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PubMed2006

Zebrafish cypher is important for somite formation and heart development.

Mammalian CYPHER (Oracle, KIA0613), a member of the PDZ-LIM family of proteins (Enigma/LMP-1, ENH, ZASP/Cypher, RIL, ALP, and CLP-36), has been associated with cardiac and muscular myopathies. Targeted deletion of Cypher in mice is neonatal lethal possibly caused by myopathies. To further investigate the role of cypher in development, we have cloned the zebrafish orthologue. We present here the gene, domain structure, and expression pattern of zebrafish cypher during development. Cypher was not present as a maternal mRNA and was absent during early development. Cypher mRNA was first detected at the 3-somite stage in adaxial somites, and as somites matured, cypher expression gradually enveloped the whole somite. Later, cypher expression was also found in the heart, in head and jaw musculature, and in the brain. We further identified 13 alternative spliced forms of cypher from zebrafish heart and skeletal muscle tissue, among them a very short form containing the PDZ domain but lacking the ZM (ZASP-like) motif and the LIM domains. Targeted gene knock-down experiments using cypher antisense morpholinos led to severe defects, including truncation of the embryo, deformation of somites, dilatation of the pericardium, and thinning of the ventricular wall. The phenotype could be rescued by a cypher form, which contains the PDZ domain and the ZM motif, but lacks all three LIM domains. These findings indicate that a PDZ domain protein is important for normal somite formation and in normal heart development. Treatment of zebrafish embryos with cyclopamine, which disrupts hedgehog signaling, abolished cypher expression in 9 somite and 15-somite stage embryos. Taken together, our data suggest that cypher may play a role downstream of sonic hedgehog, in a late stage of somite development, when slow muscle fibers differentiate and migrate from the adaxial cells.

Alternative Splicing↗
PubMed2000

Somite formation and patterning.

As a consequence of their segmented arrangement and the diversity of their tissue derivatives, somites are key elements in the establishment of the metameric body plan in vertebrates. This article aims to largely review what is known about somite development, from the initial stages of somite formation through the process of somite regionalization along the three major body axes. The role of both cell intrinsic mechanisms and environmental cues are evaluated. The periodic and bilaterally synchronous nature of somite formation is proposed to rely on the existence of a developmental clock. Molecular mechanisms underlying these events are reported. The importance of an antero-posterior somitic polarity with respect to somite formation on one hand and body segmentation on the other hand is discussed. Finally, the mechanisms leading to the regionalization of somites along the dorso-ventral and medio-lateral axes are reviewed. This somitic compartmentalization is believed to underlie the segregation of dermis, skeleton, and dorsal and appendicular musculature.

Animals↗
PubMed2001

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.

Animals↗
PubMed1996

Requirement of the paraxis gene for somite formation and musculoskeletal patterning.

The segmental organization of the vertebrate embryo is first apparent when somites form in a rostrocaudal progression from the paraxial mesoderm adjacent to the neural tube. Newly formed somites appear as paired epithelial spheres that become patterned to form vertebrae, ribs, skeletal muscle and dermis. Paraxis is a basic helix-loop-helix transcription factor expressed in paraxial mesoderm and somites. Here we show that in mice homozygous for a paraxis null mutation, cells from the paraxial mesoderm are unable to form epithelia and so somite formation is disrupted. In the absence of normal somites, the axial skeleton and skeletal muscle form but are improperly patterned. Unexpectedly, however, we found that formation of epithelial somites was not required for segmentation of the embryo or for the establishment of somitic cell lineages. These results demonstrate that paraxis regulates somite morphogenesis, and that the function of somites is to pattern the axial skeleton and skeletal muscles.

Animals↗
PubMed2000

The expression of the homeobox gene Msx1 reveals two populations of dermal progenitor cells originating from the somites.

Experimental manipulation in birds has shown that trunk dermis has a double origin: dorsally, it derives from the somite dermomyotome, while ventrally, it is formed by the somatopleure. Taking advantage of an nlacZ reporter gene integrated into the mouse Msx1 locus (Msx1(nlacZ) allele), we detected segmental expression of the Msx1 gene in cells of the dorsal mesenchyme of the trunk between embryonic days 11 and 14. Replacing somites from a chick host embryo by murine Msx1(nlacZ )somites allowed us to demonstrate that these Msx1-(beta)-galactosidase positive cells are of somitic origin. We propose that these cells are dermal progenitor cells that migrate from the somites and subsequently contribute to the dorsalmost dermis. By analysing Msx1(nlacZ) expression in a Splotch mutant, we observed that migration of these cells does not depend on Pax3, in contrast to other migratory populations such as limb muscle progenitor cells and neural crest cells. Msx1 expression was never detected in cells overlying the dermomyotome, although these cells are also of somitic origin. Therefore, we propose that two somite-derived populations of dermis progenitor cells can be distinguished. Cells expressing the Msx1 gene would migrate from the somite and contribute to the dermis of the dorsalmost trunk region. A second population of cells would disaggregate from the somite and contribute to the dermis overlying the dermomyotome. This population never expresses Msx1. Msx1 expression was investigated in the context of the onset of dermis formation monitored by the Dermo1 gene expression. The gene is downregulated prior to the onset of dermis differentiation, suggesting a role for Msx1 in the control of this process.

Animals↗
PubMed2000

Zebrafish Mesp family genes, mesp-a and mesp-b are segmentally expressed in the presomitic mesoderm, and Mesp-b confers the anterior identity to the developing somites.

Segmentation of a vertebrate embryo begins with the subdivision of the paraxial mesoderm into somites through a not-well-understood process. Recent studies provided evidence that the Notch-Delta and the FGFR (fibroblast growth factor receptor) signalling pathways are required for segmentation. In addition, the Mesp family of bHLH transcription factors have been implicated in establishing a segmental prepattern in the presomitic mesoderm. In this study, we have characterized zebrafish mesp-a and mesp-b genes that are closely related to Mesp family genes in other vertebrates. During gastrulation, only mesp-a is expressed in the paraxial mesoderm at the blastoderm margin. During the segmentation period, both genes are segmentally expressed in one to three stripes in the anterior parts of somite primordia. In fused somites (fss) embryos, in which all early somite boundary formation is blocked, initial mesp-a expression at the gastrula stage remains intact, but the expression of mesp-a and mesp-b is not detected during the segmentation period. This suggests that these genes are downstream targets of fss at the segmentation stage. Comparison with her1 expression (Müller, M., von Weizsäcker, E. and Campos-Ortega, J. A. (1996) Development 122, 2071-2078) suggests that, like her1, mesp genes are not expressed in primordia of the first several somites. Furthermore, we found that zebrafish her1 expression oscillates in the presomitic mesoderm. The her1 stripe, which first appears in the tailbud region, moves in a caudal to rostral direction, and it finally overlaps the most rostral mesp stripe. Thus, in the trunk region, both her1 and mesp transcripts are detected in every somite primordium posterior to the forming somites. Ectopic expression of Mesp-b in embryos causes a loss of the posterior identity within the somite primordium, leading to a segmentation defect. These embryos show a reduction in expression of the posterior genes, myoD and notch5, with uniform expression of the anterior genes, FGFR1, papc and notch6. These observations suggest that zebrafish mesp genes are involved in anteroposterior specification within the presumptive somites, by regulating the essential signalling pathways mediated by Notch-Delta and FGFR.

Amino Acid Sequence↗
PubMed1995

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.

Animals↗
PubMed1992

Perturbation of beta 1 integrin-mediated adhesions results in altered somite cell shape and behavior.

Cell contact and adhesion between somites and the axial extracellular matrix (ECM) is likely to play a fundamental role in vertebrate development. In a preliminary report we showed that injection of the monoclonal antibody CSAT, which recognizes the avian beta 1 integrins, causes a lateral separation of both somites and segmental plate tissue from the embryonic axis (Drake and Little, 1991). In this study we addressed the cell biological response to CSAT injection, particularly the cell-ECM interactions involved in maintaining normal somite-axial relationships. A total of 150 stage 7-10 quail embryos have been injected with CSAT and then cultured for varying periods (1-30 hr). CSAT caused somitic cells to behave abnormally. Changes include, rounding-up, extensive blebbing, and formation of retraction fibers. A majority of separated somites were able to assume normal axial position with further time culture. Whether a somite subsequently aligned at the axis was dependent on the amount of CSAT injected and the postinjection culture period. Embryos in which somites remained separated from the axis after relatively long culture intervals (18-24 hr) displayed abnormal sclerotomal cell migrations. In no case did control injected embryos exhibit cellular alterations. Similarly, the injection of RGD-containing peptides had no detectable effect on somitogenesis or somite/segmental plate adhesion to the axis. On the basis of these data, we conclude that beta 1 integrins are necessary for normal somitic cell adhesions to the axis, but not somite segmentation and differentiation.

Amino Acid Sequence↗
PubMed1989

Consequences of somite manipulation on the pattern of dorsal root ganglion development.

We have investigated dorsal root ganglion formation, in the avian embryo, as a function of the composition of the paraxial somitic mesoderm. Three or four contiguous young somites were unilaterally removed from chick embryos and replaced by multiple cranial or caudal half-somites from quail embryos. Migration of neural crest cells and formation of DRG were subsequently visualized both by the HNK-1 antibody and the Feulgen nuclear stain. At advanced migratory stages (as defined by Teillet et al. Devl Biol. 120, 329-347 1987), neural crest cells apposed to the dorsolateral faces of the neural tube were distributed in a continuous, nonsegmented pattern that was indistinguishable on unoperated sides and on sides into which either half of the somites had been grafted. In contrast, ventrolaterally, neural crest cells were distributed segmentally close to the neural tube and within the cranial part of each normal sclerotome, whereas they displayed a nonsegmental distribution when the graft involved multiple cranial half-somites or were virtually absent when multiple caudal half-somites had been implanted. In spite of the identical dorsal distribution of neural crest cells in all embryos, profound differences in the size and segmentation of DRG were observed during gangliogenesis (E4-9) according to the type of graft that had been performed. Thus when the implant consisted of compound cranial half-somites, giant, coalesced ganglia developed, encompassing the entire length of the graft. On the other hand, very small, dorsally located ganglia with irregular segmentation were seen at the level corresponding to the graft of multiple caudal half-somites. We conclude that normal morphogenesis of dorsal root ganglia depends upon the craniocaudal integrity of the somites.

Animals↗
PubMed1995

Myogenic cell migration from somites is induced by tissue contact with medial region of the presumptive limb mesoderm in chick embryos.

It is known that myogenic cells in limb buds are derived from somites. In order to examine the potential of the limb primordium (presumptive limb somatopleure) to induce myogenic cell migration, we transplanted chick presumptive limb somatopleure to the flank region of an embryo, a region that does not normally contribute myogenic cells to the limb. Somitic cell migration was examined using a vital labeling technique. When the presumptive limb somatopleure was transplanted and was in contact with the host flank somite, somitic-cell migration toward the graft was observed. The labeled somitic cells within the graft were identified as myogenic cells in two ways: first, we found that N-cadherin-expressing cells appeared in the graft. Second, after 3 further days of incubation, the somitic cells formed dorsal and ventral masses and expressed sarcomeric myosin heavy chain within the graft. Cell migration occurred only when the somite was in contact with the medial region of the presumptive limb somatopleure. When the somite was not in contact with the limb somatopleure, or when the somite was in contact with the lateral region of the limb somatopleure, migration did not occur. These observations indicate that the potential to induce myogenic cell migration is restricted to the medial region of the presumptive limb somatopleure and that tissue contact is required.

Animals↗
PubMed1998

Control of somite patterning by Sonic hedgehog and its downstream signal response genes.

In the avian embryo, previous work has demonstrated that the notochord provides inductive signals to activate myoD and pax1 regulatory genes, which are expressed in the dorsal and ventral somite cells that give rise to myotomal and sclerotomal lineages. Here, we present bead implantation and antisense inhibition experiments that show that Sonic hedgehog is both a sufficient and essential notochord signal molecule for myoD and pax1 activation in somites. Furthermore, we show that genes of the Sonic hedgehog signal response pathway, specifically patched, the Sonic hedgehog receptor, and gli and gli2/4, zinc-finger transcription factors, are activated in coordination with somite formation, establishing that Sonic hedgehog response genes play a regulatory role in coordinating the response of somites to the constitutive notochord Sonic hedgehog signal. Furthermore, the expression of patched, gli and gli2/4 is differentially patterned in the somite, providing mechanisms for differentially transducing the Sonic hedgehog signal to the myotomal and sclerotomal lineages. Finally, we show that the activation of gli2/4 is controlled by the process of somite formation and signals from the surface ectoderm, whereas upregulation of patched and activation of gli is controlled by the process of somite formation and a Sonic hedgehog signal. The Sonic hedgehog signal response genes, therefore, have important functions in regulating the initiation of the Sonic hedgehog response in newly forming somites and in regulating the patterned expression of myoD and pax1 in the myotomal and sclerotomal lineages following somite formation.

Amino Acid Sequence↗
PubMed1999

The homeobox gene Msx1 is expressed in a subset of somites, and in muscle progenitor cells migrating into the forelimb.

In myoblast cell cultures, the Msx1 protein is able to repress myogenesis and maintain cells in an undifferentiated and proliferative state. However, there has been no evidence that Msx1 is expressed in muscle or its precursors in vivo. Using mice with the nlacZ gene integrated into the Msx1 locus, we show that the reporter gene is expressed in the lateral dermomyotome of brachial and thoracic somites. Cells from this region will subsequently contribute to forelimb and intercostal muscles. Using Pax3 gene transcripts as a marker of limb muscle progenitor cells as they migrate from the somites, we have defined precisely the somitic origin and timing of cell migration from somites to limb buds in the mouse. Differences in the timing of migration between chick and mouse are discussed. Somites that label for Msx1(nlacZ )transgene expression in the forelimb region partially overlap with those that contribute Pax3-expressing cells to the forelimb. In order to see whether Msx1 is expressed in this migrating population, we have grafted somites from the forelimb level of Msx1(nlacZ )mouse embryos into a chick host embryo. We show that most cells migrating into the wing field express the Msx1(nlacZ )transgene, together with Pax3. In these experiments, Msx1 expression in the somite depends on the axial position of the graft. Wing mesenchyme is capable of inducing Msx1 transcription in somites that normally would not express the gene; chick hindlimb mesenchyme, while permissive for this expression, does not induce it. In the mouse limb bud, the Msx1(nlacZ )transgene is downregulated prior to the activation of the Myf5 gene, an early marker of myogenic differentiation. These observations are consistent with the proposal that Msx1 is involved in the repression of muscle differentiation in the lateral half of the somite and in limb muscle progenitor cells during their migration.

Animals↗
PubMed1980

Somite formation in cultured embryos of the snapping turtle. Chelydra serpentina.

A simple, reliable method for the in vitro cultivation of snapping turtle embryos was demonstrated. This technique was used to study somite formation in explants containing segmental plates. Segmental plates formed a full complement of somites whether the neural tube or the neural tube and notochord was present. Explanted snapping turtle segmental plates formed an average of 6.6 +/- 1.2 somites. Removal of the node region or tail bud from cultured intact embryos led to a cessation of somite formation after an additional 6.1 +/- 1.8 somites had formed. These results indicate the number of somites the snapping turtle segmental plate will form. Also, the number of somites formed by explanted segmental plates showed only slight variation over a wide range of segmental plate lengths. It was concluded that while snapping turtle segmental plates formed significantly fewer somites than chicken or Japanese quail segmental plates, they were similar to the avian explants in their ability to form a consistent number of somites regardless of the length of the segmental plate.

Animals↗
PubMed1981

Somitogenesis in the amphibian Xenopus laevis: scanning electron microscopic analysis of intrasomitic cellular arrangements during somite rotation.

The intrasomitic changes in cell arrangement which accompany somite rotation during somitogenesis in Xenopus laevis were analysed with the scanning electron microscope (SEM). Longitudinal, horizontal fractures of whole embryos were examined at various dorsoventral levels of stage-22 to -24 embryos. Observations of the gross morphological features of somitogenesis, and the cellular changes which accompany somite segmentation and somite rotation were made. Several of these observations lead to modifications of previous models for the cellular basis of somitogenesis in Xenopus. Individual cellular rearrangements, rather than simultaneous block rotation of a whole somite, appear to be responsible for the 90 degrees rotation of myotomal cells within a single somite. Cellular arrangements in fused somites were also examined. Some ultraviolet-irradiated embryos displayed a complete lack of a notochord. The somites in those embryos were fused across the midline beneath the neural tube. The dorsal and ventral arms of the somites are not fused. Normal rotation occurs only in the dorsal and ventral arms while, in the majority of cases, cells in the fused region fail to rotate normally. In some cases, individual cells in the fused region undergo partial rearrangement. Those observations support the notion that individual cellular rearrangements account for the rotation of the whole somite.

Animals↗
PubMed1995

Desmin transgene expression in mouse somites requires the presence of the neural tube.

Transgenic mice were used to study the effect of the neural tube on somite myogenesis. These mice express a transgene in which the 1 kb DNA 5' regulatory sequence of the desmin gene is linked to a reporter gene which codes for E. coli beta-galactosidase. In order to determine whether the developmental fate of cells, specifically the prospective myogenic population, in newly developed somites was pre-determined, newly formed somites were isolated from the caudal region of day 9.5 transgenic embryos and transplanted into 8.5 day non-transgenic host embryos. Even though the implanted somites were not oriented in the host embryos, all the specimens examined developed normally at the graft site forming a dermatome, myotome and sclerotome in the correct anatomical positions. The myotome even expressed the desmin transgene. In addition, we isolated the 3 most caudal somites, that is, the most recently developed somites, from day 9.5 transgenic embryos and maintained them on gelatin-coated coverslips in culture for up to 4 days. While these somite explants did not develop myoblasts, it was possible to induce myogenesis by introducing pieces of neural tube into the explant cultures. These results suggest that the developmental fate of cells within the newly formed somite is not predetermined, but is dependent on the microenvironment surrounding the developing somite.

Animals↗
PubMed1996

Valproic acid-induced somite teratogenesis in the chick embryo: relationship with Pax-1 gene expression.

The repeated pattern of the axial skeleton results from the segmentation and re-segmentation of the mesodermally derived somites. During these early events of somite development, the vertebrate embryonic axial skeleton is most susceptible to the teratogenic effects of a variety of pharmaceutical and environmental agents. One example is the anticonvulsant drug valproic acid (VPA), which has been shown to cause craniofacial and minor and major skeletal defects in human and animal embryos. We hypothesize that a candidate set of molecular targets of teratogens are the Pax family of pattern-forming genes, specifically Pax-1, which has been previously demonstrated to be an important regulator of axial skeletal patterning at the somite level. In this study, early developmental stage chick embryos were treated with VPA and dose-dependent malformations in somite development were observed. Two classes of anomalies were evident: class I included discrete sites of somitic fusions or mis-segmentation, and Class II included large areas of disorganized somite patterning. Northern blot analysis revealed a decreased level of Pax-1 expression in VPA-treated embryos. Whole mount in situ hybridization analysis showed that somite anomalies correlate spatially with regions of decreased Pax-1 expression. Finally, comparison of the VPA-induced somitic anomalies with those caused by gene-specific perturbation of Pax-1 gene expression through the use of an antisense oligonucleotide revealed significant similarities. Taken together, these results support the hypothesis that Pax-1 is a molecular target in VPA axial skeletal teratogenicity.

Abnormalities, Drug-Induced↗
PubMed2005

Expression patterns of collagen I (alpha1) encoding gene and muscle-specific genes reveal that the lateral domain of the fish somite forms a connective tissue surrounding the myotome.

Somites are repeated, epithelial structures that are derived from the unsegmented paraxial mesoderm located lateral to the notochord. In higher vertebrates, somites differentiate into a sclerotome that subsequently forms the vertebrae and the ribs and into a dermomyotome that gives rise to a myotome, from which arises the skeletal muscle, and to a dermatome, from which arises the dermis. Fish somites have been shown to produce a sclerotome and a myotome, but very little is known regarding their participation in the formation of connective tissues, especially at the junction between the epidermis and the myotome. To investigate the formation of connective tissues in fish somites, we have examined the expression pattern of the collagen I (alpha1) chain. As somitogenesis proceeds rostrocaudally, collagen I (alpha1) expression marks the sclerotomal cells and delineates the formation of the vertebrae. Surprisingly, after the completion of the segmentation, transcript for the collagen I (alpha1) chain appeared in a distinct epithelial-like monolayer situated at the periphery of the developing somite facing the surface epidermis. This epithelial monolayer of somitic cells that covered the superficial slow muscle cells, did not express the myogenic transcriptional regulator myogenin and was devoid of contractile filament. As the somite increased in size, these collagen-expressing epithelial cells flattened, forming a thin cellular layer underlying the epidermis and recovering the lateral surface of the myotome. In conclusion, the lateral domain of the fish somite forms a distinct epithelial cell layer sharing many characteristics with amniote dermatome.

Animals↗
PubMed1998

Control of dorsoventral somite patterning by Wnt-1 and beta-catenin.

In vertebrates, the dorsoventral patterning of somitic mesoderm is controlled by factors expressed in adjacent tissues. The ventral neural tube and the notochord function to promote the formation of the sclerotome, a ventral somite derivative, while the dorsal neural tube and the surface ectoderm have been shown to direct somite cells to a dorsal dermomyotomal fate. A number of signaling molecules are expressed in these inducing tissues during times of active cell fate specification, including members of the Hedgehog, Wnt, and BMP families. However, with the exception of the ventral determinant Sonic hedgehog (Shh), the functions of these signaling molecules with respect to dorsoventral somite patterning have not been determined. Here we investigate the role of Wnt-1, a candidate dorsalizing factor, in the regulation of sclerotome and dermomyotome formation. When ectopically expressed in the presomitic mesoderm of chick embryos in ovo, Wnt-1 differentially affects the expression of dorsal and ventral markers. Specifically, ectopic Wnt-1 is able to completely repress ventral (sclerotomal) markers and to enhance and expand the expression of dorsal (dermomyotomal) markers. However, Wnt-1 appears to be unable to convert all somitic mesoderm to a dermomyotomal fate. Delivery of an activated form of beta-catenin to somitic mesoderm mimics the effects of Wnt-1, demonstrating that Wnt-1 likely acts directly on somitic mesoderm, and not through adjacent tissues via an indirect signal relay mechanism. Taken together, our results support a model for somite patterning where sclerotome formation is controlled by the antagonistic activities of Shh and Wnt signaling pathways.

Animals↗