The specificity of motor innervation of the chick wing does not depend upon the segmental origin of muscles

In vertebrate embryos, motor axons originating from a particular craniocaudal position in the neural tube innervate limb muscles derived from myoblasts of the same segmental level. We have investigated whether this relationship is important for the formation of specific nerve-muscle connections, by altering the segmental origin of muscles and examining their resulting innervation. First, by grafting quail wing somites to a new craniocaudal position opposite the chick wing, we established that the segmental origin of a muscle can be altered: presumptive muscle cells migrated according to their new, rather than their original, somitic level, colonizing a different subset of muscles. However, after reversal of a length of brachial somitic mesoderm along the craniocaudal axis, or exchange or shift of brachial somites, the craniocaudal position of wing muscle motoneurone pools within the spinal cord was undisturbed, despite the new segmental origin of the muscles themselves. While not excluding the possibility that muscles and their motor nerves are labelled segmentally, we conclude that specific motor axon guidance in the wing does not depend upon the existence of such labels.     

The somitic level of origin of embryonic chick hindlimb muscles

Studies of avian chimeras made by transplanting groups of quail somites into chick embryos have consistently shown that the muscle cells of the hindlimb are derived from the adjacent somites, however, the pattern of cell distribution from individual somites to individual hindlimb muscles has not been characterized. I have mapped quail cell distribution in the chick hindlimb after single somite transplantation to determine if cells from an individual somite populate discrete limb muscle regions and if there is a spatial correspondence between a muscle's somitic level of origin and the known spinal cord position of its motoneuron pool. At stages 15-18 single chick somites or equivalent lengths of unsegmented somitic mesoderm adjacent to the prospective hindlimb region were replaced with the corresponding tissue from quail embryos. At stages 28-34, quail cell distribution was mapped within individual thigh muscles and shank muscle regions. A quail-specific antiserum and Feulgen staining were used to identify quail cells. Transplants from somite levels 26-33 each gave rise to consistent quail cell labeling in a unique subset of limb muscles. The anteroposterior positions of these subsets corresponded to that of the transplanted somitic tissue. For example, more anterior or anteromedial thigh muscles contained quail cells when more anterior somitic tissue had been transplanted. For the majority of thigh muscles studied and for shank muscle groups, there was also a clear correlation between somitic level of origin and motoneuron pool position. These data are compatible with the hypothesis that motoneurons and the muscle cells of their targets share axial position labels. The question of whether motoneurons from a specific spinal cord segment recognize and consequently innervate muscle cells derived from the same axial level during early axon outgrowth is addressed in the accompanying paper (C. Lance-Jones, 1988, Dev. Biol. 126, 408-419). Quail cell distribution was also mapped in chick embryos in which quail somites or unsegmented mesoderm had been placed 2-3 somites away from their position of origin. In all cases donor somitic tissues contributed to muscles in accord with their host position. These results indicate that muscle cell precursors within the somites are not specified to migrate to a predetermined target region.     

Migratory and organogenetic capacities of muscle cells in bird embryos

Summary The migratory and organogenetic capacities of muscle cells at different stages of differentiation were tested in heterospecific chick/quail recombinants. Grafts containing muscle cells were taken from the premuscular masses from 4- to 5-day quail embryos, from the limb or trunk muscles of 12-day embryonic and 4-day post-natal quails, and from experimentally produced bispecific premuscular masses in which the myoblasts are of quail origin and the connective tissue cells of chick origin. Grafts were implanted into 2-day chick embryos in place of the somitic mesoderm at the limb level. Hosts were examined 4 to 7 days after operation.After implantation of a piece of premuscular mass, quail cells were found at and around the site of the graft in the truncal region and within the limb as far as the autopod. Quail cells participated predominantly in the trunk and limb musculature, which contained a number of quail myotubes and of bispecific quail/chick myotubes. Apart from skeletal muscles, quail cells contributed sporadically to nerve envelopes and blood vessel walls in the limb.When the graft was of bispecific constitution, quail nuclei in the limb and the trunk were found exclusively in monospecific and bispecific myotubes.After implantation of differentiated embryonic or post-natal muscle tissue, quail cells in the limb contributed only sporadically to nerve envelopes and blood vessel walls, while in the trunk they also participated in the formation of muscles and tendons.It is concluded that the myogenic cells in 4 to 5-day quail premuscular masses are still able to undergo an extensive migration into the limb buds and there participate in the formation of myotubes and anatomically normal muscles. They display developmental potentialities equivalent to those of the somitic myogenic stem cells. These capacities are lost in 12-day embryonic muscles.     

Etude de la migration des cellules somitiques dans le mésoderme somatopleural de l'ébauche de l'aile

Summary In chick embryos, observations were made on serial semithin transverse sections of the wing level. In addition homo- or heterotopic replacements of the wing or leg somitic mesoderm by labelled somitic or nonsomitic mesoderm were made in 2-to 2.5-day embryos. The nuclear label used was either natural (quail donor embryos in heterotopic transplantations) or isotopic (chick donors labelled with tritiated thymidine).Histological examination revealed that the first somitic cells to leave somite 15 apparently did so at the 20 to 22 somite stage, while the last ones to leave somite 20 apparently did so shortly before the 36 somite stage.Transplantation experiments with labelled donor cells revealed the routes of migratory somitic cells and the time-course of their invasion into the outgrowing limb bud (non-somitic graft cells did not noticeably invade the limb anlage). They showed furthermore that the somitic mesoderm is not regionalized with respect to its limb myogenic properties.These results are compared with those obtained in other classes of vertebrates.

Ce travail a été subventionné en partie par la D.G.R.S.T. et le C.N.R.S.     

Fate maps of the primitive streak in chick and quail embryo: ingression timing of progenitor cells of each rostro-caudal axial level of somites

Developmental fates of cells emigrating from the primitive streak were traced by a fluorescent dye Dil both in chick and in quail embryos from the fully grown streak stage to 12-somite stage, focusing on the development of mesoderm and especially on the timing of ingression of each level of somitic mesoderm. The fate maps of the chick and quail streak were alike, although the chick streak was longer at all stages examined. The anterior part of the primitive streak predominantly produced somites. The thoracic and the lumbar somites were shown to begin to ingress at the 5 somite-stage and 10 somite-stage in a chick embryo, and 6 somite-stage and 9 somite-stage in a quail embryo, respectively. The posterior part of the streak served mainly as the origin of more lateral or extra embryonic mesoderm. As development proceeded, the fate of the posterior part of the streak changed from the lateral plate mesoderm to the tail bud mesoderm and then to extra embryonic, allantois mesoderm. The fate map of the primitive streak in chick and quail embryo presented here will serve as basic data for studies on mesoderm development with embryo manipulation, especially for transplantation experiments between chick and quail embryos.     

Supernumerary limb structures after juxtaposing dorsal and ventral chick wing bud cells

The formation of duplicated wing skeletal elements and/or extra wing muscles was studied by juxtaposing normally nonadjacent embryonic chick wing bud cells. A wedge of right or left stage 21 wing bud ectoderm and mesoderm was inserted in a slit made in a host stage 20 to 22 right wing bud at the same anteroposterior position as its position of origin. The distal edge of the donor wedge and host wing bud were aligned with each other. Donor tissue was grafted into a host wing bud in one of the following four axial relationships: both the anteroposterior and dorsoventral axes corresponded with each other (aadd); only the anteroposterior axes were opposed (apdd); only the dorsoventral axes were opposed (aadv); both the anteroposterior and dorsoventral axes were opposed (apdv). Of the 63 wings resulting from the control aadd operation and the 45 wings from the apdd operation, only 12 wings had a duplicated skeletal element; of the 69 wings sectioned from these two groups of operations, only one had an extra muscle. However, of the wings resulting from the aadv and apdv operations (48 and 52 cases, respectively), 23 had a duplicated skeletal element; of the 54 wings sectioned from these operations, 43 wings had one to four extra muscles. Furthermore, when the aadv operation was performed with a wedge of donor quail wing bud ectoderm and mesoderm or mesoderm alone, supernumerary muscles formed in these chimeric wings and they were made up of donor quail and host chick cells or only donor quail cells.     

The effect of somite manipulation on the development of motoneuron projection patterns in the embryonic chick hindlimb

Although the formation of motoneuron projections to individual muscles in the embryonic chick hindlimb has been shown to involve the specific recognition of environmental cues, the source of these cues and their mode of acquisition are not known. I show in the accompanying paper (C. Lance-Jones, 1988, Dev. Biol. 126, 394-407) that there is a correlation between the segmental level of origin of motoneurons and the somitic level of origin of the muscle cells of their targets in the chick hindlimb. These data are compatible with the hypothesis that the developmental basis for specific recognition is a positional one. Motoneurons and myogenic cells may be uniquely labeled in accord with their axial level of origin early in development and subsequently matched on the basis of these labels. To test this hypothesis, I have assessed motoneuron projection patterns in the embryonic chick hindlimb after somitic tissue manipulations. In one series of embryos, somitic mesoderm at levels 26-29 or 27-29 was reversed about the anteroposterior axis prior to myogenic cell migration and axon outgrowth. Since previous studies have shown that cells migrate from the somites in accord with their position and that somites 26-29 populate anterior thigh musculature, this operation will have reversed the somitic level of origin of anterior thigh muscles. Retrograde HRP labeling of projections to anterior thigh muscles at stage (st) 30 and st 35-38 showed that motoneuron projections were largely normal. This finding suggests that limb muscle cells or their source, the somites, do not contain the cues responsible for specific recognition prior to myogenic cell migration and axon outgrowth. To confirm that specific guidance cues were still intact after somitic mesoderm reversal, I also assessed motoneuron projections in embryos where somitic tissue plus adjacent spinal cord segments at levels 26-29 were reversed in a similar manner. Analyses of the distribution of retrogradely labeled motoneurons in reversed cord segments at st 35-36 indicated that motoneuron projections were reversed. This finding suggests that motoneurons have altered their course to project to correct targets despite the altered somitic origin of their targets and, thus, that specific guidance cues were intact. I conclude that if cues governing target or pathway choice are encoded positionally then they must be associated with other embryonic tissues such as the connective tissues or that guidance cues are acquired by myogenic cells after the onset of migration and motoneuron specification.     

The formation of leg or wing specific structures by leg bud cells grafted to the wing bud is influenced by proximity to the apical ridge

When quail or chick leg bud mesoderm was grafted to a chick wing bud, toes developed from grafts placed in direct contact with the wing apical ridge. The toes were primarily derived from quail leg cells, with variable participation of host wing cells. Donor cells also integrated into wing-specific structures, such as cartilage of the wing digits and the surrounding connective tissues. In addition to forming toes, the grafted leg mesoderm expressed its leg origin by enlarging skeletal elements in the host wing. In all cases, enlargements were derived of both quail donor and chick host cells, and were not the result of the addition of mass to the host bud. Grafts placed further than 162 microns from the ridge formed neither toes nor enlargements; rather, they integrated into wing-specific structures. Under the influence of the apical ridge, the grafted leg mesoderm cells are able to maintain their leg character and to form toes and skeletal enlargements. Grafts outside the range of ridge influence (162 microns) are affected by their surroundings to integrate into wing-specific structures. The formation of leg-specific structures by leg bud mesoderm grafted to the wing bud has been used to support the principle of nonequivalence, which states that, because of their different developmental histories, wing and leg cells are restricted to form structures specific for their respective limbs. However, we have shown that leg cells can form wing-specific structures, and therefore limb cells are not restricted in their development.     

Body wall morphogenesis: Limb-genesis interferes with body wall-genesis via its influence on the abaxial somite derivatives

The vertebrate body wall is regionalized into thoracic and lumbosacral/abdominal regions that differ in their morphology and developmental origin. The thoracic body wall has ribs and intercostal muscles, which develops from thoracic somites, whereas the abdominal wall has abdominal muscles, which develops from lumbosacral somites without ribs cage. To examine whether limb-genesis interferes with body wall-genesis, and to test the possibility that limb generation leads to the regional differentiation, an ectopic limb was induced in the thoracic region by transplanting prospective limb somatopleural mesoderm of Japanese quail between the ectoderm and somatopleural mesoderm of the chick prospective thoracic region. This ectopic limb generation induced the somitic cells to migrate into the ectopic limb mesenchyme to become its muscles and caused the loss of distal thoracic body wall (sterno-distal rib and distal intercostal muscle), without causing any significant effect on the more proximal region (proximal rib, vertebro-distal rib and proximal intercostal muscle). According to a new primaxial–abaxial classification, the proximal region is classified as primaxial and the distal region, as well as limb, is classified as abaxial. We demonstrated that ectopic limb development interfered with body wall development via its influence on the abaxial somite derivatives. The present study supports the idea that the somitic cells give rise to the primaxial derivatives keeping their own identity and fate, whereas they produce the abaxial derivatives responding to the lateral plate mesoderm.     

The embryonic origins of avian cephalic and cervical muscles and associated connective tissues

The objective of these experiments was to determine the embryonic origins of craniofacial and cervical voluntary muscles and associated connective tissues in the chick. To accomplish this, suspected primordia, including somitomeres 3-7, somites 1-7, and cephalic neural crest primordia have been transplanted from quail into chick embryos. Quail cells can be detected by the presence of a species-specific nuclear marker. The results are summarized as follows: (table; see text) These results indicate that muscles associated with branchial arch skeletal structures are derived from paraxial mesoderm, as are all other voluntary muscles in the vertebrate embryo. Thus, theories of vertebrate ontogeny and phylogeny based in part on proposed unique features of branchiomeric muscles must be critically reappraised. In addition, many of these cephalic muscles are composites of two separate primordia: the myogenic stem cells of mesodermal origin and the supporting and connective tissues derived from the neural crest or lateral plate mesoderm. Defining these embryonic origins is a necessary prerequisite to understanding how the mesenchymal primordia of cephalic muscles and connective tissues interact to form patterned, species-unique musculoskeletal systems.     

A comparative study of myogenic cell invasion of the avian wing and leg bud.

The borders of myogenic cell invasion of avian wing and leg buds were determined using the interspecific grafting technique between quail and chick embryos. Distal parts of quail limb buds were grafted ectopically into the coelomic cavity of chick embryos. The presence or absence of skeletal muscle was investigated in histological sections of the reincubated grafts. A comparison between the borders of myogenic cell invasion of the wing and leg buds showed that the differences in the position of the distal most muscles in the adult avian limbs could be a consequence of the cranio-caudal sequence of development.     

Preservation of the ability of dissociated quail wing bud mesoderm to elicit a position-related differentiative response

Previous studies showed that grafting wedges of fresh or cultured anterior quail wing mesoderm into posterior slits in chick wing buds resulted in the formation of supernumerary cartilage in a high percentage of cases. When anterior quail mesoderm, which had been dissociated into single cells and pelleted by centrifugation, was grafted into posterior slits of host chick wing buds, supernumerary rods or nodules of cartilage formed in 74.3% of the cases. Few supernumerary skeletal structures formed following control operations in which pelleted dissociated anterior or posterior mesoderm was grafted into homologous locations in host chick wing buds. When pelleted, dissociated anterior mesoderm was cultured in vitro for 1 or 2 days prior to being implanted in posterior locations, the incidence of supernumerary cartilage formation increased to 95.5% and 93.8%, respectively. The incidence of supernumerary cartilage formation following control orthotopic grafts of cultured mesoderm was 11.8% for 1-day and 31% for 2-day cultured anterior mesoderm; for 1- and 2-day cultured posterior mesoderm, the incidence of supernumerary cartilage formation was 20% and 41.7%, respectively. Longer-term culture resulted in a substantial decrease in the percentage of supernumerary cartilage after anterior to posterior grafts and an increase in the incidence of supernumerary cartilage from control grafts. The results demonstrate that quail anterior wing bud mesodermal cells do not need to maintain constant contact with one another in order to retain the ability to form or stimulate the formation of supernumerary cartilage after being grafted into a posterior location in a host wing bud. This ability is retained when the pelleted dissociated mesoderm is cultured in vitro outside the limb field for at least 1 to 2 days.     

A new marker for identifying quail cells in embryonic avian chimeras: a quail-specific antiserum

The characterization of cell behavior in quail chick chimeras has greatly increased our knowledge of the ontogeny of embryonic cell populations and the role of cell-cell interactions in development. We sought to extend the value of avian chimeras by producing a marker that would recognize cell surface components and that could be used instead of the traditional nuclear marker to identify quail cells within chimeras. We describe here a quail-specific antiserum produced by injecting chickens with a membrane fraction of 6-10-day quail embryos. By use of peroxidase coupling of a second antibody, serum reactivity was tested in tissue sections of normal quail and chick embryos and of somitic mesoderm and neural tube chimeras. The primary time period examined was 6-10 days of development. At these stages, the antiserum recognizes only quail cells and stains both plasma membrane-associated and cytoplasmic cell components. The latter characteristics allow the identification of quail axons in chimeras and facilitate visualization of quail cells at low magnification. We show that antiserum staining can also be used to identify quail cells in culture and can be combined with orthograde HRP labeling of neurons.     

A positive correlation between permissiveness of mesoderm to neural crest migration and early DRG growth.

The microenvironment created by grafting rostral somitic halves in place of normal somites leads to the formation of nonsegmented peripheral ganglia (Kalcheim and Teillet, 1989; Goldstein and Kalcheim, 1991) and is mitogenic for neural crest (NC) cells that become dorsal root ganglia (DRG) (Goldstein et al., 1990). We have now extended these studies by using three surgical manipulations to determine how additional mesodermal tissues affected DRG growth in chick embryos. The following experimental manipulations were performed: (1) unilateral deletion of epithelial somites, similar deletions followed by replacing the somites with (2) a three-dimensional collagen matrix, or (3) fragments of quail lateral plate mesoderm. When somites were absent or replaced by collagen matrix, ganglia were unsegmented, and their volumes were decreased by 21% and 12%, respectively, compared to contralateral intact DRG. In contrast, when lateral plate mesoderm was transplanted in place of somitic mesoderm, NC cells migrated into the grafted mesoderm and formed unsegmented DRG whose volumes were increased by 62.6% compared to the contralateral ganglia. These results suggest that although DRG precursors do not require sclerotome to begin migration and condensation processes, DRG size is modulated by the properties of the mesoderm. Permissiveness to migration is positively correlated with an increase in DRG volume. This volume increase observed in grafts of lateral plate mesoderm is likely to result from enhanced proliferation of neural crest progenitors, previously demonstrated for DRG cells in rostral somitic grafts.     

Position of origin of donor posterior chick wing bud tissue transplanted to an anterior host site determines the extra structures formed

The formation of supernumerary limb structures was studied by juxtaposing normally nonadjacent embryonic chick limb bud tissue. Different “wedges” (ectodern and mesoderm) of posterior donor right wing bud (stage 21) were transplanted to a slit made in stage 20–23 host right wing buds. Donor posterior tissue was transplanted to an anterior position in a host wing bud or, as a control, to the same position as its position of origin. Transplanting different wedges of posterior tissue to the same anterior host position results in wings with supernumerary structures, and different extra structures form depending on the position of origin of the donor tissue. The identification of extra limb structures formed was based on the skeletal and integumentary patterns of resulting wings and the pattern of muscles as seen in serial sections of resulting limbs. The results of experiments presented here are considered in light of current models that have been used to describe the formation of supernumerary limb structures by the embryonic chick limb bud.     

Dual origin and segmental organisation of the avian scapula

Bones of the postcranial skeleton of higher vertebrates originate from either somitic mesoderm or somatopleural layer of the lateral plate mesoderm. Controversy surrounds the origin of the scapula, a major component of the shoulder girdle, with both somitic and lateral plate origins being proposed. Abnormal scapular development has been described in the naturally occurring undulated series of mouse mutants, which has implicated Pax1 in the formation of this bone. Here we addressed the development of the scapula, firstly, by analysing the relationship between Pax1 expression and chondrogenesis and, secondly, by determining the developmental origin of the scapula using chick quail chimeric analysis. We show the following. (1) The scapula develops in a rostral-to-caudal direction and overt chondrification is preceded by an accumulation of Pax1-expressing cells. (2) The scapular head and neck are of lateral plate mesodermal origin. (3) In contrast, the scapular blade is composed of somitic cells. (4) Unlike the Pax1-positive cells of the vertebral column, which are of sclerotomal origin, the Pax1-positive cells of the scapular blade originate from the dermomyotome. (5) Finally, we show that cells of the scapular blade are organised into spatially restricted domains along its rostrocaudal axis in the same order as the somites from which they originated. Our results imply that the scapular blade is an ossifying muscular insertion rather than an original skeletal element, and that the scapular head and neck are homologous to the 'true coracoid' of higher vertebrates.     

The lateral somitic frontier: dorso-ventral aspects of anterio-posterior regionalization in avian embryos

Patterning events along the anterior-posterior (AP) axis of vertebrate embryos result in the distribution of muscle and bone forming a highly effective functional system. A key aspect of regionalized AP patterning results from variation in the migratory pattern of somite cells along the dorsal-ventral (DV) axis of the body. This occurs as somite cell populations expand around the axis or migrate away from the dorsal midline and cross into the lateral plate. The fate of somitic cells has been intensely studied and many details have been reported about inductive signaling from other tissues that influence somite cell fate and behavior. We are interested in understanding the specific differences between somites in particular AP regions and how these differences contribute to the global pattern of the organism. Using orthotopic transplants of segmental plate between quail and chick embryos, we have mapped the interface of the somitic and lateral plate mesoderm during the formation of the body wall in cervical and thoracic regions. This interface does not change dramatically in the mid-cervical region, but undergoes extensive changes in the thoracic region. Based on this regional mapping and consistent with the extensive literature, we suggest a revised method of classifying regions of the body wall that relies on embryonic cell lineages rather than adult functional criteria.     

On the non-equivalence of skeletal muscle satellite cells and embryonic myoblasts

Postnatal satellite cells, isolated from normal or previously denervated skeletal muscles of juvenile quails, were tested as to their capacity to participate in embryonic muscle ontogeny. They were grafted into 2-day chick embryo hosts, in place of a piece of brachial somitic mesoderm. Satellite cell implants were prepared from pellets either of freshly isolated cells or of cells precultured in vitro under proliferative conditions. Myogenic capacity of the implanted cells was attested by their ability to fuse into myotubes when cultured under differentiation conditions. In no case did the implanted satellite cells invade the adjacent wing bud or participate in wing muscle morphogenesis. They did not either give rise to myotubes at the site of implantation, nor did they even survive longer than 3 days in the embryonic environment. These negative results indicate that postnatal satellite cells, unlike embryonic myoblasts, are unable to take part in muscle embryogenesis. Although they derive from the same somitic myogenic cell line as the embryonic myoblasts, they therefore represent a differentiated non-totipotent type of myogenic cell.     

Attempt to produce a chick/mouse heteroclass musculature

Summary Heteroclass chick/mouse chimaeras were prepared by transplanting somitic presumptive myogenic cells or limb bud myoblasts from donor mouse embryos into chick hosts, to replace (1) previously extirpated brachial somitic mesoderm or (2) experimentally deleted limb premuscular masses. Since mouse and chick cells can be distinguished by differential staining affinities, this parameter was used to verify the viability of the implant and to assess its fate. Our analyses showed that transplanted mouse somitic myogenic stem cells or limb bud myoblasts did not participate in the host brachial musculature, whatever the experimental conditions.     

The meso-angioblast: a multipotent,self-renewing cell that originates from the dorsal aorta and differentiates into most mesodermal tissues

We have previously reported the origin of a class of skeletal myogenic cells from explants of dorsal aorta. This finding disagrees with the known origin of all skeletal muscle from somites and has therefore led us to investigate the in vivo origin of these cells and, moreover, whether their fate is restricted to skeletal muscle, as observed in vitro under the experimental conditions used. To address these issues, we grafted quail or mouse embryonic aorta into host chick embryos. Donor cells, initially incorporated into the host vessels, were later integrated into mesodermal tissues, including blood, cartilage, bone, smooth, skeletal and cardiac muscle. When expanded on a feeder layer of embryonic fibroblasts, the clonal progeny of a single cell from the mouse dorsal aorta acquired unlimited lifespan, expressed hemo-angioblastic markers (CD34, Flk1 and Kit) at both early and late passages, and maintained multipotency in culture or when transplanted into a chick embryo. We conclude that these newly identified vessel-associated stem cells, the meso-angioblasts, participate in postembryonic development of the mesoderm, and we speculate that postnatal mesodermal stem cells may be derived from a vascular developmental origin.