PROJECT SUMMARY/ABSTRACT Peripheral neurons extend axons that reside in a collagenous extracellular matrix (ECM) called the endoneurium that is produced by endoneurial fibroblast-like cells (EFLCs). Axons are further assembled into fascicles, each surrounded by the perineurium, a cellular structure that provides critical protection from toxins and mechanical stress. The signaling molecule Desert Hedgehog (Dhh) is secreted by Schwann Cells and binds to target cells to activate Gli1, a transcriptional effector of the sonic hedgehog pathway. Dhh is known to be critical for the development of the perineurium but its other roles in PNS development and maintenance are not known. In settings where Dhh expression is reduced or absent, including nerve injury, peripheral nerve fascicles reorganize into many smaller compartments called minifascicles. These structures are thought to be protective in the injury setting and possibly aid in nerve repair ? their precise roles in regeneration and the mechanisms responsible for their development remain unclear. By genetically labeling Gli1-expressing cells, we have identified PG and, unexpectedly, a population of EFLCs, as hedgehog-responsive in the PNS. Knocking out Gli1 leads to a reduction in endoneurial collagen and a phenotypic switch in EFLCs to form minifascicles. Both of these findings were also noted in Dhh knockouts, supporting a model in which Dhh-Gli1 signaling normally inhibits minifascicle formation by EFLCs. The goal of this proposal is to characterize the function of Gli1 in EFLCs in the developing, mature, and injured peripheral nerve. First, we will examine Gli1 knockout nerves for changes in composition of the endoneurial ECM and defects in axon-SC relationships. Second, we will use genetic tools to activate or block hedgehog signaling in this population in the adult PNS and look for changes in EFLC morphology and ECM composition. Third, we address the role of Gli1 signaling in these cells after sciatic nerve injury. We will perform nerve crush in wild-type mice and characterize changes in Dhh and Gli1 at both the transcript and protein levels. We will then compare peripheral nerve regeneration following crush injury in the setting of both Gli1 loss-of function and genetically sustained Gli1 expression. Finally, to definitively interrogate the role of these cells in the injury response, we will selectively express the diphtheria toxin receptor to allow their deletion prior to nerve injury. This work will substantially enhance our understanding of hedgehog signaling in the PNS and how perturbations in this signaling following nerve injury may affect repair and regeneration. !