New experimental evidence for somite resegmentation

According to the concept of resegmentation, the boundaries of vertebrae are shifted one half a segment compared with somite boundaries. This theory has been experimentally confirmed by interspecific transplantations of single somites. Due to the difficulty of exactly orientating individual somites in the host embryo, the outcome and interpretations of these experiments have occasionally been questioned. This is especially true for the formation of neural arches, their processes, and the ribs. We reinvestigated the formation of vertebrae in the avian embryo by grafting one and one half somites from quail to chick embryos. This method eliminates the possibility of a wrong somite orientation in the host embryo. Results show that the vertebral body, the neural arch and its processes are made up of material of two adjacent somites. This is also true for the rib, with the exception of the costal head, which is formed by only one somite. Whereas in the proximal part of the costal body the chick and quail cell regions border on each other in the middle of the rib, in its distal part quail cells gradually begin to mix with chick cells. The intersegmental muscles and their skeletal attachments sites are formed from the same somite. These results support and complete the data of previous studies and confirm the resegmentation concept. Accepted: 3 May 2000     

From somites to vertebral column.

We report on the development and differentiation of the somites with respect to vertebral column formation in avian and human embryos. The somites, which are made up of different compartments, establish a segmental pattern which becomes transferred to adjacent structures such as the peripheral nervous system and the vascular system. Each vertebra arises from three sclerotomic areas. The paired lateral ones give rise to the neural arches, the ribs and the pedicles of vertebrae, whereas the vertebral body and the intervening disc develop from the axially-located mesenchyme. The neural arches originate from the caudal half of one somite, whereas the vertebral body is made up of the adjacent parts of two somites. Interactions between notochord and axial mesenchyme are a prerequisite for the normal development of vertebral bodies and intervening discs. The neural arches form a frame for the neural tube and spinal ganglia. The boundary between head and vertebral column is located between the 5th and 6th somites. In the human embryo, proatlas, body of the atlas segment, and body of the axis fuse to form the axis.     

Early stages of chick somite development

We report on the formation and early differentiation of the somites in the avian embryo. The somites are derived from the mesoderm which, in the body (excluding the head), is subdivided into four compartments: the axial, paraxial, intermediate and lateral plate mesoderm. Somites develop from the paraxial mesoderm and constitute the segmental pattern of the body. They are formed in pairs by epithelialization, first at the cranial end of the paraxial mesoderm, proceeding caudally, while new mesenchyme cells enter the paraxial mesoderm as a consequence of gastrulation. After their formation, which depends upon cell-cell and cell-matrix interactions, the somites impose segmental pattern upon peripheral nerves and vascular primordia. The newly formed somite consists of an epithelial ball of columnar cells enveloping mesenchymal cells within a central cavity, the somitocoel. Each somite is surrounded by extracellular matrix material connecting the somite with adjacent structures. The competence to form skeletal muscle is a unique property of the somites and becomes realized during compartmentalization, under control of signals emanating from surrounding tissues. Compartmentalization is accompanied by altered patterns of expression of Pax genes within the somite. These are believed to be involved in the specification of somite cell lineages. Somites are also regionally specified, giving rise to particular skeletal structures at different axial levels. This axial specification appears to be reflected in Hox gene expression. MyoD is first expressed in the dorsomedial quadrant of the still epithelial somite whose cells are not yet definitely committed. During early maturation, the ventral wall of the somite undergoes an epithelio-mesenchymal transition forming the sclerotome. The sclerotome later becomes subdivided into rostral and caudal halves which are separated laterally by von Ebner's fissure. The lateral part of the caudal half of the sclerotome mainly forms the ribs, neural arches and pedicles of vertebrae, whereas within the lateral part of the rostral half the spinal nerve develops. The medially migrating sclerotomal cells form the peri-notochordal sheath, and later give rise to the vertebral bodies and intervertebral discs. The somitocoel cells also contribute to the sclerotome. The dorsal half of the somite remains epithelial and is referred to as the dermomyotome because it gives rise to the dermis of the back and the skeletal musculature. The cells located within the lateral half of the dermomyotome are the precursors of the muscles of the hypaxial domain of the body, whereas those in the medial half are precursors of the epaxial (back) muscles. Single epithelial cells at the cranio-medial edge of the dermomyotome elongate in a caudal direction, beneath the dermomyotome, and become anchored at its caudal margin. These post-mitotic and muscle protein-expressing cells form the myotome. At limb levels, the precursors of hypaxial muscles undergo an epithelio-mesenchymal transition and migrate into the somatic mesoderm, where they replicate and later differentiate. These cells express the Pax-3 gene prior to, during and after this migration. All compartments of the somite contribute endothelial cells to the formation of vascular primordia. These cells, unlike all other cells of the somite, occasionally cross the midline of the developing embryo. We also suggest a method for staging somites according to their developmental age.     

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. Accepted: 25 May 2000     

Developmental dynamics of occipital and cervical somites

Development of somites leading to somite compartments, sclerotome, dermomyotome and myotome, has been intensely investigated. Most knowledge on somite development, including the commonly used somite maturation stages, is based on data from somites at thoracic and lumbar levels. Potential regional differences in somite maturation dynamics have been indicated by a number of studies, but have not yet been comprehensively examined. Here, we present an overview on the developmental dynamics of somites at occipital and cervical levels in the chicken embryo. We show that in these regions, the onset of sclerotomal and myotomal compartment formation is later than at thoracolumbar levels, and is initiated simultaneously in multiple somites, which is in contrast to the serial cranial‐ to‐ caudal progression of somite maturation in the trunk. Our data suggest a variant spatiotemporal regulation of somite development in occipitocervical somites.     

背根神经节分节格式的形成和体节再分节

把分散的鹌鹑体节板细胞移植到鸡的体节板区。移植后１７ｈ，在移植区形成大小不等、排列杂乱的ＤＣ体节。有些体节靠近神经管，有些较靠外侧。移植后２９～３２ｈ，靠近神经管的ＤＣ体节向中壁破裂，外迁的间充质细胞在生肌节与神经管和脊索之间，形成生骨节（即原位生骨节）。每个生骨节再分为前后两半，位于外侧的ＤＣ体节，在移植后３３～３８ｈ，其侧壁或外侧壁破裂。外迁的间充质细胞在生肌节的外侧或生肌节之间，数量、大小和     

Morphological analysis of the role of the neural tube and notochord in the development of somites

The differentiation of avian somites and skeletal muscles, which themselves are derived from somites, was studied in ovo after the isolation of the unsegmented segmental plate from the notochord and/or neural tube by surgical operations at the level of the segmental plate. In each experiment, the newly formed somites had a normal histological structure, with an outer epithelial somite and core cells in the somitocoeles. Thereafter, the three derivatives of the somites (dermatome, myotome and sclerotome) reacted differently to the different operations. When the somites developed without the notochord, only somitocoele cells showed massive cell death, and muscles developed regardless of the presence or absence of the neural tube. On the contrary, no cell death was observed in any part of the somites that were formed with the neural tube or the notochord present, and muscle cells developed. However, in those embryos that retained only the notochord, striated muscles developed only in the lateral body wall. In each of the experimental operations, the surface ectoderm always covered the somites, and, regardless of the state of sclerotome and/or myotome differentiation, the dermatome always survived. These histological observations indicate that the first step in somite formation is independent of axial structures. The results further suggest that the notochord may produce diffusible factors that are necessary for the survival and further development of sclerotomal cells, and that both the neural tube and notochord can support muscle differentiation. However, it is likely that each structure has a relationship to the development of epaxial muscles and hypaxial muscles respectively. Furthermore, an intimate relationship may also exist between the surface ectoderm and the development of the dermatome.     

The role of the notochord in vertebral column formation

The backbone or vertebral column is the defining feature of vertebrates and is clearly metameric. Given that vertebrae arise from segmented paraxial mesoderm in the embryo, this metamerism is not surprising. Fate mapping studies in a variety of species have shown that ventromedial sclerotome cells of the differentiated somite contribute to the developing vertebrae and ribs. Nevertheless, extensive studies in amniote embryos have produced conflicting data on exactly how embryonic segments relate to those of the adult. To date, much attention has focused on the derivatives of the somites, while relatively little is known about the contribution of other tissues to the formation of the vertebral column. In particular, while it is clear that signals from the notochord induce and maintain proliferation of the sclerotome, and later promote chondrogenesis, the role of the notochord in vertebral segmentation has been largely overlooked. Here, we review the established role of the notochord in vertebral development, and suggest an additional role for the notochord in the segmental patterning of the vertebral column.     

The role of the notochord in vertebral column formation

The backbone or vertebral column is the defining feature of vertebrates and is clearly metameric. Given that vertebrae arise from segmented paraxial mesoderm in the embryo, this metamerism is not surprising. Fate mapping studies in a variety of species have shown that ventromedial sclerotome cells of the differentiated somite contribute to the developing vertebrae and ribs. Nevertheless, extensive studies in amniote embryos have produced conflicting data on exactly how embryonic segments relate to those of the adult. To date, much attention has focused on the derivatives of the somites, while relatively little is known about the contribution of other tissues to the formation of the vertebral column. In particular, while it is clear that signals from the notochord induce and maintain proliferation of the sclerotome, and later promote chondrogenesis, the role of the notochord in vertebral segmentation has been largely overlooked. Here, we review the established role of the notochord in vertebral development, and suggest an additional role for the notochord in the segmental patterning of the vertebral column.     

Urokinase expression during the epithelial-mesenchymal transformation of the avian somite.

Early events in the morphogenesis of the axial skeleton involve an epithelial-mesenchymal transformation of the somites. Cells of the ventromedial wall of the somite (the sclerotome) migrate to regions surrounding the notochord and neural tube and condense to form the cartilage model of the vertebrae. Urokinase activity in the axial region of the quail embryo trunk was found to increase during these stages. In situ hybridization localized urokinase mRNA expression in this region and suggests an important role for this protease in the process of cell migration and matrix remodeling during development of the axial skeleton.     

Development of somites,muscle, and skeleton is independent of signals from the wolffian duct

Background : In the vertebrate embryo, skeletal muscle and the axial skeleton arise from the somites. Patterning of the somites into the respective somite compartments, namely dermomyotome, myotome, and sclerotome, depends on molecular signals from neighboring structures, including surface ectoderm, neural tube, notochord, and lateral plate mesoderm. A potential role of the intermediate mesoderm, notably the Wolffian or nephric duct, in somite development is poorly understood. Results : We studied somite compartmentalization as well as muscular and skeletal development after surgical ablation of the early Wolffian duct anlage, which lead to loss of the Wolffian duct and absence of the mesonephros, whereas Pax2 expression in the nephrogenic mesenchyme was temporarily maintained. We show that somite compartments, as well as the somite derivatives, skeletal muscle and the cartilaginous skeleton, develop normally in the absence of the Wolffian duct. Conclusions : Our results indicate that development of the musculoskeletal system is independent of the Wolffian duct as a signaling center. Developmental Dynamics 242:941–948, 2013. © 2013 Wiley Periodicals, Inc.     

The migration and distribution of somite cells after labelling with the carbocyanine dye, Dil: the relationship of this distribution to segmentation in the vertebrate body

Summary The cells of individual somites in 2-day-old chick embryos were marked by injecting a fluorescent dye into the somitocoele. This procedure permanently marked the cells and allowed their subsequent development and distribution to be followed. The cells were found to remain in close association with each other within limited boundaries and did not mix to any great extent with similar cells from adjacent somites. Fluorescent cells from single somites were found in the intervertebral disc, connective tissue surrounding two adjacent neural arches, all the tissues between the neural arches, the dermatome, and the associated myotome. No fluorescent cells were found in the notochord or in any nervous tissue apart from accompanying connective tissue. Surprisingly, the vertebral bodies and neural arches did not contain any fluorescent cells apart from those in the connective tissue surrounding them, but this absence of fluorescent cells was thought to be due to the dilution of the fluorescence following cell proliferation. These results provide further experimental support for the theory of resegmentation in vertebral formation, and also provide evidence of a compartmental method of development along the rostrocaudal axis in vertebrates, similar to that already discovered in insects. On the basis of cell lineage criteria, the sclerotome might be considered as a developmental compartment.     

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.     

Low resistance junctions between mesoderm cells during development of trunk muscles.

1. Electrical connexions between mesoderm cells have been examined during the formation of somites in Xenopus laevis, Bombina orientalis and Ambystoma mexicanum. 2. In Xenopus the resting potentials of presumptive myotome cells (-65 + 2 mV, S.E. of mean) and somite muscle cells (-65 +/- 0-6 mV S.E. of mean) were 40 mV, greater than dermatome cells (-25 +/- 0-6 mV, S.E. of mean). Similar differences were found in Bombina and Ambystoma. 3. In all three species cells of the dermatome layer of the mesoderm were electrically coupled to each other. Cells of the presumptive myotome layer in the unsegmented region of the mesoderm were also electrically coupled. 4. In Xenopus dermatome and myotome layers of the mesoderm were not electrically coupled to each other either before or after somite formation. In the other two species dermatome and myotome layers were uncoupled once the somites had formed. 5. In all three species the position of the intersomite border in the unsegmented mesoderm region is marked by the breaking of electrical contracts between those cells destined to form the next somite and the rest of the unsegmented mesoderm. 6. In the axolotl each somite remains electrically insulated from its neighbour. In Xenopus and Bombina electrical connexions are re-established between somite muscle cells once the morphogenetic movements underlying somite formation are complete. 7. Presumptive myotome cells in Xenopus and Ambystoma acquire the membrane property of inward-going rectification before incorporation into a somite. 8. Once Xenopus and Bombina embryos show spontaneous movements large end-plate potentials are recorded from the head somites. Excitation spreads from somite to somite along the low resistance intercellular pathway allowing simultaneous contraction of several somites before extensive somite innervation. 9. The structure of developing somite muscle of Xenopus has been studied with the electron microscope. 10. Close membrane contacts of the gap junction type have been seen between undifferentiated presumptive myotome cells, muscle cells in the same somite and between muscle cells in adjacent somites. 11. Myofilament organization first begins in mesoderm cells when they are forming a new somite. Complete sarcomeres appear in the head somites when the embryo begins spontaneous flexion movements.     

Segmentation in staged human embryos: the occipitocervical region revisited

The first seven somites, the rhombomeres, and the pharyngeal arches were reassessed in 145 serially sectioned human embryos of stages 9-23, 22 of which were controlled by precise graphic reconstructions. Segmentation begins in the neuromeres, somites and aortic arches at stage 9. The following new observations are presented. (1) The first somite in the human, unlike that of the chick, is neither reduced in size nor different in structure, and it possesses sclerotome, somitocoel and dermatomyotome. (2) Somites 1-4, unlike those of the chick, are related to rhombomere 8 (rather than 7 and 8) and are caudal to pharyngeal arch 4 (rather than in line with 3 and 4). (3) Occipital segment 4 resembles a developing vertebra more than do segments 1-3. (4) The development of the basioccipital resembles that of the first two cervical vertebrae in that medial and lateral components arise in a manner that differs from that in the rest of the vertebral column. (5) The two groups of somites, occipital 1-4 and cervical 5-7, each form a median skeletal mass. (6) An 'S-shaped head/trunk interface', described for the chick and unjustifiably for the mouse, was not found because it is not compatible with the topographical development of the otic primordium and somite 1, between which neural crest migrates without hindrance in mammals. (7) Occipital segmentation and related features are documented by photomicrographs and graphic interpretations for the first time in the human. It is confirmed that the first somite, unlike that of the chick, is separated from the otic primordium by a distance, although the otic anlage undergoes a relative shift caudally. The important, although frequently neglected, distinction between lateral and medial components is emphasized. Laterally, sclerotomes 3 and 4 delineate the hypoglossal foramen, 4 gives rise to the exoccipital and participates in the occipital condyle, 5 forms the posterior arch of the atlas and 6 provides the neural arch of the axis, which is greater in height than the arches of the other cervical vertebrae. Medially, the perinotochord and migrated sclerotomic cells give rise to the basioccipital as well as to the vertebral centra, including the tripartite column of the axis. Registration between (1) the somites and (2) the occipital and cervical medial segments becomes interrupted by the special development of the axis, the three components of which come to occupy the height of only 2 1/2 segments.     

Activity of acetylcholinesterase and unspecific cholinesterase during differentiation of somites in mouse embryos

During the development of somites in mouse embryos, widespread activity of unspecific cholinesterase (BuChE) was demonstrated after prolonged incubation. Independent of their position, all somite cells and their derivatives (dermatome, myotome and sclerotome) exhibited enzyme activity in the perinuclear space and in the endoplasmic reticulum. The plasmalemma did not show any enzyme activity. Differentiation of the sclerotome into vertebrae was accompanied by a reduction of BuChE. However, a low enzyme reaction was still present in the first typical differentiated chondroblasts. Notochordal cells were detectable by their high BuChE content. This was also found in cells already showed severe degeneration. In addition to BuChE, acetylcholinesterase (AChE) was first visible on day 9 of embryonic development in newly formed myotubes of the myotomes. Some hypotheses on the functional significance of embryonic BuChE are discussed in the light of these results.     

The binding pattern of peanut lectin associated with sclerotome migration and the formation of the vertebral axis in the chick embryo

Summary Lectins have been used extensively to detect changes in carbohydrate moieties on the surface of embryonic cells during early development. Peanut agglutinin (PNA) in particular has been used to investigate changes related to cell differentiation. PNA has also been used to differentiate between the rostral and caudal sclerotome halves which have been shown to be functionally different, with neural crest cells and neurites traversing only the rostral half during their migration. In this study, we have sectioned and stained chick embryos between 3 and 8 days of age with PNA to examine the distribution of PNA binding sites associated with the vertebral column during this period and also to determine the fates of the rostral and caudal sclerotome halves. Ultrastructural localisation of PNA-gold conjugate showed that binding sites for this lectin were present intracellularly and extracellularly both on cell surfaces and in the matrix. At the light microscope level, a clear banding pattern emerged after staining with PNA which consisted of alternating light and dark staining along the entire length of the vertebral axis of the embryo. In the younger embryos, a simple banding pattern emerged where the rostral sclerotome half of each segment stained only lightly while the caudal half stained darkly. This banding pattern was present throughout the 6 day period of development and could be traced continuously but grew more complex as the sclerotome cells migrated to surround the notochord and neural tube and as the dorsal root ganglia developed. The rostral sclerotome half was found to contribute to the caudal part of one vertebral body and its neural arch, while the caudal sclerotome half was found to contribute to the intervertebral disc, the rostral half of the next caudal vertebra, and part of its neural arch.