Myogenin-dependent nAChR clustering in aneural myotubes

During development of the neuromuscular junction, nerve-derived agrin and the cell substrate laminin stimulate postsynaptic nAChR clustering. This clustering is dependent on activation of the tyrosine kinase, MuSK, which signals receptor clustering via a rapsyn-dependent mechanism. Myogenin is a muscle-specific transcription factor that controls myoblast differentiation and nAChR gene expression. Here, we used RNA interference to investigate if myogenin is also necessary for nAChR clustering. We find that myogenin expression is essential for robust nAChR clustering and cannot be compensated by the muscle regulatory factors MyoD, myf5, and MRF4. In addition, we show that clustering cannot be rescued in myogenin-depleted myotubes by simply overexpressing the essential clustering molecules MuSK, rapsyn, and nAChRs. These data suggest that myogenin controls the expression of molecules crucial to nAChR clustering in addition to its role in regulating nAChR gene expression.