Experimental studies have shown that the myogenic stem cells in birds migrate from the somite into the lateral plate mesoderm, where they later differentiate into muscle cells. Muscles being made up of myocytes and connective tissue cells, the interactions between these two types of cells of different embryological origins have been considered during the development of the musculature. In particular, our purpose was to focus on the genesis of the spatial organization of the musculature; we have taken advantage in this field of research of an embryological muscular dysgenesis in which the muscles lose their patterning.

Type I collagen is the major collagenous component of embryonic and early postnatal musculature. It is found in tendons, muscle envelopes around individual myofibers (endomysium), around muscle bundles (perimysium), and around muscle bulks (epimysium), as well as in loose connective tissue of intermuscular spaces and perimuscular tunica. Type III collagen deposition follows with some delay that of type I collagen in all mentioned locations. This type of collagen, however, is not a permanent component of tendons, from which it disappears during the second half of the incubation. Fibronectin, at first ubiquitously present around connective tissue cells and myogenic cells, becomes progressively restricted to the basal lamina zone, so as to delineate each myofiber. Laminin at first surrounds groups of muscle cells as long as the latter have not reached the stage of myofiber. At that time, laminin becomes restricted to the immediate surrounding of each myofiber.

The crooked neck dwarfism (cn/cn) is characterized, among other anomalies, by a muscular hypoplasia, particularly conspicuous in the tibiotarsal segment. Histological observations were performed between day 6 and day 12.5 of incubation. They show, in the tibiotarsal segment, that the hereditary muscular hypoplasia is not caused by a defect of the normal muscular splitting pattern. Indeed, in the mutant, the splitting of muscle masses proceeds normally up to the last partition (day 7-7.5), but is followed by the secondary fusion of individuated muscles into an unpatterned muscle tissue. Thus the mutant phenotype is the result of an inability of the muscle pattern to become stabilized into definitive structures.

Recent investigations on a hereditary muscular dysgenesis (cn/cn) in the chicken (Kieny, Mauger, Hedayat & Goetinck, 1983) have suggested that limb muscle pattern development and subsequent maintenance are two independent steps in the formation of the musculature. The respective activities or muscle cells and connective tissue cells in the ontogeny of the musculature have been investigated in avian embryos 1) by in ovo administration of drugs interfering with collagen biosynthesis, and 2) by heterogenetic somite-exchange experiments between normal and mutant embryos. None of the drugs administered to the chick embryo caused any disturbance of muscle pattern formation or maintenance whether treatment occurred before (5 days) or after (7.5 days) the muscle splitting period. Heterogenetic implantations were performed at 2 days of incubation either at the leg or at the wing level. Somitic mesoderm from non-mutant quail embryo was grafted to replace a piece of somitic mesoderm in putative mutant (cn/cn) chick embryos. The introduction of normal myogenic cells into a mutant leg or wing led to a normally patterned musculature, which demonstrates that the muscular dysgenesis cn/cn results from a defect of the somitic myogenic cell line.

Using chimeras consisting of chick embryos that had received substitution grafts of quail somites, we have determined the distalmost extension of the myogenic primordia in the outgrowing wing bud at 5 days of incubation. At Hamburger-Hamilton stage 25 the most distal premuscle cell is consistently 300 μm or more from the apex of the wing mesoblast. The stage 25 wing tip resembles very early whole limb buds in not having proceeded beyond the mesenchymal state or having expressed markers of terminal differentiation. However, unlike early whole limb buds it is free of a myogenic subpopulation. We therefore propose that the stage 25 wing tip is the appropriate system for in vitro and molecular studies of cartilage differentiation.

Since 1977, CHEVALLIER et al. had the idea that, although limb muscle cells are usually of somitic origin, somatopleural cells could give rise to myocytes under certain experimental conditions. New experiments were undertaken in order to investigate further the differentiating capacities of the limb somatopleural mesoderm. Two-day quail limb somatopleural mesoderm was ectopically implanted into two-day chick hosts (into the flank, in place of the neural tube or between the neural alar plates) and the specificity of the muscle cells in the resulting ectopic limbs was analyzed 7 to 10 days later. The grafts were grouped as a function of the stage of segmentation of the adjacent somitic mesoderm at the time of excision. Grafts, whose adjacent somitic mesoderm was still unsegmented (before stage 15 pairs of somites for the wing grafts and 26 pairs of somites for the leg grafts) or in process of segmentation (stages 15 to 20 pairs of somites for the wing grafts, only) were considered, after histological investigation, as devoid of somitically derived myogenic cells at the time of transplantation. In 6 out of 29 cases, such grafts expressed the ability to differentiate quail muscle cells. It can therefore be admitted that the limb somatopleural mesoderm contains a labile population of cells able to adjust its differentiative capacities to the environment.

The origin of smooth muscles in the skin of bird embryos has been analyzed in heterospecific quail/chick recombinants. The somitic mesoderm of the wing level of 2-day chick embryos was replaced by homotopic or heterotopic somitic mesoderm from quail embryos. The cellular constitution of tissues was observed in twelve recombinant embryos at 17 or 18 days of incubation. Results show that feather smooth muscles and vascular smooth muscles have the same origin as the cutaneous mesenchyme in which they differentiate. They are of somatopleural origin in the wing integument and of somitic (dermatomal) origin in the dorsal integument. This study further reveals that the muscular and connective tissue wall of blood vessels does not have the same embryonic origin as the endothelium. It is suggested that the latter originates from the primitive aorta.

The nuclear specificity of 12 day embryonic skeletal muscle cells has been studied in ectopic limbs that developed after implantation of quail limb somatopleural mesoderm into chick hosts. In eleven cases, the nuclearity of the myotubes was homospecifically of chick type. In the remnant eight cases, the musculature was heterospecific, the myotubes being homospecific and constituted by chick or quail nuclei or the myotubes being heterospecific and containing at random chick and quail nuclei. The heterospecific multinuclearity was analyzed in 85 portions of myotubes. The degeneration of numerous heterospecific muscle cells was attested by the shrinked and hyperchromatic states of the nuclei and by the fragmentation of the sarcoplasm that contained cellular debris. Nevertheless, there is no correlation between myotube degeneration and heterospecificity, because the same necrotic figures are observed in chick host homospecific myotubes that have reached the same degree of development.

The aim of this study is to test the ability of the intrinsic wing musculature to develop in the absence of somitic mesoderm. The experiments were performed on 2- to 2.5-day chick embryos either by replacing the somitic mesoderm adjacent to the wing field with a piece of 9-day chick embryonic midgut or by destroying, through local X-irradiation, not only the somitic mesoderm of the wing level, but also at least three somites (or presumptive somites) anterior and/or three presumptive somites posterior to the wing level. The replacemnt of somitic tissue scarcely affected the organogenesis of the forearm musculature, at least when both bones were present. In the other experiments, radio-destruction severely impaired the development of the forearm muscles, which were seldom all present and in most cases were entirely missing. The absence of a given muscle involves the simultaneous absence of the corresponding tendons. The possible origins of the muscles that formed despite the removal of the somitic mesoderm are discussed.

Quail-to-chick grafting experiments performed during the third day of incubation demonstrate that somites can contribute to limb development. In orthotopic recombinations, migrating cells originating from the grafted unsegmented or segmented somitic mesoderm adjacent to the wing or leg field end up in the musculature respectively of the wing or the leg, where they express exclusively myogenic properties. Thus, in these heterospecific recombinations, the anatomical muscle has a double origin: muscle bulk of somitic origin; tendons and connective tissues of somatopleural origin. Similar features are observed in heterotopic recombinations with (segmented or unsegmented) somitic mesoderm located cranially or caudally to the limb levels. In the reverse chick-to-quail grafting experiments, the somitc participation to the limb mesoderm can also be observed. But it is less regular than that obtained in the quail-to-chick recombinations, and the muscle bulk is made up in various proportions of graft-originated somitic cells and of host somatopleural cells. The possible existence of juxtaposed and interdigitated myogenic and tendinogenic compartments is discussed in view of the dissimilarity between the results of the two kinds of heterospecific recombinations.

The somitic mesoderm of the wing level was replaced in a two-day chick embryo by quail somitic mesoderm obtained from the level of the wing, the leg or the neck. The musculature of the host's wing on the operated side was exclusively or almost exclusively constituted by quail cells, whereas the skeleton, the dermis, the tendons and the muscular envelopes were formed by chick cells. This result demonstrates that, under the present experimental conditions, the wing musculature is originated from the somitic mesoderm of any level of the cephalocaudal axis.

In order to support the demonstration of the regulative capacity of the chick limb bud, already stressed by one of us (Kieny, 1964, 1967), heterospecific combinations were made between chick and quail tissues, the cells of the latter bearing a distinctive nuclear marker. A Japanese quail whole limb bud (stage-18 to 21 of H. H., wing or leg) was grafted distally onto the prospective zeugopod of a chick (stage-22) wing bud sectioned at the prospective wrist level. Thus, from a heterospecific surplus recombinant containing five prospective limb segments (stylopod and zeugopod from the chick host; stylopod, zeugopod and autopod from the quail graft), it was possible to obtain a normally shaped appendage that comprised either upper arm, lower arm and hand in the case of a wing bud graft, or heteromorphic upper arm, lower leg and foot in the case of a hind-limb bud graft. In these cases, regulation for excess appeared to take place mainly within the host tissues. The three proximal segments of the recombinant, namely the chick stylopod and zeugopod of the host's stump and the quail stylopod of the graft, became reorganized and gave rise to a single stylopodial segment, which usually contained a double stylopodial bone element, one of chick, the other of quail origin.The absence of development of the squeezed prospective zeugopod can be interpreted as follows: owing to an interaction with the stylopodial graft tissues, the zeugopodial cells of the juxtaposed stump boundary have shifted proximally their originally more distal positional values, so that they changed their prospective pattern of differentiation to that of stylopod. These reset zeugopodial cells combine with the stylopodial cells of host and graft and form a huge composite stylopod, in which, due to an asynchronous determination in the two species, chick and quail tissues do not cooperate fully for the development of a single bone.

The excision of a portion of unsegmented somitic mesoderm from the thoracic region has no or little effect on the development of vertebrae and ribs. Its orthotopic replacement by thoracic somitic mesoderm obtained from another embryo does not cause important deficiencies of the rib basket. But its replacement by nonsomitic tissue (neural tube, midgut, somatic mesoderm) not only causes the formation of a costal gap within the thoracic region, but also leads to the production of a defective spine on the operated side.

When prospective limb mesoderm is implanted into the prospective flank of another embryo, a supernumerary limb develops on the flank of the host. Three different criteria by which the presence of host cells could be ascertained within the extra limb have been used, namely, labeling of the graft with 3H-thymidine for the study of its evolution during the first 2 days after implantation, distinctive nuclear feature of implanted quail cells for the study of histogenetic steps 2 and 3 days later, and finally specific distinctive integumentary characters for the study of late organogenesis between 13 days of incubation and hatching.