target cell surface glycoconjugates and neural induction in an … · with fitc or tritc as...

/. Embryol. exp. Morph. 86, 39-51 (1985) 3 9Printed in Great Britain. © Company of Biologists Limited 1985

Target cell surface glycoconjugates and neuralinduction in an amphibian

LYDIE GUALANDRIS, PIERRE ROUGE AND ANNE-MARIE DUPRAT*Laboratoire de Biologie ginerale and Laboratoire de Biologie Cellulaire, FacultySciences Pharmaceutiqu.es, Universite Paul Sabatier, 118 Route de Narbonne,31062 Toulouse Cedex, France

SUMMARY

The possible involvement of target membrane specific receptor(s) in the transmission of theneural signal leading to activation of the intracellular machinery involved in the process of neuraldetermination, has been examined using lectin probes (Con A, succinylated-ConA, LcA, PsAand SB A). Not only Con A binding sites but many different glycoconjugated molecules (a-D-galactose, N-acetyl-D-galactosamine, a-D-fucose, N-acetyl-D-glucosamine, etc.) would have to beinvolved, if neural receptor(s) are invoked to explain initiation of neural induction. We show herethat the close involvement of such receptor molecules in neural induction is so far hypotheticaland remains to be demonstrated. Moreover we are inclined to the view of Barth and others whosuggested that ionic fluxes and physicochemical and electrophysiological properties of the targetmembrane could play a crucial role in neural induction.

INTRODUCTION

The molecular mechanism of the neuralization of ectodermal cells during gastru-lation is an important and yet unsolved problem of neuroembryology.

It is now recognized that a neuralizing factor (or stimulus) exerts its first effectonly at the cell surface of target cells (Tiedemann & Born, 1978; Yamamoto &Ozawa, 1981). Several authors have shown recently the important role played bythe target cell membrane in the onset of the neural induction process (Grunz &Staubach, 1979; Takata etal. 1981; Takato, Yamamoto, Ishii & Takahashi, 1984;Duprat, Gualandris & Rouge, 1982; Gualandris, Rouge & Duprat, 1983). Recently,using in vitro association of blastoporal lip with presumptive ectoderm covered(inner side) or not (outer side) by extracellular material, we have shown that theextracellular matrix (covering the neural target tissue) is not necessary for thetransmission of the neuralizing signal and seems therefore not directly involved inthe process of neural induction (Duprat & Gualandris, 1984).

The molecular components of the competent presumptive ectoderm surface(neural target tissue) have a specific pattern especially in their glycoconjugatedcomponents as visualized by binding to labelled lectin probes (Nosek, 1978;

*For reprints.Keywords: Neural induction, cell surface glycoconjugates, lectins, amphibian embryo, celldetermination.

40 L. GUALANDRIS, P. ROUGE AND A.-M. DUPRAT

Barbieri, Sanchez & Delpino, 1980; Gualandris etal. 1983). The process of neuralinduction is impaired by molecular reorganization after Soybean lectin treatmentof the target plasmalemma prior its in vitro association with the blastoporal lip(Duprat et al. 1982). This impairment is reversed after reconstitution of the normalmolecular organization of the membrane, due to normal turnover of glycocon-jugates (Gualandris etal. 1983). Therefore, glycoconjugates and /or the structuralorganization of the plasma membrane of target cells play a role in the onset of themolecular events in the neural inductive machinery which ultimately lead to neuraldetermination.

The aim of the present work was to discuss, in the light of new findings using lectinprobes (PsA, LcA, Con A and succinylated - Con A) which bind to the sameoligosaccharide residues (a-D-mannose, a-D-glucose residues), the hypotheticalexistence of a specific neural receptor on the competent target plasma membraneand its relationship to the molecules which bind lectins (Takata et al. 1984).

MATERIAL AND METHODS

Experimental procedurePresumptive ectoderm was isolated from early gastrulae (stage 8) of Pleurodeles waltl staged

according to Gallien & Durocher (1957). Experiments were carried out in Holtfreter solution,pH8-0, Tris 5mM, containing penicillin (lOOi.u.mF1) and streptomycin (lOOjugml"1).Gastrulae were manually dejellied and the vitelline membrane removed. The microsurgicallyexcised ectoderm was treated with different lectin solutions (50 jug ml"1 or 300 [ig ml"1) for 30 minor 3 h. The explants were then washed several times in Holtfreter solution and dissociated withBarth dissociation medium (88 mM-NaCl, 1 mM-KCL, 2-4 mM-NaHCO3,2 mM-Na2 HPO4,0-1 miu-KH2 PO4, 0-5 mM EDTA, pH8-5). The isolated cells were cultured on dried collagen substratein Falcon or Nunc dishes with Barth balanced salt solution (Barth & Barth, 1959) for up to 10 daysat 20 °C. For control experiments, the ectoderm was combined with the blastoporal lip accordingto the now classical Holtfreter 'sandwich-method' (Gualandris & Duprat, 1981).

The test to score neural induction was the differentiation of neurones which expressed neuro-filament polypeptide markers detected by immunocytochemistry (Duprat & Gualandris, 1984).

LectinsThe lectins used (Table 1) were:Con A - Concanavalin A (Canavalia ensiformis agglutinin)S-Con A - succinylated Con A.PsA - Pisum sativum agglutinin.LcA - Lens culinaris agglutinin.These lectins (except S-Con A supplied by IBF-France) were isolated, purified and labelled

with FITC or TRITC as previously described (Duprat et al. 1982; Gualandris et al. 1983) PsA,LcA and Con A are known to involve the capping of membrane glycoconjugates whereas S-ConA did not involve such a reorganization (Gunther et al. 1973).

Tests of lectin specificity

(1) Specificity of lectins for sugarsThe specificity of the purified lectins was tested by an haemagglutination inhibition assay

(Table 2).The haemagglutinating activity of native and succinylated lectins was determined by two-fold

serial dilution in 0-1 M-phosphate-buffered saline (pH7-2) on standard microtitration plates. To

Cell surface glycoconjugates and neural induction 41

Table 1. Characteristics oflectins.

number of subunits

number of sugar bindingsites per moleculerelative molecular mass

carbohydrate percentage

metal requirement

sugar specificity

Con A S. Con A

4y4 2y2

4 2

120000 55000

0 0

+ +

PSA

4^202

2

49000

0-3 Glc

+Sugars of Makela's group III (cf.

Glc, galactose; -NAc, N-acetyl-D-galactosamine.

LCA

2^2/32

2

48000

2 Glc, Glc-NAc

+Table 2)

each lectin solution (50 (A) were added 200/i of a 1% solution of thrice-washed rabbit eryth-rocytes in PBS. Agglutination was estimated macroscopically 12h later.

Inhibition of haemagglutination by sugars was tested by two-fold serial dilution in 50/ul. Toeach sugar dilution 50 fA of PBS containing 50 jug ml"1 of native or succinylated lectin were added.After a 1 h incubation, 200 jul of a 1% solution of thrice washed rabbit erythrocytes in PBS wereadded and agglutination was estimated macroscopically 12 h later.

The data of the inhibition test with simple sugars clearly indicate that Con A, S-Con A, PsAand LcA constitute one group of lectins which bind specifically a-D-mannosyl and a-D-glucosylresidues, a-methyl-D-mannoside being their best inhibitor.

(2) Absorption of lectinsThe specificity of lectin absorption was tested by competitive inhibition. Lectins were prein-

cubated with their hapten inhibitor (a-D-mannose) in order to prevent their haemagglutininactivity. For a preincubation, 50/zg or 300 pig of lectin was used in lml of a 0-1M solution of

Table 2. Minimum concentration (mM) of sugar giving complete inhibition ofhaemagglutination

Sugar PsA LcA Con-A S-Con A

D-mannoseD-glucoseD-fructoseD-glucosaminea-methyl-D-glucoside/3-methyl-D-glucosidea-methyl-D-mannosideN-acetyl-D-glucosaminesucrose

The following sugars were not inhibitory at final concentration of 200 mM: D-arabinose, L-fucose, L-rhamnose and D-ribose, D-galactose, D-galactosamine, a-methyl-D-galactoside, j8-methyl-D-galactoside, N-acetyl-D-galactosamine. The lectin concentration used (SOjugml"1) is4- to 8-fold higher than that producing complete haemagglutination of rabbit erythrocytes at thelast 2-fold dilution.

6.252550

10012-5

3-122525

2550

10020050

—12-55050

6-252525

1003-12

—0-78

2525

1-563-12

25500-78

1000-396-256-25


inhibitory carbohydrate for 15 and 30 min at room temperature. This sugar concentration reducedthe haemagglutinin activity to zero and was sufficient to obtain maximal saturation of lectin-binding sites.

None of the biological effects of lectins were observed when the hapten inhibitor was co-present in the solution.

(3) Saturation of binding sites (SOjUgmr1 final concentration)The saturation of lectin-binding sites on neural target cells was tested by use of unlabelled and

then fluorescent (FITC or TRITC) lectins.Explants were first incubated in a solution of unlabelled lectin (Con A for example) 50 //g ml"1

for 30 min, thoroughly washed and incubated in a solution of the fluorescent lectin (Con A-FITCor TRITC) SOjugmF1 for 30 min. After several washings the control of the fluorescence ofexplants was observed with a Leitz Dialux microscope equipped with HBO 50, filters I2 (BP450-490; LP 515) and M2 (BP 546/14; LP 580). The saturation of binding sites for S-Con A, PsAand LcA was tested in a similar way.

No fluorescence was detected on the explants. All the lectin binding sites were saturated by thefirst incubation. Con A, S-Con A, PsA and LcA (50 jUgml for 30 min) were therfore in saturat-ing concentration.

(4) Homology of binding sitesThe homology of the binding sites for these four lectins studied was checked in the same way,

using first unlabelled and then labelled lectins (SOOjugmF1 for 15 or 30min).Explants were first incubated with one lectin (Con A for example), washed, then incubated

with another lectin (PsA for example) labelled with FITC or TRITC, washed and observed inepillumination.

All the following combinations were carried out:

1st treatment1

washings 2nd treatment(300 pig ml"1)

washings Fluorescenceobservation

epillumination

Con A

S-Con A

PsA

LcA

Fluorescent PsALcA

S.ConA

Fluorescent PsALcACon A

Fluorescent Con ALcA

S.ConA

Fluorescent Con AS.ConA

PsA

Observation

Observation

•*- Observation

- • Observation

In all cases, these double-labelling experiments, showed no fluorescence on the treated ex-plants, thus indicating that PsA, LcA, S-Con A, Con A bind to the same plasmalemma-bindingsites.

(5) Tests of cell viabilityIn order to validate our results, the viability of cells following lectin treatments (50 and

SOOjiigml"1) was carefully checked using exclusion test with trypan-blue dye, ultrastructuralcytology, cell behaviour and differentiation over a 10-day period in vitro.


Fig. 1. Electron microscope micrograph. No nuclear or cytoplasmic abnormalities weredetected after a treatment of 24h with lectin. Bar = 0-5./xm. (N, nucleus; G, Golgiapparatus; me, melanin granule; m, mitochondria; nm, nuclear membrane; np, nuclearpores).

The exclusion test with trypan blue indicated a similar % of dead cells between treated and controlbatches(<5%)exceptfora300jugml~1 Con A treatment (*£1O%). No cytoplasmic or nuclear ab-normality was detected at an ultrastructural level after 30 min, 4 h and 24 h of treatment (Fig. 1).Moreover in culture, the treated cells spread and differentiated normally as did the control cells.

RESULTS

Table 3 shows the percentage of neural induction in control series.(a) Isolated gastrula ectodermal cells of P. waltl (stage 8) always differentiated intotypical epidermis (Fig. 2). No autoneuralization was observed in this species.(b) (c) After 3 h and 4 h of association between presumptive ectoderm and blas-toporal lip, neural induction occurs in approximately 80% of the cases after 3 h andin 90% of the cases after 4 h (Fig. 3).


Fig. 2. Phase contrast micrograph. Culture of isolated cells from non-induced ectoderm(cultured for 5 days): strong reaggregation and typical epidermal sheet ©. Bar =100 jum.Fig. 3. Phase contrast micrograph. Isolated cells from induced ectoderm cultured for12 days: neural differentiation is observed (—•), presence of melanocytes (^).Bar =100/an.


Table 3. Percentage of neural induction in the control association: ectoderm/blasto-poral lip

InductionNo. of cultures Induction frequence

(a) Culture of isolated ectodermalcells without previous contact withblastoporal lip (inducer). 126 0 0 %(b) Culture of ectodermal cellspreviously associated for 3 h with theblastoporal lip. 66 55 83%

(c) Culture of ectodermal cellspreviously associated for 4 h with theblastoporal lip. 106 97 92%

Neural induction is checked in culture by the differentiation of neurones, immunocytochemi-cally identified by neurofilament polypeptide markers.

(1) Effects oflectins on isolated presumptive ectoderm (stage 8)(A) Lectin-treatments for 3 h, 50 fig ml ~*These experiments were performed in saturating conditions for lectin-binding

sites on target cells. Table 4 shows the absence of an inducing effect by lectinsthemselves. The treated cells behaved and differentiated in the same way as controlcells (Table 3, batch a) into epidermal cells (Fig. 4). Under saturating concentrationof SOjUgml"1 for 3 h, none of the studied lectins had neural inducing properties.

(B) Lectin treatments for 3 h, 300 fig ml'1

Table 5 shows the percentage of neural induction occurring after treatment ofpresumptive ectoderm with lectins (at a high concentration),(a) Con A at high concentration (300 //gmr1 for 3h) induced neural structures in80% of the cases (differentiation of neurones, (Fig. 5), melanocytes, etc. could beeasily observed). These results were in agreement with those obtained by Takataetal. (1981, 1984).

Table 4. Comparison of the inducing effect of lectins, 50 fig ml'1.3h on isolatedneural target tissue (presumptive ectoderm).

LectinsSh) No. of cultures Induction Induction frequency

Con AS-Con APSALCA

The absence of neural induction is observed in all series.

35465730

0000

0%0%0%0%


Table 5. Comparison of the inducing effect of lectins, 300 pig ml*1,3h on isolatedneural target tissue (presumptive ectoderm).

Lectins1 .3h) No. of cultures Induction Induction frequency

(a) Con A(b) S-ConA(c) PSA(d) LCA

Only Con A treatment provokes neural induction.

51603129

41000

80%0%0%0%

(b) No ectoderms treated with S-Con A were induced, only epidermal differen-tiation was observed. Thus S-Con A did not exhibit a neuralizing effect.(c) (d) No neutralization could be detected when gastrula ectoderm was treatedwith PsA or LcA. In both these series, all cells differentiated into epidermalcells in the same way as after S-Con A treatment and for the non-induced controlseries.

Among the four studied lectins which bind to oligosaccharides with mannose andglucose residues, only Con A had a neuralizing effect.

(2) Lectin effects on neural induction obtained by association of presumptiveectoderm and blastoporal lip

We have previously observed (Duprat et al. 1982) that the treatment of presump-tive ectoderm by PsA or SBA (soybean agglutinin) prior to its association withblastoporal lip, led to a large reduction in the percentage of neural induction (10%of induction after SBA treatment, 20% for PsA treatment; control experimentwithout treatment: 90%). Table 6 shows the percentage of induction obtainedwhen the ectoderm was preincubated for 30 min with Con A or S-Con A or LcA(50/igml"1) prior to association for 4 h with blastoporal lip.

In the same way as for PsA, LcA inhibited the inductive machinery when thetreated ectoderm was associated with the natural inducer. The induction frequencyfell from 90% in the control batch to 19% in the treated batch. Con A and S-ConA did not have such an inhibitory effect.

Fig. 4. Electron micrograph. Cultured cells from target neural tissue (presumptiveectoderm) treated with ConA SOjUgml"1 for 3h: the behaviour and differentiation oftreated cells were identical to non-induced control cells (Fig. 2), only epidermal dif-ferentiation is observed. Bar = 1-35 jum. (c, cilia; mv, microvilli; ecm, extracellularmaterial; desmosomes (—-^-), m, melanin granule; L, lipid droplet).

Fig. 5. Electron micrograph. Neural induction occurs when presumptive ectoderm istreated with Con A SOOjUgml"1 for 3h. Bar = 0-5jum. (nf, neurofilaments; nt,neurotubules; dv, dense cored vesicles; cv, clear vesicles).


#&**•$$?•

?. &

Figs 4-5


Table 6. Effects oflectins on neural induction involved by the blastoporal lip.

Lectin pretreatment(SO^gmr^SOmin)prior association to the blastoporallip for 4 h

(a) LcA(b) Con A(c) S-ConA

PsASBA

No. of cultures

213016

8679

Induction

42215

177

Inductionfrequency

19%72%92%

19-7 %8-8%

To summarize these experiments, the effects of four lectins with affinity for thesame carbohydrate residues were studied under saturating conditions:

(A) PsA and LcA were not found to have neural inducing properties (50 /ig ml"1

and SOOjUgmr1). Moreover as previously reported they reversibly inhibited theprocess of induction by the natural inducing tissue (Gualandris et al. 1983).

(B) Con A was not a neural inducer at 50 /ig ml"1 (saturating concentration) andmoreover did not prevent neural induction by the blastoporal lip. As opposed toPsA and LcA, when used at high concentration (300 //g ml"1) it had inducingproperties.

(C) S-Con A like PsA and LcA did not present an inducing effect (50/ig and300jUgml~1). It did not inhibit the natural inductive process.

DISCUSSION

On amphibian neural target tissue, the use of double-labelled (FITC or TRITC)lectin probes, as well as the experiments with hapten inhibitors, suggested thatthese lectins react with identical carbohydrate residues on the cell surface. Con A,S-Con A, PsA and LcA are therefore assumed to bind to a common structure onthe plasma membrane.

Under rigorously identical conditions (300/igml"1 for 3h) only Con A had aneuralizing effect on the competent presumptive ectoderm (induction in 80% ofcases). Although the occurrence of this inducing action as the result of a cytolyticeffect of high concentration of Con A cannot be totally excluded (^ 10% deadcells), this inducing effect of Con A seems not to be such a consequence since asimilar death rate was sometimes found in control cultures without involving neuralinduction.

Several comments arose from the results obtained in these experiments. If weaccept the hypothesis of neural receptor existence for the neural inducing signal;Con A binding to a-D-mannose and glucose-containing sites, then such glycocon-jugates would be good candidates for such a role (Takata et al. 1981, 1984).Nevertheless the saturation of these sites with S-Con A, a dimeric chemical

Cell surface glycoconjugates and neural induction 49derivative of Con A which binds to the same sugar residues, does not lead to aninducing effect. Likewise saturation with other lectins (PsA and LcA) does not leadto induction either. In addition, Con A has no inducing properties at lower con-centrations (50 fig ml"1 for 3 h) although the competitive inhibition method showedthat all binding sites were saturated.

Another possible explanation of these results lies perhaps in the fact that twokinds of lectin-binding sites might exist on the membrane surface: (A) main sitesunrelated to induction, to which all the tested lectins would be strongly directed;(B) weak-binding sites related to induction which only bind Con A at sufficientconcentration.

However, this hypothesis cannot explain why S-Con A whose sugar specificity isclosely related to that of Con A, remains ineffective.

Moreover, in the experiments in vitro on the association of target tissue(presumptive ectoderm) with the natural inducer (blastoporal lip) we observed thatpretreatment of the target tissue with SBA, PsA or LcA gave rise to a failure ofneural induction in explants. Although these experiments cannot indicate whetherit is a problem of receiving the stimulus or giving the response, if the inductive signalrequires specific membrane receptor(s), these lectin-binding molecules could bedirectly concerned; thus many different glycoconjugates would seem to be involved,namely: a-D-galactose, N-acetyl-a-D-galactosamine, a-D-mannose, a-D-glucose,a-D-glucosamine, etc.

The possibility of close involvement of oligosaccharides in the neural inducingmechanism itself and the existence of such neural specific receptor (s) is still hypotheti-cal and there is as yet no direct evidence for them.

Whatever the hypothesis one can suppose that it is not the binding itself of ConA which initiates neural induction but that Con A possesses properties over andabove those of S-Con A, PsA, LcA, which could be responsible for this inducingeffect.

In this respect, due to the fact that only tetravalent Con A but not divalent ConA produces neural induction, is the crosslinking of the cell-surface-binding sites dueto the multivalence of this lectin (Trowbridge, 1973, Gunther et al. 1973) involvedfor this inducing activity?

Moreover, it had been shown that Con A involved ionic fluxes in treated cells.Thus Inoue et al. (1977) reported that Con A but not divalent S-Con A caused amarked induction of K+ release from cells such as rabbit reticulocytes, Wolff &Akerman (1982), Dufresne-Dube, Metivier, Dube & Guerrier (1983) have demon-strated that Con A elicites Ca2+ fluxes. On the other hand, in the light of experi-ments performed on Ranapipiens gastrulae, Barth (1965,1966), Barth and Barth(1967,1968 and 1974) proposed the following scheme for neural induction:- 'duringgastrulation, ion (Na+, K+, Ca2+, Mg2+) diffusing from cells are trapped betweenthe two surfaces of the ectoderm and the underlying chordamesoderm. It would bethe resulting increase in concentration of ions which could initiate induction ofneural plate'. Warner and coll. (for reviews see Warner, 1984) have shown in


Xenopus neurulae that the intracellular concentration of cations (Na+, K+) controlsneuronal differentiation. It is not yet clear if these changes in cation content act asa trigger or whether they are a co-factor. A stimulating effect of the cationionophore A 23187 on in vitro neuroblast differentiation has also been observed inPleurodeles waltl (Duprat & Kan, 1981). Recently Stern (1984) has proposed aninteresting model for early morphogenesis involving an ionic mechanism.

Whatever the mechanisms of action of the numerous inducing factors known upuntil now, it is therefore quite possible that the competent target tissue itself containsthe capacity and the specificity needed for neural induction. All that these neuralizingfactors so far studied would have in common is the capability to initiate the samesignal which sets in motion the machinery of neural determination.

Modification in membrane potentials and/or the initiation of ionic fluxes couldbe crucial factors in this process.

This work was supported by grants from the CNRS and the M.E.N. We thank Dr. S. Jarmanfor reviewing the English manuscript.

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(Accepted 14 November 1984)

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