Myogenic BHLH Transcription Factors: Their Overlapping Functions and Direct Regulation of MEF2C Provide a Regulatory Network for the Maintenance and Amplification of Vertebrate Myogenesis

Abstract

The myogenic basic helix-loop-helix (bHLH) genes - Myf5, MyoD, myogenin and MRF4 - exhibit distinct, but overlapping expression patterns during vertebrate myogenesis. Loss-of-function mutations in these genes have defined an in vivo model for myogenesis in which MyoD and Myf5 have redundant functions in myoblast specification, whereas myogenin acts to control myoblast differentiation. A role for MRF4 in differentiation has been suggested by various studies, but not defined. Through the analysis of MyoD-/-MRF4-/- and myogenin-/-MRF4-/- mutants, we show that MRF4 plays a role in differentiation which it shares with MyoD, but not myogenin, thereby defining a novel myogenin-independent differentiation pathway. The functional redundancy of the myogenic bHLH factors demonstrated in these and other studies led us to investigate the ability of a single factor to direct the myogenic program in the absence of the other myogenic bHLH proteins. Analysis of myogenin-/-MyoD-/-MRF4-/- mutant animals showed that alone, Myf 5 was unable to bring about differentiation, although specification of myoblasts was not affected. These results suggest that these myogenic factors possess specialized functions. However, the remarkably low level of Myf5 available in triple mutant neonatal muscle leaves open the possibility that it is the total level of myogenic bHLH transcription factors that is critical to the completion of muscle differentiation. The auto- and cross-regulation that the myogenic bHLH factors provide for one another, combined with their functional redundancy, comprises a mechanism whereby myogenesis is induced and maintained. Members of the MEF2 family of transcription factors cooperate with the myogenic bHLH factors to control the expression of muscle specific genes, thereby contributing to the maintenance and amplification of muscle development. To determine the mechanisms that regulate the expression of MEF2C, the earliest of the MEF2 factors expressed in the myogenic lineage, the mouse MEF2C gene was analyzed for cis-regulatory elements that direct its expression in the skeletal muscle lineage in vivo. As described herein, such a control region was identified, characterized and shown to be a direct transcriptional target of myogenic bHLH and MEF2 proteins. These results further define the regulatory circuit that induces, amplifies and maintains myogenesis in vivo.