Somite Maturation Research Articles

Zebrafish protocadherin 10 is involved in paraxial mesoderm development and somitogenesis

Here, we present the first report of the molecular cloning of zebrafish protocadherin 10 (Pcdh10, OL-protocadherin) and describe its functional analyses in the development of segmental plate. Epitope-tagged Pcdh10 expressed in embryos was localized on cell peripheries of adjacent cells. In situ hybridization showed that pcdh10 was expressed in the paraxial mesoderm (PAM) and developing somites, and in the pineal body, the diencephalon, and the vicinity of otocysts. Expression in PAM increased in the last few presumptive somites, reached the maximum level in the latest segmenting somites, and decreased thereafter during somite maturation. These expression patterns suggested that Pcdh10 is involved in development of PAM and somites. This was confirmed by morpholino knockdown and dominant-negative inhibition of Pcdh10 in embryos, which disturbed movements of PAM cells and somite segmentation. Comparative studies showed that pcdh10 expression lasted up to approximately three times longer in maturing somites than that of paraxial protocadherin (pcdh8). They also indicated that the adaxial cells expressed pcdh8 but not pcdh10. We propose that Pcdh10 is involved in the morphogenic movements of PAM cells and somite segmentation and that differential adhesion of Pcdh8 and Pcdh10 plays a role in the morphogenic machinery of somites and adaxial cells.

Expression of avian C‐terminal binding proteins (Ctbp1 and Ctbp2) during embryonic development

C-terminal binding proteins (CtBPs) are transcriptional corepressors of mediators of Notch, Wnt, and other signalling pathways. Thus, they are potential players in the control of several developmentally important processes, including segmentation, somitogenesis, and neural tube and limb patterning. We have cloned the avian orthologues of Ctbp1 and Ctbp2 and examined their expression pattern by whole-mount in situ hybridization between Hamburger and Hamilton (HH) stages 3 and 24. Both Ctbp genes show similar expression patterns during embryonic development, and both are detected from HH stage 3 in the developing central nervous system, by HH stage 7 in the paraxial mesoderm and later in the limb bud. In most places, Ctbp1 and Ctbp2 are expressed in overlapping domains. However, there are interesting domains and/or temporal expression patterns that are specific to each Ctbp gene. For instance, Ctbp1 is predominantly expressed in the epiblast, whereas Ctbp2 is in the primitive streak at HH stage 3. However, by HH stage 4, both genes are found in the primitive streak and in the ectoderm. Similarly, although both genes display similar expression patterns in early somitogenesis, in mature somites, Ctbp1 transcripts are located in myotomal cells, whereas Ctbp2 transcripts are observed in dermomyotomal cells. Finally, we found that emigrating neural crest cells express Ctbp2, whereas dorsal root ganglia express Ctbp1. These data suggest that Ctbp1 and Ctbp2 may be functionally redundant in some tissues and have unique functions in other tissues.

Heparin-binding EGF-like growth factor shows transient left–right asymmetrical expression in mouse myotome pairs

Heparin-binding EGF-like growth factor (HB-EGF) is a potent mitogen and chemoattractant for diverse cell types including, keratinocytes, fibroblasts and vascular smooth muscle cells. In adult mice, skeletal muscle and endothelial cells prominently express HB-EGF, although analysis of embryonic expression has been limited to studies of heart and kidney development. Here we survey HB-EGF mRNA expression in E7.5–E15 mouse embryos and show that HB-EGF is expressed in branchial arches, limb buds and, transiently, in mature somites between E9.25 and E11. This somitic expression is restricted to the myotomal compartment. Intriguingly, within myotome pairs, the expression of HB-EGF is stronger on the left side of the body, whilst cognate receptors, ErbB1 and ErbB4, are symmetrically expressed in left and right somite pairs. In iv/iv mutant embryos, with inverted left–right body axis, the expression of HB-EGF was also inverted, now being stronger in myotomes on the right side of the body. Thus, the expression of HB-EGF in myotome pairs is regulated by global cues that define the left–right body axis.

Formation and differentiation of the avian dermomyotome.

During somite maturation, the ventral half of the epithelial somite disintegrates into the mesenchymal sclerotome, whereas the dorsal half forms a transitory epithelial sheet, the dermomyotome, lying in between the sclerotome and the surface ectoderm. The dermomyotome is the source of most of the mesodermal tissues in the body, giving rise to cell types as different as muscle, connective tissue, endothelium, and cartilage. Thus, the dermomyotome is the most important turntable of mesodermal cell fate choice in the vertebrate embryo. Here, we discuss the current knowledge on the formation of the dermomyotome and the mechanisms leading to the development of the various dermomyotomal derivatives, with special emphasis on the development of musculature and dermis.

Developmentally regulated expression and functional role of alpha 7 integrin in the chick embryo.

Integrin alpha 7 beta 1 is a specific cellular receptor for laminin. In the present work, we studied the distribution pattern of the alpha 7 subunit by immunofluorescence and immunoprecipitation and the role of the integrin by blocking antibodies in early chick embryos. alpha 7 immunoreactivity was first detectable in the neural plate during neural furrow formation (stage HH5, early neurula, Hamburger & Hamilton 1951) and its expression was upregulated in the neural folds during primary neurulation. The alpha 7 expression domain spanned the entire neural tube by stage HH8 (4 somites), and was then downregulated and confined to the neuroepithelial cells in the germinal region near the lumen and the ventrolateral margins of the neural tube in embryos by the onset of stage HH17 (29 somites). Expression of alpha 7 in the neural tube was transient suggesting that alpha 7 functions during neural tube closure and axon guidance and may not be required for neuronal differentiation or for the maintenance of the differentiated cell types. alpha 7 immunoreactivity was strong in the newly formed epithelial somites, although this expression was restricted only to the myotome in the mature somites. The most intense alpha 7 immunoreactivity was detectable in the paired heart primordia and the endoderm apposing the heart primordia in embryos at stage HH8. In the developing heart, alpha 7 immunoreactivity was: (i) intense in the myocardium; (ii) milder in the endocardial cushions of the ventricle; (iii) intense in the sinus venosus; (iv) distinct in the associated blood vessels; and (v) undetectable in the dorsal mesocardium of embryos at stage HH17. Inhibition of function of alpha 7 by blocking antibodies showed that alpha 7 integrin-laminin signaling may play a critical role in tissue organization of the neural plate and neural tube closure, in tissue morphogenesis of the heart tube but not in the directional migration of pre-cardiac cells, and in somite epithelialization but not in segment formation in presomitic mesoderm. In embryos treated with alpha 7 antibody, the formation of median somites in place of a notochord was intriguing and suggested that alpha 7 integrin-laminin signaling may have played a role in segment re-specification in the mesoderm.

Expression pattern of VEGFR-2 (Quek1) during quail development.

The growth and maintenance of the blood and lymphatic vascular systems is to a large extent controlled by members of the vascular endothelial growth factor (VEGF) family via the tyrosine kinase receptors (VEGFRs) expressed in angioblastic cells. Here, we analyzed the Quek1 (VEGFR-2) expression pattern by whole mount in situ hybridization during quail development. During early embryogenesis, Quek1 expression was detected at the caudal end of the blastoderm and primitive streak and in the head paraxial mesoderm. In somites, expression was observed from HH-stage 9 onwards in the dorsolateral region of both the forming and recently formed somites. During somite maturation, expression persists in the lateral portion of the somitic compartments, the dermomyotome and the sclerotome. Additionally, a second expression domain in the maturing somite was observed in the medial part of the sclerotome adjacent to the neural tube. This expression domain extended medially and dorsally and surrounded the neural tube during later stages. In the notochord, expression was observed from HH-stage 23 onwards. In the limb bud, expression was initiated in the mesenchyme at HH-stage 17. During organogenesis, expression was detected in the pharyngeal arches and in the anlagen of the esophagus, trachea, stomach, lungs, liver, heart and gut. Expression was also seen in feather buds from day 7 onwards. Our results confirm the angiogenic potential of the mesoderm and suggest that VEGFR-2 expressing cells represent multiple pools of mesodermal precursors of the hematopoietic and angiopoietic lineages.

The T-box transcription factor Tbx18 maintains the separation of anterior and posterior somite compartments.

The compartmentalization of somites along their anterior-posterior (AP) axis is pivotal to the segmental organization of the vertebrate axial skeleton and the peripheral nervous system. Anterior and posterior somite halves contribute to different vertebral elements. They are also characterized by different proliferation rates and properties with respect to neural crest cell migration and spinal nerve passage. AP-somite polarity is generated in the anterior presomitic mesoderm by Mesp2 and Delta/Notch signaling. Here, we demonstrate that maintenance of AP-somite polarity is mediated by the T-box transcription factor Tbx18. Mice deficient for Tbx18 show expansion of pedicles with transverse processes and proximal ribs, elements derived from the posterior lateral sclerotome. AP-somite polarity is established in Tbx18 mutant embryos but is not maintained. During somite maturation, posterior somite compartments expand most likely because of posterior cells invading the anterior somite half. In the anterior lateral sclerotome, Tbx18 acts as an antiapoptotic factor. Ectopic expression experiments suggest that Tbx18 can promote anterior at the expense of posterior somite compartments. In summary, Tbx18 appears to act downstream of Mesp2 and Delta/Notch signaling to maintain the separation of anterior and posterior somite compartments.

Tbx18 and boundary formation in chick somite and wing development

The chicken Tbx gene, Tbx18, is expressed in lateral plate mesoderm, limb, and developing somites. Here we show that Tbx18 is expressed transiently in axial mesenchyme during somite segmentation. We present evidence from overexpression and transplantation experiments that Tbx18 controls fissure formation in the late stages of somite maturation. In presumptive wing lateral plate mesoderm, ectopic Tbx18 expression leads to anterior extension of the wing bud. These results suggest that Tbx18 is involved in producing mesodermal boundaries, generating in paraxial mesoderm morphological boundaries between somites and in lateral plate mesoderm a wing- or non-wing-forming boundary.

Identification and developmental expression of Xenopus paraxis

During vertebrate embryogenesis, the paraxial mesoderm becomes segmented in a rostro-caudal progression and gives rise to the somites. In this paper we report the isolation of a Xenopus orthologue of paraxis, a member of a family of basic helix-loop-helix proteins, which has been suggested to play a role in paraxial mesoderm development. Xenopus paraxis is initially expressed in the presomitic paraxial mesoderm and later in the dorsal portion of the developing somites. Finally, paraxis expression becomes restricted to the most dorso-lateral region of mature somites.

Ventral axial organs regulate expression of myotomal Fgf-8 that influences rib development

Fgf-8 encodes a secreted signaling molecule mediating key roles in embryonic patterning. This study analyzes the expression pattern, regulation, and function of this growth factor in the paraxial mesoderm of the avian embryo. In the mature somite, expression of Fgf-8 is restricted to a subpopulation of myotome cells, comprising most, but not all, epaxial and hypaxial muscle precursors. Following ablation of the notochord and floor plate, Fgf-8 expression is not activated in the somites, in either the epaxial or the hypaxial domain, while ablation of the dorsal neural tube does not affect Fgf-8 expression in paraxial mesoderm. Contrary to the view that hypaxial muscle precursors are independent of regulatory influences from axial structures, these findings provide the first evidence for a regulatory influence of ventral, but not dorsal axial structures on the hypaxial muscle domain. Sonic hedgehog can substitute for the ventral neural tube and notochord in the initiation of Fgf-8 expression in the myotome. It is also shown that Fgf-8 protein leads to an increase in sclerotomal cell proliferation and enhances rib cartilage development in mature somites, whereas inhibition of Fgf signaling by SU 5402 causes deletions in developing ribs. These observations demonstrate: (1) a regulatory influence of the ventral axial organs on the hypaxial muscle compartment; (2) regulation of epaxial and hypaxial expression of Fgf-8 by Sonic hedgehog; and (3) independent regulation of Fgf-8 and MyoD in the hypaxial myotome by ventral axial organs. It is postulated that the notochord and ventral neural tube influence hypaxial expression of Fgf-8 in the myotome and that, in turn, Fgf-8 has a functional role in rib formation.

Somite-specific expression of a novel fibronectin variant FN3 is negatively regulated by SHH

Fibronectins (FNs) are major extracellular proteins in blood plasma and many tissues of vertebrates, and play important roles in adhesion, migration and differentiation of cells. We have identified a novel variant (FN3) of fibronectin in zebrafish. FN3 mRNA is abundant, as detected by whole-mount in situ hybridization, in the presomitic mesoderm and the newly formed somites, but less abundant in mature somites. Ectopic expression of Sonic Hedgehog (SHH) results in a decrease of FN3 expression, whereas the expression level of FN3 increases in the flh mutants that lack the notochord. Our results suggest that FN3 may be involved in the formation of somites, but during somite differentiation its expression needs to be downregulated by signals derived from the axial tissues.

HSP90alpha, HSP90beta, and p53 expression following in vitro hyperthermia exposure in gestation day 10 rat embryos.

The studies presented here are aimed at understanding the expression of p53, HSP90alpha, and HSP90beta in gestation day (GD) 10 CD rat embryos. GD 10 rat embryos were exposed in vitro to 37 degrees C or 42 degrees C for 15 min, then cultured at 37 degrees C for 0.5, 1, 3, or 5 h. Immunohistochemistry was performed on formalin-fixed, paraffin embedded, sectioned embryos for p53, HSP90alpha, or HSP90beta expression. p53 expression was minimal in control embryos but was induced with heat exposure. Maximum expression of p53 was observed in rostral tissues, e.g., the optic vesicle, rostral neuroepithelium, and mature (rostral) somites 3 and 5 h after heat exposure. Expression of p53 in the caudal region, such as in mid and caudal neuroepithelium, immature (caudal) somites, and presomitic mesoderm, was moderate compared to rostral areas. No p53 expression was observed in the heart under any condition. The rostral-caudal gradient of p53 expression was not observed for HSP90alpha expression. HSP90alpha was induced in heat-exposed embryos beginning at 1 h, predominantly in neural tube and optic vesicle. Moderate but increased expression was observed in the somites of heat-exposed embryos at 3 and 5 h. Expression of p53 was primarily nuclear while HSP90alpha expression was mostly cytoplasmic. No clear association was observed between heat-induced HSP90alpha and p53 expression. HSP90beta was expressed extensively in control and heat-exposed embryos. Results indicate that heat induces p53 and HSP90alpha expression, but not HSP90beta expression, and that HSP90alpha induction is not likely to be involved in p53 regulation in mammalian embryos.

Spatial and temporal pattern of Fgf-8 expression during chicken development.

This study analyzes the temporal and spatial expression pattern of Fgf-8 over a continuous series of developmental stages. Special emphasis is laid on the paraxial mesoderm where Fgf-8 expression is highly dynamic. Whereas the anterior portion of the unsegmented mesoderm is devoid of expression, Fgf-8 is upregulated in the posterior half of a newly formed somite. Soon after somite formation, this highly localized expression gives way to a more diffuse pattern of Fgf-8 expression at low levels in presumptive sclerotomal cells. During later somite maturation, transcripts become restricted to the myotome. Co-staining with the myotome marker MyoD reveals that Fgf-8 expression defines a subpopulation of muscle precursor cells.

Homologues of c-hairy1 (her9) and lunatic fringe in zebrafish are expressed in the developing central nervous system, but not in the presomitic mesoderm.

A number of genes that are involved in somitogenesis in vertebrates are cyclically expressed in the presomitic mesoderm. These include homologues of the Drosophila genes fringe and hairy. We have analysed here two genes that belong to these classes in the zebrafish, namely the apparent orthologues of lunatic fringe (l-fng) and of c-hairy1 (called her9). However, unlike the respective mouse and chicken genes, they are not expressed cyclically in the presomitic mesoderm. Instead, both genes are mainly expressed in the central nervous system. her9 is predominantly expressed in the fore- and midbrain, and transiently in the hindbrain. Thus, the previously identified and only very distantly related her1 gene of zebrafish has more similarities to the expression of the c-hairy1 gene than its apparent orthologue her9, indicating that sequence similarity and similarity of function are not necessarily linked in this case. l-fng expression is found in alternating pre-rhombomeres, comparable to the equivalent mouse gene expression and in the anterior compartments of the mature somites, which was also shown for the chicken l-fng gene. The latter expression indicates that it might be involved in boundary definition and cell fate decision processes, rather than in pre-patterning of the somites. Interestingly, a similar role has previously been inferred for the grasshopper homologue of l-fng. This suggests that the function of l-fng in boundary definition of the somites might be ancestral, while its recruitment to the pre-patterning process of the somites might be a derived feature in higher vertebrates.

Rainbow trout glucose transporter (OnmyGLUT1): functional assessment inXenopus laevisoocytes and expression in fish embryos

Recently, we reported the cloning of a putative glucose transporter (OnmyGLUT1) from rainbow trout embryos. In this paper, we describe the functional characteristics of OnmyGLUT1 and its expression during embryonic development of rainbow trout. Transport of D-glucose was analysed in Xenopus laevis oocytes following microinjection of mRNA transcribed in vitro. These experiments confirmed that OnmyGLUT1 is a facilitative Na(+)-independent transporter. Assessment of substrate selectivity, sensitivity to cytochalasin B and phloretin and kinetic parameters showed that the rainbow trout glucose transporter was similar to a carp transporter and to mammalian GLUT1. Embryonic expression of OnmyGLUT1 was studied using whole-mount in situ hybridization. Ubiquitous distribution of transcripts was observed until the early phase of somitogenesis. During the course of organogenesis, somitic expression decreased along the rostro-caudal axis, finally ceasing in the mature somites. The OnmyGLUT1 transcripts were detected in the neural crest during the whole study period. Transcripts were also found in structures that are likely to originate from the neural crest cells (gill arches, pectoral fins, upper jaw, olfactory organs and primordia of mouth lips). Hexose transport activity was detected at all developmental stages after blastulation. Cytochalasin B blocked the accumulation of phosphorylated 2-deoxy-D-glucose by dissociated embryonic cells, suggesting an important role for transport in glucose metabolism.

In situ detection of transcripts of the myogenic factor MyoD in whole chicken embryos

In situ hybridization has provided insights into the molecular basis of skeletal myogenesis during embryonic development. In situ detection of different muscle-specific regulatory factors in whole embryos has been described. Spatial and temporal expression patterns of these factors differed among species. The expression pattern of MyoD in whole chicken embryos was studied via in situ hybridization using a probe obtained by the reverse transcription - polymerase chain reaction (RT-PCR) technique. In newly formed somites (embryos of stage 12), MyoD mRNA transcripts were detected along the anterior to posterior axis of somites immediately adjacent to the neural tube, whereas in mature somites (embryos of stage 24), MyoD transcripts were detected throughout the entire somite. These results indicate that MyoD expression is important for initiating and maintaining the avian myogenic system.

Transplacental delivery of the Wnt antagonist Frzb1 inhibits development of caudal paraxial mesoderm and skeletal myogenesis in mouse embryos.

Axial structures (neural tube/notochord) and surface ectoderm activate myogenesis in the mouse embryo; their action can be reproduced, at least in part, by several molecules such as Sonic hedgehog and Wnts. Recently, soluble Wnt antagonists have been identified. Among those examined only Frzb1 was found to be expressed in the presomitic mesoderm and newly formed somites and thus its possible role in regulating myogenesis was investigated in detail. When presomitic mesoderm or newly formed somites were cultured with axial structures and surface ectoderm on a feeder layer of C3H10T1/2 cells expressing Frzb1, myogenesis was abolished or severely reduced in presomitic mesoderm and the three most recently formed somites. In contrast, no effect was observed on more mature somites. Inhibition of myogenesis did not appear to be associated with increased cell death since the final number of cells in the explants grown in the presence of Frzb1 was only slightly reduced in comparison with controls. In order to examine the possible function of Frzb1 in vivo, we developed a method based on the overexpression of the soluble antagonist by transient transfection of WOP cells with a Frzb1 expression vector and injection of transfected cells into the placenta of pregnant females before the onset of maternofoetal circulation. Frzb1, secreted by WOP cells, accumulated in the embryo and caused a marked reduction in size of caudal structures. Myogenesis was strongly reduced and, in the most severe cases, abolished. This was not due to a generalized toxic effect since only several genes downstream of the Wnt signaling pathway such as En1, Noggin and Myf5 were downregulated; in contrast, Pax3 and Mox1 expression levels were not affected even in embryos exhibiting the most severe phenotypes. Taken together, these results suggest that Wnt signals may act by regulating both myogenic commitment and expansion of committed cells in the mouse mesoderm.

MEF-2 function is modified by a novel co-repressor, MITR.

The MEF-2 proteins are a family of transcriptional activators that have been detected in a wide variety of cell types. In skeletal muscle cells, MEF-2 proteins interact with members of the MyoD family of transcriptional activators to synergistically activate gene expression. Similar interactions with tissue or lineage-specific cofactors may also underlie MEF-2 function in other cell types. In order to screen for such cofactors, we have used a transcriptionally inactive mutant of Xenopus MEF2D in a yeast two-hybrid screen. This approach has identified a novel protein expressed in the early embryo that binds to XMEF2D and XMEF2A. The MEF-2 interacting transcription repressor (MITR) protein binds to the N-terminal MADS/MEF-2 region of the MEF-2 proteins but does not bind to the related Xenopus MADS protein serum response factor. In the early embryo, MITR expression commences at the neurula stage within the mature somites and is subsequently restricted to the myotomal muscle. In functional assays, MITR negatively regulates MEF-2-dependent transcription and we show that this repression is mediated by direct binding of MITR to the histone deacetylase HDAC1. Thus, we propose that MITR acts as a co-repressor, recruiting a specific deacetylase to downregulate MEF-2 activity.

Calcium Signaling in the Developing Xenopus Myotome

Embryonic Xenopus myocytes generate spontaneous calcium (Ca2+) transients during differentiation in culture. Suppression of these transients disrupts myofibril organization and the formation of sarcomeres through an identified signal transduction cascade. Since transients often occur during myocyte polarization and migration in culture, we hypothesized they might play additional roles in vivo during tissue formation. We have tested this hypothesis by examining Ca2+ dynamics in the intact Xenopus paraxial mesoderm as it differentiates into the mature myotome. We find that Ca2+ transients occur in cells of the developing myotome with characteristics remarkably similar to those in cultured myocytes. Transients produced within the myotome are correlated with somitogenesis as well as myocyte maturation. Since transients arise from intracellular stores in cultured myocytes, we examined the functional distribution of both IP3 and ryanodine receptors in the intact myotome by eliciting Ca2+ elevations in response to photorelease of caged IP3 and superfusion of caffeine, respectively. As in culture, transients in vivo depend on Ca2+ release from ryanodine receptor (RyR) stores, and blocking RyR during development interferes with somite maturation.

Pax3 functions in cell survival and in pax7 regulation.

In developing vertebrate embryos, Pax3 is expressed in the neural tube and in the paraxial mesoderm that gives rise to skeletal muscles. Pax3 mutants develop muscular and neural tube defects; furthermore, Pax3 is essential for the proper activation of the myogenic determination factor gene, MyoD, during early muscle development and PAX3 chromosomal translocations result in muscle tumors, providing evidence that Pax3 has diverse functions in myogenesis. To investigate the specific functions of Pax3 in development, we have examined cell survival and gene expression in presomitic mesoderm, somites and neural tube of developing wild-type and Pax3 mutant (Splotch) mouse embryos. Disruption of Pax3 expression by antisense oligonucleotides significantly impairs MyoD activation by signals from neural tube/notochord and surface ectoderm in cultured presomitic mesoderm (PSM), and is accompanied by a marked increase in programmed cell death. In Pax3 mutant (Splotch) embryos, MyoD is activated normally in the hypaxial somite, but MyoD-expressing cells are disorganized and apoptosis is prevalent in newly formed somites, but not in the neural tube or mature somites. In neural tube and somite regions where cell survival is maintained, the closely related Pax7 gene is upregulated, and its expression becomes expanded into the dorsal neural tube and somites, where Pax3 would normally be expressed. These results establish that Pax3 has complementary functions in MyoD activation and inhibition of apoptosis in the somitic mesoderm and in repression of Pax7 during neural tube and somite development.