The transition from G0 to GAlert is regulated by GLI3 processing by the primary cilia

Satellite stem cells are required for the growth, maintenance, and regeneration of skeletal muscle. Quiescent satellite stem cells possess a primary cilium, a structure that regulates the processing of the GLI-family of transcription factors. We find that GLI3, specifically, plays a critical role in satellite cell activation. Strikingly, primary cilia-mediated processing of GLI3, independent of canonical Hedgehog signaling, is required to maintain satellite cells in a G0 dormant state, as satellite cells lacking GLI3 enter GAlert in absence of injury. Furthermore, GLI3 depletion or inhibition of its processing stimulates symmetrical division in satellite cells and expansion of the stem cell pool. As a result, satellite cells lacking GLI3 display rapid cell-cycle entry, increased proliferation and augmented self-renewal, and markedly enhanced long-term regenerative capacity. Therefore, our results reveal an essential role for primary cilia processing of GLI3 in regulating muscle stem cell activation. Muscle stem cells were isolated from hindlimb muscles of 3-4 month old Pax7-CreERT2/+;Gli3+/+;R26R-YFP (control) and Pax7-CreERT2/+;Gli3fl/fl;R26R-YFP (Gli3-cKO) mice. Quiescent muscle stem cells (alpha7-Integrin+, CD34+ sorting) were obtained from uninjured hindlimb muscles, while activated muscle stem cells (alpha7-Integrin+, VCAM1+ sorting) were obtained from cardiotoxin-injured muscles 3 days after the induced-injury.