Perspectives on skeletal muscle stem cells.

Skeletal muscle has remarkable regeneration capabilities, mainly due to its resident muscle stem cells (MuSCs). In this review, we introduce recently developed technologies and the mechanistic insights they provide to the understanding of MuSC biology, including the re-definition of quiescence and Galert states. Additionally, we present recent studies that link MuSC function with cellular heterogeneity, highlighting the complex regulation of self-renewal in regeneration, muscle disorders and aging. Finally, we discuss MuSC metabolism and its role, as well as the multifaceted regulation of MuSCs by their niche. The presented conceptual advances in the MuSC field impact on our general understanding of stem cells and their therapeutic use in regenerative medicine.

Alveolar rhabdomyosarcoma-associated proteins PAX3/FOXO1A and PAX7/FOXO1A suppress the transcriptional activity of MyoD-target genes in muscle stem cells.

Rhabdomyosarcoma (RMS) is the commonest soft-tissue sarcoma in childhood and is characterized by expression of myogenic proteins, including the transcription factors MyoD and myogenin. There are two main subgroups, embryonal RMS and alveolar RMS (ARMS). Most ARMS are associated with chromosomal translocations that have breakpoints in introns of either PAX3 or PAX7, and FOXO1A. These translocations create chimeric transcription factors termed PAX3/FOXO1A and PAX7/FOXO1A respectively. Upon ectopic PAX3/FOXO1A expression, together with other genetic manipulation in mice, both differentiating myoblasts and satellite cells (the resident stem cells of postnatal muscle) can give rise to tumours with ARMS characteristics. As PAX3 and PAX7 are part of transcriptional networks that regulate muscle stem cell function in utero and during early postnatal life, PAX3/FOXO1A and PAX7/FOXO1A may subvert normal PAX3 and PAX7 functions. Here we examined how PAX3/FOXO1A and PAX7/FOXO1A affect myogenesis in satellite cells. PAX3/FOXO1A or PAX7/FOXO1A inhibited myogenin expression and prevented terminal differentiation in murine satellite cells: the same effect as dominant-negative (DN) Pax3 or Pax7 constructs. The transcription of MyoD-target genes myogenin and muscle creatine kinase were suppressed by PAX3/FOXO1A or PAX7/FOXO1A in C2C12 myogenic cells again as seen with Pax3/7DN. PAX3/FOXO1A or PAX7/FOXO1A did not inhibit the transcriptional activity of MyoD by perturbing MyoD expression, localization, phosphorylation or interaction with E-proteins. Chromatin immunoprecipitation on the myogenin promoter showed that PAX3/FOXO1A or PAX7/FOXO1A did not prevent MyoD from binding. However, PAX3/FOXO1A or PAX7/FOXO1A reduced occupation of the myogenin promoter by RNA polymerase II and decreased acetylation of histone H4, but did not directly bind to the myogenin promoter. Together, these observations reveal that PAX3/FOXO1A and PAX7/FOXO1A act to prevent myogenic differentiation via suppression of the transcriptional activation of MyoD-target genes.

P-cadherin is a direct PAX3-FOXO1A target involved in alveolar rhabdomyosarcoma aggressiveness.

Alveolar rhabdomyosarcoma (ARMS) is an aggressive childhood cancer of striated muscle characterized by the presence of the PAX3-FOXO1A or PAX7-FOXO1A chimeric oncogenic transcription factor. Identification of their targets is essential for understanding ARMS pathogenesis. To this aim, we analyzed transcriptomic data from rhabdomyosarcoma samples and found that P-cadherin expression is correlated with PAX3/7-FOXO1A presence. We then show that expression of a PAX3 dominant negative variant inhibits P-cadherin expression in ARMS cells. Using mouse models carrying modified Pax3 alleles, we demonstrate that P-cadherin is expressed in the dermomyotome and lies genetically downstream from the myogenic factor Pax3. Moreover, in vitro gel shift analysis and chromatin immunoprecipitation indicate that the P-cadherin gene is a direct transcriptional target for PAX3/7-FOXO1A. Finally, P-cadherin expression in normal myoblasts inhibits myogenesis and induces myoblast transformation, migration and invasion. Conversely, P-cadherin downregulation by small hairpin RNA decreases the transformation, migration and invasive potential of ARMS cells. P-cadherin also favors cadherin switching, which is a hallmark of metastatic progression, by controlling N- and M-cadherin expression and/or localization. Our findings demonstrate that P-cadherin is a direct PAX3-FOXO1A transcriptional target involved in ARMS aggressiveness. Therefore, P-cadherin emerges as a new and attractive target for therapeutic intervention in ARMS.

Regulation of skeletal muscle stem cell behavior by Pax3 and Pax7.

Pax genes have important roles in the regulation of stem cell behavior, leading to tissue differentiation. In the case of skeletal muscle, Pax3 and Pax7 perform this function both during development and on regeneration in the adult. The myogenic determination gene Myf5 is directly activated by Pax3, leading to the formation of skeletal muscle. Fgfr4 is also a direct Pax3 target and Sprouty1, which encodes an intracellular inhibitor of fibroblast growth factor (FGF) signaling, is under Pax3 control. Orchestration of FGF signaling, through Fgfr4/Sprouty1, modulates the entry of cells into the myogenic program, thus controling the balance between stem cell self-renewal and tissue differentiation. This and other aspects of Pax3/7 function in regulating the behavior of skeletal muscle stem cells are discussed.

The in vivo form of the murine class VI POU protein Emb is larger than that encoded by previously described transcripts.

The class VI POU domain family member known as Emb in the mouse (rat Brn5 or human mPOU/TCFbeta1) is present in vivo as a protein migrating at about 80 kDa on western blots, considerably larger than that predicted (about 42 kDa) from previously cloned coding sequences. By RT-PCR and 5' RACE strategies a full-length Emb sequence, Emb FL, is now identified. Shorter sequences encoding the -COOH terminal, and an -NH(2) terminal isoform, EmbN, were also isolated. Comparisons of Emb coding sequences between species, including the full-length zebra fish, POU(c), are presented, together with a compilation of the multiple transcripts produced by alternative splicing and the presence of different transcriptional start and stop sites, from the Emb gene.

A novel complex regulates cardiac actin gene expression through interaction of Emb, a class VI POU domain protein, MEF2D, and the histone transacetylase p300.

Expression of the mouse cardiac actin gene depends on a distal enhancer (-7 kbp) which has been shown, in transgenic mice, to direct expression to embryonic skeletal muscle. The presence of this distal sequence is also associated with reproducible expression of cardiac actin transgenes. In differentiated skeletal muscle cells, activity of the enhancer is driven by an E box, binding MyoD family members, and by a 3' AT-rich sequence which is in the location of a DNase I-hypersensitive site. This sequence does not bind MEF2 proteins, or other known muscle transcription factors, directly. Oct1 and Emb, a class VI POU domain protein, bind to consensus sites on the DNA, and it is the binding of Emb which is important for activity. Emb binds as a major complex with MEF2D and the histone transacetylase p300. The form of Emb present in this complex and as a major form in muscle cell extracts is longer (80 kDa) than that previously described. These results demonstrate the importance of this novel complex in the transcriptional regulation of the cardiac actin gene and suggest a potential role in chromatin remodeling associated with muscle gene activation.

Pw1/Peg3 is a potential cell death mediator and cooperates with Siah1a in p53-mediated apoptosis.

Induction of wild-type p53 in mouse fibroblasts causes cell cycle arrest at the G(1) phase, whereas coexpression of p53 and the protooncogene c-myc induces apoptosis. Although p53 transcriptional activity generally is required for both pathways, the molecular components mediating p53-dependent apoptosis are not well understood. To identify factors that could mediate p53-induced cell death, we used a comparative RNA differential display procedure. We have identified Pw1/Peg3 as a gene product induced during p53/c-myc-mediated apoptosis. Pw1/Peg3 is not induced during p53-mediated G(1) growth arrest nor by c-myc alone. Although it is not clear whether the induction of Pw1/Peg3 depends on p53 activity, we show that Pw1/Peg3 interacts with a p53-inducible gene product Siah1a. We demonstrate that coexpression of Pw1/Peg3 with Siah1a induces apoptosis independently of p53 whereas expression of Pw1/Peg3 or Siah1a separately has no effect on cell death. These data suggest that Siah1a and Pw1/Peg3 cooperate in the p53-mediated cell death pathway. Furthermore, we show that inhibiting Pw1/Peg3 activity blocks p53-induced apoptosis. The observation that Pw1/Peg3 is necessary for the p53 apoptotic response suggests a pivotal role for this gene in determining cell death versus survival.

Peg3/Pw1 is an imprinted gene involved in the TNF-NFkappaB signal transduction pathway.

Tumor necrosis factor (TNF) mediates a variety of biological activities including cell proliferation, differentiation and programmed cell death. The specific response to TNF depends upon cell type and reflects the presence of specific regulatory proteins that participate in the TNF response pathway. TNF signal transduction is mediated by TRAF2 which binds the TNF Receptor2 (TNFR2) and activates NFkappaB. We previously identified a gene Pw1, which encodes a large zinc-finger containing protein. We have determined that Pw1 is identical to Peg3, a paternally expressed gene of unknown function (and will therefore be referred to as Peg3 throughout this text). We report here that Peg3 associates specifically with TRAF2 but not with other TRAF family members. Peg3 expression activates NFkappaB via IkappaB-NFkappaB dissociation and acts synergistically with TRAF2. Transfection of a truncated Peg3 containing the TRAF2 interaction site, abolishes NFkappaB activation by TRAF2 and/or TNF. We conclude that Peg3 is a regulator of the TNF response. These data reveal the involvement of an imprinted gene in this pathway.

Transcription of XLPOU3, a brain-specific gene, during Xenopus laevis early embryogenesis.

XLPOU3 is a member of the Xenopus POU domain family of genes, encoding a class of homeodomain-containing transcription factors implicated in cell differentiation during development. Here we describe the isolation and characterisation of XLPOU3b, an allelic genomic counterpart of the XLPOU3 cDNA previously described (Baltzinger et al. (1992) Nucleic Acids Res. 20, 1993), and the spatio-temporal transcription patterns of this gene during development, as determined by Northern blotting and whole-mount in situ hybridisation. Like other genes encoding POU domains of class III, XLPOU3b is intronless. XLPOU3 and XLPOU3b proteins share 82% amino acid identity with the mammalian N-Oct-3/Brn-2 proteins. XLPOU3 mRNA, which is first detected at the neurula stage, is expressed in the developing brain and spinal cord. In addition, XLPOU3 transcription is observed in a restricted region of the auditory vesicle during development. The results suggest that XLPOU3 may participate in patterning the central nervous system during early Xenopus development.

Pw1, a novel zinc finger gene implicated in the myogenic and neuronal lineages.

The cellular and molecular processes leading to the establishment of the skeletal muscle lineage in the vertebrate are not well understood. The MyoD-related family of myogenic regulatory factors (MRFs) are expressed during somitogenesis although cells with myogenic capacity are present prior to gastrulation. We propose that regulatory genes exist that guide the skeletal muscle lineage during early development. In an effort to identify these regulatory genes, we performed a differential screening to isolate transcripts that are present in myogenic cells and in the embryo prior to MRF expression but absent in nonmyogenic fibroblasts. We report here the identification of Pw1. The Pw1 transcript is approximately 8.5 kb long and encodes a large protein containing 12 widespread C2H2 zinc fingers and 3 motifs containing periodic prolines and acidic residues. Consistent with the possibility that Pw1 is a transcription factor, we observe nuclear localization of the protein. Pw1 is strongly expressed upon gastrulation and subsequently becomes restricted to skeletal muscle and subregions of the central nervous system. Pw1 is initially expressed in all mesodermal cells early in development; however, its maintained expression in adult differentiated muscle suggests a specific role in the skeletal muscle lineage. Pw1 expression is cell cycle specific with levels highest during late M-phase. The gene is intronless which may facilitate transcription during cell division. At present, the precise function of Pw1 is not understood; however, we note that Pw1 maps to the proximal region of chromosome 7 near the axial segmentation mutant pudgy which shows severe perturbation of axial skeletal and muscle structures.