Project Summary This project’s long-term objective is to elucidate mechanisms that control hair-bundle development and function, show how defects in these mechanisms cause deafness and vestibular disorders, and identify pathways that could be modulate to ameliorate these syndromes. Because the ability to control bundle development may assist in hair-cell regeneration strategies, our project aims to uncover the mechanisms a hair cell uses to regulate and modulate steps in bundle growth. Although our past work focused on postnatal development of bundles of apical inner hair cells (IHCs) from the mouse cochlea, here we expand our scope here to include embryonic development, outer hair cells (OHCs), and hair cells from apex to base of the cochlea. In the first Aim, we will collect (and share) datasets of stereocilia actin and key row-specific proteins for IHCs from mid cochlear regions to the base and OHCs from apex to base. Our goal is to discern the broad developmental plan for cochlea bundles, and to identify the steps that individual hair cells modulate to create the distinct arrangements of stereocilia in these diverse bundles. In the second Aim, we will characterize the first stage of stereocilia growth, stage II in Tilney’s categorization, by measuring stereocilia dimensions, determining locations and concentrations of key proteins, and revealing the presence of interstereocilia links during embryonic development. Importantly, we will use mouse mutants to determine roles for the tip-link cadherins CDH23 and PCDH15, as well as the molecular motors MYO15A, MYO7A, MYO3A, and MYO3B. In the final Aim, we address how the pattern of stereocilia arrangement on the hair cell’s apical surface is determined. Our hypothesis is that spacing is initially set when stereocilia grow during stage II; as they are tightly packed together then with their spacing set by stereocilia width, their pattern is set and stabilized by growth of the rootlets and their interconnections through rootlet-rootlet filaments and the cuticular plate. Mice lacking ANKRD24, GRXCR2, MYO7A, or SPTAN1 have substantially altered stereocilia packing in mature hair cells, suggesting that these four proteins—and likely many others—participate in setting stereocilia spacing.