Functional analysis of MRF4 during mouse embryogenesis

MRF4 is one of four skeletal muscle specific regulatory genes, (the other three genes being MyoD, myf5, and myogenin), each of which has the unique ability to orchestrate an entire program of muscle-specific transcription when introduced into diverse cell types. These findings have led to the notion that these factors function as master regulators of muscle cell fate. Analysis of mice lacking MyoD, myf5, and myogenin have further defined their roles in the commitment and differentiation of myotomal progenitor cells. Current data strongly supports the model that MyoD and myf5 share functional redundancy in determining the muscle cell lineage, while myogenin acts downstream of MyoD and myf5, to initiate myoblast differentiation. Unlike other myogenic bHLH genes, MRF4 is expressed predominantly in the adult, suggesting that it may function to regulate adult muscle maturation and maintenance. To test this hypothesis and to eventually incorporate MRF4 into a general model for muscle specification, differentiation, maturation and maintenance, I deleted the MRF4 gene. MRF4-null mice are viable and fertile, however, they show mild rib anomalies. In addition, the expression of myogenin is dramatically upregulated only in the adult, suggesting that myogenin may compensate for the loss of MRF4 in the adult, and MRF4 may normally suppress the expression of myogenin after birth. MRF4 is also required during muscle regeneration after injury. To determine the degree of genetic redundancy between MRF4-myogenin; and MRF4-MyoD, I crossed the MRF4-null mice with MyoD- and myogenin-null mice respectively. There are no additional muscle phenotypes in double-null progeny from a MRF4 and myogenin cross, suggesting that the existence of residual fibers in myogenin-null mice is not due to the presence of MRF4. MRF4 expression also cannot account for the ability of myogenin-null myoblasts to differentiate in vitro. However, the combination of the MRF4-null mutation with the myogenin-null mutation results in a novel rib phenotype. This result suggests that MRF4 modifies the myogenin-null rib phenotype, and MRF4 and myogenin play redundant roles in rib development. MRF4 also shares dosage effects with MyoD during mouse development. (MyoD+/$-$;MRF4$-$/$-$)mice are fertile and viable, while (MyoD$-$/$-$;MRF4+/$-$) mice die between birth and two weeks after birth, and have a small skeletal structure. The double homozygous mice for MRF4 and MyoD mutations are embryonic lethal and die at around E10.5. These results suggest that MRF4 and MyoD share overlapping functions during mouse embryogenesis