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.     

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.     

Formation of the dorsal root ganglia in the avian embryo: Segmental origin and migratory behavior of neural crest progenitor cells

The segmental origin and migratory pattern of neural crest cells at the trunk level of avian embryos was studied, with special emphasis on the formation of the dorsal root ganglia (DRG) which organize in the anterior half of each somite. Neural crest cells were visualized using the quail-chick marker and HNK-1 immunofluorescence. The migratory process turned out to be closely correlated with somitic development: when the somites are epithelial in structure few labeled cells were found in a dorsolateral position on the neural tube, uniformly distributed along the craniocaudal axis. Following somitic dissociation into dermomyotome and sclerotome labeled cells follow defined migratory pathways restricted to each anterior somitic half. In contrast, opposite the posterior half of the somites, cells remain grouped in a dorsolateral position on the neural tube. The fate of crest cells originating at the level of the posterior somitic half was investigated by grafting into chick hosts short segments of quail neural primordium, which ended at mid-somitic or at intersomitic levels. It was found that neural crest cells arising opposite the posterior somitic half participate in the formation of the DRG and Schwann cells lining the dorsal and ventral root fibers of the same somitic level as well as of the subsequent one, whereas those cells originating from levels facing the anterior half of a somite participate in the formation of the corresponding DRG. Moreover, crest cells from both segmental halves segregate within each ganglion in a distinct topographical arrangement which reflects their segmental origin on the neural primordium. Labeled cells which relocate from posterior into anterior somitic regions migrate longitudinally along the neural tube. Longitudinal migration of neural crest cells was first observed when the somites are epithelial in structure and is completed after the disappearance of the last cells from the posterior somitic region at a stage corresponding to the organogenesis of the DRG.     

Autonomy of differentiation in avian branchial somites and the influence of adjacent tissues

This study investigates the differentiative abilities of avian brachial somites at stages of development before, during and after the migration of somitic cells into the wing primordium. These somites are the source of cells that migrate into the forelimbs and there give rise exclusively, and totally, to the skeletal muscle lineage of the wing and yet show no morphological evidence of commitment to that fate when they leave the somites. The aim of the study was to see if the brachial somitic cells are committed to particular developmental pathways at these stages. The brachial somites were isolated from HH stage-12, -15 and -18 chick embryos, either by microdissection or enzymatic dissociation, and grown in organ culture, in explant culture on different substrata or on the chorioallantoic membrane (CAM) of host chicks, either alone or in combination with adjacent tissues. Myogenesis and chondrogenesis occurred in all stage-18 enzymatically separated somites, regardless of the growth environment. Myogenesis was reduced in stage-15 somites and unobservable in stage-12 somites; however, recombination of stage-12 somites with epithelium or neural tube increased the incidence of myogenesis at this stage. The incidence of chondrogenesis was also less in the younger explants. Unlike its effect on myogenic expression, recombination with epithelium resulted in a dramatic decrease in chondrogenesis in both stage-12 and -15 somites. The recombination experiments suggest that conditions that maintain the normal spatial relationships within isolated somites permit expression of a preexisting specification to a particular fate. They also show that the overlying epithelium can inhibit chondrogenesis in these somites. Overall, the results suggest that by the time migration of somitic cells into wing regions is finishing, brachial somitic cells have become stabilized in their ability to undergo both myogenesis and chondrogenesis for they will do so under a variety of growth conditions and independently of adjacent tissues. However, immediately before (stage 12) and shortly after (stage 15) the onset of migration, both myogenic and chondrogenic expression by brachial somitic cells are still under the influence of interactions with adjacent tissues.     

Met and Hgf signaling controls hypaxial muscle and lateral line development in the zebrafish

Somites give rise to a number of different embryonic cell types, including the precursors of skeletal muscle populations. The lateral aspect of amniote and fish somites have been shown to give rise specifically to hypaxial muscle, including the appendicular muscle that populates fins and limbs. We have investigated the morphogenetic basis for formation of specific hypaxial muscles within the zebrafish embryo and larvae. Transplantation experiments have revealed a developmentally precocious commitment of cells derived from pectoral fin level somites to forming hypaxial and specifically appendicular muscle. The fate of transplanted somites cannot be over-ridden by local inductive signals, suggesting that somitic tissue may be fixed at an early point in their developmental history to produce appendicular muscle. We further show that this restriction in competence is mirrored at the molecular level, with the exclusive expression of the receptor tyrosine kinase met within somitic regions fated to give rise to appendicular muscle. Loss-of-function experiments reveal that Met and its ligand, hepatocyte growth factor, are required for the correct morphogenesis of the hypaxial muscles in which met is expressed. Furthermore, we demonstrate a requirement for Met signaling in the process of proneuromast deposition from the posterior lateral line primordia.     

Local extrinsic signals determine muscle and endothelial cell fate and patterning in the vertebrate limb

Both the muscle and endothelium of the vertebrate limb derive from somites. We have used replication-defective retroviral vectors to analyze the lineage relationships of these somite-derived cells in the chick. We find that myogenic precursors in the somites or proximal limb are not committed to forming slow or fast muscle fibers, particular anatomical muscles, or muscles within specific proximal/distal or dorsal/ventral limb regions. Somitic endothelial precursors are uncommitted to forming endothelium in particular proximal/distal or dorsal/ventral limb regions. Surprisingly, we also find that myogenic and endothelial cells are derived from a common somitic precursor. Thus, local extrinsic signals are critical for determining muscle and endothelial patterning as well as cell fate in the limb.     

Origin of cutaneous smooth muscles in birds (author's transl)

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.     

Distinct regulatory cascades for head and trunk myogenesis

Most head muscles arise from the pre-otic axial and paraxial head mesoderm. This tissue does not form somites, yet expresses the somitic markers Lbx1, Pax7 and Paraxis in a regionalised fashion. The domain set aside by these markers provides the lateral rectus muscle, the most caudal of the extrinsic eye muscles. In contrast to somitic cells that express Lbx1, lateral rectus precursors are non-migratory. Moreover, the set of markers characteristic for the lateral rectus precursors differs from the marker sets indicative of somitic muscle precursors. This suggests distinct roles for Lbx1/Pax7/Paraxis in the development of head and trunk muscles. When grafted to the trunk, the pre-otic head mesoderm fails to activate Lbx1, Pax7 or PARAXIS: Likewise, somites grafted into the region of the lateral rectus precursors fail to activate the lateral rectus marker set. This suggests that distinct regulatory cascades act in the development of trunk and head muscles, possibly reflecting their distinct function and evolution.     

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.     

The neural tube/notochord complex is necessary for vertebral but not limb and body wall striated muscle differentiation.

The aim of this work was to investigate the role played by the axial organs, neural tube and notochord, on the differentiation of muscle cells from the somites in the avian embryo. Two of us have previously shown that neuralectomy and notochordectomy is followed by necrosis of the somites and consecutive absence of vertebrae and of most muscle cells derived from the myotomes while the limbs develop normally with muscles. Here we have focused our attention on muscle cell differentiation by using the 13F4 mAb that recognizes a cytoplasmic antigen specific of all types of muscle cells. We show that differentiation of muscle cells of myotomes can occur in the absence of notochord and neural tube provided that the somites from which they are derived have been in contact with the axial organs for a defined period of time, about 10 hours for the first somites formed at the cervical level, a duration that progressively reduces caudalward (i.e. for thoracic and lumbar somites). Either one or the other of the two axial organs, the neural tube or the notochord can prevent somitic cell death and fulfill the requirements for myotomal muscle cell differentiation. Separation of the neural tube/notochord complex from the somites by a surgical slit on one side of the embryo gave the same results as extirpation of these organs and provided a perfect control on the non-operated side. A striking finding was that limb and body wall muscles, although derived from the somites, differentiated in the absence of the axial organs. However, limb muscles that develop after excision of the neural tube started to degenerate from E10 onward due to lack of innervation. In vitro explantation of somites from different axial levels confirmed and defined precisely the chronology of muscle cell commitment in the myotomes as revealed by the in vivo experiments.     

Muscular derivatives of the cranialmost somites revealed by long-term fate mapping in the Mexican axolotl (Ambystoma mexicanum)

The fate of single somites has not been analyzed from a comparative perspective with modern cell-marking methods. Most of what we know is based on work using quail-chick chimeras. Consequently, to what degree cell fate has been conserved despite the anatomical differences among vertebrates is unknown. We have analyzed the cell fate of the cranialmost somites, with the focus on somite two, in the Mexican axolotl (Ambystoma mexicanum). Somite cells were marked by injection of dextran-fluorescein and detected using immunofluorescence after 2 months of development in paraffin sections. Our data confirm and extend earlier studies based on classical histology in salamanders. We show that somite two contributes to different muscles, skeletal elements, and connective tissues of the head and cranial trunk region. Cells from somites two and three migrate latero-ventrally and contribute to the hypobranchial muscles mm. geniohyoideus and rectus cervicis. We provide evidence that the specific formation of the hypobranchial musculature from ventral processes of the somites might be variable in different classes of vertebrates. We further demonstrate that mm. cucullaris and dilatator laryngis, which were earlier thought to have a branchial origin, arise from somitic material in a manner very similar to the findings in quail-chick chimeras. Our findings indicate that the pattern of somitic derivatives is highly conserved within tetrapods.     

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 contribution made by cells from a single somite to tissues within a body segment and assessment of their integration with similar cells from adjacent segments

Somites represent the first visual evidence of segmentation in the developing vertebrate embryo and it is becoming clear that this segmental pattern of the somites is used in the initial stages of development of other segmented systems such as the peripheral nervous system. However, it is not known whether the somites continue to contribute to the maintenance of the segmental pattern after the dispersal of the somitic cells. In particular, the extent to which cells from a single somite contribute to all of the tissues of a single body segment and the extent to which they mix with cells from adjacent segments during their migration is not known. In this study, we have replaced single somites in the future cervical region of 2-day-old chick embryos with equivalent, similarly staged quail somites. The chimerae were then allowed to develop for a further 6 days when they were killed. The cervical region was dissected and serially sectioned. The sections were stained with the Feulgen reaction for DNA to differentiate between the chick and quail cells. The results showed that the cells from a single somite remained as a clearly delimited group throughout their migration. Furthermore, the sclerotome, dermatome and myotome portions from the single somites could always be recognised as being separate from similar cells from other somites. The somitic cells formed all of the tissues within a body segment excluding the epidermis, notochord and neural tissue. There was very little mixing of the somitic cells between adjacent segments. The segmental pattern of the somites is therefore maintained during the migration of the somitic cells and this might be fundamental to a mechanism whereby the segmentation of structures, such as the peripheral nervous system, is also maintained during development.     

The expression of the homeobox gene Msx1 reveals two populations of dermal progenitor cells originating from the somites

Experimental manipulation in birds has shown that trunk dermis has a double origin: dorsally, it derives from the somite dermomyotome, while ventrally, it is formed by the somatopleure. Taking advantage of an nlacZ reporter gene integrated into the mouse Msx1 locus (Msx1(nlacZ) allele), we detected segmental expression of the Msx1 gene in cells of the dorsal mesenchyme of the trunk between embryonic days 11 and 14. Replacing somites from a chick host embryo by murine Msx1(nlacZ )somites allowed us to demonstrate that these Msx1-(beta)-galactosidase positive cells are of somitic origin. We propose that these cells are dermal progenitor cells that migrate from the somites and subsequently contribute to the dorsalmost dermis. By analysing Msx1(nlacZ) expression in a Splotch mutant, we observed that migration of these cells does not depend on Pax3, in contrast to other migratory populations such as limb muscle progenitor cells and neural crest cells. Msx1 expression was never detected in cells overlying the dermomyotome, although these cells are also of somitic origin. Therefore, we propose that two somite-derived populations of dermis progenitor cells can be distinguished. Cells expressing the Msx1 gene would migrate from the somite and contribute to the dermis of the dorsalmost trunk region. A second population of cells would disaggregate from the somite and contribute to the dermis overlying the dermomyotome. This population never expresses Msx1. Msx1 expression was investigated in the context of the onset of dermis formation monitored by the Dermo1 gene expression. The gene is downregulated prior to the onset of dermis differentiation, suggesting a role for Msx1 in the control of this process.     

Wnt 6 regulates the epithelialisation process of the segmental plate mesoderm leading to somite formation

In higher vertebrates, the paraxial mesoderm undergoes a mesenchymal to epithelial transformation to form segmentally organised structures called somites. Experiments have shown that signals originating from the ectoderm overlying the somites or from midline structures are required for the formation of the somites, but their identity has yet to be determined. Wnt6 is a good candidate as a somite epithelialisation factor from the ectoderm since it is expressed in this tissue. In this study, we show that injection of Wnt6-producing cells beneath the ectoderm at the level of the segmental plate or lateral to the segmental plate leads to the formation of numerous small epithelial somites. Ectopic expression of Wnt6 leads to sustained expression of markers associated with the epithelial somites and reduced or delayed expression of markers associated with mesenchymally organised somitic tissue. More importantly, we show that Wnt6-producing cells are able to rescue somite formation after ectoderm ablation. Furthermore, injection of Wnt6-producing cells following the isolation of the neural tube/notochord from the segmental plate was able to rescue somite formation at both the structural (epithelialisation) and molecular level, as determined by the expression of marker genes like Paraxis or Pax-3. We show that Wnts are indeed responsible for the epithelialisation of somites by applying Wnt antagonists, which result in the segmental plate being unable to form somites. These results show that Wnt6, the only known member of this family to be localised to the chick paraxial ectoderm, is able to regulate the development of epithelial somites and that cellular organisation is pivotal in the execution of the differentiation programmes. We propose a model in which the localisation of Wnt6 and its antagonists regulates the process of epithelialisation in the paraxial mesoderm.     

Hox genes and morphological identity: axial versus lateral patterning in the vertebrate mesoderm

The successful organization of the vertebrate body requires that local information in the embryo be translated into a functional, global pattern. Somite cells form the bulk of the musculoskeletal system. Heterotopic transplants of segmental plate along the axis from quail to chick were performed to test the correlation between autonomous morphological patterning and Hox gene expression in somite subpopulations. The data presented strengthen the correlation of Hox gene expression with axial specification and focus on the significance of Hox genes in specific derivatives of the somites. We have defined two anatomical compartments of the body based on the embryonic origin of the cells making up contributing structures: the dorsal compartment, formed from purely somitic cell populations; and the ventral compartment comprising cells from somites and lateral plate. The boundary between these anatomical compartments is termed the somitic frontier. Somitic tissue transplanted between axial levels retains both original Hox expression and morphological identity in the dorsal compartment. In contrast, migrating lateral somitic cells crossing the somitic frontier do not maintain donor Hox expression but apparently adopt the Hox expression of the lateral plate and participate in the morphology appropriate to the host level. Dorsal and ventral compartments, as defined here, have relevance for experimental manipulations that influence somite cell behavior. The correlation of Hox expression profiles and patterning behavior of cells in these two compartments supports the hypothesis of independent Hox codes in paraxial and lateral plate mesoderm.     

A somitic contribution to the pectoral girdle in the axolotl revealed by long-term fate mapping

The pectoral girdle is a unique skeletal element that underwent drastic morphological changes during its evolution, especially in association with the fin-to-limb transition. Comparative studies of its development are needed to gain a deeper understanding of its evolution. Transplantation experiments using the quail-chick chimeric system have revealed that not only lateral plate mesoderm but also somites contribute to the pectoral girdle in birds. Studies in mice and turtles also document somitic contributions to the pectoral girdle, but extirpation experiments in a salamander did not affect shoulder girdle development. Somitic contributions to the pectoral girdle therefore have been interpreted as a feature unique to amniotes. Here, we present a long-term fate map of single somites in the Mexican axolotl, based on transplantations of somites two to six from GFP-transgenic donors into wild-type hosts, as well as injections of fluorescein dextran into single somites. The results show a somitic derivation of the dorsal region of the suprascapula, demonstrating that somitic contributions to the pectoral girdle are not restricted to amniotes. Comparison with the few other species studied so far leads us to suggest a position-dependent origin of the pectoral girdle. We propose that embryonic origin is determined by the proximity of the developing pectoral girdle to the somites or to the lateral plate mesoderm, respectively. This position-dependent origin and the diversity of the anatomy of the pectoral girdle among vertebrates implies that the embryonic origin of the pectoral girdle is too variable to be useful for defining homologies or for phylogenetic analysis.