Scanning electron microscopic evidence for physical segmental boundaries in the anterior presomitic mesoderm.

The metameric pattern of the axial skeleton is established during embryogenesis by somite formation from the unsegmented paraxial mesoderm (presomitic mesoderm). Here, we have investigated the morphology of the anterior presomitic mesoderm of chick embryos using scanning electron microscopy. We found periodically arranged transverse clefts in the anterior region of the presomitic mesoderm. These gaps can be regarded as physical boundaries between prospective somites in the determined zone of the presomitic mesoderm. This study provides additional evidence suggesting that prospective somite boundaries are not only marked by defined zones of gene expression, but are also accompanied by changes in cellular morphology that give rise to identifiable morphological segments.

Temporal sequence in the formation of midline dermis and dorsal vertebral elements in avian embryos.

Somites compartmentalize into a dorsal epithelial dermomyotome and a ventral mesenchymal sclerotome. While sclerotomes give rise to vertebrae and intervertebral discs, dermomyotomes contribute to skeletal muscle and epaxial dermis. Bone morphogenetic protein (BMP)-signals from the lateral mesoderm induce the lateral portion of the dermomyotome to form chondrogenic precursor cells, forming the cartilage of the scapula blade. The fact that BMPs are expressed in the roof plate of the neural tube where they induce cartilage formation led to the question why cells migrating from the medial part of the dermomyotome do not undergo chondrogenic differentiation and do not contribute to the dorsal part of the vertebrae. In the present study, we traced dermomyotomal derivatives by using the quail-chick marker technique. Our study reveals a temporal sequence in the formation of the vertebral cartilage and the midline dermis. The dorsal mesenchyme overlying the roof plate of the neural tube is formed prior to the de-epithelialization of the dermomyotome. Dermomyotomal cells start to migrate medially into the sub-ectodermal space to form the midline dermis after chondrogenesis of the dorsal mesenchyme has occurred. This time delay between chondrogenesis of the dorsal vertebra and dermal formation allows an undisturbed development of these two tissue components within a narrow region of the embryo.

Cellular and molecular investigations into the development of the pectoral girdle.

The forelimbs of higher vertebrates are composed of two portions: the appendicular region (stylopod, zeugopod and autopod) and the less prominent proximal girdle elements (scapula and clavicle) that brace the limb to the main trunk axis. We show that the formation of the muscles of the proximal limb occurs through two distinct mechanisms. The more superficial girdle muscles (pectoral and latissimus dorsi) develop by the "In-Out" mechanism whereby migration of myogenic cells from the somites into the limb bud is followed by their extension from the proximal limb bud out onto the thorax. In contrast, the deeper girdle muscles (e.g. rhomboideus profundus and serratus anterior) are induced by the forelimb field which promotes myotomal extension directly from the somites. Tbx5 inactivation demonstrated its requirement for the development of all forelimb elements which include the skeletal elements, proximal and distal muscles as well as the sternum in mammals and the cleithrum of fish. Intriguingly, the formation of the diaphragm musculature is also dependent on the Tbx5 programme. These observations challenge our classical views of the boundary between limb and trunk tissues. We suggest that significant structures located in the body should be considered as components of the forelimb.

Commitment of chondrogenic precursors of the avian scapula takes place after epithelial-mesenchymal transition of the dermomyotome.

BACKGROUND: Cells of the epithelially organised dermomyotome are traditionally believed to give rise to skeletal muscle and dermis. We have previously shown that the dermomyotome can undergo epithelial-mesenchymal transition (EMT) and give rise to chondrogenic cells, which go on to form the scapula blade in birds. At present we have little understanding regarding the issue of when the chondrogenic fate of dermomyotomal cells is determined. Using quail-chick grafting experiments, we investigated whether scapula precursor cells are committed to a chondrogenic fate while in an epithelial state or whether commitment is established after EMT. RESULTS: We show that the hypaxial dermomyotome, which normally forms the scapula, does not generate cartilaginous tissue after it is grafted to the epaxial domain. In contrast engraftment of the epaxial dermomyotome to the hypaxial domain gives rise to scapula-like cartilage. However, the hypaxial sub-ectodermal mesenchyme (SEM), which originates from the hypaxial dermomyotome after EMT, generates cartilaginous elements in the epaxial domain, whereas in reciprocal grafting experiments, the epaxial SEM cannot form cartilage in the hypaxial domain. CONCLUSIONS: We suggest that the epithelial cells of the dermomyotome are not committed to the chondrogenic lineage. Commitment to this lineage occurs after it has undergone EMT to form the sub-ectodermal mesenchyme.

The occipital lateral plate mesoderm is a novel source for vertebrate neck musculature.

In vertebrates, body musculature originates from somites, whereas head muscles originate from the cranial mesoderm. Neck muscles are located in the transition between these regions. We show that the chick occipital lateral plate mesoderm has myogenic capacity and gives rise to large muscles located in the neck and thorax. We present molecular and genetic evidence to show that these muscles not only have a unique origin, but additionally display a distinct temporal development, forming later than any other muscle group described to date. We further report that these muscles, found in the body of the animal, develop like head musculature rather than deploying the programme used by the trunk muscles. Using mouse genetics we reveal that these muscles are formed in trunk muscle mutants but are absent in head muscle mutants. In concordance with this conclusion, their connective tissue is neural crest in origin. Finally, we provide evidence that the mechanism by which these neck muscles develop is conserved in vertebrates.

Ectodermal Wnt6 is an early negative regulator of limb chondrogenesis in the chicken embryo.

BACKGROUND: Pattern formation of the limb skeleton is regulated by a complex interplay of signaling centers located in the ectodermal sheath and mesenchymal core of the limb anlagen, which results, in the forelimb, in the coordinate array of humerus, radius, ulna, carpals, metacarpals and digits. Much less understood is why skeletal elements form only in the central mesenchyme of the limb, whereas muscle anlagen develop in the peripheral mesenchyme ensheathing the chondrogenic center. Classical studies have suggested a role of the limb ectoderm as a negative regulator of limb chondrogenesis. RESULTS: In this paper, we investigated the molecular nature of the inhibitory influence of the ectoderm on limb chondrogenesis in the avian embryo in vivo. We show that ectoderm ablation in the early limb bud leads to increased and ectopic expression of early chondrogenic marker genes like Sox9 and Collagen II, indicating that the limb ectoderm inhibits limb chondrogenesis at an early stage of the chondrogenic cascade. To investigate the molecular nature of the inhibitory influence of the ectoderm, we ectopically expressed Wnt6, which is presently the only known Wnt expressed throughout the avian limb ectoderm, and found that Wnt6 overexpression leads to reduced expression of the early chondrogenic marker genes Sox9 and Collagen II. CONCLUSION: Our results suggest that the inhibitory influence of the ectoderm on limb chondrogenesis acts on an early stage of chondrogenesis upsteam of Sox9 and Collagen II. We identify Wnt6 as a candidate mediator of ectodermal chondrogenic inhibition in vivo. We propose a model of Wnt-mediated centripetal patterning of the limb by the surface ectoderm.

Pleiotrophin is expressed in avian somites and tendon anlagen.

Pleiotrophin (Ptn) is a secreted, developmentally regulated growth factor associated with the extracellular matrix. During mammalian embryogenesis, Ptn has been suggested to play a role in the development of various embryonic structures including nervous system and skeleton. In the avian embryo, Ptn has been proposed to be involved in limb cartilage development, but embryonic Ptn expression has not been comprehensively studied. We isolated a cDNA fragment containing the full-length coding sequence of chick Ptn and studied the expression of Ptn in detail until embryonic day 10. We, furthermore, isolated a 6,385-bp phage clone containing the Ptn cDNA of 2,551 bp and additional 3,787 bp downstream of the published Ptn cDNA sequence classifying a yet Ptn-unrelated chEST clone as the 3' untranslated region of Ptn. Our studies revealed novel expression domains in developing somites and during limb formation. We found prominent expression in the somitocoel cells of epithelial somites, and in a sclerotomal subcompartment, the syndetome, which gives rise to the axial tendons in the vertebral motion segment. In the limbs, Ptn was markedly expressed in tendon anlagen and in phalangeal joints. Our results introduce Ptn as a novel marker gene in avian somite and tendon development.

Remodeling of aortic smooth muscle during avian embryonic development.

The dorsal aorta is the earliest formed intraembryonic blood vessel in vertebrates composed of an inner lining of endothelial cells (ECs) and a slightly later-forming outer wall consisting of vascular smooth muscle cells (SMCs) and pericytes. We previously identified the sclerotome as the only somitic compartment contributing to aortic SMCs in the trunk of the avian embryo. However, we demonstrated that the first SMCs in the aortic floor are not of somitic origin and must be derived from a different source. Here, we show that the primary SMCs are a transient population of aortic wall cells originating from the splanchnic mesoderm. A model is presented suggesting that wall formation of the early dorsal aorta in chick is a two-step process: The primary, transient SMCs in the aortic floor originate in the splanchnic mesoderm, whereas the secondary, definitive SMCs of the entire aortic wall originate in the sclerotome.

Wnt signaling in somite development.

During vertebrate embryogenesis, specialized mesodermal structures, called somites, give rise to a variety of mesodermal tissues including skeletal muscles, vertebrae and dermis. Development of the somites is a rhythmic process that involves a series of steps including segmentation of the paraxial mesoderm, epithelialization, somite formation, somite maturation, somite patterning and differentiation of somitic cells into different lineages. Wnt signaling has been found to play crucial roles in multiple steps of somite development. In this review, we present a brief overview of current knowledge on Wnt signaling events during the development of somites and their derivatives.

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.

Formation and differentiation of avian somite derivatives.

During somite maturation, the ventral half of the epithelial somite disintegrates into the mesenchymal sclerotome, whereas the dorsal half forms a transitory epithelial sheet, the dermomyotome, lying in between the sclerotome and the surface ectoderm. The dermomyotome is the source of the majority of the mesodermal tissues in the body, giving rise to cell types as different as muscle, connective tissue, endothelium and cartilage. Thus, the dermomyotome is the most important turntable of mesodermal cell fate choice in the vertebrate embryo. Sclerotome development is characterized by a cranio-caudal polarization, resegmentation and axial identity. Its formation is controlled by signals from the notochord, the neural tube, the lateral plate mesoderm and the myotome. These signals and cross-talk between somite cells lead to the separation of various subdomains, like the central, ventral, dorsal and lateral sclerotome. Here, we discuss the current knowledge on the formation of the dermomyotome and the mechanisms leading to the development of the various dermomyotomal derivatives, with special emphasis on the development of musculature and dermis. We further discuss the molecular control of sclerotomal subdomain formation and cell type specification.

Sclerotomal origin of smooth muscle cells in the wall of the avian dorsal aorta.

The dorsal aorta is the earliest formed intraembryonic blood vessel. It is composed of an inner lining consisting of endothelial cells and an outer wall consisting of smooth muscle cells (SMCs) and fibrocytes. Aortic SMCs have been suggested to arise from several developmental lineages. Cephalic neural crest provides SMCs of the proximal part of the aorta, and SMCs of the distal part are derived from the paraxial mesoderm. Here, we show by using quail-chick chimerization that in the avian embryo, SMCs in the wall of the dorsal aorta at trunk level arise from the sclerotome. Our findings indicate a two-step process of aortic wall formation. First, non-paraxial mesoderm-derived mural cells accumulate at the floor of the aorta. We refer to these cells as primary SMCs. Second, SMCs from the sclerotome are recruited to the roof and sides of the aorta, eventually replacing the primary SMCs in the aortic floor.

Expression pattern of Dll4 during chick embryogenesis.

Notch and Delta signaling regulates cell-fate decisions in a variety of tissues in diverse organisms through cell-to-cell interactions. In this study we isolated a 696 bp fragment of chick Delta-like 4 (Dll4) cDNA and analyzed its expression pattern during chick development by in situ hybridization. We report a detailed description of cDll4 expression from HH-stage 8-30. Expression is seen in extraembryonic tissues and in the dorsal aorta throughout development but is absent from venules. Dll4 is expressed in the embryonic blood vessels, heart, somites, neural tube, limb, pharyngeal arches, esophagus, and in the developing eye. In accordance with the report from mice, cDll4 is a marker of the arterial type of endothelial cells. These analyses show that Dll4 is expressed in a wide range of tissues and organs suggesting its role in vascular development during chick embryogenesis.

Amniote somite derivatives.

Somites are segments of paraxial mesoderm that give rise to a multitude of tissues in the vertebrate embryo. Many decades of intensive research have provided a wealth of data on the complex molecular interactions leading to the formation of various somitic derivatives. In this review, we focus on the crucial role of the somites in building the body wall and limbs of amniote embryos. We give an overview on the current knowledge on the specification and differentiation of somitic cell lineages leading to the development of the vertebral column, skeletal muscle, connective tissue, meninges, and vessel endothelium, and highlight the importance of the somites in establishing the metameric pattern of the vertebrate body.

FGFs, Wnts and BMPs mediate induction of VEGFR-2 (Quek-1) expression during avian somite development.

Regulation of VEGFR-2 (Quek1) is an important mechanism during blood vessel formation. In the paraxial mesoderm, Quek1 expression is restricted to the lateral portion of the somite and later to sclerotomal cells surrounding the neural tube. By implanting FGF 8b/8c or SU 5402 beads into the paraxial mesoderm, we show that FGF8 in addition to BMP4 from the intermediate mesoderm (IM) is a positive regulator of VEGFR-2 (Quek1) expression in the quail embryo. The expression of Quek1 in the medial somite half is normally repressed by the notochord and Sfrps-expression in the neural tube. Over-expression of Wnt 1/3a also results in an up-regulation of Quek1 expression in the somites. We also show that up-regulation of FGF8/Wnt 1/3a leads to an increase in the number of endothelial cells, whereas inhibition of FGF and Wnt signaling by SU 5402 and Sfrp-2 results in a loss of endothelial cells. Our results demonstrate that the regulation of Quek1 expression in the somites is mediated by the cooperative actions of BMP4, FGF8 and Wnt-signaling pathways.

Expression pattern of Vasohibin during chick development.

Vasohibin is an angiogenesis inhibitor that is induced in endothelial cells in an autocrine manner. In this study, we cloned a 500-bp fragment of chick Vasohibin cDNA and analyzed its expression pattern by in situ hybridization during chick development. From HH-stage 3, expression of Vasohibin is observed in the area opaca and it is expressed throughout the primitive streak during later stages. At HH-stage 11, Vasohibin is expressed in head paraxial mesoderm, in the vitelline vein, dorsal neural tube, intermediate and lateral plate mesoderm, Wolffian duct, and blood islands at the caudal part of the embryo. In epithelial somites, expression is seen in the region around the somitocoel, and after somite maturation, expression is observed in the myotome, which becomes stronger with development. Expression is detected in fore and hind brain, also in the retina and lens vesicle of the developing eye. In the early limb bud, expression is initiated in the mesenchyme and becomes stronger during later stages. Expression in the limb mesoderm remains strong at the margins but decreases in the central mesenchyme. At day 7, expression is seen in interdigital grooves of the digits and digit-demarcating regions. During organogenesis, expression is seen in the anlagen of the esophagus, trachea, duodenum, lungs, liver, heart, and gut. Our analysis shows that Vasohibin is expressed in a wide range of tissues and organs suggesting that Vasohibin acts as a physiological regulator of vascular development during chick embryogenesis.

Regulation of scapula development.

The scapula is a component of the shoulder girdle. Its structure has changed greatly during evolution. For example, in humans it is a large quite flat triangular bone whereas in chicks it is a long blade like structure. In this review we describe the mechanisms that control the formation of the scapula. To assimilate our understanding regarding the development of the scapula blade we start by addressing the issue concerning the origin of the scapula. Experiments using somite extirpation, chick-quail cell marking system and genetic cell labelling techniques in a variety of species have suggested that the scapula had its origin in the somites. For example we have shown in the chick that the scapula blade originates from the somite, while the cranial part, which articulates with the upper limb, is derived from the somatopleure of the forelimb field. In the second and third part of the review we discuss the compartmental origin of this bone and the signalling molecules that control the scapula development. It is very interesting that the scapula blade originates from the dorsal compartment, dermomyotome, which has been previously been associated as a source of muscle and dermis, but not of cartilage. Thus, the development of the scapula blade can be considered a case of dermomyotomal chondrogenesis. Our results show that the dermomyotomal chondrogenesis differ from the sclerotomal chondrogenesis. Firstly, the scapula precursors are located in the hypaxial domain of the dermomyotome, from which the hypaxial muscles are derived. The fate of the scapula precursors, like the hypaxial muscle, is controlled by ectoderm-derived signals and BMPs from the lateral plate mesoderm. Ectoderm ablation and inhibition of BMP activity interfers the scapula-specific Pax1 expression and scapula blade formation. However, only somite cells in the cervicothoracic transition region appear to be committed to form scapula. This indicates that the intrinsic segment specific information determines the scapula forming competence of the somite cells. Taken together, we conclude that the scapula forming cells located within the hypaxial somitic domain require BMP signals derived from the somatopleure and as yet unidentified signals from ectoderm for activation of their coded intrinsic segment specific chondrogenic programme. In the last part we discuss the new data that provides evidence that neural crest contributes for the development of the scapula.

Regulation of ectodermal Wnt6 expression by the neural tube is transduced by dermomyotomal Wnt11: a mechanism of dermomyotomal lip sustainment.

Ectodermal Wnt6 plays an important role during development of the somites and the lateral plate mesoderm. In the course of development, Wnt6 expression shows a dynamic pattern. At the level of the segmental plate and the epithelial somites, Wnt6 is expressed in the entire ectoderm overlying the neural tube, the paraxial mesoderm and the lateral plate mesoderm. With somite maturation, expression becomes restricted to the lateral ectoderm covering the ventrolateral lip of the dermomyotome and the lateral plate mesoderm. To study the regulation of Wnt6 expression, we have interfered with neighboring signaling pathways. We show that Wnt1 and Wnt3a signaling from the neural tube inhibit Wnt6 expression in the medial surface ectoderm via dermomyotomal Wnt11. We demonstrate that Wnt11 is an epithelialization factor acting on the medial dermomyotome, and present a model suggesting Wnt11 and Wnt6 as factors maintaining the epithelial nature of the dorsomedial and ventrolateral lips of the dermomyotome, respectively, during dermomyotomal growth.

Avian stanniocalcin-2 is expressed in developing striated muscle and joints.

The glycoprotein hormone stanniocalcin (STC) has originally been described in the teleost kidney. Since then, STC homologs have been identified in various genomes including human, mouse, rat, Xenopus and zebrafish. In mammals, two STC genes, STC1 and STC2, are known. We cloned a chicken STC homolog to analyze its expression pattern during chick development. Sequence analyses revealed a high sequence similarity of the chicken STC (cSTC) clone to mammalian STC2. Interestingly the expression pattern of cSTC2 largely resembles those of murine STC1: we found expression of cSTC2 in the nephric tubules, in the myocardium, in skeletal muscle cells from the onset of differentiation, and in synovial joint anlagen of the limbs.