Gli1 marks a sentinel muscle stem cell population for muscle regeneration.

Adult skeletal muscle regeneration is mainly driven by muscle stem cells (MuSCs), which are highly heterogeneous. Although recent studies have started to characterize the heterogeneity of MuSCs, whether a subset of cells with distinct exists within MuSCs remains unanswered. Here, we find that a population of MuSCs, marked by Gli1 expression, is required for muscle regeneration. The Gli1 + MuSC population displays advantages in proliferation and differentiation both in vitro and in vivo. Depletion of this population leads to delayed muscle regeneration, while transplanted Gli1 + MuSCs support muscle regeneration more effectively than Gli1- MuSCs. Further analysis reveals that even in the uninjured muscle, Gli1 + MuSCs have elevated mTOR signaling activity, increased cell size and mitochondrial numbers compared to Gli1 - MuSCs, indicating Gli1 + MuSCs are displaying the features of primed MuSCs. Moreover, Gli1 + MuSCs greatly contribute to the formation of G Alert cells after muscle injury. Collectively, our findings demonstrate that Gli1 + MuSCs represents a distinct MuSC population which is more active in the homeostatic muscle and enters the cell cycle shortly after injury. This population functions as the tissue-resident sentinel that rapidly responds to injury and initiates muscle regeneration.