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Limb-Somite Relationship: Origin of the Limb Musculature

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Limb-Somite Relationship: Origin of the Limb Musculature /. Embryo/, exp. Morph. Vol. 41, pp. 245-258, 1977 245 Printed in Great Britain © Company of Biologists Limited 1977 Limb-somite relationship: origin of the limb musculature By ALAIN CHEVALLIER, MADELEINE KIENY AND ANNICK MAUGER1 From the Laboratoire de Zoologie et Biologie animale, Universite scientifique et medicate de Grenoble (Equipe de recherche associee au CNRS n° 621, 'Morphogenese experimentale') SUMMARY Quail-to-chick grafting experiments performed during the third day of incubation demon- strate 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 recom- binations, 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 somitic 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 com- partments is discussed in view of the dissimilarity between the results of the two kinds of heterospecific recombinations. INTRODUCTION The limb-bud arises as a local thickening in the somatopleural mesoderm of the body wall at somite level 15-20 for the wing-bud and 26-32 for the leg- bud. These thickenings subsequently elongate along their proximo-distal axis, i.e. perpendicularly to the cranio-caudal axis of the embryo. During this early period of development the mesenchyme of the limb-bud looks homogeneous and appears to be composed of a single cell type. Later, from stage 24 of Ham- burger & Hamilton (1951) on, these mesenchymal cells differentiate into muscle, cartilage, bone and connective tissue cells. Pursuing our investigations on the relationships between the somites and the limb-bud (Kieny, 1969, 1970, 1971, 1972), the question arose whether bird 1 Author's address: Laboratoire de Zoologie et Biologie animale, Universite scientifique et medicale de Grenoble, 38041 Grenoble Cedex, France. 246 A. CHEVALLIER, M. KIENY AND A. MAUGER somite cells participate in limb myogenesis in the same manner as that des- cribed in lower vertebrates [Selachians: Dohrn (1884); Braus (1899, 1906); Borisov (1970), Teleosts: Corning (1894), Amphibians: Borisov (1970)]. While it is well established that the bird limb skeleton and the dermis (Dhou- ailly & Kieny, 1970, 1972; Kieny, 1960, 1971; Pinot, 1970) arise from the somatopleural mesoderm, it is not clear yet whether the musculature is of somatopleural or somitic origin or whether limb muscles form from a combina- tion of somitic and somatopleural cells. Up to a recent date, the assumption of the somitic participation in limb musculature, hence the previous penetration of somite cells into the limb-buds, could not be confirmed experimentally. Grafting (Hamburger, 1938) as well as carbon-marking (Saunders, 1948) and tritium-labelling (Seichert, 1971) experiments failed to demonstrate a somitic contribution to the development of the limb. All these experiments were carried out at late stages, when the limb- bud was, at the earliest, about to bulge out. Despite the in vitro recombination experiments of Gumpel-Pinot (1974) which indicated that somitic material takes part in the formation of the wing-bud, the participation of somite cells in limb myogenesis remained unsolved. Since the presumptive myoblasts are histologically indistinguishable from other mesenchymal limb-bud cells at early stages of development, only longlasting labelling experiments could reveal the origin of the limb musculature. Using the biological cell labelling technique advocated by Le Douarin & Barq (1969), Christ, Jacob & Jacob (1974#) on the one hand, and our group [Chevallier, Kieny & Mauger (1976)] on the other hand, performed similar experiments that consisted in replacing the somitic mesoderm of the wing level of the chick host by somitic mesoderm from a quail donor. These experi- ments showed that indeed somitic cells participate in limb myogenesis. The experiments reported in this paper were undertaken to answer the following questions. (1) What is the extent of the somitic participation in the morphogenesis of the limb musculature ? (2) Are the somite-limb musculature relationships restricted to the somitic mesoderm of the corresponding limb level ? (3) Are these relationships the same in both kinds of xenoplastic grafting experiments between chick and quail ? MATERIAL AND METHODS The experiments were carried out on chick embryos either of a White Leghorn strain or of a crossing of Wyandotte x Rhode Island Red and on Japanese quail (Coturnix coturnix japonica) embryos, during the 3rd day of incubation. The exact stage attained at theJime of operation was specified by the number of pairs of somites. The experimental design comprised replacing the somitic mesoderm of the wing or the leg level of one species by somitic mesoderm from the other species Origin of limb musculature 247 Table 1. Distribution of experimental cases according to origin and stage {in pairs of somites) of grafted tissues and host embryos Grafts Hosts operated on the Wing level Leg level Cephalo- caudal Limit stages of donors Limit stages Number Limit stages Number level of of of origin Chick Quail Chick Quail cases Chick Quail cases Cervical — 14-18 14-23 — 8 — — — Wing 12-20 12-17 9 — 17-24 3 10-23 12-22 20 Flank — 20-24 13-15 — 6 — — — Leg 16-22 15-20 4 — 18-22 4 18-26 15-16 3 21-25 — 5 according to a procedure that has become routine and basic to many experi- ments in our group (Chevallier, 1975; Kieny, 1972; Mauger, 1912a, b). Figs. 1 and 5). When the translocations were performed at the wing level, the chick or quail hosts ranged in developmental stages from 12 to 17 pairs of somites (with the exception of 5 out of 50 cases ranging in stages from 20 to 23 pairs of somites) so that unsegmented or only partially segmented somitic mesoderm was excised; whereas, when performed at the leg level, only unsegmented somi- tic mesoderm was extirpated, the hosts ranging in developmental stages be- tween 17 and 25 pairs of somites. The grafts composed of a row of five to seven somites or presumptive somites were obtained from the cervical region (somites 5 through 10 inclusive), the wing region (presumptive somites or somites 15 through 20 inclusive), the flank region (presumptive somites or somites 21-25 inclusive) or the leg region (presumptive somites 26 through 32 inclusive). The donors ranged in develop- mental stages between 10 and 26 pairs of somites (Table 1). The orientation of the graft was recorded for each operation and only aadd homopleural operations were made on the left or right side of the embryos so that in no case was the medio-lateral axis of the graft reversed. The operated embryos were allowed to develop until day 6 to 12 of incubation. They were then sacrificed, fixed in Helly's solution and immediately embedded in paraffin. Longitudinal or transverse sections of the limbs, 7/*m thick, were stained according to the nuclear reaction of Feulgen and Rossenbeck. 248 A. CHEVALLIER, M. KIENY AND A. MAUGER Donor Somite no. 14 Graft: somite Somite no. 14 mesoderm from level Ectoderm Discarded Fig. 1. Experimental scheme of orthotopic replacement of the somitic mesoderm from the wing level by heterospecific somitic mesoderm. Combinations between chick host and quail donor, and vice versa. RESULTS In this paper, we focus our histological investigations on the intrinsic limb musculature. The observations that were made simultaneously on the limb girdles will be published separately (Chevallier, 1977). A. Quail grafts to chick hosts 1. Orthotopic transplantations Wing level. There were 20 chick embryos in which the somitic mesoderm of the wing level was orthotopically (Fig. 1) replaced by quail somitic mesoderm. FIGURES 2-4 Quail grafts to chick host. Fusiform muscles. Fig. 2. Forearm muscle six days after the orthotopic replacement of the somitic mesoderm at the wing level. Quail donor and chick host: 17 pairs of somites, x 284. Figs. 3 and 4. Two cases of forearm muscles seven days after the heterotopic replace- ment of the somitic mesoderm of the wing level by somitic mesoderm from the leg level. Quail donors: respectively 16 and 20 pairs of somites. Chick hosts: respec- tively 15 and 16 pairs of somites, x 672. The interrupted lines represent the limit between chick (C) and quail (Q) cells. CT, surrounding muscular connective tissue comprising chick cells. 250 A. CHEVALLIER, M. KIENY AND A. MAUGER In all wings of the operated side, quail cells of somitic origin were found in the upper arm, lower arm and hand. While the skeleton and dermis were always and uniformly composed of chick somatopleural cells, the musculature, on the contrary, was made of both somite-originating quail cells and somatopleural chick cells. However, these cells were not distributed at random, but the bispeci- ficity was highly organized. Indeed, 4-7 days after the operation, the bulk of the fusiform muscles were mainly composed of quail cells whereas the attached tendons, at both ends, were constituted by chick cells (Fig. 2). Moreover, in the bulk, the presence of quail nuclei was selectively restricted to the differentiating muscle fibres proper. We found only one exception to this picture; namely, in one of these 17 speci- mens, the bulk and the tendons of one fusiform muscle from the zeugopod were completely made up by chick cells.
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