The peripheral nervous system (PNS) serves the essential function of relaying information between the central nervous system (CNS) and the body. This information is transmitted in the form of electrical signals, which travel down the axons of PNS neurons. When peripheral nerves become injured, the connection between the CNS and tissues is severed, causing sensation and motor control to be lost. PNS injuries are common, and although these nerves are capable of regenerating, patients often do not regain full functional recovery. The complete nerve is formed of axons surrounded by several layers of ensheathing glia and connective tissue. Regeneration requires all the cells in these layers to precisely coordinate their behaviors to ensure debris is cleared and new axons re-grow. Axons are wrapped by Schwann cells, which are then surrounded by an endoneurium, perineurium, and epineurium. The perineurium is a protective cellular sheath essential for nerve function, and is formed by flattened, interdigitated perineurial glial cells. These cells play an essential role n nerve development, but how they respond to nerve injury and participate in regeneration is not known. The long-term goal of this project is to elucidate the role of perineurial glia in PNS regeneration. This will be investigated through use of a novel injury assay designed to view the dynamic behaviors of individual glial cells following a nerve transection in vivo. In this assay, a Micropoint laser ablation system is used to transect the motor nerve in live, transgenic zebrafish, and the behaviors perineurial glia are visualized using time-lapse confocal microscopy. Preliminary data from this assay suggests that perineurial glia send membrane processes toward injury sites, form bridges between divided proximal and distal nerve stumps, and may aid in phagocytizing debris. This will be investigated further by: 1) Fully characterizing the perineurial glial response to nerve injury, and how this response is coordinated with Schwann cells and macrophages through the use of vital dyes, immunohistochemistry, and in vivo imaging, 2) Investigating if axonal damage is the cue that initiates perineurial glial injury responses using in vivo imaging, genetic mutants, and injections of degenerating axonal material, and 3) Using genetic manipulation to determine if perineurial glia are essential for nerve degeneration and regeneration. This work will lead to a more complete understanding of how multiple cell types coordinate their actions to rebuild the nervous system after disease or injury.