Interplay between Pitx2 and Pax7 temporally governs specification of extraocular muscle progenitors

Gene regulatory networks that act upstream of skeletal muscle fate determinants are distinct in different anatomical locations. Despite recent efforts, a clear understanding of the cascade of events underlying the emergence and maintenance of the stem cell pool in specific muscle groups remains unresolved and debated. Here, we invalidated Pitx2 with multiple Cre-driver mice prenatally, postnatally, and during lineage progression and showed that this gene becomes progressively dispensable for specification and maintenance of the extraocular muscle (EOM) stem cell pool, yet it is the major EOM upstream regulator during early development. Moreover, constitutive inactivation of Pax7 postnatally showed a greater loss of muscle stem cells in the EOM compared to the limb, pointing to a relay between Pitx2, Myf5 and Pax7 for maintenance of the EOM stem cells. Further, we demonstrate that EOM stem cells adopt a quiescent state earlier that those in limb muscles and do not spontaneously re-enter in proliferation in the adult as previously suggested, yet EOMs have a significantly higher content of Pax7+ muscle stem cells per area pre- and post-natally. This unique feature could result from different dynamics of lineage progression in vivo, given the lower fraction of committed and differentiating EOM myoblasts. Finally, significantly less MuSCs are present in EOM compared to the limb in the mdx mouse model for Duchenne muscular dystrophy. Overall, our study provides a comprehensive in vivo characterization of muscle stem cell heterogeneity along the body axis and brings further insights into the unusual sparing of EOM during muscular dystrophy.