Pitx2 defines alternate pathways acting through MyoD during limb and somitic myogenesis.

The MyoD gene is part of the core regulatory network that governs skeletal myogenesis and acts as an essential determinant of the myogenic cell fate. Although generic regulatory networks converging on this gene have been described, the specific mechanisms leading to MyoD expression in muscles of different ontology remain misunderstood. We now show that the homeobox gene Pitx2 is required for initial activation of the MyoD gene in limb muscle precursors through direct binding of Pitx2 to the MyoD core enhancer. Whereas Myf5 and Mrf4 are dispensable for limb muscle progenitor fate, inactivation of Myf5 and Mrf4 in Pitx2 mutants results in a drastic decrease of limb MyoD expression. Thus, Pitx2 and Myf5 define parallel genetic pathways for limb myogenesis. We show a similar dependence on Pitx2 and Myf5(Mrf4) in myotome, where MyoD expression is initially activated by Myf5 and Mrf4. In their absence, MyoD expression is eventually rescued by a Pax3-dependent mechanism. We now provide evidence that Pitx2 contributes to the rescue of MyoD expression and that it acts downstream of Pax3. We thus propose that myogenic differentiation of somite-derived muscle cells relies on two parallel genetic pathways, with the Pitx2 pathway being of primary importance for limb myogenesis but the Myf5 and Mrf4 pathway predominating in myotome. Muscle-specific wiring of regulatory networks composed of similar transcription factors thus underlies development of distinct skeletal muscles.