Regulation of Muscle Stem Cells by Netrin Signaling

Skeletal muscle regenerates following injury, with muscle stem cells (MuSCs) the source of new myofibers. MuSCs are quiescent during homeostasis. Quiescence is an actively maintained state, supported by signals from the MuSC niche. Upon muscle injury, MuSCs activate and enter the cell cycle, proliferate as myoblasts,

and differentiate and fuse to form new myofibers. How MuSCs maintain quiescence is not well understood, and there is little known about the earliest events in the transition from quiescence to activation (the Q-A transition). Quiescent MuSCs in vivo have long, heterogeneous cellular projections that rapidly retract in response to

muscle injury. Projections may therefore act as direct sensors of the niche environment. Projection retraction is driven by a Rac-to-Rho GTPase activity switch that promotes downstream MuSC activation events. These observations lead to several hypotheses: 1) MuSC projections are morphologically dynamic at quiescence,

providing a surveillance function for muscle damage; 2) MuSC dynamics during quiescence are regulated by the relative balance of Rac and Rho activities promoted by niche-derived cues; and 3) there exist factors in muscle tissue that signal to stimulate MuSC projection outgrowth and, consequently, promote MuSC

quiescence. Such factors are anticipated to be critical regulators of MuSC quiescence and the Q-A transition, but their identities are unknown. Moreover, new approaches are required to screen for candidate factors. We have developed an ex vivo live imaging assay for MuSCs within muscle bundles and used it to identify the

axonal chemoattractant, netrin-1, as a candidate niche-derived regulator of MuSC projection dynamics. Multiple cell types in adult muscle express netrin-1, and MuSCs express the netrin-1 receptors, Neogenin (Neo1) and Dcc. Furthermore, MuSCs extend projections in response to recombinant netrin-1, as visualized in

the ex vivo live imaging assay. It is proposed here to determine the role of netrin-1 signaling in MuSC quiescence and the Q-A transition. We will use conditional genetic removal of netrin-1 receptors from MuSCs to test directly the role of netrin-1 signaling in MuSC morphology, quiescence, and the Q-A transition. A

combination of in vivo and ex vivo techniques will be used, including tissue clearing, in vivo analyses of uninjured and injured muscles, analysis of MuSCs on single myofibers; and live imaging of MuSCs within muscle bundles. Successful completion of the proposed work will provide strong evidence that: 1) regulation of

MuSC projection dynamics is a key process in maintenance of quiescence; and 2) netrin-1 is the first member of a new class of MuSC quiescence regulator. Such findings will validate the use of our new ex vivo live imaging assay as a means to identify secreted signaling cues involved in this process.