This study investigates the differentiative abilities of avian brachial somites at stages of development before, during and after the migration of somitic cells into the wing primordium. These somites are the source of cells that migrate into the forelimbs and there give rise exclusively, and totally, to the skeletal muscle lineage of the wing and yet show no morphological evidence of commitment to that fate when they leave the somites. The aim of the study was to see if the brachial somitic cells are committed to particular developmental pathways at these stages.

The brachial somites were isolated from HH stage-12, -15 and -18 chick embryos, either by microdissection or enzymatic dissociation, and grown in organ culture, in explant culture on different substrata or on the chorioallantoic membrane (CAM) of host chicks, either alone or in combination with adjacent tissues. Myogenesis and chondrogenesis occurred in all stage-18 enzymatically separated somites, regardless of the growth environment. Myogenesis was reduced in stage-15 somites and unobservable in stage-12 somites; however, recombination of stage-12 somites with epithelium or neural tube increased the incidence of myogenesis at this stage. The incidence of chondrogenesis was also less in the younger expiants. Unlike its effect on myogenic expression, recombination with epithelium resulted in a dramatic decrease in chondrogenesis in both stage-12 and -15 somites.

The recombination experiments suggest that conditions that maintain the normal spatial relationships within isolated somites permit expression of a pre-existing specification to a particular fate. They also show that the overlying epithelium can inhibit chondrogenesis in these somites. Overall, the results suggest that by the time migration of somitic cells into wing regions is finishing, brachial somitic cells have become stabilized in their ability to undergo both myogenesis and chondrogenesis for they will do so under a variety of growth conditions and in-dependently of adjacent tissues. However, immediately before (stage 12) and shortly after (stage 15) the onset of migration, both myogenic and chondrogenic expression by brachial somitic cells are still under the influence of interactions with adjacent tissues.

A fundamental feature of the vertebrate body plan, established early in embryonic life, is the ordered segmentation of paraxial mesenchyme into pairs of somites arranged along the anteroposterior axis. Initially, somites are localized condensations of mesenchymal cells. Subsequently, each somite passes through a transient ‘epithelial’ phase in which it consists of a ball of polarized, epithelial cells, oriented with their basal aspect externally and surrounded by a basal lamina (Mestres & Hinrichsen, 1976). This epithelial phase is soon lost as each individual somite subdivides into three parts, each with a different fate; the sclerotome, myotome and dermatome subsequently give rise to vertebral cartilage, trunk musculature and dermal connective tissue, respectively (Williams, 1910; Langman & Nelson, 1968). In fact, covert differences exist well before this morphological regionalization. For instance, during the initial condensation stage, ventrolaterally migrating trunk neural crest cells move exclusively through the anterior portion of each somite (Rickmann, Fawcett & Keynes, 1985), reflecting differences in either extracellular matrix composition or cell contact relationships within the somite. An additional feature of somite development is seen at presumptive limb levels along the anteroposterior axis, in that some ventrolateral somitic cells migrate laterally to establish a myogenic lineage within that region of the somatopleure that will grow out to form the limb (Christ, Jacob & Jacob, 1974, 1977; Chevallier, Kieny & Mauger, 1976, 1977). Furthermore, although this capacity to migrate, colonize other mesenchymes and establish myogenic lineages outside of the somites themselves, normally is displayed only by somitic cells at limb levels, heterotopic grafting reveals that it is a potential possessed by all somites (Chevallier et al. 1977; Christ, Jacob & Jacob, 1978; Chevallier, 1979).

Thus, somites undergo dramatic morphogenetic changes and give rise to a range of tissue types. Surprisingly, little is known about specification and commitment to particular differentiation pathways or about environmental factors that influence expression of committed somitic cells. Most work on differentiation in somites has focused on somite chondrogenesis (reviewed by Hall, 1977). The major drawback of most of these studies was the lack of precision concerning which somites were used. Such definition is crucially important given the meristic, anteroposterior progression of somite formation and maturation, for the differentiative abilities of somites at one level of the embryonic axis do not reflect the abilities of somites at other levels.

In the study reported here, we have chosen to work with avian brachial somites (somite pairs 15-20). These somites are of particular interest because, despite past controversy, it has now been established unequivocally that the myogenic cell lineage within the developing limb bud is derived exclusively from these somites (Christ, Jacob & Jacob, 1979; Kenny-Mobbs & Hall, 1983; Kenny-Mobbs, 1985). The lineage derives from cells that leave the somites between 48 and 72 h of incubation (Christ et al. 1977; Chevallier, 1978). Clearly, the differentiative abilities of the brachial somitic cells over this period are of pivotal importance to an understanding of myogenesis within the limb and of how commitment to a myogenic fate might occur.

Two general questions have been addressed in this study. First, at which stage are cells in the brachial somites first capable of autonomous myogenic and chondrogenic differentiation? Isolated somites of different ages have been organ cultured, explant cultured or CAM grafted; differentiation has been monitored using morphology, alcian-blue staining of cartilage matrix and immunocytochemical staining of skeletal muscle-specific forms of actin and myosin. The ability to differentiate autonomously in these various (permissive) environments we interpret as a reflection of ‘specification’, but not necessarily ‘determination’, of a particular differentiation pathway (Mohun, Tilly, Mohun & Slack, 1980; Slack, 1983). Verification of an irreversible determination could only be made if attempts to switch cells into an altered differentiation pathway had failed; no such experiments were undertaken in the present work. Second, do tissues adjacent to the somites in vivo influence the expression of a particular differentiation pathway in vitro”! Isolated somites have been cultured and/or grafted in recombination with normally adjacent neural tube or dorsolateral ectoderm, and differentiation evaluated as described above. Evidence of either an inhibitory or stimulatory influence has been related to the possible roles of the tissues, in vivo, in normal somite maturation and development.

Our results indicate that by the time migration of somitic cells into wing regions is finishing, cells within the brachial somites have become stabilized in their ability to undergo both myogenesis and chondrogenesis for they will do so under a variety of growth conditions and independently of adjacent tissues. However, immediately before, and shortly after, onset of migration, expression of both phenotypes is still under the influence of interactions with adjacent tissues.

Fertilized eggs of a White Leghorn x Light Sussex strain of Gallus domesticus, obtained from a local hatchery, were incubated at 38°C and at a relative humidity of 50-60 %, to Hamburger & Hamilton (1951) stages 12, 15 and 18. HH stage 12 is a stage before somitic cells migrate into presumptive wing somatopleure; HH stage 15 is after onset of that migration and HH stage 18 is nearing completion of somitic cell migration. Embryos used as hosts for chorioallantoic grafting were incubated for 9-10 days.

Dissections

Embryos were removed from underlying yolk with the aid of filter paper rings, washed in phosphate-buffered saline (PBS) and transferred to fresh PBS for dissection. Somites were dissected from the brachial region of embryos, i.e. from the level of the 14-15th pair of somites to that of the 20th-21st pair, giving a string of six to seven somites with their covering ectodermal and endodermal layers. In some dissections neural tube and notochord were also included (Fig. 1).

Fig. 1.

A diagram illustrating the various combinations of brachial tissues that were cultured, grafted or explanted. The brachial region was taken to be that between somite pairs 15 (shaded) and 20 (not yet segmented in this stage-12 embryo). The pieces of tissue obtained from this region by microdissection, shown in the top part of the diagram, included the string of brachial somites with covering epithelial layers, adjacent neural tube and notochord (S+NT+N+E) or the brachial somites with the covering epithelial layers only (S+E). Enzymatically isolated somites, shown in the bottom part of the diagram, were grown either alone (S) or recombined with neural tube (S+NT) or with the dorsal ectoderm (S+E).

Fig. 1.

A diagram illustrating the various combinations of brachial tissues that were cultured, grafted or explanted. The brachial region was taken to be that between somite pairs 15 (shaded) and 20 (not yet segmented in this stage-12 embryo). The pieces of tissue obtained from this region by microdissection, shown in the top part of the diagram, included the string of brachial somites with covering epithelial layers, adjacent neural tube and notochord (S+NT+N+E) or the brachial somites with the covering epithelial layers only (S+E). Enzymatically isolated somites, shown in the bottom part of the diagram, were grown either alone (S) or recombined with neural tube (S+NT) or with the dorsal ectoderm (S+E).

Somites were also enzymatically-isolated from all adjacent tissues. In these experiments the entire brachial region was removed (to the edges of the area pellucida) and placed in a mixture of 1·25% pancreatin and 0·25% trypsin in Tris-buffered Tyrode’s solution, for 20-30min at 4°C. The digested tissue was then transferred to a 1:1 solution of alpha Eagles’ Minimal Essential Medium (a-MEM) and horse serum, and then into fresh α -MEM for mechanical separation of the tissue components (Fig. 2)

Fig. 2.

A photomicrograph showing pieces of stage-12 brachial region tissue, after they have been enzymatically dissociated and then mechanically separated. The ectoderm has partially folded over on itself, e, dorsal ectoderm; nt, neural tube; s, somites. Bar, 200μm.

Fig. 2.

A photomicrograph showing pieces of stage-12 brachial region tissue, after they have been enzymatically dissociated and then mechanically separated. The ectoderm has partially folded over on itself, e, dorsal ectoderm; nt, neural tube; s, somites. Bar, 200μm.

Culture of isolated somites

Microdissected and enzymatically isolated somites, the latter either alone or recombined with adjacent neural tube or epithelium, were grown in organ culture or as grafts to the chorioallantoic membrane (CAM) of host chick embryos. Enzymatically separated somites were also explanted to various substrata (see below).

Tissues to be organ cultured were placed on small squares of 0·45μm pore size Millipore filter which, in turn, were placed on stainless steel mesh supports in 35 mm plastic tissue-culture dishes (Sterilin). To each dish was added 1·5 ml of culture medium consisting of α-MEM, heat-inactivated horse serum and 50 % embryo extract in a 75:15:10 v/v mixture. Cultures were grown at 37°C, in an atmosphere of 5 % CO2 in air, in a humidified incubator. Culture medium was replaced every other day. Tissues to be CAM-grafted were set up on Millipore filters as de-scribed for organ culture and then grafted to the CAM of 9 to 10-day host chick embryos as described previously (Kenny-Mobbs, 1985).

In the tissue recombination experiments, enzymatically isolated somites were positioned on filters as described above. For the somites-plus-epithelium (S+E) combinations, enzymatically isolated epithelium from the brachial region was stretched across a string of somites on the filter. In the somites-plus-neural-tube (S+NT) recombinations, isolated neural tube was positioned immediately alongside a string of somites on the filter (Fig. 1). Recombinations were then cultured or grafted as outlined above.

All cultures and grafts were grown for 6 or 7 days, a period chosen to be a compromise between maximum differentiation and necrosis in cultures as the tissue mass increased and breakdown of CAM tissues as the host neared hatching. Cultures and grafts were then recovered, rinsed in PBS and fixed in 4% formaldehyde, 1 % glutaraldehyde in phosphate buffer (McDowell & Trump, 1976) and processed for paraffin embedding.

All organ cultures and CAM grafts were serially sectioned and stained with haematoxylin, eosin and alcian blue. Every section was examined for evidence of chondrogenic and myogenic expression. Chondrogenesis was assessed by the presence of alcian-blue-stained rods or irregularly shaped nodules discretely defined by a covering of perichondrium (e.g. Figs 3, 5). Assessment of myogenesis was based on the presence of multinucleated myotubes, whether they were simple and few in number (e.g. Fig. 6) or organized into well-defined muscle bundles (e.g. Fig. 10). A culture or graft was scored as containing cartilage and/or muscle irrespective of the size or number of cartilage elements or muscle bundles, hence the values on the tables indicate the incidence of differentiation expressed as a percentage of the total number of cultures or grafts examined. These data were statistically analysed using 2x2 contingency tables and the χ2 distribution. Differences in the amount of cartilage and/or muscle formed are described in the text of the Results and shown in the figures.

Fig. 3.

(A) A 7-day CAM graft of stage-12 microdissected brachial somites that included the axial tissues. Both the neural tube (nt) and notochord (n) have differentiated along with somitic cartilage (c), muscle (m) and kidney tubules (k). (B) A 7-day CAM graft of stage-12 microdissected somites (somites with covering epithelial layers intact). Cartilage rods (c) and feather germs (fg) were the only tissues differentiated in this graft. Bars, 50μm.

Fig. 3.

(A) A 7-day CAM graft of stage-12 microdissected brachial somites that included the axial tissues. Both the neural tube (nt) and notochord (n) have differentiated along with somitic cartilage (c), muscle (m) and kidney tubules (k). (B) A 7-day CAM graft of stage-12 microdissected somites (somites with covering epithelial layers intact). Cartilage rods (c) and feather germs (fg) were the only tissues differentiated in this graft. Bars, 50μm.

Fig. 4.

(A) A piece of stage-12 microdissected brachial tissue, similar to Fig. 3A, which was grown in organ culture for 7 days. Axial neural tube (nt) and notchord (n) are seen as well as kidney tubules (Æ), somitic cartilage (c) and muscle (m). (B) In the absence of the axial tissue, stage-12 microdissected brachial somites in organ culture rarely formed cartilage. Occasionally what appeared to be small myotubes (arrows) could be seen; kidney tubules (k). (C) A 6-day organ culture of stage-18 microdissected brachial somites. Note that cartilage (c) and muscle (m) formation are similar to that in A. Bars, 40γm.

Fig. 4.

(A) A piece of stage-12 microdissected brachial tissue, similar to Fig. 3A, which was grown in organ culture for 7 days. Axial neural tube (nt) and notchord (n) are seen as well as kidney tubules (Æ), somitic cartilage (c) and muscle (m). (B) In the absence of the axial tissue, stage-12 microdissected brachial somites in organ culture rarely formed cartilage. Occasionally what appeared to be small myotubes (arrows) could be seen; kidney tubules (k). (C) A 6-day organ culture of stage-18 microdissected brachial somites. Note that cartilage (c) and muscle (m) formation are similar to that in A. Bars, 40γm.

Fig. 5-7.

. Histological sections of organ cultures of brachial somites only (Fig. 5; bars, 30μm), somites recombined with ectoderm (Fig. 6; bars, 15μm), and somites recombined with neural tube (Fig. 7; (A) bar 15gm; (B) bar, 30μm). Tissues were enzymatically isolated from stage-12 (A), -15 (B) and -18 (C) embryos and grown in culture for 6-8 days, c, cartilage; e, epithelium; m, muscle; arrows, myotubes.

Fig. 5-7.

. Histological sections of organ cultures of brachial somites only (Fig. 5; bars, 30μm), somites recombined with ectoderm (Fig. 6; bars, 15μm), and somites recombined with neural tube (Fig. 7; (A) bar 15gm; (B) bar, 30μm). Tissues were enzymatically isolated from stage-12 (A), -15 (B) and -18 (C) embryos and grown in culture for 6-8 days, c, cartilage; e, epithelium; m, muscle; arrows, myotubes.

Enzymatically isolated somites were also grown on glass coverslips or tissue-culture plastic, which was either untreated or coated with a collagen substratum that was a mixture of types I and II collagen (Vitrogen 100, Flow Laboratories, UK). Isolated somites were transferred to the substrata in a small volume of cr-MEM and allowed to attach. Additional medium was added (1·5ml/dish) and expiants incubated as described above for organ cultures. During the culture period, expiants were examined using inverted-phase-contrast microscopy and photographed with Kodak Technical Pan film.

Some expiants were treated with rabbit antibodies raised against frog skeletal muscle myosin and actin, but known to cross react with equivalent chick antigens. These antibodies were a gift of Dr Burr Atkinson, University of Western Ontario, and were prepared as described by Dhanarajan & Atkinson (1980). The staining protocol used has been described in detail elsewhere (Kenny-Mobbs, 1985; Kenny-Mobbs & Thorogood, 1986); briefly it involved incubation for Ih, in light-proof conditions at 37 °C, in the primary antibody (against skeletal muscle antigens) and then in the secondary antibody (goat anti-rabbit IgG, conjugated to fluorescein isothiocyanate). Negative controls consisted of substituting PBS for the primary antibody; expiants from HH stage-18 embryos served as positive controls.

(A) Differentiation in microdissected brachial somites

Strings of somites from the level of the 14—15th pair to that of the 20th-21st pair, when microdissected from embryos of stages 12, 15 and 18, were found to undergo comparable growth and differentiation of the derivatives of the three germ layers when axial tissues were included in the organ cultures and CAM grafts. Differentiated neural tube, notochord, vertebral cartilage and muscle, and gut tissue invariably formed, while kidney tubules, ganglia and feather germs were often present (Figs 3A, 4A).

If somites were microdissected free of adjacent axial and lateral plate tissues, but retained the covering ectodermal and endodermal epithelia, tissue differentiation in the recovered organ cultures and CAM grafts was markedly different, in relation to both initial age of the explant and environment in which it was grown (Table 1). Differentiation of cartilage and muscle in somites microdissected from stage-18 embryos was not noticeably affected by the absence of axial tissues (Fig. 4C). In cultures and grafts of somites from stage-15 embryos the incidence of skeletal myogenesis was not appreciably affected, but the amount of muscle formed in these expiants decreased substantially. Chondrogenesis did not occur in organ cultures from this stage but was essentially unchanged in CAM grafts. In expiants from stage-12 somites, both chondrogenesis and myogenesis were greatly reduced, in both incidence and amount (Figs 3B, 4B). At both stages 12 and 15 the incidence of chondrogenesis was significantly greater (P<0·05, P<0·001, respectively) on the CAM than in organ culture but, otherwise, differentiation was not influenced by the growth environment.

Table 1.

Incidence of differentiation in brachial somites microdissected free of axial and lateral plate tissues, from stage-12, -15 and -18 embryos, and organ cultured or CAM grafted for 6-7 days

Incidence of differentiation in brachial somites microdissected free of axial and lateral plate tissues, from stage-12, -15 and -18 embryos, and organ cultured or CAM grafted for 6-7 days
Incidence of differentiation in brachial somites microdissected free of axial and lateral plate tissues, from stage-12, -15 and -18 embryos, and organ cultured or CAM grafted for 6-7 days

When isolating somites by microdissection, it is difficult to ensure the complete removal of adjacent tissues, especially small pieces of neural tissue and intermediate mesoderm. Consequently, somites were isolated from the brachial region by enzymatic digestion followed by mechanical separation. As described in the following sections, chondrogenesis and myogenesis in cultures and grafts of these enzymatically isolated somites varied depending on the age of isolated somites, the conditions under which they were grown and the tissues with which they were associated in recombinations.

Chondrogenesis in enzymatically isolated and recombined somites

Organ-cultured somites removed from stage-12, -15 and -18 embryos showed a stage-related increase in the incidence of cartilage in all combinations of tissues (Table 2). On their own, isolated somites underwent chondrogenesis in about 50 % of stage-12 cultures, rising to 100 % for stage-18 cultures (Fig. 5). However, chondrogenesis appeared to be inhibited by the presence of epithelial tissue, for the incidence of cartilage formation dropped substantially (P< 0·001 for stage 12 and P<0·01 for stage 15) when somites were recombined with brachial region epithelium (Table 2; Fig. 6). Similarly, microdissected somites, which retained their ectodermal and endodermal coverings, had a very low incidence of chondrogenesis for stages 12 and 15 (see Table 1). Recombinations of stage-12 and -15 somites with neural tube had no significant effect on the incidences of chondrogenesis compared to somites on their own (Fig. 7). By stage 18 nearly all combinations of cultured somites formed cartilage.

Table 2.

Incidence of chondrogenesis in enzymatically separated somites from HH stage-12, -15 and -18 embryos grown in organ culture alone (S) or recombined with epithelium (S+E) or neural tube (S+NT).

Incidence of chondrogenesis in enzymatically separated somites from HH stage-12, -15 and -18 embryos grown in organ culture alone (S) or recombined with epithelium (S+E) or neural tube (S+NT).
Incidence of chondrogenesis in enzymatically separated somites from HH stage-12, -15 and -18 embryos grown in organ culture alone (S) or recombined with epithelium (S+E) or neural tube (S+NT).

Chondrogenesis in CAM-grafted somites and somite recombinations presented a different, pattern of expression (Table 3). Isolated somites from stage-12 and -15 embryos grown on the CAM formed no cartilage (Fig. 8) whereas in organ culture they had done so in over 50 % of the cases. In recombinations of somites and brachial epithelium (Fig. 9), cartilage formation was very low in stage-12 grafts (as also seen in organ culture) but was significantly greater (P< 0·001) in stage-15 grafts compared with cultures of the same stage. Similarly, chondrogenesis in epithelia-covered, microdissected somites was noticeably greater on the CAM than in organ culture for stage-12 and -15 somites (see Table 1). Recombination with neural tube significantly increased the incidence of cartilage formation in somites from stages 12 and 15 grown on the CAM (Table 3, Fig. 10). All combinations of somite grafts from stage-18 embryos contained cartilage.

Table 3.

Incidence of chondrogenesis in enzymatically separated somites from HH stage-12, -15 and -18 embryos grown on the CAM alone (S) or recombined with epithelium (S+E) or neural tube (S+NT)

Incidence of chondrogenesis in enzymatically separated somites from HH stage-12, -15 and -18 embryos grown on the CAM alone (S) or recombined with epithelium (S+E) or neural tube (S+NT)
Incidence of chondrogenesis in enzymatically separated somites from HH stage-12, -15 and -18 embryos grown on the CAM alone (S) or recombined with epithelium (S+E) or neural tube (S+NT)
Fig. 8-10.

Histological sections of CAM grafts of brachial somites only (Fig. 8; (A,B) bars, 20μm; (C) bar, 40 μm), somites recombined with ectoderm (Fig. 9; bars, 40μm), and somites recombined with neural tube (Fig. 10; bars, 60μm). Tissues were enzymatically isolated from stage-12 (A), -15 (B) and -18 (C) embryos and grafted for 6-7 days. Arrows in A indicate small myotubes, c, cartilage; g, ganglion; m. muscle; nt, neural tube.

Fig. 8-10.

Histological sections of CAM grafts of brachial somites only (Fig. 8; (A,B) bars, 20μm; (C) bar, 40 μm), somites recombined with ectoderm (Fig. 9; bars, 40μm), and somites recombined with neural tube (Fig. 10; bars, 60μm). Tissues were enzymatically isolated from stage-12 (A), -15 (B) and -18 (C) embryos and grafted for 6-7 days. Arrows in A indicate small myotubes, c, cartilage; g, ganglion; m. muscle; nt, neural tube.

Myogenesis in enzymatically isolated and recombined somites

Somites enzymatically isolated from stage-18 embryos formed muscle in almost all organ cultures and grafts whether grown alone or recombined with ectoderm or neural tube (Tables 4, 5; Figs 5C-10C). This was also the case for somites isolated from stage-15 embryos (Figs 5B-10B), with the exception of isolated somites grafted to the CAM in which the incidence of myogenesis was only 8 %.

Table 4.

Incidence of myogenesis in enzymatically separated somites from HH stage-12, -15 and -18 embryos grown in organ culture alone (S) or recombined with epithelium (S+E) or neural tube (S+NT)

Incidence of myogenesis in enzymatically separated somites from HH stage-12, -15 and -18 embryos grown in organ culture alone (S) or recombined with epithelium (S+E) or neural tube (S+NT)
Incidence of myogenesis in enzymatically separated somites from HH stage-12, -15 and -18 embryos grown in organ culture alone (S) or recombined with epithelium (S+E) or neural tube (S+NT)
Table 5.

Incidence of myogenesis in enzymatically separated somites from HH stage-12, -15 and -18 embryos grown on the CAM alone (S) or recombined with epithelium (S+E) or neural tube (S+NT)

Incidence of myogenesis in enzymatically separated somites from HH stage-12, -15 and -18 embryos grown on the CAM alone (S) or recombined with epithelium (S+E) or neural tube (S+NT)
Incidence of myogenesis in enzymatically separated somites from HH stage-12, -15 and -18 embryos grown on the CAM alone (S) or recombined with epithelium (S+E) or neural tube (S+NT)

Myogenesis in stage-12 somites, however, varied depending on the tissue cultured or grafted but was not significantly affected by the growth environment, i.e. cultures versus grafts (Tables 4, 5; Figs 5A-10A). Enzymatically isolated somites, organ cultured or grafted on their own, did not form muscle. Recombinations with epithelium (S+E) resulted in an increased incidence of myogenesis, especially in organ culture (Table 4; Fig. 6A). The percentage of CAM grafts of S+E recombinations forming muscle was similar to grafts of microdissected somites (see Table 1) and, although organ-cultured S+E recombinations displayed a higher incidence of myogenesis than organ-cultured, microdissected somites, this difference was not significant. The presence of neural tube greatly increased (P< 0·001) the incidence of myogenesis in stage-12 somites, both in organ culture and on the CAM (Figs 7A, 10A).

Immunofluorescence of muscle antigens in explanted somites

Enzymatically isolated somites from the three stages under study were also explanted directly onto tissue-culture plastic or glass coverslip surfaces that were either coated with collagen or left untreated. Regardless of the type of substratum, somites from stage-18 embryos invariably formed large myotubes in such expiants (Fig. 11C). Myotubes could also be detected in about 50% of stage-15 explanted somites, with expression not greatly influenced by the substratum (Table 6; Fig. 11B). Somites from stage-12 embryos, on the other hand, did not form myotubes under these conditions although, based on solely morphological criteria, one or two may have been present when somites were explanted to collagen-coated substrata (Table 6; Fig. 11A).

Table 6.

Incidence of myogenesis in enzymatically separated somites explanted to different substrata

Incidence of myogenesis in enzymatically separated somites explanted to different substrata
Incidence of myogenesis in enzymatically separated somites explanted to different substrata
Fig. 11.

Phase-contrast micrographs of living cultures of brachial somites enzymatically separated from stage-12 (A), -15 (B) and -18 (C) embryos and explanted to collagen-coated coverslips for 6 days. Arrows indicate stuctures that appear to be myotubes. Bars, 20μm.

Fig. 11.

Phase-contrast micrographs of living cultures of brachial somites enzymatically separated from stage-12 (A), -15 (B) and -18 (C) embryos and explanted to collagen-coated coverslips for 6 days. Arrows indicate stuctures that appear to be myotubes. Bars, 20μm.

Expiants of somites cultured for 6 to 7 days were incubated with rabbit antibodies to skeletal muscle myosin and actin in an attempt to determine the earliest stage at which these muscle-specific antigens could be detected (Table 7). Expiants of stage-18 somites, which always formed myotubes, were used as positive controls (Figs 12C-14C). It was found that all stage-15 somites reacted positively, but less in-tensely than controls, to both myosin and actin antibodies (Figs 12B-14B), although previously myotubes could only be identified clearly in about half of these expiants (see Table 6). However, none of the stage-12 expiants reacted positively to the skeletal muscle antibodies (Figs 12A-14A). Clearly, the few ‘multinucleate cells’ tentatively identified as myotubes on the basis of morphological criteria alone (see Table 6) did not contain detectable levels of skeletal muscle myosin and actin and therefore were concluded not to be myotubes.

Table 7.

Intensity of immunofluorescent staining of explanted somites from HH stage-12, -15 and -18 embryos using rabbit antibodies to skeletal muscle myosin and actin

Intensity of immunofluorescent staining of explanted somites from HH stage-12, -15 and -18 embryos using rabbit antibodies to skeletal muscle myosin and actin
Intensity of immunofluorescent staining of explanted somites from HH stage-12, -15 and -18 embryos using rabbit antibodies to skeletal muscle myosin and actin
Fig. 12-14.

Immunofluorescence of whole mounts of expiants of brachial somites from stage-12 (A), -15 (B) and -18 (C) embryos, stained with rabbit antibodies against skeletal muscle myosin (Fig. 12), and skeletal muscle actin (Fig. 13). Fig. 14 shows control expiants which were incubated with normal nonimmune rabbit serum as a first layer. Myotubes in stage-15 and -18 expiants reacted positively with both skeletal muscle antibodies; no reaction above background was seen in expiants derived from stage-12 embryos. Bars, 10μm.

Fig. 12-14.

Immunofluorescence of whole mounts of expiants of brachial somites from stage-12 (A), -15 (B) and -18 (C) embryos, stained with rabbit antibodies against skeletal muscle myosin (Fig. 12), and skeletal muscle actin (Fig. 13). Fig. 14 shows control expiants which were incubated with normal nonimmune rabbit serum as a first layer. Myotubes in stage-15 and -18 expiants reacted positively with both skeletal muscle antibodies; no reaction above background was seen in expiants derived from stage-12 embryos. Bars, 10μm.

To summarize the results, somites from stage-18 embryos underwent both myogenesis and chondro-genesis under all conditions of in vitro culture or CAM grafting, whether grown alone or in combination with adjacent tissues. Myogenesis occurred in most combinations of stage-15 somites tested except in enzymatically isolated somites grown on the CAM. Chondrogenesis in stage-15 somites varied with both the combination of tissues and the growth conditions and was inhibited by association with epithelial tissues. Experiments with stage-12 somites showed myogenesis to be influenced primarily by the combination of tissues cultured or grafted, association with neural tube being the most conducive to myogenesis. Chondrogenesis in stage-12 somites seemed to be inhibited mainly by association with epithelia, as was the case with stage-15 somites.

Earlier studies on somites (e.g. Murray & Selby, 1933; Avery, Chow & Holtzer, 1956; Ellison, Ambrose & Easty, 1969a,b; O’Hare, 1972a,b,c) are of limited value for information on the onset of differentiation in the brachial region (or any other specific region), because the somites cultured or grafted were often drawn from a pool of somites from various anteroposterior levels. Given the anteroposterior gradient of somite segmentation and differentiation, data on the differentiative abilities of somites at any one level cannot be extrapolated to somites at all levels. For example, during the stages of development examined in this study, the somites in the brachial region (see Table 8) segment from the paraxial mesoderm, differentiate into dermatomal, myotomal and sclerotomal regions (Chevallier, 1978), lose cells to lateral plate mesoderm as somitic cells invade the wing primordia (Christ et al. 1974, 1977; Chevallier et al. 1976, 1977) and are invaded, in turn, by neural crest cells and outgrowing nerve fibres as they migrate towards lateral plate somatopleure (Rickmann et al. 1985). Most of these events have occurred already in more anterior somites and have yet to occur more posteriorly. The results of this study show that (1) the brachial somitic cells at these stages are also becoming stabilized in their ability to express myogenic and chondrogenic phenotypes, and (2) brachial region neural tube and epithelia play a role in this stabilization process.

Table 8.

Normal developmental events in the brachial somites

Normal developmental events in the brachial somites
Normal developmental events in the brachial somites

Autonomy of differentiation

Analysis of the autonomy of differentiation in brachial somites is valid only in those experiments in which the somites were enzymatically isolated and thus removed from influences of all adjacent tissues. There are no other studies, comparable to the present one, directed specifically to differentiation in enzymatically isolated brachial somites; for instance, the recent report by Swalla & Solursh (1984) describing in vitro differentiation in stage 16/17 somites (including those from brachial levels) is based on cultures that included ectoderm and notochord. However, information gleaned from a few studies suggests that the earliest stage at which enzymatically isolated brachial somites have been found to form skeletal muscle in organ culture is stage 13/14 (Ellison et al. 1969b) and, on the CAM, not before stage 15 (O’Hare, 1972a; but the level of origin of the somites was not specified in this case). In a study in which quail somites were grafted to chick wing buds, brachial somites from embryos stage 13 and older formed skeletal muscle (Wachtler, Christ & Jacob, 1982). These observations support the present findings and, compared with a myosin antibody study of somites in normal embryos (Holtzer, Marshall & Finck, 1957), suggest that commitment to myogenesis in brachial somites occurs shortly before the onset of differentiation and shortly before, or concomitant with, the onset of migration of somitic cells into the wing primordium (see Table 8).

Similar analysis of previously published data on chondrogenesis indicates that differentiation may be elicited from CAM-grafted somites as early as stage 13 (O’Hare, 1972b), but these grafts included more anterior somites which may be (chondrogenically) committed as early as stage 9 (Ellison et al. 1969a). However, other reports describe a ‘rare’ incidence of chondrogenesis from cultured, isolated somites (anterior to and including somite 17) from as late as stage 17 (Gordon & Lash, 1974). Our results show that autonomy of chondrogenic expression is also stage-related and, on the basis of differentiation on the CAM, appears to lag slightly behind onset of myogenic expression.

Somites cultured and grafted from stage-12 embryos did not form muscle under any of the conditions of growth used in this study. It might be argued that the methods of culture and CAM grafting were not conducive to growth and differentiation of these somites. However, the isolated somites were growing, as evidenced by differentiation of cartilage in over half of the organ cultures and spreading and growth of cells in the explant cultures. Viability on the CAM was less apparent, since neither cartilage nor muscle formed and, if connective tissue were the only tissue undergoing differentiation, it would be impossible to distinguish its presence from host CAM tissues, given the lack of appropriate markers. However, the fact that somites isolated from embryos just 6h older (stage 15) did form muscle with a low frequency on the CAM (8%), and that by stage 18 the brachial somites formed well-developed muscle bundles and cartilage, shows that the CAM, per se, does not limit myogenic or chondrogenic expression. We regard differentiation on the CAM as a more accurate measure of the degree of autonomy of expression achieved by somites since they are completely isolated from any normally adjacent influences in this environment. The presence of cartilage in most of the organ and explant cultures of all stages, and of small amounts of muscle in all organ and explant cultures from stage 15 (compared with only 8% of CAM grafts), could be attributed to the stimulatory effects of some factor(s) in the embryo extract supplement to the culture medium. That only small amounts of muscle and cartilage formed in culture suggests that commitment to these pathways had been initiated only in the most anterior somites of the brachial region. The number of somites cultured or grafted does not seem to affect differentiation either, for half a somite strip (3 somites) or two somite strips (12 somites) showed the same type of differentiation although the amount of a particular tissue might be lesser or greater (unpublished observations).

The trend towards autonomy of myogenic and chondrogenic expression, within brachial somites, with increasing developmental stage most likely reflects a stabilization of pre-existing specification for each phenotype by the extended period of in vivo interaction with adjacent tissues. The recombination experiments of this study indicate that two of these adjacent tissues, neural tube and epithelium, have different influences on expression of the two phenotypes.

Role of the neural tube

The precise configuration of somites, whether in culture or as a graft, is fundamental to their sub-sequent development. At early stages of development, removal of axial tissues has been shown to block the transition from an epithelioid somite to one consisting of dermatome, myotome and sclerotome (Packard & Jacobson, 1976). Furthermore, the collagenous matrix attaching the somite to axial structures is regarded as maintaining the stability of somite configuration and preventing cells migrating away (Packard & Jacobson, 1976; Bellairs, 1979; Bellairs & Veini, 1980). By stage 18, the transition to sclerotome, myotome and dermatome has been completed before culturing or grafting. We suggest that the decreased incidence of differentiation at stages 12 and 15 reflects either a failure to maintain the configuration of the individual somite (i.e. the cells disperse), or a blocking of the transition from a hollow epithelioid sphere to a regionally organized somite. In the recombination experiments the neural tube, which retains its elongated, tubular form and appears to undergo normal differentiation in culture or on the CAM, should maintain a relatively normal somite configuration and, hence, normal tissue relationships within individual somites, thus facilitating expression of myogenic and chondrogenic phenotypes. We as sume that muscle and cartilage formed in these experiments is derived either from pre-existing myotome and sclerotome, respectively, or from cells destined to give rise to these regions.

A further factor promoting myogenesis may be the production of some trophic or mitogenic stimulus emanating from the axial structures (Maden, 1978; Oh & Markelonis, 1980; Oh et al. 1981). Langman & Nelson (1968) showed that there is a high level of mitotic activity within the somites during their regionalization and proposed that the myotome arises by division in, and displacement from, the dermatomal layer resulting in a mass of cells subjacent to the dermatome. The absence of the axial structures might reduce cell division within isolated somites and thereby inhibit myotomal formation. Indeed, the ‘hollow vesicle’ appearance of somites cultured in the absence of axial tissues (e.g. Packard & Jacobson, 1976) suggests that the myotome is missing.

The in vivo establishment of the ability to differentiate into cartilage autonomously in vitro reflects not only the transition from epithelioid to regionalized somite, but a further influence of axial tissues. A matrix-mediated interaction between notochord/ ventral neural tube and sclerotome cells stabilizes the somitic cells’ ability to differentiate and thus promotes the formation of cartilage in vivo and in vitro (reviewed by Hall, 1977); all of the present results from the recombination experiments display this promoting effect. However, maintenance of a normal somite configuration may be especially important for chondrogenesis. It has been shown in limb mesenchyme that conditions that cause a flattening of chondrogenically committed cells can reversibly inhibit chondrogenic expression (Solursh, Linsenmayer & Jensen, 1982; Zanetti & Solursh, 1984). The present study, and that of Carlson & Kenney (1980), have shown that enzymatically isolated somites at stages 12 and 15 have a tendency to disperse. The marked absence of chondrogenesis in CAM grafts of such somites indicates that the tendency to disperse, with a concomitant cell flattening, may occur on the CAM; clearly recombination with neural tube completely reverses this result (see Table 3).

Ellison et al. (1969a, p. 338) suggested that ‘.. .the most favourable conditions [for differentiation] are those which most adequately replace the stabilization provided in vivo by the tissues which normally surround the developing primordium’. The neural tube may be especially effective in ‘mimicking’ the in vivo conditions not only by providing a mitotic/trophic stimulus but also by imparting a structural stability to the isolated somites.

Role of the epithelium

The role of epithelium in differentiation of cartilage and muscle in brachial somites appears complex. Cultures and grafts of enzymatically isolated somites recombined with epithelium (S+E) have a greater incidence of myogenesis than somites grown on their own. Chondrogenesis, on the other hand, seems to be inhibited by the presence of epithelium overlying somites grown in organ culture. In the same combinations grown on the CAM, the presence of epithelium had no effect on chondrogenesis at stage 12 but increased the incidence at stage 15.

As was suggested for neural tube influences, the stimulatory effect of the epithelium on somitic myogenesis may be related to association with a tissue that can produce a collagenous matrix (Bellairs, 1979; see also O’Hare, 1973) and thus maintain some structural integrity in isolated somites. If this is the case, then the similarity in incidence of myogenesis in CAM grafts of stage-12 and -15 microdissected somites (with ectoderm and endoderm present) versus enzymatically isolated and recombined somites plus ectoderm, suggests that the separation technique has not permanently impaired the function of the ectoderm.

The role of epithelium in chondrogenic expression by brachial somites is clearly inhibitory when somites are grown in organ culture. With increasing developmental stage, however, this inhibitory effect decreases. If the inhibition depends on proximity of chondrogenic cells to the epithelium (Zanetti & Solursh, 1986), with increasing regional differentiation of the somites with time, the somitic cells may tend to disperse less (see above) and also the chondrogenic cells will become more ventrally aggregated within the somite. Either way they are less likely to come under the direct influence of epithelium.

On the CAM, epithelium seems to increase the incidence of chondrogenesis in stage-15 somites. Possible explanations of this result are (1) that the epithelium is resorbed by, or incorporated into, the CAM and hence unable to exert its inhibitory influence or (2) the CAM environment allows more normal growth of somitic tissue and, as regional segregation occurs, chondrogenic cells are displaced from the proximity of the epithelium, and its inhibitory influences, to form the sclerotome of the somite where, over the graft period, they undergo cartilage differentiation.

Conclusions

Taken as a whole, these experiments show that brachial somites acquire the ability for autonomous myogenic and chondrogenic expression over the 24 h or so between stages 12 and 18. It seems likely that this acquisition proceeds in an anteroposterior sequence, is related to the differentiation of the somites into derma-, myo- and sclerotomal regions and is dependent on the maintenance of contacts with epithelial tissues, either their cells or cell products. Under the conditions of this study, the isolated brachial somites either did not differentiate, differentiated into cartilage or muscle, or differentiated into cartilage and muscle. We interpret these findings to mean that cells specified to form skeletal muscle and cartilage are present in the brachial somites from the earliest stage we examined. Whether they are irreversibly ‘determined’ was not tested.

The establishment of autonomous differentiation with increasing developmental age is not a surprising finding. The value of this particular study, however, is that it provides data on the differentiative abilities of the somites of a particular region (the brachial region) over a specific developmental period. This is of interest in terms of the relationship between somite formation (segmentation and regionalization) and commitment to particular differentiation pathways, and provides essential data on which to base detailed studies using molecular probes for the onset of gene expression within these somites. These results are also important in the context of the migration of somitic cells into the wing primordia (Chevallier, 1978; Jacob, Christ & Jacob, 1978) and the possible state of commitment of the migrating cells. The results of our experiments indicate that shortly before, or concomitant with, the onset of somitic cell migration, the brachial somitic cells acquire the ability to undergo autonomous myogenic expression. While not providing direct evidence for the myogenic determination of the cells that invade the wing primordia, these results suggest that this subpopulation of cells is specified for a myogenic fate at the time of its departure from the brachial somites.

The authors would like to acknowledge support for this study from the Nuffield Small Grants Scheme and the Natural Sciences and Engineering Research Council of Canada who provided a postdoctoral fellowship for T.K-M. We would like to thank H. I. Roach for advice on statistical analysis of the data. Fig. f was drawn by Peter Jack from the Department of Teaching Media, Southampton University. Part of this work was presented at the 7th European Anatomical Congress in Innsbruck (Kenny-Mobbs & Thorogood, 1986).

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