/ . Embryol. exp. Morph., Vol. 18, J,pp. 143-53, August 1967 143With 2 plates

Printed in Great Britain

Effects of histories on morphogenesis anddifferentiation in chick embryos

By P. MALPOIX1 & A. EMELINCKX1

From the Laboratoire de Morphologie Animale, Universite Libre de Bruxelles

The transcription of DNA is known to be inhibited in vitro by the presenceof histones. This has been shown to be true for isolated nuclei, isolated chromatin,or for DNA alone (Bonner & Huang, 1962; Bonner, Huang & Gilden, 1963;Allfrey & Mirsky, 1963; Allfrey, Faulkner & Mirsky, 1964; Sonnenberg & Zubay,1965). The masking of genomic sites in adult differentiated tissues seems the mostprobable explanation of the expression of only a fraction of the adult genome(Paul & Gilmour, 1966). Affinities have been observed between certain basesequences in DNA and certain fractions of histones (Liau, Hnilica & Hurlbert,1965; Skalka, Fowler, Andre & Hurwitz, 1966; Tan, 1966), yet the remarkablesimilarity in composition between the histones of different species and organsof the same species does not encourage the idea that histones are specificrepressors of genomic function. Many authors (Zalokar, 1964; Goldberg &Atchley, 1966; Frenster 1967) have proposed hypotheses tending to reconcilethe apparent non-specificity of the relation between DNA and histones andtheir intervention in gene function: it is thought that polyanions (RNAs, phos-phoproteins, phospholipids, non-histone proteins or hormones) may removehistones from binding sites and release specific single-stranded sequences ofDNA for transcription.

Biological proof is required before we can be sure that histones act asrepressors; specificity of action will have to be demonstrated in living cells inorder to consolidate the picture obtained from in vitro experiments. Efficientbiological tests of histone specificity are difficult to devise, since it has not beenpossible to obtain pure and completely homogeneous fractions of histones sofar, and little is known of the degree of penetration of different histone fractionsinto living cells. Parallel to studies of the synthesis of basic proteins in thenucleus and cytoplasm of differentiating cells (Malpoix & Limbosch, 1966, andwork in progress), we thought it might be interesting to check the effects ofdifferent histones or fractions of histones on chick morphogenesis and on thedifferentiation of hematopoietic tissues, with which we are particularly ac-quainted.

The present paper describes the morphostatic action of lysine-rich calf1 Authors' address: Laboratoire de Morphologie Animale, Universite Libre de Bruxelles,

Belgique.

144 P. MALPOIX & A. EMELINCKX

thymus histones on chick embryo development, and pinpoints particular anoma-lies which suggest a certain analogy between histone and actinomycin action.Embryonic erythroblasts incubated in vitro according to the method describedelsewhere (Malpoix & Limbosch, 1966) have been used to test possible inhibitoryeffects of histones extracted from mature adult erythrocytes on RNA synthesisin immature cells, with the aim of inhibiting the 'nome' (cf. Paul & Gilmour,1966) of the immature erythrocyte. If histones are indeed specific repressors ofRNA synthesis, adult erythrocytes should contain a specific fraction able toinhibit the immature, functional genome. Different fractions of erythrocytehistones, rich in arginine or lysine, have also been used. Heterologous histonesextracted from calf thymus or Asterias gonads were tested in the same way.Further experiments of the same nature were carried out on nuclei isolatedfrom immature embryonic erythroblasts or from 2-day chick embryos, withthe intention of further specifying the nature of the effects observed.

MATERIALS AND METHODS

(a) Study of morphogenesis

Chick embryos explanted at stages 4 to 6 were cultivated on agar accordingto the method of Britt & Hermann (1959). Histones were added to the culturemedium in concentrations varying from 50 fi% to 2 mg/ml, and growth anddevelopment were followed for a 20 h period, and compared with controls.Embryos were then photographed, fixed and embedded for histological exami-nation.

(b) Biochemical effects in whole embryos3H-Cytidine and 3H-phenylalanine incorporation was measured in treated

and control embryos by autoradiography (Ficq, 1961).

(c) Chick erythroblasts cultivated in vitro

Blood was taken from chick embryos aged 5 days 17 h, as described elsewhere(Malpoix & Limbosch, 1966) and incubated for 1-4 h in presence and absenceof exogenous histones in varied concentrations. The incorporation of 3H-uridineinto RNA was measured by scintillation counting as described in the samepaper.

(d) Isolated nuclei {from chick embryo blood and from 2-day embryos)

The method used is based on that of Hammel and Bessman (1964), with theslight modifications required to obtain pure nuclei from immature red cells(the relationship between nucleus and cytoplasm being much more intimate thanin mature cells). For instance, the addition of polyvinylpyrrolidone (PVP) to aconcentration of 0-5 % during the last stage of the isolation procedure ensures theobtaining of pure nuclei, uncontaminated by cytoplasm (cf. Birnstiel, Chipchase &

Histories and differentiation 145

Hayes, 1962). The technique, briefly, was as follows: blood was washed twicewith Ringer saline and centrifuged. To the pellet a solution of 0-25 M sucrose,0-003 M-CaCl2 containing 1 % saponin was added and gently stirred with amagnetic stirrer for 8 minutes to release the nuclei, which were then centrifugedfor 5 minutes at lOOOg. The nuclear pellet was washed by resuspension in0-25M sucrose, 0-003 M-CaCl2 several times and finally resuspended in the samemedium containing 0-5 % PVP. The incubation medium was as follows: 0-25Msucrose, 0-0065 M-MgCl2. 6H20,0-004 M-NaCl, 0-003 M-CaCl2 in 001 M phosphatebuffer, pH 6-7. In some experiments PVP was added to the incubation mediumat a concentration of 0-5 %, in others serum albumin to a concentration of 0-1 %.

Table 1. Amino acid composition of histories and fractions ofhistones used

Samples hydrolysed in 6 N - H C I , dissolved in pH 2-2 citrate buffer, analysed in aBeckman/Spinco amino acid analyser.

Amino acids

LysineHistidineArginine

Total % of basic amino acids

Aspartic acidThreonineSerineGlutamic acidProlineGlycineAlanineValineMethionineIsoleucineLeucineTyrosinePhenylalanine

Ratio lysine/arginine

Histones

Total(a)

13-41-879-92

2519

4-895 316-248-664-528-68

12-61617—4-398-342-491-79

1-3

Micromoles 1

from erythrocytesA

Arginine-rich(b)

11-59201

10-43

2403

5-505-685-929-54-299-61

12-84-60-843-58-982-69205

111

Lysine-rich(c)

17-472-1J6-92

26-52

4-525-889-487-815-45612

12186-440-824-745-632-791-61

2-52

:%)

thymushistones

(d)

26-270-312-45

2903

3-624-765-555-838-557-47

22-584-72

Traces1-314-790-551-23

10-72

A ci"AT*l ilO/\o lei Ida

gonadhistones

(e)

13-721-937-73

23-38

6-995-585-84

10095-937-98

12-62602—4035-972072-22

106

(e) Histone preparations

The composition of the histones and fractions ofhistones used is indicatedin Table 1. Sample (a) was supplied by R. Vendrely, sample {d) by N. Herremans,sample (e) by A. Ficq, to whom we should like to express our thanks. Sample(d) was prepared from calf thymus by the method of Johns (1964), giving afraction rich in lysine. Samples (b) and (c) were prepared according to the

IO JEEM l 8

146 P. MALPOIX & A. EMELINCKX

method of Hnilica (1964), samples (a) and (e) by the method of Vendrely,Genty & Coirault (1965). Arginine-rich and lysine-rich erythrocyte histoneswere subjected to disk electrophoresis according to the method of Ornstein &Davis (1961) and McAllister, Wan & Irwin (1963).

(a) Morphostatic effects

Certain anomalies are frequent among treated embryos, notably microcephalyand malformation of the brain, which remains small and undifferentiated. Theneural folds often fail to close in embryos treated in early stages (stages 4 or5—Hamburger & Hamilton (1951)) and fore, mid and hind brain may remainopen. These anomalies are often associated with failure to form lens or oticplacodes. Some of the treated embryos, on the other hand, show no particulardeformation, but only a general slowing down of development resulting indecreased size.

Table 2. Relative frequency of the different types of anomaly observed in chickembryos treated from stage 4 or 5 onward with calf thymus lysine-rich histones

Concentrationof calf thymushistones used

500 /4g/ml1 mg/ml

No. ofembryostreated

8018

Completeblock

1518

No. of anomalies observed

Microcephaly

11

Openneuralplate

16

Decreasedlength and

size

38

A complete block, affecting every part of the embryo, was sometimes observedwith 500 /ig histones/ml. 1 and 2 mg of histones/ml were totally inhibitory, butthe effects observed were completely unspecific.

Usually, however, even in embryos in which nervous differentiation is seriouslyaffected, somites segregate in a normal way, and heart and vascular tissuesdevelop fairly well. Plate 1 illustrates these phenomena. Table 2 summarizesthe frequency with which such disorders are observed in young embryos.Embryos treated from stage 7 onwards are but little affected, if at all. Thewide range and variability of effect should be stressed.

Cytological anomalies include loss of basophilia and, in the flattened neuralplate regions of blocked embryos, cells were rounded instead of cubiform.

(b) Biochemical effects on whole embryos3H-Cytidine incorporation was inhibited by an average of 50 % by a con-

centration of 500 [ig histones/ml and by 80 % in the presence of 1 mg/ml after6 h treatment. Phenylalanine incorporation fell by about 45 % after a similarperiod in the presence of 500 fig histones/ml.

Histones and differentiation 147

(c) Inhibition of RNA synthesis in erythroblasts cultivated in vitro

Total erythrocyte histones have little effect on RNA synthesis by erythro-blasts incubated in vitro. A maximum of 40 % inhibition of 3H-uridine incorp-oration was recorded after 3 h incubation in vitro. Calf thymus ly sine-richhistones were without significant effect (an inhibition of 18 % was observed

Table 3. Counts per minute are expressed after subtraction of background

A

B

C

D

E

Duration of treatment (h)

Controls

1

4690Treated: total erythrocyte histones

125/*g/ml250300500650

Controls

48854726476347214926

1215Treated: lysine-rich calf thymus histones

50/tg/ml250500

1 mg/ml

Duration of treatment (h)Controls:

12041076

9611472

2

Cpm

11323

101329092983280928668

3742

3364317936513077

H1020

Treated: arginine-rich erythrocyte histones2-4 mg/ml5-8

ControlsTreated: lysine rich histones

21 mg/ml3-2 mg

ControlsTreated

Polylysine 450 /*g/mlPolylysine 550 /*g/ml

499388

—

——

506

736914

3

18664

1445112987135131283411002

6233

5993601463555099

3

1769

13221129

2457

25852129

2965

33533456

2 3 .

% inhibition

1019132823

————

—

5062

—

——

2230273741

———18

3—

2536

—

——

% stimulationA

+ 45+ 81

+ 13+ 17

after 3 h). Using different fractions of erythrocyte histones, inhibition wasfound to be particularly effective with the arginine-rich fraction after l | h( - 6 2 %) but this effect was transitory: after 3 h, the degree of inhibition hadfallen to 36 %. Lysine-rich histones, on the contrary, had no significant inhibitoryaction (see Table 3 A-D). Polylysine was found to have a short-lived stimulatoryeffect (Table 3 E).

148 P. MALPOIX & A. EMELINCKX

(d) The effects of exogenous histones on zH-uridine incorporation in isolatednuclei

In incubation media containing serum albumin (0-1 %), all the histones used(histones from erythrocytes, calf thymus or asterias gonads) were inhibitory,though to a variable extent (Table 4A, B). On the contrary, in the presence of

Table 4. Incorporation of zH-uridine in isolated nuclei of (A) young chick embryos,(B) erythroblasts from young chick embryos, in the presence of homologous andheterologous histones, as compared with controls

4A

4B

Controls

Treated1 mg erythrocyte histones/ml2 mg erythrocyte histones/ml1 mg calf thymus histones/ml2 mg calf thymus histones/ml

TreatedErythrocyte histones, 1 mg/mlErythrocyte histones, 2 mg/mlCalf thymus histones, 1 mg/ml

Cpm431

7620

24971

672

305221399

% inhibition as comparedwith controls

-82- 9 5-42-83

- 5 4-67-40

Table 5. Incorporation of 3H-uridine in the isolated nuclei of chick embryos aged5 days 17 h, in the presence of homologous histones, compared with controls

o//oCpm stimulation

Controls 59 —3 mg lysine-rich erythrocyte histones/ml 221 2743 mg arginine-rich erythrocyte histones/ml 102 743 mg total histones from erythrocytes/ml 239 300

Table 6. Incorporation of sH-uridine in isolated erythroblast nuclei in the presenceof homologous and heterologous histones, as compared with controls

0/

/oCpm stimulation

Controls2 mg lysine rich erythrocyte histones/ml2 mg arginine-rich erythrocyte histones/ml2 mg erythrocyte histones/ml2 mg Asterias gonad histones/ml

1 fig actinomycin/ml

64246157346224

20

284145445251

inhibition-68

J. Embryol. exp. Morph., Vol. 18, Part 1 PLATE 1

A B

D

A. Control embryos after 20 h incubation. B, C, D. Treated embryos. B. Microcephaly;heart and somite development relatively normal. C. Undifferentiated, open neural plate;six somites. D. Small abnormal embryo with open brain (neural folds have failed to close),whereas the vascular area is relatively well-developed.

P. MALPOIX & A. EMELINCKX facing p. 148

/. Embryol. exp. Morph., Vol. 18, Part 1 PLATE 2

B

D

Incubation from stage 4 onwards in the presence and in the absence of calf thymus histones(500/^g/ml).

A. Control embryo, section fore brain; lens induction. B. Treated embryo, section forebrain, which is small and underdeveloped; lens induction has not occurred. C. Treatedembryo; open neural plate in the region of the fore brain. D. As for C. E. Control embryo,section in region of hind brain, to be compared with F. Presence of otic placodes. F. Treatedembryo, section of hind brain (not closed); absence of otic placodes.

P. MALPOIX & A. EMELINCKX

Histones and differentiation 149

polyvinylpyrrolidone, all of them apparently increased the incorporation ofuridine, or of phenylalanine (Tables 5-7).(e) Disk electrophoresis of arginine-rich and ly sine-rich histones revealed thatboth fractions were heterogeneous and contained four different components(Text-fig. 1).

Table 7. Incorporation of zH-phenylalanine in isolated nuclei of erythrocytesfrom chick embryos aged 14 days, in the presence of homologous and heterologoushistones, as compared with controls

0//o

Cpm stimulationControls 1490 —1 mg erythrocyte histones/ml 6217 +3181 mg thymus histone/ml 5434 +264

A - , B

Text-fig. 1. Disk electrophoresis. (A) Fraction rich in arginine.(B) Fraction rich in lysine.

DISCUSSION AND CONCLUSIONS

Although the morphostatic effects of exogenous histones have been found tobe variable, abnormal embryos are characterized by the undifferentiated stateof the brain and central nervous system. This effect is strikingly similar to thatobtained with actinomycin in chicks (Heilporn-Pohl, 1964) and in amphibians(Flickinger, 1963; Brachet, Denis & de Vitry, 1964). Moreover, these resultsconfirm those of Sherbet (1966), who has recorded similar, variable, morpho-

150 P. MALPOIX & A. EMELINCKX

logical effects in chick embryos, using calf thymus histones; this author wasnevertheless able to demonstrate the penetration of labelled histones intonucleus and cytoplasm of all cells of the treated embryos. The varied effects weretherefore unlikely to be due to irregular penetration, and may even demonstratea certain degree of specificity related to mRNA synthesis and time of applicationof treatment. Lack of effect on older embryos could thus be due to the priorsynthesis of the mRNAs required for differentiation; failure of lens inductionmay result from inhibited mRNA synthesis. Lysine-rich calf thymus histonesdo not inhibit hematopoiesis in the chick vascular area to any great extent,perhaps because of the existence of previously synthesized mRNA, perhapsbecause of the lack of affinity between these histones and the 'nome' of thepotential blood-forming cells. The otic placodes are thought to possess relativelyautonomous differentiating potentiality (Flickinger, 1963); our results suggestthat the expression of the inherent capacity, in this case, requires the normaldevelopment and closure of the rhombencephalon.

Actinomycin is known to act through inhibition of DNA-dependent RNAsynthesis, and we have shown that exogenous histones also diminish cytidineincorporation. The possibility therefore exists that exogenous histones act, atleast in part, at gene level (cf. Brachet, 1964; Brachet et al. 1964). Furtherresearch will be required to confirm or confute this hypothesis, however, sincehistones are also known to inhibit mitochondrial enzymes and to alter membraneproperties.

Total erythrocyte histones inhibit RNA synthesis in immature erythroblaststo a slight extent (40 % inhibition after 3 h). High concentrations of arginine-richhistones from erythrocytes were somewhat more effective ( -60%) , but theinhibition observed was transitory. Calf thymus histone had little inhibitoryeffect, perhaps because the fraction used was rich in lysine and contained littlearginine. Polylysine actually stimulated RNA synthesis in isolated embryonicerythroblasts. The slight differences observed suggest that analysis of thebiological effects of homogeneous fractions may reveal specificity. Histonefractions prepared by known methods show heterogeneity (cf. arginine-richand lysine-rich fractions subjected to electrophoresis).

The results obtained with nuclei were contradictory, in that all the histonesexamined were stimulatory when the incubation medium contained poly-vinylpyrrolidone, and inhibitory when the medium contained serum albumin.According to Michaels, Waddel, Zinner & Sigel (1966) polyvinylpyrrolidone isable to combine with certain substances and alter their properties; this mayexplain our results. Serum albumin may accelerate histone penetration bypinocytosis or act in an additive way (cf. Sluyser, 1966). In fact, many authorshave stressed that the composition of the incubation medium for isolatednuclei or DNA can alter the histone-DNA association and the nature of theresponse vis-a-vis RNA synthesis (Sonnenberg & Zubay, 1965; Leng & Felsen-feld, 1966; Skalka et al 1966). A simple change in histone concentration can

Histories and differentiation 151

change the response from stimulation to inhibition (Goodwin & Sizer, 1965).Uridine may enter cells or nuclei more readily under certain experimentalconditions, as in the presence of added histones. Some basic proteins areknown to stabilize or protect certain types of mRNA (Monroy, Maggio &Rinaldi, 1965; Spirin, 1966) and it is therefore quite possible that certain basicprotein fractions are without inhibitory function in respect of certain types ofmRNA.

SUMMARY

1. The morphostatic effects of lysine-rich calf thymus histones have beenfound comparable to those produced by actinomycin, in that nervous differen-tiation is strongly inhibited. Lens induction and the development of oticplacodes were often prevented. Haematopoiesis and heart and somite formationwere much less affected.

2. The incorporation of 3H-uridine into RNA is inhibited in immatureembryonic erythroblasts incubated in vitro in the presence of 650/£g/ml ofhistones from mature adult erythrocytes. An arginine-rich adult erythrocytehistone fraction produced more marked inhibition, but of a transitory nature.Lysine-rich calf thymus histone was without significant inhibitory effect. Poly-lysine actually stimulated RNA synthesis in isolated erythroblasts.

3. In isolated nuclei from chick embryos or immature erythroblasts, exogenoushistones of diverse origin were found to stimulate RNA synthesis in the presenceof polyvinylpyrrolidone and to inhibit it in the presence of serum albumin.

RESUME

Les ejfets des histones sur la morphogenese et sur la differenciation

1. Les effets morphostatiques des histones de thymus de veau riches enlysine ressemblent a ceux observes en presence d'actinomycine; en effet, ladifferenciation du systeme nerveux central est particulierement inhibee. L'in-duction du cristallin et le developpement des placodes otiques sont souventinhibes. L'hematopoiese, le developpement du cceur et la segregation des somitessont peu affectes.

2. L'incorporation d'uridine-3H dans le RNA est inhibee dans les erythro-blastes embryonnaires incubes in vitro en presence de 650 /*g d'histones d'ery-throcytes adultes/ml. La fraction d'histones d'erythrocytes riches en arginine estplus inhibitrice, mais l'effet est transitoire. Une fraction d'histones de thymus deveau riche en lysine n'inhibe pas cette incorporation d'une maniere significative.La polylysine stimule la synthese de RNA dans les erythroblastes isoles.

3. Dans les noyaux isoles, les effets d'histones exogenes sont paradoxaux: eneffet, en presence de polyvinylpyrrolidone elles sont stimulatrices, en presencede serum albumine elles sont inhibitrices, vis a vis de la synthese du RNA.

This work has been carried out under Euratom-Universite Libre de Bruxelles contractno. 016-61-ABIB.

152 P. MALPOIX & A. EMELINCKX

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