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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 <jats:italic>Pitx2<\/jats:italic> with multiple <jats:italic>Cre<\/jats:italic>-driver mice prenatally, postnatally, and during lineage progression. We showed that this gene becomes progressively dispensable for specification and maintenance of the muscle stem (MuSC) cell pool in extraocular muscles (EOMs) despite being, together with Myf5, a major upstream regulator during early development. Moreover, constitutive inactivation of <jats:italic>Pax7<\/jats:italic> postnatally led to a greater loss of MuSCs in the EOMs compared to the limb. Thus, we propose a relay between <jats:italic>Pitx2<\/jats:italic>, <jats:italic>Myf5<\/jats:italic> and <jats:italic>Pax7<\/jats:italic> for EOM stem cell maintenance. We demonstrate also that MuSCs in the EOMs adopt a quiescent state earlier that those in limb muscles and do not spontaneously proliferate in the adult, yet EOMs have a significantly higher content of Pax7+ MuSCs per area pre- and post-natally. Finally, while limb MuSCs proliferate in the <jats:italic>mdx<\/jats:italic> mouse model for Duchenne muscular dystrophy, significantly less MuSCs were present in the EOMs of the <jats:italic>mdx<\/jats:italic> mouse model compared to controls, and they were not proliferative. Overall, our study provides a comprehensive <jats:italic>in vivo<\/jats:italic> characterisation of MuSC heterogeneity along the body axis and brings further insights into the unusual sparing of EOMs during muscular dystrophy.<\/jats:p>","DOI":"10.1371\/journal.pgen.1010935","type":"journal-article","created":{"date-parts":[[2024,6,14]],"date-time":"2024-06-14T17:37:44Z","timestamp":1718386664000},"page":"e1010935","update-policy":"https:\/\/doi.org\/10.1371\/journal.pgen.corrections_policy","source":"Crossref","is-referenced-by-count":11,"title":["Interplay between Pitx2 and Pax7 temporally governs specification of extraocular muscle stem cells"],"prefix":"10.1371","volume":"20","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-5259-3408","authenticated-orcid":true,"given":"Mao","family":"Kuriki","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2602-3190","authenticated-orcid":true,"given":"Amaury","family":"Korb","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3244-3378","authenticated-orcid":true,"given":"Glenda","family":"Comai","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1809-7202","authenticated-orcid":true,"given":"Shahragim","family":"Tajbakhsh","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2024,6,14]]},"reference":[{"key":"pgen.1010935.ref001","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/B978-0-12-405943-6.00001-4","article-title":"Molecular and cellular regulation of skeletal myogenesis","volume":"110","author":"G Comai","year":"2014","journal-title":"Curr Top Dev Biol"},{"key":"pgen.1010935.ref002","doi-asserted-by":"crossref","first-page":"110","DOI":"10.1016\/j.ceb.2021.06.005","article-title":"Diversity in cranial muscles: Origins and developmental programs","volume":"73","author":"A Grimaldi","year":"2021","journal-title":"Curr Opin Cell Biol"},{"key":"pgen.1010935.ref003","doi-asserted-by":"crossref","DOI":"10.7554\/eLife.70235","article-title":"Identification of bipotent progenitors that give rise to myogenic and connective tissues in mouse.","volume":"11","author":"A Grimaldi","year":"2022","journal-title":"Elife."},{"issue":"5","key":"pgen.1010935.ref004","doi-asserted-by":"crossref","first-page":"1194","DOI":"10.1002\/dvdy.20697","article-title":"The differentiation and morphogenesis of craniofacial muscles","volume":"235","author":"DM Noden","year":"2006","journal-title":"Dev Dyn"},{"key":"pgen.1010935.ref005","doi-asserted-by":"crossref","first-page":"10","DOI":"10.1186\/s40851-016-0046-3","article-title":"Comparative morphology and development of extra-ocular muscles in the lamprey and gnathostomes reveal the ancestral state and developmental patterns of the vertebrate head","volume":"2","author":"DG Suzuki","year":"2016","journal-title":"Zoological Lett"},{"issue":"6\u20137","key":"pgen.1010935.ref006","doi-asserted-by":"crossref","first-page":"e23097","DOI":"10.1002\/dvg.23097","article-title":"Neural crest and the patterning of vertebrate craniofacial muscles","volume":"56","author":"JM Ziermann","year":"2018","journal-title":"Genesis"},{"issue":"6","key":"pgen.1010935.ref007","doi-asserted-by":"crossref","first-page":"451","DOI":"10.1016\/S0039-6257(05)80055-4","article-title":"Extraocular muscles: basic and clinical aspects of structure and function","volume":"39","author":"JD Porter","year":"1995","journal-title":"Surv. 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