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In mammals, several genetic pathways have been characterized that govern engagement of multipotent embryonic progenitors into the myogenic program through the control of the key myogenic regulatory gene  Myod . Here we demonstrate the involvement of Six homeoproteins. We first targeted into a  Pax3  allele a sequence encoding a negative form of Six4 that binds DNA but cannot interact with essential Eya co-factors. The resulting embryos present hypoplasic skeletal muscles and impaired  Myod  activation in the trunk in the absence of  Myf5/Mrf4 . At the axial level, we further show that  Myod  is still expressed in compound  Six1/Six4:Pax3  but not in  Six1/Six4:Myf5  triple mutant embryos, demonstrating that  Six1/4  participates in the  Pax3-Myod  genetic pathway.  Myod  expression and head myogenesis is preserved in  Six1/Six4:Myf5  triple mutant embryos, illustrating that upstream regulators of  Myod  in different embryonic territories are distinct. We show that  Myod  regulatory regions are directly controlled by Six proteins and that, in the absence of Six1 and Six4, Six2 can compensate.

Six Homeoproteins Directly Activate Myod Expression in the Gene Regulatory Networks That Control Early Myogenesis