PROJECT SUMMARY Approximately one quarter of patients with vertigo or dizziness have central vestibular disorders. In addition, hearing loss or tinnitus can have central origins. Despite the prevalence of central deficits in the auditory/vestibular system in patients, our understanding of central dysfunction at the molecular or cellular level in vertebrates is lacking. Here, we propose to characterize a novel class of zebrafish mutants that have central auditory/vestibular deficits to gain insights into this understudied area of research. This proposal focuses on two mutants: raumschiff and starliner, which were isolated from chemical mutagenesis screens for hearing and balance defects. Unlike our previously characterized mutants, raumschiff and starliner mutants have normal vestibular induced eye movements despite presenting with an obvious balance defect while swimming or at rest. In addition, a defect in hearing is present in both mutants. We have identified mutations in two genes: in starliner mutants, the split ends (spen) gene harbors a nonsense mutation and in raumschiff we identified a missense mutation in vacuolar protein sorting 4a (vps4a). RNAseq anaylsis of mutant and sibling transcripts indicate that both mutants have striking misregulation of gene expression in the hindbrain and midbrain regions, yet their development and gross brain morphology is normal. These results suggest that the defects are functional in nature and may involve circuit level or synaptic changes. To gain a better understanding of the central defects, we will take advantage of imaging whole fish expressing relevant transgene markers and use newly developed methods for brain-wide imaging of cellular responses to auditory and vestibular stimuli. These experiments will focus on the regions or cell types where misregulation of gene expression is most prevalent. Collectively our studies will enhance our understanding of the genes and regulatory networks involved in central function.