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,

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.

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).

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.

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 1.

Proportion of somite explants differentiating to muscle on different media

Proportion of somite explants differentiating to muscle on different media
Proportion of somite explants differentiating to muscle on different media

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.

Table 2.

Proportion of somite explants differentiating to muscle in explants of different sizes

Proportion of somite explants differentiating to muscle in explants of different sizes
Proportion of somite explants differentiating to muscle in explants of different sizes

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 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.

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 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.

  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 expiants 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 expiants.

  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.

Myogenèse dans des somites d’ embryon de Poulet cultivés in vitro

  1. La différenciation de cellules musculaires dans des somites cultivés en culture organotypique a été étudiée parallèlement au développement de cartilage dans le même système en vue d’ analyser des facteurs de la stabilization de la différenciation, principalement pour savoir s’ il existe une spécificité.

  2. Des somites d’ embryons de Poulet des stades 9 à 17 sont cultivés sur des filtres Millipore flottant sur un milieu nutritif gélosé. On observe la présence de fibres musculaires plurinuclées.

  3. Des fibres musculaires plurinuclées sont observées dans des explants provenant d’ embryons des stades 11 à 17.

  4. Le fréquence de la formation de muscles variée avec les conditions de culture. Elle est favorisée par l’ addition d’ œuf au milieu, par le serum de veau ploutôt que par le serum de cheval, et par l’ augmentation de volume de l’ explant,

  5. La comparaison des résultats avec ceux qu’ on obtient au sujet de la différenciation du cartilage dans le même système montre une variation dans les détails entre les réactions des muscles et du cartilage aux conditions externes, particulièrement par rapport aux constituants protéiques du milieu.

  6. On conclut que le phase de stabilization de la différenciation cellulaire dépend, dans une certaine mesure, d’ influences spécifiques de l’ environnement et que, par conséquent, des facteurs externes doivent avoir un certain contrôle de la différenciation définitive 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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