OVERLAPPING FUNCTIONS OF THE MYOGENIC BHLH GENES MRF4 AND MYOD REVEALED IN DOUBLE MUTANT MICE

The myogenic basic helix-loop-helix (bHLH) genes - MyoD, Myf5, myogeni n and MRF4 - exhibit distinct, but overlapping expression patterns dur ing development of the skeletal muscle lineage and loss-of-function mu tations in these genes result in different effects on muscle developme nt. MyoD and Myf5 have been shown to act early in the myogenic lineage to establish myoblast identity, whereas myogenin acts later to contro l myoblast differentiation. In mice lacking myogenin, there is a sever e deficiency of skeletal muscle, but some residual muscle fibers are p resent in mutant mice at birth. Mice lacking MRF4 are viable and have skeletal muscle, but they upregulate myogenin expression, which could potentially compensate for the absence of MRF4. Previous studies in wh ich Myf5 and MRF4 null mutations were combined suggested that these ge nes do not share overlapping myogenic functions in vivo. To determine whether the functions of MRF4 might overlap with those of myogenin or MyoD, we generated double mutant mice lacking MRF4 and either myogenin or MyoD. MRF4/myogenin double mutant mice contained a comparable numb er of residual muscle fibers to mice lacking myogenin alone and myobla sts from those double mutant mice formed differentiated multinucleated myotubes in vitro as efficiently as wild-type myoblasts, indicating t hat neither myogenin nor MRF4 is absolutely essential for myoblast dif ferentiation. Whereas mice lacking either MRF4 or MyoD were viable and did not show defects in muscle development, MRF4/MyoD double mutants displayed a severe muscle deficiency similar to that in myogenin mutan ts. Myogenin was expressed in MRF4/MyoD double mutants, indicating tha t myogenin is insufficient to support normal myogenesis in vivo. These results reveal unanticipated compensatory roles for MRF4 and MyoD in the muscle differentiation pathway and suggest that a threshold level of myogenic bHLH factors is required to activate muscle structural gen es, with this level normally being achieved by combinations of multipl e myogenic bHLH factors.