/. Embryol. exp. Morph. Vol. 21, 2, pp. 341-46, April 1969
341
Printed in Great Britain
Myogenesis in chick embryo somites in vitro
By M. L. ELLISON, 1 E. J. AMBROSE 1 & G. C. EASTY 1
Chester Beatty Research Institute, London
Evidence from a number of studies (e.g. Holtzer, 1964; Coon & Cahn, 1966;
Konigsberg, 1963) has suggested that the differentiation of some types of cells
in vitro may be influenced by environmental factors. Culture conditions can
permit or prevent the phenotypic expression of characteristics already determined in the cells.
The length of time involved in the stabilization of differentiation appears to
vary widely according to the conditions of culture. Coon (1966), using chondrocytes, and Simpson & Cox (1967), using lizard tail regenerate, among other
examples, have demonstated that determination could be maintained over many
mitotic divisions in a latent form, before being expressed eventually under the
stabilizing conditions.
A range of environmental factors has been shown to be effective during
stabilization, including protein constituents of the medium (Coon, 1966; Cahn
& Cahn, 1966), conditioned medium (Konigsberg, 1963), cell density (Abbott &
Holtzer, 1966; Umansky, 1966) and tissue mass (Grobstein, 1964; Wessells &
Cohen, 1967).
Little is known, however, of how these factors act on the mechanism of
expression within the developing cells. A basic question concerns the specificity
of the stabilization. Will conditions which are found to be favourable to the
differentiation of one cell type, necessarily enhance differentiation equally in all
tissues, or has the environment some selective action ?
Umansky (1966) showed that during the growth of dispersed limb bud cells,
at some initial cell population densities cartilage differentiation was favoured,
while at others muscle was also able to develop. Although he interprets this as a
determination of developmental fate by density-dependent aggregation, it could
equally be seen as a selection of pre-existing populations of cells, and undoubtedly demonstrates that an environmental factor influences different tissues in
different ways.
In order to study this question in more detail, the differentiation of muscle
in chick embryo somite explants was compared over a range of conditions with
the incidence of cartilage differentiation as previously reported (Ellison,
1
Authors' address: Chester Beatty Research Institute, Institute of Cancer Research, Royal
Cancer Hospital, London, S.W.3.
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M. L. ELLISON, E. J. AMBROSE & G. C. EASTY
Ambrose & Easty, 1969). The variables against which the ability of somites to
produce muscle was tested were: different nutrient media, different sizes of
explant, and the presence of an oily layer on top of the somites.
MATERIALS AND METHODS
Since the same fixed and stained explants which were previously scored for
cartilage were scored for muscle, the culture methods were identical over the
two series of results, and are detailed in the previous paper (Ellison et al. 1969).
Briefly, these methods involved the maintenance of groups of varying numbers
of somites from stages 9 to 17 chick embryos on an agar medium consisting of
NCTC109 and Simms BSS with antibiotics, plus 20% horse serum (HS) or
or foetal calf serum (FCS), or 16 % of either serum with 20 % of an homogenate
of unincubated hen's egg. The explants, each on a TA Millipore filter raft,
consisted of eight somites when the influence of the medium was under study,
but varied between 1 somite and 32 when the influence of explant size was being
tested. After 8-11 days incubation in 5 % CO2 in air, the cultures were fixed in
glutaraldehyde, stained in Bismarck Brown and Toluidine Blue and mounted in
XAM for microscopy.
The scoring criterion for muscle was the presence of myotubes containing
three or more aligned nuclei (see Okazaki & Holtzer, 1966).
Table 1. Proportion of somite explants differentiating to
muscle on different media
Medium
Chick stages
9
10
11
12
13
14
15
16
17
Horse serum
—
—
—
—
—
—
0/11
0/22
0/14
HS + egg
—
—
—
—
—
6/6
8/12
12/13
12/12
Foetal calf
serum
0/6
0/9
0/29
0/16
0/10
2/16
14/24
19/23
14/25
FCS + egg
0/6
0/7
2/23
3/13
8/15
9/14
8/9
11/11
12/12
RESULTS
Somites isolated from stages 11-17 chick embryos and cultured in clusters on
nutrient agar were found to differentiate muscle over a culture period of 8-11
days. No muscle was detected in somites explanted from stages 9 and 10 chick
embryos. The incidence of muscle differentiation in the stages in which it
occurred was observed to vary with the conditions of culture.
Myogenesis in chick embryo
343
Medium constituents
The incidence of muscle differentiation in a range of somites cultured as
8-somite clusters on four different media is summarized in Table 1. No muscle
was formed where horse serum was the only protein constituent, but the
presence of 20 % egg permitted muscle development in more than half of the
explants at each of stages 14-17. Although foetal calf serum was not so markedly
favourable as egg, its presence in the medium in place of horse serum did enable
muscle to differentiate in the four later stages, while the combination of both
egg and foetal calf serum permitted differentiation in earlier material back as
far as stage 11.
Table 2. Proportion of somite explants differentiating to muscle
in explants of different sizes
No. of somites per explant
Chick
stages
9
10
11
12
13
14
15
16
17
1
—
—
—
—
—
0/5
0/12
0/15
1/11
2
4
6
8
12
16
—
—
—
—
—
0/7
2/16
3/14
1/15
—
—
0/5
0/7
0/5
0/9
3/13
6/14
8/12
—
—
0/6
0/6
0/5
0/9
4/10
6/11
8/10
0/6
0/9
0/29
0/16
0/10
2/16
14/24
19/23
14/25
0/5
0/5
0/8
0/12
0/6
0/10
10/11
—
—
0/5
0/6
0/10
0/12
1/7
3/7
—
—
—
24
0/5
0/6
0/9
—
—
—
—
—
—
32
0/5
0/9
0/7
—
—
—
—
—
—
Explant size
When clusters of different numbers of somites were cultured on a constant
medium, that containing foetal calf serum (FCS medium), a variation of muscle
incidence with the size of the explant occurred. Since the mean somite size
increases with the age of the chicks, the relationship between somite number and
cluster size is complex from stage to stage, but a direct comparison can be made
within any one stage.
Thus, as can be seen in Table 2, within any one stage increase in size of the
explant tends to give an increase in proportion of positives. The tendency for
increased incidence of muscle with increased age of somites in the vertical
columns of Table 2, each representing a given somite number per cluster, is
probably also a reflexion of the effect of increased explant size, but a simple
comparison between size and age in these explants cannot be made.
Liquid paraffin layer
Explants consisting of one stage 15, eight stage 9 and eight stage 10 somites
were used to test the effect of a layer of liquid paraffin lying over the tissue and
the medium surface (FCS medium). Although this factor (which eliminated the
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M. L. ELLISON, E. J. AMBROSE & G. C. EASTY
explant/air interface) enhanced the survival of all of these explants and favoured
differentiation of cartilage in them, muscle development was stimulated hardly
at all. None of 22 stage 10 explants, or five stage 9 explants, and only two of 15
stage 15 explants showed any muscle. However, this may be due to the over-all
relatively low incidence of muscle on FCS medium, and to the fact that the test
explants were therefore not sufficiently near to positive experimental points on
Table 2, either as regards chick stage or explant size.
DISCUSSION
The general conclusions for muscle differentiation suggested by these results
are similar to those reached for cartilage differentiation in the same experimental system (see Ellison et al. 1969), that is, that the somites are already
determined for muscle at an early stage (at least by stage 11) and that subsequent
to determination there is a phase of development during which the actual
expression of the characteristic is open to environmental influence. Again the
observation that several favourable factors supplement each other's activity
indicates that the phenomenon is not in vitro 'induction' of muscle, but a less
specific environmental control.
However, it is clear from a comparison of Tables 1 and 2 in this report with
the tables in the previous paper (Ellison et al. 1969) that myogenesis responds
differently in detail from chondrogenesis to some of the factors tested. The
difference in response in the two cell types was a matter of degree; each tissue
responded favourably to both factors, but the more markedly stimulatory of
the two factors for cartilage was the replacement of HS with FCS, while for
muscle the more marked stimulus came with the addition of egg to the serum.
In other words, starting with almost negative results for both tissues on HS
medium, more explants had muscle than had cartilage where egg was added to
HS, while more had cartilage than had muscle when FCS replaced the HS.
Another difference between the differentiation of the two tissues was seen in
the effect of the oily layer—cartilage was strikingly stimulated, while muscle
cells were apparently not much affected. As discussed earlier, this may be a
reflexion of the difference in response of the two cell types to medium constituents, but it does emphasize that, for a given explant, the actual appearance of one
cell type or another can be dictated by conditions of culture.
On the other hand, the response of both types of cell to increased explant size
was the same, although the incidence of differentiation was depressed for muscle
compared with that for cartilage, since the medium (FCS medium) was relatively
more favourable to cartilage. But that both muscle and cartilage could differentiate on FCS medium, and both show the same response to increased
explant size, does demonstrate that chick somites of the stages under study
contain both muscle and cartilage cell populations. It does not seem likely that
these populations are being determined in vitro, because the evidence for each
Myogenesis in chick embryo
345
cell type taken individually suggests that determination is an early event,
occurring before explantation.
But again, the evidence for both cell types taken together clearly shows a
selective enhancement of the differentiation of one or another of the cell types
by some of the environmental factors studied. This indicates that the stabilization phase, which is operative in both muscle and cartilage differentiating
systems in the somites, is open to a selective control by the environment. Thus
extrinsic factors, active during this phase, may play a part in the ultimate
differentiated state of the organ or tissue area.
SUMMARY
1. The differentiation of muscle cells from somites in organ culture has been
compared with the development of cartilage in the same system, in an attempt
to analyse the specificity of environmental effects on the stabilization of differentiation.
2. Stages 9-17 chick embryo somites were cultured on Millipore filter rafts
on nutrient agar and scored for the presence of myotubes.
3. Myotubes were detected in explants from stages 11-17.
4. The incidence of muscle varied with the culture conditions, being favoured
by the addition of egg to the medium, by foetal calf serum rather than horse
serum in the medium and by an increased volume of the somite explants.
5. Comparison of these results with those obtained for cartilage differentiation in the same system indicated a variation in detail between the response of
muscle and of cartilage to environmental conditions, particularly to the protein
constituents of the medium.
6. It is concluded that the stabilization phase of cell differentiation is open
to a degree of selective influence from the environment, and that extrinsic
factors may have some control over the ultimate differentiated fate of the tissue.
RESUME
Myogenese dans des somites d'embryon de Poulet cultives in vitro
1. La differenciation de cellules musculaires dans des somites cultives en
culture organotypique a ete etudiee parallelement au developpement de cartilage
dans le meme systeme en vue d'analyser des facteurs de la stabilization de la
differenciation, principalement pour savoir s'il existe une specificite.
2. Des somites d'embryons de Poulet des stades 9 a 17 sont cultives sur des
filtres Millipore flottant sur un milieu nutritif gelose. On observe la presence de
fibres musculaires plurinuclees.
3. Des fibres musculaires plurinuclees sont observees dans des explants
provenant d'embryons des stades 11 a 17.
4. Le frequence de la formation de muscles variee avec les conditions de
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M. L. ELLISON, E. J. AMBROSE & G. C. EASTY
culture. Elle est favorisee par l'addition d'ceuf au milieu, par le serum de veau
ploutot que par le serum de cheval, et par l'augmentation de volume de l'explant,
5. La comparaison des resultats avec ceux qu'on obtient au sujet de la
differenciation du cartilage dans le meme systeme montre une variation dans
les details entre les reactions des muscles et du cartilage aux conditions externes,
particulierement par rapport aux constituants proteiques du milieu.
6. On conclut que le phase de stabilization de la differenciation cellulaire
depend, dans une certaine mesure, d'influences specifiques de l'environnement
et que, par consequent, des facteurs externes doivent avoir un certain controle
de la differenciation definitive du tissu.
This investigation has been supported by grants to the Chester Beatty Research Institute
(Institute of Cancer Research: Royal Cancer Hospital) from the Medical Research Council
and the British Empire Cancer Campaign for Research, and by the Public Health Service
Research Grant No. CA-03188-08 from the National Cancer Institute, U.S. Public Health
Service.
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(Manuscript received 30 September 1968)