Myotome Of Somites Research Articles

Disabled-2: a positive regulator of the early differentiation of myoblasts

Dab2 is an adaptor protein and a tumor suppressor. Our previous study has found that Dab2 was expressed in early differentiating skeletal muscles in mouse embryos. In this study, we determined the role of Dab2 in the skeletal muscle differentiation using C2C12 myoblasts in vitro and Xenopus laevis embryos in vivo. The expression of Dab2 was increased in C2C12 myoblasts during the formation of myotubes in vitro. Knockdown of Dab2 expression in C2C12 myoblasts resulted in a reduction of myotube formation, whereas the myotube formation was enhanced upon overexpression of Dab2. Re-expression of Dab2 in C2C12 myoblasts with downregulated expression of Dab2 restored their capacity to form myotubes. Microarray profiling and subsequent network analyses on the 155 differentially expressed genes after Dab2 knockdown showed that Mef2c was an important myogenic transcription factor regulated by Dab2 through the p38 MAPK pathway. It was also involved in other pathways that are associated with muscular development and functions. In Xenopus embryos developed in vivo, XDab2 was expressed in the myotome of somites where various myogenic markers were also expressed. Knockdown of XDab2 expression with antisense morpholinos downregulated the expression of myogenic markers in somites. In conclusion, this study is the first to provide solid evidence to show that Dab2 is a positive regulator of the early myoblast differentiation.

Migrating cells mediate long-range WNT signaling

In amniotes, it is widely accepted that WNTs secreted by the dorsal neural tube form a concentration gradient that regulates early somite patterning and myotome organization. Here we demonstrate in the chicken embryo that WNT protein is not secreted to act at a distance, but rather loaded onto migrating neural crest cells that deliver it to somites. Inhibiting neural crest migration or ablating their population hasaprofoundimpactontheWNTresponseinsomites.Furthermore, we show that a central player in the efficient delivery of WNT to somites is the heparan sulfate proteoglycan GPC4, expressed by neural crest. Together, our data describe a novel mode of signaling whereby WNT proteins hitch a ride on migratory neural crest cells to pattern the somites at a distance from its source.

Itm2a Is a Pax3 Target Gene, Expressed at Sites of Skeletal Muscle Formation In Vivo

The paired-box homeodomain transcription factor Pax3 is a key regulator of the nervous system, neural crest and skeletal muscle development. Despite the important role of this transcription factor, very few direct target genes have been characterized. We show that Itm2a, which encodes a type 2 transmembrane protein, is a direct Pax3 target in vivo, by combining genetic approaches and in vivo chromatin immunoprecipitation assays. We have generated a conditional mutant allele for Itm2a, which is an imprinted gene, by flanking exons 2–4 with loxP sites and inserting an IRESnLacZ reporter in the 3′ UTR of the gene. The LacZ reporter reproduces the expression profile of Itm2a, and allowed us to further characterize its expression at sites of myogenesis, in the dermomyotome and myotome of somites, and in limb buds, in the mouse embryo. We further show that Itm2a is not only expressed in adult muscle fibres but also in the satellite cells responsible for regeneration. Itm2a mutant mice are viable and fertile with no overt phenotype during skeletal muscle formation or regeneration. Potential compensatory mechanisms are discussed.

Genesis of muscle fiber-type diversity during mouse embryogenesis relies on Six1 and Six4 gene expression

Adult skeletal muscles in vertebrates are composed of different types of myofibers endowed with distinct metabolic and contraction speed properties. Genesis of this fiber-type heterogeneity during development remains poorly known, at least in mammals. Six1 and Six4 homeoproteins of the Six/sine oculis family are expressed throughout muscle development in mice, and Six1 protein is enriched in the nuclei of adult fast-twitch myofibers. Furthermore, Six1/Six4 proteins are known to control the early activation of fast-type muscle genes in myocytes present in the mouse somitic myotome. Using double Six1:Six4 mutants (SixdKO) to dissect in vivo the genesis of muscle fiber-type heterogeneity, we analyzed here the phenotype of the dorsal/epaxial muscles remaining in SixdKO. We show by electron microscopy analysis that the absence of these homeoproteins precludes normal sarcomeric organization of the myofiber leading to a dystrophic aspect, and by immunohistochemistry experiments a deficiency in synaptogenesis. Affymetrix transcriptome analysis of the muscles remaining in E18.5 SixdKO identifies a major role for these homeoproteins in the control of genes that are specifically activated in the adult fast/glycolytic myofibers, particularly those controlling Ca2+ homeostasis. Absence of Six1 and Six4 leads to the development of dorsal myofibers lacking expression of fast-type muscle genes, and mainly expressing a slow-type muscle program. The absence of restriction of the slow-type program during the fetal period in SixdKO back muscles is associated with a decreased HDAC4 protein level, and subcellular relocalization of the transcription repressor Sox6. Six genes thus behave as essential global regulators of muscle gene expression, as well as a central switch to drive the skeletal muscle fast phenotype during fetal development.

Klhl31 is associated with skeletal myogenesis and its expression is regulated by myogenic signals and Myf-5

Klhl31 is an orthologue of Drosophila Kelch and belongs to a family of Kelch-like proteins in vertebrates. Members of this family contain multiple protein domains, including an amino-terminal broad complex/ tram-track/ bric-a-brac (BTB) or poxvirus and zinc finger (POZ) domain, carboxy-terminal Kelch repeats and a central linker region. We show that Klhl31 is highly expressed in the developing heart, the somite myotome and later in differentiated skeletal muscle and the myocardium. In developing somites expression of Klhl31 was initiated in the epaxial domain of the myotome, shortly after the skeletal muscle specific bHLH transcription factor, MyoD, was first expressed. Klhl31 remained expressed in skeletal muscle throughout embryonic and fetal development. Tissue ablations and rescue experiments that regulate myogenesis also govern expression of Klhl31 expression in somites. In particular, axial tissues, neural tube, floor plate and notochord, and surface ectoderm, provide combinatorial cues for myogenesis and the appropriate expression of Klhl31. We show that a combination of myogenic signals, Shh and either Wnt-1 or Wnt-6, are sufficient for Klhl31 expression in the dorsal somite. Furthermore, ectopic expression of Myf-5 led to expression of Klhl31 in the developing neural tube, indicating that Klhl31 is a novel and integral part of vertebrate myogenesis.

Regulation of vertebrate myotome development by the p38 MAP kinase–MEF2 signaling pathway

Biochemical and cell culture studies have characterized the myocyte enhancer factor 2 (MEF2) transcriptional regulatory proteins as obligatory partners for the myogenic regulatory factors (MRFs) in the differentiation of myogenic cells in culture. However, the role of MEF2 activation in somitic myogenesis has not been fully characterized. Here, we report a critical interaction between the p38 mitogen-activated protein kinase (p38 MAPK) and MEF2 in the developing somite myotome. We document expression of MEF2A and p38 MAPK proteins in the somite of 9.5 dpc mouse embryos concurrent with Myf 5 protein expression. We also observed that abrogation of p38 MAPK signaling blocks MEF2 activation using a MEF2 transgenic ‘sensor’ mouse. Inhibition of p38 MAPK signaling concurrently inhibited myogenic differentiation in somite cultures and in embryos in vivo using transplacental injection of a p38 inhibitor (SB203580). Finally, we document that commitment to the myogenic lineage is not appreciably affected by p38 MAPK inhibition since the activation of an early marker of myogenic commitment (Myf 5) occurs normally when p38 MAPK signaling is inhibited. Thus, we present novel evidence indicating a crucial role for p38 MAPK signaling to the MEF2 transcriptional regulators during early mammalian somite development and myotome formation.

Regulation of myosin expression during myotome formation.

The first skeletal muscle fibers to form in vertebrate embryos appear in the somitic myotome. PCR analysis and in situ hybridization with isoform-specific probes reveal differences in the temporal appearance and spatial distribution of fast and slow myosin heavy chain mRNA transcripts within myotomal fibers. Embryonic fast myosin heavy chain was the first isoform expressed, followed rapidly by slow myosin heavy chains 1 and 3, with slow myosin heavy chain 2 appearing several hours later. Neonatal fast myosin heavy chain is not expressed in myotomal fibers. Although transcripts of embryonic fast myosin heavy chain were always distributed throughout the length of myotomal fibers, the mRNA for each slow myosin heavy chain isoform was initially restricted to the centrally located myotomal fiber nuclei. As development proceeded, slow myosin heavy chain transcripts spread throughout the length of myotomal fibers in order of their appearance. Explants of segments from embryos containing neural tube, notochord and somites 7-10, when incubated overnight, become innervated by motor neurons from the neural tube and express all four myosin heavy chain genes. Removal of the neural tube and/or notochord from explants prior to incubation or addition of d-tubocurare to intact explants prevented expression of slow myosin chain 2 but expression of genes encoding the other myosin heavy chain isoforms was unaffected. Thus, expression of slow myosin heavy chain 2 is dependent on functional innervation, whereas expression of embryonic fast and slow myosin heavy chain 1 and 3 are innervation independent. Implantation of sonic-hedgehog-soaked beads in vivo increased the accumulation of both fast and slow myosin heavy chain transcripts, as well as overall myotome size and individual fiber size, but had no effect on myotomal fiber phenotype. Transcripts encoding embryonic fast myosin heavy chain first appear ventrolaterally in the myotome, whereas slow myosin heavy chain transcripts first appear in fibers positioned midway between the ventrolateral and dorsomedial lips of the myotome. Therefore, models of epaxial myotome formation must account for the positioning of the oldest fibers in the more ventral-lateral region of the myotome and the youngest fibers in the dorsomedial region.

VITO-1, a novel vestigial related protein is predominantly expressed in the skeletal muscle lineage

In order to identify novel genes expressed in skeletal muscle we performed a subtractive hybridization for genes expressed in human skeletal muscle but not in other tissues. We identified a novel scalloped interaction domain (SID) containing protein in humans and in the mouse, which we named VITO-1. Highest homology of VITO-1 was found with the Drosophila v est i gial and the human TO NDU proteins in the SID (54 and 40%, respectively). Using whole-mount hybridzation and Northern blot analysis, we showed that VITO-1 is expressed in the somitic myotome from E8.75 mouse embryos onwards and later on in skeletal muscle but not in the heart. Additional expression domains during development were detected in the pharyngeal pouches and clefts starting at E8.0 as well as in the cranial pharynx and in Rathkes pouch. By Northern blot analysis, we found VITO-1 to be up-regulated in C2C12 myotubes although some expression can be detected in proliferating C2C12 myoblasts. No expression was spotted in other adult mouse tissues. Likewise, expression of human Vito-1 during fetal and adult human development was found exclusively in skeletal muscle preferentially in fast skeletal muscles. These data suggest a role of VITO-1 for the development of skeletal muscles and of pharyngeal clefts/Rathkes’ pouch derived structures.

The dynamic expression pattern of B lymphocyte induced maturation protein-1 (Blimp-1) during mouse embryonic development

We have analyzed the spatial and temporal patterns of B lymphocyte-induced maturation protein-1 (Blimp-1) expression during mouse embryonic development. Blimp-1 expression is induced early in the anterior definitive endoderm, mesoderm of head process, and prechordal plate. In ectoderm-derived tissues at later stages, Blimp-1 expression is found in the primitive photoreceptors of neural retina, in differentiated epithelial cells of epidermis, tongue, oral and nasal cavities, and in the precursors of internal root sheaths of hair follicles. In mesoderm-derived tissues, Blimp-1 expression is observed in splanchnopleure, a subset of somatopleure-derived cells in limb buds, and myotomes of somites. Blimp-1 is also expressed in mesenchyme of developing hand plates, digits, branchial arches, nasal processes, and external genitalia. Blimp-1 is present in mesenchyme-derived chondroblasts, supporting cells of taste buds, and papilla of teeth, hair follicles and taste buds. In endoderm-derived tissues, Blimp-1 expression in the foregut region is restricted to a subset of epithelial cells at the headfold stage while expression in the endodermal epithelium of midgut and hindgut persists from the headfold stage to birth. Finally, Blimp-1 is expressed in the migrating primordial germ cells.

Early developmental expression pattern of retinoblastoma tumor suppressor mRNA indicates a role in the epithelial-to-mesenchyme transformation of endocardial cushion cells.

The earliest stages of embryonic development are characterized by the generation of precursor cell populations that differentiate and coalesce into tissue and organ primordia. To provide sufficient numbers of differentiated cells for tissue and organ formation, the differentiative as well as the proliferative processes of cells must be controlled and coordinated. Potential regulators of the proliferative process include molecules that control the cell cycle, in particular, the tumor suppressor proteins. To begin to understand the role such molecules can play in development, we have studied the expression of the retinoblastoma tumor suppressor (Rb) gene in early chicken development. Our studies in early chicken embryos show that Rb is encoded by a single gene that gives rise to several Rb mRNA isoforms through alternative splicing of a primary transcript. These mRNA isoforms potentially encode Rb proteins that differ with respect to the number of sequence motifs known to target cyclin-dependent kinases to Rb, suggesting dynamic control of Rb phosphorylation and function during development. This complex expression pattern of Rb mRNA begins as early as the blastoderm stage of chicken development (stage 3) and continues through stage 18, the latest stage examined. Despite this early embryonic expression of Rb mRNA as detected by reverse transcription polymerase chain reaction, Rb mRNA levels sufficient to be detected by in situ hybridization were not expressed until after stage 14 of development. Rb mRNA was found to be localized to cells of the endocardial cushions of the early heart tube, cells of the epicardium, and myogenic cells of the somitic myotome. Interestingly, each of these cell types undergoes an epithelial-to-mesenchyme transformation to form a migratory and/or invasive population of mesenchymal cells. We have focused our studies on the expression of Rb mRNA in endocardial cells of the early heart tube, because the transition of these cells to mesenchyme initiates the important process of septation, an early step in the formation of heart valves.

Expression of sprouty2 during early development of the chick embryo is coincident with known sites of FGF signalling

The Drosophila sprouty protein is a recently-identified intracellular modulator of FGF and EGF receptor tyrosine kinase activity which antagonises ras/MAP kinase signalling. In a differential display analysis to identify genes involved in patterning the mid/hindbrain region of the chick neural tube, we have identified a sprouty orthologue, sprouty2. Here we report expression of sprouty2 transcripts in the developing chick embryo. We find a close correlation with known sites of FGF activity but little correlation with expression patterns of members of the EGF family. Initially, transcripts are associated with the primitive streak. During the period of neural tube patterning expression is detected in the anterior neuropore, in the isthmic region and in neural plate and posterior spinal cord. Transcripts are also detected in the otic placode, tail bud, mesoderm of the branchial arches, somitic myotome, retina, limb buds and gut mesenchyme; all known sites of FGF action.

Filamin isogene expression during mouse myogenesis.

The developmental pattern of filamin gene expression has been studied in mouse embryos by using in situ hybridization. The probes used were isoform specific, (35)S-labeled antisense complementary ribonucleic acids (cRNAs) to the 3; untranslated region (3; UTR) of muscle-specific and nonmuscle-specific filamin genes. Northern blot and in situ hybridization results showed that nonmuscle-specific filamin transcripts had a size of 9.5 kb and were expressed in all nonmuscle tissues. Labeling was most intense in tissues containing a substantial proportion of epithelial and smooth muscle cells. Muscle-specific filamin transcripts had a size of 10 kb and were expressed primarily in cardiac and skeletal muscle. The expression of muscle-specific filamin messenger ribonucleicacids (mRNAs) was detected in heart at 8.0 days after coitum, whereas that in the myotomes of somites was not detected until 10.5 days after coitum. The expression of muscle-specific filamin mRNAs in heart and in skeletal muscle continued through the subsequent days of myogenesis. The results showed that muscle-specific filamin gene transcripts are detected before the formation of myotubes in vivo. This is the first study of filamin gene expression at the early stages of skeletal muscle development. Dev Dyn 2000;217:99-108.

Filamin isogene expression during mouse myogenesis

The developmental pattern of filamin gene expression has been studied in mouse embryos by using in situ hybridization. The probes used were isoform specific, 35S-labeled antisense complementary ribonucleic acids (cRNAs) to the 3` untranslated region (3` UTR) of muscle-specific and nonmuscle-specific filamin genes. Northern blot and in situ hybridization results showed that nonmuscle-specific filamin transcripts had a size of 9.5 kb and were expressed in all nonmuscle tissues. Labeling was most intense in tissues containing a substantial proportion of epithelial and smooth muscle cells. Muscle-specific filamin transcripts had a size of 10 kb and were expressed primarily in cardiac and skeletal muscle. The expression of muscle-specific filamin messenger ribonucleicacids (mRNAs) was detected in heart at 8.0 days after coitum, whereas that in the myotomes of somites was not detected until 10.5 days after coitum. The expression of muscle-specific filamin mRNAs in heart and in skeletal muscle continued through the subsequent days of myogenesis. The results showed that muscle-specific filamin gene transcripts are detected before the formation of myotubes in vivo. This is the first study of filamin gene expression at the early stages of skeletal muscle development. Dev Dyn 2000;217:99–108. © 2000 Wiley-Liss, Inc.

Slow troponin T mRNA in striated muscles is expressed in both cell type and developmental stage specific manner.

We have cloned cDNA sequences of both rat and mouse slow troponin T gene. These sequences share a high level of homology with each other and with the human slow troponin T gene although we were unable to detect an alternatively spliced exon present at 3' end of human slow troponin T cDNA in either mouse or rat cDNAs. Northern blot analysis detected a high level expression of slow troponin T in adult mouse Soleus with a lower level expression in mixed postnatal skeletal muscles. Unlike late fetal and postnatal skeletal muscles in which slow troponin T expression is restricted to slow muscle fibre rich regions only, in situ hybridisation analysis detected this isoform to be highly expressed in somitic myotome and all muscle masses at 10-14 days of gestation after which its expression was rapidly downregulated. The unexpected expression of slow troponin T mRNA in fetal heart was apparent by both northern blotting and in situ hybridisation analyses. Slow troponin T mRNA in fetal heart was first detected at 10 day in utero reaching maximum levels of expression at 12-15 days gestation. The slow troponin T in the heart was mainly expressed in the ventral ventricles until day 15 after which low level expression was also observed in both atria. Slow troponin T mRNA in both atrium and ventricle was mainly expressed in outer wall of the myocardium although it was also expressed in interventricular septum. This study therefore shows that in addition to being a cell type specific marker during later fetal and postnatal skeletal muscle development, slow troponin T represented one of the major developmental isoforms expressed in embryonic and fetal skeletal muscle as well as in the cardiac muscle.

Cloning of the genes encoding mouse cardiac and skeletal calsequestrins: expression pattern during embryogenesis.

Calsequestrin is a low-affinity and high-capacity calcium-binding protein in the sarcoplasmic reticulum (SR). In the present study, we have cloned and sequenced mouse cardiac and skeletal calsequestrin cDNAs. The deduced amino acid sequences are highly homologous to those of other mammalian calsequestrins. As expected, the cardiac and skeletal calsequestrins are expressed specifically and exclusively in adult heart and skeletal muscles, respectively. In-situ hybridization was performed to examine the expression pattern of the calsequestrins in the developing mouse and rat embryos. During early organogenesis, the cardiac and skeletal calsequestrin transcripts were detected exclusively in the heart primordium and the myotome of somites, respectively. The cardiac calsequestrin transcripts were later detected in fetal heart and skeletal muscles, whereas the skeletal calsequestrin transcripts were only found in fetal skeletal muscles. These data suggest that the cardiac calsequestrin plays a role in the differentiation and function of heart, and in the function of fetal skeletal muscles in conjunction with the skeletal calsequestrin, but not in the early differentiation of the myotome of somites. The expression of the skeletal calsequestrin in the myotome is regulated probably by myogenin, a myogenic regulatory gene.

A Potential Role of R-cadherin in Striated Muscle Formation

We have examined the murine embryonic expression pattern of the cell adhesion molecule R-cadherin in muscle, kidney, thymus, and lung. In developing muscle, R-cadherin was first seen at 10.5–11.5 days postcoitum in the somitic myotome. Consistently, we found R-cadherin expressed at the highest levels in the myotome, early skeletal muscle, and smooth muscle (both vascular and visceral), while very low levels of R-cadherin were detected in the heart. The expression pattern and subcellular localization of R-cadherin in developing skeletal muscle indicate a possible role in myoblast cell–cell interactions during both primary and secondary myogenesis. In the developing kidney, R-cadherin was first detected at 10.5 days postcoitum in the mesonephric epithelial tubule cells. In the metanephric kidney, it was specifically expressed in the pretubular aggregates, comma- and S-shaped bodies, proximal tubules, and collecting ducts. Thus, in the kidney, R-cadherin was associated with the mesenchymal–epithelial transition. R-cadherin was also found in other developing epithelia, for example in the thymic epithelial cells. In the lung, R-cadherin was expressed at the highest levels in the smooth muscle surrounding the lung epithelial tubules. To test whether R-cadherin can direct formation of tissues, we constitutively expressed R-cadherin inE-cadherin-/-ES cells and examined histogenesis in teratomas derived from these cells. R-cadherin exclusively rescued formation of striated muscle and epithelia in the teratomas. R-cadherin's ability to form epitheliain vivowas substantiated by its ability to rescue formation of cystic embryoid bodiesin vitro.By comparing our data with the previously reported embryonic expression patterns and histogenetic activities of E- and N-cadherin, we suggest that R-cadherin plays an important role in the formation of striated muscle and possibly also of epithelia.

Fibroblast growth factor receptor 1 in skeletal and heart muscle cells: expression during early avian development and regulation after notochord transplantation.

Basic fibroblast growth factor (bFGF, FGF-2) mediates several biological functions during embryonic development. With regard to skeletal muscle formation, it has been suggested that FGF-2 is involved in the growth and differentiation of myogenic precursor cells. To identify the FGF-responsive cells we studied the expression of FGF receptor type I (FGFR-1) during early embryonic development of the chick. FGFR-1 immunoreactivity is present at all stages examined (embryonic day [E] 2-E5). Expression of FGFR-1 is found in the somite myotome, limb bud muscle cells, eye and tongue muscle cells, and myocardium. Transplantation of an additional notochord into the paraxial mesoderm, which prevents the formation of a myotome, reveals the absence of FGFR-1 immunoreactivity on the operated side. The distinct expression pattern of FGFR-1 in migrating and differentiating muscle cells indicates that in addition to the stimulation of proliferation of myoblasts, FGF-2 exerts other (nonmitogenic) effects on postmitotic myocytes.

Sequence and Expression of Amphioxus Alkali Myosin Light Chain (AmphiMLC-alk) Throughout Development: Implications for Vertebrate Myogenesis

The lower chordate amphioxus, widely considered the closest living invertebrate relative of the vertebrates, is a key organism for understanding the relationship between gene duplications and evolution of the complex vertebrate body plan. In tetrapod vertebrates, the alkali myosin light chain genes (MLC-alk), which code for proteins associated with the globular head of the myosin heavy chain, constitute a large family with stage-, tissue-, and fiber-type-specific expression of different isoforms thought to have arisen by duplication of a single ancestral gene. In protostome invertebrates, e.g., arthropods, molluscs, and nematodes, only one MLC-alk gene has been found, but the number of such genes in deuterostome invertebrates and lower vertebrates is unknown. The present report, describing the sequence and expression throughout development of the amphioxus gene for alkali myosin light chain (AmphiMLC-alk), thus fills a major gap in understanding the relation between gene duplication and increasing diversity of muscle-cell types. A full-length clone (1 kb) of AmphiMLC-alk was isolated from a larval amphioxus cDNA library. It coded for a 149-amino-acid protein most closely related to the vertebrate embryonic form of MLC-alk. Southern blot analysis revealed only one copy of AmphiMLC-alk and suggested that it is the only MLC-alk gene in amphioxus. Northern blot analysis indicated that this gene produces only one transcript, which is expressed at all stages of development and in adults. In situ hybridizations showed expression initially in the myotomes of somites 2-5 of neurula embryos and soon thereafter in the myotomes of somite 1 and of newly forming somites progressively added posteriorly. Myotomal expression continues throughout larval development and into the adult stage as the myotomal cells differentiate into striated, mononucleate muscle cells—unlike vertebrate striated muscle cells, those of amphioxus never become multinucleate. In late larvae and adults myotomal expression of AmphiMLC-alk is localized along the medial edge of the myotome and at the ends of the cells. This is the first demonstration of intracellular localization of MLC transcripts in muscle cells of any animal. Expression of AmphiMLC-alk was also detected in smooth muscles as well as in striated muscles not derived from the myotome. These expression data are consistent with the Southern blot analysis in suggesting that there is only one MLC-alk gene in amphioxus. Thus, duplication of an ancestral vertebrate MLC-alk gene probably occurred after the vertebrate and amphioxus lineages split. We conclude that development of a segmented axial musculature preceded the evolution of multiple MLC-alk isoforms, which evidently arose about the time of multinucleation. Since myogenesis in amphioxus is similar to but far simpler than myogenesis in vertebrates at both the structural and gene levels, an understanding of myogenesis in amphioxus can give insights into both the evolutionary history and the detailed mechanisms of vertebrate myogenesis.

Inactivation of the myogenic bHLH gene MRF4 results in up-regulation of myogenin and rib anomalies.

The myogenic basic helix-loop-helix (bHLH) proteins MyoD, myf5, myogenin, and MRF4 can initiate myogenesis when expressed in nonmuscle cells. During embryogenesis, each of the myogenic bHLH genes is expressed in a unique temporospatial pattern within the skeletal muscle lineage, suggesting that they play distinct roles in muscle development. Gene targeting has shown that MyoD and myf5 play partially redundant roles in the genesis of myoblasts, whereas myogenin is required for terminal differentiation. MRF4 is expressed transiently in the somite myotome during embryogenesis and then becomes up-regulated during late fetal development to eventually become the predominant myogenic bHLH factor expressed in adult skeletal muscle. On the basis of its expression pattern, it has been proposed that MRF4 may regulate skeletal muscle maturation and aspects of adult myogenesis. To determine the function of MRF4, we generated mice carrying a homozygous germ-line mutation in the MRF4 gene. These mice showed only a subtle reduction in expression of a subset of muscle-specific genes but showed a dramatic increase in expression of myogenin, suggesting that it may compensate for the absence of MRF4 and demonstrating that MRF4 is required for the down-regulation of myogenin expression that normally occurs in postnatal skeletal muscle. Paradoxically, MRF4-null mice exhibited multiple rib anomalies, including extensive bifurcations, fusions, and supernumerary processes. These results demonstrate an unanticipated regulatory relationship between myogenin and MRF4 and suggest that MRF4 influences rib outgrowth through an indirect mechanism.

Neural tube can induce fast myosin heavy chain isoform expression during embryonic development

We investigated the role of the neural tube in muscle cell differentiation in developing somitic myotome of chick embryo, particularly through fast myosin heavy chain (MHC) isoform expression. An embryonic fast MHC labeled with EB165 mAb was expressed in somitic cells from stage 15 of Hamburger and Hamilton (H.H.) (24 somites). Moreover, a distinct early embryonic fast MHC was expressed only from stage 15 of H.H. to stage 36 (E10). Like neonatal MHC, this isoform was labeled with 2E9 mAb but differed in its immunopeptide mapping. Expression of EB165-labeled embryonic fast MHC occurred in somitic myotomes deprived of neural tube influence by in ovo ablation as well as in somite explants cultured alone in vitro. Conversely, ablation of the neural tube prevented somitic expression of MHC labeled with 2E9 mAb. The neural tube induced in vitro expression of this MHC in explants of somites which failed to express it when cultured alone. These results indicate that signals emanating from the neural tube are required for the expression of early embryonic fast MHC isoform in developing somitic myotome.