Evolutionarily conserved regulation of embryonic fast-twitch skeletal muscle differentiation by Pbx factors

Vertebrate skeletal muscles are composed of both slow-twitch and fast-twitch fiber types. How the differentiation of distinct fiber types is activated during embryogenesis is not well characterized. Skeletal muscle differentiation is initiated by the activity of the myogenic basic helix-loop-helix (bHLH) transcription factors Myf5, Myod1, Myf6, and Myog. Myod1 functions as a muscle master regulatory factor and directly activates muscle differentiation genes, including those specific to both slow and fast muscle fibers. Our previous studies showed that Pbx TALE-class homeodomain proteins bind with Myod1 on the promoter of the zebrafish fast muscle gene mylpfa and are required for proper activation of mylpfa expression and the fast-twitch muscle-specific differentiation program in zebrafish embryos. Pbx proteins have also been shown to bind regulatory regions of muscle differentiation genes in mammalian muscle cells in culture. Here, we use new zebrafish mutant strains to confirm the essential roles of zebrafish Pbx factors in embryonic fast muscle differentiation. Furthermore, we examine the requirements for Pbx genes in mouse embryonic skeletal muscle differentiation, an area that has not been investigated in the mammalian embryo. Removing Pbx1 function from skeletal muscle in Myf5Cre/+;Pbx1fl/fl mouse embryos has minor effects on embryonic muscle development. However, concomitantly deleting Pbx2 function in Myf5Cre/+;Pbx1fl/fl;Pbx2-/- mouse embryos causes delayed activation and reduced expression of fast muscle differentiation genes. In the mouse, Pbx1/Pbx2-dependent fast muscle genes closely match those that have been previously shown to be dependent on murine Six1 and Six4. This work establishes evolutionarily conserved requirements for Pbx factors in embryonic fast muscle differentiation. Our studies are revealing how Pbx homeodomain proteins help direct specific cellular differentiation pathways.