Deletion of TECRL promotes skeletal muscle repair by up-regulating EGR2.

Myogenic regeneration relies on the proliferation and differentiation of satellite cells. TECRL (trans-2,3-enoyl-CoA reductase like) is an endoplasmic reticulum protein only expressed in cardiac and skeletal muscle. However, its role in myogenesis remains unknown. We show that TECRL expression is increased in response to injury. Satellite cell-specific deletion of TECRL enhances muscle repair by increasing the expression of EGR2 through the activation of the ERK1/2 signaling pathway, which in turn promotes the expression of PAX7. We further show that TECRL deletion led to the upregulation of the histone acetyltransferase general control nonderepressible 5, which enhances the transcription of EGR2 through acetylation. Importantly, we showed that AAV9-mediated TECRL silencing improved muscle repair in mice. These findings shed light on myogenic regeneration and muscle repair.

Interplay between Pitx2 and Pax7 temporally governs specification of extraocular muscle stem cells.

Gene regulatory networks that act upstream of skeletal muscle fate determinants are distinct in different anatomical locations. Despite recent efforts, a clear understanding of the cascade of events underlying the emergence and maintenance of the stem cell pool in specific muscle groups remains unresolved and debated. Here, we invalidated Pitx2 with multiple Cre-driver mice prenatally, postnatally, and during lineage progression. We showed that this gene becomes progressively dispensable for specification and maintenance of the muscle stem (MuSC) cell pool in extraocular muscles (EOMs) despite being, together with Myf5, a major upstream regulator during early development. Moreover, constitutive inactivation of Pax7 postnatally led to a greater loss of MuSCs in the EOMs compared to the limb. Thus, we propose a relay between Pitx2, Myf5 and Pax7 for EOM stem cell maintenance. We demonstrate also that MuSCs in the EOMs adopt a quiescent state earlier that those in limb muscles and do not spontaneously proliferate in the adult, yet EOMs have a significantly higher content of Pax7+ MuSCs per area pre- and post-natally. Finally, while limb MuSCs proliferate in the mdx mouse model for Duchenne muscular dystrophy, significantly less MuSCs were present in the EOMs of the mdx mouse model compared to controls, and they were not proliferative. Overall, our study provides a comprehensive in vivo characterisation of MuSC heterogeneity along the body axis and brings further insights into the unusual sparing of EOMs during muscular dystrophy.

Loss of Tob1 promotes muscle regeneration through muscle stem cell expansion.

Muscle stem cells (MuSCs) play an indispensable role in postnatal muscle growth and hypertrophy in adults. MuSCs also retain a highly regenerative capacity and are therefore considered a promising stem cell source for regenerative therapy for muscle diseases. In this study, we identify tumor-suppressor protein Tob1 as a Pax7 target protein that negatively controls the population expansion of MuSCs. Tob1 protein is undetectable in the quiescent state but is upregulated during activation in MuSCs. Tob1 ablation in mice accelerates MuSC population expansion and boosts muscle regeneration. Moreover, inactivation of Tob1 in MuSCs ameliorates the efficiency of MuSC transplantation in a murine muscular dystrophy model. Collectively, selective targeting of Tob1 might be a therapeutic option for the treatment of muscular diseases, including muscular dystrophy and age-related sarcopenia.

SIX1+PAX3+ identify a progenitor for myogenic lineage commitment from hPSCs.

The earliest skeletal muscle progenitor cells (SMPCs) derived from human pluripotent stem cells (hPSCs) are often identified by factors expressed by a diverse number of progenitors. An early transcriptional checkpoint that defines myogenic commitment could improve hPSC differentiation to skeletal muscle. Analysis of several myogenic factors in human embryos and early hPSC differentiations found SIX1+PAX3+ co-expression was most indictive of myogenesis. Using dCas9-KRAB hPSCs, we demonstrate that early inhibition of SIX1 alone significantly decreased PAX3 expression, reduced PAX7+ SMPCs, and myotubes later in differentiation. Emergence of SIX1+PAX3+ precursors can be improved by manipulating seeding density, monitoring metabolic secretion and altering the concentration of CHIR99021. These modifications resulted in the co-emergence of hPSC-derived sclerotome, cardiac and neural crest that we hypothesized enhanced hPSC myogenic differentiation. Inhibition of non-myogenic lineages modulated PAX3 independent of SIX1. To better understand SIX1 expression, we compared directed differentiations to fetal progenitors and adult satellite cells by RNA-seq. Although SIX1 continued to be expressed across human development, SIX1 co-factor expression was dependent on developmental timing. We provide a resource to enable efficient derivation of skeletal muscle from hPSCs.

Pax3/7 gene function in Oikopleura dioica supports a neuroepithelial-like origin for its house-making Fol territory.

Larvacean tunicates feature a spectacular innovation not seen in other animals - the trunk oikoplastic epithelium (OE). This epithelium produces a house, a large and complex extracellular structure used for filtering and concentrating food particles. Previously we identified several homeobox transcription factor genes expressed during early OE patterning. Among these are two Pax3/7 copies that we named pax37A and pax37B. The vertebrate homologs, PAX3 and PAX7 are involved in developmental processes related to neural crest and muscles. In the ascidian tunicate Ciona intestinalis, Pax3/7 plays a role in the development of cells deriving from the neural plate border, including trunk epidermal sensory neurons and tail nerve cord neurons, as well as in the neural tube closure. Here we have investigated the roles of Oikopleura dioica pax37A and pax37B in the development of the OE, by using CRISPR-Cas9 mutant lines and analyzing scRNA-seq data from wild-type animals. We found that pax37B but not pax37A is essential for the differentiation of cell fields that produce the food concentrating filter of the house: the anterior Fol, giant Fol and Nasse cells. Trajectory analysis supported a neuroepithelial-like or a preplacodal ectoderm transcriptional signature in these cells. We propose that the highly specialized secretory epithelial cells of the Fol region either maintained or evolved neuroepithelial features. This is supported by a fragmented gene regulatory network involved in their development that also operates in ascidian epidermal neurons.

Divergent transcriptional and transforming properties of PAX3-FOXO1 and PAX7-FOXO1 paralogs.

The hallmarks of the alveolar subclass of rhabdomyosarcoma are chromosomal translocations that generate chimeric PAX3-FOXO1 or PAX7-FOXO1 transcription factors. Overexpression of either PAX-FOXO1s results in related cell transformation in animal models. Yet, in patients the two structural genetic aberrations they derived from are associated with distinct pathological manifestations. To assess the mechanisms underlying these differences, we generated isogenic fibroblast lines expressing either PAX-FOXO1 paralog. Mapping of their genomic recruitment using CUT&Tag revealed that the two chimeric proteins have distinct DNA binding preferences. In addition, PAX7-FOXO1 binding results in greater recruitment of the H3K27ac activation mark than PAX3-FOXO1 binding and is accompanied by greater transcriptional activation of neighbouring genes. These effects are associated with a PAX-FOXO1-specific alteration in the expression of genes regulating cell shape and the cell cycle. Consistently, PAX3-FOXO1 accentuates fibroblast cellular traits associated with contractility and surface adhesion and limits entry into S phase. In contrast, PAX7-FOXO1 drives cells to adopt an amoeboid shape, reduces entry into M phase, and causes increased DNA damage. Altogether, our results argue that the diversity of rhabdomyosarcoma manifestation arises, in part, from the divergence between the genomic occupancy and transcriptional activity of PAX3-FOXO1 and PAX7-FOXO1.

Primary cilia in satellite cells are the mechanical sensors for muscle hypertrophy.

Skeletal muscle atrophy is commonly associated with aging, immobilization, muscle unloading, and congenital myopathies. Generation of mature muscle cells from skeletal muscle satellite cells (SCs) is pivotal in repairing muscle tissue. Exercise therapy promotes muscle hypertrophy and strength. Primary cilium is implicated as the mechanical sensor in some mammalian cells, but its role in skeletal muscle cells remains vague. To determine mechanical sensors for exercise-induced muscle hypertrophy, we established three SC-specific cilium dysfunctional mouse models-Myogenic factor 5 (Myf5)-Arf-like Protein 3 (Arl3)-/-, Paired box protein Pax-7 (Pax7)-Intraflagellar transport protein 88 homolog (Ift88)-/-, and Pax7-Arl3-/--by specifically deleting a ciliary protein ARL3 in MYF5-expressing SCs, or IFT88 in PAX7-expressing SCs, or ARL3 in PAX7-expressing SCs, respectively. We show that the Myf5-Arl3-/- mice develop grossly the same as WT mice. Intriguingly, mechanical stimulation-induced muscle hypertrophy or myoblast differentiation is abrogated in Myf5-Arl3-/- and Pax7-Arl3-/- mice or primary isolated Myf5-Arl3-/- and Pax7-Ift88-/- myoblasts, likely due to defective cilia-mediated Hedgehog (Hh) signaling. Collectively, we demonstrate SC cilia serve as mechanical sensors and promote exercise-induced muscle hypertrophy via Hh signaling pathway.

Inhibitor of Myom3 inhibits proliferation and promotes differentiation of sheep myoblasts.

Skeletal muscle quality and yield are important production traits in livestock, and improving skeletal muscle quality while increasing its yield is an important goal of economic breeding. The proliferation and differentiation process of sheep myoblasts directly affects the growth and development of their muscles, thereby affecting the yield of mutton. Myomesin 3 (Myom3), as a functional gene related to muscle growth, currently lacks research on its function in myoblasts. This study aims to investigate the effect of the Myom3 gene on the proliferation and differentiation of sheep myoblasts and its potential molecular mechanisms. The results showed that inhibitor of Myom3 in the proliferation phase of myoblasts resulted in significant downregulation of the proliferation marker gene paired box 7 (Pax7) and myogenic regulatory factors (MRFs; Myf5, Myod1, Myog, P < 0.01), a significant decrease in the EdU-positive cell rate (P < 0.05), and a significant increase in the cell apoptosis rate (P < 0.01), which inhibited the proliferation of myoblasts and promoted their apoptosis. During the differentiation phase of myoblasts, the inhibitor of Myom3 resulted in significant downregulation of the Pax7 gene, upregulation of MRFs (Myod1, Myog, P < 0.05), and a significant increase in fusion index (P < 0.05), promoting the differentiation of myoblasts. Further transcriptome sequencing revealed that differentially expressed genes in the Myom3 interference group were mainly enriched in the MAPK signaling pathway, TNF signaling pathway, and IL-17 signaling pathway. In summary, the inhibitor of Myom3 inhibits myoblast proliferation and promotes myoblast differentiation. Therefore, Myom3 has a potential regulatory effect on the growth and development of sheep muscles, and in-depth functional research can be used for molecular breeding practices in sheep.

Isolation of a persistently quiescent muscle satellite cell population.

Although studies have identified characteristics of quiescent satellite cells (SCs), their isolation has been hampered by the fact that the isolation procedures result in the activation of these cells into their rapidly proliferating progeny (myoblasts). Thus, the use of myoblasts for therapeutic (regenerative medicine) or industrial applications (cellular agriculture) has been impeded by the limited proliferative and differentiative capacity of these myogenic progenitors. Here we identify a subpopulation of satellite cells isolated from mouse skeletal muscle using flow cytometry that is highly Pax7-positive, exhibit a very slow proliferation rate (7.7 ± 1.2 days/doubling), and are capable of being maintained in culture for at least 3 mo without a change in phenotype. These cells can be activated from quiescence using a p38 inhibitor or by exposure to freeze-thaw cycles. Once activated, these cells proliferate rapidly (22.7 ± 0.2 h/doubling), have reduced Pax7 expression (threefold decrease in Pax7 fluorescence vs. quiescence), and differentiate into myotubes with a high efficiency. Furthermore, these cells withstand freeze-thawing readily without a significant loss of viability (83.1 ± 2.1% live). The results presented here provide researchers with a method to isolate quiescent satellite cells, allowing for more detailed examinations of the factors affecting satellite cell quiescence/activation and providing a cell source that has a unique potential in the regenerative medicine and cellular agriculture fields.NEW & NOTEWORTHY We provide a method to isolate quiescent satellite cells from skeletal muscle. These cells are highly Pax7-positive, exhibit a very slow proliferation rate, and are capable of being maintained in culture for months without a change in phenotype. The use of these cells by muscle researchers will allow for more detailed examinations of the factors affecting satellite cell quiescence/activation and provide a novel cell source for the regenerative medicine and cellular agriculture fields.

TLE4 regulates muscle stem cell quiescence and skeletal muscle differentiation.

Muscle stem (satellite) cells express Pax7, a key transcription factor essential for satellite cell maintenance and adult muscle regeneration. We identify the corepressor transducin-like enhancer of split-4 (TLE4) as a Pax7 interaction partner expressed in quiescent satellite cells under homeostasis. A subset of satellite cells transiently downregulate TLE4 during early time points following muscle injury. We identify these to be activated satellite cells, and that TLE4 downregulation is required for Myf5 activation and myogenic commitment. Our results indicate that TLE4 represses Pax7-mediated Myf5 transcriptional activation by occupying the -111 kb Myf5 enhancer to maintain quiescence. Loss of TLE4 function causes Myf5 upregulation, an increase in satellite cell numbers and altered differentiation dynamics during regeneration. Thus, we have uncovered a novel mechanism to maintain satellite cell quiescence and regulate muscle differentiation mediated by the corepressor TLE4.

The axolotl genome and the evolution of key tissue formation regulators.

Salamanders serve as important tetrapod models for developmental, regeneration and evolutionary studies. An extensive molecular toolkit makes the Mexican axolotl (Ambystoma mexicanum) a key representative salamander for molecular investigations. Here we report the sequencing and assembly of the 32-gigabase-pair axolotl genome using an approach that combined long-read sequencing, optical mapping and development of a new genome assembler (MARVEL). We observed a size expansion of introns and intergenic regions, largely attributable to multiplication of long terminal repeat retroelements. We provide evidence that intron size in developmental genes is under constraint and that species-restricted genes may contribute to limb regeneration. The axolotl genome assembly does not contain the essential developmental gene Pax3. However, mutation of the axolotl Pax3 paralogue Pax7 resulted in an axolotl phenotype that was similar to those seen in Pax3-/- and Pax7-/- mutant mice. The axolotl genome provides a rich biological resource for developmental and evolutionary studies.

MyoD Over-Expression Rescues GST-bFGF Repressed Myogenesis.

During embryogenesis, basic fibroblast growth factor (bFGF) is released from neural tube and myotome to promote myogenic fate in the somite, and is routinely used for the culture of adult skeletal muscle (SKM) stem cells (MuSC, called satellite cells). However, the mechanism employed by bFGF to promote SKM lineage and MuSC proliferation has not been analyzed in detail. Furthermore, the question of if the post-translational modification (PTM) of bFGF is important to its stemness-promoting effect has not been answered. In this study, GST-bFGF was expressed and purified from E.coli, which lacks the PTM system in eukaryotes. We found that both GST-bFGF and commercially available bFGF activated the Akt-Erk pathway and had strong cell proliferation effect on C2C12 myoblasts and MuSC. GST-bFGF reversibly compromised the myogenesis of C2C12 myoblasts and MuSC, and it increased the expression of Myf5, Pax3/7, and Cyclin D1 but strongly repressed that of MyoD, suggesting the maintenance of myogenic stemness amid repressed MyoD expression. The proliferation effect of GST-bFGF was conserved in C2C12 over-expressed with MyoD (C2C12-tTA-MyoD), implying its independence of the down-regulation of MyoD. In addition, the repressive effect of GST-bFGF on myogenic differentiation was almost totally rescued by the over-expression of MyoD. Together, these evidences suggest that (1) GST-bFGF and bFGF have similar effects on myogenic cell proliferation and differentiation, and (2) GST-bFGF can promote MuSC stemness and proliferation by differentially regulating MRFs and Pax3/7, (3) MyoD repression by GST-bFGF is reversible and independent of the proliferation effect, and (4) GST-bFGF can be a good substitute for bFGF in sustaining MuSC stemness and proliferation.

Pax7 is involved in leucophore formation in goldfish and gene knockout improves the transparency of transparent goldfish.

Lines with few or no pigment cells have been established in fishes, and these lines are useful for bioimaging. The transparent goldfish (tra) line previously established by N-ethyl-N-nitrosourea (ENU) mutagenesis is also suitable for such experiments. However, in the case of tra, leucophores form in the adult fish, making it difficult to observe the organs inside body from outside the body. In this study, we attempted to create a knockout line of the pax7a and pax7b genes, which are thought to be involved in the formation of leucophores, to further improve the transparency of tra strain.Mutations were introduced by microinjection of the CRISPR/Cas9 mixture into single-cell embryos, mutant individuals were found in F0, and the next generation was generated to confirm the mutation patterns. As a result, multiple mutation patterns, including knockout, were obtained. The same pattern of knockout F1 with pax7a and pax7b mutations was crossed to generate a homozygous knockout in F2.In the resulting pax7b-/- (tra) fish but not in pax7a-/- (tra) fish, the number of leucophores was reduced compared to that in tra, and the transparency of the body was improved. It was suggested that pax7b plays an important role in leucophore formation in goldfish. The established transparent pax7b-/- (tra) goldfish line will be a useful model for bioimaging of the body interior.

Expression atlas of avian neural crest proteins: Neurulation to migration.

Neural crest (NC) cells are a dynamic population of embryonic stem cells that create various adult tissues in vertebrate species including craniofacial bone and cartilage and the peripheral and enteric nervous systems. NC development is thought to be a conserved and complex process that is controlled by a tightly-regulated gene regulatory network (GRN) of morphogens, transcription factors, and cell adhesion proteins. While multiple studies have characterized the expression of several GRN factors in single species, a comprehensive protein analysis that directly compares expression across development is lacking. To address this lack in information, we used three closely related avian models, Gallus gallus (chicken), Coturnix japonica (Japanese quail), and Pavo cristatus (Indian peafowl), to compare the localization and timing of four GRN transcription factors, PAX7, SNAI2, SOX9, and SOX10, from the onset of neurulation to migration. While the spatial expression of these factors is largely conserved, we find that quail NC cells express SNAI2, SOX9, and SOX10 proteins at the equivalent of earlier developmental stages than chick and peafowl. In addition, quail NC cells migrate farther and more rapidly than the larger organisms. These data suggest that despite a conservation of NC GRN players, differences in the timing of NC development between species remain a significant frontier to be explored with functional studies.

The thymus regulates skeletal muscle regeneration by directly promoting satellite cell expansion.

The thymus is the central immune organ, but it is known to progressively degenerate with age. As thymus degeneration is paralleled by the wasting of aging skeletal muscle, we speculated that the thymus may play a role in muscle wasting. Here, using thymectomized mice, we show that the thymus is necessary for skeletal muscle regeneration, a process tightly associated with muscle aging. Compared to control mice, the thymectomized mice displayed comparable growth of muscle mass, but decreased muscle regeneration in response to injury, as evidenced by small and sparse regenerative myofibers along with inhibited expression of regeneration-associated genes myh3, myod, and myogenin. Using paired box 7 (Pax7)-immunofluorescence staining and 5-Bromo-2'-deoxyuridine-incorporation assay, we determined that the decreased regeneration capacity was caused by a limited satellite cell pool. Interestingly, the conditioned culture medium of isolated thymocytes had a potent capacity to directly stimulate satellite cell expansion in vitro. These expanded cells were enriched in subpopulations of quiescent satellite cells (Pax7highMyoDlowEdUpos) and activated satellite cells (Pax7highMyoDhighEdUpos), which were efficiently incorporated into the regenerative myofibers. We thus propose that the thymus plays an essential role in muscle regeneration by directly promoting satellite cell expansion and may function profoundly in the muscle aging process.

Differentiation of the human PAX7-positive myogenic precursors/satellite cell lineage in vitro.

Satellite cells (SC) are muscle stem cells that can regenerate adult muscles upon injury. Most SC originate from PAX7+ myogenic precursors set aside during development. Although myogenesis has been studied in mouse and chicken embryos, little is known about human muscle development. Here, we report the generation of human induced pluripotent stem cell (iPSC) reporter lines in which fluorescent proteins have been introduced into the PAX7 and MYOG loci. We use single cell RNA sequencing to analyze the developmental trajectory of the iPSC-derived PAX7+ myogenic precursors. We show that the PAX7+ cells generated in culture can produce myofibers and self-renew in vitro and in vivo Together, we demonstrate that cells exhibiting characteristics of human fetal satellite cells can be produced in vitro from iPSC, opening interesting avenues for muscular dystrophy cell therapy. This work provides significant insights into the development of the human myogenic lineage.

SIX1 and SIX4 homeoproteins regulate PAX7+ progenitor cell properties during fetal epaxial myogenesis.

Pax7 expression marks stem cells in developing skeletal muscles and adult satellite cells during homeostasis and muscle regeneration. The genetic determinants that control the entrance into the myogenic program and the appearance of PAX7+ cells during embryogenesis are poorly understood. SIX homeoproteins are encoded by the sine oculis-related homeobox Six1-Six6 genes in vertebrates. Six1, Six2, Six4 and Six5 are expressed in the muscle lineage. Here, we tested the hypothesis that Six1 and Six4 could participate in the genesis of myogenic stem cells. We show that fewer PAX7+ cells occupy a satellite cell position between the myofiber and its associated basal lamina in Six1 and Six4 knockout mice (s1s4KO) at E18. However, PAX7+ cells are detected in remaining muscle masses present in the epaxial region of the double mutant embryos and are able to divide and contribute to muscle growth. To further characterize the properties of s1s4KO PAX7+ cells, we analyzed their transcriptome and tested their properties after transplantation in adult regenerating tibialis anterior muscle. Mutant stem cells contribute to hypotrophic myofibers that are not innervated but retain the ability to self-renew.

Defining and identifying satellite cell-opathies within muscular dystrophies and myopathies.

Muscular dystrophies and congenital myopathies arise from specific genetic mutations causing skeletal muscle weakness that reduces quality of life. Muscle health relies on resident muscle stem cells called satellite cells, which enable life-course muscle growth, maintenance, repair and regeneration. Such tuned plasticity gradually diminishes in muscle diseases, suggesting compromised satellite cell function. A central issue however, is whether the pathogenic mutation perturbs satellite cell function directly and/or indirectly via an increasingly hostile microenvironment as disease progresses. Here, we explore the effects on satellite cell function of pathogenic mutations in genes (myopathogenes) that associate with muscle disorders, to evaluate clinical and muscle pathological hallmarks that define dysfunctional satellite cells. We deploy transcriptomic analysis and comparison between muscular dystrophies and myopathies to determine the contribution of satellite cell dysfunction using literature, expression dynamics of myopathogenes and their response to the satellite cell regulator PAX7. Our multimodal approach extends current pathological classifications to define Satellite Cell-opathies: muscle disorders in which satellite cell dysfunction contributes to pathology. Primary Satellite Cell-opathies are conditions where mutations in a myopathogene directly affect satellite cell function, such as in Progressive Congenital Myopathy with Scoliosis (MYOSCO) and Carey-Fineman-Ziter Syndrome (CFZS). Primary satellite cell-opathies are generally characterised as being congenital with general hypotonia, and specific involvement of respiratory, trunk and facial muscles, although serum CK levels are usually within the normal range. Secondary Satellite Cell-opathies have mutations in myopathogenes that affect both satellite cells and muscle fibres. Such classification aids diagnosis and predicting probable disease course, as well as informing on treatment and therapeutic development.

Recapitulating human myogenesis ex vivo using human pluripotent stem cells.

Human pluripotent stem cells (hPSCs) provide a human model for developmental myogenesis, disease modeling and development of therapeutics. Differentiation of hPSCs into muscle stem cells has the potential to provide a cell-based therapy for many skeletal muscle wasting diseases. This review describes the current state of hPSCs towards recapitulating human myogenesis ex vivo, considerations of stem cell and progenitor cell state as well as function for future use of hPSC-derived muscle cells in regenerative medicine.

NEDD4-1 deficiency impairs satellite cell function during skeletal muscle regeneration.

BACKGROUND: Satellite cells are tissue-specific stem cells primarily responsible for the regenerative capacity of skeletal muscle. Satellite cell function and maintenance are regulated by extrinsic and intrinsic mechanisms, including the ubiquitin-proteasome system, which is key for maintaining protein homeostasis. In this context, it has been shown that ubiquitin-ligase NEDD4-1 targets the transcription factor PAX7 for proteasome-dependent degradation, promoting muscle differentiation in vitro. Nonetheless, whether NEDD4-1 is required for satellite cell function in regenerating muscle remains to be determined. RESULTS: Using conditional gene ablation, we show that NEDD4-1 loss, specifically in the satellite cell population, impairs muscle regeneration resulting in a significant reduction of whole-muscle size. At the cellular level, NEDD4-1-null muscle progenitors exhibit a significant decrease in the ability to proliferate and differentiate, contributing to the formation of myofibers with reduced diameter. CONCLUSIONS: These results indicate that NEDD4-1 expression is critical for proper muscle regeneration in vivo and suggest that it may control satellite cell function at multiple levels.