Myf5 Transcripts Research Articles

Assessing Myf5 and Lbx1 contribution to carapace development by reproducing their turtle-specific signatures in mouse embryos.

The turtle carapace is an evolutionary novelty resulting from changes in the processes that build ribs and their associated muscles in most tetrapod species. Turtle embryos have several unique features that might play a role in this process, including the carapacial ridge, a Myf5 gene with shorter coding region that generates an alternative splice variant lacking exon 2, and unusual expression patterns of Lbx1 and HGF. We investigated these turtle-specific expression differences using genetic approaches in mouse embryos. At mid-gestation, mouse embryos producing Myf5 transcripts lacking exon 2 replicated some early properties of turtle somites, but still developed into viable and fertile mice. Extending Lbx1 expression into the hypaxial dermomyotomal lip of trunk somites to mimic the turtle Lbx1 expression pattern, produced fusions in the distal part of the ribs. Turtle-like Myf5 activity might generate a plastic state in developing trunk somites under which they can either enter carapace morphogenetic routes, possibly triggered by signals from the carapacial ridge, or still engage in the development of a standard tetrapod ribcage in the absence of those signals. In addition, trunk Lbx1 expression might play a later role in the formation of the lateral border of the carapace.

HMEJ-mediated site-specific integration of a myostatin inhibitor increases skeletal muscle mass in porcine

As a robust antagonist of myostatin (MSTN), follistatin (FST) is an important regulator of skeletal muscle development, and the delivery of FST to muscle tissue represents a potential therapeutic strategy for muscular dystrophies. The N terminus and FSI domain of FST are the functional domains for MSTN binding. Here, we aimed to achieve site-specific integration of FSI-I-I, including the signal peptide, N terminus, and three FSI domains, into the last codon of the porcine MSTN gene using a homology-mediated end joining (HMEJ)-based strategy mediated by CRISPR-Cas9. Based on somatic cell nuclear transfer (SCNT) technology, we successfully obtained FSI-I-I knockin pigs. H&E staining of longissimus dorsi and gastrocnemius cross-sections showed larger myofiber sizes in FSI-I-I knockin pigs than in controls. Moreover, the Smad and Erk pathways were inhibited, whereas the PI3k/Akt pathway was activated in FSI-I-I knockin pigs. In addition, the levels of MyoD, Myf5, and MyoG transcription were upregulated while that of MRF4 was downregulated in FSI-I-I knockin pigs. These results indicate that the FSI-I-I gene mediates skeletal muscle hypertrophy through an MSTN-related signaling pathway and the expression of myogenic regulatory factors. Overall, FSI-I-I knockin pigs with hypertrophic muscle tissue hold great promise as a therapeutic model for human muscular dystrophies.

Lack of a skeletal muscle phenotype in adult human bone marrow stromal cells following xenogeneic-free expansion

BackgroundMany studies have elegantly shown that murine and rat bone marrow-derived mesenchymal stromal cells (bmMSCs) contribute to muscle regeneration and improve muscle function. Yet, the ability of transplanted human bmMSCs to manifest myogenic potential shows conflicting results. While human adipose- and umbilical cord-derived MSCs can be differentiated into a skeletal muscle phenotype using horse serum (HS), bmMSCs have only been shown to differentiate towards the skeletal muscle lineage using a complex mixture of cytokines followed by transfection with notch intracellular domain.MethodsSince xenogeneic-free growth supplements are increasingly being used in the expansion of bmMSCs in clinical trials, we investigated the effects of human plasma and platelet lysate (P/PL) on the expression of neuromuscular markers and whether P/PL-expanded human bmMSCs could be differentiated towards a skeletal myogenic phenotype. Neuromuscular markers were measured using the highly sensitive droplet digital polymerase chain reaction for measuring the expression of Myf5, MyoD, MyoG, ACTA1, Desmin, GAP-43, and Coronin 1b transcripts, by performing immunofluorescence for the expression of Desmin, GAP-43, and MEF2, and flow cytometry for the expression of CD56/neural cell adhesion molecule (NCAM).ResultsDespite that bmMSCs expressed the myogenic regulatory factor (MRF) MEF2 after expansion in P/PL, bmMSCs cultured under such conditions did not express other essential MRFs including Myf5, MyoD, MyoG, or ACTA1 needed for myogenesis. Moreover, HS did not induce myogenesis of bmMSCs and hence did not induce the expression of any of these myogenic markers. P/PL, however, did lead to a significant increase in neurogenic GAP-43, as well as Desmin expression, and resulted in a high baseline expression of the neurogenic gene Coronin 1b which was sustained under further P/PL or HS culture conditions. Fetal bovine serum resulted in equally high levels of GAP-43 and Coronin 1b. Moreover, the proportion of CD56/NCAM-positive bmMSCs cultured in P/PL was 5.9 ± 2.1.ConclusionsThese data suggest that P/PL may prime a small portion of bmMSCs towards an early neural precursor cell type. Collectively, this shows that P/PL partially primes the cells towards a neurogenic phenotype, but does not prime adult human bmMSCs towards the skeletal muscle lineage.

The homeoprotein Msx1 cooperates with Pkn1 to prevent terminal differentiation in myogenic precursor cells

The homeoprotein Msx1 plays a critical role in embryonic patterning, particularly to maintain myogenic progenitor cell fate. However, the mechanisms underlying Msx1-mediated inhibition of myogenesis remain largely unknown. Here, we show that Msx1 cooperates with the protein kinase C-related kinase 1 (Pkn1), a member of the protein kinase C-related kinase family, to prevent the terminal differentiation of myogenic precursor cells. In mouse C2C12 cells, Pkn1 knockout partly impaired Msx1-mediated inhibition of myogenic differentiation, indicating a role for Pkn1 in this process. Furthermore, we found that Pkn1 was required for Msx1 enrichment at the promoter of Myf5, a myogenic regulatory gene. In Pkn1 knockout cells, this reduced Msx1 enrichment at the Myf5 promoter coincided with attenuated repression of Myf5 transcription. Together with our observation that Msx1 and Pkn1 were associated in a protein complex, these findings strongly suggest that Msx1 cooperates with Pkn1 to down-regulate Myf5 and, therefore, prevent the differentiation of myogenic precursor cells. Collectively, our data provide key insights into the mechanisms underlying Msx1 function in the prevention of myogenic differentiation.

The Dystrophin Glycoprotein Complex Regulates the Epigenetic Activation of Muscle Stem Cell Commitment

Asymmetrically dividing muscle stem cells in skeletal muscle give rise to committed cells, where themyogenic determination factor Myf5 is transcriptionally activated by Pax7. This activation isdependent on Carm1, which methylates Pax7 onmultiple arginine residues, to recruit the ASH2L:MLL1/2:WDR5:RBBP5 histone methyltransferase complex to the proximal promoter of Myf5. Here, we found that Carm1 is a specific substrate of p38γ/MAPK12 and that phosphorylation of Carm1 prevents its nuclear translocation. Basal localization of the p38γ/p-Carm1 complex in muscle stem cells occurs via binding to the dystrophin-glycoprotein complex (DGC) through β1-syntrophin. In dystrophin-deficient muscle stem cells undergoing asymmetric division, p38γ/β1-syntrophin interactions are abrogated, resulting in enhanced Carm1 phosphorylation. The resulting progenitors exhibit reduced Carm1 binding to Pax7, reduced H3K4-methylation of chromatin, and reduced transcription of Myf5 and other Pax7 target genes. Therefore, our experiments suggest that dysregulation of p38γ/Carm1 results in altered epigenetic gene regulation in Duchenne muscular dystrophy.

Salidroside Inhibits Myogenesis by Modulating p-Smad3-Induced Myf5 Transcription.

Aim: Salidroside is an active compound extracted from Rhodiola rosea which is used to alleviate fatigue and enhance endurance in high altitude regions. Some studies have demonstrated that salidroside can affect precursor cell differentiation in hematopoietic stem cells, erythrocytes, and osteoblasts. The aim of this study was to investigate the effect of salidroside on myoblast differentiation and to explore the underlying molecular mechanisms of this effect.Methods: C2C12 myoblast cells were treated with different concentrations of salidroside in differentiation media. Real-time PCR, Western blotting, and immunofluorescence assay were employed to evaluate the effects of salidroside on C2C12 differentiation. RNA interference was used to reveal the important role of Myf5 in myogenesis inhibited by salidroside. Chromatin Immunoprecipitation and dual-luciferase reporter assay were utilized to explore the underlying mechanisms of salidroside-induced upregulation of Myf5.Results: We found that salidroside inhibits myogenesis by downregulating MyoD and myogenin, preserves undifferentiated reserve cell pools by upregulating Myf5. Knocking down Myf5 expression significantly rescued the myogenesis inhibited by salidroside. The effect of salidroside on myogenesis was associated with increased phosphorylated Smad3 (p-Smad3). Both SIS3 (Specific inhibitor of p-Smad3) and dominant negative Smad3 plasmid (DN-Smad3) attenuated the inhibitory effect of salidroside on C2C12 differentiation. Moreover, the induction of Myf5 transcription by salidroside was dependent on a Smad-binding site in the promoter region of Myf5 gene.Conclusion and Implications: Our findings identify a novel role and mechanism for salidroside in regulating myogenesis through p-Smad3-induced Myf5 transcription, which may have implications for its further application in combating degenerative muscular diseases caused by depletion of muscle stem cells, such as Duchenne muscular dystrophy or sarcopenia.

Fragile X mental retardation protein regulates skeletal muscle stem cell activity by regulating the stability of Myf5 mRNA

BackgroundRegeneration of adult tissues relies on adult stem cells that are primed to enter a differentiation program, while typically remaining quiescent. In mouse skeletal muscle, these features are reconciled by multiple translational control mechanisms that ensure primed muscle stem cells (MuSCs) are not activated. In quiescent MuSCs, this concept is illustrated by reversible microRNA silencing of Myf5 translation, mediated by microRNA-31 and fragile X mental retardation protein (FMRP).MethodsIn this work, we take advantage of FMRP knockout (Fmr1−/−) mice to support the role for FMRP in maintaining stem cell properties of the MuSC. We compare the activity of MuSCs in vivo after acute injury and engraftment, as well as ex vivo during culture. We use RNA immunoprecipitation and 3’UTR poly-adenine (poly(A)) length assays to assess the impact of FMRP on the stability of transcripts for myogenic regulatory factors.ResultsWe show that RNA-binding FMRP is required to maintain the MuSC pool. More specifically, FMRP is required for stem cell properties of muscle stem cells, which include MuSC capacity to prime the myogenic program, their self-renewal, and their capacity to efficiently regenerate muscle. We provide evidence that FMRP regulation of MuSC activity occurs in part by the capacity of FMRP to directly bind Myf5 transcripts and impact rates of Myf5 deadenylation.ConclusionsOur results provide further evidence supporting a role for post-transcriptional silencing platforms by RNA-binding proteins in maintaining stemness properties of adult stem cells. In addition, deregulated MuSC activity in the absence of Fmr1 may have implications for fragile X syndrome, which is associated with muscle hypotonia during infancy.

Catechins activate muscle stem cells by Myf5 induction and stimulate muscle regeneration

Muscle weakness is one of the most common symptoms in aged individuals and increases risk of mortality. Thus, maintenance of muscle mass is important for inhibiting aging. In this study, we investigated the effect of catechins, polyphenol compounds in green tea, on muscle regeneration. We found that (−)-epicatechin gallate (ECG) and (−)-epigallocatechin-3-gallate (EGCG) activate satellite cells by induction of Myf5 transcription factors. For satellite cell activation, Akt kinase was significantly induced after ECG treatment and ECG-induced satellite cell activation was blocked in the presence of Akt inhibitor. ECG also promotes myogenic differentiation through the induction of myogenic markers, including Myogenin and Muscle creatine kinase (MCK), in satellite and C2C12 myoblast cells. Finally, EGCG administration to mice significantly increased muscle fiber size for regeneration. Taken together, the results suggest that catechins stimulate muscle stem cell activation and differentiation for muscle regeneration.

Fine-tuning the onset of myogenesis by homeobox proteins that interact with the Myf5 limb enhancer.

ABSTRACTSkeletal myogenesis in vertebrates is initiated at different sites of skeletal muscle formation during development, by activation of specific control elements of the myogenic regulatory genes. In the mouse embryo, Myf5 is the first myogenic determination gene to be expressed and its spatiotemporal regulation requires multiple enhancer sequences, extending over 120 kb upstream of the Mrf4-Myf5 locus. An enhancer, located at −57/−58 kb from Myf5, is responsible for its activation in myogenic cells derived from the hypaxial domain of the somite, that will form limb muscles. Pax3 and Six1/4 transcription factors are essential activators of this enhancer, acting on a 145-bp core element. Myogenic progenitor cells that will form the future muscle masses of the limbs express the factors necessary for Myf5 activation when they delaminate from the hypaxial dermomyotome and migrate into the forelimb bud, however they do not activate Myf5 and the myogenic programme until they have populated the prospective muscle masses. We show that Msx1 and Meox2 homeodomain-containing transcription factors bind in vitro and in vivo to specific sites in the 145-bp element, and are implicated in fine-tuning activation of Myf5 in the forelimb. Msx1, when bound between Pax and Six sites, prevents the binding of these key activators, thus inhibiting transcription of Myf5 and consequent premature myogenic differentiation. Meox2 is required for Myf5 activation at the onset of myogenesis via direct binding to other homeodomain sites in this sequence. Thus, these homeodomain factors, acting in addition to Pax3 and Six1/4, fine-tune the entry of progenitor cells into myogenesis at early stages of forelimb development.

MicroRNA-3906 Regulates Fast Muscle Differentiation through Modulating the Target Gene homer-1b in Zebrafish Embryos

A microRNA, termed miR-In300 or miR-3906, suppresses the transcription of myf5 through silencing dickkopf-related protein 3 (dkk3r/dkk3a) during early development when myf5 is highly transcribed, but not at late stages when myf5 transcription is reduced. Moreover, after 24 hpf, when muscle cells are starting to differentiate, Dkk3a could not be detected in muscle tissue at 20 hpf. To explain these reversals, we collected embryos at 32 hpf, performed assays, and identified homer-1b, which regulates calcium release from sarcoplasmic reticulum, as the target gene of miR-3906. We further found that either miR-3906 knockdown or homer-1b overexpression increased expressions of fmhc4 and atp2a1 of calcium-dependent fast muscle fibrils, but not slow muscle fibrils, and caused a severe disruption of sarcomeric actin and Z-disc structure. Additionally, compared to control embryos, the intracellular calcium concentration ([Ca2+]i) of these treated embryos was increased as high as 83.9–97.3% in fast muscle. In contrast, either miR-3906 overexpression or homer-1b knockdown caused decreases of [Ca2+]i and, correspondingly, defective phenotypes in fast muscle. These defects could be rescued by inducing homer-1b expression at later stage. These results indicate that miR-3906 controls [Ca2+]i homeostasis in fast muscle through fine tuning homer-1b expression during differentiation to maintain normal muscle development.

Direct molecular regulation of the myogenic determination gene Myf5 by Pax3, with modulation by Six1/4 factors, is exemplified by the −111 kb-Myf5 enhancer

The Myf5 gene plays an important role in myogenic determination during mouse embryo development. Multiple genomic regions of the Mrf4–Myf5 locus have been characterised as enhancer sequences responsible for the complex spatiotemporal expression of the Myf5 gene at the onset of myogenesis. These include an enhancer sequence, located at −111kb upstream of the Myf5 transcription start site, which is responsible of Myf5 activation in ventral somitic domains (Ribas et al., 2011. Dev. Biol. 355, 372–380). We show that the −111kb-Myf5 enhancer also directs transgene expression in some limb muscles, and is active at foetal as well as embryonic stages. We have carried out further characterisation of the regulation of this enhancer and show that the paired-box Pax3 transcription factor binds to it in vitro as in vivo, and that Pax binding sites are essential for its activity. This requirement is independent of the previously reported regulation by TEAD transcription factors. Six1/4 which, like Pax3, are important upstream regulators of myogenesis, also bind in vivo to sites in the −111kb-Myf5 enhancer and modulate its activity. The −111kb-Myf5 enhancer therefore shares common functional characteristics with another Myf5 regulatory sequence, the hypaxial and limb 145bp-Myf5 enhancer, both being directly regulated in vivo by Pax3 and Six1/4 proteins. However, in the case of the −111kb-Myf5 enhancer, Six has less effect and we conclude that Pax regulation plays a major role in controlling this aspect of the Myf5 gene expression at the onset of myogenesis in the embryo.

A necrotic stimulus is required to maximize matrix-mediated myogenesis in mice

SUMMARYBiomaterials that are similar to skeletal muscle extracellular matrix have been shown to augment regeneration in ischemic muscle. In this study, treatment with a collagen-based matrix stimulated molecular myogenesis in an mdx murine model of necrosis. Matrix-treated animals ran ≥40% further, demonstrating functional regeneration, and expressed increased levels of myogenic transcripts. By contrast, matrix treatment was unable to induce transcriptional or functional changes in an MLC/SOD1G93A atrophic mouse model. In vitro, satellite cells were cultured under standard conditions, on matrix, in the presence of myocyte debris (to simulate a necrotic-like environment) or with both matrix and necrotic stimuli. Exposure to both matrix and necrotic stimuli induced the greatest increases in mef2c, myf5, myoD and myogenin transcripts. Furthermore, conditioned medium collected from satellite cells cultured with both stimuli contained elevated levels of factors that modulate satellite cell activation and proliferation, such as FGF-2, HGF and SDF-1. Application of the conditioned medium to C2C12 myoblasts accelerated maturation, as demonstrated by increased mef2c, myf5 and myogenin transcripts and fusion indexes. In summary, the collagen matrix required a necrotic stimulus to enhance the maturation of satellite cells and their secretion of a myogenic cocktail. Considering that matrix treatment supports myogenesis only in in vivo models that exhibit necrosis, this study demonstrates that a necrotic environment is required to maximize matrix-mediated myogenesis.

Sonic hedgehog acts cell-autonomously on muscle precursor cells to generate limb muscle diversity

How muscle diversity is generated in the vertebrate body is poorly understood. In the limb, dorsal and ventral muscle masses constitute the first myogenic diversification, as each gives rise to distinct muscles. Myogenesis initiates after muscle precursor cells (MPCs) have migrated from the somites to the limb bud and populated the prospective muscle masses. Here, we show that Sonic hedgehog (Shh) from the zone of polarizing activity (ZPA) drives myogenesis specifically within the ventral muscle mass. Shh directly induces ventral MPCs to initiate Myf5 transcription and myogenesis through essential Gli-binding sites located in the Myf5 limb enhancer. In the absence of Shh signaling, myogenesis is delayed, MPCs fail to migrate distally, and ventral paw muscles fail to form. Thus, Shh production in the limb ZPA is essential for the spatiotemporal control of myogenesis and coordinates muscle and skeletal development by acting directly to regulate the formation of specific ventral muscles.

Injected matrix stimulates myogenesis and regeneration of mouse skeletal muscle after ischaemic injury

Biomaterial-guided regeneration represents a novel approach for the treatment of myopathies. Revascularisation and the intramuscular extracellular matrix are important factors in stimulating myogenesis and regenerating muscle damaged by ischaemia. In this study, we used an injectable collagen matrix, enhanced with sialyl LewisX (sLeX), to guide skeletal muscle differentiation and regeneration. The elastic properties of collagen and sLeX-collagen matrices were similar to those of skeletal muscle, and culture of pluripotent mESCs on the matrices promoted their differentiation into myocyte-like cells expressing Pax3, MHC3, myogenin and Myf5. The regenerative properties of matrices were evaluated in ischaemic mouse hind-limbs. Treatment with the sLeX-matrix augmented the production of myogenic-mediated factors insulin-like growth factor (IGF)-1, and IGF binding protein-2 and -5 after 3 days. This was followed by muscle regeneration, including a greater number of regenerating myofibres and increased transcription of Six1, M-cadherin, myogenin and Myf5 after 10 days. Simultaneously, the sLeX-matrix promoted increased mobilisation and engraftment of bone marrow-derived progenitor cells, the development of larger arterioles and the restoration of tissue perfusion. Both matrix treatments tended to reduce maximal forces of ischaemic solei muscles, but sLeX-matrix lessened this loss of force and also prevented muscle fatigue. Only sLeX-matrix treatment improved mobility of mice on a treadmill. Together, these results suggest a novel approach for regenerative myogenesis, whereby treatment only with a matrix, which possesses an inherent ability to guide myogenic differentiation of pluripotent stem cells, can enhance the endogenous vascular and myogenic regeneration of skeletal muscle, thus holding promise for future clinical use.

302 A Necrotic Mechanism is Required To Maximize Matrix-Augmented Regeneration

302 A Necrotic Mechanism is Required To Maximize Matrix-Augmented Regeneration

Carm1 Regulates Pax7 Transcriptional Activity through MLL1/2 Recruitment during Asymmetric Satellite Stem Cell Divisions

In skeletal muscle, asymmetrically dividing satellite stem cells give rise to committed satellite cells that transcribe the myogenic determination factor Myf5, a Pax7-target gene. We identified the arginine methyltransferase Carm1 as a Pax7 interacting protein and found that Carm1 specifically methylates multiple arginines in the N terminus of Pax7. Methylated Pax7 directly binds the C-terminal cleavage forms of the trithorax proteins MLL1/2 resulting in the recruitment of the ASH2L:MLL1/2:WDR5:RBBP5 histone H3K4 methyltransferase complex to regulatory enhancers and the proximal promoter of Myf5. Finally, Carm1 is required for the induction of de novo Myf5 transcription following asymmetric satellite stem cell divisions. We defined the C-terminal MLL region as a reader domain for the recognition of arginine methylated proteins such as Pax7. Thus, arginine methylation of Pax7 by Carm1 functions as a molecular switch controlling the epigenetic induction of Myf5 during satellite stem cell asymmetric division and entry into the myogenic program.

Postprandial expression of growth-related genes in Atlantic salmon (Salmo salar L.) juveniles fasted for 1 week and fed a single meal to satiation

We investigated postprandial changes in transcript abundance following a single satiating meal in juvenile Atlantic salmon (Salmo salar L.) (about 70g body mass) following fasting for 1 week at 12°C. The expression of twenty-three growth-related genes was determined in fast myotomal muscle using quantitative real-time PCR at the following postprandial time points: -12, 0, 1, 3, 6, 12, 24, 48 and 96h. The gut was fullest 1-6h after feeding and emptied within 48-96h. IGF-I, MyoD1c, MRF4 and myf5 transcripts were sharply up-regulated within 1h of refeeding and are promising candidate genes involved in a fast-response signalling system that regulates fish myotomal muscle growth. These genes clustered together with MyoD1b and suggest a coordinated regulation to favour resumption of myogenesis as an early response to feeding. Insulin-like growth factor (IGF)-II and the ubiquitin ligase MAFbx/atrogin-1 were initially down-regulated but restored to initial values after 12h. It is also suggested that local production of IGF-I within the muscle might suppress catabolic pathways depressing MAFbx/atrogin-1.

In vitro expression profiling of myostatin, follistatin, decorin and muscle-specific transcription factors in adult caprine contractile myotubes

Skeletal muscle is one of the several adult postmitotic tissues that retain the capacity to regenerate, which relies on a population of quiescent precursors, termed satellite cells. Proliferation and differentiation of myoblasts to form mature myotubes in vitro has been a valuable tool in the characterization of the cellular events during myogenesis, which is a multistep process starting with progenitor cell proliferation, followed by their exit from the cell cycle, differentiation, alignment, and fusion to form multinucleated myotubes. A typical feature during muscle differentiation is the variation in expression of various genes along with myogenic factors. In this experiment, mRNA level of myostatin, follistatin, decorin and three muscle-specific transcription factors in adult caprine contractile myotubes have been studied through quantitative real time PCR. We observed that the expression level of myostatin, decorin, Myf5 and myogenin transcripts were significantly higher in contractile myotubes compared to myoblast monolayer (P < 0.05), and follistatin level was similar in both types of cells, whereas MyoD transcript level was significantly high in monolayer culture which might be due heterogeneity of myoblast population. It is concluded that the information generated would provide the base line information as well as monitoring markers to undertake experiments aimed at modulating muscle growth.

Essential roles of basic helix-loop-helix transcription factors, Capsulin and Musculin, during craniofacial myogenesis of zebrafish.

Capsulin and Musculin are basic helix-loop-helix transcription factors, but their biophysiological roles in zebrafish cranial myogenesis are unclear. Expressions of endogenous capsulin transcripts are detected at the central- (~24-hpf) and at dorsal- and ventral-mesoderm cores (~30-72hpf) of branchial arches. In contrast, musculin transcripts are expressed as a two-phase manner: early phase (20-22hpf) expressions of musculin are detected at the head mesoderm, whereas late-phase (36-72hpf) are detected at all presumptive head-muscle precursors. Knockdown of either capsulin or musculin leads to loss of all cranial muscles without affecting trunk muscle development. The defective phenotypes of Capsulin- and Musculin-morphant can be rescued by co-injection of mRNA of each other. Both myf5 and myod transcripts are down-regulated in the Capsulin-morphant while myod transcripts are up-regulated in the Musculin-morphant. Therefore, we propose a putative regulatory network to understand how capsulin/musculin regulate distinctly either myf5 or myod during zebrafish craniofacial myogenesis.

Dickkopf-3-related Gene Regulates the Expression of Zebrafish myf5 Gene through Phosphorylated p38a-dependent Smad4 Activity

Myf5 is a myogenic regulatory factor that functions in myogenesis. An intronic microRNA, miR-In300, located within zebrafish myf5 intron I, has been reported to silence myf5 through the targeting of dickkopf-3-related gene (dkk3r). However, the molecular mechanism underlying the control of myf5 expression by dkk3r is unknown. By injecting dkk3r-specific morpholino-oligonucleotide (dkk3r-MO) to knock down Dkk3r, we found that the phosphorylated p38a protein was reduced. Knockdown of p38a resulted in malformed somites and reduced myf5 transcripts, which photocopied the defects induced by injection of dkk3r-MO. To block the MAPK pathway, phosphorylation of p38 was inhibited by introduction of SB203580, which caused the down-regulation of myf5 expression. The GFP signal was dramatically decreased in somites when we injected p38a-MO into embryos derived from transgenic line Tg(myf5(80K):GFP), in which the GFP was driven by the myf5 promoter. Although these p38a-MO-induced defects were rescued by co-injection with p38a mRNA, they were not rescued with p38a mRNA containing a mutation at the phosphorylation domain. Moreover, overexpression of Smad2 or Smad3a enhanced myf5 expression, but the defects induced by the dominant negative form of either Smad2 or Smad3a equaled those of embryos injected with either dkk3r-MO or p38a-MO. These results support the involvement of Smad2·Smad3a in p38a mediation. Overexpression of Smad4 enabled the rescue of myf5 defects in the dkk3r-MO-injected embryos, but knockdown of either dkk3r or p38a caused Smad4 protein to lose stability. Therefore, we concluded that Dkk3r regulates p38a phosphorylation to maintain Smad4 stability, in turn enabling the Smad2·Smad3a·Smad4 complex to form and activate the myf5 promoter.