PROJECT SUMMARY Death of sensory hair cells in the inner ear is a common cause of auditory and vestibular dysfunction in humans. This damage is permanent due to the inability of mammals to effectively regenerate inner ear sensory patches beyond the neonatal stage. In contrast, non-mammalian vertebrate species such as birds and fish can regenerate hair cells throughout life. This discrepancy in regeneration capacity between mammals and non- mammalian vertebrates is not well understood. In species that can regenerate hair cells, the nonsensory support cells that surround hair cells can act as hair cell progenitors. In vivo studies of inner ear hair cell regeneration and support cells are limited by the inner ear’s location within the bony skull of mammals and birds. As a result, the zebrafish lateral line, an external sensory system for detecting changes in water flow, has been developed as an alternative model for studying support cells and hair cell regeneration in living animals. However, the degree to which the mechanisms of regeneration in the lateral line are similar to those of the ear is unclear. The zebrafish inner ear is an understudied system that has the potential to bridge the gap between mammalian inner ear and lateral line studies. This project examines hair cell regeneration in the zebrafish inner ear during the larval stage, at which point the sensory organs become fully functional and remain accessible for in vivo imaging. We aim to identify hair cell precursors in the zebrafish inner ear and determine when and how they give rise to new hair cells during regeneration. In the inner ear of birds and mammals, support cells regenerate hair cells by proliferating and differentiating or by direct transdifferentiation. In the zebrafish lateral line, only the former mechanism is used. In Aim 1 of this proposal, we will use a novel method of hair cell ablation to determine the timecourse of regeneration and whether cell proliferation, transdifferentiation, or both are used in the zebrafish inner ear. Hair cells in the lateral line are regenerated primarily by a spatially and functionally distinct group of support cells. Whether a similar support cell subtype exists in the zebrafish inner ear is unknown. In Aim 2, we will validate putative inner ear support cell subtypes identified through single-cell RNA-seq analysis. We will then generate transgenic lines labeling support cell subtypes and use fate mapping to identify hair cell precursors. Together these experiments will characterize the timing and mechanism of hair cell regeneration and the diversity of support cell subtypes in the zebrafish inner ear. This work will help to establish the zebrafish inner ear as an outstanding in vivo model system for future in-depth studies of support cells and their roles in conserved mechanisms of regeneration. Expanding our knowledge of the mechanisms of inner ear hair cell regeneration in animals such as the zebrafish will ultimately lead to the advancement of treatments for auditory and vestibular decline in humans.

PROJECT NARRATIVE Mammals are severely limited in their ability to regenerate inner ear sensory hair cells compared to non- mammalian vertebrates such as zebrafish; consequently, damage to the auditory and vestibular systems in humans leads to permanent deafness and vestibular dysfunction. The zebrafish inner ear provides a highly regenerative and evolutionarily conserved model system in which hair cell regeneration can be studied in intact, live animals. This project aims to develop the larval zebrafish as a model system by quantifying and determining the mechanism of hair cell regeneration and characterizing inner ear cell subtypes that are involved in hair cell regeneration.