Pax7 reporter mouse models: a pocket guide for satellite cell research.

Since their discovery, satellite cells have showcased their need as primary contributors to skeletal muscle maintenance and repair. Satellite cells lay dormant, but when needed, activate, differentiate, fuse to fibres and self-renew, that has bestowed satellite cells with the title of muscle stem cells. The satellite cell specific transcription factor Pax7 has enabled researchers to develop animal models against the Pax7 locus in order to isolate and characterise satellite cell-mediated events. This review focuses specifically on describing Pax7 reporter mouse models. Here we describe how each model was generated and the key findings obtained. The strengths and limitations of each model are also discussed. The aim is to provide new and current satellite cell enthusiasts with a basic understanding of the available Pax7 reporter mice and hopefully guide selection of the most appropriate Pax7 model to answer a specific research question.

Antagonism Between DUX4 and DUX4c Highlights a Pathomechanism Operating Through ß-Catenin in Facioscapulohumeral Muscular Dystrophy.

Aberrant expression of the transcription factor DUX4 from D4Z4 macrosatellite repeats on chromosome 4q35, and its transcriptome, associate with pathogenesis in facioscapulohumeral muscular dystrophy (FSHD). Forced DUX4 expression halts skeletal muscle cell proliferation and induces cell death. DUX4 binds DNA via two homeodomains that are identical in sequence to those of DUX4c (DUX4L9): a closely related transcriptional regulator encoded by a single, inverted, mutated D4Z4 unit located centromeric to the D4Z4 macrosatellite array on chromosome 4. However, the function and contribution of DUX4c to FSHD pathogenesis are unclear. To explore interplay between DUX4, DUX4c, and the DUX4-induced phenotype, we investigated whether DUX4c interferes with DUX4 function in human myogenesis. Constitutive expression of DUX4c rescued the DUX4-induced inhibition of proliferation and reduced cell death in human myoblasts. Functionally, DUX4 promotes nuclear translocation of ß-CATENIN and increases canonical WNT signalling. Concomitant constitutive expression of DUX4c prevents ß-CATENIN nuclear accumulation and the downstream transcriptional program. DUX4 reduces endogenous DUX4c levels, whereas constitutive expression of DUX4c robustly suppresses expression of DUX4 target genes, suggesting molecular antagonism. In line, DUX4 expression in FSHD myoblasts correlates with reduced DUX4c levels. Addressing the mechanism, we identified a subset of genes involved in the WNT/ß-CATENIN pathway that are differentially regulated between DUX4 and DUX4c, whose expression pattern can separate muscle biopsies from severely affected FSHD patients from healthy. Finally, blockade of WNT/ß-CATENIN signalling rescues viability of FSHD myoblasts. Together, our study highlights an antagonistic interplay whereby DUX4 alters cell viability via ß-CATENIN signalling and DUX4c counteracts aspects of DUX4-mediated toxicity in human muscle cells, potentially acting as a gene modifier for FSHD severity. Importantly, direct DUX4 regulation of the WNT/ß-CATENIN pathway informs future therapeutic interventions to ameliorate FSHD pathology.

Fine-Tuning of Piezo1 Expression and Activity Ensures Efficient Myoblast Fusion during Skeletal Myogenesis.

Mechanical stimuli, such as stretch and resistance training, are essential in regulating the growth and functioning of skeletal muscles. However, the molecular mechanisms involved in sensing mechanical stress during muscle formation remain unclear. Here, we investigated the role of the mechanosensitive ion channel Piezo1 during myogenic progression of both fast and slow muscle satellite cells. We found that Piezo1 level increases during myogenic differentiation and direct manipulation of Piezo1 in muscle stem cells alters the myogenic progression. Indeed, Piezo1 knockdown suppresses myoblast fusion, leading to smaller myotubes. Such an event is accompanied by significant downregulation of the fusogenic protein Myomaker. In parallel, while Piezo1 knockdown also lowers Ca2+ influx in response to stretch, Piezo1 activation increases Ca2+ influx in response to stretch and enhances myoblasts fusion. These findings may help understand molecular defects present in some muscle diseases. Our study shows that Piezo1 is essential for terminal muscle differentiation acting on myoblast fusion, suggesting that Piezo1 deregulation may have implications in muscle aging and degenerative diseases, including muscular dystrophies and neuromuscular disorders.

MBNL1-Associated Mitochondrial Dysfunction and Apoptosis in C2C12 Myotubes and Mouse Skeletal Muscle.

We explored the interrelationship between a tissue-specific alternative splicing factor muscleblind-like 1 (MBNL1) and peroxisome proliferator-activated receptor-γ coactivator 1-α (PGC-1α), B-cell lymphoma 2 (Bcl-2) or Bcl-2-associated X protein (Bax) in C2C12 myotubes and mouse skeletal muscle to investigate a possible physiological role of MBNL1 in mitochondrial-associated apoptosis of skeletal muscle. Expression level of PGC-1α and mitochondrial membrane potential evaluated by the fluorescence ratio of JC-1 aggregate to monomer in C2C12 myotubes were suppressed by knockdown of MBNL1. Conversely, the ratio of Bax to Bcl-2 as well as the apoptotic index in C2C12 myotubes was increased by MBNL1 knockdown. In plantaris muscle, on the other hand, not only the minimum muscle fiber diameter but also the expression level of MBNL1 and PGC-1α in of 100-week-old mice were significantly lower than that of 10-week-old mice. Furthermore, the ratio of Bax to Bcl-2 in mouse plantaris muscle was increased by aging. These results suggest that MBNL1 may play a key role in aging-associated muscle atrophy accompanied with mitochondrial dysfunction and apoptosis via mediating PGC-1α expression in skeletal muscle.

DEPDC1B is a key regulator of myoblast proliferation in mouse and man.

OBJECTIVES: DISHEVELLED, EGL-10, PLECKSTRIN (DEP) domain-containing 1B (DEPDC1B) promotes dismantling of focal adhesions and coordinates detachment events during cell cycle progression. DEPDC1B is overexpressed in several cancers with expression inversely correlated with patient survival. Here, we analysed the role of DEPDC1B in the regulation of murine and human skeletal myogenesis. MATERIALS AND METHODS: Expression dynamics of DEPDC1B were examined in murine and human myoblasts and rhabdomyosarcoma cells in vitro by RT-qPCR and/or immunolabelling. DEPDC1B function was mainly tested via siRNA-mediated gene knockdown. RESULTS: DEPDC1B was expressed in proliferating murine and human myoblasts, with expression then decreasing markedly during myogenic differentiation. SiRNA-mediated knockdown of DEPDC1B reduced myoblast proliferation and induced entry into myogenic differentiation, with deregulation of key cell cycle regulators (cyclins, CDK, CDKi). DEPDC1B and ß-catenin co-knockdown was unable to rescue proliferation in myoblasts, suggesting that DEPDC1B functions independently of canonical WNT signalling during myogenesis. DEPDC1B can also suppress RHOA activity in some cell types, but DEPDC1B and RHOA co-knockdown actually had an additive effect by both further reducing proliferation and enhancing myogenic differentiation. DEPDC1B was expressed in human Rh30 rhabdomyosarcoma cells, where DEPDC1B or RHOA knockdown promoted myogenic differentiation, but without influencing proliferation. CONCLUSION: DEPDC1B plays a central role in myoblasts by driving proliferation and preventing precocious myogenic differentiation during skeletal myogenesis in both mouse and human.

Myogenin promotes myocyte fusion to balance fibre number and size.

Each skeletal muscle acquires its unique size before birth, when terminally differentiating myocytes fuse to form a defined number of multinucleated myofibres. Although mice in which the transcription factor Myogenin is mutated lack most myogenesis and die perinatally, a specific cell biological role for Myogenin has remained elusive. Here we report that loss of function of zebrafish myog prevents formation of almost all multinucleated muscle fibres. A second, Myogenin-independent, fusion pathway in the deep myotome requires Hedgehog signalling. Lack of Myogenin does not prevent terminal differentiation; the smaller myotome has a normal number of myocytes forming more mononuclear, thin, albeit functional, fast muscle fibres. Mechanistically, Myogenin binds to the myomaker promoter and is required for expression of myomaker and other genes essential for myocyte fusion. Adult myog mutants display reduced muscle mass, decreased fibre size and nucleation. Adult-derived myog mutant myocytes show persistent defective fusion ex vivo. Myogenin is therefore essential for muscle homeostasis, regulating myocyte fusion to determine both muscle fibre number and size.