Ruijin Huang

Formation and differentiation of the avian sclerotome.

The avian sclerotome forms by epitheliomesenchymal transition of the ventral half-somite. Sclerotome development is characterized by a craniocaudal 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 crosstalk between somite cells lead to the separation of various subdomains, such as the central and ventral sclerotomes that express Pax1 under the control of Sonic hedgehog and Noggin, and the dorsal and lateral sclerotome that do not express Pax1 and are controlled by Bmp-4. Further subdomains that give rise to specific derivatives are the syndetome, neurotome, meningotome, and arthrotome. The molecular control of subdomain formation and cell type specification is discussed.

The development of the avian vertebral column.

Segmentation of the paraxial mesoderm leads to somite formation. The underlying molecular mechanisms involve the oscillation of ”clock-genes” like c-hairy-1 and lunatic fringe indicative of an implication of the Notch signaling pathway. The cranio-caudal polarity of each segment is already established in the cranial part of the segmental plate and accompanied by the expression of genes like Delta1, Mesp1, Mesp2, Uncx-1, and EphA4 which are restricted to one half of the prospective somite. Dorsoventral compartmentalization of somites leads to the development of the dermomyotome and the sclerotome, the latter forming as a consequence of an epithelio-to-mesenchymal transition of the ventral part of the somite. The sclerotome cells express Pax-1 and Pax-9, which are induced by notochordal signals mediated by sonic hedgehog (Shh) and noggin. The craniocaudal somite compartmentalization that becomes visible in the sclerotomes is the prerequisite for the segmental pattern of the peripheral nervous system and the formation of the vertebrae and ribs, whose boundaries are shifted half a segment compared to the sclerotome boundaries. Sclerotome development is characterized by the formation of three subcompartments giving rise to different parts of the axial skeleton and ribs. The lateral sclerotome gives rise to the laminae and pedicles of the neural arches and to the ribs. Its development depends on signals from the notochord and the myotome. The ventral sclerotome giving rise to the vertebral bodies and intervertebral discs is made up of Pax-1 expressing cells that have invaded the perinotochordal space. The dorsal sclerotome is formed by cells that migrate from the dorso-medial angle of the sclerotome into the space between the roof plate of the neural tube and the dermis. These cells express the genes Msx1 and Msx2, which are induced by BMP-4 secreted from the roof plate, and they later form the dorsal part of the neural arch and the spinous process. The formation of the ventral and dorsal sclerotome requires directed migration of sclerotome cells. The regionalization of the paraxial mesoderm occurs by a combination of functionally Hox genes, the Hox code, and determines the segment identity. The development of the vertebral column is a consequence of a segment-specific balance between proliferation, apoptosis and differentiation of cells.

SEGMENTATION OF THE VERTEBRATE BODY

Abstract The segmental character of the vertebrate body wall is reflected by metamerically arranged tissues that are patterned during embryonic life as a consequence of somite formation, compartmentalization and differentiation. The somites bud off the paraxial mesoderm in a cranio-caudal sequence and are compartmentalized by local signals from adjacent structures. These signals may be mediated by diffusible substances such as Sonic hedgehog (Shh), Wnts and Bone morphogenetic protein (BMPs) or by cell–cell interactions via membrane-bound receptors and ligands such as Delta and Notch. Compartmentalization of the somites and their derivatives is reflected by the differential expression of developmental regulatory genes such as Pax-1, 3, 7 and 9, MyoD, paraxis, twist and others. Secondary segmentation is imposed upon other tissues, such as blood vessels and nerves, by the rearrangement and regionalization of the somitic derivatives, especially the sclerotome. Early cranio-caudal identity is determined by the expression of different Hox genes. Finally, fusion of segmental anlagen occurs to form segment-overbridging skeletal elements and muscles. The expression of homologous genes indicates that the process of segmentation in vertebrates and invertebrates is homologous, derived by descent from a common ancestor.

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. Developmental Dynamics 236:2382–2396, 2007.

The fate of somitocoele cells in avian embryos.

The early somite of avian embryos is made up of an epithelial wall and mesenchymal cells located within the somitocoele. We have studied the fate of somitocoele cells for a period of up to 6 days, using the quailchick marker technique. We also applied the QH-1 antibody, which specifically stains hemangiopoietic cells of quail origin, and studied the proliferative activity of epithelial somites with the BrdU anti-BrdU method. Our results show that somitocoele cells mainly give rise to the ribs and peripheral parts of the intervertébral discs. After 1 and 2 days of reincubation, the grafted somitocoele cells were located in the lateral part of the sclerotome, and only a few cells migrated axially towards the notochord. In frontal sections, the cells were located in a triangular area within the cranial part of the caudal sclerotome half. After 3 days of reincubation, some of the cells had migrated cranially along the myotome. After longer reincubation periods, cells grafted into one somite could be found in two adjacent ribs. The studies with the QH-1 antibody show that a subpopulation of somitocoele cells has angiogenic potency. Endothelial cells originating from the mesenchyme of the somitocoele migrated actively and even invaded the ipsilateral half of the neural tube. In the epithelial wall of the somite, BrdU-labelled nuclei were found basally, whereas more apically the nuclei were not stained, but mitotic figures were frequently present. The somitocoele cells also showed a high proliferative activity with about 26% of nuclei labelled with BrdU.

Origin and development of the avian tongue muscles.

 The musculature of the vertebrate tongue is composed of cells recruited from the somites. In this paper we have investigated the migration and organisation of the muscle cells that give rise to the tongue muscle during chick embryogenesis. At the molecular level, our data suggests that a population of Tbx-3 expressing cells migrate away from the occipital somites prior to the migration of muscle precursors that express Pax-3. Both populations take the same pathway and form the hypoglossal cord. The first signs of muscle cell differentiation were not detected until cells had migrated some distance from the somites. We have determined the contribution of single somites to the musculature of the tongue and show in contrast to previous data that somites 2–6 take part in the formation of all glossal and infrahyoid muscles to the same extent but do not contribute to suprahyoid muscle. This is particularly interesting since glossal and infrahyoid muscle differ from the suprahyoid muscles not only in their morphology, but also in their developmental origin. Furthermore we show that myocytes cross the midline and contribute to the contralateral glossal and infrahyoid muscles. This is supported from our molecular data, which showed that the migratory precursor population was maintained primarily at the rostral tip of the developing hypoglossal cord.

The fate of the first avian somite

Abstract We have studied the derivatives of the first somite using the quail-chick marking technique. After transplantation of the somite, the chick embryos were reincubated for periods ranging from 4 h to 11 days. Coronal and sagittal sections of the embryos were prepared for parallel staining with Feulgen-reaction, anti-quail antibody, anti-desmin antibody and QH-1 antibody. The first somite consists of an epithelial envelope surrounding somitocoele cells. Like other somites, it forms sclerotome, dermatome and myotome. Cells contribute to the occipital and parasphenoid bone, the meninges, the dermis in the occipital region and the pharyngeal connective tissue. The contribution of the first somite to bones, meninges, dermis and pharyngeal connective tissue is characterised by sharp anterior and posterior boundaries. In contrast, other derivatives such as connective tissue surrounding the vagus nerve, the carotid artery, and jugular vein exceed 10 to 18 segments. This is also true for myogenic cells participating in the formation of the cucullaris capitis muscle that extends from the temporal bone to the shoulder. In one third of the embryos, myocytes of the intrinsic laryngeal muscles are derived from the grafted first somite. Moreover, endothelial cells originate from this somite and migrate into the head (hindbrain, meninges, dermis), neck (pharynx, connective tissue surrounding the vagus nerve, carotid artery and jugular vein) and thorax. With respect to differentiation and derivatives the first somite is similar to other somites.

Function of Somite and Somitocoele Cells in the Formation of the Vertebral Motion Segment in Avian Embryos

We have studied the distribution of thoracic somite and somitocoele-derived cells using homotopical grafting between quail and chicken embryos and reincubation periods of 2-6 days. Serial sections were evaluated with antibodies against quail cells, quail hemangiopoietic cells and desmin. With the exception of neural crest cells in the cranial sclerotome half, all cells of the operated segment are quail cells derived from a single somite. These cells differentiate into sclerotome, myotome and the anlage of the dermis of the back. After longer reincubation periods, the somite-derived quail cells form the neighboring halves of 2 adjacent vertebral bodies and the intervening (disc-homologous) tissue. Resegmentation is furthermore visible in the lamina and the spinous process. Somite cells also form the articular and transverse processes, and the intertransverse muscle including its insertion to the next cranial transverse process. One thoracic somite forms the proximal part of 1 rib. In more distal parts, 1 somite forms the cranial half of 1 rib and the caudal half of the next cranial rib, and the intercostal muscle and part of the connective tissue. Somite-derived quail cells are found in muscle that bridges over 2 segments cranial and caudal from the operated segment. The craniocaudal distribution of endothelial cells is approximately the same. Somitocoele cells that are located centrally in the epithelial somite express the sclerotome-markers Pax-1 and Pax-9. After 2-3 days of reincubation, grafted thoracic somitocoele cells are found mainly in the cranial part of the caudal sclerotome half. They form an area representing the anlagen of the intervertebral disc and the rib. After longer reincubation periods, the grafted quail somitocoele cells form the intervertebral disc-homologous tissue and the proximal part of the rib. In more distal parts of the rib they are located in the cranial half of 1 rib and the caudal half of the next cranial rib. The somitocoele cells also form the surface of the intervertebral joint, and give rise to a small number of endothelial cells that are found up to 1 segment cranial and caudal to the operation site. Our studies show that resegmentation is found in most parts of the vertebra and in the distal ribs. One somite forms the origin and insertion of the segmental muscle. Therefore, the somite can be regarded as the ancestor of the vertebral motion segment. Somitocoele cells are located centrally both in the epithelial somite and in the vertebral motion segment.

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.

Contribution of single somites to the skeleton and muscles of the occipital and cervical regions in avian embryos

Controversy has surrounded the process of resegmentation of cervico-occipital somites. We have reinvestigated this topic by grafting single somites of quail embryos homotopically into chick embryos. Somites one to five contribute to the skull. Somites one and two contribute to the parasphenoid, which develops by direct ossification in a non-segmental fashion. All cartilaginous derivatives of the somites are segmental. Somite two forms a stripe of cells in the basioccipital, exoccipital and supraoccipital. Somites three to five give rise to the subsequent caudal parts of the basioccipital and exoccipital. Somite five forms the first motion segment including the occipital condyle, the cranial part of the atlas and the tip of the dens axis. Therefore, the border between head and neck is in the centre of somite five, and corresponds to the expression boundary of Choxb-3. Somite six forms the caudal part of the atlas and the cranial part of the axis. Somites two to eight all contribute to the cranio-cervical muscles with the exception of the Mm. rectus capitis dorsalis and ventralis and the M. biventer cervicis, which do not receive contributions from somite two. In contrast, the M. cucullaris capitis is exclusively formed by myogenic cells from somite two, which parallels its exclusive innervation by the accessory nerve. Our data confirm the segmental nature of the occiput, and show that resegmentation is a very regular process involving all except the four cranialmost somites. Except for somites one and two, all of the somites contribute to the muscles located at the appropriate levels.