Mediolateral somitic origin of ribs and dermis determined by quail-chick chimeras

Somites are transient mesodermal structures giving rise to all skeletal muscles of the body, the axial skeleton and the dermis of the back. Somites arise from successive segmentation of the presomitic mesoderm (PSM). They appear first as epithelial spheres that rapidly differentiate into a ventral mesenchyme, the sclerotome, and a dorsal epithelial dermomyotome. The sclerotome gives rise to vertebrae and ribs while the dermomyotome is the source of all skeletal muscles and the dorsal dermis. Quail-chick fate mapping and diI-labeling experiments have demonstrated that the epithelial somite can be further subdivided into a medial and a lateral moiety. These two subdomains are derived from different regions of the primitive streak and give rise to different sets of muscles. The lateral somitic cells migrate to form the musculature of the limbs and body wall, known as the hypaxial muscles, while the medial somite gives rise to the vertebrae and the associated epaxial muscles. The respective contribution of the medial and lateral somitic compartments to the other somitic derivatives, namely the dermis and the ribs has not been addressed and therefore remains unknown. We have created quail-chick chimeras of either the medial or lateral part of the PSM to examine the origin of the dorsal dermis and the ribs. We demonstrate that the whole dorsal dermis and the proximal ribs exclusively originates from the medial somitic compartment, whereas the distal ribs derive from the lateral compartment.     

Determination of somite cells: independence of cell differentiation and morphogenesis

Somites are mesodermal structures which appear transiently in vertebrates in the course of their development. Cells situated ventromedially in a somite differentiate into the sclerotome, which gives rise to cartilage, while the other part of the somite differentiates into dermomyotome which gives rise to muscle and dermis. The sclerotome is further divided into a rostral half, where neural crest cells settle and motor nerves grow, and a caudal half. To find out when these axes are determined and how they rule later development, especially the morphogenesis of cartilage derived from the somites, we transplanted the newly formed three caudal somites of 2.5-day-old quail embryos into chick embryos of about the same age, with reversal of some axes. The results were summarized as follows. (1) When transplantation reversed only the dorsoventral axis, one day after the operation the two caudal somites gave rise to normal dermomyotomes and sclerotomes, while the most rostral somite gave rise to a sclerotome abnormally situated just beneath ectoderm. These results suggest that the dorsoventral axis was not determined when the somites were formed, but began to be determined about three hours after their formation. (2) When the transplantation reversed only the rostrocaudal axis, two days after the operation the rudiments of dorsal root ganglia were formed at the caudal (originally rostral) halves of the transplanted sclerotomes. The rostrocaudal axis of the somites had therefore been determined when the somites were formed. (3) When the transplantation reversed both the dorsoventral and the rostrocaudal axes, two days after the operation, sclerotomes derived from the prospective dermomyotomal region of the somites were shown to keep their original rostrocaudal axis, judging from the position of the rudiments of ganglia. Combined with results 1 and 2, this suggested that the fate of the sclerotomal cells along the rostrocaudal axis was determined previously and independently of the determination of somite cell differentiation into dermomyotome and sclerotome. (4) In the 9.5-day-old chimeric embryos with rostrocaudally reversed somites, the morphology of vertebrae and ribs derived from the explanted somites were reversed along the rostrocaudal axis. The morphology of cartilage derived from the somites was shown to be determined intrinsically in the somites by the time these were formed from the segmental plate. The rostrocaudal pattern of the vertebral column is therefore controlled by factors intrinsic to the somitic mesoderm, and not by interactions between this mesoderm and the notochord and/or neural tube, arising after segmentation.     

Somite development without influence of the surface ectoderm in the chick embryo: the compartments of a somite responsible for distal rib development

In the development of the somite, signals from neighboring tissues have been suggested to play critical roles. We have found that when interaction between the ectoderm and the somite is blocked by inserting a piece of polyethylene terephatalate film between them in 2-day-chicken embryo, one of the derivatives of somite, the distal rib, did not form. We examined somite development after the operation, to know the correlation between somite development and distal rib formation. In the operated embryo, the dermomyotome was medio-laterally shorter than in the normal embryo, and Pax3 and Sim1 expressions that are seen in the lateral part of normal dermomyotomes were not found, suggesting that the lateral part of the dermomyotome was missing. Although the sclerotome appeared to be normal in its histology and Pax1 expression pattern in the operated embryo, we could not detect the expression of either Scleraxis nor γ-FBP that are expressed in the cells around the boundaries between the adjacent dermomyotomes in normal embryos. Thus, under the influence of surface ectoderm, the lateral part of dermomyotome and/or the mesenchyme around rostral and caudal edges of dermomyotomes are suggested to play an important role in the distal rib development.     

Contribution of somitic cells to the avian ribs

The traditional view that all parts of the ribs originate from the sclerotome of the thoracic somites has recently been challenged by an alternative view suggesting that only the proximal rib derives from the sclerotome, while the distal rib arises from regions of the dermomyotome. In view of this continuing controversy and to learn more about the cell interactions during rib morphogenesis, this study aimed to reveal the precise contributions made by somitic cells to the ribs and associated tissues of the thoracic cage. A replication-deficient lacZ-encoding retrovirus was utilized to label cell populations within distinct regions of somites 19-26 in stage 13-18 chick embryos. Analysis of the subsequent contributions made by these cells revealed that the thoracic somites are the sole source of cells for the ribs. More precisely, it is the sclerotome compartment of the somites that contributes cells to both the proximal and distal elements of the ribs, confirming the traditional view of the origin of the ribs. Results also indicate that the precursor cells of the ribs and intercostal muscles are intimately associated within the somite, a relationship that may be essential for proper rib morphogenesis. Finally, the data from this study also show that the distal ribs are largely subject to resegmentation, although cell mixing may occur at the most sternal extremities.     

Three developmental compartments involved in rib formation

When the thoracic somitic mesoderm was separated from the neural tube and the notochord with a piece of aluminum foil in two-day chick embryos, seven days after the operation ribs lacked their proximal part. The embryos were rescued by co-transplanting the notochord, the ventral half of neural tube, or QT6 cells transformed with Shh, on the somite side of the aluminum foil insert. Thus, proximal rib development depends on the notochord and the ventral neural tube, an effect which might be mediated through Shh secreted by these axial tissues. On the other hand, when the thoracic somitic mesoderm was separated from the surface ectoderm by a piece of polyethylene terephthalate film, the distal parts of the ribs were missing, suggesting that distal rib development depends on surface ectoderm. In these embryos, expression of Pax3 was weak and perturbed showing that the dermomyotome developed abnormally. It is not clear whether the development of distal rib is mediated by the dermomyotome, or the ectoderm. It has previously been shown that sternal rib development depends on lateral plate mesoderm. As to the distal rib, it is considered to be composed of two parts. Thus, the rib is composed of three developmental compartments, in agreement with a recently presented classification of somite derivatives as primaxial and abaxial.     

Paradox segmentation along inter- and intrasomitic borderlines is followed by dysmorphology of the axial skeleton in the open brain (opb) mouse mutant

In open brain (opb) mutant embryos, developmental defects of the trunk spinal cord were spatially correlated with severe defects of the epaxial somite derivatives including sclerotomes, whereas hypaxial somite derivatives are much less affected. Later in development, the neural arches (epaxial sclerotome derivatives) formed but were severely disorganized, and also the distal ribs (hypaxial sclerotome derivatives) were malformed. Adjacent neural arches and vertebral bodies were often fused where joints should have formed suggesting defects of the intrasomitic borderlines. Moreover, neural arches frequently and ribs sometimes were split into halves at distinct levels along the dorso-ventral body axis. This suggests that ‘resegmentation’ of sclerotomes across the somite borders did not completely occur. These prominent skeletal defects were preceded by reduced expression of Pax1 along the intrasomitic borderlines, and incomplete maintenance of somite borders between central sclerotome moieties. The defects of the axial skeleton were accompanied by segmentation defects of the myotomes which were split distally, and also partly fused from adjacent segments across somite borders. The segmentation defects observed suggest that in opb mutants both segmental borderlines, the somite borders and the intrasomitic borderlines (fissures), were affected and behaved paradoxically. Dev. Genet. 22:359–373, 1998. © 1998 Wiley-Liss, Inc.     

Developmental Plasticity of the Prospective Dermatome and the Prospective Sclerotome Region of an Avian Somite

The ventro-medial wall of a somite gives rise to the sclerotome and then to cartilaginous axial skeleton, while the dorso-lateral wall differentiates into the dermomyotome to form dermal mesenchyme and muscle. Although previous studies suggested pluri-potency of somite cell differentiation, apparent pluri-potency may be the result of migration of predetermined cells. To investigate whether the developmental fate of any region is determined, I isolated fragments of a region of a quail somite and transplanted them into chick embryos. When a fragment of the ventral wall of a quail somite, the prospective sclerotome, was transplanted into a chick embryo between the ectoderm and a newly formed somite, the transplanted quail cells were shown to form myotome and mesenchyme in 4-day chimera embryos and to form muscle and dermal tissue in 9-day chimeras. On the other hand, when a fragment of the dorsal wall of a quail somite, the prospective dermomyotome, was transplanted into a chick embryo between the neural tube and a newly formed somite, the graft gave rise to mesenchyme around the neural tube and notochord and then to vertebral cartilage. Thus the developmental fate of a region of a somite was shown not to be determined at the time of somite segmentation, confirming previous observations.     

The concerted action of Meox homeobox genes is required upstream of genetic pathways essential for the formation,patterning and differentiation of somites

The paraxial mesoderm of the somites of the vertebrate embryo contains the precursors of the axial skeleton, skeletal muscles and dermis. The Meox1 and Meox2 homeobox genes are expressed in the somites and their derivatives during embryogenesis. Mice homozygous for a null mutation in Meox1 display relatively mild defects in sclerotome derived vertebral and rib bones, whereas absence of Meox2 function leads to defective differentiation and morphogenesis of the limb muscles. By contrast, mice carrying null mutations for both Meox genes display a dramatic and wide-ranging synthetic phenotype associated with extremely disrupted somite morphogenesis, patterning and differentiation. Mutant animals lack an axial skeleton and skeletal muscles are severely deficient. Our results demonstrate that Meox1 and Meox2 genes function together and upstream of several genetic hierarchies that are required for the development of somites. In particular, our studies place Meox gene function upstream of Pax genes in the regulation of chondrogenic and myogenic differentiation of paraxial mesoderm.     

Differential regulation of the chick dorsal thoracic dermal progenitors from the medial dermomyotome

The chick dorsal feather-forming dermis originates from the dorsomedial somite and its formation depends primarily on Wnt1 from the dorsal neural tube. We investigate further the origin and specification of dermal progenitors from the medial dermomyotome. This comprises two distinct domains: the dorsomedial lip and a more central region (or intervening zone) that derives from it. We confirm that Wnt1 induces Wnt11 expression in the dorsomedial lip as previously shown, and show using DiI injections that some of these cells, which continue to express Wnt11 migrate under the ectoderm, towards the midline, to form most of the dorsal dermis. Transplantation of left somites to the right side to reverse the mediolateral axis confirms this finding and moreover suggests the presence of an attractive or permissive environment produced by the midline tissues or/and a repellent or inadequate environment by the lateral tissues. By contrast, the dorsolateral dermal cells just delaminate from the surface of the intervening space, which expresses En1. Excision of the axial organs or the ectoderm, and grafting of Wnt1-secreting cells, shows that, although the two populations of dermal progenitors both requires Wnt1 for their survival, the signalling required for their specification differs. Indeed Wnt11 expression relies on dorsal neural tube-derived Wnt1, while En1 expression depends on the presence of the ectoderm. The dorsal feather-forming dermal progenitors thus appear to be differentially regulated by dorsal signals from the neural tube and the ectoderm, and derive directly and indirectly from the dorsomedial lip. As these two dermomyotomal populations are well known to also give rise to epaxial muscles, an isolated domain of the dermomyotome that contains only dermal precursors does not exist and none of the dermomyotomal domains can be considered uniquely as a dermatome.     

Sequential activation of butyrylcholinesterase in rostral half somites and acetylcholinesterase in motoneurones and myotomes preceding growth of motor axons

By applying double-staining procedures that combine cholinesterase histochemistry (acetyl- and butyrylcholinesterase, respectively) as indicators of neuronal and myotomal tissue differentiation on longitudinal sections, together with detection of motor axons with antibodies to G4 antigen, we here describe the spatiotemporal expression of all components of the segmental motor units along the trunk of chicken embryos between stages 16-20. In particular, BChE expression is spatially elevated on the rostral part of the differentiating somite. About 2-3 somites more rostrally (and thus developmentally later), AChE is expressed almost simultaneously in a nonsegmented fashion in neuronal cell bodies of the ventral horn and in the corresponding dermomyotomes. There it is first detectable in a rostromedial sector. With a delay (4-6 somites compared with AChE in motoneurones), motor axons begin to grow exclusively through the BChE-rich sclerotomal space towards the AChE-activated myotome anlage. On motor axons, AChE detection is significantly retarded. We conclude that the rostrocaudal segmental asymmetry is not restricted to the sclerotomes (which other authors have described before by using different markers), but it extends into the dermomyotome, in which cholinesterases introduce an early subdivision. Hence, the entire process of first myotome differentiation, motor axon growth and establishment of first target contacts are taking place within the rostral half somite. We suggest that both cholinesterases might be involved in processes of motor unit differentiation and fibre guidance.     

Extirpation experiments on embryonic rudiments of the carapace of Chelydra serpentina

Somites, along with adjacent neural tube and overlying ectoderm, were extirpated unilaterally from embryos of Chelydra serpentina. Mesoderm of three somites was removed from various levels. The operations included the last formed somite and were done on embryos with 12 to 22 pairs of somites. In practice it was found that ventromedial portions of the somites were not included in the extirpation. The animals were preserved before pigmentation became heavy. The cartilaginous skeleton was stained selectively. The extirpations resulted in depletions of ribs consonant with relating the second rib to the fourteenth somite. The somites behaved as mosaics; they did not reconstitute each other nor did they regenerate after partial extirpation. The rudiments for the ribs were separable from the rudiments of the vertebrae, the sclerotomes, and were found to arise from a more lateral portion of the somite. The scutes are ectodermal derivatives, which are held to be dependent upon underlying somitic mesoderm for their differentiation. The extirpations resulted in abnormalities and depletions of scutes.     

Pathways and mechanisms of avian trunk neural crest cell migration and localization

Crest cells individualized at the dorsal border of the neural tube, while they became surrounded by a fibronectin-rich matrix. Crest cells initiated their migration between the basement membranes of the neural tube and the ectoderm. In the vagal region, crest cells migrated in a fibronectin-rich environment between the ectoderm and the dermomyotome, very rapidly reaching the apex of the pharynx. In the trunk region, crest cells opposite the bulk of the somite accumulated at the junction between the somite, the neural tube, and the ectoderm; they resumed their migration at the onset of the dissociation of the somite into dermomyotome and sclerotome. Migration occurred more ventrally along the neural tube; nevertheless, the formation of the rapidly expanding sclerotome prevented crest cells from reaching the paranotochordal region. Thereafter, crest cells accumulated between the neural tube, the dermomyotome, and the sclerotome, where ultimately they formed the dorsal root ganglia. In contrast, cells opposite the intersomitic space did not encounter these obstacles and utilized a narrow pathway formed between the basement membranes of the two adjacent somites. This pathway allowed crest cells to reach the most ventral regions of the embryo very rapidly; they accumulated along the aorta to form the aortic plexuses, the adrenal medulla, and the sympathetic ganglia. The basic features of the migration pathways are (1) a strict delimitation by the fibronectin-rich basement membranes of the surrounding tissues, (2) a formation of space concomitant with the migration of crest cells, (3) a transient existence: continued migration is correlated with the presence of fibronectin, whereas cessation is correlated with its focal disappearance. The crest cells are characterized by their inability to traverse basement membranes and penetrate within tissues. We propose that the combination of active proliferation, unique motility properties, and the presence of narrow pathways are the major mechanisms ensuring correct directionality. Morphologically defined transient routes of migration along with developmentally regulated changes in the extracellular matrix and in the adhesive properties of crest cells are most probably involved in their stabilization in defined territories and their aggregation into ganglia.     

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° vSMC of the body wall including those of the limbs originate from the somite. 2° Like vSMC, aortic pericytes originate from the somite. 3° 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-β 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.     

The axial skeleton of the labyrinthodont Eogyrinus attheyi

An articulated length of vertebral column is used as a basis for the reconstruction of the salient features of the axial skeleton of the embolomerous anthracosaur Eogyrinus attheyi Watson, together with other material, including the holotype, in the Hancock Museum, Newcastle upon Tyne.
The trunk vertebrae are typically emboloinerous, with disoshaped notochordal pleuro-centra, firmly attached by broad facets to this neural arches, and much thinner intercentra. Regional variation is chiefly concerned with the span of the transverse processes, which diminishes posteriorly, and the associated separation of the two heads of each rib. A longitudinal series of trunk ribs, of diminishing length from the mid-trunk backwards, is reconstructed.
Eogyrinus has a normal tetrapod sacrum with one characteristic sacral rib. The first few caudal vertebrae bear ribs of unusual form, (of which four are preserved in sequence in the articulated specimen. The fifth caudal intercentrum bears the first and largest haemal arch and the pleurocentrum of the seventh caudal is distinguished by marked muscle origins presumably for the caudifemoral muscles.
The probability that Eogyrinus, like the few other embolomeres known, had an unusually long vertebral column for a labyrinthodont, is supported by an orthometric comparison using Romer's data on the American form Archeria.