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.     

Embryonic development of the emu,Dromaius novaehollandiae

The chick, Gallus gallus, is the traditional model in avian developmental studies. Data on other bird species are scarce. Here, we present a comparative study of the embryonic development of the chick and the emu Dromaius novaehollandiae, a member of Paleognathae, which also includes the ostrich, rhea, tinamou, kiwi, and cassowary. Emu embryos ranging from Hamburger and Hamilton (HH) equivalent stages 1 to 43 were collected and their gross morphology analyzed. Its early development was studied in detail with time‐lapse imaging and molecular techniques. Emu embryos in general take 2–3 times longer incubation time to reach equivalent chicken stages, requiring 1 day for HH2, 2.5 days for HH4, 7 days for limb bud initiation, 23 days for feather germ appearance, and approximately 50–56 days for hatching. Chordin gene expression is similar in emu and chick embryos, and emu Brachyury is not expressed until HH3. Circulation is established at approximately the 27‐ to 30‐somite stage. Forelimb buds are formed and patterned initially, but their growth is severely retarded. The size difference between an emu and a chick embryo only becomes apparent after limb bud formation. Overall, emu and chick embryogenesis proceeds through similar stages, but developmental heterochrony between these two species is widely observed. Developmental Dynamics, 2011. © 2010 Wiley‐Liss, Inc.     

Paraxis is required for somite morphogenesis and differentiation in Xenopus laevis

Background : In most vertebrates, the segmentation of the paraxial mesoderm involves the formation of metameric units called somites through a mesenchymal‐epithelial transition. However, this process is different in Xenopus laevis because it does not form an epithelial somite. Xenopus somitogenesis is characterized by a complex cells rearrangement that requires the coordinated regulation of cell shape, adhesion, and motility. The molecular mechanisms that control these cell behaviors underlying somite formation are little known. Although the Paraxis has been implicated in the epithelialization of somite in chick and mouse, its role in Xenopus somite morphogenesis has not been determined. Results : Using a morpholino and hormone‐inducible construction approaches, we showed that both gain and loss of function of paraxis affect somite elongation, rotation and alignment, causing a severe disorganization of somitic tissue. We further found that depletion or overexpression of paraxis in the somite led to the downregulation or upregulation, respectively, of cell adhesion expression markers. Finally, we demonstrated that paraxis is necessary for the proper expression of myotomal and sclerotomal differentiation markers. Conclusions : Our results demonstrate that paraxis regulates the cell rearrangements that take place during the somitogenesis of Xenopus by regulating cell adhesion. Furthermore, paraxis is also required for somite differentiation. Developmental Dynamics 244:973–987, 2015. © 2015 Wiley Periodicals, Inc.     

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.     

Expression of myogenic regulatory factors in chicken embryos during somite and limb development

The expression of the myogenic regulatory factors (MRFs), Myf5, MyoD, myogenin (Mgn) and MRF4 have been analysed during the development of chicken embryo somites and limbs. In somites, Myf5 is expressed first in somites and paraxial mesoderm at HH stage 9 followed by MyoD at HH stage 12, and Mgn and MRF4 at HH stage 14. In older somites, Myf5 and MyoD are also expressed in the ventrally extending myotome prior to Mgn and MRF4 expression. In limb muscles a similar temporal sequence is observed with Myf5 expression detected first in forelimbs at HH stage 22, MyoD at HH stage 23, Mgn at HH stage 24 and MRF4 at HH stage 30. This report describes the precise time of onset of expression of each MRF in somites and limbs during chicken embryo development, and provides a detailed comparative timeline of MRF expression in different embryonic muscle groups.     

Sonic hedgehog enhances somite cell viability and formation of primary slow muscle fibers in avian segmented mesoderm

 Primary skeletal muscle fibers first form in the segmented portions of paraxial mesoderm called somites. Although the neural tube and notochord are recognized as crucial in patterning myogenic cell lineages during avian and mammalian somitic myogenesis, the source, identities, and actions of the signals governing this process remain controversial. It has been shown that signals emanating from the ventral neural tube and/or notochord alone or Shh alone serve to activate MyoD expression in somites. However, beyond a role in initiating MyoD expression, little is known about the effects of Shh on primary muscle fiber formation in somites of higher vertebrates. The studies reported here investigate how the ventral neural tube promotes myogenesis and compare the effects of the ventral neural tube with those of purified Shh protein on fiber formation in somites. We show that purified Shh protein mimics actions of the ventral neural tube on somites including initiation of muscle fiber formation, enhancement of numbers of primary muscle fibers, and particularly, the formation of primary fibers that express slow myosin. There is a marked increase in slow myosin expression in fibers in response to Shh as somites mature. The effects of ventral neural tube on fiber formation can be blocked by disrupting the Shh signaling pathway by increasing the activity of somitic cyclic AMP-dependent protein kinase A. Furthermore, it was demonstrated that apoptosis is a dominant fate of somite cells, but not somitic muscle fibers, when cultured in the absence of the neural tube, and that application of Shh protein to somites reduced apoptosis. The block to apoptosis by Shh is a manifestation of the maturity of the somite with a progressive increase in the block as somites are displaced rostrally from somite III forward. We conclude that purified Shh protein in mimicking the effects of the ventral neural tube on segmented mesoderm can exert pleiotropic effects during primary myogenesis, including: control of the proliferative expansion of myogenic progenitor cells, antagonism of cell death pathways within the precursors to muscle fibers, and during the crucial process of primary myogenesis, can exert an effect on diversification of muscle fiber types. Accepted: 1 March 1999     

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.     

Churchill and Sip1a repress fibroblast growth factor signaling during zebrafish somitogenesis

Cell‐type specific regulation of a small number of growth factor signal transduction pathways generates diverse developmental outcomes. The zinc finger protein Churchill (ChCh) is a key effector of fibroblast growth factor (FGF) signaling during gastrulation. ChCh is largely thought to act by inducing expression of the multifunctional Sip1 (Smad Interacting Protein 1). We investigated the function of ChCh and Sip1a during zebrafish somitogenesis. Knockdown of ChCh or Sip1a results in misshapen somites that are short and narrow. As in wild‐type embryos, cycling gene expression occurs in the developing somites in ChCh and Sip1a compromised embryos, but expression of her1 and her7 is maintained in formed somites. In addition, tail bud fgf8 expression is expanded anteriorly in these embryos. Finally, we found that blocking FGF8 restores somite morphology in ChCh and Sip1a compromised embryos. These results demonstrate a novel role for ChCh and Sip1a in repression of FGF activity. Developmental Dynamics 239:548–558, 2010. © 2009 Wiley‐Liss, Inc.     

Embryonic development of the house shrew (Suncus murinus)

Summary The embryonic development during the period from 1 to 12 pairs of somites was observed in an insectivore species, the house shrew (Suncus murinus), which has been bred within a closed colony. Embryos were staged by the number of somite pairs. Each stage was punctuated at every addition of three pairs of somites and numbered after the Carnegie system. The first somite became apparent between 8 and 9.0 days after fertilization, and the 12th somite appeared between 9.5 and 10.0 days. The rate of somite formation was one pair in every 3–4 h on average. The embryonic events during this period were as follows: 1. From the beginning of stage 9, the embryonic body consistently displayed a kyphosis, and as development progressed, the caudal portion of the embryo spiralled clockwise. 2. The first and second pharyngeal arches formed; their development was precocious among mammalian embryos in relation to somitic count. 3. The segmental pattern of the neural fold was similar to that of laboratory rodents and primates. The first fusion of the cranial neural folds took place in the occipital somite region, the second fusion in the diencephalic region, and the third at the end of the neural plate, thus leaving two neuropores in the cephalic region. 4. The timing of appearance of the optic sulcus was similar to that of human embryos but was delayed in comparison with that of laboratory rodents. 5. The heart always showed a more advanced state than that of other mammalian embryos. From the beginning of stage 9, an unpaired endocardial tube was seen in the bulbo-ventricular region, and deflection from a symmetrical appearance soon took place. 6. The differentiation of foregut was also precocious, and the thyroid and respiratory primordia appeared earlier than in other mammals. The present study emphasizes that there are considerable variations in timing and manner of morphogenesis among early mammalian embryos.     

From the primitive streak to the somitic mesoderm: labeling the early stages of chick embryos using <Emphasis Type="Italic">EGFP</Emphasis> transfection

Mesoderm is derived from the primitive streak. The rostral region of the primitive streak forms the somitic mesoderm. We have previously shown the developmental origin of each level of the somitic mesoderm using DiI fluorescence labeling of the primitive streak. We found that the more caudal segments were derived from the primitive streak during the later developmental stages. DiI labeled several pairs of somites and showed the distinct rostral boundary; however, the fluorescence gradually disappeared in the caudal region. This finding can be explained in two ways: the primitive streak at a specific developmental stage is primordial of only a certain number of pairs of somites, or the DiI fluorescent dye was gradually diluted within the primitive streak by cell division. Here, we traced the development of the primitive streak cells using enhanced green fluorescent protein (EGFP) transfection. We confirmed that, the later the EGFP transfection stage, the more caudal the somites labeled. Different from DiI labeling, EGFP transfection performed at any developmental stage labeled the entire somitic mesoderm from the anterior boundary to the tail bud in 4.5-day-old embryos. Furthermore, the secondary neural tube was also labeled, suggesting that not only the somite precursor cells but also the axial stem cells were labeled.     

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     

A scanning electron microscope survey of the origin of the primordial pronephric duct cells in the avian embryo

In the avian embryo, the pronephric duct derives from the anterior part of a ridge that develops just lateral to the somites and segmental plate. The ridge extends from the sixth somite to Hensen's node and begins to form as the sixth somite is condensing. By the nine-somite stage, the cranial end of the ridge (for a length roughly equivalent to four or five somite diameters) is seen as a duct primordium of smooth, elongated cells that resemble the migrating cells of the caudal tip of the duct as it extends to the cloaca. These cells show a decreased attachment to the fibers of the interstitial matrix and an increased adhesion to other duct cells. By 10 somites, there is a well-formed pronephric duct rudiment at a time when the pronephric tubules have not yet begun to develop. Therefore, the avian pronephric duct has a separate origin from the pronephric tubules and may play an inductive role in the formation of pronephric tubules.     

Increases in the number of cells in different areas of epithelial somites related to changes in morphology and development

There are two distinct groups of cells in the epithelial somite: cells in the epithelial ball that form the periphery, and loose mesenchymal cells found in the central cavity (somitocoele). Recent work has produced evidence to show that these two groups of cells have significant differences (morphology, origin, fibronectin content, reaction to peanut lectin, communication properties) but the significance of these differences has yet to be established. It is not yet clear whether the epithelial somite stage of development is merely a time for cell proliferation, or whether it is a time when significant differences develop which have consequences in subsequent morphogenesis. Certainly, there are indications that the two groups of cells might form different structures related to the vertebral column based on their position in the subsequent sclerotome. In this study, we have examined the number of cells that are present in both the epithelial ball and the somitocoele at various stages of maturity. The results show that later-formed somites contain significantly more cells in both the epithelial ball and the somitocoele. Furthermore, while the density of cells in the epithelial ball remains constant (accounting for an increase in dimensions of the somite), there is a significant increase in density of cells in the somitocoele. This suggests that there is an important distinction being created between the cells of the epithelial ball and those in the somitocoele. The results also illustrate that somite development is not the same at all segmental levels and that development of each might need to be considered on an individual basis, especially as the later-formed somites are known not to remain in this stage of development for as long as the earlier-formed somites.     

A resegmentation‐shift model for vertebral patterning

Segmentation of the vertebrate body axis is established in the embryo by formation of somites, which give rise to the axial muscles (myotome) and vertebrae (sclerotome). To allow a muscle to attach to two successive vertebrae, the myotome and sclerotome must be repositioned by half a segment with respect to each other. Two main models have been put forward: ‘resegmentation’ proposes that each half‐sclerotome joins with the half‐sclerotome from the next adjacent somite to form a vertebra containing cells from two successive somites on each side of the midline. The second model postulates that a single vertebra is made from a single somite and that the sclerotome shifts with respect to the myotome. There is conflicting evidence for these models, and the possibility that the mechanism may vary along the vertebral column has not been considered. Here we use DiI and DiO to trace somite contributions to the vertebrae in different axial regions in the chick embryo. We demonstrate that vertebral bodies and neural arches form by resegmentation but that sclerotome cells shift in a region‐specific manner according to their dorsoventral position within a segment. We propose a ‘resegmentation‐shift’ model as the mechanism for amniote vertebral patterning.     

Developmental study on reduction and kinks of the tail in a new mutant knotty-tail mouse

The knotty-tail (knt/knt) mouse has a short and knotty tail. The tail deformity is caused by a decrease in the number of caudal vertebrae and a deformity of them in the distal part of the tail. The objective of the study was to determine how reduction and kinks of the tail region were formed during secondary body formation. By day 12.0?pc, the somitogenesis of knt/knt embryos was completed; the number of caudal somites more or less agreed with those of the caudal vertebrae in knt/knt mice and were similar to those of knt/+ embryos. On the other hand, the somitogenesis of knt/+ embryos continued up to day 12.5?pc. The somites below about the sixth caudal somite were wedge-shaped with a dorsal apex in knt/knt embryos. The location of abnormal somites also corresponded well to that of deformed caudal vertebrae. Abnormal somitogenesis was always preceded by abnormalities in the presomitic region. Under gross observation, this could be seen to become markedly thickened, and histologically its dorsoventral diameter increased in the transverse plane on days 10.5–12.0?pc. In the mesenchyme there was often obvious cell death at the boundary of the unsegmented area and the tail bud after day 10.5?pc. These results suggested that the shortness of tail was primarily caused by the agenesis of distal caudal vertebrae following the agenesis of distal caudal somites, and partly by the disappearance of the presomitic part due to cell death, while the tail kinks were caused by the deformation of each caudal vertebra following disturbances of the caudal somites. Also, it is highly probable that the prominent cell death at the boundary of the unsegmented area and the tail bud may involve a defect or deformity of somites in this mutant.     

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. Developmental Dynamics 239:1162–1177, 2010.© 2010 Wiley‐Liss, Inc.     

The ventralizing effect of the notochord on somite differentiation in chick embryos

The dorso-ventral pattern formation of the somites becomes manifest by the formation of the epithelially organized dorsal dermomyotome and the mesenchymal ventrally situated sclerotome. While the dermomyotome gives rise to dermis and muscle, the sclerotome differentiates into cartilage and bone of the axial skeleton. The onset of muscle differentiation can be visualized by immunohistochemistry for proteins associated with muscle contractility, e.g. desmin. The sclerotome cells and the epithelial ventral half of the somite express Pax-1, a member of a gene family with a sequence similarity to Drosophila paired-box-containing genes. In the present study, changes of Pax-1 expression were studied after grafting an additional notochord into the paraxial mesoderm region. The influence of the notochord and the floor-plate on dermomyotome formation and myotome differentiation has also been investigated. The notochord is found to exert a ventralizing effect on the establishment of the dorso-ventral pattern in the somites. Notochord grafts lead to a suppression of the formation and differentiation of the dorsal somitic derivatives. Simultaneously, a widening of the Pax-1-expressing domain in the sclerotome can be observed. In contrast, grafted roof-plate and aorta do not interfere with dorso-ventral patterning of the somitic derivatives.