Vestibular hair cells - vestibular deficits are highly prevalent and cause debilitating symptoms

Degeneration and ototoxin- induced injury of vestibular hair cells (HCS) are underlying causes of some vestibular deficits. Remarkably, adult mammals, including humans, can replace some vestibular HCS once they are lost. However, in rodents and likely in humans, only one type of vestibular HC - type II (HCII) - is naturally replaced, and this degree of regeneration does not restore vestibulo-motor functions. Functional testing by our group and others' provides strong evidence that the other type of vestibular HC - type I (HCI) - must be replaced to reverse vestibular deficits. HCI are very different from HCII; for instance, they have longer and thicker stereocilia (the mechanosensitive organelle of HCS), and they synapse on a single large calyx-shaped afferent terminal of a vestibular ganglion neuron (VGN) rather than on the VGN's bouton terminals, as HCII do. These features are thought to endow HCI with functional specializations such as the ability to detect fast-onset, high-frequency stimuli. However, we understand very little about how these specializations develop or which molecular mechanisms control their genesis, differentiation, and maintenance in adult mammals. This lack of knowledge hampers scientists' efforts to determine how to drive functional regeneration in mammals after HC loss. Recently, we found that conditional knockout (CKO) of the transcription factor SOX2 from normal HCII and from naturally regenerated HCII in adult mice drives many of them to partially convert toward the HCI fate, including establishing the long, thick stereocilia unique to HCI. SOX2 CKO from adult HCII also triggers vgns to remove bouton terminals and extend a full or partial HCI-specific calyx terminal on the converting HCII. However, reprogramming and synaptic remodeling of regenerated HCII after SOX2 deletion are insufficient to recover vestibulo-motor functions. The objective of the proposed studies is to exploit these new findings to investigate molecular mechanisms by which hcis establish and maintain their type-specific hair bundle and innervation phenotypes, which are critical for function. Aim 1 will use mouse multiomics and cut & run to define gene regulatory networks by which SOX2 in HCII blocks acquisition of HCI features, and IT will begin to define SOX2-independent mechanisms that promote the HCI fate. Aim 2 will use targeted gene deletion in mice to identify novel SOX2-regulated genes that are required to develop and maintain the stereocilia morphology of HCI. Aim 3 will use targeted loss and gain of gene function in mice to define novel SOX2- regulated genes that are necessary in HCI to establish and maintain the calyx-type terminal and will employ snrnaseq to identify new ligand-receptor pairs that may regulate VGN innervation of HCI and HCII. This project will engage experts from four laboratories to generate new fundamental knowledge about how vestibular HCS develop and maintain structures that are critical for sensory transduction and synaptic transmission, providing new insights for strategies to regenerate vestibular HCS in adult mammals.