PROJECT SUMMARY Loss of hearing is a prevalent sensory pathology in the United States that affects over 30 million people. A significant proportion of deafness is attributed to sensorineural hearing loss, which often involves the damage of afferent nerve fibers which relay auditory information from the mechanosensitive hair cells of the inner ear to the brain. The restoration of physiologic hearing would require the regeneration of afferent fibers into the sensory epithelium of the cochlea, followed by the reinnervation of appropriate hair cell targets. Nerve regeneration studies in humans and other mammalian models are lacking due to the limited accessibility of the inner ear. The zebrafish lateral line system, composed of superficial fluid-flow detecting hair cells and afferent nerve fibers, offers a simple and accessible model of nerve regeneration. In this model, there are likely various paracrine, juxtacrine, and neuron-autonomous signaling mechanisms working in coordination to guide axon pathfinding and target selection. Aim 1 of this proposal will determine the molecular cues expressed by target sensory hair cells to guide reinnervation by regenerating afferent axons of the lateral line. Following transection of the lateral line nerve, hair cells from the zebrafish will be isolated at multiple timepoints. In these hair cells, the expression changes of canonical and non-canonical molecular cues that may be used to attract axonal growth cones will be quantified through transcriptome sequencing. Aim 2 will investigate the neuronal bias for reinnervation of developmentally related hair cell populations. Although studies suggest that neurons retain a memory for their original hair cell targets, how this memory is established or maintained is unknown. A transgenic imaging technique will be used to label and trace clonal populations of regenerating axons following transection of the lateral line nerve. It is hypothesized that neurons prefer reinnervating hair cells that arose from a shared sensory placode during development. Aim 3 will reveal changes in the local physical environment of the regenerating nerve to allow entry of individual afferent fibers into their target organ. By imaging transgenic fish with fluorescently labeled Schwann cells and collagen, changes will be shown in Schwann cell tracts and the epithelial basement membrane to permit entry of individual axons into the zebrafish neuromast, which contains target hair cells. It is hypothesized that physical gaps form in Schwann cell and basement membrane layers in close proximity to denervated hair cells to allow passage of regenerating axons branching off the main nerve bundle. Together, these studies will elucidate the mechanisms governing afferent nerve regeneration and the reinnervation of hair cells, which may provide insight towards restoring hearing in the deafened human cochlea. These studies will be carried out under the direct mentorship of Dr. A. J. Hudspeth at The Rockefel...