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J. Embryol. exp. Morph. 90, 415-436 (1985) 415 Printed in Great Britain © The Company of Biologists Limited 1985 Myogenic differentiation in early chick wing mesenchyme in the absence of the brachial somites TERRY KENNY-MOBBS Department of Biology, Dalhousie University, Halifax, Nova Scotia, Canada, B3H 4J1 SUMMARY A controversy exists in the literature over the ability of wing mesenchyme of somatopleural origin to form skeletal muscle. Experimental approaches used in such studies leave open the possibility of postoperative accessibility of the experimental wings to somitic cell invasion. In the present study wing somatopleural tissue was isolated from HH stage-12 to -21 chick embryos and grown either in organ culture (OC) or on the chorioallantoic membrane (CAM) of host chicks, conditions under which postoperative entry of somitic cells is impossible. In the presence of axial and somitic tissues of the brachial region, the wing territories from all four stages underwent comparable growth and tissue differentiation. However, isolated wing regions showed a stagedependency in the differentiation of skeletal muscle but not of other limb tissues. The incidence and amount of skeletal muscle was markedly reduced in HH stage-12 and -15 isolated wing regions while myogenesis in HH stage-18 and -21 wing buds was not affected by the absence of the somitic tissues. These results are consistent with reported stages of somitic cell migration into wings with the exception of HH stage-12 explants which should have been muscleless if somitic cells are the sole source of wing myofibres. The possibility that somitic cells had been included in these explants was investigated by 1) testing the myogenic potential of lateral plate tissue adjacent to the wing, 2) altering the dissection procedure for isolating wing territories and 3) using antibodies to skeletal muscle myosin and actin to detect myotubes. The results from this series of experiments illustrate the need for extraordinary care in the isolation of wing regions when investigating limb-somite relationships and suggest that the myogenic capacity attributed to wing somatopleural cells in the past can be accounted for by either postoperative entry of somitic cells into experimental wings or inadvertent inclusion of somitic cells in the primordia when dissected. Overall, the results show that somitic cells are the sole source of wing myofibres for, in their absence, somatopleural cells form all mesodermally-derived wing cell types except skeletal myofibres. INTRODUCTION It has been clearly established from the chick/quail recombination experiments of Christ, Jacob & Jacob (1974, 1977, 1979) and Chevallier, Kieny & Mauger (1976, 1977; Chevallier, 1978; Kieny & Chevallier, 1980; Mauger, Kieny & Chevallier, 1980) that cells from the somites adjacent to the limb territories of chick and quail embryos migrate into the somatopleural mesoderm of the limb and there differentiate exclusively into skeletal myofibres. Morphological studies of Present address: Medical Oncology Unit, Centre Block, Southampton General Hospital, Southampton, SO9 4XY, U.K. Key words: limb-somite, myogenesis, wing bud, mesenchyme, somatopleural, chick embryo. 416 T. KENNY-MOBBS normal embryos (Fischel, 1895; Grim, 1970; Christ et al. 1977; Chevallier, 1978; Jacob etal. 1978) and other experimental approaches (Chevallier etal. 1911,1978; Chevallier, 1979; Christ etal. 1977,1979; McLachlan & Hornbruch, 1979; Mauger etal. 1980) have corroborated this conclusion. All other mesenchymal limb tissues, including the connective tissues of the skeletal muscles, develop from somatopleurally-derived cells. Taken as a whole, these studies show that there are at least two distinct populations of cells within the undifferentiated limb mesenchyme - one that gives rise to skeletal muscle and one that forms the rest of the mesenchymally-derived limb tissues. While all evidence indicates that the somitically-derived cells will give rise only to skeletal myofibres, the developmental fates of the somatopleurallyderived cells are not so clearly established. When the brachial somites were extirpated and replaced by a piece of 9-day gut tissue or destroyed by X-irradiation (Chevallier etal. 1977, 1978; Chevallier, 1979), 100% and 3 3 % , respectively, of the experimental wings contained muscle. Replacement of chick brachial somites with those of quail resulted in the presence of quail nuclei in all skeletal myofibres of the wing on the operated side (Christ et al. 1977; Chevallier et al. 1977, 1978). However, when quail brachial somites were replaced by chick somites, chick nuclei were found only in skeletal myofibres but some myofibres were of quail origin and some of both chick and quail origin. These various experimental results suggest that somatopleurally-derived limb mesenchyme may also have skeletal myogenic potential, at least under the experimental conditions that prevailed in these studies. More direct tests of the developmental potentials of somatopleurally-derived limb cells have been made by isolating wing regions and grafting them to the coelomic cavities of chick hosts (Christ etal. 1979; McLachlan & Hornbruch, 1979; Mauger et al. 1980) or into other embryonic sites (Mauger et al. 1980), but these experiments, too, gave contradictory results. Christ et al. (1979) maintained that the somatopleural cells did not have the ability to form skeletal muscle while both McLachlan & Hornbruch (1979) and Mauger et al. (1980) concluded that the somatopleural cells were developmentally labile and capable of expressing a myogenic phenotype under certain experimental conditions. Before one can ascribe a myogenic potential to the somatopleurally-derived cells of the limb mesenchyme, it is essential that inclusion or postoperative entry of somitic cells into the wing territory be obviated. Removal of the brachial somites or their destruction by X-irradiation are not infallible methods for ensuring complete removal or destruction of all somitic cells (Mauger, 1970; Kieny et al. 1972; Chevallier etal. 1978). Furthermore, it is important that the sites in which the isolated wing tissues are grown are not accessible to somitic cell invasion. For many of the intraembryonic graft sites (e.g., the coelom) this cannot be guaranteed, as shown by the results of Mauger et al. (1980). The experiments described in this paper attempt to resolve the question of the possible myogenic potential of the wing mesenchyme of somatopleural origin by isolating wing territories at early stages and growing them either in organ culture Myogenic potential of early wing mesenchyme All or grafted to the chorioallantoic membrane of host chicks, conditions under which postoperative entry of somitic cells into the wing region is impossible. The results show a stage-related dependence of limb myogenesis on the presence of the brachial somites and also show that somatopleurally-derived limb mesenchymal cells do not have skeletal myogenic potential - thai: the somitic cells that migrate into the limb regions are the sole source of limb myofibres. Some of these results were presented at the Third International Conference on Limb Development and Regeneration (Kenny-Mobbs & Hall, 1983). MATERIALS AND METHODS Fertile eggs of the White Leghorn variety of Gallus domesticus were obtained from a local hatchery. The eggs were incubated to the required stage of development at 37°C and a relative humidity of 50-60 %. Embryonic development was staged according to Hamburger & Hamilton (1951) using the number of somites as the main criterion. Tissues were dissected from embryos of four specific stages of development; HH stages 12,15, 18 and 21. Wing primordia from these embryos should have contained different numbers of somitic cells. Migration into wing primordia had not yet begun in the HH stage-12 embryos; HH stage-15 wing primordia should have contained a few somitic cells since migration would have been under way for a few hours before isolation; HH stage-18 wing buds should have contained nearly a full complement of somitic cells since migration was nearing completion (Christ et al. 1977; Chevallier, 1978) and, in HH stage-21 wing buds, the onset of histological differentiation was imminent. (i) Dissections Embryos were removed from the yolk with the aid of filter paper discs and transferred to saline. Wing regions were then dissected free of surrounding tissues as illustrated in Fig. 1A. For the two younger stages this resulted in a rectangular piece of tissue that included the three germ layers of the lateral plate opposite somites 12/13 to'20/21 (Fig. IB) and sometimes included portions of the intermediate mesoderm. In the older embryos only the wing buds were removed (lateral plate somatopleure only), although occasionally some intermediate mesoderm was included (Fig. 1C). Wing territories that included the somitic and axial tissues were also dissected from embryos of each of the four stages. In these cases, the embryo was cut up the midline of the neural tube (Fig. 1A) so that each dissected piece contained the wing territory and the tissues medial to it, viz., the intermediate mesoderm, half of the neural tube and portions of the notochord (Fig. ID). The covering epithelial layers were also present. In a second series of experiments, two areas of lateral plate tissue were dissected from HH stage-12 embryos (Fig. 2A). The first of these (diagonally-crossed in Fig. 2A) was wing lateral plate and included all three germ layers adjacent to somites 14/15 to presumptive somites 20/21. Care was taken to ensure that the medial cut of this block of tissue was such that intermediate mesoderm was excluded from the dissected tissues. The second piece of tissue (stippled in Fig. 2A) included the lateral plate tissue (all three layers) adjacent to somites 10/11 to somites 13/14. In both cases the distal boundary of the dissected tissues was at the margin of the areas pellucida and opaca. At both levels some dissections were made that included medial tissues - the intermediate mesoderm, somitic mesoderm and axial tissues. Lateral plate tissues of the wing region and that posterior to the wing level were dissected from embryos of HH stage-15 embryos. In HH stage-12 embryos the posterior boundary of the wing territory can be established by estimating where the remaining brachial somites will segment; however, it is difficult to determine the exact location of more posterior tissues, in this 418 T. KENNY-MOBBS 1A Myogenic potential of early wing mesenchyme 419 case the flank region of the embryo (at the level of somites 21-24). Consequently, the ability of lateral plate tissue at this level to form skeletal muscle was assessed in slightly older embryos. The dissected area is shown stippled in Fig. 2B. The anterior cut was made at the level of somites 21/22 and the posterior at the level of somites 24/25. Again some tissues were dissected to include the more medial tissues. (ii) Organ cultures Each wing territory was mounted on a small square of Millipore filter and placed, filter side down, on wire mesh supports in 35 mm plastic dishes (Falcon Type 3001). Three tofivepieces of tissue were placed in each dish and 1-5 ml of culture medium was added. The culture medium consisted of BGJb with 15 % horse serum and 5 % embryo extract. All components of the medium were purchased from GIBCO. The cultures were maintained at 37 °C in an atmosphere of 5 % carbon dioxide in air. The medium was changed every other day over the culture period which varied from 4 to 9 days. At the end of the culture period the explants were rinsed in saline and fixed as described below. Fig. 2. A schematic diagram of (A) an HH stage-12 embryo and (B) an HH stage-15 embryo illustrating the lateral plate tissues dissected from the level of the wing (wlp), the region anterior to the wing (alp) and that posterior to the wing (pip). Somite 15 is shaded. Bar = 1 mm. Fig. 1. (A) A schematic diagram of an HH stage-12 embryo illustrating the boundaries of areas dissected when (a) the wing territory was isolated with the adjacent somitic and axial tissues and (b) only wing region lateral plate was isolated. The 15th somite pair (cross-hatched) marks the beginning of the wing (or brachial) region of the embryo. Bar = lmm. (B-D) Paraffin sections of the isolated tissues before explantation. d, distal; p, proximal. Bars = 50/an. (B) Lateral plate tissue of the brachial region of an HH stage-12 embryo. Both somato- and splanchnopleural layers of the lateral plate were included in the dissected wing regions from HH stage-12 and -15 embryos. (C) Isolated wing bud of an HH stage-18 embryo. Only the wing buds (lateral plate somatopleure) were isolated from HH stage-18 and -21 embryos. (D) Wing region of an HH stage-15 embryo that was dissected with the adjacent somitic and axial tissues. /, intermediate mesoderm; nt, neural tube; ,?, somitic mesoderm; wlp, wing region lateral plate. 420 T. KENNY-MOBBS (iii) Chorioallantoic grafts Host embryos for chorioallantoic (CAM) grafting were incubated for 8 to 10 days. The host eggs were windowed using a fine-toothed hacksaw blade and were then returned to the incubator until required for grafting. Tissues to be CAM-grafted were dissected and set up for organ culture as described in the previous sections. They were held in the CO2 incubator until grafted. The tissue to be grafted was positioned on the CAM at a bifurcation of the blood vessels, with either the filter or the tissues abutting the CAM. The window was replaced and sealed in position with Scotch tape. The hosts were then returned to the incubator for a further 4 to 7 days. (iv) Histological techniques All cultures, grafts and normal tissues prepared for light-microscopic examination were fixed in one of three fixatives; neutral buffered formalin (Drury & Wallington, 1967), half-strength Karnovsky's (Karnovsky, 1965) or phosphate-buffered 4 % formaldehyde, 1 % glutaraldehyde (McDowell & Trump, 1976). Fixed tissues were processed for paraffin embedding through an ascending series of alcohol and xylene and were infiltrated and embedded with Paraplast Plus (m.p. 56-57°C; Canlab.). 6 jum serial sections were prepared and stained by one of three staining procedures; Mallory's triple stain (modification of Pantin, 1960), haematoxylin/alcian blue/chlorantine fast red (modified from Lison, 1954), or haematoxylin/eosin/alcian blue, and were mounted in DPX synthetic mountant (BDH Chemicals). Photomicrographs were taken on Kodak Panatomic X film (ASA 32) with a Zeiss Photomicroscope II. (v) Indirect immunofluorescence Antibodies to skeletal muscle myosin and actin were generously supplied by Dr B. G. Atkinson, Department of Zoology, The University of Western Ontario. The antibodies were raised in rabbits to skeletal muscle proteins isolated from frog hindlimb muscles and have been found to cross-react with their antigens in various species, including chick (Atkinson, pers. comm.). The procedures for the isolation and purification of these proteins and for the raising and purification of the antibodies have been described by Dhanarajan (1979) and Dhanarajan & Atkinson (1980). Grafts or cultures of HH stage-12 wing regions were rinsed in saline and immediately frozen in O.C.T. Compound (Tissue-Tek II, Canlab). 10/zm sections were cut and serially mounted on precleaned microscope slides. The sections were air-dried for 5-15 min and then fixed for 30 min in 3 % paraformaldehyde in phosphate-buffered saline (PBS). They were then rinsed in PBS and permeabilized in absolute acetone at —20°C. Sections were exposed to the skeletal muscle antibodies, diluted 1:1 with PBS, for 1 h at 37°C, rinsed in PBS, and exposed to goat anti-rabbit globulin conjugated tofluoresceinisothiocyanate (FITC-GaR-IgG; GIBCO) for 30min at 37°C. After several rinses in PBS and distilled water, the sections were mounted in 6 % polyvinyl alcohol. As controls the skeletal muscle antibody was replaced by serum from pre-immune rabbits, also diluted 1:1 with PBS, or the first layer was omitted altogether and the sections exposed only to the second layer - the FITC-GaR-IgG. The sections were examined with a Zeiss Universal fluorescence microscope, equipped for epifluorescence, using an HBO 200 W/4 light source, exciter filter BG12/4, barrier filters 44 and 50 and Neofluar objectives. Photographs were taken on Kodak Tri-X film. RESULTS (i) Explants of wing territories and adjacent somitic and axial tissues In the first series of experiments, wing territories, along with the somites and portions of the neural tube and notochord of the brachial region (e.g., Fig. ID) Myogenic potential of early wing mesenchyme All were isolated from HH stage-12 to -21 embryos. 40 such explants were cultured and 42 were grafted onto the CAM. Little stage-related difference was found in the growth and differentiation of explants grown in culture or on the CAM. Generally though, growth was considerably greater on the CAM than in culture and some grafts from all four stages exhibited some evidence of a wing morphology (Fig. 3A) while none of the cultures did so. Feather germs, kidney tubules and gut were common features of both cultures and grafts, with the exception of HH stage-21 explants in which no gut tissue differentiated. Neural tissue was present in all and notochord in most of the explants. In the CAM grafts, in which vertebral cartilages underwent considerable morphogenesis, ganglia were observed on the neural side of these cartilages and nerve bundles and blood vessels were seen to extend into the surrounding tissues through gaps in the vertebral cartilage (Fig. 4A). Cartilage differentiated in all cultures and grafts (Fig. 3). At least three distinct cartilage elements were observed in all cultures and 65-75 % contained four or more elements. Cartilage morphogenesis was greater in the CAM grafts and, when the grafts formed identifiable wings, elements could be identified as intrinsic limb cartilages, including autopodial elements. Membranous bone of the clavicle was found in 23 % of HH stage-18 grafts (Fig. 4B). Discrete muscle bundles with readily identified myotubes were present in both vertebral and limb regions of the explants (Fig. 4A,C). In some sections, especially of more distal wing regions, early stages of tendon formation could be seen (Fig. 4C). From these results it is clear that explants of wing primordia from all four stages, when grown in the presence of the brachial somites and portions of the neural tube and notochord, exhibited a similar pattern of growth and differentiation of the wing region. The incidence of a recognizable wing morphology increased with increasing age of the explant but tissue differentiation was the same. (ii) Explants of isolated wing territories (a) In this series of experiments, wing primordia were dissected free of adjacent tissues and grown in culture or on the CAM. Growth on the CAM was more substantial than in culture and, like explants that included the axial tissues, grafts with a wing-like morphology were more commonly obtained from the older explants (Fig. 5). Differentiation in isolated wing primordia differed from the explants described above in that there was a decrease in the number of cartilage elements that formed and in the incidence of kidney differentiation for all four stages (Tables 1 and 2). Grafts of HH stage-18 and -21 wing buds contained more cartilage elements than cultures of the same age. Differences in the presence of skeletal muscle in these explants were found to be stage-related; while all explants of HH stage-18 and -21 wing buds contained welldeveloped skeletal muscle bundles (Tables 1 and 2; Figs 5,6), HH stage-12 and -15 explants showed a marked decrease in the incidence of skeletal muscle formation, although a few explants from both stages contained one or two muscle bundles (Tables 1 and 2; Fig. 7). Most of the HH stage-12 and -15 explants contained 422 T. KENNY-MOBBS Myogenic potential of early wing mesenchyme 423 Table 1. Differentiation in explants of HH stage-12 to -21 wing primordia grown in culture for 4 to 9 days* Cartilage elements Stage 1-2 3-4 HH12 HH15 HH18 HH21 69% 47% 0% 18% 31% 53% 62% 77% Muscle Condensations Myotubes 0% 0% 38% 5% 75% 65% 25% 35% 100% 100% Gut 94% 82% 15% 0% Kidney n 94 % 59 % 23 % 13% 16 17 13 44 * Differentiation expressed as a percentage of samples (n) containing the tissue. Table 2. Differentiation in grafts of HH stage-12 to- 21 wing primordia grown on the CAM for 4 to 7 days* Cartilage elements Stage 1-2 3-4 HH12 HH15 HH18 HH21 73% 73% 0% 0% 27% 27% 19% 12% Muscle Condensations Myotubes 0% 0% 81% 88% 72% 82% 15% 18% 100% 100% Gut 91% 91% 52% 0% Kidney n 48 % 55 % 57 % 12% 33 11 21 16 * Differentiation expressed as a percentage of samples (n) containing the tissue. condensations in 'myogenic' areas (Tables 1 and 2; Fig. 8A,B), but no myotubes could be found within these condensations. The low incidence of skeletal myogenesis in the explants from HH stage-12 and -15 embryos prompted a careful examination of the differentiation of other mesodermally-derived tissues in these explants. It was found that differentiation of kidney tubules occurred in 94 % of cultured and 48 % of CAM-grafted explants from HH stage-12 embryos (see Tables 1 and 2). Myotubes were present in four of the cultures and in five of the grafts and all nine of these explants contained kidney tubules. This coincidence of kidney tubule differentiation with the presence of Fig. 3. Survey sections of CAM-grafts of wing regions explanted with the adjacent somitic and axial tissues. (A) Wing region of a graft from an HH stage-12 embryo grown on the CAM for 6 days showing a wing-like morphology and the presence of cartilage (c) and skeletal muscle (m) bundles. Bar = 200jum. (B) An amorphouslyshaped graft of an HH stage-18 wing bud grown on the CAM for 6 days. Differentiation of axial and somitic tissues (top) and lateral plate tissues (bottom), including cartilage (c) and muscle (m) can be seen, nt, neural tube; k, kidney. Bar = 300/an. Fig. 4. Paraffin sections of wing regions CAM-grafted with adjacent somitic and axial tissues. Bars = 100 jum. (A) The axial region of an HH stage-15 graft grown for 6 days on the CAM showing the differentiation of vertebral cartilage (c) and muscle (m) and spinal ganglia (g). (B) A graft from an HH stage-18 embryo, grown for 6 days on the CAM, in which the clavicle (b) had formed, c, cartilage. (C) A graft from an HH stage12 embryo left on the CAM for 10 days showing tendon formation (arrow), c, cartilage; m, muscle. 424 T. KENNY-MOBBS Myogenic potential of early wing mesenchyme 425 Fig. 8. Paraffin sections through an HH stage-12 isolated wing region grown on the CAM for 5-5 days. (A) Cartilage (c) and feather germs (f) are seen and, in the loose connective tissue between these two, there is an area of condensation. Bar = 100 /mi. (B) A higher magnification of the condensed area. No myotubes could be identified in this region. The asterisk (*) marks the area indicated at the top of the arrow in Fig. 8A. Bar = 25 skeletal muscle raised the possibility that somitic cells had been inadvertently included in the initial dissections. Such 'contaminating' somitic cells might have been included i) by making the medial cut of the dissection of the wing regions too close to the intermediate mesoderm and thereby accidentally including some somitic cells which lie medial to the intermediate mesoderm, or ii) by including lateral plate tissue anterior and/or posterior to the wing territories in the initial dissections. Such tissues already may have contained cells capable of myogenic Fig. 5. A survey section of an isolated HH stage-18 wing bud that had been grown on the CAM for 6 days. Note the wing-like morphology and the differentiation of cartilage elements (c), muscle (m) and bone (b). Bar = 1000ym. Fig. 6. A section through an HH stage-18 isolated wing bud grown in culture for 6 days, c, cartilage; m, muscle. Bar = 200 jum. Fig. 7. A survey section of an HH stage-12 isolated wing region grown on the CAM for 6-5 days. Cartilage elements (c), kidney tubules (k) and one small muscle bundle (m) formed in this graft. Bar = 200 urn. 426 T. KENNY-MOBBS Table 3. Differentiation in CAM grafts of HH stage-12 and -15 wing territories explantedfor 6 days* Stage 12 15 Cartilage elements 3-4 5 1-2 41% 23% 30% 27 % 30% 50 % Muscle Condensations Myotubes 96% 82% 0% 9% Gut Kidney n 85% 95% 4% 5% 27 22 'Differentiation expressed as a percentage of samples (n) containing the tissue. expression. Two series of experiments were carried out to check these possibilities. (b) Lateral plate tissue of the wing territory was dissected from HH stage-12 and -15 embryos (Fig. 2) and grafted to the CAM. These explants differed from those described in section (a) above in that the region dissected was restricted to the lateral plate tissue opposite somites 15-20 and the medial cut for the dissection avoided not only the somites but also the intermediate mesoderm. Tissue differentiation in these explants was not appreciably different from that observed in the HH stage-12 and -15 CAM grafts described before (cf. Tables 2 and 3) with the notable exceptions of a marked decrease in the incidence of kidney tubule formation and a reduction in the incidence of skeletal muscle differentiation in both HH stage-15 explants (9 % compared with 18 % in the previous dissections) and HH stage-12 grafts, in which no myotubes could be found (Table 3; Figs 9,10). Areas of cellular condensation in the loose connective tissues adjacent to cartilage elements were common in grafts from both stages (Figs 9, 10). No myotubes could be found within these condensations in HH stage-12 grafts but because they were such a common feature of the areas in which muscle would normally form, they were investigated further with antibodies to skeletal muscle myosin and actin using indirect immunofluorescence. Eleven explants of HH stage-12 wing regions (seven grafts and four cultures) and five explants of wing regions with the adjacent axial and somitic tissues (three grafts and two cultures) were studied using these antibodies, the latter serving as positive controls. All grafts and cultures that included the somitic and axial tissues in the initial dissection were strongly positive for the presence of skeletal muscle myosin (Fig. 11) and gave a fainter, though positive, reaction with skeletal muscle actin antibodies. In the absence of somitic tissue, all cultures and grafts gave a negative reaction for both skeletal muscle myosin (Fig. 12A) and actin (Fig. 12B). Smooth Figs 9, 10. Sections through CAM grafts showing tissue differentiation in explants of wing lateral plate from HH stage-12 (Fig. 9) and HH stage-15 (Fig. 10) embryos. Cartilage (c), feather germs (/), loose connective tissue and areas of condensation (arrows) were common features of these grafts. Within the condensations (Figs 9B, 10B), cells were more densely packed but no myotubes could be identified. Bars = 100(im (A); bars = 50/an (B). Myogenic potential of early wing mesenchyme \v 427 428 T. KENNY-MOBBS muscle cells of the gut wall did not react with either antibody. The controls with pre-immune serum (Fig. 13A) and the application of only the second layer (FITCGaR-IgG) (Fig. 13B) were negative. (iii) Grafts of lateral plate tissue anterior and posterior to the wing regions In the final series of experiments in this study, lateral plate tissues anterior to the wing region (in HH stage-12 embryos) and posterior to the wing region (in HH stage-15 embryos) were analysed for their ability to form skeletal muscle when isolated from the adjacent somites and grafted to the CAM. In the majority of anterior lateral plate grafts (aLP) one or two cartilage elements formed (Table 4, Fig. 14). Membranous bone of the clavicle, and gut, liver, and kidney tissues also differentiated in many of the grafts. However, none of the grafts of aLP contained myotubes or condensations in myogenic areas. Grafts of posterior lateral plate (pLP) formed large mounds of tissue consisting mainly of a vascularized loose connective tissue in which one S-shaped cartilage element was frequently seen (Table 4; Fig. 15A). Feather germs and gut were the only other tissues identified in these grafts. In two samples an area of condensation adjacent to the cartilage element was seen (Fig. 15B) but no myotubes were found. In grafts of aLP and pLP in which the axial and somitic tissues were included in the initial dissection, neural tissue, notochord, vertebral cartilage and kidney tubules differentiated and all explants contained well-developed skeletal muscle bundles in both vertebral and lateral plate regions of the grafts (Fig. 16). DISCUSSION The results of this study demonstrate a stage-related dependence of limb myogenesis on the presence of somites in the brachial region consistent with the reported stages of somitic cell migration into wing primordia (Chevallier, 1978; Christ et al. 1977). They not only support a somitic origin for wing skeletal muscle but also show that these somitic cells are the sole source of such muscle for, in their absence, cells of somatopleural origin form all mesodermally-derived cell types of the wing except skeletal myofibres. There are two critical requirements for an ectopic site in these limb-somite studies, namely, i) that the site supports the growth and differentiation of the explanted limbs and ii) that it is isolated from the somitic tissues. Another obvious criterion is that no somitic cells are included in the initially dissected tissues. What Figs 11-13. Immunofluorescence in frozen sections of HH stage-12 CAM grafts of wing regions explanted with (controls) and without adjacent somitic tissues. All negatives were given equal exposure on printing. Bars = 200 /im. Fig. 11. Positive reaction to skeletal muscle myosin antibodies in a control graft. Fig. 12. In the absence of somitic tissue, explants of wing regions gave negative reactions to antibodies both to skeletal muscle myosin (12A) and actin (12B). Fig. 13. Controls in which explants containing somitic tissues were treated with preimmune serum (13A) instead of antibody or treated only with FITC-GaR-IgG (13B). c, cartilage; m, muscle. 429 Myogenic potential of early wing mesenchyme <7 .1'" 12A —- 12 ft <* 13 : 430 T. KENNY-MOBBS 5»£ Myogenic potential of early wing mesenchyme 431 Table 4. Differentiation in grafts ofHHstage-12 lateral plate opposite somites 10/11 to 13/14 (aLP) and HH stage-15 lateral plate opposite somites 21-25 (pLP) grown on the CAM for 6 and 5 days, respectively Stage Cartilage elements 1-2 3 12 (aLP) 15 (pLP) 73% 92% 7% 0% Clavicle Muscle Gut Liver Kidney n 40% 0% 0% 0% 87% 85% 73% 0% 27% 0% 15 13 is not so obvious is the special care that must be taken to ensure that this does not occur. Each of these points will now be considered in detail. (i) Differentiation in explanted wing regions None of the ectopic sites used in the different limb-somite studies appears to be appreciably better than any other in permitting normal myogenesis to occur. McLachlan & Hornbruch suggested that the CAM and the coelom may not be equivalent 'in their ability to evoke differentiation of cell types' (p. 216, McLachlan & Hornbruch, 1979), implying that the CAM, while fostering cartilage differentiation, might not be conducive for muscle differentiation. This is clearly not the case as large, healthy muscle bundles were found in CAM grafts of wing buds isolated from older embryos (HH 18 and 21), as well as in wing regions from all four stages when adjacent axial and somitic tissues were included. Differentiation of skeletal muscle in explanted limbs might also be affected by the lack of innervation of the explants since skeletal muscle eventually degenerates without innervation, although initial differentiation of skeletal muscle is not nervedependent (e.g., Hunt, 1932; Eastlick, 1943). It has been suggested that Christ et al. observed no skeletal muscle in their coelomic grafts because it had degenerated (McLachlan & Hornbruch, 1979; Mauger et al. 1980). The lack of skeletal muscle in the explants of the present study cannot be attributed to degenerative breakdown; although the explants were not innervated, there was no evidence of degeneration over the periods of explantation used. (ii) Exclusion of somitic cells from explanted wing regions As already stressed in the Introduction, to assign a myogenic capacity to wing somatopleural cells, one must be sure that there are no somitic cells present in the experimental limbs as they develop. An alternative explanation for the presence of Figs 14-16. Paraffin sections of 5- to 6-day-old CAM grafts of lateral plate tissue anterior (aLP) and posterior (pLP) to the wing region. Fig. 14. An HH stage-12 aLP explant showing cartilage (c), bone (b) and a feather germ(/). Bar = 200 ^m. Fig. 15. Grafts of HH stage-15 pLP in which (A) an S-shaped cartilage element (c) and loose connective tissue formed. Bar = 200jum. (B) A condensation (*) in which myotubes could not be identified, c, cartilage. Bar = 200 ^m. Fig. 16. An explant of HH stage-12 aLP that included axial and somitic tissues. Neural tissue (nt), cartilage (c) and muscle (m) differentiated in these grafts. Bar =100 jan. 432 T. KENNY-MOBBS skeletal muscle in the explants of earlier studies and the first series of experiments in this study lies in the assumption that somitic cells were present in the experimental limbs. This could have happened if a) the explanted wing regions were .accessible to somitic cell invasion after grafting or b) somitic cells were included inadvertently in the initially dissected tissues. (a) Access to postoperative invasion by somitic cells The first of these possibilities has no bearing on the results of the present study since explants grown in culture or on the CAM of host chicks are completely isolated from somitic regions. However, it does have a bearing on past studies for all explants to different ectopic sites within host chicks were made to regions in and around the limb territories and in hosts in which somitic cell migration was under way, i.e., between HH stages 13 and 17 (Christ et al. 1979; McLachlan & Hornbruch, 1979; Mauger et al. 1980). Several observations suggest that these graft sites are less than ideal for studies on limb-somite relationships because they do not ensure isolation of the grafted primordium from host somitic cells. In order to survive, grafts must attach to the host tissue and become vascularized (Hamburger, 1938; Rudnick, 1945). The location and extent of this attachment varies but in many cases it forms in the somatopleural layer and sometimes very close to the lateral edges of the somites (e.g., fig. 1, Dhouailly & Kieny, 1972). Such continuity clearly makes the graft susceptible to invasion by host cells. That such an invasion occurs has been shown in grafts to the flank (Dhouailly & Kieny, 1970, 1972; Mauger et al. 1980) and in grafts in place of the neural tube (Mauger etal. 1980). Dhouailly & Kieny (1970,1972) assumed that flank somatopleural cells had invaded the grafts; however, evidence suggests that somitic cells also contributed to the ectopic limbs. First, the distribution of host cells in the ectopic limbs (fig. 1, Dhouailly & Kieny, 1970) was exactly that seen for somitic cells in limbs formed after chick somites had been replaced by those of quail (e.g., Chevallier, 1978). Second, somitic cells from all cephalocaudal levels tested were found to be capable of entering limb territories and giving rise to normal limb musculature when placed adjacent to limb regions (Chevallier et al. 1977; Christ et al. 1978). Third, limb territories seem to elicit the migration of adjacent somitic cells (Mauger & Kieny, 1980). Fourth, myotubes of host origin were found in ectopic limbs after grafting to the flank and in place of the neural tube (Mauger et al. 1980). Considering these observations, and the fact that somitic cell migration is under way in the hosts, it must be assumed that host somitic cells will invade limb primordia grafted to these sites. McLachlan & Hornbruch (1979) did not describe the distribution of host cells in their grafts of quail wing primordia to the chick coelom but showed host cells in muscle bundles in the recovered grafts. Likewise, Christ etal. (1979) made no mention of the presence of host cells in their coelomic grafts but later stated that such invasion probably occurs (Christ & Jacob, 1980). Grafts between the folds of the neural tube, the third site used by Mauger et al. (1980), can be considered impervious to somitic cell invasion and such grafts were found to be muscleless when recovered. Myogenic potential of early wing mesenchyme 433 There is an important difference in the grafting procedure used by these three groups of workers that should be noted. Both McLachlan & Hornbruch (1979) and Christ et al. (1979) grafted intact limb primordia. On the other hand, Mauger et al. (1980) stripped the ectoderm from the primordia before grafting them. In the latter grafts the limb somatopleure induced the formation of an ectopic limb that incorporated both host and graft mesodermal cells and was covered by host ectoderm (Dhouailly & Kieny, 1970, 1972). These explants may have been more susceptible than the intact primordia to invasion by host somitic cells since the absence of the graft ectoderm may have eliminated any physical barrier to such invasion. In itself, the presence of host somitic cells does nor negate the fact that graft cells formed myotubes in wing primordia explanted before somitic migration had begun in the donor (McLachlan & Hornbruch, 1979; Mauger et al. 1980) but the findings of Mauger et al. (1980) suggest that this potential can only be expressed in the presence of somitic myogenic cells since no graft-only myotubes were found. The majority of myotubes (>69%) were of host origin only; 26% of the myotubes contained graft-originated nuclei, but always in association with host nuclei (Mauger etal. 1980, Table 1). Again, however, the critical point is the origin of the graft cells that form muscle. These observations do not support the suggestion of McLachlan & Hornbruch (1979) that the presence of somitic myogenic cells in normal limbs suppresses skeletal myogenesis in limb somatopleural cells. (b) Inclusion of somitic cells in dissected wing regions The presence of muscle in the first series of HH stage-12 grafts in this study was not immediately interpreted as evidence that somatopleurally-derived cells have myogenic potential because both the amount and incidence were so low, especially considering that the grafts had otherwise differentiated normally (compared with controls). In attempting to explain these results, one was led to the conclusion that the routine dissection procedure for the isolation of wing primordia used by workers studying limb-somite relationships may not be sufficient to ensure complete exclusion of somitic cells. There are two possible ways that somitic cells might be included in the isolated wing regions; by including adjacent lateral plate tissue that had myogenic potential (already invaded by somitic cells) or by including parts of the somites themselves. In the first case one must consider the timing of the onset of somitic migration into lateral plate tissues. Studies by Chevallier (1978), Christ et al (1977) and Mauger et al. (1980) show that it is unlikely that migration into wing lateral plate begins before all brachial somites have segmented, so wing regions from embryos with 14 to 19 somite pairs (HH stages 11 to 13") can be considered devoid of somitic cells. However, migration of somitic cells into the lateral plate occurs in a cephalocaudal sequence, for example, reaching the level of somite 12 at HH stage 12 (Mauger et al. 1980), somite 19 at HH stage 14 (Pinot, 1969) and somite 26 by HH stage 16 (Jacob et al. 1979). Given this sequential initiation of somitic cell migration it seemed possible that lateral plate tissue anterior or, less likely, 434 T. KENNY-MOBBS posterior to the wing region might already contain myogenic cells. In the first experiments of the present study portions of this adjacent tissue were included in the dissected pieces to ensure that the wing primordium was dissected intact. The results from two different types of grafts showed that inclusion of this adjacent tissue does not account for the differentiation of skeletal muscle in the initial HH stage-12 cultures and grafts. CAM grafts of HH stage-12 lateral plate anterior to the wing (aLP) and that posterior to the wing (pLP) from HH stage-15 embryos showed that these regions do not have myogenic potential at these stages. Obviously, in limb-somite studies one takes special care not to include somitic tissue in the dissected wing regions, but the results of the present study suggest that it is necessary to exclude tissue even more lateral to the somites, namely, the intermediate mesoderm, to be absolutely sure of not including some somitic cells. In the first series of experiments, the nine explants that contained skeletal muscle also contained kidney tubules (e.g., Fig. 7). However, in the second series, in which not only the somitic tissue but also the intermediate mesoderm was excluded from the dissected pieces, kidney differentiated in one of 27 explants and none contained skeletal muscle. Whether accidental inclusion could be the source of somitic cells in the grafts of McLachlan & Hornbruch (1979) and Mauger et al. (1980) cannot be determined from their papers; however, both groups of workers explanted wing primordia as young as HH stages 10/11 and Pinot (1969) has shown that the boundaries between the somitic, intermediate and lateral plate mesoderms at the wing level cannot be distinguished until HH stage 12. Without some indication of these boundaries, there is a strong possibility that some somitic cells were included in the dissected primordia. Since the differentiation of other tissue types in the experimental wings was not described in these papers, a coincidence of kidney tubule differentiation with the presence of skeletal muscle would not have been noticed and the possibility of inclusion of somitic cells in the explant not recognized. To summarize, of the four possible explanations for the presence of somitic cells in experimental wings that contained skeletal muscle, namely, postoperative entry, early migration, derivation from adjacent lateral plate and inadvertent inclusion during dissection of the explants, the last seems the most likely for the explants in the first series of experiments in this study and, albeit on circumstantial evidence, for the explants of McLachlan & Hornbruch (1979) and Mauger et al. (1980). In the experiments involving the destruction or replacement of brachial somites (e.g., Chevallier et al. 1911,1978), a combination of incomplete removal of host somitic cells, regulation, and developmental asynchrony between chick and quail cells could reasonably account for the presence of somitic cells and, hence, skeletal muscle in the experimental wings. In conclusion, the results of this study argue against the possibility that limb cells of somatopleural origin have skeletal myogenic potential. In all cases in which skeletal myotubes were found, there was evidence that somitic cells entered or were included in the experimental wings. When these possibilities were excluded, Myogenic potential of early wing mesenchyme 435 as in the second series of HH stage-12 grafts in this study, no skeletal muscle differentiated in the explants. Therefore, limb cells of somatopleural origin have the potential to form all mesodermal derivatives of the limb except the skeletal myofibres, which differentiate from somitic cells that invade the limb as it begins its outgrowth from the body wall. The author gratefully acknowledges scholarship support from the Natural Sciences and Engineering Research Council (NSERC) of Canada and from the I.W. Killam Memorial Fund at Dalhousie University. The work was done in the laboratory of Dr Brian Hall and was funded by an NSERC grant to Dr Hall. My thanks to Drs Brian Hall, Ian Mobbs and Peter Thorogood for comments on the manuscript. REFERENCES A. (1978). Etude de la migration des cellules somitiques dans le m6soderme somatopleural de l'6bauche de l'aile. Wilhelm Roux' Arch. devlBiol. 184, 57-73. CHEVALLIER, A. (1979). Role of the somitic mesoderm in the development of the thorax in bird embryos. II. Origin of the thoracic and appendicular musculature. /. Embryol. exp. Morph. 49, 73-88. CHEVALLIER, A., KIENY,M. &MAUGER, A. (1976). Surl'originede la musculature de l'aile chezles oiseaux. C. r. hebd. Sianc. Acad. ScL, Paris, D. 282, 309-311. CHEVALLIER, A., KIENY, M. & MAUGER, A. (1977). Limb-somite relationship: origin of the limb musculature. J. Embryol. exp. Morph. 41, 245-258. CHEVALLIER, A., KIENY, M. & MAUGER, A. (1978). Limb-somite relationship: effect of removal of somitic mesoderm on the wing musculature. /. Embryol. exp. Morph. 43, 263-278. CHRIST, B. & JACOB, H. J. (1980). Origin, distribution, and determination of chick limb mesenchymal cells. In Teratology of the Limbs (ed. H.-.J. Merker, H. Nau & D. Neubert), pp. 67-77. Berlin: Walter de Gruyter & Co. CHRIST, B., JACOB, H. J. & JACOB, M. (1974). Uber den Ursprung der Fliigelmuskalatur. Experimentelle Untersuchungen mit Wachtel- und Hiihnerembryonen. Experientia, 30, 1446-1449. CHRIST, B., JACOB, H. J. & JACOB, M. (1977). Experimental analysis of the origin of the wing musculature in avian embryos. Anat. Embryol. 150,171-186. CHRIST, B. JACOB, H. J. & JACOB, M. (1978). Zur Frage der regionalen Determination der friihembryonalen Muskelanlagen. Experimentelle Untersuchungen an Wachtel- und Hiihnerembryonen. Verh. Anat. Ges. 72, 353-357. CHRIST, B., JACOB, H. J. & JACOB, M. (1979). Differentiating abilities of avian somatopleural mesoderm. Experientia 35, 1376-1378. DHANARAJAN, Z. C. (1979). Thyroid hormone-induced muscle differentiation in Rana catesbeiana tadpole hind limbs. Ph.D. Thesis, The University of Western Ontario, pp. 24-34. DHANARAJAN, Z. C. & ATKINSON, B. G. (1980). M-line protein preparations from frog skeletal muscle: isolation and localization of an M-line protein and a 105,000 dalton polypeptide contaminant. Can. J. Biochem. 58, 516-526. DHOUAILLY, D. & KIENY, M. (1970). Participation du m6soderme somatopleural du flanc a l'6dification d'un membre suppl6mentaire chez les oiseaux. C. r. hebd. Seanc. Acad. ScL, Paris, D. 271, 519-522. DHOUAILLY, D. & KIENY, M. (1972). The capacity of the flank somatic mesoderm of early bird embryos to participate in limb development. Devi Biol. 28,162-175. DRURY, R. A. B. & WALLINGTON, E. A. (1967). Carleton'sHistologicalTechnique, 4thed. London: Oxford University Press. EASTLICK, H. L. (1943). Studies on transplanted embryonic limbs of the chick. I. The development of muscle in nerveless and in innervated grafts. /. exp. Zool. 93, 27-49. FISCHEL, A. (1895). Zur Entwicklung der ventralen Rumpf- und der Extremitatenmuskulatur der Vogel und Saugethiere. Morph. Jahrb. 23, 544-561. CHEVALLIER, 436 T. KENNY-MOBBS GRIM, M. (1970). Differentiation of myoblasts and the relationship between somites and the wing bud of the chick embryo. Z. Anat. Entwicklungsgesch. 132, 260-271. HAMBURGER, V. (1938). Morphogenetic and axial self-differentiation of transplanted limb primordia of 2-day chick embryos. /. exp. Zool. 77, 279-399. HAMBURGER, V. & HAMILTON, H. L. (1951). A series of normal stages in the development of the chick embryo. /. Morph. 88, 49-92. HUNT, E. A. (1932). The differentiation of chick limb buds in chorioallantoic grafts, with special reference to the muscles. J. exp. Zool. 62, 57-91. JACOB, M., CHRIST, B. & JACOB, H. J. (1978). On the migration of myogenic stem cells into the prospective wing region of chick embryos. A scanning and transmission electron microscope study. Anat. Embryol. 153, 179-193. JACOB, M., CHRIST, B. & JACOB, H. J. (1979). The migration of myogenic cells from the somites into the leg region of avian embryos. Anat. Embryol. 157, 291-309. KARNOVSKY, M. J. (1965). A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. /. Cell Biol. 27,137A-138A. KENNY-MOBBS, T. & HALL, B. K. (1983). Muscle differentiation in cultures and chorioallantoic grafts of pre-bud wing territories. In Limb Development and Regeneration, part B. Prog. Clin. Biol. Res. HOB, (ed. R. O. Kelley, P. F. Goetinck & J. A. MacCabe), pp. 323-333. New York: Alan R. Liss, Inc. KIENY, M. & CHEVALLIER, A. (1980). Existe-t-il une relation spatiale entre le niveau d'origine des cellules somitiques myogenes et leur localisation terminale dans l'aile? Archs Anat. microsc. Morph. exp. 69, 35-46. KIENY, M., MAUGER, A. & SENGEL, P. (1972). Early regionalization of the somitic mesoderm as studied by the development of the axial skeleton of the chick embryo. Devi Biol. 28,142-161. LISON, L. (1954). Alcian Blue 8 G with Chlorantine Fast Red 5 B. A technic for selective staining of mucopolysaccharides. Stain Technol. 29, 131-138. MAUGER, A. (1970). Le deVeloppement du plumage dorsal de l'embryon de Poulet 6tudi6 a l'aide d'irradiations aux rayons X. Devi Biol. 22, 412-432. MAUGER, A. & KIENY, M. (1980). Migratory and organogenetic capacities of muscle cells in bird embryos. Wilhelm Roux' Arch, devl Biol. 189, 123-134. MAUGER, A., KIENY, M. & CHEVALLIER, A. (1980). Limb-somite relationship: myogenic potentialities of somatopleural mesoderm. Archs Anat. microsc. Morph. exp. 69, 175-196. MCDOWELL, E. M. & TRUMP, B. F. (1976). Histologic fixatives suitable for diagnostic light and electron microscopy. Arch. Pathol. lab. Med. 100, 405-414. MCLACHLAN, J. C. & HORNBRUCH, A. (1979). Muscle-forming potential of the non-somitic cells of the early avian limb bud. /. Embryol. exp. Morph. 54, 209-217. PANTTN, C. F. A. (1960). Notes on Microscopical Technique for Zoologists. Cambridge: Cambridge University Press. PINOT, M. (1969). Culture in vitro de l'ebauche ou du territoire pr6somptif de l'aile de l'embryon de poulet: Diff6renciation du cartilage. Archs Anat. microsc. Morphol. exp. 58, 123-144. RUDNICK, D. (1945). Limb-forming potencies of the chick blastoderm: including notes on associated trunk structures. Conn. Acad. Arts Sci. Trans. 36, 353-377. (Accepted 9 July 1985)