Inactivation of the myogenic bHLH gene MRF4 results in up-regulation of myogenin and rib anomalies.

The myogenic basic helix-loop-helix (bHLH) proteins MyoD, myf5, myogenin, and MRF4 can initiate myogenesis when expressed in nonmuscle cells. During embryogenesis, each of the myogenic bHLH genes is expressed in a unique temporospatial pattern within the skeletal muscle lineage, suggesting that they play distinct roles in muscle development. Gene targeting has shown that MyoD and myf5 play partially redundant roles in the genesis of myoblasts, whereas myogenin is required for terminal differentiation. MRF4 is expressed transiently in the somite myotome during embryogenesis and then becomes up-regulated during late fetal development to eventually become the predominant myogenic bHLH factor expressed in adult skeletal muscle. On the basis of its expression pattern, it has been proposed that MRF4 may regulate skeletal muscle maturation and aspects of adult myogenesis. To determine the function of MRF4, we generated mice carrying a homozygous germ-line mutation in the MRF4 gene. These mice showed only a subtle reduction in expression of a subset of muscle-specific genes but showed a dramatic increase in expression of myogenin, suggesting that it may compensate for the absence of MRF4 and demonstrating that MRF4 is required for the down-regulation of myogenin expression that normally occurs in postnatal skeletal muscle. Paradoxically, MRF4-null mice exhibited multiple rib anomalies, including extensive bifurcations, fusions, and supernumerary processes. These results demonstrate an unanticipated regulatory relationship between myogenin and MRF4 and suggest that MRF4 influences rib outgrowth through an indirect mechanism.