Muscle Regulatory Factors MyoD Research Articles

The Satellite Cell at 60: The Foundation Years.

The resident stem cell for skeletal muscle is the satellite cell. On the 50th anniversary of its discovery in 1961, we described the history of skeletal muscle research and the seminal findings made during the first 20 years in the life of the satellite cell (Scharner and Zammit 2011, doi: 10.1186/2044-5040-1-28). These studies established the satellite cell as the source of myoblasts for growth and regeneration of skeletal muscle. Now on the 60th anniversary, we highlight breakthroughs in the second phase of satellite cell research from 1980 to 2000. These include technical innovations such as isolation of primary satellite cells and viable muscle fibres complete with satellite cells in their niche, together with generation of many useful reagents including genetically modified organisms and antibodies still in use today. New methodologies were combined with description of endogenous satellite cells markers, notably Pax7. Discovery of the muscle regulatory factors Myf5, MyoD, myogenin, and MRF4 in the late 1980s revolutionized understanding of the control of both developmental and regerenative myogenesis. Emergence of genetic lineage markers facilitated identification of satellite cells in situ, and also empowered transplantation studies to examine satellite cell function. Finally, satellite cell heterogeneity and the supportive role of non-satellite cell types in muscle regeneration were described. These major advances in methodology and in understanding satellite cell biology provided further foundations for the dramatic escalation of work on muscle stem cells in the 21st century.

Oscillations of Delta-like1 regulate the balance between differentiation and maintenance of muscle stem cells

Cell-cell interactions mediated by Notch are critical for the maintenance of skeletal muscle stem cells. However, dynamics, cellular source and identity of functional Notch ligands during expansion of the stem cell pool in muscle growth and regeneration remain poorly characterized. Here we demonstrate that oscillating Delta-like 1 (Dll1) produced by myogenic cells is an indispensable Notch ligand for self-renewal of muscle stem cells in mice. Dll1 expression is controlled by the Notch target Hes1 and the muscle regulatory factor MyoD. Consistent with our mathematical model, our experimental analyses show that Hes1 acts as the oscillatory pacemaker, whereas MyoD regulates robust Dll1 expression. Interfering with Dll1 oscillations without changing its overall expression level impairs self-renewal, resulting in premature differentiation of muscle stem cells during muscle growth and regeneration. We conclude that the oscillatory Dll1 input into Notch signaling ensures the equilibrium between self-renewal and differentiation in myogenic cell communities.

Basal Signalling Through Death Receptor 5 and Caspase 3 Activates p38 Kinase to Regulate Serum Response Factor (SRF)-Mediated MyoD Transcription.

We have previously reported that stable expression of a dominant negative Death Receptor 5 (dnDR5) in skeletal myoblasts results in decreased basal caspase activity and decreased mRNA and protein expression of the muscle regulatory transcription factor MyoD in growth medium (GM), resulting in inhibited differentation when myoblasts are then cultured in differentiation media (DM). Further, this decreased level of MyoD mRNA was not a consequence of altered message stability, but rather correlated with decreased acetylation of histones in the distal regulatory region (DRR) of the MyoD extended promoter known to control MyoD transcription. As serum response factor (SRF) is the transcription factor known to be responsible for basal MyoD expression in GM, we compared the level of SRF binding to the non-canonical serum response element (SRE) within the DRR in parental and dnDR5 expressing myoblasts. Herein, we report that stable expression of dnDR5 resulted in decreased levels of serum response factor (SRF) binding to the CArG box in the SRE of the DRR. Total SRF expression levels were not affected, but phosphorylation indicative of SRF activation was impaired. This decreased SRF phosphorylation correlated with decreased phosphorylation-induced activation of p38 kinase. Moreover, the aforementioned signaling events affected by expression of dnDR5 could be appropriately recapitulated using either a pharmacological inhibitor of caspase 3 or p38 kinase. Thus, our results have established a signaling pathway from DR5 through caspases to p38 kinase activation, to SRF activation and the basal expression of MyoD.

The muscle regulatory transcription factor MyoD participates with p53 to directly increase the expression of the pro-apoptotic Bcl2 family member PUMA.

The muscle regulatory transcription factor MyoD is a master regulator of skeletal myoblast differentiation. We have previously reported that MyoD is also necessary for the elevated expression of the pro-apoptotic Bcl2 family member PUMA, and the ensuing apoptosis, that occurs in a subset of myoblasts induced to differentiate. Herein, we report the identification of a functional MyoD binding site within the extended PUMA promoter. In silico analysis of the murine PUMA extended promoter revealed three potential MyoD binding sites within 2kb of the transcription start site. Expression from a luciferase reporter construct containing this 2kb fragment was enhanced by activation of MyoD in both myoblasts and fibroblasts and diminished by silencing of MyoD in myoblasts. Experiments utilizing truncated versions of this promoter region revealed that the potential binding site at position - 857 was necessary for expression. Chromatin immunoprecipitation (ChIP) analysis confirmed binding of MyoD to the DNA region encompassing position - 857. The increase in MyoD binding to the PUMA promoter as a consequence of culture in differentiation media (DM) was comparable to the increase in MyoD binding at the myogenin promoter and was diminished in myoblasts silenced for MyoD expression. Finally, ChIP analysis using an antibody specific for the transcription factor p53 demonstrated that, in myoblasts silenced for MyoD expression, p53 binding to the PUMA promoter was diminished in response to culture in DM. These data indicate that MyoD plays a direct role in regulating PUMA expression and reveal functional consequences of MyoD expression on p53 mediated transcription of PUMA.

Myotome adaptability confers developmental robustness to somitic myogenesis in response to fibre number alteration

Balancing the number of stem cells and their progeny is crucial for tissue development and repair. Here we examine how cell numbers and overall muscle size are tightly regulated during zebrafish somitic muscle development. Muscle stem/precursor cell (MPCs) expressing Pax7 are initially located in the dermomyotome (DM) external cell layer, adopt a highly stereotypical distribution and thereafter a proportion of MPCs migrate into the myotome. Regional variations in the proliferation and terminal differentiation of MPCs contribute to growth of the myotome. To probe the robustness of muscle size control and spatiotemporal regulation of MPCs, we compared the behaviour of wild type (wt) MPCs with those in mutant zebrafish that lack the muscle regulatory factor Myod. Myodfh261 mutants form one third fewer multinucleate fast muscle fibres than wt and show a significant expansion of the Pax7+ MPC population in the DM. Subsequently, myodfh261 mutant fibres generate more cytoplasm per nucleus, leading to recovery of muscle bulk. In addition, relative to wt siblings, there is an increased number of MPCs in myodfh261 mutants and these migrate prematurely into the myotome, differentiate and contribute to the hypertrophy of existing fibres. Thus, homeostatic reduction of the excess MPCs returns their number to normal levels, but fibre numbers remain low. The GSK3 antagonist BIO prevents MPC migration into the deep myotome, suggesting that canonical Wnt pathway activation maintains the DM in zebrafish, as in amniotes. BIO does not, however, block recovery of the myodfh261 mutant myotome, indicating that homeostasis acts on fibre intrinsic growth to maintain muscle bulk. The findings suggest the existence of a critical window for early fast fibre formation followed by a period in which homeostatic mechanisms regulate myotome growth by controlling fibre size. The feedback controls we reveal in muscle help explain the extremely precise grading of myotome size along the body axis irrespective of fish size, nutrition and genetic variation and may form a paradigm for wider matching of organ size.

Gene profiling of embryonic skeletal muscle lacking type I ryanodine receptor Ca(2+) release channel.

In mature skeletal muscle, the intracellular Ca2+ concentration rises dramatically upon membrane depolarization, constituting the link between excitation and contraction. This process requires Ca2+ release from the sarcoplasmic reticulum via the type 1 ryanodine receptor (RYR1). However, RYR1’s potential roles in muscle development remain obscure. We used an established RyR1- null mouse model, dyspedic, to investigate the effects of the absence of a functional RYR1 and, consequently, the lack of RyR1-mediated Ca2+ signaling, during embryogenesis. Homozygous dyspedic mice die after birth and display small limbs and abnormal skeletal muscle organization. Skeletal muscles from front and hind limbs of dyspedic fetuses (day E18.5) were subjected to microarray analyses, revealing 318 differentially expressed genes. We observed altered expression of multiple transcription factors and members of key signaling pathways. Differential regulation was also observed for genes encoding contractile as well as muscle-specific structural proteins. Additional qRT-PCR analysis revealed altered mRNA levels of the canonical muscle regulatory factors Six1, Six4, Pax7, MyoD, MyoG and MRF4 in mutant muscle, which is in line with the severe developmental retardation seen in dyspedic muscle histology analyses. Taken together, these findings suggest an important non-contractile role of RyR1 or RYR1-mediated Ca2+ signaling during muscle organ development.

Muscle regulatory factors regulate T1R3 taste receptor expression

T1R3 is a T1R class of G protein-coupled receptors, composing subunit of the umami taste receptor when complexed with T1R1. T1R3 was originally discovered in gustatory tissue but is now known to be expressed in a wide variety of tissues and cell types such the intestine, pancreatic β-cells, skeletal muscle, and heart. In addition to taste recognition, the T1R1/T1R3 complex functions as an amino acid sensor and has been proposed to be a control mechanism for the secretion of hormones, such as cholecystokinin, insulin, and duodenal HCO3- and activates the mammalian rapamycin complex 1 (MTORC1) to inhibit autophagy. T1R3 knockout mice have increased rate of autophagy in the heart, skeletal muscle and liver. Thus, T1R3 has multiple physiological functions and is widely expressed in vivo. However, the exact mechanisms regulating T1R3 expression are largely unknown.Here, we used comparative genomics and functional analyses to characterize the genomic region upstream of the annotated transcriptional start of human T1R3. This revealed that the T1R3 promoter in human and mouse resides in an evolutionary conserved region (ECR). We also identified a repressive element located upstream of the human T1R3 promoter that has relatively high degree of conservation with rhesus macaque. Additionally, the muscle regulatory factors MyoD and Myogenin regulate T1R3 expression and T1R3 expression increases with skeletal muscle differentiation of murine myoblast C2C12 cells.Taken together, our study raises the possibility that MyoD and Myogenin might control skeletal muscle metabolism and homeostasis through the regulation of T1R3 promoter activity.

NEDD4 Regulates PAX7 Levels Promoting Activation of the Differentiation Program in Skeletal Muscle Precursors.

The transcription factor Pax7 regulates skeletal muscle stem cell (satellite cells) specification and maintenance through various mechanisms, including repressing the activity of the muscle regulatory factor MyoD. Hence, Pax7-to-MyoD protein ratios can determine maintenance of the committed-undifferentiated state or activation of the differentiation program. Pax7 expression decreases sharply in differentiating myoblasts but is maintained in cells (re)acquiring quiescence, yet the mechanisms regulating Pax7 levels based on differentiation status are not well understood. Here we show that Pax7 levels are directly regulated by the ubiquitin-ligase Nedd4. Our results indicate that Nedd4 is expressed in quiescent and activated satellite cells, that Nedd4 and Pax7 physically interact during early muscle differentiation-correlating with Pax7 ubiquitination and decline-and that Nedd4 loss of function prevented this effect. Furthermore, even transient nuclear accumulation of Nedd4 induced a drop in Pax7 levels and precocious muscle differentiation. Consequently, we propose that Nedd4 functions as a novel Pax7 regulator, which activity is temporally and spatially controlled to modulate the Pax7 protein levels and therefore satellite cell fate.

Effects of phytoestrogens on growth-related and lipogenic genes in rainbow trout (Oncorhynchus mykiss)

This study determined whether estradiol (E2) or the phytoestrogens genistein and daidzein regulate expression of growth-related and lipogenic genes in rainbow trout. Juvenile fish (5 mon, 65.8±1.8g) received intraperitoneal injections of E2, genistein, or daidzein (5μg/g body weight) or a higher dose of genistein (50μg/g body weight). Liver and white muscle were harvested 24h post-injection. In liver, expression of vitellogenin (vtg) and estrogen receptor alpha (era1) increased in all treatments and reflected treatment estrogenicity (E2>genistein (50μg/g)>genistein (5μg/g)=daidzein (5μg/g)). Estradiol and genistein (50μg/g) reduced components of the growth hormone (GH)/insulin-like growth factor (IGF) axis in liver, including increased expression of IGF binding protein-2b1 (igfbp2b1) and reduced igfbp5b1. In liver E2 and genistein (50μg/g) affected expression of components of the transforming growth factor beta signaling mechanism, reduced expression of ppar and rxr transcription factors, and increased expression of fatty acid synthesis genes srebp1, acly, fas, scd1, and gpat and lipid binding proteins fabp3 and lpl. In muscle E2 and genistein (50μg/g) increased era1 and erb1 expression and decreased erb2 expression. Other genes responded to phytoestrogens in a manner that suggested regulation by estrogen receptor-independent mechanisms, including increased ghr2, igfbp2a, igfbp4, and igfbp5b1. Expression of muscle regulatory factors pax7 and myod was increased by E2 and genistein. These data indicate that genistein and daidzein affect expression of genes in rainbow trout that regulate physiological mechanisms central to growth and nutrient retention.

Barx2 and Pax7 have antagonistic functions in regulation of wnt signaling and satellite cell differentiation.

The canonical Wnt signaling pathway is critical for myogenesis and can induce muscle progenitors to switch from proliferation to differentiation; how Wnt signals integrate with muscle-specific regulatory factors in this process is poorly understood. We previously demonstrated that the Barx2 homeobox protein promotes differentiation in cooperation with the muscle regulatory factor (MRF) MyoD. Pax7, another important muscle homeobox factor, represses differentiation. We now identify Barx2, MyoD, and Pax7 as novel components of the Wnt effector complex, providing a new molecular pathway for regulation of muscle progenitor differentiation. Canonical Wnt signaling induces Barx2 expression in muscle progenitors and perturbation of Barx2 leads to misregulation of Wnt target genes. Barx2 activates two endogenous Wnt target promoters as well as the Wnt reporter gene TOPflash, the latter synergistically with MyoD. Moreover, Barx2 interacts with the core Wnt effectors β-catenin and T cell-factor 4 (TCF4), is recruited to TCF/lymphoid enhancer factor sites, and promotes recruitment of β-catenin. In contrast, Pax7 represses the Wnt reporter gene and antagonizes the activating effect of Barx2. Pax7 also binds β-catenin suggesting that Barx2 and Pax7 may compete for interaction with the core Wnt effector complex. Overall, the data show for the first time that Barx2, Pax7, and MRFs can act as direct transcriptional effectors of Wnt signals in myoblasts and that Barx2 and Wnt signaling participate in a regulatory loop. We propose that antagonism between Barx2 and Pax7 in regulation of Wnt signaling may help mediate the switch from myoblast proliferation to differentiation.

Age-related changes affect rat rotator cuff muscle function

The influence of age on rotator cuff function and muscle structure remains poorly understood. We hypothesize that normal aging influences rotator cuff function, muscle structure, and regulatory protein expression in an established rat model of aging. Seventeen rats were obtained from the National Institute on Aging. The supraspinatus muscles in 11 middle-aged (12 months old) and 6 old (28 months old) rats were studied for age-related changes in rotator cuff neuromuscular function by in vivo muscle force testing and electromyography (EMG). Changes in muscle structure and molecular changes were assessed with quantitative immunohistochemistry for myogenic determination factor 1 (MyoD) and myogenic factor 5 (Myf5) expression. Old animals revealed significantly decreased peak tetanic muscle force at 0.5 N and 0.7 N preload tension (P < .05). The age of the animal accounted for 20.9% of variance and significantly influenced muscle force (P = .026). Preload tension significantly influenced muscle force production (P < .001) and accounted for 12.7% of total variance. There was regional heterogeneity in maximal compound motor action potential (CMAP) amplitude in the supraspinatus muscle; the proximal portion had a significantly higher CMAP than the middle and distal portions (P < .05). The expression of muscle regulatory factors MyoD and Myf5 was significantly decreased in old animals compared with middle-aged animals (P < .05). The normal aging process in this rat model significantly influenced contractile strength of the supraspinatus muscle and led to decreased expression of muscle regulatory factors. High preload tensions led to a significant decrease in force production in both middle-aged and old animals.

Regulation of Pax7 protein levels by caspase-3 and proteasome activity in differentiating myoblasts.

The transcription factor Pax7 negatively regulates the activity of the muscle regulatory transcription factor MyoD, preventing muscle precursor cells from undergoing terminal differentiation. In this context, the ratio between Pax7 and MyoD protein levels is thought to be critical in allowing myogenesis to proceed or to maintain the undifferentiated muscle precursor state. We have previously shown that Pax7 is subject to rapid down regulation in differentiating myoblasts, via a proteasome-dependent pathway. Here we present evidence indicating that Pax7 is also subject to caspase-3-dependent regulation. Furthermore, simultaneous inhibition of caspase-3 and proteasome activity induced further accumulation of Pax7 protein in differentiating myoblasts. These results suggest that at early stages of muscle differentiation, Pax7 levels are regulated by at least two independent mechanisms involving caspase-3 and proteasome activity.

Caspase-3, myogenic transcription factors and cell cycle inhibitors are regulated by leukemia inhibitory factor to mediate inhibition of myogenic differentiation.

BackgroundLeukemia inhibitory factor (LIF) is known to inhibit myogenic differentiation as well as to inhibit apoptosis and caspase-3 activation in non-differentiating myoblasts. In addition caspase-3 activity is required for myogenic differentiation. Therefore the aim of this study was to further investigate mechanisms of the differentiation suppressing effect of LIF in particular the possibility of a caspase-3 mediated inhibition of differentiation.ResultsLIF dependent inhibition of differentiation appeared to involve several mechanisms. Differentiating myoblasts that were exposed to LIF displayed increased transcripts for c-fos. Transcripts for the cell cycle inhibitor p21 as well as muscle regulatory factors myoD and myogenin were decreased with LIF exposure. However, LIF did not directly induce a proliferative effect under differentiation conditions, but did prevent the proportion of myoblasts that were proliferating from decreasing as differentiation proceeded. LIF stimulation decreased the percentage of cells positive for active caspase-3 occurring during differentiation. Both the effect of LIF inhibiting caspase-3 activation and differentiation appeared dependent on mitogen activated protein kinase and extracellular signal regulated kinase kinase (MEK) signalling. The role of LIF in myogenic differentiation was further refined to demonstrate that myoblasts are unlikely to secrete LIF endogenously.ConclusionsAltogether this study provides a more comprehensive view of the role of LIF in myogenic differentiation including LIF and receptor regulation in myoblasts and myotubes, mechanisms of inhibition of differentiation and the link between caspase-3 activation, apoptosis and myogenic differentiation.

The myogenic transcriptional network

Myogenesis has been a leading model for elucidating the molecular mechanisms that underlie tissue differentiation and development since the discovery of MyoD. During myogenesis, the fate of myogenic precursor cells is first determined by Pax3/Pax7. This is followed by regulation of the myogenic differentiation program by muscle regulatory factors (Myf5, MyoD, Myog, and Mrf4) to form muscle tissues. Recent studies have uncovered a detailed myogenic program that involves the RP58 (Zfp238)-dependent regulatory network, which is critical for repressing the expression of inhibitor of DNA binding (Id) proteins. These novel findings contribute to a comprehensive understanding of the muscle differentiation transcriptional program.

Regulation of Pax7 protein levels by caspase-3 and proteasome activity in differentiating myoblasts

The transcription factor Pax7 negatively regulates the activity of the muscle regulatory transcription factor MyoD, preventing muscle precursor cells from undergoing terminal differentiation. In this context, the ratio between Pax7 and MyoD protein levels is thought to be critical in allowing myogenesis to proceed or to maintain the undifferentiated muscle precursor state. We have previously shown that Pax7 is subject to rapid down regulation in differentiating myoblasts, via a proteasome-dependent pathway. Here we present evidence indicating that Pax7 is also subject to caspase-3-dependent regulation. Furthermore, simultaneous inhibition of caspase-3 and proteasome activity induced further accumulation of Pax7 protein in differentiating myoblasts. These results suggest that at early stages of muscle differentiation, Pax7 levels are regulated by at least two independent mechanisms involving caspase-3 and proteasome activity.

Increased HGF and c-Met in muscle tissues of polymyositis and dermatomyositis patients: Beneficial roles of HGF in muscle regeneration

We investigated the expression of hepatocyte growth factor (HGF), which has mitogenic and anti-fibrotic activities, in muscle tissue of polymyositis/dermatomyositis (PM/DM) patients, as well as its functional roles in cultured myoblasts. Immunohistochemistry in muscle from PM/DM patients revealed that HGF was expressed predominantly on infiltrating mononuclear cells and that muscle cells expressed the receptor c-met. Cultured myoblasts produced HGF; which was increased by IL-1alpha but suppressed by TGF-beta and dexamethasone. Exogenous HGF induced myoblast proliferation and reduced procollagen type I production. Furthermore, HGF enhanced the gene expression of muscle regulatory factors MyoD and Myf5, while suppressing expression of fibrosis-related genes, connective tissue growth factor and alpha-smooth muscle actin. Although dexamethasone showed contrasting effects to HGF on the expression of these genes, co-treatment with HGF ameliorated the effects of dexamethasone. Taking the beneficial roles of HGF into consideration, administration of HGF might contribute to muscle regeneration in PM/DM especially under corticosteroid treatment.

Increased expression of the pro-apoptotic Bcl2 family member PUMA and apoptosis by the muscle regulatory transcription factor MyoD in response to a variety of stimuli

We have previously reported that the level of MyoD expression correlates with the level of apoptosis that occurs in a subpopulation of skeletal myoblasts induced to differentiate by serum withdrawal. Herein we document that MyoD expression contributes to the level of apoptosis in myoblasts and fibroblasts in response to a variety of apoptotic stimuli. Specifically, re-expression of MyoD in skeletal myoblasts rendered defective for both differentiation and apoptosis by the expression of oncogenic Ras restores their ability to undergo both differentiation and apoptosis in response to serum withdrawal. Further, using a fibroblast cell line expressing an estrogen receptor:MyoD fusion protein, we have determined that addition of estrogen sensitizes these fibroblasts to apoptosis induced by serum withdrawal, or by treatment with etoposide or thapsigargin. RNAi mediated silencing of MyoD in either 23A2 or C2C12 myoblasts renders these cells resistant to apoptosis induced by serum withdrawal, or by treatment with etoposide or thapsigargin. Finally, MyoD mediated regulation of the apoptotic response to these various stimuli, in both myoblasts and fibroblasts, correlates with the level of induction of the pro-apoptotic Bcl2 family member PUMA.

Non-canonical role for the TRAIL receptor DR5/FADD/caspase pathway in the regulation of MyoD expression and skeletal myoblast differentiation

We report herein that the TRAIL receptor DR5/FADD/caspase pathway plays a role in skeletal myoblast differentiation through modulation of the expression of the muscle regulatory transcription factor MyoD. Specifically, treatment with the selective caspase 3 inhibitor DEVD-fmk or the selective caspase 8 inhibitor IETD-fmk in growth media (GM), prior to culture in differentiation media (DM), inhibited differentiation. Further, this treatment resulted in decreased levels of MyoD message and protein. We next explored a role for the TRAIL receptor DR5/FADD pathway. We found that expression of either dominant negative (dn) FADD or dominant negative (dn) DR5 also resulted in decreased levels of MyoD mRNA and protein and blocked differentiation. This decreased level of MyoD mRNA was not a consequence of altered stability. Treatment with TSA, an inhibitor of histone deacetylases (HDACs), allowed MyoD expression in myoblasts expressing dnDR5. Finally, acetylation of histones associated with the distal regulatory region (DRR) enhancer of MyoD was decreased in myoblasts expressing dnDR5. Thus, our data suggests a non-canonical role for the TRAIL receptor/FADD pathway in the regulation of MyoD expression and skeletal myoblast differentiation.

Localization of mRNAs and Proteins in Methyl Methacrylate-embedded Tissues

Precise localization of proteins and mRNA in histological sections is necessary for evaluating spatial gene expression patterns. Here we report sensitive detection of the gene products in fish tissues by immunohistochemistry (IHC) and in situ hybridization (ISH) assays on sections of whole specimens and vertebra embedded in methyl methacrylate (MMA) resin. This plastic resin favors easy preparation of various specimen types and enables preparation of large sections with well-preserved cell morphology. IHC analysis of the muscle regulatory factor MyoD in transverse sections of juvenile cod revealed MyoD-positive cells in the dorsolateral parts of the adaxial muscle. ISH revealed less spatially restricted signals of the bone morphogenic protein bmp4 in muscle and brain. To assess the applicability of ISH on sections of bony tissue, col1a1 and col2a1 expression was investigated in non-decalcified vertebra sections of Atlantic salmon. The former was identified in both chondrocytes and osteoblasts, whereas the latter was mostly evident in chondrocytes. We conclude that MMA resin offers easy preparation of large and problematic tissues and the possibility of carrying out both IHC and ISH analyses using standard protocols.

A distinct profile of myogenic regulatory factor detection within Pax7+ cells at S phase supports a unique role of Myf5 during posthatch chicken myogenesis

Satellite cells are skeletal muscle stem cells that provide myogenic progeny for myofiber growth and repair. Temporal expression of muscle regulatory factors (MRFs) and the paired box transcription factor Pax7 defines characteristic phases of proliferation (Pax7(+)/MyoD(+)/myogenin(-)) and differentiation (Pax7(-)/MyoD(+)/myogenin(+)) during myogenesis of satellite cells. Here, using bromodeoxyuridine (BrdU) labeling and triple immunodetection, we analyzed expression patterns of Pax7 and the MRFs MyoD, Myf5, or myogenin within S phase myoblasts prepared from posthatch chicken muscle. Essentially, all BrdU incorporation was restricted to Pax7(+) cells, of which the majority also expressed MyoD. The presence of a minor BrdU(+)/Pax7(+)/myogenin(+) population in proliferation stage cultures suggests that myogenin up-regulation is alone insufficient for terminal differentiation. Myf5 was detected strictly within Pax7(+) cells and decreased during S phase while MyoD presence persisted in cycling cells. This study provides novel data in support of a unique role for Myf5 during posthatch myogenesis.