The neural plate specifies somite size in the Xenopus laevis gastrula.

The organizer has traditionally been considered the major source of somite-inducing signals. We show here that signaling from the neural plate specifies somite tissue and regulates somite size in the Xenopus gastrula. Ectopic undifferentiated neural tissue induces massive somite expansion at the expense of intermediate and lateral plate mesoderm. Although the early expanded somite expresses muscle-specific markers, only a portion terminally differentiates, suggesting that myotome development requires additional signals. Explant assays demonstrate that neural tissue induces somite-specific marker expression even in the absence of the organizer. Finally, we demonstrate that neural tissue is required for proper somite development because elimination of neural precursors results in pronounced somite reduction. Thus, an important reciprocal interaction exists between somite and neural tissue that is mutually reinforcing and critical for normal embryonic patterning.     

Dynamic expression of lunatic fringe suggests a link between notch signaling and an autonomous cellular oscillator driving somite segmentation

The metameric organization of the vertebrate trunk is a characteristic feature of all members of this phylum. The origin of this metamerism can be traced to the division of paraxial mesoderm into individual units, termed somites, during embryonic development. Despite the identification of somites as the first overt sign of segmentation in vertebrates well over 100 years ago, the mechanism(s) underlying somite formation remain poorly understood. Recently, however, several genes have been identified which play prominent roles in orchestrating segmentation, including the novel secreted factor lunatic fringe. To gain further insight into the mechanism by which lunatic fringe controls somite development, we have conducted a thorough analysis of lunatic fringe expression in the unsegmented paraxial mesoderm of chick embryos. Here we report that lunatic fringe is expressed predominantly in somite -II, where somite I corresponds to the most recently formed somite and somite -I corresponds to the group of cells which will form the next somite. In addition, we show that lunatic fringe is expressed in a highly dynamic manner in the chick segmental plate prior to somite formation and that lunatic fringe expression cycles autonomously with a periodicity of somite formation. Moreover, the murine ortholog of lunatic fringe undergoes a similar cycling expression pattern in the presomitic mesoderm of somite stage mouse embryos. The demonstration of a dynamic periodic expression pattern suggests that lunatic fringe may function to integrate notch signaling to a cellular oscillator controlling somite segmentation.     

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

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

Critical numbers of neural crest cells are required in the pathways from the neural tube to the foregut to ensure complete enteric nervous system formation

The enteric nervous system (ENS) is mainly derived from vagal neural crest cells (NCC) that arise at the level of somites 1-7. To understand how the size and composition of the NCC progenitor pool affects ENS development, we reduced the number of NCC by ablating the neural tube adjacent to somites 3-6 to produce aganglionic gut. We then back-transplanted various somite lengths of quail neural tube into the ablated region to determine the 'tipping point', whereby sufficient progenitors were available for complete ENS formation. The addition of one somite length of either vagal, sacral or trunk neural tube into embryos that had the neural tube ablated adjacent to somites 3-6, resulted in ENS formation along the entire gut. Although these additional cells contributed to the progenitor pool, the quail NCC from different axial levels retained their intrinsic identities with respect to their ability to form the ENS; vagal NCC formed most of the ENS, sacral NCC contributed a limited number of ENS cells, and trunk NCC did not contribute to the ENS. As one somite length of vagal NCC was found to comprise almost the entire ENS, we ablated all of the vagal neural crest and back-transplanted one somite length of vagal neural tube from the level of somite 1 or somite 3 into the vagal region at the position of somite 3. NCC from somite 3 formed the ENS along the entire gut, whereas NCC from somite 1 did not. Intrinsic differences, such as an increased capacity for proliferation, as demonstrated in vitro and in vivo, appear to underlie the ability of somite 3 NCC to form the entire ENS.     

Analysis of normal somite development

We describe how the first 6 somite pairs form, using the third somites as examples. This history is based upon time-lapse movies of carbon-marked embryos and histological studies by light and electron microscopy of embryos fixed in situ with glutaraldehyde and osmium tetroxide. At head-process stage a continuous sheet of mesoblast occupies the regions of the future third somites. Mesoblast cells attach either to hypoblast or to overlying neural plate which is already a simple pseudostratified columnar epithelium. Prospective somite cells are those attached to the neuroepithelium, and they extend laterally exactly as far as the neural plate does. By head-fold stage, regression of the node down the midline is shearing the sheet of mesoblast into right and left halves. Somite cells hang from the bottom of the neural plate. As the neural plate condenses toward the midline, attached somite cells are compacted. When the somite segments, somite cells are tightly apposed to one another, and, in addition to junctions binding their basal ends, new junctions appear between their apical ends. This leads to reorganization into the typical somite rosette configuration. Spaces filled with extracellular materials form around the whole somite.     

A cascading development model for amphibian embryos

The mesodermal tissue of some amphibian gastrula develops into a dorsal-to-ventral sequence of notochord, somite, pronephros, and lateral plate cell types. The cellular proportions regulate with respect to embryo size. The dorsal blastoporal lip appears to function as an organizer for the embryo. The transplantation of a donor lip to the ventral side of a host causes a second, opposed embryo to form and the system commits similar total proportions of cells as do normally developing embryos. Transplantation of donor somite to the ventral side of a host causes a reduction in the proportion of host somite developed. A modified reaction-diffusion system governing embryo development is proposed. Developmental simulations consistent with experimental observations are presented and analyzed. The results suggest that the degree of somite inhibition is positively correlated with the size of the somite transplant. Further predictions are that sufficiently large somite transplants would induce ectopic, ventral pronephros to form and ventral pronephros transplants would inhibit host pronephros development. This paper has been reproduced directly from disc using a LA-T_EX system.     

Developmental Fate of Artificially Disordered Somite Cells after Heteroplastic Transplantation

A disordered somite pattern could be produced artificially when the segmental lateral plate of chickembryo was replaced by dissociated cells of quail segmental pate.The artificially disordered somitepattern formed at either place was used in our work as a model to analyze the mechanism of thedevelopment and differentiation of somite on chick embryo.Our conclusions include the following:1.Although the formation of somites from the dissociated segmental plate cells does not requirespecial environment,the development and differentiation of the somltes require a special environmentwhich is related to the neural tube and notochord.The effect of this special environmental factor maydecrease gradually with the increase of the distance from neural tube to lateral plate.2.The somites located on paraxial area at different distances to the axis have different fates indevelopment.3.The formation of epithelial vesicles is the property of somite cells and the epithelial vesicle is thestructural basis of somite differentiation.If and factor interferes with the differentiation of thesomite,the epithelial vesicle of the somite will be degenerated within certain period of time.4.During resegmentation of the somite,the number,size and arrangement of sclerotome in situ donot depend on the somite from which they are derived.5.Somite cells do not transdifferentiate into kidney tubule directly from their original epithelialvesicles,but are reorganized from the free cells dispersed from the disrupted somites.6.The establishment of cell commitment may involve several steps.Before commitment isestablished the of cell commitment is labile.7.The differentiation of sclerotome starts with the rupture of epithelial wall of somites and thedirection of its movement depends not only on the notochord but also on their position with respectto the neural tube and notochord.8.The disordered somite pattern doesn't influence the segmentation of dorsal root ganglia in situ,but causes the formation of the ectopic dorsal root ganglia.Key Words:Somite differentiation;Artificial disordered somite pattern;Chimeral somite;Resegmentation of sclerotome;Distribution of dorsal root ganglia     

Can tissue surface tension drive somite formation?

The prevailing model of somitogenesis supposes that the presomitic mesoderm is segmented into somites by a clock and wavefront mechanism. During segmentation, mesenchymal cells undergo compaction, followed by a detachment of the presumptive somite from the rest of the presomitic mesoderm and the subsequent morphological changes leading to rounded somites. We investigate the possibility that minimization of tissue surface tension drives the somite sculpting processes. Given the time in which somite formation occurs and the high bulk viscosities of tissues, we find that only small changes in shape and form of tissue typically occur through cell movement driven by tissue surface tension. This is particularly true for somitogenesis in the zebrafish. Hence it is unlikely that such processes are the sole and major driving force behind somite formation. We propose a simple chemotactic mechanism that together with heightened adhesion can account for the morphological changes in the time allotted for somite formation.     

Myogenesis and contraction in the early embryonic heart of the rainbow trout

Summary Myogenesis in the embryonic heart of the rainbow trout, Salmo galrdneri (Rich.), was investigated electron microscopically from the 29th to the 41st somite stage. Thick and thin myofilaments are formed simultaneously as well as precursors of Z-lines, to which the thin filaments are attached. The genesis of filaments takes place in the region around the intracellular yolk droplets. The first myofibrils appear by the 33rd somite stage, probably formed by a mechanism of self-assembly in which the binding sites of actin and myosin participate. A- and I-bands do not develop before the 38th somite stage. The contraction already begins during the 33rd somite stage in the middle of the tubular heart. Gradually, the peristaltic waves spread increasingly to other parts of the heart. In the 41st somite stage the entire heart is contractile and all myocytes contain myofibrils.     

Fgf8 drives myogenic progression of a novel lateral fast muscle fibre population in zebrafish

Fibroblast growth factors (Fgfs) have long been implicated in regulating vertebrate skeletal muscle differentiation, but their precise role(s) in vivo remain unclear. Here, we show that Fgf8 signalling in the somite is required for myod expression and terminal differentiation of a subset of fast muscle cells in the zebrafish lateral somite. In the absence of Fgf8, lateral somite cells transiently express myf5 but fail to make muscle and remain in a dermomyotome-like state characterised by pax3 and meox expression. Slow muscle fibres form and commence normal migration in the absence of Fgf8, but fail to traverse the expanded undifferentiated lateral somite. The Fgf8-independent residual population of medial fast muscle fibres is not Hedgehog dependent. However, Fgf8-independent medial fast muscle precursors are lacking in floatinghead mutants, suggesting that they require another ventral midline-derived signal. We conclude that Fgf8 drives terminal differentiation of a specific population of lateral muscle precursor cells within the early somite.     

Three-dimensional models of chick embryo somites obtained by mathematical methods applied to ultrastructural data

Summary A model of a thoracolumbar somite of a chick embryo at the 53rd incubation hour was obtained by mathematical methods, after identification of somite cell types by means of electron microscopy.Each specific district occupied by the cell types was precisely determined.On the basis of these observations, the somite was three-dimensionally reconstructed and the spatial positions of the primitive myotome, dermatome, sclerotome, undifferentiated mesoderm and myocele were precisely identified.     

Embryonic muscle development in rainbow trout (Oncorhynchus mykiss): a scanning electron microscopy and immunohistological study

Embryonic muscle development was studied in rainbow trout (Oncorhynchus mykiss) at low and high temperature using scanning electron microscopy (SEM) and immunohistology. Somite development was described starting at stage 16 (Vernier JM. 1969. Ann Embryol Morphogen 4:495-520) for both temperatures, with special interest in their shape and size. Muscle differentiation, associated with somite growth, is characterized by a larger increase in height compared to width and by acquisition of a chevron shape. Thin structures such as striation, sarcomeres, and myofibrils within muscle cells and myotubes were observed starting at the eyed stage (stage 24). Immunohistological analyses showed appearance of embryonic fast myosin at stage 20 in the deep part of the somite. The area where myosin was expressed extended in the somite throughout embryonic development and the presence of myosin was observed in the entire somite at hatching (stage 30). Slow myosin was expressed in a monolayer of superficial cells at the eyed stage and during the entire embryonic development. Then it was expressed in a few layers of cells located in the red muscle area. These results suggest that muscle differentiation, characterized by myosin expression, is engaged at stage 20. Myogenesis starts in the deep part of the somite, near the notochord and progresses laterally to cover the complete somite at hatching when the somite is composed of muscle fibres exhibiting a high degree of maturity. No significant difference was observed in terms of muscular development between low- and high-temperature conditions. J. Exp. Zool. 286:379-389, 2000.     

Interactions between muscle fibers and segment boundaries in zebrafish

The most obvious segmental structures in the vertebrate embryo are somites: transient structures that give rise to vertebrae and much of the musculature. In zebrafish, most somitic cells give rise to long muscle fibers that are anchored to intersegmental boundaries. Therefore, this boundary is analogous to the mammalian tendon in that it transduces muscle-generated force to the skeletal system. We have investigated interactions between somite boundaries and muscle fibers. We define three stages of segment boundary formation. The first stage is the formation of the initial epithelial somite boundary. The second "transition" stage involves both the elongation of initially round muscle precursor cells and somite boundary maturation. The third stage is myotome boundary formation, where the boundary becomes rich in extracellular matrix and all muscle precursor cells have elongated to form long muscle fibers. It is known that formation of the initial epithelial somite boundary requires Notch signaling; vertebrate Notch pathway mutants show severe defects in somitogenesis. However, many zebrafish Notch pathway mutants are homozygous viable suggesting that segmentation of their larval and adult body plans at least partially recovers. We show that epithelial somite boundary formation and slow-twitch muscle morphogenesis are initially disrupted in after eight (aei) mutant embryos (which lack function of the Notch ligand, DeltaD); however, myotome boundaries form later ("recover") in a Hedgehog-dependent fashion. Inhibition of Hedgehog-induced slow muscle induction in aei/deltaD and deadly seven (des)/notch1a mutant embryos suggests that slow muscle is necessary for myotome boundary recovery in the absence of initial epithelial somite boundary formation. Because we have previously demonstrated that slow muscle migration triggers fast muscle cell elongation in zebrafish, we hypothesize that migrating slow muscle facilitates myotome boundary formation in aei/deltaD mutant embryos by patterning coordinated fast muscle cell elongation. In addition, we utilized genetic mosaic analysis to show that somite boundaries also function to limit the extent to which fast muscle cells can elongate. Combined, our results indicate that multiple interactions between somite boundaries and muscle fibers mediate zebrafish segmentation.     

A spatial and temporal analysis of dorsal root and sympathetic ganglion formation in the avian embryo

The present study explores the formation of the dorsal root and sympathetic ganglia in the trunk of the avian embryo. Particular emphasis was given to the timing of gangliogenesis and the relative positions of the neural crest-derived ganglia with respect to the somites. Neural crest cells and their derivatives were recognized by the HNK-1 antibody. The time at which neural crest cell coalesced to form ganglia was assessed by the state of cellular aggregation. The state of ganglionic differentiation was assessed by the expression of neurofilament proteins and the neural cell adhesion molecule (N-CAM). At the level of the 15th somite, neural crest cells were observed in the rostral half of the somite at stage 15, during active neural crest migration, and occupied the rostral two-thirds of the somite at progressive stages. HNK-1 positive cells appeared to be organized in three to four streams of cells oriented mediolaterally and dorsoventrally. The dorsal root ganglia and sympathetic ganglia were first detectable at stages 20 and 21, respectively. Both ganglionic rudiments were aligned with the rostral portion of the somite. The dorsal root ganglia occupied the rostral two-thirds of each somite, whereas cells in the sympathetic ganglia occupied a region corresponding to approximately one-third of each somite. At the time of condensation of the dorsal root ganglia, abundant neurofilament staining was observed within the ganglia. However, no N-CAM immunoreactivity was detected until three stages later at stage 23. In contrast, the sympathetic ganglia demonstrated both neurofilament and N-CAM immunoreactivity at the time of condensation. The observation that both dorsal root and sympathetic ganglia form in register with the rostral portion of somite suggests that cues localized at these axial levels, perhaps within the rostral somite, may influence the position where neural crest cells condense to form ganglia. In sensory ganglia, N-CAM expression does not correlate with the onset of gangliogenesis, suggesting that molecules other than N-CAM may play an important role in the aggregation of some neuronal populations.     

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

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

Diisopropylfluorophosphate inhibits acetylcholinesterase activity and disrupts somitogenesis in the zebrafish.

Acetylcholinesterase (AChE) activity, localized histochemically, appeared in the nuclei of presumptive somitic mesodermal cells prior to the onset of somitogenesis. AChE activity appeared in a rostro-caudal sequence, in cells located the equivalent of five somite lengths caudal to the last formed somite. To investigate whether AChE activity was required for somitogenesis, several inhibitors of AChE activity were tested for their ability to block somitogenesis. Diisopropylfluorophosphate (DFP), a broad spectrum inhibitor of serine proteases and related enzymes, was the only AChE inhibitor tested that disrupted somitogenesis. Gastrulae at 50% epiboly exposed continuously to DFP at concentrations between 40 microM and 90 microM completed epiboly, but exhibited a dose-dependent decrease in the number of somites formed, and a parallel decrease in the caudal extent of somite innervation, by 24 hours post-fertilization (h). Fifteen somite (15h) embryos exposed to DFP at the ED50 of 70 microM for 3 hours, followed by recovery to 24h, developed abnormal somites. Approximately five normal somites formed after drug treatment before the first abnormal somite formed. The abnormal somites corresponded in location to that area of the presumptive somitic mesoderm that would have initiated AChE activity while the DFP was present. While exposed to 70 microM DFP, presumptive somites formed and motoneurons extended processes that had initiated AChE activity at the time of treatment with DFP, although at a slower than normal rate. However, embryos exposed to 1 mM DFP for 30 minutes at both the 5 and 15 somite stages, followed by recovery to 24h, developed the normal number of somites but were reduced in the caudal extent of somite innervation, and occasionally developed abnormal primary motoneurons. As with the abnormal somites, the abnormal motoneurons would have initiated AChE activity while the DFP was present. Presumptive somitic mesoderm unable to initiate AChE activity due to inhibition by DFP developed abnormally. While the effects of DFP are not limited to inhibiting AChE, the data support the "clock and wavefront" model proposed for somite formation, and support the hypothesis that AChE activity has a role in somitogenesis in zebrafish.     

THE DIFFERENTIAL SENSITIVITY OF CULTURED CHICK MESODERMAL CELLS TO ACTINOMYCIN D

Cells were isolated from the somite mesoderm and from the unsegmented (presomite) mesoderm of early chick embryos and exposed to actinomycin D in single cell culture. Actinomycin D inhibited proliferation in cell cultures derived from the unsegmented mesoderm, although the same concentrations of this antibiotic did not inhibit cultures derived from the somite mesoderm. This differential sensitivity parallels the regionally specific necrosis and degeneration observed in the unsegmented mesoderm of intact chick embryos exposed to actinomycin D. In culture, both cell types exhibited approximately the same permeability to labeled actinomycin D and showed comparable inhibition of RNA, DNA, and protein syntheses in the presence of the antibiotic. However, freshly isolated mesodermal cells from the somite region had a higher content of RNA than did cells from the unsegmented region, and the somite cells maintained a higher rate of macromolecular synthesis in untreated cultures.