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Abstract

Myogenesis in development as well as during muscle regeneration involves a coordi- nated interplay of various cell and tissue types. Careful orchestration of immune cells, satellite cells, connective tissue and resident cell types is required for effective tissue repair. The muscle interstitium comprises various resident cell types such as intersti- tial fibroblasts, pericytes or fibro/ adipogenic progenitors (FAPs). While FAPs reside quiescent in the muscle interstitium in homeostatic tissue they are activated upon injury and provide a pro-myogenic environment necessary for regeneration. However, due to the lack of markers to particularly identify activated FAPs the clarification of exact underlying cell specific mechanisms is impeded, and a clear cell discrimi- nation challenging. FAPs are proposed as originating from mesenchymal cells, but confirmation remains elusive. Odd skipped related-1 ( Osr1 ) is expressed in muscle connective tissue derived from the lateral plate mesoderm during embryonic and fetal myogenesis in the limbs. Unpublished data demonstrate its crucial functional role for muscle patterning and contribution to muscle interstitial cells at the time of birth. However, postnatal ex- pression of Osr1 was not clear, and functional studies in the adult require a conditional Osr1 allele due to the lethality of a constitutive knock-out of Osr1 at early fetal stage. Here, the generation of two novel tools for investigating Osr1 expression in adult mice was performed. The Osr1 promotor-driven lacZ-reporter mouse model Osr1- MFA enables identification of Osr1+ cells by histological and immunohistochemical methods as well as by fluorescent-activated cell sorting (FACS) technique. The con- ditional line Osr1-flox carries a floxed allele of Osr1 thus allows for a conditional knock-out of Osr1 . Previous reports described a transient muscle interstitial cell population present during the first 3 weeks after birth, comprising myogenic non-satellite cell progenitors and juvenile FAPs. Here, expression of Osr1 in the FAP subpopulation is demonstrated which allows for specific identification of these cells in muscle tissue. Genetic lineage tracing demonstrated that these Osr1+ juvenile FAPs, at least in part, give rise to quiescent FAPs in adult muscle, where Osr1 expression is downregulated. These data contribute to the clarification of the origin of FAPs in the adult. Muscle damage triggers Osr1 expression in FAPs after cell activation, where it remains upregulated during the early phase of muscle regeneration. In a model of muscle injury that promotes fat formation, limited adipocytic contribution of Osr1+ FAPs was observed after lineage tracing. These data are in contrast to previous reports attributing a strong contribution of FACS isolated and engrafted FAPs to ectopic fatty accumulation. This highlights the importance of the tissue environment and the proneness of FAPs to external cues when removed from the tissue context. Moreover, in regenerated muscle the majority of FAPs had expressed Osr1 during the early phase of regeneration. These results provide evidence for Osr1 expression as a specific marker for activated FAPs after muscle injury. A large fraction of TCF4+ fibroblasts in regenerated muscle is derived from TCF4–/ Osr1+ FAPs. This observation elucidates TCF4+ fibroblasts as progenies of FAPs. A conditional knock-out of Osr1 after injury results in significantly reduced pro- inflammatory macrophage abundance and abnormal accumulation of aggregates in myofibers during an early stage of regeneration. These results suggest a functional involvement in the plasmin system. It is hypothesized that Osr1 indirectly regulates the conversion of plasminogen to plasmin, thus mediates extracellular matrix deposi- tion, macrophage recruitment and myofiber degeneration. This hypothesis assigns an essential role to Osr1 expression in FAPs for an effective muscle regeneration after injury.