Prednisolone rescues Duchenne muscular dystrophy phenotypes in human pluripotent stem cell-derived skeletal muscle in vitro.

Duchenne muscular dystrophy (DMD) is a devastating genetic disease leading to degeneration of skeletal muscles and premature death. How dystrophin absence leads to muscle wasting remains unclear. Here, we describe an optimized protocol to differentiate human induced pluripotent stem cells (iPSC) to a late myogenic stage. This allows us to recapitulate classical DMD phenotypes (mislocalization of proteins of the dystrophin-associated glycoprotein complex, increased fusion, myofiber branching, force contraction defects, and calcium hyperactivation) in isogenic DMD-mutant iPSC lines in vitro. Treatment of the myogenic cultures with prednisolone (the standard of care for DMD) can dramatically rescue force contraction, fusion, and branching defects in DMD iPSC lines. This argues that prednisolone acts directly on myofibers, challenging the largely prevalent view that its beneficial effects are caused by antiinflammatory properties. Our work introduces a human in vitro model to study the onset of DMD pathology and test novel therapeutic approaches.

TGFß signalling acts as a molecular brake of myoblast fusion.

Fusion of nascent myoblasts to pre-existing myofibres is critical for skeletal muscle growth and repair. The vast majority of molecules known to regulate myoblast fusion are necessary in this process. Here, we uncover, through high-throughput in vitro assays and in vivo studies in the chicken embryo, that TGFß (SMAD2/3-dependent) signalling acts specifically and uniquely as a molecular brake on muscle fusion. While constitutive activation of the pathway arrests fusion, its inhibition leads to a striking over-fusion phenotype. This dynamic control of TGFß signalling in the embryonic muscle relies on a receptor complementation mechanism, prompted by the merging of myoblasts with myofibres, each carrying one component of the heterodimer receptor complex. The competence of myofibres to fuse is likely restored through endocytic degradation of activated receptors. Altogether, this study shows that muscle fusion relies on TGFß signalling to regulate its pace.

Phosphorylation of Lbx1 controls lateral myoblast migration into the limb.

The migration of limb myogenic precursors from limb level somites to their ultimate site of differentiation in the limb is a paradigmatic example of a set of dynamic and orchestrated migratory cell behaviours. The homeobox containing transcription factor ladybird homeobox 1 (Lbx1) is a central regulator of limb myoblast migration, null mutations of Lbx1 result in severe disruptions to limb muscle formation, particularly in the distal region of the limb in mice (Gross et al., 2000). As such Lbx1 has been hypothesized to control lateral migration of myoblasts into the distal limb anlage. It acts as a core regulator of the limb myoblast migration machinery, controlled by Pax3. A secondary role for Lbx1 in the differentiation and commitment of limb musculature has also been proposed (Brohmann et al., 2000; Uchiyama et al., 2000). Here we show that lateral migration, but not differentiation or commitment of limb myoblasts, is controlled by the phosphorylation of three adjacent serine residues of LBX1. Electroporation of limb level somites in the chick embryo with a dephosphomimetic form of Lbx1 results in a specific defect in the lateral migration of limb myoblasts. Although the initial delamination and migration of myoblasts is unaffected, migration into the distal limb bud is severely disrupted. Interestingly, myoblasts undergo normal differentiation independent of their migratory status, suggesting that the differentiation potential of hypaxial muscle is not regulated by the phosphorylation state of LBX1. Furthermore, we show that FGF8 and ERK mediated signal transduction, both critical regulators of the developing limb bud, have the capacity to induce the phosphorylation of LBX1 at these residues. Overall, this suggests a mechanism whereby the phosphorylation of LBX1, potentially through FGF8 and ERK signalling, controls the lateral migration of myoblasts into the distal limb bud.

The chicken embryo as an efficient model to test the function of muscle fusion genes in amniotes.

The fusion of myoblasts into multinucleated myotubes is a crucial step of muscle growth during development and of muscle repair in the adult. While multiple genes were shown to play a role in this process, a vertebrate model where novel candidates can be tested and analyzed at high throughput and relative ease has been lacking. Here, we show that the early chicken embryo is a fast and robust model in which functional testing of muscle fusion candidate genes can be performed. We have used known modulators of muscle fusion, Rac1 and Cdc42, along with the in vivo electroporation of integrated, inducible vectors, to show that the chicken embryo is a suitable model in which their function can be tested and quantified. In addition to nuclei content, specific characteristics of the experimental model allow a fine characterization of additional morphological features that are nearly impossible to assess in other model organisms. This study should establish the chicken embryo as a cheap, reliable and powerful model in which novel vertebrate muscle fusion candidates can be evaluated.

Cytoplasmic NOTCH and membrane-derived ß-catenin link cell fate choice to epithelial-mesenchymal transition during myogenesis.

How cells in the embryo coordinate epithelial plasticity with cell fate decision in a fast changing cellular environment is largely unknown. In chick embryos, skeletal muscle formation is initiated by migrating Delta1-expressing neural crest cells that trigger NOTCH signaling and myogenesis in selected epithelial somite progenitor cells, which rapidly translocate into the nascent muscle to differentiate. Here, we uncovered at the heart of this response a signaling module encompassing NOTCH, GSK-3ß, SNAI1 and ß-catenin. Independent of its transcriptional function, NOTCH profoundly inhibits GSK-3ß activity. As a result SNAI1 is stabilized, triggering an epithelial to mesenchymal transition. This allows the recruitment of ß-catenin from the membrane, which acts as a transcriptional co-factor to activate myogenesis, independently of WNT ligand. Our results intimately associate the initiation of myogenesis to a change in cell adhesion and may reveal a general principle for coupling cell fate changes to EMT in many developmental and pathological processes.