In vertebrates, skeletal muscle is a model for the acquisition of cell fate from stem cells. Two determination factors of the basic helix-loop-helix myogenic regulatory factor (MRF) family, Myf5 and Myod, are thought to direct this transition because double-mutant mice totally lack skeletal muscle fibres and myoblasts. In the absence of these factors, progenitor cells remain multipotent and can change their fate. Gene targeting studies have revealed hierarchical relationships between these and the other MRF genes, Mrf4 and myogenin, where the latter are regarded as differentiation genes. Here we show, using an allelic series of three Myf5 mutants that differentially affect the expression of the genetically linked Mrf4 gene, that skeletal muscle is present in the new Myf5:Myod double-null mice only when Mrf4 expression is not compromised. This finding contradicts the widely held view that myogenic identity is conferred solely by Myf5 and Myod, and identifies Mrf4 as a determination gene. We revise the epistatic relationship of the MRFs, in which both Myf5 and Mrf4 act upstream of Myod to direct embryonic multipotent cells into the myogenic lineage.

Skeletal muscle serves as a paradigm for the acquisition of cell fate, yet the relationship between primitive cell populations and emerging myoblasts has remained elusive. We identify a novel population of resident Pax3 + / Pax7 + , muscle marker-negative cells throughout development. Using mouse mutants that uncouple myogenic progression, we show that these Pax + cells give rise to muscle progenitors. In the absence of skeletal muscle, they apoptose after down-regulation of Pax7. Furthermore, they mark the emergence of satellite cells during fetal development, and do not require Pax3 function. These findings identify critical cell populations during lineage restriction, and provide a framework for defining myogenic cell states for therapeutic studies. An unresolved issue in skeletal muscle development has been the nature of a reserve population of undifferentiated cells that ensures continued prenatal growth of this tissue (Parker et al. 2003;Tajbakhsh 2003). In the early embryo, muscle progenitors and precursors have been described (Tajbakhsh and Buckingham 2000). During post-natal life, satellite cells are the principal regenerative cell type (Zammit and Beauchamp 2001). About 1 d before birth, the appearance of progenitors associated with fibers has led to the assumption that these cells will give rise to future satellite cells in the adult (Cossu et al. 1993). However, the link between progenitors in the early somite and those observed at birth has remained enigmatic. In addition, the appearance of embryonic and fetal myoblasts has raised questions regarding their relationship with prenatal progenitors and emerging satellite cells. Studies in avians had suggested that satellite cells originate from somites, yet the endothelial origin of satellite cells remained unresolved (Armand et al. 1983). Furthermore, recent observations that muscle-or bonemarrow-derived mesenchymal stem cells can give rise to satellite cells or contribute to adult muscle after injury has questioned the notion that satellite cells originate exclusively from somites (Ferrari et al. 1998;Asakura et al. 2002;LaBarge and Blau 2002;Polesskaya et al. 2003). Therefore, in spite of considerable genetic data, cell relationships in the skeletal muscle lineage remain unresolved.During embryonic development, transitory structures called somites give rise to an epithelial dermomyotome, the source of dermal and endothelial precursors, as well as all the skeletal muscles in the body (Christ and Ordahl 1995;Tajbakhsh and Buckingham 2000). Multipotent muscle progenitor cells (MPCs) arising in the dermomyotome acquire definitive identity via the myogenic regulatory factors (MRFs) Myf5, Mrf4, and Myod (Rudnicki et al. 1993;Tajbakhsh et al. 1996;Kassar-Duchossoy et al. 2004). These progenitors give rise to muscle precursors called myoblasts (Hauschka 1994;Tajbakhsh 2003). Muscle differentiation is subsequently mediated by Myogenin, Myod, and Mrf4 (Tajbakhsh and Buckingham 2000). In the somite, the first skeletal muscle mass to form is the myotome. Skeleta...

Satellite cells assure postnatal skeletal muscle growth and repair. Despite extensive studies, their stem cell character remains largely undefined. Using pulse-chase labelling with BrdU to mark the putative stem cell niche, we identify a subpopulation of label-retaining satellite cells during growth and after injury. Strikingly, some of these cells display selective template-DNA strand segregation during mitosis in the muscle fibre in vivo, as well as in culture independent of their niche, indicating that genomic DNA strands are nonequivalent. Furthermore, we demonstrate that the asymmetric cell-fate determinant Numb segregates selectively to one daughter cell during mitosis and before differentiation, suggesting that Numb is associated with self-renewal. Finally, we show that template DNA cosegregates with Numb in label-retaining cells that express the self-renewal marker Pax7. The cosegregation of 'immortal' template DNA strands and their link with the asymmetry apparatus has important implications for stem cell biology and cancer.

The myogenic determination genes Myf5, Myod and Mrf4 direct skeletal muscle cell fate prenatally. In adult myogenesis, Myod has been shown to regulate myoblast differentiation, however, our understanding of satellite cell regulation is incomplete since the roles of Myf5 and Mrf4 had not been clearly defined. Here we examine the function of Myf5 and Mrf4 in the adult using recently generated alleles. Mrf4 is not expressed in normal or Myf5 null satellite cells and myoblasts, therefore excluding a role for this determination gene in adult muscle progenitors. Skeletal muscles of adult Myf5 null mice exhibit a subtle progressive myopathy. Crucially, adult Myf5 null mice exhibit perturbed muscle regeneration with a significant increase in muscle fibre hypertrophy, delayed differentiation, adipocyte accumulation, and fibrosis after freeze-injury. Satellite cell numbers are not significantly altered in Myf5 null animals and they show a modest impaired proliferation under some conditions in vitro. Mice double mutant for Myf5 and Dystrophin were more severely affected than single mutants, with enhanced necrosis and regeneration. Therefore, we show that Myf5 is a regulator of regenerative myogenesis and homeostasis, with functions distinct from those of Myod and Mrf4.