Muscle-specific Genes Research Articles

The Notch target E(spl)mδ is a muscle-specific gene involved in methylmercury toxicity in motor neuron development

Methylmercury (MeHg) is a ubiquitous environmental toxin that has a selective and potent impact on the nervous system, particularly during neural development yet, the mechanisms for its apparent neurodevelopmental specificity are unknown. The Notch receptor pathway has been implicated as a MeHg target in several studies. Notch signaling mediates cell–cell signals in a number of developmental contexts including neurogenesis and myogenesis, where it fundamentally acts to repress differentiation. Previous work in our lab has shown that MeHg causes preferential upregulation of a canonical Notch response gene, E(spl)mδ, in Drosophila embryos. In parallel, MeHg is seen to disrupt outgrowth of embryonic intersegmental motor nerves (ISN), which can be mimicked by expression of activated Notch in embryonic neurons. However, overexpression of E(spl)mδ in developing neurons fails to elicit motor neuron outgrowth defects, pointing to a non-autonomous role for E(spl)mδ in motor axon development. In this study we investigate a role for E(spl)mδ in conveying the toxicity of MeHg in the embryo. We find that endogenous expression of the E(spl)mδ gene localizes to developing somatic muscles in embryos. Notably, E(spl)mδ expression is seen in several muscles that are known synaptic targets for both the ISN and the segmental motor nerve (SN). We also demonstrate that the SN, similar to the ISN, exhibits disrupted axon outgrowth in response to MeHg. E(spl)mδ can induce a SN motor neuron phenotype, similar to MeHg treatment; but, only when E(spl)mδ expression is targeted to developing muscles. E(spl)mδ overexpression in developing muscles also results in aberrant muscle morphology, which is not apparent with expression of the closely related E(spl)mγ in developing muscles. Our data point to a role for the Notch target E(spl)mδ in mediating MeHg toxicity in embryonic development by disrupting the coordinated targeting of motor neurons to their muscle targets.

Phenotypic Modulation of Primary Vascular Smooth Muscle Cells by Short-Term Culture on Micropatterned Substrate

Loss of contractility and acquisition of an epithelial phenotype of vascular smooth muscle cells (VSMCs) are key events in proliferative vascular pathologies such as atherosclerosis and post-angioplastic restenosis. There is no proper cell culture system allowing differentiation of VSMCs so that it is difficult to delineate the molecular mechanism responsible for proliferative vasculopathy. We investigated whether a micropatterned substrate could restore the contractile phenotype of VSMCs in vitro. To induce and maintain the differentiated VSMC phenotype in vitro, we introduced a micropatterned groove substrate to modulate the morphology and function of VSMCs. Later than 7th passage of VSMCs showed typical synthetic phenotype characterized by epithelial morphology, increased proliferation rates and corresponding gene expression profiles; while short-term culture of these cells on a micropatterned groove induced a change to an intermediate phenotype characterized by low proliferation rates, increased migration, a spindle-like morphology associated with cytoskeletal rearrangement and expression of muscle-specific genes. Microarray analysis showed preferential expression of contractile and smooth muscle cell-specific genes in cells cultured on the micropatterned groove. Culture on a patterned groove may provide a valuable model for the study the role of VSMCs in normal vascular physiology and a variety of proliferative vascular diseases.

Expression of Signaling Components in Embryonic Eyelid Epithelium

Closure of an epithelium opening is a critical morphogenetic event for development. An excellent example for this process is the transient closure of embryonic eyelid. Eyelid closure requires shape change and migration of epithelial cells at the tip of the developing eyelids, and is dictated by numerous signaling pathways. Here we evaluated gene expression in epithelial cells isolated from the tip (leading edge, LE) and inner surface epithelium (IE) of the eyelid from E15.5 mouse fetuses by laser capture microdissection (LCM). We showed that the LE and IE cells are different at E15.5, such that IE had higher expression of muscle specific genes, while LE acquired epithelium identities. Despite their distinct destinies, these cells were overall similar in expression of signaling components for the “eyelid closure pathways”. However, while the LE cells had more abundant expression of Fgfr2, Erbb2, Shh, Ptch1 and 2, Smo and Gli2, and Jag1 and Notch1, the IE cells had more abundant expression of Bmp5 and Bmpr1a. In addition, the LE cells had more abundant expression of adenomatosis polyposis coli down-regulated 1 (Apcdd1), but the IE cells had high expression of Dkk2. Our results suggest that the functionally distinct LE and IE cells have also differential expression of signaling molecules that may contribute to the cell-specific responses to morphogenetic signals. The expression pattern suggests that the EGF, Shh and NOTCH pathways are preferentially active in LE cells, the BMP pathways are effective in IE cells, and the Wnt pathway may be repressed in LE and IE cells via different mechanisms.

The Transcription Factor TEAD1 Represses Smooth Muscle-specific Gene Expression by Abolishing Myocardin Function*♦

The TEAD (transcriptional enhancer activator domain) proteins share an evolutionarily conserved DNA-binding TEA domain, which binds to the MCAT cis-acting regulatory element. Previous studies have shown that TEAD proteins are involved in regulating the expression of smooth muscle α-actin. However, it remains undetermined whether TEAD proteins play a broader role in regulating expression of other genes in vascular smooth muscle cells. In this study, we show that the expression of TEAD1 is significantly induced during smooth muscle cell phenotypic modulation and negatively correlates with smooth muscle-specific gene expression. We further demonstrate that TEAD1 plays a novel role in suppressing expression of smooth muscle-specific genes, including smooth muscle α-actin, by abolishing the promyogenic function of myocardin, a key mediator of smooth muscle differentiation. Mechanistically, we found that TEAD1 competes with myocardin for binding to serum response factor (SRF), resulting in disruption of myocardin and SRF interactions and thereby attenuating expression of smooth muscle-specific genes. This study provides the first evidence demonstrating that TEAD1 is a novel general repressor of smooth muscle-specific gene expression through interfering with myocardin binding to SRF.

Small Leucine Zipper Protein (sLZIP) Negatively Regulates Skeletal Muscle Differentiation via Interaction with α-Actinin-4

The small leucine zipper protein (sLZIP) plays a role in transcriptional regulation in various types of cells. However, the role of sLZIP in myogenesis is unknown. We identified α-actinin-4 (ACTN4) as a sLZIP-binding protein. ACTN4 functions as a transcriptional regulator of myocyte enhancer factor (MEF)2, which plays a critical role in expression of muscle-specific genes during skeletal muscle differentiation. We found that ACTN4 translocates to the nucleus, induces myogenic gene expression, and promotes myotube formation during myogenesis. The myogenic process is controlled by an association between myogenic factors and MEF2 transcription factors. ACTN4 increased expression of muscle-specific proteins via interaction with MEF2. However, sLZIP decreased myogenic gene expression and myotube formation during myogenesis via disruption of the association between ACTN4 and MEF2. ACTN4 increased the promoter activities of myogenic genes, whereas sLZIP abrogated the effect of ACTN4 on transcriptional activation of myogenic genes in myoblasts. The C terminus of sLZIP is required for interaction with the C terminus of ACTN4, based on deletion mutant analysis, and sLZIP plays a role in regulation of MEF2 transactivation via interaction with ACTN4. Our results indicate that sLZIP negatively regulates skeletal muscle differentiation via interaction with ACTN4 and that sLZIP can be used as a therapeutic target molecule for treatment of muscle hypertrophy and associated diseases.

TBX2 blocks myogenesis and promotes proliferation in rhabdomyosarcoma cells

Rhabdomyosarcomas (RMSs) are the most frequent soft tissue sarcomas in children that share many features of developing skeletal muscle. We have discovered that a T-box family member, TBX2, is highly upregulated in tumor cells of both major RMS subtypes. TBX2 is a repressor that is often overexpressed in cancer cells and is thought to function in bypassing cell growth control, including repression of p14 and p21. The cell cycle regulator p21 is required for the terminal differentiation of skeletal muscle cells and is silenced in RMS cells. We have found that TBX2 interacts with the myogenic regulatory factors MyoD and myogenin and inhibits the activity of these factors. TBX2 is expressed in primary myoblasts and C2C12 cells, but is strongly downregulated upon differentiation. TBX2 recruits the histone deacetylase HDAC1 and is a potent inhibitor of the expression of muscle-specific genes and the cell cycle regulators, p21 and p14. TBX2 promotes the proliferation of RMS cells and either depletions of TBX2 or dominant negative TBX2 upregulate p21- and muscle-specific genes. Significantly, depletion or interference with TBX2 completely inhibits tumor growth in a xenograft assay, highlighting the oncogenic role of TBX2 in RMS cells. Thus, the data demonstrate that elevated expression of TBX2 contributes to the pathology of RMS cells by promoting proliferation and repressing differentiation-specific gene expression. These results show that deregulated TBX2 serves as an oncogene in RMS, suggesting that TBX2 may serve as a new diagnostic marker or therapeutic target for RMS tumors.

The 31-kDa caspase-generated cleavage product of p130Cas antagonizes the action of MyoD during myogenesis

Myogenesis is regulated by the basic helix-loop-helix (bHLH) myogenic regulatory factor MyoD, which induces muscle-specific gene expression by binding to the E-box sequence as a heterodimer with ubiquitous bHLH E2A (E12/E47) proteins. Here, we report that a 31-kDa caspase-generated cleavage product of Crk-associated substrate (p130Cas), herein called 31-kDa, is downregulated during muscle cell differentiation. 31-kDa contains a helix-loop-helix (HLH) domain that shows greater sequence homology with Id (inhibitor of DNA binding) proteins than with bHLH proteins. This HLH domain, lacking the basic region required for DNA binding, mediated the direct interaction of 31-kDa with MyoD. Overexpression of 31-kDa in C3H10T1/2 cells inhibited not only the transcriptional activation of p21Waf1/Cip1 and E-box-dependent muscle-specific genes by MyoD and/or E2A but also MyoD-induced myosin heavy chain expression and myogenic conversion. In sum, our results suggest a role for 31-kDa as a negative regulator of MyoD in the muscle differentiation program.

Engineered Human Muscle Tissue from Skeletal Muscle Derived Stem Cells and Induced Pluripotent Stem Cell Derived Cardiac Cells.

During development, cardiac and skeletal muscle share major transcription factors and sarcomere proteins which were generally regarded as specific to either cardiac or skeletal muscle but not both in terminally differentiated adult cardiac or skeletal muscle. Here, we investigated whether artificial muscle constructed from human skeletal muscle derived stem cells (MDSCs) recapitulates developmental similarities between cardiac and skeletal muscle. We constructed 3-dimensional collagen-based engineered muscle tissue (EMT) using MDSCs (MDSC-EMT) and compared the biochemical and contractile properties with EMT using induced pluripotent stem (iPS) cell-derived cardiac cells (iPS-EMT). Both MDSC-EMT and iPS-EMT expressed cardiac specific troponins, fast skeletal muscle myosin heavy chain, and connexin-43 mimicking developing cardiac or skeletal muscle. At the transcriptional level, MDSC-EMT and iPS-EMT upregulated both cardiac and skeletal muscle-specific genes and expressed Nkx2.5 and Myo-D proteins. MDSC-EMT displayed intracellular calcium ion transients and responses to isoproterenol. Contractile force measurements of MDSC-EMT demonstrated functional properties of immature cardiac and skeletal muscle in both tissues. Results suggest that the EMT from MDSCs mimics developing cardiac and skeletal muscle and can serve as a useful in vitro functioning striated muscle model for investigation of stem cell differentiation and therapeutic options of MDSCs for cardiac repair.

Histone acetyltransferase p300 promotes MKL1-mediated transactivation of catechol-<italic>O</italic>-methyltransferase gene

Previous studies have revealed that histone acetyltransferase p300 is recruited to the promoters of certain cardiac and smooth muscle specific genes to enhance the transactivation activity of myocardin, which is a master regulator in cardiovascular differentiation and development. Here, we found that the gene encoding catechol-O-methyltransferase (COMT), an important metabolic enzyme catalyzing the conversion of estrogen, is also a target gene of myocardin-related transcription factors (MRTFs). Megakaryoblastic leukemia 1 (MKL1, also named MRTF-A) and p300 could synergistically augment the expression of COMT gene, increase the metabolic rate of estrogen, and thus reduce the proliferation of MCF-7 breast cancer cells stimulated by estrogen.

Loss of MEF2D expression inhibits differentiation and contributes to oncogenesis in rhabdomyosarcoma cells

BackgroundRhabdomyosarcoma (RMS) is a highly malignant pediatric cancer that is the most common form of soft tissue tumors in children. RMS cells have many features of skeletal muscle cells, yet do not differentiate. Thus, our studies have focused on the defects present in these cells that block myogenesis.MethodsProtein and RNA analysis identified the loss of MEF2D in RMS cells. MEF2D was expressed in RD and RH30 cells by transient transfection and selection of stable cell lines, respectively, to demonstrate the rescue of muscle differentiation observed. A combination of techniques such as proliferation assays, scratch assays and soft agar assays were used with RH30 cells expressing MEF2D to demonstrate the loss of oncogenic growth in vitro and xenograft assays were used to confirm the loss of tumor growth in vivo.ResultsHere, we show that one member of the MEF2 family of proteins required for normal myogenesis, MEF2D, is largely absent in RMS cell lines representing both major subtypes of RMS as well as primary cells derived from an embryonal RMS model. We show that the down regulation of MEF2D is a major cause for the failure of RMS cells to differentiate. We find that MyoD and myogenin are bound with their dimerization partner, the E proteins, to the promoters of muscle specific genes in RMS cells. However, we cannot detect MEF2D binding at any promoter tested. We find that exogenous MEF2D expression can activate muscle specific luciferase constructs, up regulate p21 expression and increase muscle specific gene expression including the expression of myosin heavy chain, a marker for skeletal muscle differentiation. Restoring expression of MEF2D also inhibits proliferation, cell motility and anchorage independent growth in vitro. We have confirmed the inhibition of tumorigenicity by MEF2D in a tumor xenograft model, with a complete regression of tumor growth.ConclusionsOur data indicate that the oncogenic properties of RMS cells can be partially attributed to the loss of MEF2D expression and that restoration of MEF2D may represent a useful therapeutic strategy to decrease tumorigenicity.

Expression, SNV identification, linkage disequilibrium, and combined genotype association analysis of the muscle-specific gene CSRP3 in Chinese cattle

The cysteine and glycine-rich protein 3 (CSRP3) plays an important role in the myofiber differentiation. Here, we identified five SNVs in all exon and intron regions of the CSRP3 gene using DNA sequencing, PCR-RFLP and forced-PCR-RFLP methods in 554 cattle. Four of the five SNVs were significantly associated with growth performance and carcass traits of the cattle. In addition, we evaluated haplotype frequency and linkage disequilibrium coefficient of five sequence variants. The result of haplotype analysis demonstrated 28 haplotypes present in Qinchuan and two haplotypes in Chinese Holstein. Only haplotypes 1 and 8 were being shared by two populations, haplotype 14 had the highest haplotype frequency in Qinchuan (17.4%) and haplotype 8 had the highest haplotype frequency in Chinese Holstein (94.4%). Statistical analyses of combined genotypes indicated that some combined genotypes were significantly or highly significantly associated with growth and carcass traits in the Qinchuan cattle population. qPCR analyses also showed that bovine CSRP3 gene was exclusively expressed in longissimus dorsi muscle and heart tissues. The data support the high potential of the CSRP3 as a marker gene for the improvement of growth performance and carcass traits in selection programs.

Insm1 controls development of pituitary endocrine cells and requires a SNAG domain for function and for recruitment of histone-modifying factors

The Insm1 gene encodes a zinc finger factor expressed in many endocrine organs. We show here that Insm1 is required for differentiation of all endocrine cells in the pituitary. Thus, in Insm1 mutant mice, hormones characteristic of the different pituitary cell types (thyroid-stimulating hormone, follicle-stimulating hormone, melanocyte-stimulating hormone, adrenocorticotrope hormone, growth hormone and prolactin) are absent or produced at markedly reduced levels. This differentiation deficit is accompanied by upregulated expression of components of the Notch signaling pathway, and by prolonged expression of progenitor markers, such as Sox2. Furthermore, skeletal muscle-specific genes are ectopically expressed in endocrine cells, indicating that Insm1 participates in the repression of an inappropriate gene expression program. Because Insm1 is also essential for differentiation of endocrine cells in the pancreas, intestine and adrenal gland, it is emerging as a transcription factor that acts in a pan-endocrine manner. The Insm1 factor contains a SNAG domain at its N-terminus, and we show here that the SNAG domain recruits histone-modifying factors (Kdm1a, Hdac1/2 and Rcor1-3) and other proteins implicated in transcriptional regulation (Hmg20a/b and Gse1). Deletion of sequences encoding the SNAG domain in mice disrupted differentiation of pituitary endocrine cells, and resulted in an upregulated expression of components of the Notch signaling pathway and ectopic expression of skeletal muscle-specific genes. Our work demonstrates that Insm1 acts in the epigenetic and transcriptional network that controls differentiation of endocrine cells in the anterior pituitary gland, and that it requires the SNAG domain to exert this function in vivo.

The Polycomb group (PcG) protein EZH2 supports the survival of PAX3-FOXO1 alveolar rhabdomyosarcoma by repressing FBXO32 (Atrogin1/MAFbx).

The Polycomb group (PcG) proteins regulate stem cell differentiation via the repression of gene transcription, and their deregulation has been widely implicated in cancer development. The PcG protein Enhancer of Zeste Homolog 2 (EZH2) works as a catalytic subunit of the Polycomb Repressive Complex 2 (PRC2) by methylating lysine 27 on histone H3 (H3K27me3), a hallmark of PRC2-mediated gene repression. In skeletal muscle progenitors, EZH2 prevents an unscheduled differentiation by repressing muscle-specific gene expression and is downregulated during the course of differentiation. In rhabdomyosarcoma (RMS), a pediatric soft-tissue sarcoma thought to arise from myogenic precursors, EZH2 is abnormally expressed and its downregulation in vitro leads to muscle-like differentiation of RMS cells of the embryonal variant. However, the role of EZH2 in the clinically aggressive subgroup of alveolar RMS, characterized by the expression of PAX3-FOXO1 oncoprotein, remains unknown. We show here that EZH2 depletion in these cells leads to programmed cell death. Transcriptional derepression of F-box protein 32 (FBXO32) (Atrogin1/MAFbx), a gene associated with muscle homeostasis, was evidenced in PAX3-FOXO1 RMS cells silenced for EZH2. This phenomenon was associated with reduced EZH2 occupancy and H3K27me3 levels at the FBXO32 promoter. Simultaneous knockdown of FBXO32 and EZH2 in PAX3-FOXO1 RMS cells impaired the pro-apoptotic response, whereas the overexpression of FBXO32 facilitated programmed cell death in EZH2-depleted cells. Pharmacological inhibition of EZH2 by either 3-Deazaneplanocin A or a catalytic EZH2 inhibitor mirrored the phenotypic and molecular effects of EZH2 knockdown in vitro and prevented tumor growth in vivo. Collectively, these results indicate that EZH2 is a key factor in the proliferation and survival of PAX3-FOXO1 alveolar RMS cells working, at least in part, by repressing FBXO32. They also suggest that the reducing activity of EZH2 could represent a novel adjuvant strategy to eradicate high-risk PAX3-FOXO1 alveolar RMS.

MyoD and Myf6 gene expression patterns in skeletal muscle during embryonic and posthatch development in the domestic duck (Anas platyrhynchos domestica)

The MyoD and Myf6 genes, which are muscle regulatory factors (MRFs), play major roles in muscle growth and development and initiate muscle fibre formation via the regulation of muscle-specific gene translation. Therefore, MyoD and Myf6 are potential candidate genes for meat production traits in animals and poultry. The objective of this study was to evaluate MyoD and Myf6 gene expression patterns in the skeletal muscle during early developmental stage of ducks. Gene expression levels were detected using the quantitative RT-PCR method in the breast muscle (BM) and leg muscle (LM) at embryonic days 13, 17, 21, 25, 27, as well as at 1 week posthatching in Gaoyou and Jinding ducks (Anas platyrhynchos domestica). The MyoD and Myf6 gene profiles in the two duck breeds were consistent during early development, and MyoD gene expression showed a 'wave' trend in BM and an approximate 'anti-√' trend in LM. Myf6 gene expression in BM showed the highest level at embryonic day 21, which subsequently decreased, although remained relatively high, while levels at embryonic days 13, 17 and 21 were higher in LM. The results of correlation analysis showed that MyoD and Myf6 gene expression levels were more strongly correlated in LM than in BM in both duck breeds. These results indicated that different expression patterns of the MyoD and Myf6 genes in BM and LM may be related to muscle development and differentiation, suggesting that MyoD and Myf6 are integral to skeletal muscle development.

The Drosophila wings apart Gene Anchors a Novel, Evolutionarily Conserved Pathway of Neuromuscular Development

wings apart (wap) is a recessive, semilethal gene located on the X chromosome in Drosophila melanogaster, which is required for normal wing-vein patterning. We show that the wap mutation also results in loss of the adult jump muscle. We use complementation mapping and gene-specific RNA interference to localize the wap locus to the proximal X chromosome. We identify the annotated gene CG14614 as the gene affected by the wap mutation, since one wap allele contains a non-sense mutation in CG14614, and a genomic fragment containing only CG14614 rescues the jump-muscle phenotypes of two wap mutant alleles. The wap gene lies centromere-proximal to touch-insensitive larva B and centromere-distal to CG14619, which is tentatively assigned as the gene affected in introverted mutants. In mutant wap animals, founder cell precursors for the jump muscle are specified early in development, but are later lost. Through tissue-specific knockdowns, we demonstrate that wap function is required in both the musculature and the nervous system for normal jump-muscle formation. wap/CG14614 is homologous to vertebrate wdr68, DDB1 and CUL4 associated factor 7, which also are expressed in neuromuscular tissues. Thus, our findings provide insight into mechanisms of neuromuscular development in higher animals and facilitate the understanding of neuromuscular diseases that may result from mis-expression of muscle-specific or neuron-specific genes.

C-Myb Inhibits Myoblast Fusion

Satellite cells represent a heterogeneous population of stem and progenitor cells responsible for muscle growth, repair and regeneration. We investigated whether c-Myb could play a role in satellite cell biology because our previous results using satellite cell-derived mouse myoblast cell line C2C12 showed that c-Myb was expressed in growing cells and downregulated during differentiation. We detected c-Myb expression in activated satellite cells of regenerating muscle. c-Myb was also discovered in activated satellite cells associated with isolated viable myofiber and in descendants of activated satellite cells, proliferating myoblasts. However, no c-Myb expression was detected in multinucleated myotubes originated from fusing myoblasts. The constitutive expression of c-Myb lacking the 3′ untranslated region (3′ UTR) strongly inhibited the ability of myoblasts to fuse. The inhibition was dependent on intact c-Myb transactivation domain as myoblasts expressing mutated c-Myb in transactivation domain were able to fuse. The absence of 3′ UTR of c-Myb was also important because the expression of c-Myb coding region with its 3′ UTR did not inhibit myoblast fusion. The same results were repeated in C2C12 cells as well. Moreover, it was documented that 3′ UTR of c-Myb was responsible for downregulation of c-Myb protein levels in differentiating C2C12 cells. DNA microarray analysis of C2C12 cells revealed that the expression of several muscle-specific genes was downregulated during differentiation of c-Myb-expressing cells, namely: ACTN2, MYH8, TNNC2, MYOG, CKM and LRRN1. A detailed qRT-PCR analysis of MYOG, TNNC2 and LRRN1 is presented. Our findings thus indicate that c-Myb is involved in regulating the differentiation program of myogenic progenitor cells as its expression blocks myoblast fusion.

Role of ATP and electrical stimulation on the expression of muscle-specific genes

Role of ATP and electrical stimulation on the expression of muscle-specific genes

Dysregulation of 4q35- and muscle-specific genes in fetuses with a short D4Z4 array linked to facio-scapulo-humeral dystrophy

Facio-scapulo-humeral dystrophy (FSHD) results from deletions in the subtelomeric macrosatellite D4Z4 array on the 4q35 region. Upregulation of the DUX4 retrogene from the last D4Z4 repeated unit is thought to underlie FSHD pathophysiology. However, no one knows what triggers muscle defect and when alteration arises. To gain further insights into the molecular mechanisms of the disease, we evaluated at the molecular level, the perturbation linked to the FSHD genotype with no a priori on disease onset, severity or penetrance and prior to any infiltration by fibrotic or adipose tissue in biopsies from fetuses carrying a short pathogenic D4Z4 array (n = 6) compared with fetuses with a non-pathogenic D4Z4 array (n = 21). By measuring expression of several muscle-specific markers and 4q35 genes including the DUX4 retrogene by an RT-PCR and western blotting, we observed a global dysregulation of genes involved in myogenesis including MYOD1 in samples with <11 D4Z4. The DUX4-fl pathogenic transcript was detected in FSHD biopsies but also in controls. Importantly, in FSHD fetuses, we mainly detected the non-spliced DUX4-fl isoform. In addition, several other genes clustered at the 4q35 locus are upregulated in FSHD fetuses. Our study is the first to examine fetuses carrying an FSHD-linked genotype and reveals an extensive dysregulation of several muscle-specific and 4q35 genes at early development stage at a distance from any muscle defect. Overall, our work suggests that even if FSHD is an adult-onset muscular dystrophy, the disease might also involve early molecular defects arising during myogenesis or early differentiation.

TSHZ3 and SOX9 Regulate the Timing of Smooth Muscle Cell Differentiation in the Ureter by Reducing Myocardin Activity

Smooth muscle cells are of key importance for the proper functioning of different visceral organs including those of the urogenital system. In the mouse ureter, the two transcriptional regulators TSHZ3 and SOX9 are independently required for initiation of smooth muscle differentiation from uncommitted mesenchymal precursor cells. However, it has remained unclear whether TSHZ3 and SOX9 act independently or as part of a larger regulatory network. Here, we set out to characterize the molecular function of TSHZ3 in the differentiation of the ureteric mesenchyme. Using a yeast-two-hybrid screen, we identified SOX9 as an interacting protein. We show that TSHZ3 also binds to the master regulator of the smooth muscle program, MYOCD, and displaces it from the coregulator SRF, thereby disrupting the activation of smooth muscle specific genes. We found that the initiation of the expression of smooth muscle specific genes in MYOCD-positive ureteric mesenchyme coincides with the down regulation of Sox9 expression, identifying SOX9 as a possible negative regulator of smooth muscle cell differentiation. To test this hypothesis, we prolonged the expression of Sox9 in the ureteric mesenchyme in vivo. We found that Sox9 does not affect Myocd expression but significantly reduces the expression of MYOCD/SRF-dependent smooth muscle genes, suggesting that down-regulation of Sox9 is a prerequisite for MYOCD activity. We propose that the dynamic expression of Sox9 and the interaction between TSHZ3, SOX9 and MYOCD provide a mechanism that regulates the pace of progression of the myogenic program in the ureter.

Myf5 expression during fetal myogenesis defines the developmental progenitors of adult satellite cells

Myf5 is a member of the muscle-specific determination genes and plays a critical role in skeletal muscle development. Whereas the expression of Myf5 during embryonic and fetal myogenesis has been extensively studied, its expression in progenitors that will ultimately give rise to adult satellite cells, the stem cells responsible for muscle repair, is still largely unexplored. To investigate this aspect, we have generated a mouse strain carrying a CreER coding sequence in the Myf5 locus. In this strain, Tamoxifen-inducible Cre activity parallels endogenous Myf5 expression. Combining Myf5CreER and Cre reporter alleles, we were able to evaluate the contribution of cells expressing Myf5 at distinct developmental stages to the pool of satellite cells in adult hindlimb muscles. Although it was possible to trace back the origin of some rare satellite cells to a subpopulation of Myf5+ve progenitors in the limb buds at the late embryonic stage (∼E12), a significant number of satellite cells arise from cells which expressed Myf5 for the first time at the fetal stage (∼E15). These studies provide direct evidence that adult satellite cells derive from progenitors that first express the myogenic determination gene Myf5 during fetal stages of myogenesis.