Resegmentation in the Mexican axolotl, Ambystoma mexicanum.

The segmental series of somites in the vertebrate embryo gives rise to the axial skeleton. In amniote models, single vertebrae are derived from the sclerotome of two adjacent somites. This process, known as resegmentation, is well-studied using the quail-chick chimeric system, but the presumed generality of resegmentation across vertebrates remains poorly evaluated. Resegmentation has been questioned in anamniotes, given that the sclerotome is much smaller and lacks obvious differentiation between cranial and caudal portions. Here, we provide the first experimental evidence that resegmentation does occur in a species of amphibian. Fate mapping of individual somites in the Mexican axolotl (Ambystoma mexicanum) revealed that individual vertebrae receive cells from two adjacent somites as in the chicken. These findings suggest that large size and segmentation of the sclerotome into distinct cranial and caudal portions are not requirements for resegmentation. Our results, in addition to those for zebrafish, indicate that resegmentation is a general process in building the vertebral column in vertebrates, although it may be achieved in different ways in different groups.

Development and evolution of the muscles of the pelvic fin.

Locomotor strategies in terrestrial tetrapods have evolved from the utilisation of sinusoidal contractions of axial musculature, evident in ancestral fish species, to the reliance on powerful and complex limb muscles to provide propulsive force. Within tetrapods, a hindlimb-dominant locomotor strategy predominates, and its evolution is considered critical for the evident success of the tetrapod transition onto land. Here, we determine the developmental mechanisms of pelvic fin muscle formation in living fish species at critical points within the vertebrate phylogeny and reveal a stepwise modification from a primitive to a more derived mode of pelvic fin muscle formation. A distinct process generates pelvic fin muscle in bony fishes that incorporates both primitive and derived characteristics of vertebrate appendicular muscle formation. We propose that the adoption of the fully derived mode of hindlimb muscle formation from this bimodal character state is an evolutionary innovation that was critical to the success of the tetrapod transition.

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.

Temporal and spatial patterning of axial myotome fibers in Xenopus laevis.

Somites give rise to the vertebral column and segmented musculature of adult vertebrates. The cell movements that position cells within somites along the anteroposterior and dorsoventral axes are not well understood. Using a fate mapping approach, we show that at the onset of Xenopus laevis gastrulation, mesoderm cells undergo distinct cell movements to form myotome fibers positioned in discrete locations within somites and along the anteroposterior axis. We show that the distribution of presomitic cells along the anteroposterior axis is influenced by convergent and extension movements of the notochord. Heterochronic and heterotopic transplantations between presomitic gastrula and early tail bud stages show that these cells are interchangeable and can form myotome fibers in locations determined by the host embryo. However, additional transplantation experiments revealed differences in the competency of presomitic cells to form myotome fibers, suggesting that maturation within the tail bud presomitic mesoderm is required for myotome fiber differentiation.

[The aortic endothelium in the embryo: genesis and role in hematopoiesis]. / L'endothélium aortique chez l'embryon : genèse et rôle dans l'hématopoïèse.

Intra-aortic haematopoiesis is a transient phenomenon, characterised by the emergence of Hematopoietic Stem Cells (HSC) from the ventral aortic endothelium through an endothelial cell (EC) to HSC lineage switch. HSC differentiation is followed by the colonization of definitive haematopoietic organs. Since intra-aortic haematopoiesis is born from EC of the aortic floor, we wondered how vascular integrity was maintained during hematopoietic production. We have used interspecific quail to chick grafts to study the aortic morphogenesis during hematopoiesis. We have demonstrated that: 1) before haematopoiesis, the aortic endothelium, originally entirely from splanchnic origin, was colonized by somitic EC, creating a new roof and sides derived from the somite, whereas the floor was contributed by splanchnopleural-derived EC. 2) As haematopoiesis proceeded, somite-derived EC colonized the aortic floor, where they settled underneath the HSC clusters. 3) After haematopoiesis, splanchnopleural ECs have disappeared from the aortic floor and have been replaced by somite-derived EC. At this stage, the whole aortic endothelium originated from somitic cells. 4) We have identified that the somite contributed to the vascular smooth muscle cells (VSMC). 5) Using grafts of either single quail dermomyotome or sclerotome in the chick, we showed that EC originated from the dermomyotome whereas the vascular smooth muscle cells originated from the sclerotome. Taken together, our results bring about new insights on aorta morphogenesis and the time-restricted production of HSCs.

Sclerotomal origin of vascular smooth muscle cells and pericytes in the embryo.

We previously demonstrated that progenitors of both endothelium and smooth muscle cells in the aortic wall originated from the somite in the trunk of the embryo. However whether the contribution to vascular Smooth Muscle Cells (vSMC) is restricted to the aorta or encompasses other vessels of the trunk is not known. Moreover, the somitic compartment that gives rise to vSMC is yet to be defined. Quail-chick orthotopic transplantations of either the segmental plate or the dorsal or ventral halves from single somites demonstrate that 1 degrees vSMC of the body wall including those of the limbs originate from the somite. 2 degrees Like vSMC, aortic pericytes originate from the somite. 3 degrees The sclerotome is the somite compartment that gives rise to vSMC and pericytes. PAX1 and FOXC2, two molecular markers of the sclerotomal compartment, are expressed by vSMC and pericytes during the earliest phases of vascular wall formation. Later on, PDGFR-beta and MYOCARDIN are also expressed by these cells. In contrast, the dermomyotome gives rise to endothelium but never to cells in the vascular wall. Taken together, out data point out to the critical role of the somite in vessel formation and demonstrate that vSMC and endothelial cells originate from two independent somitic compartments.

Avian pelvis originates from lateral plate mesoderm and its development requires signals from both ectoderm and paraxial mesoderm.

The pelvic girdle is composed of three skeletal elements: ilium, pubis, and ischium. In comparison with other parts of the postcranial skeleton, its development is not well known to date. To elucidate the embryonic origin of the avian pelvic girdle and the signaling centers that control its development, we have performed extirpation and quail-to-chick grafting experiments. The results reveal that the entire pelvic girdle originates from the somatopleure at somite levels 26 to 35. No somitic cell contribution to skeletal elements of the pelvis has been detected. Removal of the surface ectoderm covering the lateral plate mesoderm has revealed that ectodermal signals control the development of the pelvic girdle, especially the formation of the pubis and ischium. The impaired development of the ischium and pubis correlates with the downregulation of Pax1 and Alx4, two transcription factors that control the normal development of the ischium and pubis. Although of somatopleural origin, the development of the ilium depends on somitic signals. Insertion of a barrier between somites and somatopleure disrupts the expression of Emx2 and prevents normal development of the ilium but does not affect the expression of Pax1 or Alx4 and the development of the pubis and ischium. Thus, the development of the ilium, but not of the pubis and ischium, depends on somitic and ectodermal signals.

Role of Wnt-6 in limb myogenesis.

Cells from the ventrolateral lip of the dermomyotome at limb levels undergo epithelio-mesenchymal transition and migrate as individual and undifferentiated cells into the limb buds. The precursor cells are under the influence of various signaling factors in the limb. Dorsal and ventral ectoderm influences various aspects of limb development. In addition to our previous studies, we investigated the influence of ectoderm and Wnt-6 on somitic cells in the limb bud. We show that in the absence of ectoderm the precursor cells never form muscle cells but differentiate into endothelial cells. In addition, we show that Wnt-6 that is secreted from the ectoderm influences the precursor cells to form muscle even in the absence of ectoderm. This indicates that Wnt-6 is an ectodermal signal that induces somite-derived progenitor cells to form muscle cells during limb development.

Asymmetric cell divisions are concentrated in the dermomyotome dorsomedial lip during epaxial primary myotome morphogenesis.

To determine if somitic stem cell pools could be identified by an intrinsic difference in mitotic behaviour, the orientation of mitoses in the dermomyotome epithelium was analysed. We describe a concentration of apico-basal mitoses within the dermomyotome dorsomedial lip (DML). The occurrence of apico-basal divisions is closely associated with asymmetric localisation of the notch pathway factor numb, allowing description of such divisions as asymmetric. In contrast, planar divisions, occurring in the plane of the epithelium, are symmetric. Further, we show that the DML environmental niche is sufficient to promote numb expression in epaxial dermomyotome tissue that does not normally express this factor. These data provide, for the first time, a non-retrospective tracing analysis of the mechanism by which the DML fulfils the stem-cell pool role it plays during epaxial primary myotome morphogenesis.

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.

Normal and aberrant craniofacial myogenesis by grafted trunk somitic and segmental plate mesoderm.

Our research assesses the ability of three trunk mesodermal populations -- medial and lateral halves of newly formed somites, and presomitic (segmental plate) mesenchyme -- to participate in the differentiation and morphogenesis of craniofacial muscles. Grafts from quail donor embryos were placed in mesodermal pockets adjacent to the midbrain-hindbrain boundary, prior to the onset of neural crest migration, in chick host embryos. This encompasses the site where the lateral rectus and the proximal first branchial arch muscle primordia arise. The distribution and differentiation of graft-derived cells were assayed using QCPN and QH1 antibodies to identify all quail cells and quail endothelial cells, respectively. Chimeric embryos were assayed for expression of myf5, myod, paraxis and lbx1, and the synthesis of myosin heavy chain (MyHC), between 1 and 6 days later (stages 14-30). Heterotopic and control (orthotopic) transplants consistently produced invasive angioblasts, and contributed to the lateral rectus and proximal first branchial arch muscles; many also contributed to the dorsal oblique muscle. The spatiotemporal patterns of transcription factor and MyHC expression by these trunk cells mimicked those of normal head muscles. Heterotopic grafts also gave rise to many ectopic muscles. These were observed in somite-like condensations at the implant site, in dense mesenchymal aggregates adjacent to the midbrain-hindbrain boundary, and in numerous small condensations scattered deep to the dorsal margin of the eye. Cells in ectopic condensations expressed trunk transcription factors and differentiated rapidly, mimicking the trunk myogenic timetable. A novel discovery was the formation by grafted trunk mesoderm of many mononucleated myocytes and irregularly oriented myotubes deep to the eye. These results establish that the head environment is able to support the progressive differentiation of several distinct trunk myogenic progenitor populations, over-riding whatever biases were present at the time of grafting. The spatial and temporal control of head muscle differentiation and morphogenesis are very site specific, and head mesoderm outside of these sites is normally refractory to, or inhibited by, the signals that initiate ectopic myogenesis by grafted trunk mesoderm cells.

Differing patterns of neurotrophin-receptor expressing neurons allow distinction of the transient Frorieps' ganglia from normal DRG before morphological differences appear.

The Frorieps' ganglia are dorsal root ganglia (DRG) that form and then degenerate during normal embryonic development of amniotes. Their degeneration or survival has been shown to be modulated by modifying expression of Hox-family and other genes involved in pattern formation, and by the mesodermal microenvironment of the cranial somites in which they develop. In ovo application of the neurotrophin NGF partially rescues DRG2 from degeneration. To further examine the potential role of neurotrophins in the life cycle of Frorieps' DRG we have now quantified the numbers of neurons expressing neurotrophin receptors trkA and trkC in avian Frorieps' ganglia (DRG2) and normal cervical DRG (DRG5). We have found that the Frorieps' DRG are different from normal DRG in terms of the numbers of neurons expressing these receptors. trkC-expressing neurons are generally lacking in DRG2, this is the earliest (St 18, E2.5) described difference between DRG2 and normal DRG, preceding morphological differences between these ganglia that appear at St 20. The difference between DRG2 and DRG5 in terms of numbers of trkA-expressing neurons is evident only at later embryonic stages, where DRG2 contains a higher proportion of trkA neurons than normal cervical DRG. The few trkC+ neurons present late in DRG2 development are not concentrated in the VL portion of the ganglion, the zone where trkC+ neurons are generally found in normal DRG. We also find that DRG2 neurons are smaller than those of normal DRG, this is true for both trkA+ and trkC+ populations. These data together therefore suggest that the neurons that survive in the Frorieps' ganglia at later stages belong almost exclusively to the trkA-expressing DM class DRG neurons. We further find that the differences in the populations of trkA/trkC between DRG2 and DRG5 result from signals from the mesodermal microenvironment, since DRG arising in cranial somites transplanted caudally contain few trkC+ neurons and a higher proportion of trkA+ cells than contralateral controls.

The relationship between limb muscle and endothelial cells migrating from single somite.

Somites contribute myogenic and endothelial precursor cells to the limb bud. Transplantations of single somites have shown the pattern of muscle cell distribution from individual somites to individual limb muscles. However, the pattern of the endothelial cell distribution from individual somites to the limb has not been characterized. We have mapped quail muscle and endothelial cell distribution in the distal part of the chick limb after single somite transplantation to determine if there is a spatial relationship between muscle and endothelial cells originating from the same somite. Single brachial somites from quail donor embryos were transplanted into chick embryos, and, following incubation, serial sections were stained with a quail-endothelial cell-specific monoclonal antibody (QH-1), an anti-quail antibody (QCPN) and an anti-desmin antibody to distinguish the quail endothelial and muscle cells from chick cells. Our results show that transplants of somite 16-21 each gave rise to quail endothelial cells in the wing. The anterioposterior position of the blood vessels formed by somitic endothelial cells corresponded to the craniocaudal position of the somite from which they have originated. Endothelial cells were located not only in the peri- and endomysium but also in the subcutaneous, intermuscular, perineural and periost tissues. There was no strict correlation between the distribution of muscle and endothelial cell from a single transplanted somite. Blood vessels formed by grafted quail endothelial cells could invade the muscle that did not contain any quail muscle cells, and conversely a muscle composed of numerous quail muscle cells was lacking any endothelial cells of quail origin. Furthermore, a chimeric limb with very little quail muscle cells was found to contain numerous quail endothelial cells and vice versa. These results suggest that muscle and endothelial cells derived from the same somite migrate on different routes in the developing limb bud.

Persistent myogenic capacity of the dermomyotome dorsomedial lip and restriction of myogenic competence.

The dorsomedial lip (DML) of the somite dermomyotome is the source of cells for the early growth and morphogenesis of the epaxial primary myotome and the overlying dermomyotome epithelium. We have used quail-chick transplantation to investigate the mechanistic basis for DML activity. The ablated DML of chick wing-level somites was replaced with tissue fragments from various mesoderm regions of quail embryos and their capacity to form myotomal tissue assessed by confocal microscopy. Transplanted fragments from the epithelial sheet region of the dermomyotome exhibited full DML growth and morphogenetic capacity. Ventral somite fragments (sclerotome), head paraxial mesoderm or non-paraxial (lateral plate) mesoderm tested in this assay were each able to expand mitotically in concert with the surrounding paraxial mesoderm, although no myogenic potential was evident. When ablated DMLs were replaced with fragments of the dermomyotome ventrolateral lip of wing-level somites or pre-somitic mesoderm (segmental plate), myotome development was evident but was delayed or otherwise limited in some cases. Timed DML ablation-replacement experiments demonstrate that DML activity is progressive throughout the embryonic period (to at least E7) and its continued presence is necessary for the complete patterning of each myotome segment. The results of serial transplantation and BrdU pulse-chase experiments are most consistent with the conclusion that the DML consists of a self-renewing population of progenitor cells that are the primary source of cells driving the growth and morphogenesis of the myotome and dermomyotome in the epaxial domain of the body.

Evidence for medial/lateral specification and positional information within the presomitic mesoderm.

In the vertebrate embryo, segmentation is built on repetitive structures, named somites, which are formed progressively from the most rostral part of presomitic mesoderm, every 90 minutes in the avian embryo. The discovery of the cyclic expression of several genes, occurring every 90 minutes in each presomitic cell, has shown that there is a molecular clock linked to somitogenesis. We demonstrate that a dynamic expression pattern of the cycling genes is already evident at the level of the prospective presomitic territory. The analysis of this expression pattern, correlated with a quail/chick fate-map, identifies a 'wave' of expression travelling along the future medial/lateral presomitic axis. Further analysis also reveals the existence of a medial/lateral asynchrony of expression at the level of presomitic mesoderm. This work suggests that the molecular clock is providing cellular positional information not only along the anterior/posterior but also along the medial/lateral presomitic axis. Finally, by using an in vitro culture system, we show that the information for morphological somite formation and molecular segmentation is segregated within the medial/lateral presomitic axis. Medial presomitic cells are able to form somites and express segmentation markers in the absence of lateral presomitic cells. By contrast, and surprisingly, lateral presomitic cells that are deprived of their medial counterparts are not able to organise themselves into somites and lose the expression of genes known to be important for vertebrate segmentation, such as Delta-1, Notch-1, paraxis, hairy1, hairy2 and lunatic fringe.

Morphogenesis of gut and distribution of the progenitors of gut endocrine cells at cranial somite levels of the chick embryo.

The primary objective of this study was to establish the distribution of the progenitors of selected gut endocrine cell types at cranial somite levels. In addition, analysis of the material has provided new information about the location of the presumptive territories of certain gut regions and of the pancreas. Narrow transverse strips of full-thickness blastoderm two or three somites in length were excised at the levels of somites 1 to 5 of 8.5- to 18-somite chick embryos and cultured as chorioallantoic grafts to an age equivalent to 20 days of incubation. The grafts were analysed by immunocytochemistry, and their morphology was evaluated. Individual grafts exhibited up to five different types of gut morphology, including those of oesophagus, proventriculus, gizzard, pyloric region, small intestine, and pancreas. The morphologic survey yielded new information about the location, extent, or both, of the territories of the pyloric region, the small intestine, and the pancreas. In general, the progenitors of gut endocrine cell types identified were those expected for the different morphologic regions: in only a few instances were ectopic endocrine cell types detected. The available evidence points to the progenitors of bombesin/gastrin-releasing peptide cells being located cranial to somite 5 at the stages studied. Based on the morphology and the proportion of insulin cells, the development of pancreas in grafts appeared compromised compared with grafts of the intact dorsal pancreatic bud: this may relate to the likely exclusion of dorsal pancreatic bud mesoderm from the graft area. The results show that presumptive small intestinal endoderm in grafts can differentiate in the absence of homologous (i.e., small intestinal) mesoderm: this accords with the view that the primary source of positional information in the gut is in the endoderm.

The dermomyotome dorsomedial lip drives growth and morphogenesis of both the primary myotome and dermomyotome epithelium.

The cellular and molecular mechanisms that govern early muscle patterning in vertebrate development are unknown. The earliest skeletal muscle to organize, the primary myotome of the epaxial domain, is a thin sheet of muscle tissue that expands in each somite segment in a lateral-to-medial direction in concert with the overlying dermomyotome epithelium. Several mutually contradictory models have been proposed to explain how myotome precursor cells, which are known to reside within the dermomyotome, translocate to the subjacent myotome layer to form this first segmented muscle tissue of the body. Using experimental embryology to discriminate among these models, we show here that ablation of the dorsomedial lip (DML) of the dermomyotome epithelium blocks further primary myotome growth while ablation of other dermomyotome regions does not. Myotome growth and morphogenesis can be restored in a DML-ablated somite of a host embryo by transplantation of a second DML from a donor embryo. Chick-quail marking experiments show that new myotome cells in such recombinant somites are derived from the donor DML and that cells from other regions of the somite are neither present nor required. In addition to the myotome, the transplanted DML also gives rise to the dermomyotome epithelium overlying the new myotome growth region and from which the mesenchymal dermatome will later emerge. These results demonstrate that the DML is a cellular growth engine that is both necessary and sufficient to drive the growth and morphogenesis of the primary myotome and simultaneously drive that of the dermomyotome, an epithelium containing muscle, dermis and possibly other potentialities.