embryo.

Although these experiments strongly suggest that intracellular sodium is important, clearly it would be more convincing if direct measurements of intracellular sodium could be made under the appropr­iate experimental conditions. This was done using sodium sensitive micro-electrodes to determine intracellular sodium levels (Breckenridge and Warner, 1982). There are a number of technical problems associated with such measurements which are discussed in detail in that paper, so that the absolute values for intracellular sodium are likely to be subject to some error. Nevertheless these experiments showed: a) Prior to neurulation intracellar sodium in both ectoderm and

neural plate is around 30roM, about the level to be expected from the resting potential measurements and the potassium sensitivity of the resting potential.

b) As neurulation proceeds intracellular sodium in neural plate cells drops steeply, with approximately the same time course as the increase of resting membrane potential, reaching less than 10 roM by the late neural fold stage. Sodium in ectoderm cells remains close to 30 roM.

c) When the sodium pump is blocked with 10-SM strophanthidin before the natural activation of the sodium pump the fall in intra­cellular sodium is prevented; when Na has begun to fall, pump inhibition produces a rise in internal sodium to about 30 roM within 2 hrs.

d) When extracellular calcium is raised, or extracellular sodium lowered, in the presence of a sodium pump inhibitor, intracellu­lar sodium falls despite inhibition of the sodium pump.

These results show that changes in internal sodium do indeed take place as predicted by the experiments where neural different­iation is assessed. They provide strong support for the hypothesis that the reason for the catastrophic effect of inhibiting the sodium

INTRACELLULAR SODIUM 259

pump during neurulation on the subsequent differentiation of neurones is directly related to the changes in intracellular cation concentra­tion that follow Na pump inhibition. It is not yet possible to say whether it is the fall in intracellular sodium, approximately 3-fo1d, rise in intracellular potassium, about 20%, or the substantial fall in the ratio of intracellular sodium to intracellular potassium -about 30-fo1d - that is the operative factor during normal develop­ment. It is also not clear whether these alterations in cation content act as a trigger, setting in hand other events leading to neuronal differentiation, or whether they are an essential co-factor allowing the consequences of other changes in cell metabolism to be expressed.

It seems likely that the sodium pumps responsible for the fall in intracellular sodium are inserted some time before they are acti­vated in the normal course of development (see Blackshaw and Warner, 1976b). A search for factors that can lead to the extra synthesis of sodium pumps might therefore shed some light on the extremely poorly understood events which initiate the development of the nervous system. Similarly study of the effects of changing cation content on gene activity and the expression of intracellular enzymes may lead to a better understanding of the events which accompany expression of the neuronal phenotype.

Acknowledgements

I am grateful to Angela Messenger and Lorna Breckenridge for allowing me to quote unpublished results. The work in this article was supported by grants from the Medical Research Council.

REFERENCES

Bers, D. M., and Ellis, D., 1982, Intracellular calcium and sodium activity in sheep heart Purkinje fibres: effect of changes in external sodium and intracellular pH, Pfu1gers Arch., 393:171-178.

Blackshaw, S. E., and Warner, A. E., 1976a, Onset of acetylcholine sensitivity and end-plate activity in developing myotome muscles of Xenopus 1aevis., Nature, 262:217-218.

Blackshaw, S. E., and Warner, A. E., 1976b, Alterations in resting membrane properties at neural plate stages of development of the nervous system, J.Physio1., 255:231-247.

Blackshaw, S. E., and Warner, A. E., 1976c, Low resistance junctions between mesoderm cells during development of trunk muscles, J.Physio1., 255:209-230.

Blaustein, M. P., 1974, The inter-relationship between sodium and calcium fluxes across cell membranes, Rev.Physio1.Biochem. Pharmac., 70:33-82.

Breckenridge, L. J., and Warner, A. E., 1982, Intracellular sodium and the differentiation of amphibian embryonic neurones, J.Physio1., 332:393-413.

260 A. E. 'WARNER

Deitmer, J. W., and Ellis, D., 1978, Changes in the intracellular sodium activity of sheep heart Purkinje fibres produced by calcium and other divalent cations, J.Physiol. 277:437-453.

Ellis, D., 1977, The effect of external cations and ouabain on the intracellular sodium activity of sheep heart Purkinje fibres, J.Physiol. 273:211-240.

Ellis, D., and Deitmer, J. W., 1978, The relationship between intra­cellular and extracellular sodium activity of sheep heart Purkinje fibres during inhibition of the Na-K pump, Pflugers Arch., 377:209-315.

Glynn, I. M., 1957, The action of cardiac glycosides on sodium and potassium movements in human red cells, J.Physiol. 136: 148-173.

Jacobson, C. 0., 1964, Motor nuclei, cranial root and nerve fibre patterns in the medulla oblongata after reversal experiments on the neural plate of axolotl larvae, Zool.Bidrag.Uppsala, 37:73-160.

Jacobson, M., 1982, Origins of the nervous system in amphibia, in: Neuronal Development. N. C. Spitzer, ed., Plenum, New York, pp 45-99.

Kullberg, R. W., Cohen, M. W., and Lentz, T. L., 1977, Development of the myotomal neuromuscular junction in Xenopus laevis: an electrophysiological and ultrastructural study, Dev.Biol., 60:101-129.

Messenger, E. A., and Warner, A. E., 1977, The action of melatonin on single amphibian pigment cells in tissue culture, Brit.J. Pharmac., 61:607-614.

Messenger, E. A., and Warner, A. E., 1979, The function of the sodium pump during the differentiation of amphibian embryonic neur­ones, J.Physiol. 292:85-105.

Potter, D. D., Furshpan, E. J., and Lennox, E. S., 1966, Connections between cells of the developing squid as revealed by electro­physiological methods, Proc.Nat.Acad.Sci.USA., 55:328-336.

Vulliamy, T. J., and Messenger, E. A., 1981, Tetanus toxin: a marker of amphibian neuronal differentiation in vitro, Neurosci.Lett. 22:87-90.

Warner, A. E., 1973, The electrical properties of the ectoderm in'the amphibian embryo during induction and early development of the nervous system, J.Physiol. 355:267-286.

Warner, A. E., and Lawrence, P. A., 1982, Permeability of gap junctions at the segmental border in insect epidermis, Cell, 28:243-252. -­

Weingart, R., 1977, The actions of ouabain on intercellular coupling and conduction velocity in mammalian ventricular muscle. J.Physiol., 264:341-366.

Wolpert, L., 1978. Gap junctions: channels for communication in development. in: Intercellular Junctions and Synapses, J. Feldman, N. B. Gilula, and J. D. Pitts, eds., Chapman and Hall.

5

FACTORS INVOLVED IN NEURONAL SURVIVAL, DEVELOPMENT AND DIFFERENTIATION

PURIFICATION OF A NEUROTROPHIC PROTEIN

FROM MAMMALIAN BRAIN

Y. -A. Barde and H. Thoenen

Max-Planck-Institute for Psychiatry Dept. of Neurochemistry D-8033 Martinsried b. Munchen Federal Republic of Germany

INTRODUCTION

During the development of many parts of the vertebrate nervous system, more neurons are produced than are found in the adult, the quantitative adjustment of neuronal numbers taking place largely by their elimination (Jacobson, 1978). Evidence from transplantation and ablation experiments indicates that this process is not genetic­ally pre-progrannned, but can be modulated. If, for example, the eyeball of a chick embryo is removed early in development, most of the ciliary ganglion neurons which would have projected to it die (Landmesser and Pilar, 1974). Conversely, if an additional optic cup is grafted, more neurons than normal are found in the ciliary ganglia (Narayanan and Narayanan, 1978; Boydston and Sohal, 1979). These examples, together with many others (for review, see T. J. Cunningham, 1982) indicate that neuronal death is a general phenom­enon under epigenetic control. However, little is known about the nature of the molecules (with one exception discussed below) and the mechanisms which are responsible for this phenomenon.

Our laboratory has recently been involved in the isolation of a new molecule that is responsible for the survival of neurons. (For a review of the work currently done in this area by other laboratories, the reader is referred to comprehensive accounts, e.g. Varon and Adler, 1982; Barde, Edgar, and Thoenen, 1983). However, before we describe our own work, we will give a brief account of the infor­mation available on nerve growth factor (NGF), a well-defined neuro­trophic molecule with an established physiological function.

263

264 Y.-A. BARDE AND H. THOENEN

NGF

The fortuitous discovery more than 20 years ago of large amounts of NGF in the submandibular gland of the adult male mouse has allowed the elucidation of the decisive role played by this molecule in the regulation of neuronal death during development (For reviews see Levi-Montalcini and Angeletti, 1968; Thoenen and Barde, 1980): injection of NGF prevents the death of neurons in ganglia of the peripheral nervous system (Hamburger, Brunso-Bechtold, and Yip, 1981) and conversely, injection of antibodies to NGF dramatically increases the degree of normally occurring cell death in these ganglia (Cohen, 1960). In addition, several lines of evidence show that the target organs innervated by NGF-responsive neurons are able to regulate their density of innervation by a mechanism involving NGF. First, growing neurites have specific NGF-receptors, present at the nerve endings which make contact with target organs (Dumas, Schwab and Thoenen, 1979; Carbonetto and Stach, 1982; Rohrer and Barde, 1982). Furthermore. after binding to its receptor, NGF is transported retro­gradely within the axon to the cell body where specific effects can be triggered (Thoenen and Barde, 1980; Schwab and Thoenen, 1983). Very recently, it has been shown that the density of neurites can be regulated by the amount of NGF to which these neurites are exposed (Campenot, 1982). Finally, a very sensitive and specific enzyme immunoassay (developed by Sigrun Korsching in our laboratory, Korsching and Thoenen, 1983) has demonstrated that low levels of NGF are indeed produced by organs innervated by NGF-responsive neurons -a long missing important piece of evidence - and that NGF levels are correlated with the density of sympathetic innervation (For an exten­sive discussion of the problems linked with NGF measurements, see Thoenen and Barde, 1980). Thus, there is now no reason to doubt that NGF has a physiological function. However, these studies were only possible because of the large amounts of NGF present in the salivary gland of the male mouse, for which there is no satisfactory explan­ation. Neither salivary gland NGF nor the large quantities of NGF which have been found in the guinea pig prostate and the bovine seminal plasma seem to play any physiological role in the developm~nt of the nervous system (Harper et al., 1979; Harper, Glanville and Thoenen, 1982). So far no rich source has been found of an analogous growth factor for neurons unresponsive to NGF. This does not mean, however, that isolation of other such factors is impossible, but as discussed in the following section, it does mean that their isolation is a much more tedious problem.

OTHER NEUROTROPHIC FACTORS

It is likely that other factors exist besides NGF which support the survival and differentiation of specific neuronal populations. Indeed. in recent years, a considerable number of in vitro studies have demonstrated that such factors can be found in a wide variety of

PURIFICATION OF A NEUROTROPHIC PROTEIN 265

conditioned media or tissue extracts (For reviews, see Varon and Adler, 1981; Barde, Edgar and Thoenen, 1983). These previously undefined molecules have been shown to support the survival and neurite extension from a variety of neurons which do not respond to NGF, for example parasympathetic and motor neurons. Progress in tissue culture and protein purification techniques makes it likely that these molecules will be isolated and characterized. An example of how this can be done, together with the difficulties inherent to such projects is discussed below.

PURIFICATION OF A NEUROTROPHIC FACTOR FROM BRAIN

The Assay System

Any purification procedure requires an assay system that re­flects the characteristics of the molecule to be isolated. Culture of neurons is the method of choice as it allows precise measurements to be made on large numbers of test samples. While tissue culture is only a tool and not an end in itself, there is no a priori reason to think that in vitro effects will not be correlated with those in vivo. For example, NGF has been isolated using tissue culture~ech­niques to follow its purification, and, as discussed above, experi­ments performed in vivo have confirmed that it has a physiological function.

Our assay system was the survival in culture of spinal sensory neurons isolated from chick embryos. These neurons can easily be obtained in large amounts and when grown in the absence of other cell types, they die unless the culture medium is supplemented with tissue extracts or medium previously conditioned by other cultured cells. This is the key feature of this assay system. Sensory neurons have an additional characteristic which is that they project to the central nervous system (unlike other ganglia from the peripheral nervous system) - a point of importance when trying to isolate a molecule from the central nervous system with putative neurotrophic activity. Because spinal sensory neurons (like other neurons from the peripheral nervous system) display a typical morphology in cul­ture, they are easy to identify and to count reliably when. seeded at low density. The effect of any fraction to be tested can thus be quantified by measuring its protein content, and by determining the dilution which permits, for example, half of the maximal survival seen when the activity is saturating. The concentration of protein necessary to obtain half-maximal survival may then be defined as one unit.

The Purification Procedure

Since we were interested to know if molecules with a neuro­trophic effect could be purified from the central nervous system,

266 Y.-A. BARDE AND H. THOENEN

where NGF does not seem to have a physiological function (Thoenen and Barde, 1980), we started with pig brain as a source of material (for a detailed account of the purification procedure, the reader is referred to Barde, Edgar and Thoenen, 1982). This material has the advantage that it is obtainable in very large amounts - an essential point when the protein is expected to be present in very low amounts (as a point of reference, NGF is active at concentrations as low as 1 ng/ml and present in tissues such as the superior cervical ganglia of the rat at about 20 ng/g wet weight). The isolation procedure in­volved the following steps: extraction from pig brain of the pro­teins soluble at pH 4.0, followed by chromatography on CM-cellulose, hydroxylapatite, and phenyl sepharose. At this stage, in spite of a purification factor of about 10,000 fold (in terms of increase in specific activity over the starting material), the preparation was still very heterogenous when analyzed by polyacrylamide gel electro­phoresis in the presence of sodium dodecylsulfate. The fortunate observation that biological activity could be partially recovered after treatment of the preparation with SDS prompted us to use pre­parative gel electrophoresis as a final step of purification. The material thus isolated migrates as one band in an SDS polyacrylamide­gel. with a molecular weight of 12,300, and is a very basic protein with an isoelectric point of about 10.2. Approximately 3 pg can be isolated from one kilogram of starting material. The overall purifi­cation factor needed was over one million fold and the yield was about 10%. In terms of molecular weight (on SDS-gel), isoelectric point and final specific activity the characteristics of this mol­ecule are remarkably similar to those of NGF isolated from the sali­vary gland of the mouse (Server and Shooter, 1977), the prostate gland of the guinea pig (Chapman, Banks, Vernon, and Walker, 1981) or from the bovine seminal plasma (Harper, Glanville and Thoenen, 1982). However, the molecule isolated from brain is clearly a different entity: its effects are not blocked by antibodies to either mouse or bovine NGF. Furthermore, it is a functionally distinct molecule: at saturating concentrations 10 ng/ml) , approximately 40% of the neurons isolated from the 5th lumbar dorsal root ganglia of the 10-day old chick embryo can be kept alive in culture. Interestingly, the other 50% can be kept alive with NGF, and the combination of both agents' allows the survival of almost all the neurons plated (Barde, Edgar and Thoenen, 1982; Barde, unpublished observations). Cell counts performed on that ganglion show that this figure corresponds to about 90% of the neurons originally present in the ganglion. Why these neurons require 2 survival factors to be maintained in culture is still unclear at the moment, but based on other experiments (Barde, Edgar and Thoenen, 1980), we speculate that this is due to the fact that sensory ganglia are composed of neurons which are at different stages of maturation: early in development, most of the neurons would require NGF for their development and this would be provided by the periphery. Progressively during development, NGF may lose its ability to support survival. In this regard, we know that many neurons lose their NGF-receptors both in vivo and in vitro during

PURIFICATION OF A NEUROTROPHIC PROTEIN 267

development (Herrup and Shooter, 1975; Rohrer and Barde, 1982). Ganglion cells may then begin to depend on the newly isolated mol­ecule for survival. This molecule could originate either from the satellite cells present in dorsal root ganglia and/or in the glial cells or target neurons present in the spinal cord. Preliminary experiments have shown that the development of neurotrophic activity in rodent brain takes place at a time during which extensive prolif­eration of glia also occurs. The use of antibodies raised against the purified molecule should help to determine which cells are indeed producing this molecule in vivo. While there is very good evidence that pure glial cells produce neurotrophic molecules in vitro (Lindsay et al., 1982) it is not known if such molecules are also produced in vivo and if the molecules produced in vitro are really identical to that described here.

An additional piece of evidence that we are dealing with an entity which is functionally distinct from NGF is derived from recent experiments performed in collaboration with Dr. James E. Turner (Wake Forest University). These studies indicate that the brain neuro­trophic molecule is able to induce fiber outgrowth from rat embryonic retinal explants (Turner, Barde, Schwab and Thoenen, 1982; Turner and Barde, unpublished observation), a system (central nervous system neurons) in which NGF has not effect.

These experiments have demonstrated that it is possible to isolate a neurotrophic molecule from the brain where it is present at very low concentration, which is also likely to be the case for other neurotrophic factors. However, all the experiments so far have been performed in vitro, and the limited amounts of purified material make it difficult to envisage experiments in vivo in the near future. Such experiments will probably have to wait for the determination of the amino acid sequence of the protein, allowing the synthesis of an active peptide and/or the production of the molecule by bacteria using recombinant DNA technology. However, the production of specific antibodies appears more feasible in the near future and this should allow interesting experiments to be performed, addressing the issue of phYSiological importance and localization of this factor.

REFERENCES

Barde, Y. -A., Edgar, D., and Thoenen, H., 1980, Sensory neurons in culture: changing requirements for survival factors during embryonic development, Proc.Natl.Acad.Sci.USA, 77:1199-1203.

Barde, Y. -A., Edgar, D., and Thoenen, H., 1982, Purification of a new neurotrophic factor from mammalian brain, EMBO J., 1:549-553.

Barde, Y. -A., Edgar, D., and Thoenen, H., 1983, New neurotrophic factors, Ann.Rev.Physiol., 45:601-612.

Boydston, W. R., and Sohal, G. S., 1979, Grafting of an additional periphery reduces embryonic loss of neurons, Brain Res., 178: 403-410.

268 Y.-A. BARDE AND H. THOENEN

Campenot. R •• 1982. Development of sympathetic neurons in compart­mentalized cultures. I. Local control of neurite growth by nerve growth factor. Dev.Biol •• 93:1-12.

Carbonetto. S •• and Stach. R. W •• 1982. Localization of nerve growth factor bound to neurons growing nerve fibers in culture. Dev.Brain Res •• 3:463-473.

Chapman. C. A •• Banks. B. E. C •• Vernon. C. A •• and Walker. J. M •• 1981. The isolation and characterization of nerve growth factor from the prostate gland of the guinea pig. Eur.J. Biochem.. 115: 347-351. --

Cohen. S •• 1960. Purification of a nerve-growth promoting protein from the mouse salivary gland and its neurocytotoxic anti­serum. Proc.Natl.Acad.Sci.USA. 46:302-311.

Cunningham. T. J •• 1982. Naturally occurring neuron death and its regulation by developing neural pathways. Int.Rev.Cytol •• 74: 163-186.

Dumas. M •• Schwab. M. E •• and Thoenen. H •• 1979. Retrograde axonal transport of specific macromolecule as a tool for character­izing nerve terminal membranes. J.Neurobiol •• 10:179-197.

Hamburger. V •• Brunso-Bechtold. J •• and Yip. J •• 1981. Neuronal death in the spinal ganglia of the chick embryo and its reduction by nerve growth factor. J.Neurosci •• 1:60-71.

Harper. G. P •• Barde. Y. -A •• Burnstock. G., Carstairs. J. R., Dennison. M. E •• Suda, K •• and Vernon, C. A •• Guinea pig prostate is a rich source of nerve growth factor. Nature, 279: 160-162.

Harper. G. P •• Glanville. R. W., and Thoenen, H •• 1982. The purifi­cation of nerve growth factor from bovine seminal plasma. J.Biol.Chem •• 257:8541-8548.

Herrup. K., and Shooter. E. M •• 1975. Properties of the S-NGF receptor in development. J.Cell BioI •• 67:118-125.

Jacobson. M •• 1978. Developmental Neurobiology (2nd ed.) Plenum Press. New York.

Korsching. S •• and Thoenen. H., 1983, Nerve growth factor in sym­pathetic ganglia and corresponding target organs of the rat: correlation with density of sympathetic innervation, Proc.Natl.Acad.Sci.USA, 80:3513-3516.

Landmesser. L •• and Pilar. G •• 1974. Synaptic formation during embryogenesis on ganglia cells lacking a periphery. J.Physiol. 241:715-736.

Levi-Montalcini. R •• and Angeletti. P. U •• 1968. Nerve Growth Factor. Physiol.Rev •• 48:534-569.

Lindsay, R. M •• Barber. P. C •• Sherwood. M. R. C •• Zimmer. J •• and Raisman. G., Astrocytes cultures from adult rat brain. Derivation, characterization and neurotrophic properties of pure astroglial cells from corpus callosum. Brain Res •• 243: 329-343.

Narayanan, C. H •• and Narayanan, Y •• 1978, Neuronal adjustments in developing nuclear centers of the chick embryo following transplantation of an additional optic primordium. J.Embryol. Exp.Morphol., 44:53-70.

PURIFICATION OF A NEUROTROPHIC PROTEIN 269

Rohrer, H., and Barde, Y. -A., 1982, Presence and disappearance of nerve growth factor receptors on sensory neurons in culture, Dev.Biol., 89:309-315.

Schwab, M. E., and Thoenen, H., 1983, Retrograde axonal transport, in: "Handbook of Neurochemistry", A. Lajtha, ed., New York, Plenum, pp. 381-404.

Server, S., and Shooter, E. M., 1977, Nerve Growth Factor, Adv.in Proc.Chem., 31:339-409.

Thoenen, H., and Barde, Y. -A., 1980, Physiology of nerve growth factor, Physiol.Rev., 60:1284-1335.

Turner, J. E., Barde, Y. -A., Schwab, M. E., and Thoenen, H., 1983, Extract from brain stimulates neurite outgrowth from fetal rat retinal explants, Dev.Brain Res., 6:77-83.

Varon, S., and Adler, R., 1981, Trophic and specifying factors direct to neuronal cells, in: "Advances in cellular neurobiology", S. Fedoroff and L. Herz, ed., Academic Press, New York, pp .115-163.

STUDIES OF THE DEVELOPMENT OF CENTRAL

NORADRENERGIC NEURONS IN VITRO

Umberto di Porzio and M. Estenoz

Istituto Embriologia Molecolare C.N.R., Naples Italy

We are interested in understanding the specificity and re­liability with which neurons form connections - the molecular events that determine specific cellular interactions which give rise to stabilized synapses and form neuronal circuitry. Neurons come in a remarkable diversity of forms. For any type of neuron the pattern of possible synaptic connections and that of dendrite arborization is repetitive and preserved within each species. Neurons migrate to their final location, extend axons in search of proper targets with which they establish functional contacts, and give rise, thereafter, to dendritic outgrowth in an ordered and constant fashion[l]. Most neurons will form connections in a topographically ordered way and will form an appropriate number of contacts, specific for each neuron. How is the final differentiated or mature state acquired? Which are the processes and their sequence by which the developing nervous system unfolds?

It is well accepted that this accurate organization is acquired through the interactions of several phenomena which are genetically predetermined (e.g., the dendritic arborization of Purkinjie cells) and/or the consequence of environmental influences on the growing tissues. For example, some cells may have a set of 'labels' on their surfaces which match only those of a restricted number of other cells allowing the reciprocal recognition between presynaptic fibers and appropriate targets - as proposed to explain the specificity of retinotectal connections[2]. In other instances, the temporal order of arrival of different populations of fibers competing for the same target neurons has been evoked to explain connectivity[3]. On the other hand, a surplus of neurons is produced early in development and part of these embryonic neurons die, probably as a consequence of competition for trophic factors required for neuronal survival which

271

272 U. DI PORZIO AND M. ESTENOZ

are supplied by the target cells[4]. A portion of synaptic contacts initially formed is eliminated, maybe through a mechanism of selec­tive stabilization regulated by the target[5-8]. Environmental cues may modulate the plasticity of neurons. They may come from the nature of the extracellular matrix[9j, diffusable factorsL10,ll], cell surface components[12,13], interactions with non-neuronal cells[14,15], or electric fields[16].

From experimental data obtained in the last decade, we know that cellular behavior within the nervous system is not unique. For example, in the absence of the proper target, a neuron can form heterologous functional synapses[17] as if, at least in some cases, normally occurring synapses are determined by a hierarchical affinity for different cell types so that, in the absence of the 'best choice', a graded preference for other cell types can take place. Even the phenotypic commitment can vary: some neurons show transient phenotypes during development[18] or can be experimentally induced to change their phenotypic expression through manipulation of the environment[14,19], indicating that the developing nervous system has the capacity to regulate and a high degree of plasticity.

In vitro techniques are now being largely used in developmental studies of the nervous system. They offer the means of analyzing complex systems in simple environments and can allow the study of cellular behavior revealed, for example, by perturbation experiments. We have used cell cultures to investigate the rules that govern the cellular interactions that intervene during the development of the central aminergic neurons. In culture, in the presence or absence of glial cells, target striatal cells stimulate the maturation of as­cending dopaminergic neurons from the midbrain of mouse embryos, increasing uptake and synthesis of dopamine[20,21]. The addition of a medium conditioned on striatal cultures could not reproduce the stimulation observed in cocultures; whereas striatal membrane compon­ents specifically stimulated DA uptake in mesencephalic cultures[22]. No stimulation of DA neurons was observed with non-target cerebellar cells[23]. Similar to dopaminergic neurons, noradrenergic (NE) neurons from the embryonic brain stem of the mouse could be main­tained in vitro for several weeks in the presence of serum and showed high affinity uptake mechanisms for labelled amines (3H-DA and 3H-NE)[23j. The concomitant presence in culture of cerebellar and noradrenergic neurons resulted in an enhancement of uptake capacity for NE neurons. This effect correlates to the quantity of available target sites: increasing the concentration of cerebellar cells turther stimulated 3H-DA uptake[23]. The stimulatory capacity of the target seems developmentally regulated since more mature cerebella (E 16-E 17) exerted greater stimulation than younger structures (E14-E15)[23j, although this difference could also represent a quan­titative more than a qualitative difference since during the chosen period the number of Purkinje cells in the cerebellum differs.

CENTRAL NORADRENERGIC NEURONS 273

Neurons from the brain stem could grow in a medium free of serum, supplemented with insulin (25 ~g/ml) and transferrin (100 ~g/ml) (Figure 1). The medium had to be pre-incubated with cells growing in vitro prior to use in order to allow long term (3 weeks) survival of the cultures[211. In these cultures no glial cells could be detected at the light microscopic level in absence of serum. In most cases the uptake capacity for labelled catecholamines was greater in serum-free conditions than in cultures supplemented with serum (data not shown). Co culturing brain stem and cerebellar neurons resulted in enhancement of high affinity uptake for ~H-DA (Figure 2). The degree of stimulation could vary in different exper­iments from 180% to 300%. Striatal cells were also capable of stimu­lating 3H-DA uptake in brain stem cultures although to a lesser extent than cerebellar cells (Figure 3), as observed in serum-supple­mented cultures[231. This result could be due to specific inter­actions between NE cells and striatal neurons since a weak NE inner­vation originating in the locus coeruleus is present in the striatum[24,251.

Tyrosine Hydroxylase-like immunoreactivity (Figure 4) showed that both in cultures and cocultures with cerebellar neurons, the number of TH positive cells was not significantly different (respec­tively, 128 z 15 and 136 z 20 (n=8) in 10 day old cultures). The uptake of labelled amines was inhibited by desmethyl imipramine (DMI) of 61.5% and 76.5% in cultures and cocultures, respectively. Also, Fluoxetine, the inhibitor of uptake in serotoninergic (5-HT) fibers, partially inhibited 3H-DA uptake (42.3% and 58.2% in cultures and cocultures, respectively), indicating that 5HT neurons, which are present in our cultures since the raphe nuclei are dissected together with the locus coeruleus, in these conditions take up large amounts of catecholamines (Figure 3).

Fig. 1. Hoffman interference optics of brain stem cultures.

274

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Fig. 3. Percentage of stimulation by striatal (STR) and cerebellar (CRB) cells on brain stem neurons (b.s.) 3H-DA high affinity uptake after 5 days (A) and 11 days (B) in cultures (n=5).

CENTRAL NORADRENERGIC NEURONS 275

I

Fig. 4. TH like immunoreactivity: 16 day old cultures grown in the presence of serum were fixed 1 hr at O°C with 4% Para­formaldehyde 0.1% Glutaraldehyde, pre-incubated 20 min. with goat serum in presence of 0.1% Triton X and was incubated for 1 hr at O°C with TH antiserum (a gift from A. van den Pol) diluted 1:1000. Goat antirabbit was the second anti­serum. TH positive cells are stained with PAP (peroxidase antiperoxidase) DAB (diaminobenzidine) lightly osmicated (1% Os04 20 min) and embedded in Epon.

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Fig. 5. Total specific binding of 3H-DMI was measured by incubating brain stem (b.s.) cells ± cerebellar cells (CRB) or dopa­minergic cells (mes), at O°C for 1 hr with 1.6 nM (A) and 5 nM (B) ~H-DMI in PBS with 137 mM NaCI. Non-specific bind­ing, calculated from the binding performed in the presence of 100 ~M DMI, was subtracted to obtain total specific bind­ing, which is expressed in fmol/culture. NE, where used, was 1 mM (n=8).

276 U. DI PORZIO AND M. ESTENOZ

Binding experiments of 3H-DMI were performed in brain stem cultures and cocultures with cerebellar cells[26]. As shown in Figure 5, the total specific binding was dependent on ~H-DMI concen­trations and could be partially displaced in the presence of 1 roM NE. Very little total specific binding could be detected in dopaminergic cell cultures. 3H-DMI appears to label NE neuronal uptake sites[26]. Nevertheless, no significant differences of specific binding were observed in cultures and cocultures, both at 5 nM and 1.6 nM 3H-DMI concentrations, whereas in cocultures a significant increase of amine uptake was observed. That could indicate that the increased 3H-DA uptake in cocultures reflects a changed affinity constant of uptake sites for the amine, but this seems improbable since in the dopamin­ergic system the affinity constant for 3H-DA was not modified in cocultures[27]. Alternatively, this result may indicate that at least the total specific 3H-DMI binding can not be taken as a numer­ical index of uptake sites, or that in our cocultures cerebellar cells increase a specific uptake in fibers different from NE fibers. Further studies are required to clarify this result.

REFERENCES

1. M.

2. R. 3. D. 4. V. 5. J. 6. E. 7. M.

8. D.

9. P.

10. R.

11. F. 12. S.

13. U. 14. N.

15. P.

16. N. 17. C.

Jacobson, "Developmental Neurobiology", 2nd ed., Plenum Press, New York, (1978). W. Sperry, Proc.Natl.Acad.Sci.USA, 50:703 (1963). I. Gottlieb and W. M. Cowan, Brain Res., 41:452 (1972). Hamburger, Neurosci.Res.Pro.Bull., 15:Suppl:1 (1977). P. Changeux and A. Danchin, Nature, 264:705 (1976). Knyihar, B. Csillik, and P. Rakic, Science, 202:1206 (1978). Murray, S. Sharma, and M. A. Edwards, J.Comp.Neurol., 209:374 (1982). C. van Essen, in: "Neuronal Development", N. C. Spitzer, ed., Plenum Press, New York, pp.333-376 (1982). C. Letourneau, in: "Neuronal Development", N. C. Spitzer, ed., Plenum Press, New York, pp.213-254 (1982). Levi-Montalcini, in: "The Neurosciences: Path of Discovery',', F. G. Worden, J. ~ Sawzey, and G. Edelman, eds., MIT Press, Cambridge, Mass., pp.243-265 (1975). Collins and A. Dawson, J.Neurosci., 2:1005 (1982). Denis-Donini and G. Augusti-Tocco, Curr.Top.Dev.Biol., 16: 323 (1980). Rutishauser and G. M. Edelman, J.Cell BioI., 87:370 (1980). M. Le Douarin, in: "Development of the Autonomic Nervous System", PitmanMedical, London (Ciba Found.Symp.83), pp.19-50 (1981). H. Patterson and L. L. Chun, Proc.Natl.Acad.Sci.USA, 71: 3607 (1974). Patel and M. M. Poo, J.Neurosci., 2:483 (1982). Sotelo, Trends.Neurosci., 3:33 (1980).

CENTRAL NORADRENERGIC NEURONS 277

18. I. B. Black, M. C. Bohn, G. Miller Janakait, and J. A. Kessler, in: "Development of the autonomic nervous system", Pitman Medical, London (Ciba Found.Symp.83), pp.177-193 (1981).

19. P. H. Patterson, Ann.Rev.Neurosci., 1:1 (1978). 20. A. Prochiantz, U. di Porzio, A. Kato, B. Berger, and J.

Glowinski, Proc.Natl.Acad.Sci.USA, 76:5387 (1979). 21. U. di Porzio, M. C. Daguet, J. Glowinski, and A. Prochiantz,

Nature, 288:370 (1980). 22. A. Prochiantz, M. C. Daguet, A. Herbert, and J. Glowinski,

Nature, 293:570 (1981). 23. U. di Porzio and M. Estenoz, in: "Membranes in growth and

development", J. F. Hoffman, G. H. Giebisch, and L. Bolis, eds., Alan R. Liss, Inc., New York, (1982).

24. Coyle and Henry, J.Neurochem., 21:61 (1973). 25. M. Weinstock, A. Zaeadil, E. Muth, W. Crowly, T. o 'Donohue , D.

Jacobowitz, and I. Kopin, Euro.J.Parm., 68:422 (1980). 26. C. -M. Lee and S. H. Snyder, Proc.Natl.Acad.Sci.USA, 78:5250

(1981). 27. M. C. Daguet. U. di Porzio, A. Prochiantz, A. Kato, and J.

Glowinski, Brain Res., 191:323 (1980).

NERVE GROWTH PROMOTERS IN THE EMBRYONIC CHICK

ABSTRACT

Ted Ebendal

Department of Zoology Uppsala University, Box 561 S-751 22 Uppsala, Sweden

Current work on the assaying of neurotrophic activities in chick embryo extract is described. Characteristics from three culture systems are used for these studies: (1) Density of nerve fiber outgrowth from chick ganglia explanted to a collagen gel; (2) Sur­vival of dissociated chick ciliary neurons embedded in small gels of collagen; (3) Length of outgrowing neurite bundles from chick retinal explants placed on collagen gels. Crude extract of the 18 day-old chick embryo enhances all three responses in a dose dependent fashion. The effects are not blocked by antiserum to mouse nerve growth factor (NGF). Chromatography shows that the activities elute as an acidic protein with apparent M.W. of 40,000. However, obser­vations indicate that under certain conditions the activities may dissociate into factors of lower M.W., some of which are recognized by antibodies to mouse a-NGF.

INTRODUCTION

Several aspects of neural development, such as axonal growth and the survival of neurons, are thought to be regulated by epigenetic trophic interactions (reviews by Varon and Bunge, 1978; Varon and Adler, 1980). In most cases, however, the molecular identity of the trophic signals and the regulation of their activity during embryo­genesis remains unknown (see Barde et al., 1983).

It has been demonstrated from this laboratory that explanted tissues from the chick embryo play a supportive role in nerve fiber outgrowth from peripheral ganglia in collagen gel co-cultures

279

280 T. EBENDAL

(Ebendal and Jacobson. 1977; Ebendal. 1979). Furthermore. extracts from the chick embryo have the same effect (Ebendal et al •• 1979. 1983a).

This paper describes present work to further characterize the biological actions and chemical properties of neurotrophic factors extractable from the chick embryo. with a new understanding of their specific actions during development.

MATERIAL AND METHODS

Details of the routine procedures of this laboratory have been given elsewhere. Ciliary. sympathetic and spinal ganglia were dis­sected from the 9 day-old chick embryo (Ebendal et al •• 1979. 1983a). Dissociation of ciliary ganglia (Helfand et al •• 1976) took place as described (Ebendal et al •• 1983a) but with a pre-plating step (2 hours in a tissue culture plastic dish. 1% fetal calf serum) to reduce the number of non-neurons. Explants from the neural retina were taken from the 6 day-old chick embryo by means of a glass capil­lary (Carri and Ebendal. 1983). Collagen gels used as substrata for the bioassays were prepared following Elsdale and Bard (1972). Chromatography was essentially as reported by Ebendal et al. (1979. 1983c).

EMBRYO EXTRACT AFFECTING PERIPHERAL FIBER OUTGROWTH

These studies were carried out with intact ganglia explanted into the collagen gels (Ebendal et al •• 1982. 1983a).

A number of organ extracts were tested by serial dilutions. allowing for a quantitative estimation of the fiber-supporting ac­tivity in various regions of the chick embryo at two stages of de­velopment (Day 8 and Day 18). It was found that the fiber outgrowth followed dose response curves (Ebendal et al •• 1982) and that the responses were roughly parallel in ciliary. sympathetic and spinal' ganglia. However. the level of response varied considerably with the development stage and the organ extracted (Ebendal et al •• 1983a). Thus, the yolk sac was found to be a notably rich source for the nerve-growth-promoting activity on Day 8 whereas the carcass had become the major source (accounting for 90%) by Day 18. The total activity of the nerve growth promoters increased drastically (about 27-fold) from Day 8 to 18. A similar developmental increase in fiber supporting activity. with little evidence of specificity differences between organs, was found also by Hill et al. (1981) using dissoci­ated chick ciliary neurons.

Based on the ciliary response, and the inability of antiserum to nerve growth factor (NGF) to inhibit the effects of chick embryo

NERVE GROvITH PROMOTERS 281

extract it was concluded that the active substance differs from the known a-NGF (Ebendal et al., 1982, 1983a).

NEURON SURVIVAL ENHANCED BY EMBRYO EXTRACT

Moreover, by sectioning cultured intact ciliary ganglia it was established that the embryo extract greatly increased also the sur­vival of the neurons within the explant (Ebendal et al., 1982, 1983a; Hedlund, 1982).

In order to study this survival effect further, dissociated ciliary neurons were trapped in a gel of collagen (Figure 1). Two hundred ~l of the collagen-cell mixture was spread as a thin drop in the center of a 3~ mm culture dish (Ebendal et al., 1983a). After the gel had set, 0.8 ml of medium with the extract was added and the number of neurons counted within a strip across the gel (Figure 1) to obtain initial neuron densities. After two days of incubation neuron density was again determined.

The gels prevent reaggregation of neurons and allow fairly low neuron densities to be used for the assay. The few non-neurons which remain after preplating were also spaced out and do not survive in a medium containing only 1% fetal calf serum. As described in earlier reports (Ebendal et al., 1982, 1983a) chick embryo extracts stimulate survival of dissociated ciliary neurons in the collagen gel. The effect takes place in a saturable dose-dependent manner (Figure 2). At high concentrations of extract neurons undergo lysis, levels of survival again reaching zero (Figure 2). A unit of activity is defined at the 50% survival level (Figure 2; cf Adler et al., 1979).

So far we have found close parallels between the nerve out­growth-stimulating activity measured with intact ganglia and the survival promoting effect measured in dissociated ganglionic neurons (Ebendal et al., 1982, 1983a). It is possible that the two methods measure different aspects of the same active component(s). In this context it is striking that the survival activity also shows the marked developmental increase (Landa et al., 1980; Hill et al., 1981) noted above for fiber outgrowth.

CHICK EMBRYO EXTRACTS AS PROMOTERS OF RETINAL FIBER GROWTH

Circular plugs of the neuroretina from the 6 day-old chick embryo, explanted onto a collagen gel, were used for this study (Carri and Ebendal, 1982, 1983). After explantation, medium with tissue extract was added over the gels and the resulting fiber out­growth measured after four days of incubation. In control medium neurites were sparse, and less than 0.2 mm in length. NGF had no effect on the neurites whereas an extract of the optic lobe of the

282 T. EBENDAL

~ ...... ~ r.. __ ~.-4 • 'a-.~

Fig. 1. The bioassay for ciliary neuron survival activity. Dis­sociated neurons from the chick embryo ciliary ganglion are mixed with a collagen-medium solution, and a 200 ~l drop is spread in a 35 mm culture dish and allowed to gel. The test sample is added in a volume of 0.8 ml culture medium (Eagle's Basal Medium, 1% fetal calf serum) as shown in the lower figure. The number of neurons in the gel is initially about 2,000 (about 800 neurons per cm2 projected area). After two days of incubation (37°C, 5% CO2 ) the number of surviving neurons in the gel is determined (using phase con­trast optics) throughout the thickness of the gel over a strip passing the center (top figure). The strip covers about 5% of the projection of the gel drop.

Day 18 chick embryo stimulated fiber outgrowth in a dose-dependent, fashion with maximum fiber lengths of over 1.2 mm (Ebendal and Carri, 1983).

However, the effect is not specific for the normal target area of the retinal axons. A number of other brain areas as well as peripheral tissues had a similar effect (Carri and Ebendal, 1982; Ebendal et al., 1983b). From a definition of a unit of activity (based on half-maximum outgrowth) the total activity present in the brain and in the periphery was compared with corresponding figures for the fiber outgrowth evoked in peripheral ganglia (Ebendal et al., 1983b). Interestingly, in both cases only 1-2% of the units measured in the 18 day-old chick embryo were present in the brain. Kato et al. (1983) too found that a peripheral chick extract (made from the gizzard) gave rise to fiber outgrowth in chick retinal explants.

NERVE GROWTH PROMOTERS

100

~ en z 0 a: ::) w 50 Z

(!l Z :> :> a: ::) en

10

0 o--n I-f/ i

0 0.002 0.01 0.03 0.13

A280

\f> \ \ \ \ \ \ \

0.53

\ \ 0\

\ \ \ \ \ \ o

2.1

283

Fig. 2. Outcome of an assay for ciliary neuron survival activity in chick embryo extract (as in Figure 1). Th~ fraction of neurons surviving two days in the gel drop ~~ shown. The extract (prepared from entire Day 18 chi~k ~mbryos) was added as a series of twofold dilutions (~Qncentration ex­pressed as. absorption at 280 nm). At high concentrations inhibition resulting in cell lysis is obvious. The con­centration at 50% survival is indicated in the graph (one unit of survival promoting activity per ml).

Such findings naturally raise the question of how specific is the growth stimulation of a certain class of nerve fibers. One attractive idea is that there exists a family of closely related proteins, showing partial overlap in the bioassays, with growth regulating functions for different neurons during development. The neural retina with its highly ordered projection to the tectum seems an ideal system to study this hypothesis. Experiments are in pro­gress to test whether indeed the different parts of the tectum (optic lobe) stimulate fiber growth differentially, depending upon retinal position (Carri and Ebendal, in progress).

CHROMATOGRAPHY OF CHICK EMBRYO EXTRACT

Preliminary attempts to fractionate chick heart extract in order to characterize the promoter of nerve fiber outgrowth in chick ganglia have been presented (Ebendal et a1., 1979). It was found

284 T. EBENDAL

that the activity eluted in a position of 40,000 M.W. upon gel fil­tration.

More recently the entire Day 18 chick embryo was used for ex­traction (in view of the rich and widespread occurrence of nerve growth-promoting activity at this stage). The studies are performed in collaboration with Dr. J. Porath and Dr. M. Belew at the Bio­chemistry Department of Uppsala University, Sweden.

The basic approach uses large scale ion exchange chromatography for the initial processing of the embryo extract. Most of the fiber outgrowth-promoting activity behaves as an acidic protein (Ebendal and Belew, 1980; Ebendal et al., 1983b), but a few percent of the activity appears as a highly basic protein. The biological activity of these two forms are indistinguishable, as both stimulate fiber growth in sympathetic and ciliary ganglia and both remain unaffected by antiserum to S-NGF. Details of the elution pattern of the ac­tivity from anion exchangers are given by Ebendal et al. (1983b,c). The active fractions have been further studied by gel filtration in the presence of urea (Ebendal et al., 1983c). Under these conditions the activity elutes in a region corresponding to 15,000-40,000 M.W., the shift to a lower molecular weight possibly indicating dis­sociation phenomena. Similar findings were reported by Manthorpe et al. (1980), using selected eye tissues from the chick embryo as starting material for chromatography.

Promising methods for further purification include immobilized metal affinity chromatography (Porath et aI., 1975). Under certain conditions the activity is adsorbed to metal chelate gels and can be recovered with an increase in purity.

The application of the three different assays described above to the fractions obtained now gives us the possibility to study whether the measured growth activities separate during chromatography and thus are attributable to distinct molecular species. This study is currently in progress. Preliminary data suggest that the activiti,es co-purify during the first steps of chromatography but further frac­tionation indicated that the retinal fiber-stimulating activity may deviate from the survival and neurite promoting activity for periph­eral neurons.

A most striking observation made during the successive fraction­ations of chick embryo extract is the additional appearance of NGF-like activity after a few steps of chromatography. This finding is based on both a two-site radioimmunoassay for S-NGF (Ebendal et al., 1983c) and the NGF bioassay (Ebendal, 1979; Ebendal et al., 1983c). As stated above, no NGF is found in the crude embryo ex­tract. After the steps of ion exchange chromatography (Ebendal and Belew, 1980; Ebendal et al., 1983b) trace amounts of NGF can be identified by radioimmunoassay and by bioassay {Ebendal et al.,

NERVE GROWTH PROMOTERS 285

1983c). After treatments such as gel filtration in 6 M urea the apparent NGF activity is increased (Ebendal et al., 1983c) and the dense fiber halos evoked in smypathetic ganglia completely blocked by the inclusion of antiserum to mouse S-NGF (unpublished observations). It thus seems that antibodies to mouse S-NGF indeed recognize a chicken NGF given the right conditions, as earlier suggested by blocking experiments in co-cultures between living chicken irides and sympathetic ganglia (Ebendal et al., 1982). Possibly the NGF of the crude extract is strongly complexed and not accessible for the anti­bodies.

The relation between the neurotrophic activities found in the crude embryo extract and after fractionation is not clear at present but is being studied in our laboratory.

Acknowledgements

This work was supported by grants from the Swedish Science Research Council (B-BU 4024-102, S-FO 4024-101). Technical assistance was given by Mrs. Annika Kylberg, Mrs. Stine Soderstrom and Mr. Bo Molin and secretarial help by Mrs. Lilian Moreno.

REFERENCES

Adler, R., Landa, K. B., Manthorpe, M., and Varon, S., 1979, Cholin­ergic neuronotrophic factors: intraocular distribution of trophic activity for ciliary neurons, Science, 204:1434-1436.

Barbin, G., Manthorpe, M., and Varon, S., 1981, Molecular behaviors of the ciliary neuronotrophic factor(s), Soc.Neurosci.Abstr., 7:554.

Barde, Y. -A., Edgar, D., and Thoenen, H., 1983, New neurotrophic factors, Ann.Rev.Physiol., 45:601-612.

Carri, N. G., and Ebendal, T., 1982, Distribution of a chick embryo factor promoting growth of retinal axons, Soc.Neurosci.Abstr., 8:299.

Carri, N. G., and Ebendal, T., 1983, Organotypic cultures of neural retina: neurite outgrowth stimulated by brain extracts, Develop.Brain Res., 6:219-229.

Ebendal, T., 1979, Stage-dependent stimulation of neurite outgrowth exerted by nerve growth factor and chick heart in cultured embryonic ganglia, Develop.Biol., 72:276-290.

Ebendal, T., and Jacobson, C. -0., 1977, Tissue explants affecting extension and orientation ofaxons in cultured chick embryo ganglia, Exp.Cell Res., 105:379-387.

Ebendal, T., and Belew, M., 1980, Chick heart factor controlling neurite extension, Eur.J.Cell BioI., 22:409.

Ebendal, T., Belew, M., Jacobson, C. -0., and Porath, J., 1979, Neurite outgrowth elicited by embryonic chick heart: partial purification of the active factor, Neurosci.Lett., 14:91-95.

286 T. EBENDAL

Ebendal, T., Hedlund, K. -0., and Norrgren, G., 1982, Nerve growth factors in chick tissues, J.Neurosci.Res., 8:153-164.

Ebendal, T., Norrgren, G., and Hedlund, K. -0., 1983a, Nerve growth­promoting activity in the chick embryo: quantitative aspects, Med.Biol., 61:65-72.

Ebendal, T., Norrgren, G., Carri, N. G., and Belew, M., 1983b, Nerve growth factors in peripheral tissues and CNS, in: "Recent Achievements in Restorative Neurology", Vol.1,~. Dimitrijevic and Sir John Eccles, eds., Raven Press (in press).

Ebendal, T., Olson, L., Seiger, A., and Belew, M., 1983c, Nerve growth factors in chick and rat tissues, in: "Cellular and Molecular Biology of Neuronal Development": I. Black, ed., Plenum, New York (in press).

Elsdale, T., and Bard, J., 1972, Collagen substrata for studies on cell behavior, J.Cell BioI., 54:626-637.

Hedlund, K. -0., 1982, Growth promotion in developing ganglionic neurons: ultrastructural aspects, Acta Univ.Ups.Abstr.Upps. Diss.Fac.Sci., 639, 16 pp.

Helfand, S. L., Smith, G. A., and Wessells, N. K., 1976, Survival and development in culture of dissociated parasympathetic neurons from ciliary ganglia, Develop. BioI. , 50:541-547.

Hill, C. E., Hendry, I. A., and Bonyhady, R. E., 1981, Avian para­sympathetic neurotrophic factors: age-related increases and lack of regional specificity, Develop.Biol., 85:258-261.

Kato, S., Negishi, K., Hayashi, Y., and Miki, N., 1983, Enhancement of neurite outgrowth and aspartate-glutamate uptake systems in retinal explants cultured with chick gizzard extract, J.Neurochem., 40:929-938.

Landa, K. B.,·Adler, R., Manthorpe, M., and Varon, S., 1980, Cholin­ergic neuronotrophic factors III. Development increase of trophic activity for chick embryo ciliary ganglion neurons in their intraocular target tissues, Develop.Biol., 74:401-408.

Manthorpe, M., Skaper, S., Adler, R., Landa, K., and Varon, S., 1980, Cholinergic neuronotrophic factors: fractionation properties of an extract from selected chick embryonic eye tissues,

_ J.Neurochem., 34:69-75. Porath, J., Carlsson, J., Olsson, I., and Belfrage, G., 1975, Metal

chelate affinity chromatography, a new approach to protein fractionation, Nature, 258:598-599.

Thoenen, H., and Barde, Y. -A., 1980, Physiology of nerve growth factor, Physiol!Rev., 60:1284-1335.

Varon, S. S., and Bunge; R. P., 1978, Trophic mechanisms in the peripheral nervous system, Ann.Rev.Neurosci., 1:327-361.

Varon, S., and Adler, R., 1980, Nerve growth factors and control of nerve growth, Ctirr.Top.Develop.Biol., 16:207-252.

THE ROLES AND LIMITATIONS OF GROWTH FACTORS IN

NEURONAL DEVELOPMENT

David Edgar

Max-Planck-Institute for Psychiatry

The differentiation of neuronal and glial cells results from the partial and selective expression of their common genome. Although the molecular mechanisms quantitatively regulating gene expression in the adult are well investigated, little is known about those mechan­isms which determine the qualitative changes that occur during dev­elopment (Thoenen and Edgar, 1982). The peripheral nervous system of the chick embryo lends itself to the analysis of mechanisms involved in neural development not only because its ganglia contain compara­tively few cell types, but also because the ontogenesis of the cell lineages which constitute the peripheral ganglia is extensively described (Le Douarin, 1981).

Our work is concerned with the maturation of neurons in periph­eral ganglia and their anlagen, presumably after the neuronal stem cells from the neural crest have diverged from the glial cell line­age. During this maturation, two phenomena may be defined: the trophic support of survival of the young neurons, and the acquisition of the mature neuronal phenotype. There is good evidence that deter­mination of neuronal survival is epigenetically controlled (for review see Hamburger and Oppenheim, 1982), and there is also some evidence that the neuronal phenotype may at least partially be deter­mined by environmental factors (for review see Landis and Patterson, 1981). Consequently, we have employed tissue-culture techniques to investigate the mechanisms involved; young neurons are isolated from their normal environment in vivo, the object being to determine the requirements of the cultured neurons in order to survive, and express either the phenotype they would normally acquire in vivo or an "abnormal" phenotype, such as the expression of cholinergic proper­ties by sympathetic neurons which would normally mature as adrenergic neurons in vivo. A reservation applies to all such experiments

287

288 D. EDGAR

however, in that although the behavior of neurons in culture might mimic their behavior in vivo, this does not necessarily mean that the factors operating in vitro are the same as those responsible during normal development.

By culturing neurons isolated from the spinal sensory and sym­pathetic ganglia from chicks of different embryonic ages, it was demonstrated that such neurons have multiple and changing require­ments for macromolecular survival factors during development (Barde, et al., 1980; Edgar et al., 1981). In addition to the well charac­terized protein, nerve growth factor (NGF), which has been shown to be necessary for the survival of mammalian sympathetic and sensory neurons in vivo, it could be shown that a variety of tissue extracts also supported neuronal survival. The isolation and characterization of these new neurotrophic molecules has been discussed elsewhere (Barde et al., 1983) however the experiments which showed that the tissue extracts contained factors which are functionally distinct from NGF also revealed that there are distinct subpopulations of neurons present in both sensory and sympathetic ganglia. Thus, while maximally half the neurons from spinal sensory ganglia will survive with NGF alone, in the presence of NGF and the neurotrophic molecule present in brain extracts (Barde et al., 1982) essentially all sensory neurons survive in culture: the ganglionic neurons can there­fore be classified into subpopulations, based on their requirements for survival factors. Although somewhat unorthodox, survival requirements can be regarded as neuronal phenotypes (perhaps due to the expression of receptors for a given factor), and the correlation between survival factor requirement and other neuronal markers is of interest because it would reflect co-expression of individual parts of the neuronal genome which could be a consequence of a common regulatory mechanism.

By determination of the conventional neuronal markers choline acetyl transferase and tyrosine hydroxylase in sub-populations of chick sympathetic neurons selected in culture by their survival factor requirements, it could indeed by shown that the growth factor requirement of a given sub-population was correlated with its part­icular neurotransmitter synthesizing enzyme (Edgar et al., 1981; Rohrer et al., 1983). Thus those sympathetic neurons which survive in response to NGF alone are predominantly adrenergic, whereas that subpopulation which requires survival factors other than NGF (e.g. heart cell conditioned medium) contains neurons which have a cholinergic phenotype. It should be stressed that in these exper­iments the survival factors used are not inducing the appearance of phenotypes, but rather sel- ecting subpopulations of neurons whose distinctive phenotypes can be demonstrated in vivo.

The mechanism by which sub populations of ganglionic neurons arise, displaying different enzymic markers and survival factor requirements, is still an open question. To approach this problem we

FACTORS IN NEURONAL DEVELOPMENT 289

have recently begun to analyze the development of a spectrum of neuronal markers, in particular peptide putative neurotransmitters, during the ontogenesis of sympathetic and spinal ganglia of the chick embryo. The general conclusion emerging from these observations is that expression of the peptides substance P, somatostatin and vaso­active intestinal polypeptide is differentially regulated throughout embryogenesis (Hayashi et al., 1983). While neuronal survival factors are unlikely to be responsible for such differentiation they can, however, modulate the expression of a given phenotype once it has been induced, demonstrated by the increased levels of tyrosine hydroxylase and substance P in the sympathetic and sensory nervous systems brought about by increased levels of NGF (Thoenen and Edgar, 1982). Similarly, local environmental factors within the ganglia alone are unlikely to be responsible for the production of different neuronal phenotypes, although conditions within the ganglion can markedly affect development in other ways: cell contact phenomena are "permissive" in that they can determine if a neuron will survive in response to a given growth factor (Edgar and Thoenen, 1982), and also determine the levels of expression of particular genes, presumably once they are induced (Thoenen and Edgar, 1982). The possible mechanisms responsible for the induction of heterogeneous neuronal phenotypes within the same ganglion to be considered include analysis of the dates of birth (and possible lineage) of distinct neuronal sUbpopulations, together with determination of their afferent connec­tions and sites of projection during development.

REFERENCES

Barde, Y.-A., Edgar, D., and Thoenen, H. , 1980, Proc.Natn.Acad.Sci. USA. , 77: 1199-1203.

Barde, Y .-A., Edgar, D., and Thoenen, H. , 1982, EMBO J., 1:549-553. Barde, Y.-A., Edgar, D., and Thoenen, H. , 1983, Annu.Rev.PhIsiol.,

45:601-612. Edgar, D., Barde, Y.-A., and Thoenen, H., 1981, Nature (Lond.) 289:

294-295. Edgar, D., and Thoenen, H., 1982, Dev.Brain Res., 5:89-92. Hamburger, V., and Oppenheim, R. W., 1982, Neuroscience Commentaries,

Vol.I, pp 39-55. Hayashi, M., Edgar, D., and Thoenen, H., 1983, Neuroscience,

10:31-39. Landis, S. C., and Patterson, P. H., 1981, Trends in NeuroSci.,

4:172-175. Rohrer, H., Thoenen, H., and Edgar, D., 1983, Develop.Biol.,

99:34-40.

CELL INTERACTIONS DURING FORMATION OF THE NEUROMUSCULAR JUNCTION.

THE SEARCH FOR MUSCLE-DERIVED MOTONEURON GROWTH FACTORS

Christopher E. Henderson

Neurobiologie Moleculaire Institut Pasteur. 25 rue du Dr. Roux 75015 Paris. France

The cellular environment of the spinal motoneuron during dev­elopment of the nervous system is particularly complex. Its cell body and dendrites. enveloped by central glia within the spinal cord, receive many presynaptic afferents. while the axon. during its growth into the periphery, crosses several different tissues before contact­ing myoblasts in the differentiating muscle masses and before itself being myelinated. It is conceivable that all these cell types play a role in regulating the differentiation of the motoneuron during embryogenesis.

One of this complex set of reciprocal interactions has often been singled out for study: that between motoneurons and skeletal muscle. Cell interactions at the nerve-muscle junction may be classed either as "anterograde" or "retrograde" (Changeux. 1979). Examples of anterograde interactions would be the effect of nerve­derived substances on acetylcholine receptor aggregation or the effect of the pattern of motoneuron activity on the synthesis of different contractile proteins. In this article. however. I shall discuss only the retrograde effects of muscle on motoneuron development.

In the chick embryo. if a limb bud is extirpated before it has been innervated. all of the motoneurons destined to innervate it die during the subsequent wave of naturally occuring cell death. as opposed to about 50% in controls (Hamburger. 1934). On the other hand. if a supernumerary limb is implanted. about 25% of the neurons that would normally die can be rescued (Hollyday and Hamburger. 1976). If synaptic transmission at the neuromuscular junction is blocked. then many more motoneurons than normal survive (Pittman and Oppenheim, 1978). These results and others gave rise to the idea

291

292 C. E. HENDERSON

(which has been contested; see Lamb, 1980) that muscles produce a growth factor(s) upon which motoneurons depend for their survival at certain stages of development. Following the formation of the first synapses, the amount of growth factor produced might decrease, lead­ing to competition between motoneurons for a limited stock of factor. The number of motoneurons surviving this competition would increase in situations where more target tissue was present (supernumerary limb) or where the decrease in production of the factor was prevented (synaptic blockade).

The importance of the target in the regulation of axon outgrowth is intrinsically more difficult to test in vivo. Early stages of axon outgrowth may occur normally even after limb extripation (Oppenheim et al., 1978), but it has been reported that in chick wings devoid of muscle, the muscle nerve branches do not develop (Lewis et al., 1981).

In other systems, two molecules have been shown to have proper­ties that fit them for the role of retrograde growth factor: the Nerve Growth Factor, NGF, and a factor purified from pig brain (see Y.-A. Barde, this volume). The purified NGF molecule enhances sur­vival, neurotransmitter synthesis and neurite outgrowth in cultures of sensory neurons. Neither factor has any reported effect on spinal motoneurons.

Much less is known about factors acting at the neuromuscular junction, although considerable progress has been made in the study of molecules.affecting neurons of the cholinergic ciliary ganglion (see C. R. Bader et al., T. Ebendal, this volume). Various workers have reported effects of tissue extracts and conditioned media on three parameters of the development of spinal neurons or identified motoneurons in vitro: (i) cell survival; (ii) acetylcholine syn­thesis; and (iii) neurite outgrowth (for refs., see Henderson et al., 1983b). We have used dissociated chicken spinal neurons to investi­gate the effects or muscle-derived substances on one of these para­meters: neurite outgrowth.

NEURITE-PROMOTING ACTIVITY IN EMBRYONIC MUSCLE-CONDITIONED MEDIUM ("ECM")

When dissociated spinal neurons from 4~-day chicken embryos were cultured in serum-free F12 medium on uncoated plastic dishes, about 5% of the cells developed neurites after 20 h in vitro (Figure 1: F12). In parallel cultures to which serum-free medium conditioned over primary cultures of embryonic myotubes had been added, the percentage of cells with neurite was about 35% (Fig. 1: ECM). This response was dose-dependent and allowed the determination for any tissue extract or conditioned medium of a specific activity value, expressed in units/mg protein. No effect on cell survival was ob­served under these conditions (Henderson et al., 1981).

MOTONEURON GROHTH FACTORS 293

Fig. 1. Effects of embryonic muscle-conditioned medium on neurite outgrowth in cultures of embryonic chicken spinal neurons. Phase-contrast micrographs are shown of cells cultured for 21 h in: "F12", F12 medium without serum; "ECM", F12 medium supplemented with muscle-conditioned medium (dilution 1:20). Culture methods were as described in Henderson et al., 1983a.

Using this rapid quantitative assay, a preliminary character­ization of the active factor(s) in embryonic muscle-conditioned medium ("ECM") was performed. It was associated with macromolecular species of molecular weights 40 kDa and greater, was trypsin­sensitive but resistant to 0.1% (w/v) sodium dodecyl sulphate. Activity was not found in media conditioned over primary cultures of several other tissues from the same embryos. Moreover, the activity in ECM did not significantly affect neurite outgrowth from dorsal root ganglion explants or PC12 cells (R. Defez and C. E. Henderson, unpublished). It thus showed at least some specificity for the interaction between spinal neurons and muscle. Contrary to what has been observed for certain other neurite-promoting activities (Collins, 1978), the factor in ECM was active even under conditions in which it did not bind to the culture substratum.

We have no direct evidence that the responsive cells in our cultures were motoneurons, although the use of early embryos (Berg

294 C. E. HENDERSON

and Fischbach, 1978) and numerical arguments (Henderson, 1983) make it conceivable that they were. More than 93% of the cells in these spinal cord cultures contained neurofilament subunits, as revealed by indirect immunofluorescence, and it is likely that similar popu­lations of cells survived in all culture conditions tested (Henderson et al., 1983b).

NEURITE-PROMOTING ACTIVITY IN POSTNATAL MUSCLE EXTRACTS ("PNME")

High-speed supernatants of homogenates of neonatal chick leg muscle (PNME) also contained a neurite-promoting activity for these spinal neurons (Henderson et al., 1983b). The specific activity in extracts from chicks of different ages increased sharply (ca. 10-fold) after hatching up to a peak at 3-5 days, and by 7 days had fallen back nearly to its original levels (not shown).

The active factor(s) in PNME had physicochemical properties that seemed to distinguish it from the factor in ECM. Also resistant to 0.1% SDS, it nevertheless was considerably more resistant to trypsin and bound to the culture substratum under all cell culture conditions tested. Similar levels of activity were found in soluble extracts of many organs of the same chick; its distribution was thus less tissue­specific than that of the factor in ECM.

EFFECTS OF DENERVATION

Partial denervation of mammalian muscles under certain con­ditions leads to "sprouting" of the remaining intramuscular axons and reinnervation of those fibres denervated. It has been proposed that the denervated fibres release a sprouting factor, capable of diffus­ing a limited distance within the muscle (Brown et al., 1981).

After transection of the sciatic nerve of 6-d-old chicks, the specific neurite-promoting activity in extracts of the denervated ' muscles increased up to 15-fold in the 3-5 days following denervation (Fig. 2; Henderson et al., 1983a). This increase was not observed in the contralateral leg or in sham-operated chicks. Its appearance at the high specific activity of 10,000 units/mg protein was thus a specific and local response to denervation whose time course in chicks was comparable to that of the sprouting observed in mammalian muscles. It is not yet known whether the activity produced in re­sponse to denervation has the physicochemical properties of the factor in ECM or of that in PNME.

Similar results have been obtained concerning the production of NGF by the rat iris (see T. Ebendal, this volume), and of factors affecting the survival of identified spinal motoneurons (Slack and Pockett, 1982) or ciliary neurons (Hill and Bennett, 1983). It would

MOTONEURON GROWTH FACTORS 295

8 4

3

4\ 1/ 2 ir------1

days a Iter denervat ion

Fig. 2. Changes in the specific neurite-promoting activity of muscle extracts following denervation. 6-day-old chicks from (A) Warren or (B) SV15 Vedette strains were denervated (e) or maintained as unoperated controls (0). Specific activities were determined from dose-response curves (1 unit = amount of extract required to give 50% of maximal neurite­outgrowth response). Results are expressed as the mean ± range of results from two different chicks.

thus seem reasonable to suppose that the synthesis of retrograde growth factors is regulated according to the state of innervation of the target tissue.

CONCLUSIONS

We have detected neurite-promoting activities for embryonic spinal neurons at three different stages of development, with bio­logical properties that are suggestive of a physiological role for each of them. In the case of the activity in ECM, only on cell type other than muscle cells produced significant amounts in vitro (Henderson et al., 1981). Levels of activity in PNME were develop­mentally regulated, reaching a peak in muscles of the lower leg at 3 days after hatching (Henderson et al., 1983b). The production of a similar activity was closely linked to the state of innervation of the muscle (Henderson et al., 1983a).

It is tempting to correlate these growth-promoting activities with certain stages of motoneuron development in vivo: initial in­vasion of the differentiating muscle masses, stabilization of nerve

296 C. E. HENDERSON

terminals during the regression of multiple innervation, and regener­ation after injury, for instance. However, only after purification of the active factors involved and characterization of the responsive neurons will it be possible seriously to test these hypotheses.

Acknowledgements

I thank Jean-Pierre Changeux, in whose laboratory this work was done, and Monique Huchet, who helped to perform all experiments described. Anti~neurofilament antibodies were the generous gift of Denise Paulin, Institut Pasteur.

REFERENCES

Berg, D. K., and Fischbach, G. D., 1978, Enrichment of spinal cell cultures with motoneurons, J.Cell BioI., 77:83-98.

Brown, M. C., Holland, R. L., and Hopkins, W. G., 1981, Motor nerve sprouting. Ann.Rev.Neurosci., 4:17-42.

Changeux, J. P., 1979, Molecular interactions in adult and developing neuromuscular junction, in: The Neurosciences Fourth Study Program (MIT Press, Cambridge, MA), pp.749-778.

Collins, F., 1978, Induction of neurite outgrowth by a conditioned medium factor bound to culture substratum, Proc.Natl.Acad. Sci., 75:5210-5213.

Ebendal~., Olson, L., Seiger, A., and Hedlund, K.-O., 1980, Nerve growth factors in rat iris, Nature, 286:25-28.

Hamburger, V., 1934, The effects of wing bud extirpation on the development of the central nervous system in chick embryos, J.Exp.Zool., 68:449-494.

Henderson, C. E., 1983, Roles for retrograde factors in synapse formation at the nerve-muscle junction, Prog.Brain Res., 58:369-373.

Henderson, C. E., Huchet, M., and Changeux, J. P., 1981. Neurite outgrowth from embryonic chicken spinal neurons is promoted by media conditioned by muscle cells. 'Proc.Na~l.Acad.Sci •• USA.· 78:2625-2629.

Henderson. C. E •• Huchet, M., and Changeux. J. P •• 1983a. Denervation increases a neurite-promoting activity in extracts of skeletal muscle. Nature. 302:609-611.

Henderson. C. E •• Huchet. M., and Changeux. J. P., 1983b, Neurite promoting activities for embryonic spinal neurons and their developmental changes in the chick. Dev.Biol •• (submitted).

Hill. M. A •• and Bennett. M. R •• 1983. Cholinergic growth factor from skeletal muscle elevated following denervation. Neurosci. Lett., 35:31-35.

Hollyday. M •• and Hamburger. V •• 1976. Reduction of the naturally occurring motor neuron loss by enlargement of the periphery. J.Comp.Neurol., 170:311-320.

MOTONEURON GROWTH FACTORS 297

Lamb, A. H., 1980, Motoneurone counts in Xenopus frogs reared with one bilaterally-innervated hindlimb, Nature, 284:347-350.

Lewis, J., Chevallier, A., Kieny, M., and Wolpert, L., 1981, Muscle nerve branches do not develop in chick wings devoid of muscle, J. Embryol.Exp.Morph., 64:211-232.

Oppenheim, R.W., Chu-Wang, I.-W., and Maderdrut, J. L., 1978, Cell death of motoneurons in the chick embryo spinal cord. The differentiation of motoneurons prior to their induced degener­ation following limb-bud removal. J.Comp.Neurol., 177:87-112.

Pittman, R., and Oppenheim, R. W., 1978, Neuromuscular blockade increases motoneurone survival during normal cell death in the chick embryo. Nature, 271:364-366.

Slack, J. R., and Pockett, S., 1982, Motor neurotrophic factor in denervated adult skeletal muscle. Brain Res., 247:138-140.

A SPINAL CORD DERIVED NEUROTROPHIC GROWTH FACTOR FOR

SPINAL NERVE SENSORY NEURONS

SUMMARY

Ronald M. Lindsay and Caroline Peters

Laboratory of Neurobiology National Institute for Medical Research The Ridgeway, Mill Hill, London

It has previously been shown that glioma cells[l], brain ex­tracts[2] and astroglial cells from adult brain[3,4,5] contain neuro­trophic activity that supports survival and outgrowth of neurites from sensory neurons of the chick embryo dorsal root ganglion main­tained in culture. Surprisingly during these studies we were unable to detect similar ·neurotrophic activity in spinal cord extracts of embryonic chicks of up to age E16[2]. Considering spinal cord to be a more appropriate source than brain of neurotrophic activity for spinal nerve sensory neurons, we have extended our study to examine the neurotrophic properties of extracts of spinal cord from chicks of embryonic age E18 to 12 weeks post-hatching.

Our findings indicate that spinal cord tissue does in fact contain a survival/neurotrophic growth factor for sensory neurons, but that this activity is not readily detectable until after hatching whereupon it increases rapidly towards an adult level. The neuro­trophic growth factor present in spinal cord extracts is immunologic­ally and functionally distinct from the well characterized mouse submandibular gland nerve growth factor (NGF). In contrast to the latter, spinal cord neurotrophic activity has little effect upon the survival of early post-mitotic (E6-E8) sensory neurons but is capable of promoting survival and neurite outgrowth from most neurons of the E12-E16 chick embryo dorsal root ganglion in tissue culture. We have found similar activity in adult rat and adult human spinal cord, where again the levels in perinatal tissue are below detection. The neurotrophic activity of spinal cord extracts seems to be at least partially specific for sensory neurons as it has little or no effect upon comparable age sympathetic neurons.

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300 R. M. LINDSAY AND C. PETERS

INTRODUCTION

There is now substantial evidence to support the hypothesis that some if not all types of neurons depend for their survival and normal maintenance upon exogenous growth promoting substances. Such neuro­or neuronotrophic growth factors are thought to be derived from the target cells which neurons innervate or from the non-neuronal, es­pecially glial, component of neural tissue. Until recently, however, this theory has hinged upon a single, albeit well documented case, that of nerve growth factor (NGF), a now fully characterized protein isolated in high yield from salivary glands of the adult male mouse. It has been demonstrated in tissue culture and to some extent in vivo that NGF promotes the survival and outgrowth of neurites from sym­pathetic neurons at all stages of development and survival and neurite extension from spinal nerve sensory neurons at early stages in embryonic development (see ref. 6 for a recent review of NGF). NGF is not, however, a ubiquitous neurotrophic agent, as few if any central nervous system neurons respond to it, as do neither para­sympathetic neurons nor sensory neurons of cranial nerve ganglia derived from the neural placodes (as opposed to those of neural crest origin).

Given the restricted specificity of NGF, its limited develop­mental role in the maintenance of sensory neurons of neural crest origin[7] (dorsal root ganglion cells), and the apparent lack of NGF in appropriate peripheral target tissues[8], we have been interested in looking for novel neurotrophic growth factors in peripheral and central target tissues. We have compared the response of sensory neurons from cranial and spinal nerve ganglia to NGF and such neuro­trophic substances detectable in tissue extracts[2], co-cultured cellsL3,4,5] and conditioned medium from cultured cells[l]. This report deals with our recent finding of neurotrophic activity dis­tinct from NGF in extracts of chick, rat and human spinal cord.

METHODS

Our studies have centered on the use of tissue culture as a convenient method for the detection and quantitation of neurotrophic activity in tissue extracts, conditioned medium, etc. Most of our experiments are described within the range provided by altering two simple parameters - (a) the test neuron (b) the source of neuro­trophic activity.

Test Neurons

Dorsal root ganglia (DRG), nodose sensory ganglia (NG = inferior ganglion of the Xth cranial nerve), trigeminal sensory ganglia (TG) of the Vth cranial nerve and paravertebral sympathetic chain ganglia (SC) were dissected from chick embryos of 6 - 16 days incubation (E6-E16). Cultures were established in which ganglia were maintained

GRm-lTH FACTOR FOR SENSORY NEURONS 301

as whole explants (neurons + non-neuronal cells) or in which the ganglia were dissociated into a suspension of single cells and en­riched for neurons prior to plating. In the former cultures 4 - 6 ganglia were explanted into 1 ml of collagen gel solution in a 35mm culture dish, and medium (Eagle's minimum essential medium - MEM) with 2% chick serum, 5% horse serum and any test neurotrophic sub­stance (10 - 500 ~l of tissue extract etc.) to a total volume of 1.5 ml was overlaid after the gel had set. The extent of fibre outgrowth was assessed after 1 - 7 days, using an arbitrary scale of 0 - 5 with reference to a scale of standards for a dose response of E10 DRG to NGF. For "pure" neuronal cultures, pooled ganglia treated with 0.1 -0.25% trypsin for 10 - 30 min. depending on age, were washed 3x with MEM and dissociated by a combination of gentle vortexing and tritura­tion through the bore of a siliconized glass pipette. Neuronal enrichment was achieved by the preplatihg procedure of McCarthy and Partlow[9]. The neuron-enriched cell suspension was plated, after counting, at 5,000 - 15,000 viable neurons per 35mm culture dish, the latter having been first precoated with poly-L-Iysine followed by a thin film of collagen gel. Medium, serum and test growth factor were added to a total volume of 1.5 ml as for explant cultures. Neuronal survival (phase-bright neurons with neurites at least 4 - 5 x longer than the diameter of the cell perikaryon) was determined by strip­counting of the culture dish at a minimum of 48h after plating. This parameter is used as a quantitative measure of neurotrophic activity.

Source of Neurotrophic Activity

Spinal cord tissue was dissected from chicks of age E16 to 12 weeks post-hatching, taking great care to ensure that no spinal ganglia remained attached and removing as much of the meninges as possible. Tissue was washed twice in phosphate-buffered saline, frozen directly on dry-ice and stored at -80°C until required. Tissue extracts were prepared by homogenizing 1 gm of tissue (wet wt.) per 10ml of MEM in a Sorval Omnimix for 30 secs., followed by 3 - 4 strokes in a Thomas teflon coated tissue grinder. Homogenates were centrifuged first at 30,000g for 30 min. and then at 170,000g for 20 h in a Beckman 42.1 rotor. Floating lipid material was re­moved from on top of the high-speed supernatant, before carefully withdrawing the upper 80% of the clear supernatant. This was fil­tered through a sterile 0.2 ~ Millipore filter and stored at -20°C until used.

RESULTS

Explant Culture

In accordance with previous reports[2,10,11] spinal cord extract from chick embryos of age E16 - E18 or younger produced little or no fibre outgrowth from explanted DRG from chicks between E8 - E16. As

302 R. M. LINDSAY AND C. PETERS

shown in Figure 1, however, significant neurotrophic activity was found in the extract of P5 chick spinal cord (P = post-hatching) and by P10 to P14 sufficient activity was present to produce maximal fibre outgrowth (halo) from the E12 DRG.

In contrast to the activity of NGF but comparable to that of brain extract[12], the neurotrophic growth factor present in post­hatched spinal cord has no effect upon early post-mitotic neurons (E8). Some fibre outgrowth is elicited from E10 DRG, but maximal response to spinal cord extract is only seen with E12 or older DRG. Spinal cord extract differs from NGF in other ways (i) NGF elicits a fibre outgrowth from responsive ganglia that is readily detectable within 12 hours of explantation, and a maximal halo of fibres is often seen at 24 h. Halo formation by spinal cord extract seems to be delayed by several hours in comparison to NGF, with maximal response only appearing at 36 - 48 h. (ii) after a week or more in culture (with or without a medium change) fibre outgrowth production by E12 ganglia in response to NGF is usually obscured by the migration and proliferation of ganglionic non-neuronal cells. We believe it important to note that with chick spinal cord extract, however, this migration and proliferation of non-neuronal cells appears to be partially inhibited and a vigorous outgrowth of fibres is still detectable in explants maintained in culture for 10 days or more, Figure 3.

,..... :::> .... as

5

~ 4 .s= i o 3 "­til -:::> o 2

E 12 DRG Explant Cultures

400J,J1

200J,J1

50111

50 eo 70 eo

Age of Chick Spinal Cord Extract Prep.

Fig. 1. Developmental appearance of neurotrophic activity in chick spinal cord. Response of E12 chick dorsal root ganglion explants to 50, 200 and 400 J.il of spinal cord extract from chicks of E16 to 12 weeks post-hatching. Results are a measure of fibre outgrowth (halo size in arbitrary units) after 48 h, mean + s.e.m. (n=6).

GROWTH FACTOR FOR SENSORY NEURONS 303

Fig. 2. Comparison of E12 DRG explants after 10 days in culture with either (A) spinal cord extract, 300 ~l of 4 wk chick cord or (B) nerve growth factor, 20 ng mouse NGF. Note massive fibre outgrowth still visible in (A) with little non-neuronal cell spread. In (B) neurites are only· visible at the extremity of the explant due to migration and proliferation of non-neuronal cells.

The developmental increase in neurotrophic activity observed in spinal cord extracts from P5 and older chicks is not accounted for by any dramatic change in the total protein content of cord tissue over this period, as this value fluctuates by less than 20% (2.5 - 2.8 mg protein/ml extract) over the entire age range of Figure 1. In all ages where detectable the activity in spinal cord extract produces a dose dependent response. In PlO or older chicks increasing activity is seen between 50 - 400 ~l of extract, Figure 1, after which a plateau is reached. With E16 DRG in explant culture the response to spinal cord extract is greater than with any concentration of NGF, but not as pronounced as that seen with E12 DRG. If, however, the ganglia are pulsed with anti-mitotic agents (lO-SM cytosine arabino­side, fluorodeoxyuridine and uridine) during the first 24 h in ex­plant culture, then both E12 and E16 explants (not shown) show equally massive fibre outgrowth in response to active spinal cord extract. E8 ganglia, in contrast, are unresponsive to spinal cord neurotrophic activity under any conditions.

304 R. M. LINDSAY AND C. PETERS

Dissociated Neuronal Cultures

To confirm that the activity of spinal cord extracts seen in explant cultures is neurotrophic and not acting through some indirect action on ganglionic non-neuronal cells, all experiments with explant cultures were repeated with dissociated neuron-enriched cultures, freed of fibroblasts and Schwann cells. As shown partly in Figure 3 a very similar pattern of results was seen under these conditions (i) neurotrophic activity is not detectable in spinal cord extracts of chick embryos up to E18, but is readily detectable within a week of hatching and reaches an adult level at P14. (ii) the activity pre­sent in spinal cord extract is directed towards maturing DRG neurons, at a time when their known response to NGF is declining. (iii) dissociated neuronal cultures show a dose response to active spinal cord extract that parallels observations with explant cultures.

-CIS

CIS C o ~

:::> Q)

Z

Dissociated DRG Cultures

3

2

E8 neurons

~10L!~:dO~==1~O~=;2~O==~30~===4~OE=~SO~==~80~==~7;O==~80;=~~

Age of Chick Spinal Cord Extract Prep.

Fig. 3. Comparative response of dissociated, enriched neurons from. E8 or E12 chick DRG to spinal cord extract (100 pI) from E16 to 12 week-old chicks. Prior to hatching there is little neurotrophic activity in spinal cord extracts for spinal nerve sensory neurons. Early embryonic neurons (E8) are not responsive to spinal cord extract from any age but extracts from P5 or older chicks support survival and neurite out­growth from more than 50% of E12 DRG neurons. Results are from triplicate dishes in which 7,000 - 8,000 viable neurons were originally seeded. Neuronal survival was determined at 48 h as described in Methods. In all the above experiments the addition of antiserum to mouse NGF, at up to 50 pg/ml (enough to block the activity of 100 - 200 ng of NGF) had no effect on the ability of spinal cord extract to elicit fibre outgrowth from responsive DRG.

GROWTH FACTOR FOR SENSORY NEURONS 305

In addition to testing spinal cord extracts from chick tissue on leuronal cultures, extracts from perinatal and adult rat and human ;pinal cord were examined for neurotrophic activity. Neither rat or luman perinatal tissue (all human tissue was 12 - 14 h post-mortem) :ontained detectable activity but extracts of adult spinal cord from loth species promoted the survival and outgrowth of neurites from E12 :hick DRG. Adult rat tissue contained 85 - 90% of the activity found In 4 wk post-hatched chick spinal cord, whilst two samples of adult luman tissue contained 30 - 40% of this level. The lower level in luman tissue may well be due to the 12 - 24 hour delay before tissue :ould be frozen.

Finally, a comparison was made of the response of spinal, no­lose, and trigeminal sensory neurons and paravertebral chain sympath­~tic neurons to spinal cord extract from 4 wk (P28) chick. Sympath­~tic neurons of E12 chick embryo showed little response to spinal :ord extract, whereas NGF was capable of supporting the survival of ilmost all of these neurons in dissociated culture. Neurons of the ~12 trigeminal ganglion were almost as responsive as DRG neurons to ;pinal cord extract, and although survival of nodose ganglion neurons In vitro was promoted by the same neurotrophic activity this was less narked than with sensory neurons from the other ganglionic sources. ~ detailed comparative age series with cranial nerve ganglia is still In progress.

;ONCLUSIONS

In a previous study[2] we noted that neurotrophic activity for )eripheral neurons could be detected in many tissues of the develop­Lng chick embryo, with the notable exception of the spinal cord. ~ilst the level of neurotrophic activity for DRG neurons varied ~reatly from tissue to tissue, embryonic spinal cord extract appeared :0 have an almost inhibitory effect on fibre outgrowth from explants )r dissociated neurons. This observation is somewhat in conflict v.ith other results of our own[1-5] and others[7,12] which cumulat­lvely suggest that survival and maintenance of maturing DRG neurons Ls not dependent on NGF[13] but rather on neurotrophic activity lerived from central nervous tissue[2,7] and specifically from astro­~lial cells[1,3]. That neurotrophic activity for spinal nerve sen­;ory neurons should be present in brain, in most cases at consider­ible distance from the responsive neuron, and not in the obvious !entral target i.e. spinal cord, seemed contradictory to current ldeas. We believe that we have now solved this apparent dilemma with )ur present observations which indicate that spinal cord does contain leurotrophic activity for spinal nerve sensory neurons but that this lctivity is only detectable at a late stage in development. The leurotrophic activity of spinal cord appears to be functionally ~imilar to that found in brain[12], but is immunologically and funct­lonally distinct from NGF.

306 R. M. LINDSAY AND C. PETERS

The physiological relevance of spinal cord derived neurotrophic growth factor remains to be established, but we would predict that with refined technique such a growth factor will be found even earlier in development than suggested by our current results. This prediction takes into account the inevitable loss of neurotrophic activity due to proteolysis during our sample preparation and the limitations of our simple bio-assay. We would speculate, however, that the apparently late :appearance of neurotrophic activity in spinal cord compared to brain may be due to a later appearance of astrog1ia1 cells in the ,former. Given our bias that astrocytes are the source of this neurotrophic growth factor, it is interesting to note that the astrog1ia1 marker, glial fibrillary protein (GFAP) is only first detected in the chick embryo spinal cord at E12[14] and it is not until the day of hatching that this marker is found uniformly distributed throughout the spinal cord.

REFERENCES

1. Y.-A. Barde, R. M. Lindsay, D. Monard, and H. Thoenen, Nature, 274:818 (1978).

2. R. M. Lindsay and J. Tarbit, NeuroscLLett., 12:195-200 (1979). 3. R. M. Lindsay, Nature, 282:80-82 (1979). 4. R. M. Lindsay, P. C. Barber, M. R. C. Sherwood, J. Zimmer and

Raisman, Brain Res., 243:329-343 (1982). 5. R. M. Lindsay, Biochem.Soc.Trans., 10:429-431 (1982). 6. H. Thoenen and Y. -A. Barde, Physio1.Rev., 60:1284-1335 (1980). 7. Y.-A. Barde, D. Edgar, and H. Thoenen, Proc.Nat1.Acad.Sci.USA,

77:1199-1203 (1980). 8. G. Harper, F. L. Pearce, and C. A. Vernon, Dev.Bio1., 77:391-402

(1980) • 9. K. D. McCarthy and L. M. Partlow, Brain Res., 114:391-414

(1976) • 10. T. Ebenda1 and c.-o Jacobson, Exp.Ce11 Res., 105:379-387 (1977). 11. R. J. Riopelle and D. A. Cameron, J.Neurobio1., 12:175-186

(1981). 12. Y.-A. Barde, D. Edgar, and H. Thoenen, Embo J., 1:549-553

(1982). 13. P. D. Gorin and E. M. Johnson, Brain Res., 198:27-42 (1980). 14. A. Bignami and D. Dahl, Dev.Bio1., 44:204-209 (1975).

INHIBITION OF PROTEOLYTIC ACTIVITY AS MODULATION

OF NEURITE OUTGROWTH

Denis Monard and Joachim Gunther

Friedrich Miescher-Institut P.O. Box 2543, CH 4002-Basel Switzerland

Glial cells, including glioma cells, grown in tissue culture release macromolecular factors which can promote neurite extension and/or survival of neuronal cells[1,2]. One of these factors induces neurite extension in neuroblastoma cells[1]. A similar neurite promoting activity is also found in the medium conditioned by rat brain primary cultures{3]. In such media, there is a correlation between the presence of the biological activity and the age of the animal from which the primary culture was derived[3]. This sharp rise in neurite promoting activity released by brain primary cultures derived from 3-5 days old animals coincides with the period of rat brain development at which the burst of glial cell multiplication takes place. Since neural arborization strongly expands at, or just following this phase, these results have suggested the relevance of such an glia-derived activity in postnatal rat brain maturation. They also revealed the existence of a type of molecular mediators in glia-neuronal interactions.

Knowledge about the precise origin and function of such mol­ecules requires, as first step, their biochemical identification. A purification procedure involving anion exchange, Affi-gel Blue and Carboxymethylsepharose chromatography has been outlined[4]. The purified material is optimally active at 1-2 nanograms per milliliter of culture medium. We also found out that a strong inhibitory activ­ity to urokinase and plasminogen activator co-purifies with the neurite promoting activity[4]. The biological activity is adsorbed on immobilized urokinase and cannot be eluted. The use of a modified urokinase coupled to Sepharose beads allows recovery of both the protease inhibitory activity and the neurite promoting activity. Both activities can be attributed to the same protein band upon silver staining of polyacrylamide gels.

307

308 D. MONARD AND J. GUNTHER

We have investigated the effect of other, well known serine protease inhibitors on neurite outgrowth. Only two of them, hirudin[5] and a synthetic peptide[6] strongly promote neurite exten­sion at concentrations as low as 5x10- 11M, that is in the same range at which the purified factor is optimally active. Since hirudin and the synthetic peptide are considered to be inhibitors rather specific for thrombin, the effect of this serine protease on the glia-induced neurite extension has been studied. Thrombin concentrations as low as 2,5 ng/ml are sufficient to completely block the neurite extension promoted by an optimal dose of concentrated serum-free glia con­ditioned medium. Other serine proteases do not show this potency. As example, an 100 fold higher concentration of trypsine is required to antagonize glial factor activity. Urokinase and plasmin remain without effect, even when tested at 10 ~g/ml.

These data suggest a thrombin-like nature of the cell associated protease which is inhibited by glial factor. The fact that urokinase is inhibited by the factor but is not able to antagonize the glia­induced neurite outgrowth could indicate that the inhibitory protein has a much greater affinity for the protease which is associated with the cell surface.

These results allow to postulate an important function for the inhibition of cell surface associated proteolytic activity at early stages of neuronal cell maturation. Cellular migration represents the first event concomitant or following neuroblasts' proliferation. It is generally assumed that the migration and the factors which could modulate it, would have a key influence on the final location of the individual neuronal cells, and therefore on their subsequent abilities to establish adequate connections with target cells in order to finally lead to an operative specific wiring. The role of cell surface protease activity in cell migration has been stressed in many developmental systems[7,8]. Migrating cells are considered to have more cell surface proteolytic activity than stationary, differ­entiated cells[9,10]. In neuronal cells, an increase in cell surface associated proteolytic activity has been attributed to granule cells at the time of their migration during cerebellum development[ll] •. Recently, the synthetic peptide inhibitor which is able to mimic glial factor in inducing neurite outgrowth at 5x10- 11M has been tested in cultured paraflocculi of rat cerebellum. A high concen­tration of this peptide (10-4M) inhibits the migration of the granule cells which can be followed in this tissue culture model[12]. If cell surface proteolytic activity is positively implicated in the migration of neuroblasts, events leading to an inhibition of this proteolytic activity would modulate the extent of the migration. Our results indicate that the vicinity of the neuronal cells, especially surrounding glial cells, would have, through the release of factors as the one we have characterized, the possibility to impair or block neuroblast migration and, thus create conditions compatible with neurite outgrowth. Such a regulation through glial cells or local

MODULATION OF NEURITE OUTGROWTH 309

concentrations of factor(s) released by them would even provide a way of controlling migration and onset of neurite outgrowth to a dif­ferent degree and at a different timing for each individual neuro­blast. Such phenomena would certainly create the conditions leading to the final specific location of each neuronal cell. They would therefore be implicated in the distribution of th~ cellular units which will have to be wired together through subsequent steps in neuronal differentiation.

REFERENCES

1. D.

2. Y.

3. Y.

4. D.

5. F.

6. C.

7. M.

Monard, F. Solomon, M. Rentsch, and R. Gysin, Glia-induced morphological differentiation in neuroblastoma cells, Proc. Nat.Acad.ScLUSA, 70:1894-1897 (1973). --A. Barde, R. M. Lindsay, D. Monard, and Thoenen, H., New factor released by cultured glioma cells supporting survival and growth of sensory neurones, Nature, 274:818 (1978). Schfirch-Rathgeb and D. Monard, Brain development influences the appearance of glial factor-like activity in rat brain primary cultures, Nature, 273:308-309 (1978). Monard, E. Niday, A. Limat, and F. Solomon, Inhibition of protease activity can lead to neurite extension in neuro­blastoma cells, Prog.Brain Res., 58:359-364 (1983). Markwardt, Untersuchungen fiber Hirudin, Naturwissenschaften, 42:537-538 (1955). Kettner and E. Shaw, D-PHE-PRO-ARGCH2 CI, a selective affinity label for thrombin, Thromb.Res., 14:969-973 (1979). I. Sherman, S. Strickland, and E. Reich, Differentiation of early mouse embryonic and teratocarcinoma cells in vitro: plasminogen activator production, Cancer Res., 36:4208-4216 (1976) •

8. W. Topp, J. D. Hall, M. Marsden, A. K. Teresky, D. Rifkin, A. J. Levine, and R. Pollack, In vitro differentiation of teratomas and plasminogen activator in teratocarcinoma-derived cells, Cancer Res., 36:4217-4223 (1976).

9. J. C. Unkeless, A. Tobia, L. Ossowski, J. P. Quigley, D. B. Rifkin, and E. Reich, An enzymatic function associated with transformation of fibroblasts by oncogenic viruses, J.exp. Med., 137:85-111 (1973).

10. L. Ossowski, J. P. Quigley, and E. Reich, Fibrinolysis associated with oncogenic transformation, J.Biol.Chem., 249: 4312-4320 (1974).

11. A. Krytosek and N. W. Seeds, Plasminogen activator secretion by granule neurons in cultures of developing cerebellum, Proc. Nat1.Acad.ScLUSA, 78:7810-7814 (1981). --

12. G. Moonen, M. P. Grau-Wagemans, and I. Selak, Plasminogen acti­vator-plasmin system and neuronal migration, Nature, 298: 753-755 (1982).

SURFACE-BOUND AND RELEASED NEURONAL GLYCOPROTEINS

AND GLYCOLIPIDS

Paul H. Patterson

Biology Division California Institute of Technology Pasadena, California 91125, USA

The establishment of the complex network of synaptic connections which occurs during vertebrate neural development is the result of a sequential series of cellular interactions between neurons and their targets. These interactions occur throughout the periods of cell migration, axonal outgrowth, recognition of target cells, different­iation and pre- and postsynaptic elements, competition between and reorganization of connections, and stabilization of the final con­nections in maturity (cf Patterson and Purves, 1982). A number of these stages, particularly the later ones. are controlled primarily by synaptic transmission. For example. distribution of acetylcholine receptors in adult skeletal muscle is determined largely by the activity induced in the muscle by synaptic transmission (Lomo and Westgaard. 1976). On the other hand. a number of interactions. particularly during the early phases of synapse formation. do not appear to be mediated by the transmitter. The initial organization of muscle acetylcholine receptors under the nerve. for example. can occur in the absence of acetylcholine binding to its receptor (Anderson et al •• 1977). Cellular interactions in this case must involve other secreted or cell surface molecules. The identifi­cation. purification and characterization of these molecules is a particularly challenging area of contemporary neurobiology.

SURFACE GLYCOCONJUGATES

In a effort to identify molecules which would be used by neurons in interactions with other cells, we have identified surface-bound and released glycoproteins and glycolipids which are highly enriched in specific neuron types. For example, the axons of cultured ad­renergic sympathetic neurons bind the lectin soybean agglutinin (SBA)

311

312 P. H. PATTERSON

at a 5-fold high density than the axons of cultured cholinergic sympathetic neurons (Schwab and Landis, 1981). Zurn (1982) identi­fied the neuronal receptors for SBA as two neutral glycolipids, one comigrating with globoside in thin layer chromatography. SBA binds specifically to these lipids on polyacrylamide gels, thin layer chromatography plates and on the surfaces of living neurons. Neuronal proteins, on the other hand, labeled poorly with SBA. In addition, when the surfaces of living neurons were labeled with galactose oxidase, Zurn found that the adrenergic neurons had more of the two neutral glycolipids accessible for labeling than did the cholinergic neurons, which is consistent with the lectin binding data. Of direct relevance to these findings is the work of Raff et al. (1979) which showed that only half of dissociated rat dorsal root ganglion neurons in culture bound an anti-globoside serum. Thus there is a difference in glycolipid composition or accessibility among sensory neurons as well.

Cultured adrenergic and cholinergic sympathetic neurons also exhibit several differences in their profiles of major surface glyco­proteins. Braun et al. (1981) labeled neuronal surface proteins by metabolic and surface-specific methods, and then analyzed the pro­teins by two-dimensional gel electrophoresis and autoradiography. The expression of two ot the surface proteins was correlated with the transmitter phenotype, one being greater in adrenergic neurons (A155) and one elevated in cholinergic neurons (C55). It will be of interest to develop antibodies against these proteins in order to assess their distribution in vivo.

Chun and colleagues (1980; in preparation) have isolated mono­clonal antibodies which bind preferentially to either adrenergic or cholinergic sympathetic neurons grown in culture. As a first step towards determining the function of such surface antigens, Chun et al., grew sympathetic neurons in the presence of a number of mono­clonal antibodies. Thus far, 14 antibodies have been used in chronic incubations and their effects on development assessed by biochemical analysis of a variety of neuronal functions. Of these, three had nb detectable effects on the neurons, five had marginal effects, and six caused reproducible and specific functional changes in the neurons. Alterations, both positive and negative, were seen in catecholamine uptake as well as synthesis and accumulation. Only one ot these antibodies had detectable deleterious effects on neuronal survival or growth. For the most part, therefore, nonspecific, gross effects caused by antibody binding or internalization were not a problem. We do not know, however, whether the observed developmental alterations were caused by the antibody blocking the activity of a given antigen or by causing the internalization of the antigen.

One of the antibodies which has been studied in the most detail, ASCS4, binds to a variety of neurons in both the peripheral and central nervous systems. Immunohistochemical analysis of frozen

GLYCOPROTEINS AND GLYCOLIPIDS 313

sections also revealed that the antibody does not bind detectably to a variety of glands including the adrenal medulla (Chun and Patterson, in preparation). When sympathetic neurons are grown in the presence of this antibody, their ability to synthesize and ac­cumulate both catecholamine and acetylcholine is increased. How could an antibody binding on the cell surface have a positive effect on transmitter synthesis and storage? One possibility is that its binding alters the process of vesicle turnover during the exocytosis­endocytosis cycle. This idea acquired some plausibility when Chun found that the surface antigen to which the antibody binds is a protein that had been independently studied by K. Sweadner. The latter's findings (see below) had suggested the hypothesis that this protein may be involved in vesicle turnover (Sweadner and Patterson, 1981; Sweadner. 1983).

The antigen in question is one of a pair of acidic. major sur­face membrane glycoproteins of high molecular weight (230 K and 200 K daltons. called Bl and B3. respectively). Bl is recognized by Chun's monoclonal antibody ASCS4. and its size. charge. and precipitability by monoclonal and conventional antibodies identify it as the sym­pathetic neuron equivalent of the PC12 cell line protein termed NILE (for NGF Inducible, Large External protein) by Greene and colleagues (McGuire. et al., 1978; Salton, et al., 1981). Bl and B3 are first detected in pulse-chase experiments in more basic. lower molecular weight precursor forms. The Bl precursor is precipitated by antibody ASCS4, and is termed PI (Figure 1). The precursors PI and P3 in­itially can be labeled by incubation of the sympathetic neurons with 3H-mannose, but this label is lost with a half-life of about 1 hr. As the 3H-mannose is lost. the precursors are inserted into the surface membrane. and labeling with 3H-fucose can be detected (Pl+Bl, P3+B3; Figure 1) (Sweadner. 1983). The conversion of PI to Bl involves a shift to a more acidic pI and the addition of an apparent 15K daltons. The actual change in molecular weight could be less than 15K daltons because the changes in carbohydrate composition indicated by the labeling experiments could alter the behavior of PI and Bl in the SDS gels. A parallel sequence of labeling steps occurs in the synthesis of B3, but no antibody is yet available to confirm a relationship between P3 and B3.

The appearance of Bl and B3 in the surface membrane is indicated by susceptibility to a number of external agents added to intact neuronal cultures (trypsin. neuraminidase. lactoperoxidase and galac­tose oxidase) (Sweadner and Patterson. 1981; Sweadner. 1983). In Figure 1. Bl is depicted as having a tail inserted in the membrane. This is hypothetical, as we have no information as yet on the actual disposition of this protein in or on the membrane. The sugar label­ing studies suggest. however, that Bl is an intrinsic membrane glycoprotein, rather than an extracellular matrix proteoglycan (cf Lennarz, 1980). The pulse-chase studies further show that after their appearance on the cell surface. Bl and B3 each undergo an

314

81 230k acidic

P1 215k basic

-15k t1/. 5 hr

2

o mannose * sialic • fucose 1< other

P. II. PATTERSON

82 215k acidic

Fig. 1. Diagram summarizing the processing of one of the two sets of proteins described in the text. Monoclonal antibody ASCS4 specifically precipitates proteins P1, B1, B2 and S2. Pulse-chase experiments suggest that P1 is a precursor of B1. P1 can be labeled with 3II-mannose at early times but quickly loses the label during a chase. At the same time it loses its mannose label, P1 apparently is inserted into the surface membrane, acquiring fucose, sialic acid, an apparent 15 K daltons of mass, a more acidic charge, and suscept­ibility to extracellular trypsin, neuroaminidase, and surface iodination. With a slower half-time, B1 loses about 15 K daltons of mass, yielding B2. B2 is then apparently released from the neuronal surface (as S2) following de­polarization. The carbohydrate structures are shown solely for illustrative purposes; nothing is yet known about the number or structure of these chains.

apparently spontaneous conversion (t! _ 5 hrs) to two lower molecular weight forms, termed B2 and B4. Again, the apparent change in mol-, ecular weight is about 15 K daltons. In Figure 1, this conversion is depicted as a loss of the tail in the membrane, but this again is hypothetical; we have no information as yet on the chemical nature of the conversion.

The next stage in the life cycle of these surface proteins occurs when the neurons are depolarized. When ~ransm~ter release is evoked with any of a variety of agents (50 mM K + Ba ,A23187, black widow spider venom (BWSV), veratridine, PCMBS, alomethecin, or monensin), B2 and B4 are released from the cell at the expense not only of B2 and B4, but also Bl and B3. The loss of these proteins is selective; many other membrane and cytoplasmic proteins are not released under the same conditions. The interpretation that the released proteins (S2 and S4) are in fact derived from B2 and B4 is supported by the finding that antibody ASCS4 can precipitate S2 (as

GLYCOPROTEINS AND GLYCOLIPIDS 315

well as Bl and B2). It is interesting that although the apparent sequence of P3+B3+B4+S4 completely parallels the Pl+Bl+B2+S2 se­quence, with very similar changes in molecular weights and charge, antibody ASCS4 fails to recognize any of the P3, B3, B4 and S4 mol­ecules.

-1+ -1+ The ~elease of S2 and S4 requires Ca (Co blocks the effect

of high K , A23187 and BWSV). This would be expected if the protein release was related to exocytosi~ Sweadner also found, however, that protein release requires Ca even under circumstances where _ transmitter release does not. That is, while norepinephrine r~ease evoked from the neuro~by BWSV can occur in the absence o~a or in~he presence of La ,protein release is blocked by La or low Ca Thus, while protein release is always accompanied by trans-mitter release, the reverse is not true. ~tein release is there-fore not required for exocytosis. That La bloc~protein release is interesting in light of the observation that La also blocks the recovery of motor nerve terminals after heavy stimulation and vesicle depletion (Clark et al., 1972). Our working hypothesis is that protein release follows exocytosis, and may be involved in vesicle membrane recovery during endocytosis.

To test the endocytosis hypothesis, W. Matthew (unpublished) has mutagenized PC12 cells and selected for cells which lack Bl and B2, using antibody ASCS4 and complement. These cells are being cloned, and their exo- and endocytotic capabilities will be assayed. It will be important to determine the nature of the surface changes in these mutant lines, as well as to attempt the potentially difficult experi­ment of adding back B2 or S2 to the mutant cells in an effort to correct whatever deficits they display.

SPONTANEOUSLY RELEASED PROTEINS

In addition to the depolarization-induced protein release, cultured neurons also release or secrete well-defined and restricted sets of intracellular glycoproteins. This spontaneous secretion is not affected by increased or decreased transmitter release (Sweadner, 1981). Furthermore, there are striking differences in the families of glycoproteins spontaneously secreted into the medium by cultured adrenergic vs. cholinergic sympathetic neurons. After labeling with 3H-Ieucine or 3H-fucose, the neurons secrete 16-18 major soluble proteins which differ from the surface membrane glycoproteins in two-dimensional gel analysis. Five of these proteins are correlated with the neuronal transmitter phenotype and are thus candidates for extracellular functions specific to one or the other type of neuron (Sweadner, 1981).

Recently, Pittman (1983) has found that several types of sensory neurons also spontaneously secrete characteristic families of glyco­proteins into the culture medium. Neuronal cultures from rat dorsal

316 P. R. PATTERSON

root, nodose and trigeminal ganglia each secrete at least one protein which appears not to overlap on two-dimensional gels with proteins from the other sensory ganglia, or with the proteins secreted by sympathetic neurons. These findings may be of interest in the con­text of developmental trophic interactions. It is well known that a variety of sensory receptors are dependent on their sensory inner­vation for initial differentiation, as well as maintenance of differ­entiated phenotype in the adult (cf Patterson and Purves, 1982). In addition, there is evidence that various sensory neurons differ in their ability to induce particular receptors (Zalewski, 1969). It is possible that some of the spontaneously secreted proteins could subserve such specific trophic roles.

To examine this possibility further, and to develop probes for localizing these proteins in vivo, Matthew and Pittman are attempting to raise monoclonal antibodies against the secreted proteins. Al­though these proteins are major cellular products, comprising 1-3% of total ongoing protein synthesis, the actual amounts of protein re­leased from primary neuron cultures is quite small. Thus alternative approaches are being pursued. In vitro immunization (Luben and Mohler, 1980) has the advantage that very small amounts of antigen are necessary. Another strategy is to use neuronal cell lines as the source pf released protein. Matthew (unpublished) has successfully used ~h~ i~ vitro immunization method to generate monoclonal anti­bodies ~g~1nst as little as 10 ng of PC12 cell secreted protein. This app~oach therefore offers promise in the search for develop­mental sig~ls produced by neurons.

Acknowled&~~en~~

The recent research reported in this article was supported by the NINCDS, the Rita Allen and McKnight Foundations, the Charles A. King Trust, the Jane Coffin Childs Memorial Fund, and the Swiss National Foundation for Scientific Research. In addition to the authors cited, others contributing to this work were: A. Doupe, S. Landis, D. McDowell, E. Silvesto, G. Spencer, E. Wolinsky and V. yee.

REFERENCES

Anderson, M. J., Cohen, M. W., and Zorchyta, E., 1977, Effects of innervation on the distribution of acetyicholine receptors on cultured muscle cells, J.Physiol., 268:731-756.

Braun, S., Sweadner, K. J., and Patterson, P. R., 1981, Neuronal surfaces: distinctive glycoproteins of cultured adrenergic and cholinergic sympathetic neuro~s, J.Neurosci., 1:1397-1406.

Chun, L. L. Y., Patterson, P. R., a~d Cantor, R., 1980, Preliminary studies on the use of monoclonal antibodies as probes for sym­pathetic development. J.Exp.Biol., 89:73-83.

GLYCOPROTEINS AND GLYCOLIPIDS

Clark, A. W., Mauro, A., Longevecker, H. E., and Hurlbut, W. P., 1972, Changes in the fine structure of the neuromuscular junction of the frog caused by black widow spider venom. J.Cell BioI., 52:1-14.

317

Lennarz. W. J., (ed.), 1980, The Biochemistry of Glycoproteins and Proteoglycans, Plenum Press. New York.

Lomo, T., and Westgaard, R. H., 1975. Control of ACh sensitivity in rat muscle fibers, Cold Spring Harbor S¥ffip.Quant.Biol., 40: 263-274.

Luben, R. A., and Mohler. M. A., 1980, In vitro immunization as an adjunct to production of hybridoma producing antibodies against the lymphokine osteoclast activating factor, Molec. Immunol •• 17:635-639.

McGuire, J. L., Greene, L. A., and Furano, A. V., 1978, NGF stimu­lates incorporation of fucose or glucoasamine into an external glycoprotein in cultured rat PC12 pheochromocytoma cells, Cell, 15:357-365.

Patterson, P. H., and Purves, D., 1982. Readings. in :p~velopmental Neurobiology, Cold Spring Harbor. New York.

Pittman, R. N., 1983. Spontaneously released proteins from cultures of sensory ganglia include plasminogen activator and a calcium dependent protease, 13th Ann.Mtg.Soc.Neur~s~t., 9:5.4.

Raff, M. C., Fields, K. L., Hakomori, S., Mirsky, R., Pruss, R., and Winter, J., 1979, Cell-type specific markers for distinguish­ing and studying the major classes of neurons and the major classes of glial cells in culture, Brain Res., 174:283-308.

Schwab, M., and Landis, S. C., 1981, Membrane properties of cultured rat sympathetic neurons: morphological studies of adrenergic and cholinergic differentiation, Devel.Biol., 84:67-78.

Salton, S. R. J., Richter-Landsberg, C., Greene, L. A., and Shelanski, M. L., 1983, Nerve growth factor-inducible large external (NILE) glycoprotein: studies of a central and peripheral neuronal marker, J.Neurosci., 3:441-454.

Sweadner, K. J., 1981, Environmentally regulated expression of soluble extracellular proteins of sympathetic neurons, J.Biol. Chem., 256:4063-4070.

Sweadner, K. J., 1983, Post-transitional modification and evoked release ot two large surface proteins of sympathetic neurons, J.Neurosci., in press.

Sweadner, K. J., and Patterson, P. H., 1981, Neuronal surface protein release accompanies transmitter release. Soc.Neurosci.Abstr •• 7:227.15.

Zalewski, A. A., 1969, Combined effects of testosterone and motor. sensory. or gustatory nerve reinnervation in the regeneration of taste buds, Exp.Neurol •• 24:285-297.

Zurn. A •• 1982, Identification of glycolipid binding sites for soy­bean agglutinin and differences in the surface glycolipids of cultured adrenergic and cholinergic sympathetic neurons, Devel.Biol., 94:483-498.

IN VITRO STUDIES ON THE MATURATION OF THE ASCENDING

MESENCEPHALIC DOPAMINERGIC NEURONS

A. Prochiantz, S. Denis-Donini, M. -C. Daguet-de Montety M. Mallat, A. Herbet and J. Glowinski

Groupe NB, INSERM U.114, College de France 11 place Marcelin Berthelot 75231 Paris cedex 5, France

INTRODUCTION

The formation of the brain proceeds through several stages: cell division and migration, cellular aggregation and structure formation, neuritic elongation, progression of growth cones in direc­tion of the appropriate target, target recognition, synaptogenesis, cell death, collateral elimination etc. The result is the establish­ment of a series of highly specific anatomo-physiological inter­actions. How this specificity is obtained remains one of the central non-answered problems in brain embryology. Schematically, three principal possible mechanisms are postulated. Firstly purely spatio­temporal models: each developmental event is strictly determined by the preceding one. For example, neurite elongation occurs in a definite direction due to the absence of other possibilities at the time of its initiation (Figure lA). Secondly, the existence of gradients of diffusible chemotrophic molecules. As in the example of the elongating neurite, its growth cone is able to measure dif­ferences in concentrations of factors and therefore to orient its growth (Figure IB). Lastly (Figure IB), the existence of surface molecules allowing cellular labelling; such molecules can eventually be disposed in a gradient-like manner. In principle, three gradients of either soluble or membrane bound signals should be sufficient for a cell body, a neurite or a growth cone to know exactly its position in a tridimensional environment. The coexistence of more molecular species can be postulated, the extreme situation being the individual molecular recognition of each target cell by its specific afferente as proposed by Sperry.

319

320 A. PROCHIANTZ ET AL.

A B

I'; . ::

Fig. 1. A. Neurite growth towards its target is the only possi­bility. B. The existence of two possible pathways neces­sitates the presence of soluble or membrane associated in­formative molecules.

Evidence favoring these hypothetical models have been obtained by several groups. It is likely that different mechanisms intervene separately or simultaneously according to species, structure or developmental period. In this short review of our work, we shall mainly focus our attention on the possible role of membrane bound molecules in the differentiation of ascending dopaminergic (DA) neurons of the mouse brain. As an introduction, we shall recall some results obtained by other authors in the domain of neuronal cell recognition.

CELLULAR INTERACTIONS AND THE ESTABLISHMENT OF SPECIFIC NETWORKS (short review)

In spite of the fact that several recognition mechanisms occur­ring between fibers and cell bodies may also account for soma-soma interactions, I shall mainly discuss the aspects concerning neurite­neurite and neurite-soma recognition processes.

Fasciculation

Some neurites tend to remain inside the same structure which include their cell bodies. They contribute either to local dendritic arborization or to short axons in the case of interneurons. Other neurites are able to leave the structure and to seek out a target. Very often, the outgrowth of neurites is associated with the forma­tion of bundles. It has been shown that the neuronal cell adhesion molecule (N-CAM), known to promote cellular adhesion, also enables neurite association[I]. This association may render the fibers more

MATURATION OF DOPAMINERGIC NEURONS 321

sensitive to the presence of chemotrophic signals[2]. Bundle out­growth outside cellular aggregates is specific. For example dif­ferent branches can be formed even if the cells are at the origin located in the same region of the brain. The occurrence of selective fasciculation has been demonstrated by Bray et al.[3]. Neurites from two homologous explants (retina-retina, or superior cervical ganglion-superior cervical ganglion) can intermingle in vitro. On the contrary neurites emerging from the retina and from the superior cervical ganglion cannot. If cells from the retina and from the superior cervical ganglion are dissociated andco-reaggregated, sympathetic and retinal bundles leave the aggregate separately.

Directional Growth and Pathway Recognition

In the peripheral nervous system, nerve growth factor (NGF) acts as a chemotactic signal both in vivo and in vitro. Evidence for such soluble factors having the same properties in the CNS is still sparse and often contradictory[4,S]. On the contrary, it is well documented that specific pathway recognition might occur through recognition and/or adhesion between membrane bound molecules. The role of dif­ferential adhesion in the specification of neuronal pathways has been postulated by Fraser[6] and is substantiated in several experimental situations[7-9]. Growth cone adhesion can occur with other neurites (the pioneering fiber hypothesis) or with cell bodies (glial or neuronal).

Neurite-Target Tissue Interaction

Before the fine definition of synapses, afferent fibers have to penetrate the target structure. In some experimental situations it seems that this penetration is specific. In vitro sensory fibers from the dorsal root ganglion penetrate in the dorsal but not in the ventral horn of the spinal cord[lO]. In vivo grafted embryonic neurons reinnervate previously denervated tissues along the appropri­ate layers[ll]. Growth cones from retinal ganglionic neurites can distinguish between tectal target cells and non-target cells[12]. Moreover gradients of adhesion/recognition molecules may exist, a naso-temporal gradient in the retina corresponding to an antero­posterior one in the tectum[13]. This recognition is mediated through membrane-membrane interactions as demonstrated by the specific binding of tectal membranes to the appropriate ganglion cell neurites[14]. Recently, the existence of a membrane molecule dis­posed in a gradient-like manner has been demonstrated in the ret­ina[lS]. This molecule may act as a positional signal.

~2 A. PROCHIANTZ ET AL.

THE MODEL OF THE NIGRO-STRIATAL DOPAMINERGIC PATHWAY

Ascending DA neurons are located in the mesencephalic flexure of the mouse brain embryo. They send non-myelinated axons towards rostral structures, mainly the striatum, sub-cortical limbic struc­tures, the cerebral cortex. Locally they develop a dense dendritic network. DA cells are generated on the 12th day of embryonic life near the midline of the mesencephalon from where they migrate lat­erally. Axon growth starts immediately and the first DA terminals can be observed in the striatum on the 14th day of embryonic life. Innervation reaches its mature stage four weeks after birth. In our study we have chosen to concentrate on the nigro-striatal system because of the very dense innervation of the striatum by DA affer­ents. This model allows us to ask the main questions raised earlier. 1) How do DA cell bodies migrate from the median part to more lateral parts of the mesencephalon? 2) What are the signals that allow one to differentiate between neurites remaining in the mesencephalon and those able to leave the structure in the direction of rostral struc­tures? 3) Is there a specific recognition of target cells by DA growth cones?

Pharmacological Evidence of Striatal Target Cells Being Recognized by DA Neurites

Dissociated mesencephalic DA neurons can be cultivated alone (l.5x10o per cm2 ) or with striatal cells (co-culture 1.5x105 mesen­cephalic, 105. striatal cells per cm2 ) on a substratum of collagen plus polyornithine (PORN). In these conditions it was observed that the ability of the cells to take up and synthesize DA was increased when measured after 8 days and 15 days in vitro[16]. This was inter­preted as resulting from an enhanced maturation of the cells in the presence of their target. This result was confirmed in a chemically define medium, conditions in which neurons constitute 95 per cent of the cellular population[17,18]. No effect of soluble compounds was observed. This effect was not due to differences in cellular conc~n­tration between culture and co-culture since 3H-DA uptake was linear in culture between 5x10 5 and 3.5x10~ cells per cm2 • From this it was envisaged that neuro-neuronal connections could influence DA cell maturation.

Morphological Evidence for Specific Contacts Between DA Terminals and Their Target Elements

In order to analyse eventual morphological modifications of DA cells in co-culture, cells were plated on PORN at low density (5x104 cells per cm2 ) to prevent cell aggregation, and cultures (5x104 mesencephalic cells per cm2 ) were compared to co-cultures (2.5x10 4 mesencephalic cells plus 2.5x10 4 striatal cells per cm2 ). In these

MATURATION OF DOPAMINERGIC NEURONS 323

conditions 3H-DA uptake per DA cell was hardly increased in co-cul­ture. This led us to analyse the length of the DA neurites in cul­ture and co-culture. It appeared that the presence of striatal cells resulted in a decreased length of DA neurites observed by autoradio­graphy after specific 3H- DA uptake. Such a decrease in the mean length of DA neurites was seen after 2,4 and 6 days in vitro, and did not occur when striatal cells were replaced by cerebellar cells (Table 1). This result was considered as an indirect proof that DA cell terminals had a higher affinity for striatal cells than for PORN, mensecephalic or cerebellar cells[19]. Striatal glia or medium conditioned on striatal glia or striatal neurons did not affect the mean length of DA neurites. We suggested therefore that specific adhesion and/or recognition may occur between DA terminals and their striatal target neurons in vitro. Final confirmation of this possi­bility awaits ultrastructural studies which are in progress.

As for the non-enhanced uptake ability of DA cells in co-culture at low density on PORN substratum, it could be envisaged that in mesencephalic culture the higher neuritic length may compensate for the better maturational state of the cells in co-culture with stri­atal cells. The differences between biochemical and morphological behavior of DA cells in co-culture in the previous conditions (high cell density, collagen plus PORN substratum) and the later ones (low cell density and PORN substratum) are presently being investigated.

Influence of Astroglial Cells on DA Cell Morphogenesis

In order to analyse the possible influence of glial cells on DA cell maturation, mesencephalic DA neurons were plated on astroglial mono layers generated either from the mesencephalon or from the stri­atum. The astroglial nature of the mono layers was checked by

Table 1. Distribution of Dopaminergic Neurons with a Neuritic Length Comprised between (in pm)

0-100 100-200 200-300 300-400 400-500 500-600 600-700 700-S00 >SOO

Mesencephal Cells

o o 7

IS 19 19 7 S 7

Mesencephalic + Striatal Cells

1 16 22 IS 10

1 o o o

Mesencephalic + Cerebellar Cells

o o o 5

13 10 5 5 6

324 A. PROCHIM{TZ ET AL.

specific labelling with an antibody against glial fibrillary acidic protein (GFAP). Already after 2 days in culture it was shown that on striatal glia, the DA neurons adopted a very simple morphology with one main single long, thin and poorly branched neurite. On the contrary on mesencephalic glia although half of the neurons behaved as on striatal glia half of them displayed a highly branched and varicose array of neurites (Figure 2)[20]. The total number of surviving DA neurons on striatal or mesencephalic glial substratum was identical. Three hypothesis were proposed to explain these differences. Firstly, highly branched neurites belong to a specific subclass of DA neurons which do not survive on striatal astroglia. Secondly, growth factors exist at the surface of some mesencephalic glial cells and neurons plated on these cells develop better than others plated on striatal cells or some mesencephalic glial cells. Ihirdly neurites with different morphologies would be qualitatively different; namely some mesencephalic glial cells would allow den­dritic outgrowth and differenciation, striatal and some mesencephalic glial cells allowing axonal growth only. The first hypothesis was rejected by experiments showing that all neurons were 'plastic' and could adopt both morphologies. DA cells grown on striatal or mesen­cephalic glia for 2 days and transferred on the other glial type have indeed been shown to modify their morphological pattern. We have not yet been able for the time being to distinguish between the growth factor and the axon/dendrite hypothesis.

Since both morphologies could be obtained on mesencephalic glia, we looked for a heterogeneity in these cells. It was shown that DA neurons plated on glia prepared from rostral or caudal regions of the mesencephalon had the poorly branched phenotype. The very highly branched pattern could develop only on glial monolayers generated from the precise region of the mesencephalon where DA cells are located.

Lastly it was shown by co-culturing striatal and mesencephalic glial cells in separate compartments but in the same culture medium that glial factors responsible for this morphogenetic effect were not diffusible on long distances and were therefore likely to be mem- ' brane-associated. Indeed, both morphologies were found on the cor­responding glial substratum.

Migratory Behavior of DA Cells on Specific Astroglial Monolayers

Experiments were performed in which mesencephalic explants instead of dissociated cells were cultured on striatal or mesen­cephalic astroglia. After 2 days in vitro we found that on mesen­cephalic glia, a large number of cells had migrated outside the explant. Most of them (if not all) had a highly branched arboriz­ation. On striatal glia almost no DA neurons were able to migrate. It seems therefore that triggering the- highly branched phenotype and

MATURATION OF DOPAMINERGIC NEURONS 325

Fig. 2. Autoradiography of dopaminergic neuron grown for 2 days on striatal glia (A) or mesencephalic glia (B). X 200.

allowing DA cell migration might be two properties of the same sub­class of mesencephalic astrocytes. Indeed, specific interactions between neurons and astroglia have been proposed to mediate granule cell migration in the cerebellum[21].

326 A. PROCHIANTZ ET AL.

Maturation of DA Neurons is Enhanced by Striatal and Mesencephalic Membrane Preparation

In the preceding experiments, the biochemical and morphological behavior of DA neurons could be modified in co-culture with nerve or glial cells. In no case could these modifications be attributed to the presence of soluble compounds. On the contrary several lines of evidence support the important role of direct neuro-neuronal or neuro-glial interactions. In order to directly assess the presence of differentiation signals at the cell surface, mesencephalic cells were cultured in the presence of membranes from the striatum, mesen­cephalon, parietal cortex, hippocampus and cerebellum. Analysis of the ability of the cells to take up 3H- DA was performed after 3 days in these different culture conditions[22]. It was found that stri­atal membranes prepared from striata of 2 week and 3 week-old ani­mals, stimulated 3H-DA uptake by DA neurons in culture, but had no influence 'on cultures containing no DA neurons (such as striatal cultures) (Figure 3). Membranes prepared from younger or adult animals did not share this property; mesencephalic membranes also had a slight effect, but this was never the case for membranes prepared from non-target structures (Figure 3 and 4). It was suggested that

E 0..

.g

J! 0

Ci.. => ~ I

I C")

Fig. 3.

ABC D

es.encepha lic Cell 5 Striatal Cell5 Mes.encephalic Cell Striatal Cell5

2000

1000

+BZ +BZ :tBZ

Striatal Membranes(~1) Mesencephalic Membranes(~I)

Effect of striatal (A,B) and mesencephalic (C,D) membranes from 3 week-old animals on 3H-DA uptake in 3 day-old mesencephalic (A,C) and striatal (B,D) cultures. BZ: benz tropine , a specific inhibitor of 3H-DA uptake in dopa­minergic cells.

MATURATION OF DOPAMINERGIC NEURONS

c .Q "0 "3 E .~

~ u L.

~

Hippocampal Membranes

I ~~~~0~~~~:-___ 9 100 t'" 20 Ad

o 10 20 30

1-'9 Membrane Proteins

327

Fig. 4. Effect of striatal and hippocampal membranes of new-born, 9 day-old, 20 day-old and adult animals on 3H-DA uptake in 3 day-old mesencephalic cultures.

at the age of maximal innervation of the striatum, molecules appear at the surface of striatal cells that enhance the spreading of nerve terminals inside this structure. Such molecules would disappear once innervation was accomplished.

CONCLUSION

The series of experiments briefly summarized here encourage us to propose an hypothetical model on the cellular interactions that may intervene in the formation of the DA nigrostriatal pathway (Figure 5).

1. Divisions occur near the midline of a definite mesencephalic region.

2. Migration takes place laterally and is directed by local membrane interactions with a specific subclass of mesencephalic astro­cytes.

3. During or after migration, highly branched neurites develop locally but are not able to leave the cell body environment. This phenomenon is triggered through neuro-glial interactions with local astrocytes.

4. One neurite only can leave this region. The cause of its asym­metric directional growth is unknown.

328 A. PROCHIANTZ ET AL.

Fig. 5. Schematic representation of the different stages in the formation of the dopaminergic nigro-striatal pathway. 1 - cell division. 2 - cell migration. 3 - local neurite development (dendrites?). 4 - outgrowth of the axon. 5 - target recognition and spreading. 6 - synapse for­mation.

5. Growth cones specifically recognize the target striatal structure and invade it. This invasion is accompanied by branching phenom­ena and is enhanced by surface molecules that are mainly present during the second and third week of post-natal development.

6. Nerve terminals can form synaptic contacts with their target striatal neurons. .

Further studies, especially at the ultrastructural level are needed. They will allow us to determine the nature of the highly branched and poorly branched neurites as well as the existence in vitro of synaptic connections between DA cells and striatal neurons.

MATURATION OF DOPA11INERGIC NEURONS 329

REFERENCES

1. U. Rutishauser, W. E. Gall, and G. M. Edelman, J.Cell BioI., 79: 382 (1978).

2. U. Rutishauser and G. M. Edelman, J.Cell BioI., 87:370 (1980). 3. D. Bray, P. Wood, and R. P. Bunge, Exp.Cell Res., 130:241

(1980). 4. B. Bjerre, A. Bjorklund, and U. Stenevi, Brain Res., 74:1

5. C.

6. D. 7. F.

8. P. 9. M.

10. N.

11. E. 12. F. 13. F. 14. W. 15. G.

16. A.

17. U.

18. A.

19. S.

(1974). F. Dreyfus, E. R. Peterson, and S. M. Crain, Brain Res., 194: 540 (1980). E. Fraser, Dev.Biol., 79:453 (1980). Collins and J. E. Garrett Jr., Proc.Natl.Acad.Sci.USA, 77: 6226 (1980). C. Letourneau, Exp.Cell Res., 124:127 (1979). J. Katz, R. J. Lasek, and H. J. W. Nauta, Neuroscience, 5:821 (1980). R. Smalheiser, E. R. Peterson, and S. M. Crain, Dev.Brain Res., 2:383 (1982). ~Lewis and C. W. Cotamn, J.Neuroscience, 2:66 (1982). Bonhoeffer and J. Huf, Nature, 288:162 (1980). Bonhoeffer and J. Huf, The EMBO Journal, 1:427 (1982). Halfter, M. Claviez, and U. Schwarz, Nature, 292:67 (1981). D. Trisler, M. D. Schneider, and M. Nirenberg, Proc.Natl. Acad.Sci.USA, 78:2145 (1981). Prochiantz, U. di Porzio, A. Kato, B. Berger, and J. Glowinski, Proc.Natl.Acad.Sci.USA, 76:5387 (1979). di Porzio, M. -C. Daguet, J. Glowinski, and A. Prochiantz, Nature, 288:370 (1980). Prochiantz, A. Delacourte, M. -C. Daguet, and D. Paulin, Exp.Cell Res., 139:404 (1982). Denis-Donini, J. Glowinski, and A. Prochiantz, submitted (1983).

20. S. Denis-Donini, J. Glowinski, and A. Prochiantz, submitted (1983).

21. R. L. Sidman and P. Rakic, Brain Res., 62:1 (1973). 22. A. Prochiantz, M. -C. Daguet, A. Herbet, and J. Glowinski,

Nature, 293:570 (1981).

BRAIN FACTORS SUPPORTING PROLIFERATION OF

NEURONAL CELLS IN CULTURE

M. Sensenbrenner, I. Barakat and G. Labourdette

Centre de Neurochimie du CNRS 5 rue Blaise Pascal - 67084 Strasbourg Cedex - France

During embryogenesis the proliferation and the maturation of nerve cells are influenced by the surrounding environment. Cell interactions as well as trophic factors may be involved in the suc­cessive steps of neuronal evolution. The use of dissociated nerve cells in culture has facilitated investigations on the influence of growth factors on the development of these cells.

Several reports from our laboratory and from others have described the presence of neurotrophic factors in brain extracts which support the survival and maturation of neuronal cells in cul­ture[1,2,6,7,8,9,10,12]. More recently, we have shown that brain extracts contain substances which control the proliferation of chick neuroblasts[3,5].

The proliferative activity of cultured neuroblasts from cerebral hemispheres of 6 day-old chick embryos in the absence as well as in the presence of brain extracts and the characterization of the active brain factors will be presented in this report.

Dissociated cells trom cerebral hemispheres of 6 day-old chick embryos were cultured in 60 mm diameter Falcon Petri dishes (1.5 x 106 cells or 3 x 106 cells/dish) on a collagen substrate in standard nutrient medium. The nutrient medium consisted of Eagle's minimum essential medium supplemented with either 5% fetal calf serum or 5% horse serum and 50 units/ml of penicillin. The cultures were incu­bated at 37°C in a humidified atmosphere of 97% air and 3% CO 2 •

After 24 h the medium was removed. Some cultures received again standard medium, while others received a nutrient medium to which meningeal extract or brain extract was added. The meningeal extract

331

332 M. SENSENBRENNER ET AL.

was prepared from 8 day-old chick embryo meningeal membranes[5]. The brain extracts were prepared from whole brains of chick embryos and of adult chicken, rat and beef[3].

In some experiments the dissociated brain cells were cultured in association with meningeal cells obtained from surrounding mesodermal membranes of 8 day-old chick embryo brains[5].

RESULTS

In control cultures small sized clumps of cells formed during the first 5 days. The neuronal nature of these cells was demon­strated by the presence of acetylcholinesterase activity revealed by histochemistry, by the presence of the specific neuronal membrane protein D2 and the neuron-specific enolase shown by immunohisto­chemistry.

Autoradiographic analysis have shown that precursors of neurons (neuroblasts) proliferate immediately after the onset of the culture and during the first week[ll]. In nutrient medium containing fetal calf serum, glial cells (astroblasts) started to proliferate actively only after 4 days in culture. In the presence of horse serum this cellular type seldom remained over a 7 day period. Thus, cultures derived from 6 day-old chick embryos contain mainly neuronal cells during the first 4 or 7 days, depending on the culture conditions.

We have shown that the cell density and cell-cell interactions were important and favored neuroblast proliferation[4].

Cell counts, determination of DNA content and measurement of 3H-thymidine incorporation have indicated quantitatively a prolifer­ation of neuroblasts during the first week in culture, before the occurrence of extensive growth of non-neuronal elements[3].

When brain cells were mixed with meningeal cells or were grown on preformed monolayers of meningeal cells, the contact with these cells produced a stimulatory effect on neuroblast multiplication[5]. These observations may indicate a trophic role exerted by brain mesodermal cells in the nervous system during embryonic development. The addition of meningeal extract also enhanced the multiplication of neuroblasts. indicating the presence of diffusible stimulatory sub­stances in meningeal membranes.

It was also demonstrated that in the presence of brain extract from 8 day-old chick embryos, the proliferative activity of the neuroblasts was stimulated[3]. When the nutrient medium contained fetal calf serum a 2-fold increase was elicited on day 3 by the treatment with the embryonic brain extract, while in the presence of horse serum a 20-fold stimulation was obtained. In this latter culture condition there was a 5-fold increase with adult chicken

BRAIN FACTORS 333

brain extract and a 2-and 3-fold increase with adult rat and beef brain extract, respectively.

Active factors from beef brains were purified from a chloroform­methanol precipitate, by an aqueous extraction and chromatographic procedures. In beef brain extracts nucleic acids were found to be active on neuroblast proliferation.

From chick brain extracts prepared by an aqueous extraction and submitted to chromatographic procedures, a protein fraction was isolated which elicited a stimulatory effect on the multiplication of neuroblasts grown in the presence of horse serum. The active mole­cule is heat and trypsin sensitive with an apparent molecular weight of roughly 70.000 daltons.

CONCLUSIONS

In dissociated brain cell cultures from 6 day-old chick embryos neuronal precursors (neuroblasts) were capable of proliferation during the first week. The addition of meningeal cells as well as meningeal or brain extracts stimulated the proliferative activity of these cells. Our results suggest that factors are present in the brain which are mitogenic for neuroblasts. In beef brain extracts nucleic acids were active. while in chick brain extracts an active protein fraction of about 70.000 daltons was isolated.

REFERENCES

1. P. Athias. M. Sensenbrenner. and P. Mandel. Differentiation. 2:99-106 (1974).

2. Y. A. Barde. D. Edgar. and H. Thoenen. The EMBO Journal, Vol. I, nO 5:549-553, (1982).

3. I. Barakat and M. Sensenbrenner. Dev.Brain Res., 1:355-368, (1981).

4. I. Barakat, M. Sensenbrenner, and G. Vincendon, Neurochem.Res., 7:287-300, (1982).

5. I. Barakat. E. Wittendorp-Rechenmann, R. V. Rechenmann, and M. Sensenbrenner, Dev.Neurosci •• 4:363-372, (1981).

6. K. A. Crutcher and F. Collins, Science, 217:67-68, (1982). 7. A. G. Hyndman and R. Adler, Dev.Neurosci •• 5:40-53, (1982). 8. R. M. Lindsay and J. Tarbit, Neuroscience Letters, 12:195-200.

(1979). 9. M. Schwartz, Y. Mizrachi. and N. Eshhar, Dev.Brain Res.,

3:29-35, (1982). 10. M. Sensenbrenner, N. Springer. J. Booher, and P. Mandel.

Neurobiology, 2:49-60, (1972). 11. M. Sensenbrenner, E. Wittendorp, I. Barakat, and R. V.

Rechenmann. Dev.Biol. 75:268-277. (1980). 12. H. Tanaka and K. Obata, Dev.Brain Res., 4:313-321, (1982).

PLASTICITY IN THE NEUROTRANSMITTER PHENOTYPE OF RAT

SYMPATHETIC NEURONS IN PRIMARY CULTURE

Jean-Paul Swerts, Marie-Claude Giess, Colette Mathieu Elizabeth Sauron, Agathe Le Van Thai and Michel Weber

Laboratoire de Pharmacologie et de Toxicologie Fondamentales, CNRS 205, route de Narbonne 31400 Toulouse

Plasticity in the neurotransmitter phenotype of immature neurons or neuron precursors in the developing autonomic nervous system (ANS) has been demonstrated in vivo and in primary cultures. It is known that the nature of the neurotransmitter synthesized by neurons of the avian ANS is not predetermined at the level of the neural crest, but is controlled by the cellular environment through which cells of the neural crest migrate[l]. Populations of neurons or neuroblasts have been described in rodent ANS which during development express in a transitory manner a adrenergic phenotype before switching to another (possibly cholinergic) phenotype[2-4].

Studies performed in primary cultures of neurons seem suitable for the identification of the extracellular signals involved in this epigenetic regulation of neurotransmitter phenotype. Various culture conditions have been described which cause a preferential expression of the cholinergic phenotype among sympathetic neurons dissociated from the superior cervical ganglia of new-born rats. These include conditioned medium (CM) by certain rat non-neuronal cells[5,6], the substratum prepared by p-formaldehyde fixed rat heart cells[7] as well as human placental serum and chick embryo extract[8-10].

During the past few years, we have been purifying the 'cholin­ergic factor' from C6 glioma or muscle CM[11,12]. This partially purified factor has been used to study the mechanism of neurotrans­mitter plasticity in cultures of sympathetic neurons from new-born rat superior cervical ganglia.

335

336 J.-P. SWERTS ET AL.

EFFECT OF CM FACTOR ON NEUROTRANSMITTER SYNTHESIS AND ACCUMULATION BY NEURON CULTURES

Sympathetic neuron cultures grown for 10-20 days in the presence of purified CM factor displayed an increased ability to synthesize and store [3H]Ach from [3H]cho1ine, as compared to sister cultures grown in the absence of factor. A marked decrease in [3H]CA syn­thesis and accumulation from [3H]tyrosine was also consistently observed (Figure 1). The purified factor thus reproduced the effects of CM described by Patterson and Chun[5,6]. The first two steps of CA synthesis were by-passed by examining the synthesis and accumu­lation of [3H]norepinephrine (NE) is sister cultures incubated with 95 ~M [3H]dopamine (DA). After a 4 hr incubation, similar amounts of

pmoley

dish 200

100

3H-acetylchoJine from 3H-choJine

-+

100

50

3H-catecholamines from 3H-tyrosine

norepinephrine

[l dopamine

Cb -+ -+

100

50

3H-catecholamines from 3H-dopamine

norepinephrine

dopamine

CIJ -+ -+

Fig. 1. Effects of purified cholinergic factor on neurotransmitter synthesis and accumulation from exogenous choline, tyrosine and dopamine. Cholinergic factor was purified from muscle CM by ammonium sulfate precipitation and chromatography on DEAE- and CM-ce11u10se, as described[ll] and mixed with culture medium to a final concentration of 30 ~g/m1. Twelve sister neuron cultures were grown between days 3-20 with or without factor with medium changed every second day. On day 20, neurons were incubated for 6.5 hrs in the presence of either [~HJtyrosine (80 ~M, 0.8 Ci/mmo1) and [3H]cho1ine (83 ~M, 0.7 Ci/mmo1) (3 cultures of each group) or [3H]dopamine (95 ~M, 1.0 Ci/mmo1) supplemented with 80 ~M unlabelled tyrosine and 80 ~M unlabelled choline (3 cultures of each group). The cultures were then carefully washed before the [3H]neurotransmitter synthesized and accumulated by the neurons were separated by high voltage paper electrophoresis at pH 1.9. (-): cultures grown without factor. (+): sister cultures grown with factor.

PLASTICITY IN THE NEUROTRANSMITTER PHENOTYPE 337

[3H]DA were found in cultures grown without and with the purified factor. However, the amount of [3H]NE synthesized and accumulated by the latter cultures was reduced by 99% (Figure 1). Interestingly, we consistently found that cultures grown without factor accumulated similar amounts of [3H]DA after an incubation with either [3H]tyro­sine or [3H]DA. The experiment of Figure 1 also suggested that neurons synthesized and accumulated more [~H]NE from exogenous [~H]DA than from [3H]tyrosine, but this phenomenon was not observed in other experiments of the same design.

In another series of experiments, sister cultures were grown for 15 days with or without muscle CM and tested for the synthesis and accumulation of labeled neurotransmitters from [3H]choline, [3H]ace­tate and [14 C]tyramine (Figure 2). CM caused a similar 7.2-10 fold increase in [3H]Ach synthesis and storage from [3H]choline and from [3H]acetate. However the amount of [3H]Ach formed from [3H]choline was 12-16 fold higher than from [3H]acetate, in the absence and in the presence of CM, suggesting that acetate is not a major precursor of the acetyl CoA pool used for Ach synthesis. As found with [3H]DA as precursor, cultures grown with and without CM accumulated identi­cal amounts of exogenous [14C]tyramine, but the synthesis and accumu­lation of [14 C]octopamine was reduced by 64% in CM cultures grown with CM. In addition to [14C]octopamine, another radioactive com­pound (referred to as 'metabolite' on Figure 2) accumulated in the neurons, which migrated approximately as DA during high voltage electrophoresis. This product, whose nature is unknown, was reduced by 81% in CM cultures. It was probably not DA, whose formation from octopamine only takes place in the liver[13]. One likely hypothesis is that this unknown compound was a metabolite of octopamine, which would explain its decrease in CM cultures.

These experiments thus demonstrated effects of both CM and purified CM factor at three different levels: 1) increase in [3H]Ach synthesis and accumulation from both [3H]choline and [3H]acetate, 2) decrease in [3H]DA and [3H]NE synthesis and accumulation from [3H]tyrosine and 3) decrease in the synthesis and accumulation of 8-hydroxylated products from both[3H]DA and [14C]tyramine.

REGULATION BY CM FACTOR OF ENZYMES INVOLVED IN NEUROTRANSMITTER SYNTHESIS AND DEGRADATION BY SYMPATHETIC NEURON CULTURES

The differences in neurotransmitter synthesis and accumulation observed by comparing cultures grown with and without CM may result theoretically from differences in the uptake of the precursors, in the rates of synthesis and degradation of the neurotransmitters, in the storage capacity of the neurons and in the release of the neuro transmitters during the incubation with the precursors. To gain a better understanding of the level(s) at which the neurotransmitter phenotype of sympathetic neurons is modified by conditioned medium,

338

pmoley /c!ish

150

100

50

o

"C-octopamine from "C-tyramine

tyramine octopamine metabolite

- + - + - +

100

50

J.-P. SWERTS ET AL.

3H-acetylcholine 3H-acetylcholine from 3H-choline from 3H-acetate

-+ -+

Fig. 2. Effects of conditioned medium on neurotransmitter synthesis and accumulation from exogenous choline, acetate and tyramine. Eight sister neuron cultures were grown for 15 days with (+) or without (-) muscle CM. At day 15, the cultures were incubated for 4.5 hrs with 83 ~M [3H]choline (0.72 Ci/mmole) and 59 ~M [14 C]tyramine (50 mCi/mmole) in the presence of 19 ~M L-tyrosine and 1 mM pargyline (2 cultures of each group). Sister cultures were simul­taneously incubated with 190 ~M [3H]acetate (3.0 Ci/mmole), in a modified L10-COL medium containing 1 mM choline and lacking glucose, galactose and pyruvate. The cultures were then processed as for Figure 1.

we have undertaken a systematic comparison of vario~s §nzymatic activities present in homogenates from sister neuron c~ltures grown for 10-28 days in the presence or in the abs~nce ~f s~scle CM. These homogenates are thereafter referred to as CM and CM homogenates.

In agreement with the results of Patterson and Chun[5], CM had no effect on neuronal survival and on neuronal growth, as measured by the total amount of protein per dish. In addition the specific activity of lactate dehydrogenase, a+cytopla~mic enzyme common to many cell types, was identical in CM and CM homogenates.

By using the Fonnum assay[14], we found that the specific ac~ivity of ch~line acetyltransferase (CAT) was 25-100 fold higher in CM than in CM homogenates, a result consistent with earlier data of Patterson and Chun[5] (Table 1). Most remarkably, we also found that the activities of the three enzymes of NE biosynthesis pathway, tyrosine hydroxylase, dopa deca~boxylase an~ dopamine-S-hydroxylase were about 2-fold smaller in CM than in eM homogenates. This is

PLASTICITY IN THE NEUROTRANSMITTER PHENOTYPE 339

the first demonstration of a common regulation of these three enzymes. Nerve Growth Factor injections to new-born rodents and electrically or reflexively elicited increases in preganglionic activity are well-known situations in which tyrosine-hydroxylase and dopamine-S-hydroxylase are induced in the superior cervical ganglion, but in none of these cases has an increase in dopa decarboxylase specific activity been observed[15-18].

As far as neurotransmitter degradation is concerned, Table 1 demonstrate+that acetyl~holinesterase (AcChE) activity was 3-4-fold lower in CM than in CM homogenates. The physiological significance of this result is unclear, because high levels of AcChE activity have been associated with the cholinergic phenotype in cat sympathetic ganglia [see discussion in Reference 19]. The development of AcChE as revealed by cytochemical staining has also been taken as an index of adrenergic to cholinergic neurotransmitter switch in sympathetic nerve fibers innervating rat sweat glands[3,4]. The comparison between data obtained in vivo and in culture thus clearly deserves further study.

For all enzymes studied, variations in activities similar to those shown on Table 1 have been demonstrated by comparing sister neuron cultures grown with and without 3-5 ~g/~l of cholinergic factor partially purified from muscle CM by ammonium sulfate precipi­tation, chromatography on DEAE and CM-cellulose and adsorption on hydroxyapatite[II,12]. These data make more likely our working hypothesis that the same macromolecular factor released by a variety of non-neuronal cells is involved in the regulation of the enzymes listed on Table 1.

Several lines of evidence strongly sugges~ that t~e difference in tyrosine hydroxylase (TOH) activities in CM and CM cultures results from a difference in the number of enzyme molecules[20J:

1) the purified factor has no effect on TOH activity when directly added to the enzyme assay cocktail. The factor itself is thus not an inhibitor of this enzyme. + _

2) mixing experiments have shown that TOH ac~ivities in CM and CM homogenates are additive, showing that CM homogenates do not con­tain higher concentrations of a hypothetical enzyme inhibitor (or lower concentrations of an activator). This in fact has been verified for all enzymes listed on Table 1.

3) the apparent KM'S of TOH ~or both_L-tyrosine and for the cofactor DMPH~ are identical in CM and CM homogenates.

4) the difference in enzyme activity between the two homogenates is observed whether TOH is assayed under phosphorylating or non-phos­phorylating conditions[21]. This difference is thus not due to a modification of the phosphorylation state of the e¥zyme. _

5) immunoprecipitation curves of TOH activities in CM and CM homo­genates by a specific anti-TOH anti-serum[22] are strictly parallel.

Tab

le 1

. S

iste

r cu

ltu

res

of

sym

pat

het

ic n

euro

ns

wer

e gr

own

for

12-2

4 da

ys w

ith

or

wit

ho

ut

CM

and

and

hom

ogen

ized

in

150

mM

N

aCI,

5

mM

N

a p

ho

sph

ate

bu

ffer

pH 6

.8,

0.2%

T

rito

n X

-100

b

efo

re e

nzym

e acti

vit

ies w

ere

assa

yed

by

pu

bli

shed

met

hods

[201

. A

ll v

alu

es a

re

exp

ress

ed i

n p

mol

/min

x ~

g p

rote

in.

Num

ber

of

CM

hom

ogen

ates

+

CM

ho

mog

enat

es

ind

epen

den

t N

umbe

r se

ries

of

of

mea

n ra

ng

e m

ean

ran

ge

cu

ltu

res

det

erm

inat

ion

s ±S

EM

±SEM

Ch

oli

ne

acety

l-tr

an

sfera

se

4 12

0

.16

±0

.02

0

.09

-0.2

8

9.2

±

1.5

5.0

-1

8.6

T

yro

sin

e h

yd

rox

yla

se

10

25

1.2

3±

0.0

9

0.4

8-2

.42

0

.61

±0

.08

0

.07

-1

.42

D

opa

dec

arb

ox

yla

se

5 11

0

.99

±0

.15

0

.35

-1.9

6

0.5

4±

0.1

0

0.1

4-

1.0

7

Dop

amin

e 8

-hy

dro

xy

lase

4

8 3

.04

±0

.19

2

.15

-3.9

8

1.5

3±

0.1

0

1.2

1-

2.1

0

Ace

tylc

ho

-li

nest

era

se

4 14

30

7±33

10

5-45

3 85

±7

52-1

49

w

.jO­

o c.... I 0-0

V'l ~ ~

CIl tt:I

t-':l ~

PLASTICITY IN THE NEUROTRANSMITTER PHENOTYPE 341

These+data th~s demonstrate that the difference in TOH activi­ties in CM and CM cultures can be accounted for by a decrease in the number of enzyme molecules without significant modifications of their catalytic properties. It is unknown if this+difference origin­ates from a smaller rate of enzyme synthesis in CM neurons, and/or from a larger rate of enzyme degradation. Regulations at both levels are indeed known for enzymes involved in catecholamine synthesis in neural crest derivatives.

REGULATION OF ACETYLCHOLINE METABOLISM BY CM FACTOR IN CULTURES FROM RAT SPINAL CORD AND NODOSE GANGLIA

The effects of muscle CM were studied on cultures prepared from embryonic spinal cord (a derivative of the neural tube) and from the nodose ganglia, whose neurons derive from a placode. In agreement with earlier data[23-2S], CM increased both [3H]acetylcholine syn­thesis and accumulation and CAT activity in spinal cord cultures. In addition, we found that AcChE activity was also increased under the same conditions (data not shown). These effects of CM were repro­duced by the CM factor we have partially purified by following its activity on sympathetic neuron cultures. In the experiment shown in Table 2, CAT and AcChE activities were mUltiplied by 4.8 and 2.2 respectively. Neurons from rat nodose ganglia survived for several weeks in the absence of NGF. As found with sympathetic neurons cultures[S]. CM had no effects on neuronal survival in these cul-

Table 2. The spinal cord of rat embryos (E14) has been dissociated by trituration and the cells grown on poly-L-Iysine in a modified LIS-C02 medium containing S% horse serum and S% fetal calf serum. The medium was changed three times a week. Sister cultures were grown in the same medium sup­plement with S ~g/ml CM factor. Nodose ganglia from new­born rats were dissociated with 0.2% dispase and grown on collagen in LIS-C0 2 medium[S] with S% rat serum but no NGF. Ara C (10 ~M) was present for the first 6 days. Sister cultures were grown with SO% muscle CM. All cultures were tested for CAT and AcChE activity at day 21. Data are all expressed in pmol/min x ~g protein as mean ± SEM for 4 determinations

Control SO% CM or S ~g/ml CM

* p < 0.001 **p < O.OS

Spinal cord cultures Nodose ganglion cultures CAT AcChE CAT AcChE

O.OS±O.OO 37.4±1.6 0.16±0.01 7.31±0.8S

factor 0.24±0.00* 81.8±1.8* 0.63±0.01* 4.26±0.26**

vs control conditions vs control conditions (student's t-test)

342 J.-P. SWERTS ET AL.

tures, but increased [3H]acetylcholine synthesis and storage from [3H] choline. This effect correlated with an increase in CAT activity and a decrease in AcChE activity (Table 2). A 2-3 fold increase in [3HJacetylcholine synthesis and accumulation was also observed in cultures grown for 15 days in the presence of 5 ~g/ml CM factor.

As the CM factor has not yet been purified to homogeneity, our results do not demonstrate that the same macromolecule is actually acting on neurons from sympathetic and nodose ganglia and from the spinal cord, but it is a reasonable working hypothesis. It is how­ever puzzling that this partially purified factor causes a decrease in AcChE activity in cultures from sympathetic and nodose ganglia, and an increase in spinal cord cultures. This enzyme may thus be differently regulated in different cell types.

Acknowledgement

This work was supported by grants from the Centre National de la Recherche Scientifique (PIRMED) and Institut National de la Sante et de la Recherche Medicale.

KEFERENCES

1. N. M. Le Douarin, The ontogeny of the neural crest in avian embryo chimaeras, Nature, 286:663-669 (1980).

2. 1. B. Black, Stages of neurotransmitter development in autonomic neurons, Science, 215:1198-1204 (1982).

3. s. C. Landis and D. Keefe, Development of cholinergic sym­pathetic innervation of eccrine sweat glands in rat footpad, Soc.for Neurosci.Abstr., Vol.6, p.379 (1980).

4. s. C. Landis and D. Keefe, Evidence for neurotransmitter plasticity in vivo. Developmental changes in properties of cholinergic sympathetic neurons, Submitted.

5. P. H. Patterson and L. L. Y. Chun, The induction of acetyl­choline synthesis in primary cultures of dissociated rat sym­pathetic neurons. I. Effects ot conditioned medium, Dev. BioI., 56:263-280 (1977a).

6. P. H. Patterson and L. L. Y. Chun, The induction of acetyl­choline synthesis in primary cultures of dissociated rat sym­pathetic neurons. II. Developmental aspects. Dev.Biol •• 60: 473-481 (1977b).

7. E. Hawrot, Cultured sympathetic neurons: Effects of cell-derived and synthetic substrata on survival and development. Dev. BioI •• 74:136-151 (1980). ----

8. M. Johnson. D. Ross, M. Meyers. R. Rees. R. Bunge, E. Wakshull, and H. Burton. Synaptic vesicle cytochemistry changes when cultured sympathetic neurons develop cholinergic inter­actions, Nature, 262:308-310 (1976).

PLASTICITY IN THE NEUROTRANSMITTER PHENOTYPE 343

9. M. I. Johnson, C. D. Ross, M. Meyers, E. L. Spitznagel, and R. P. Bunge, Morphological and biochemical studies on the development of cholinergic properties in cultured sympathetic neurons. I. Correlative changes in choline acetyltransferase and synaptic vesicle cytochemistry, J.Cell BioI., 84:680-691 (1980) •

10. L. Iacovitti, T. H. Joh, D. H. Park, and R. P. Bunge, Dual ex­pression of neurotransmitter synthesis in cultured neurons, J.Neurosci., 1:685-690 (1981).

11. M. Weber, A diffusible factor responsible for the determination of cholinergic functions in cultured sympathetic neurons. Partial purification and characterization, J.Biol.Chem., 256: 3447-3453 (1981).

12. M. J. Weber and A. Le Van Thai, Progress in the purification of a factor involved in the neurotransmitter choice made by cul­tured sympathetic neurons, in: "Embryonic Development", M. M. Burger and R. Weber, eds., Vol.85 B, pp.473-483 (1982).

13. J. Axelrod, J. K. Inscoe, and J. Daly, Enzymatic formation of O-methylated dihydroxy derivatives from phenolic amine~ and indoles, J.Pharm.Exp.Ther., 149:16-22 (1965).

14. F. Fonnum, A rapid radiochemical method for the determination of choline acetyltransferase, J.Neurochem., 24:407-409 (1975).

15. I. B. Black, I. A. Hendry, and L. L. Iversen, Differences in the regulation of tyrosine hydroxylase and DOPA-decarboxylase in sympathetic ganglia and adrenal, Nature, 231:27-29 (1971).

16. H. Thoenen, P. U. Angeletti, R. Levi-Montalcini, and R. Kettler, Selective induction by Nerve Growth Factor of tyrosine-hydroxylase and dopamine-S-hydroxylase in the rat superior cervical ganglia, Proc.Natl.Acad.Sci.USA, 68:1598-1602 (1971).

17. H. Thoenen, Comparison between the effect of neuronal activity and Nerve Growth Factor on the enzymes involved in the synthesis of norepinephrine, Pharmacol.Rev., 24:255-267 (1972) •

18. A. Chalazonitis, P. J. Rice, and R. E. Zigmond, Increased ganglionic tyrosine-hydroxylase and dopamine-S-hydroxylase activities following preganglionic nerve stimulation: role of nicotinic receptors, J.Pharm.Exp.Ther., 213:139-143 (1980).

19. J. M. Lundberg, T. Hokfelt, M. Schultzberg, K. Uvnas-Wallenstein, C. Kohler, and S. I. Said, Occurrence of vasoactive intestinal polypeptide (VIP)-like immunoreactivity in certain cholinergic neurons of the cat: evidence for combined immunohistochemistry and acetylcholinesterase staining, Neuroscience, 4:1539-1559 (1979).

20. J. P. Swerts, A. Le Van Thai, A. Vigny, and M. Weber, Regulation of enzymes responsible for neurotransmitter synthesis and degradation in cultured rat sympathetic neurons. I. Effects of conditioned medium, Dev.Biol., in the press.

21. P. R. Vulliet, T. A. Langan, and N. Weiner, Tyrosine hydroxylase: a substrate of cAMP-dependent protein kinase, Proc.Natl.Acad.Sci.USA, 77:92-96 (1980).

344 J.-P. SWERTS ET AL.

22. B. Berger, U. Di Porzio, M. C. Daguet, M. Gay, A. Vigny, J. Glowinski, and A. Prochiantz, Long-term development of mesencephalic dopaminergic neurons of mouse embryos in dissociated primary cultures: morphological and histochemical characteristics, Neuroscience, 7:193-205 (1982).

23. E. L. Giller, J. H. Neale, P. N. Bullock, B. K. Schrier, and P. G. Nelson, Choline acetyltransferase activity of spinal cord cell cultures increased by co-culture with muscle and by muscle-conditioned medium, J.Cell Biol., 74:16-29 (1977).

24. E. W. Godfrey, B. K. Schrier, and P. G. Nelson, Source and target cell specificities of a conditioned medium factor that increases choline acetyltransferase activity in cultured spinal cord cells, Dev.Biol., 77:403-418 (1980).

25. N. Brookes, D. R. Burt, A. M. Goldberg, and G. G. Bierkamper. The influence of muscle-conditioned medium on cholinergic maturation in spinal cord cell cultures, Brain Research, 186:474-479 (1980).

INDEX

A23187, 23, 61, 102, 231, 314, 315

A5E3, 183-190 Acetate (transmitter precursor),

337 Acetylcholine

accumulation neural tissue, 15-19 synthetic neurons, 311,

336-342 sensitivity, 225, 244 synthesis

neural tissue, 15-19 sympathetic neurons, 311,

336-342 Acetylcholine receptor

embryonic amphibian myotubes,

Adhesion (continued) neural migration, 140

Adrenergic neurons amphibian, 63 avian, 109-120, 140 mouse (in vit~o), 272 rat, 109-120, 311-316 vertebrates, 140

Aggregation (neural in vivo), 143 Albumin, 191-198 Alomethecin (transmitter release),

314 Ancestral cell group, 42 Archenteron (amphibian), 59 Astrocyte

chick embryo (spinal cord), 305, 306

225-227 mouse embryonic mammalian myotubes, brain, 202, 204

225-227 mesencephalon, 323-327 muscarinic (cerebellar), 135 striatum, 323-327 Xenopus (translation in vit~o), rat, 186-190

133, 134 Autonomic neurons, 227 Acetylcholinesterase, 12, 19, 145, avian

146, 226, 332, 339-342 in vit~o, 109-120, 215-222 Xenopus (translation in ovo), in vivo, 109-120

133, 134 human (in vivo), 177-180 ACh (see Acetylcholine) AChE (see Acetylcholinesterase) AChR (see Acetylcholine receptor) Actin, 157 Adhesion, 177, 179, 320-323

early morphogenesis, 46-48 neural induction, 24

rat in vit~o, 109-120, 207-210,

311-316, 335-342 in vivo, 109-120

vertebrates (in vivo), 140 Axons, 324

pioneer, 51, 52

345

346

Axonal outgrowth, 50, 52 Axonal transport, 157, 161

Ba2+ (transmitter release), 314 Basal lamina, 57, 140, 227 Blastocoel (amphibian), 89 Blastocyst, 169 Blastomere

amphibian, 233, 237 xenopus, 39-50

Blastoporal lip (amphibian), 5, 10, 21, 29

Blastula amphibian, 67, 71, 75 axolotl, 57 cynops, 85 xenopus, 39, 49

Brain (development), 319 bovine

extract, 133, 331-333 in vitro, 202

chick (extract), 331-333 fetal chick, 177, 178 fetal human, 177, 178, 191-198 fetal mouse

in vitro, 123-128 in vivo, 191-198

fetal pig, 191-198 fetal rat

in vitro, 177, 191-198 in vivo (mRNA) , 132, 133

fetal sheep, 191-198 mouse

glia, 203 in vitro, 272-276

Brain pig (extract), 265-267 rat, 187, 299

development, 307 extract, 288, 331-333

Ca 2+ differentiation, 257, 258 neural induction, 102 protein release, 315 second messenger, 230, 231

CA (see Catecholamines) Calmodulin, 231

INDEX

cAMP (see cyclic mononucleotides) Capping (ectoderm), 8 Carbonic anhydrase II (see also

Marker, glial, central), 128

Carcinoma (mouse), 145-152 Cardiotonic steroid, 254-258 CAT (see choline acetyltrans-

ferase) Catecholamine

accumulation (sympathetic neurons), 110, 117, 312, 336, 341

biosynthetic enzymes, 110 fluorescence, 12, 19, 146, 147 synthesis

neural tissue, 16, 19, 255 sympathetic neurons, 110,

113, 312, 336, 341 uptake

brain stem cells, 273 sympathetic neurons, 110,

113, 312 Caudalization (amphibian), 69 Cell commitment, 46 Cell death, 263-265 Cephalo spinal fluid (protein

localization), 133, 178, 191-198

Cerebellar neurons bovine, 202 mouse, 272-276, 323

Cerebellum, 131-135, 187, 203, 325

Cerebral cortex (mouse), 322 Cerebral ganglion (amphibian

development), 235 Cesium (channel-blocker), 219 CG (see Ciliary ganglion) cGMP (see Cyclic mononucleotides) Channel

chemically sensitive, 239-246 endplate, 225-227 gap junctional, 253

INDEX

Channel (continued) ionic, 215-219, 225, 232,

239-246 neurotransmitter response, 243 voltage-dependent, 239-246

ChE (see Cholinesterase) Cholera toxin, 231 Cholesterol, 128 Choline (neurotransmitter

precursor), 15, 336, 337 Choline acetyl transferase , 288,

338-342 Cholinergtc neurons, 227

amphibii¥l, 63 avian, 215-222 rat, 312, 315

Cholinesterase, 255 translation in OVO, 134, 135

Chondroitin sulfate, 61, 140 Chordamesoderm (amphibian), 10-19,

21-33 Chorio-allantoic membrane

(transmitter phenotypic expression), 114, 117

Choroid plexus, 191-198

Conditioned medium heart cells, 288 muscle cells, 292, 293,

335-342 non-neuronal cells, 208,

335-342 pig brain, 265 rat brain, 307-309 striatal glia, 323 striatal neurons, 323

Conductance, 243-244 Cortical plate, 192 Corticosterone, 117

347

Coupling (electrical), 243-245, 253, 255

CSF (see Cephalo spinal fluid) Culture conditions

mesencephalic dopaminergic cells (mouse), 322

sensory neurons (chick embryo), 301

striatal cells (mouse), 322 sympathetic neurons (chick

embryo), 301 Curare, 225, 244

Chromatin, 93, 104 Ciliary ganglion (chick),

279-285 Clonal domain, 42

Cyclic mononucleotides 215-222, differentiation, 145-148

induction, 23, 61, 102, 145-148, 230, 231

Clonal restriction (Xenopus), 39-52

CM (see Conditioned medium) Cobalt (channel-blocker), 218,

219 Collagen

ECM, 57, 140, 141 neural tissue, 12, 13 substratum, 279-281

Cytoplasm distribution, 230, 233 neural induction, 83-85, 169 transfer, 230, 233

Cytoskeleton (neural induction), 33, 101, 102

02, 177-180, 332 0-600, 231

Compartment (early morphogenesis),DBH (see Oopamine-S-hydroxylase) 39-50 Dendritic arborization, 271,

Compartmentation, 42 319-327 Competence (amphibian ectoderm), Denervation, 225-227, 291-294

21-33, 67-72 Depolarization, 314 Con A (see Lectin, Concanavalin

A) Desmethylimipramine, 14, 272,

276

348 INDEX

Desmin, 158, 160 Dextra sulfate, 61 Differentiation

DRG (see Dorsal root ganglion)

auto-, 61, 67 cell, 229-237 ectoderm, 21-33, 83-87 glial, 201-204, 287

ECM (see Matrix, extracellular) Ectoderm

amphibian, 4-10, 21-33, 55-66, 67-72, 75-80, 89-104, 251-258

mesoderm, 70, 71 cynops, 83-87 muscle, 239-247 Electrical excitability neural, 3-19, 22-33, 41, 55-66, development, 221, 226, 229-237,

70, 75, 83-87, 89-104, 240-246, 255 139-143, 145-152 ionic dependence, 215-222,

neuronal, 157-171, 251-259, 240-246 264, 287-289, 316, 324 Endocrine cells, 140

neuronal membrane, 239-247 Endoderm neurotransmitter, 109-120 amphibian, 22, 55-66, 91 stimulated, 59 teratocarcinoma, 145-152 subsynaptic, 226, 227 Endodermalization (amphibian), 24 transcription, 55, 246, 247 Endocylosis, 315 translation, 246, 247 Endothelial cells (cerebral), 194

Division Endplate cell, 229, 230, 319 current, 227 early morphogenesis, 42, 48 motor, 225-227 transcription, 230 Enolase (neuron specific), 124, translation, 230 125, 332

DMI (see Desmethylimipramine) Enteric glia, 182-190 Dopa decarboxylase, 338 Dopamine

neurotransmitter precursor, 336, 337

synthesis (brain dopaminergic neurons) 272, 322

uptake, 337 brain dopaminergic neurons,

273, 276, 322-326 neural plate, neural fold,

14 Dopamine-S-hydroxylase, 110, 338 Dopaminergic neurons (mouse),

272, 276, 319-328 Dorsal root ganglion

chick embryo, 266, 267, 293, 299-306

human, 179 rat, 183-187, 316 vertebrates, 140

Enteric neurons, 110, 113 Ependymal cells (glial ependymal

tanycytes), 123 Epidermis

amphibian, 4, 22, 55-66 Xenopus, 50

Epithelial cells choroid plexus, 192, 194 neural tissue, 12, 165

Exocytosis, 315 Extract

brain, 128, 265, 331-333 chick embryo, 120, 279-285 chick muscle, 294, 295 spinal cord, 299-305 tissue, 288

Factor AChR synthesis, 227 AChR clustering, 227

INDEX

Factor (continued) cell surface components, 272 CM (see Conditioned medium) cytoplasmic, 63, 230 density, 322, 323 developmental stage-dependent,

279-285 ECM, 272 electric fields, 272 environment, 50, 55, 109-120,

128, 140, 145, 182, 229, 331, 335

epigenetic, 207, 229, 263, 271, 279, 287-289, 335

extract (see Extract) glia (see Glia) gradient, 319, 321 growth, 279-285, 287-289,

Fetuin, 191-198 Fibroblast, 141 Fibronectin

ECM, 140-143 enteric glia, 186, 189 fibroblast, 202 neural tissue, 10, 12, 50

Filament intermediate, 12, 157-171,

183-190 neurofilament, 12-19, 110,

146-148, 158-171, 255, 294

microfilament, 27, 49, 157 Fluoxetine, 273 Follicle cells, 230 Forebrain (amphibian differen­

tiation), 23, 70, 71

349

299-306, 331-333 hormonal, 113 maturation promoting, 230 motor neuron growth, 291-296 Na , 257-259 NGF (see NGF)

GABA (see y-aminobutyric acid) Galactocerebroside (see also

Marker, glial) oligodendrocyte-specific, 148,

202, 204 non-neuronal cells, 19, 272, peripheral glia, 183, 186

300, 304 Galactose oxidase, 209, 210, 312, neural-inducing, 5-12, 75-80, 313

83-87, 89-104 y-aminobutyric acid, 243, 245 neuralizing (see Inductor) Gangliogenesis, 143 neurite extension (see Neurite Ganglioside, 208-210

extension) Gastrulation pH, 230 amphibian, 3-19, 21-29, 57, phenotype (see Phenotype) 67-72, 76-80, 89-103, retinal, 128 251, 252 retinoic acid (see Retinoic

acid) serum, 120

cynops, 84, 87 Xenopus, 41-52

Germ cells (amphibian, survival (see Survival) invertebrates), 230 tissue, 113 GFAP (see Glial fibrillary trophic, 263, 279-285, 299-306, acidic protein)

331-333 Glia Fasciculation (neurite), 10, 12,

177, 179 Fertilization (amphibian,

invertebrate), 232 a-Fetoprotein, 191-198

Bergmann, 132, 187 differentiation, 124, 128,

201-204, 287 early morphogenesis, 46 enteric, 182-190, 186-190

350

Glia (continued) factor, 267, 300-306, 323 formation, 307-309 glioma, 307-309, 335 peripheral, 181-190, 186-190 proliferation, 267, 332

Glial fibrillary acidic protein (see also Marker, glial, central), 123, 125, 146, 148, 158, 185-190, 202, 324

Glucagon, 61 Glucocorticoid, 117-119 Glutamate (neurotransmitter),

244 GlutaIDlne synthetase (peripheral

glia), 185-190 Glycine (neurotransmitter),

243-246 Glycoaminoglycan, 140 Glycolipid, 204, 207-210,

311-316 Glycopeptidase, 85 Glycoprotein, 27-33, 50, 83-87,

140, 177-180, 208-210, 311-316

Glycosphingolipid, 208 Granule cells (migration),

132-135, 308, 325 Growth cone, 319-328 Gut

amphibian, 59 rat, 110-118

Heparan sulfate, 60, 61 Hindbrain (amphibian differen-

tiation), 69-71 Hippocampal dentate gyrus, 133 Hippocampus, 326 Hirudin, 308 Hormone, 61, 117, 118, 124,

229-231, 273

Horseradish peroxidase, 39-50, 195

Hydrocortisone, 117 Hypothalamic neurons (mouse),

123-128

INDEX

Hypothalamus (mouse development), 123-128

Immunization (in vitro), 316 Immunocytolysis, 202 Implantation (amphibian-gastrula),

22, 89 Induction (amphibian)

archencephalic, 93-97 neural, 3-19, 21-33, 75-80,

83-87, 89-104, 240, 245 primary, 21-33, 55-66, 75-80,

253 sequential, 67-72

Inductor caudalizing, 69 mesodermalizing, 24, 69, 72 neuralizing, 21-33, 69, 70,

89-103 substance, 24, 61, 93-104 tissue, 4, 61, 67-72, 75-80,

83 vegetalizing, 23-33, 68, 90

Innervation muscle, 291-296 striatum, 322, 327 sympathetic, 264

Insulin, 273 Interneuron (Xenopus-spinal), 50 Ionophore, 61

Junction gap, 253, 257 neuromuscular, 291-296 tight, 194, 195

K+ (differentiation), 254, 256 a-Keratin, 158, 165 Kinase (cAMP-dependent protein),

230

HRP (see Horseradish peroxidase) Lactate dehydrogenase, 338 Hyaluronic acid, 63, 66, 140

INDEX 351

Lactopero~idase ,125I-labeling), Matrix 178, 313

(extracellular), 10, 33, 101, 102, 139-143, 151, 272, 313 Laminin, 140

Layer ependymal (xenopus), 255 germ

amphibian, 56, 57, 68 Xenopus, 41, 43

granular (Xenopus), 132 molecular (Xenopus), 132 subventricular (mammalian), 192 ventricular (mammalian), 192

Lectin, 3-9, 26-32, 61, 83-87, 178, 208, 209, 232

concanavalin A, 4, 26-32, 83-85, 178, 232

Dolichos biflorus, 85, 208 Lens culinaris, 4 Pisum sativum, 4 Ricinus Communis agglutinin,

208, 209 Soybean agglutinin, 4, 208,

209, 311, 312

Maturation brain

mouse, 124 vertebrates, 167

glia (peripheral), 183 neurons

mesencephalic dopaminergic, 319-328

sensory, 244, 246, 266, 287 sympathetic, 338

Medium (defined), 198, 322 Meiosis (reinitiation), 229-232 Melanin, 59, 63 Melanocyte, 140 Melatonin, 252, 255 Membrane (neural induction),

83-85 Meningeal cells

chick, 331-333 Xenopus, 132

Wheat germ agglutinin, 27, 178 Mesencephalic neurons Limbic structures, 322 mouse, 272, 319-328 Lithium (vegeta1ization), 60, 61 Lymphocyte (stimulation), 83-85

MAP, 160 Marker

cerebellum, 132 fibroblast, 202 glial

central, 123-125, 128, 146, 148, 158, 185-190, 201-204, 324

peripheral, 148, 182-190 intracellular, 39 neuronal, 124, 332

enzyme, 288, 289 intracellular, 12, 110,

146-148, 157-171, 255 membrane channel, 215 surface, 14, 177-180, 202,

204, 208, 209, 255, 271, 312-316, 319

quail, 215-222 Mesenchyme cells, 60 Mesoderm, 16, 22, 57-63, 67-72,

75, 91, 100, 233, 237, 243

Mesodermalization, 24, 75, 76 Metaphase, 234 Met-enkephalin, 125 I-Methy1adenine, 229-234 Microtubule, 157, 161 Migration, 133, 134

glial, 124 granular, 134, 308, 309 malignant, 133 neural, 10, 42, 139-143 neuronal, 109-119, 124, 308,

309, 319-328 Mitogen (lectin), 83 Monensin (transmitter release),

314

352

Morphogenesis amphibian, 56-66, 230, 233 vertebrates, 139-143 Xenopus, 39-52

Morula, 67 Mosaicism, 229-235 Motility, 47, 48, 140-143 Motor neurons, 225, 241, 244,

265 primary (Xenopus), 46-52 spinal (embryonic chick),

291-295 MPF (see Factor, maturation

promoting) mRNA, 131-135 Muscle

regulation, 225-227 smooth, 186

Muscle cells amphibian, 255, 256 Xenopus, 50

Mutant jimpy, 201, 203 staggerer, 132, 135

Myelin, 183, 201 basic myelin protein, 128,

148, 183 Myelinisation, 182-187 Myotube

amphibian, 225 ma=alian, 225 Xenopus, 51

Na+ (differentiation), 252-259 N-CAM (see Neural cell adhesion

molecule) NE (see Norepinephrine) Nerve

peripheral, 178, 185, 203 terminal, 225, 226, 295, 296,

323, 328 Neural arborization, 307 Neural cell adhesion molecule,

143, 177, 179 Neural crest, 139-143

complex, 55, 109-120

Neural crest (continued) derivatives, 341

amphibian, 251, 256 human, 178

mesencephalic, 215-222

INDEX

Neural fold, 10-16, 253-258 Neuralization, 23, 24, 69, 75-78,

102 Neural plate, 10-16, 55, 68, 71,

100, 240-245, 251-258 Neural receptor (induction), 6,

9, 22, 23, 83 Neural tube

amphibian, 240, 243, 251-257, 255

mouse, 123 Xenopus, 50

Neuraminidase, 313 Neurite

density, 264 extension, 10, 12, 198, 245,

246, 264, 265, 279-285, 319-328

neuroblastoma, 307-309 sensory neurons, 292-294,

299-305 transport, 161

Neuroblast, 166, 167 cerebral hemispheres, 331-333 differentiation, 110-118 gastrulation, 19 migration, 307-309 proliferation, 307-309

Neuroblastoma, 133, 167, 171, 307-309

Neuroderm, 75-80 Neuroectoderm, 25, 68, 71, 75-80 Neuroependyma, 195 Neurofilament, 12-19, 110,

146-148, 255, 294 Neurogenesis

amphibian, 3-19, 21-33, 67-72, 75-80, 89-104, 229-237, 239-247, 251-259

cynops, 83-87

INDEX

Neurogenesis (continued) vertebrates, 139-143, 165-167,

271-276, 308, 309 Neuro-glia-relationship, 182,

307-309 Neuronal cells

competition, 271-291 epithelial, 124 morphogenesis, 46, 56-66 secretory, 124, 128 specificity, 202, 271, 272,

319 Neuropeptide, 124, 125 Neurophysin, 124, 128 Neurotubule, 12 Neurulation

amphibian, 10-16, 22, 70, 77-80, 102, 253-259

Xenopus, 49 NGF, 119, 204, 207, 263-267,

279-285, 299-305, 339 NGF inducible, large external

protein, 313 Nigro-striatal dopaminergic

system, 322-328 NILE protein (see NGF inducible,

large external protein) Norepinephrine

accumulation, 336, 337 release, 315 synthesis, 336, 337 uptake

Organizer amphibian, 3, 68, 75 cynops, 86, 87

Ouabain, 61

353

neural induction, 23, 102 neuronal differentiation, 254

Paraendocrine cells, 140 Parasympathetic neurons

chick embryo, 265, 279-285, 300, 305

human, 179 Parenchymal cells, 180 Parenchyme (transmitter pheno­

typic expression), 114 Parietal cortex, 326 PC 12 cells (see Pheochromocy-

toma) Perineurial cells, 186 Permeability, 254 Phenotype

modulation glia (peripheral), 182, 183 neurons, 125, 207-210, 272

transmitter, 109-120, 171, 207-210, 246, 255, 287-289, 312-316, 335-342

Pheochromocytoma, 167, 171, 293, 315

Picrotoxin, 244 Pigment cells (amphibian

Schwann cells, 204 development), 252, 255 sympathetic neurons, 110, 113 Plasma protein, 191-198

Notochord Plasminogen activator, 133, 134, amphibian, 55, 63, 91, 255 307 transmitter phenotypic

expression, 113-117

Octopamine, 337 Oligodendroglia

cell line, 128 differentiation, 201-204 surface antigens, 201-204

Oocyte, 232-234

Plasticity, 109, 207-210, 272, 324, 335-342

Plate neural, 68 precordal, 68

Plexus (myenteric), 114, 185, 186 Polarity (animal-vegetal), 67,

232-234

354 INDEX

Potential Ribonucleoprotein particles (RNP), action 33, 93-104

development, 240-244 Rohon-Beard neurons, 46-52, ionic basis of, 218-221, 233, 240-246

234, 240-246 endplate, 255 fertilization, 232 resting, 232, 234, 246,

253-258 Potentiality, 124 Progesterone, 230 Proliferation

nervous cell line, 128 neural crest cells, 142 neuroblast, 308 neuronal cells, 331-333

Prophase (amphibian), 230 Prostaglandin El, 61 Protease (serine), 308 Proteoglycan, 313 Pump, 61, 254-259

Ran-I, 183-190 Ran-II, 183-190 Raphe nucleus (mouse), 273 RCA (see Lectin, Ricinus

Communis Agglutinin) Reaggregation

neural induction, 79, 80 plating, 281, 322

Recognition cell-cell, 39-52, 201, 207-210,

236, 237, 271, 311, 319-328

tissue, 25, 79 Reflex (primary), 50 Regeneration, 242, 291-296 Regulation (posttranscriptional),

131-135 Reticulocyte lysate (post­

transcriptional regulation), 131

Retinal cells chick, 279-284

S-IOO (see also Marker, glial, central), 124

Sandwich method (gastrula), 22, 90

Sarcolemma amphibian, 225, 227 mammalian, 225, 227

Satellite cells chick, 267 rat, 182-190

SBA (see Lectin, Soybean Agglutinin)

SCG (see Superior cervical ganglion)

Schwann cells chick, 204 rat, 182-187

Sciatic nerve chick, 294 rat, 183-187

Secretion (glycoprotein), 315 Sensitivity (neurotransmitter),

244 Sensory ganglion, 182, 183 Sensory neurons

cranial human, 178, 179 chick, 300

nodose chick, 300, 305 rat, 315, 316, 341, 342

spinal amphibian, 240-246 chick, 265, 266, 280, 288,

289, 291-295, 299-306 Xenopus, 46

trigeminal chick, 300, 305 rat, 315, 316

rat, 267 Serotoninergic fibers, 85, 273 Retinoic acid, 145-148, 169, 171 Sialic acid, 208

INDEX

Somatic cells, 230 Somatostatin, 125, 289 Somite, 255 Sperm, 231-234 Spinal cord

amphibian, 71, 240-246 chick, 299-306 human, 178, 299-306 mouse, 203 rat, 299-306, 341, 342 Xenopus, 52

Stem cells, 123

355

Sympathetic ganglion (glia), 182 Sympathetic neurons

avian, 110, 113 cat, 339 chick, 280-285, 287-289, 299,

305 human, 179 rat, 113, 177, 207-210, 311-316,

335-342 Synapse, 207-210, 271, 272, 289,

Striatal cells (mouse), 272, 273,

292, 308, 319-328 chemical, 240, 245 electrical, 240, 245 folds, 226 neuromuscular, 226, 227 regulation, 311

322, 323 Striatum (mouse), 322-327 Strophanthidin, 254-258 Substance P, 289 Substratum

mesencephalic dopaminergic cells, 322

Tail (formation), 68, 69 TEA (see Tetraethylammonium) Tectum (chick), 283 Teratocarcinoma, 145-152 Testosterone, 61

neural crest cells, 114 parasympathetic neurons,

279-281 sensory neurons, 301 striatal cells, 322 sympathetic neurons, 279-281,

Tetanus Toxin (see also Marker, neuronal, surface), 14, 19, 202, 204, 255

Tetraethylammonium, 218, 219 301, 335

Sulfatide (glia) central, 204 peripheral, 183

Superior cervical ganglion 186, 207-210, 266, 335-341

Survival, 331-333 dopaminergic neurons, 324 neuroblastoma, 307-309 parasympathetic neurons,

279-284

Tetrodotoxin, 218, 219, 245 Thrombin, 308 TOH (see Tyrosine hydroxylase) Transferrin, 191-198, 273

(rat),Transglutaminase, 231 Transplantation (gastrula), 76 Trunk (formation), 68 TT (see Tetanus Toxin) TTX (see Tetrodotoxin) Tubulin, 157-161 Turnover (neurotransmitter

vesicle), 313 sensory neurons, 263-267, Tyramine (neurotransmitter

287-289, 291, 292, precursor), 337 299-305 Tyrosine (neurotransmitter

stem cells, 271, 273 precursor), 16, 336, 337 sympathetic neurons, 312, 338, Tyrosine hydroxylase, 110, 273,

341 288, 289, 338-341 Sweat glands (rat), 339

356

Umbilical cord (transmitter phenotypic expression), 114

Uncoupling, 243 Urokinase, 307, 308

Valinomycin, 61 Vasoactive intestinal polypeptide,

289 Vasopressin, 124 Ventricle, 123-125, 193, 194 Veratridine (transmitter release),

314

INDEX

Vimentin, 158-171, 183-190, 204 VIP (see Vasoactive intestinal

polypeptide) Volume (neural induction), 76-78

WGA (see Lectin, Wheat germ agglutinin)

White matter, 203

Xenopus development, 39-52, 254 ectoderm, 26-29, 254 post transcriptional regulation,

131-135