/ . Embryol. exp. Morph., Vol. 18, J,pp. 143-53, August 1967
With 2 plates
Printed in Great Britain
143
Effects of histories on morphogenesis and
differentiation in chick embryos
By P. MALPOIX 1 & A. EMELINCKX 1
From the Laboratoire de Morphologie Animale, Universite Libre de Bruxelles
The transcription of DNA is known to be inhibited in vitro by the presence
of 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 most
probable explanation of the expression of only a fraction of the adult genome
(Paul & Gilmour, 1966). Affinities have been observed between certain base
sequences in DNA and certain fractions of histones (Liau, Hnilica & Hurlbert,
1965; Skalka, Fowler, Andre & Hurwitz, 1966; Tan, 1966), yet the remarkable
similarity in composition between the histones of different species and organs
of the same species does not encourage the idea that histones are specific
repressors of genomic function. Many authors (Zalokar, 1964; Goldberg &
Atchley, 1966; Frenster 1967) have proposed hypotheses tending to reconcile
the apparent non-specificity of the relation between DNA and histones and
their intervention in gene function: it is thought that polyanions (RNAs, phosphoproteins, phospholipids, non-histone proteins or hormones) may remove
histones from binding sites and release specific single-stranded sequences of
DNA for transcription.
Biological proof is required before we can be sure that histones act as
repressors; specificity of action will have to be demonstrated in living cells in
order to consolidate the picture obtained from in vitro experiments. Efficient
biological tests of histone specificity are difficult to devise, since it has not been
possible to obtain pure and completely homogeneous fractions of histones so
far, and little is known of the degree of penetration of different histone fractions
into living cells. Parallel to studies of the synthesis of basic proteins in the
nucleus and cytoplasm of differentiating cells (Malpoix & Limbosch, 1966, and
work in progress), we thought it might be interesting to check the effects of
different histones or fractions of histones on chick morphogenesis and on the
differentiation of hematopoietic tissues, with which we are particularly acquainted.
The present paper describes the morphostatic action of lysine-rich calf
Authors' address: Laboratoire de Morphologie Animale, Universite Libre de Bruxelles,
Belgique.
1
144
P. MALPOIX & A. EMELINCKX
thymus histones on chick embryo development, and pinpoints particular anomalies which suggest a certain analogy between histone and actinomycin action.
Embryonic erythroblasts incubated in vitro according to the method described
elsewhere (Malpoix & Limbosch, 1966) have been used to test possible inhibitory
effects of histones extracted from mature adult erythrocytes on RNA synthesis
in immature cells, with the aim of inhibiting the 'nome' (cf. Paul & Gilmour,
1966) of the immature erythrocyte. If histones are indeed specific repressors of
RNA synthesis, adult erythrocytes should contain a specific fraction able to
inhibit the immature, functional genome. Different fractions of erythrocyte
histones, rich in arginine or lysine, have also been used. Heterologous histones
extracted from calf thymus or Asterias gonads were tested in the same way.
Further experiments of the same nature were carried out on nuclei isolated
from immature embryonic erythroblasts or from 2-day chick embryos, with
the 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 according
to the method of Britt & Hermann (1959). Histones were added to the culture
medium in concentrations varying from 50 fi% to 2 mg/ml, and growth and
development were followed for a 20 h period, and compared with controls.
Embryos were then photographed, fixed and embedded for histological examination.
(b) Biochemical effects in whole embryos
3
H-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 absence
of exogenous histones in varied concentrations. The incorporation of 3H-uridine
into RNA was measured by scintillation counting as described in the same
paper.
(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 the
slight modifications required to obtain pure nuclei from immature red cells
(the relationship between nucleus and cytoplasm being much more intimate than
in mature cells). For instance, the addition of polyvinylpyrrolidone (PVP) to a
concentration of 0-5 % during the last stage of the isolation procedure ensures the
obtaining of pure nuclei, uncontaminated by cytoplasm (cf. Birnstiel, Chipchase &
Histories and differentiation
145
Hayes, 1962). The technique, briefly, was as follows: blood was washed twice
with 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 a
magnetic stirrer for 8 minutes to release the nuclei, which were then centrifuged
for 5 minutes at lOOOg. The nuclear pellet was washed by resuspension in
0-25M sucrose, 0-003 M-CaCl2 several times and finally resuspended in the same
medium containing 0-5 % PVP. The incubation medium was as follows: 0-25M
sucrose, 0-0065 M-MgCl2. 6H20,0-004 M-NaCl, 0-003 M-CaCl2 in 001 M phosphate
buffer, pH 6-7. In some experiments PVP was added to the incubation medium
at 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 a
Beckman/Spinco amino acid analyser.
Micromoles 1:%)
Histones from erythrocytes
A
A ci"AT*l ilO
/\o lei
Ida
Arginine- Lysine- thymus gonad
rich
rich
histones histones
(b)
(e)
(d)
(c)
Amino acids
Total
(a)
Lysine
Histidine
Arginine
Total % of basic amino acids
13-4
1-87
9-92
2519
11-59
201
10-43
2403
17-47
2-1J
6-92
26-52
26-27
0-31
2-45
2903
13-72
1-93
7-73
23-38
Aspartic acid
Threonine
Serine
Glutamic acid
Proline
Glycine
Alanine
Valine
Methionine
Isoleucine
Leucine
Tyrosine
Phenylalanine
Ratio lysine/arginine
4-89
5 31
6-24
8-66
4-52
8-68
12-61
617
—
4-39
8-34
2-49
1-79
1-3
5-50
5-68
5-92
9-5
4-29
9-61
12-8
4-52
5-88
9-48
7-81
5-45
3-62
4-76
5-55
5-83
8-55
7-47
22-58
4-72
Traces
1-31
4-79
0-55
1-23
10-72
6-99
5-58
5-84
1009
5-93
7-98
12-62
602
—
403
5-97
207
2-22
106
4-6
0-84
3-5
8-98
2-69
205
111
612
1218
6-44
0-82
4-74
5-63
2-79
1-61
2-52
(e) Histone preparations
The composition of the histones and fractions ofhistones used is indicated
in 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 a
fraction rich in lysine. Samples (b) and (c) were prepared according to the
IO
JEEM
l8
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 histones
were 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 microcephaly
and malformation of the brain, which remains small and undifferentiated. The
neural folds often fail to close in embryos treated in early stages (stages 4 or
5—Hamburger & Hamilton (1951)) and fore, mid and hind brain may remain
open. These anomalies are often associated with failure to form lens or otic
placodes. Some of the treated embryos, on the other hand, show no particular
deformation, but only a general slowing down of development resulting in
decreased size.
Table 2. Relative frequency of the different types of anomaly observed in chick
embryos treated from stage 4 or 5 onward with calf thymus lysine-rich histones
No. of anomalies observed
Concentration
of calf thymus
histones used
No. of
embryos
treated
500 /4g/ml
1 mg/ml
80
18
Complete
block
Microcephaly
15
18
11
Open
neural
plate
Decreased
length and
size
16
38
A complete block, affecting every part of the embryo, was sometimes observed
with 500 /ig histones/ml. 1 and 2 mg of histones/ml were totally inhibitory, but
the effects observed were completely unspecific.
Usually, however, even in embryos in which nervous differentiation is seriously
affected, somites segregate in a normal way, and heart and vascular tissues
develop fairly well. Plate 1 illustrates these phenomena. Table 2 summarizes
the frequency with which such disorders are observed in young embryos.
Embryos treated from stage 7 onwards are but little affected, if at all. The
wide range and variability of effect should be stressed.
Cytological anomalies include loss of basophilia and, in the flattened neural
plate regions of blocked embryos, cells were rounded instead of cubiform.
(b) Biochemical effects on whole embryos
3
H-Cytidine incorporation was inhibited by an average of 50 % by a concentration of 500 [ig histones/ml and by 80 % in the presence of 1 mg/ml after
6 h treatment. Phenylalanine incorporation fell by about 45 % after a similar
period 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 erythroblasts incubated in vitro. A maximum of 40 % inhibition of 3H-uridine incorporation was recorded after 3 h incubation in vitro. Calf thymus ly sine-rich
histones were without significant effect (an inhibition of 18 % was observed
Table 3. Counts per minute are expressed after subtraction of background
Duration of treatment (h)
1
2
3
% inhibition
Cpm
A
B
C
Controls
4690
Treated: total erythrocyte histones
125/*g/ml
4885
250
4726
300
4763
500
4721
650
4926
11323
18664
10132
9092
9832
8092
8668
14451
12987
13513
12834
11002
3742
1215
Controls
Treated: lysine-rich calf thymus histones
50/tg/ml
3364
1204
250
1076
3179
961
500
3651
3077
1 mg/ml
1472
6233
Duration of treatment (h)
H
1020
Controls:
Treated: arginine-rich erythrocyte histones
499
2-4 mg/ml
3
5-8
D
Controls
Treated: lysine rich histones
21 mg/ml
3-2 mg
3 .
2
10
19
13
28
23
22
—
—
—
—
—
—
—
18
1769
—
3
—
50
62
25
36
—
1322
1129
2457
—
—
—
—
2585
2129
—
—
—
—
388
5993
6014
6355
5099
30
27
37
41
% stimulation
A
E
Controls
Treated
Polylysine 450 /*g/ml
Polylysine 550 /*g/ml
506
2965
736
914
3353
3456
+ 45
+ 81
+ 13
+ 17
after 3 h). Using different fractions of erythrocyte histones, inhibition was
found 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 had
fallen to 36 %. Lysine-rich histones, on the contrary, had no significant inhibitory
action (see Table 3 A-D). Polylysine was found to have a short-lived stimulatory
effect (Table 3 E).
148
P. MALPOIX & A. EMELINCKX
(d) The effects of exogenous histones on zH-uridine incorporation in isolated
nuclei
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 and
heterologous histones, as compared with controls
Controls
4A
Cpm
431
% inhibition as compared
with controls
76
20
-82
249
71
-42
-83
Treated
1 mg erythrocyte histones/ml
2 mg erythrocyte histones/ml
1 mg calf thymus histones/ml
2 mg calf thymus histones/ml
-95
672
4B
Treated
Erythrocyte histones, 1 mg/ml
Erythrocyte histones, 2 mg/ml
Calf thymus histones, 1 mg/ml
305
-54
221
-67
399
-40
Table 5. Incorporation of 3H-uridine in the isolated nuclei of chick embryos aged
5 days 17 h, in the presence of homologous histones, compared with controls
o/
/o
Controls
3 mg lysine-rich erythrocyte histones/ml
3 mg arginine-rich erythrocyte histones/ml
3 mg total histones from erythrocytes/ml
Cpm
stimulation
59
221
102
239
—
274
74
300
Table 6. Incorporation of sH-uridine in isolated erythroblast nuclei in the presence
of homologous and heterologous histones, as compared with controls
0/
/o
Controls
2 mg lysine rich erythrocyte histones/ml
2 mg arginine-rich erythrocyte histones/ml
2 mg erythrocyte histones/ml
2 mg Asterias gonad histones/ml
1 fig actinomycin/ml
Cpm
64
246
157
346
224
20
stimulation
284
145
445
251
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 fore
brain, which is small and underdeveloped; lens induction has not occurred. C. Treated
embryo; 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. Treated
embryo, 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 of
uridine, or of phenylalanine (Tables 5-7).
(e) Disk electrophoresis of arginine-rich and lysine-rich histones revealed that
both fractions were heterogeneous and contained four different components
(Text-fig. 1).
Table 7. Incorporation of zH-phenylalanine in isolated nuclei of erythrocytes
from chick embryos aged 14 days, in the presence of homologous and heterologous
histones, as compared with controls
0/
/o
Cpm
1490
6217
5434
Controls
1 mg erythrocyte histones/ml
1 mg thymus histone/ml
A
-
,
stimulation
—
+318
+264
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 to
be variable, abnormal embryos are characterized by the undifferentiated state
of the brain and central nervous system. This effect is strikingly similar to that
obtained with actinomycin in chicks (Heilporn-Pohl, 1964) and in amphibians
(Flickinger, 1963; Brachet, Denis & de Vitry, 1964). Moreover, these results
confirm 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 was
nevertheless able to demonstrate the penetration of labelled histones into
nucleus and cytoplasm of all cells of the treated embryos. The varied effects were
therefore unlikely to be due to irregular penetration, and may even demonstrate
a certain degree of specificity related to mRNA synthesis and time of application
of treatment. Lack of effect on older embryos could thus be due to the prior
synthesis of the mRNAs required for differentiation; failure of lens induction
may result from inhibited mRNA synthesis. Lysine-rich calf thymus histones
do not inhibit hematopoiesis in the chick vascular area to any great extent,
perhaps because of the existence of previously synthesized mRNA, perhaps
because of the lack of affinity between these histones and the 'nome' of the
potential blood-forming cells. The otic placodes are thought to possess relatively
autonomous differentiating potentiality (Flickinger, 1963); our results suggest
that the expression of the inherent capacity, in this case, requires the normal
development and closure of the rhombencephalon.
Actinomycin is known to act through inhibition of DNA-dependent RNA
synthesis, and we have shown that exogenous histones also diminish cytidine
incorporation. The possibility therefore exists that exogenous histones act, at
least in part, at gene level (cf. Brachet, 1964; Brachet et al. 1964). Further
research will be required to confirm or confute this hypothesis, however, since
histones are also known to inhibit mitochondrial enzymes and to alter membrane
properties.
Total erythrocyte histones inhibit RNA synthesis in immature erythroblasts
to a slight extent (40 % inhibition after 3 h). High concentrations of arginine-rich
histones from erythrocytes were somewhat more effective ( - 6 0 % ) , but the
inhibition observed was transitory. Calf thymus histone had little inhibitory
effect, perhaps because the fraction used was rich in lysine and contained little
arginine. Polylysine actually stimulated RNA synthesis in isolated embryonic
erythroblasts. The slight differences observed suggest that analysis of the
biological effects of homogeneous fractions may reveal specificity. Histone
fractions prepared by known methods show heterogeneity (cf. arginine-rich
and lysine-rich fractions subjected to electrophoresis).
The results obtained with nuclei were contradictory, in that all the histones
examined were stimulatory when the incubation medium contained polyvinylpyrrolidone, and inhibitory when the medium contained serum albumin.
According to Michaels, Waddel, Zinner & Sigel (1966) polyvinylpyrrolidone is
able to combine with certain substances and alter their properties; this may
explain our results. Serum albumin may accelerate histone penetration by
pinocytosis or act in an additive way (cf. Sluyser, 1966). In fact, many authors
have stressed that the composition of the incubation medium for isolated
nuclei or DNA can alter the histone-DNA association and the nature of the
response vis-a-vis RNA synthesis (Sonnenberg & Zubay, 1965; Leng & Felsenfeld, 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 experimental
conditions, as in the presence of added histones. Some basic proteins are
known to stabilize or protect certain types of mRNA (Monroy, Maggio &
Rinaldi, 1965; Spirin, 1966) and it is therefore quite possible that certain basic
protein fractions are without inhibitory function in respect of certain types of
mRNA.
SUMMARY
1. The morphostatic effects of lysine-rich calf thymus histones have been
found comparable to those produced by actinomycin, in that nervous differentiation is strongly inhibited. Lens induction and the development of otic
placodes were often prevented. Haematopoiesis and heart and somite formation
were much less affected.
2. The incorporation of 3H-uridine into RNA is inhibited in immature
embryonic erythroblasts incubated in vitro in the presence of 650/£g/ml of
histones from mature adult erythrocytes. An arginine-rich adult erythrocyte
histone fraction produced more marked inhibition, but of a transitory nature.
Lysine-rich calf thymus histone was without significant inhibitory effect. Polylysine actually stimulated RNA synthesis in isolated erythroblasts.
3. In isolated nuclei from chick embryos or immature erythroblasts, exogenous
histones of diverse origin were found to stimulate RNA synthesis in the presence
of 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 en
lysine ressemblent a ceux observes en presence d'actinomycine; en effet, la
differenciation du systeme nerveux central est particulierement inhibee. L'induction du cristallin et le developpement des placodes otiques sont souvent
inhibes. L'hematopoiese, le developpement du cceur et la segregation des somites
sont peu affectes.
2. L'incorporation d'uridine-3H dans le RNA est inhibee dans les erythroblastes embryonnaires incubes in vitro en presence de 650 /*g d'histones d'erythrocytes adultes/ml. La fraction d'histones d'erythrocytes riches en arginine est
plus inhibitrice, mais l'effet est transitoire. Une fraction d'histones de thymus de
veau 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: en
effet, en presence de polyvinylpyrrolidone elles sont stimulatrices, en presence
de 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 contract
no. 016-61-ABIB.
152
P. MALPOIX & A. EMELINCKX
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{Manuscript received 20 March 1967)