. About 1 child for every 500 to 1000 newborns is affected by sensorineural hearing loss, making it one of the most common birth defects. Genetic factors underlie most cases of hearing loss in young people. Low- cost genomic sequencing technologies continually identify new mutations associated with human hearing loss, but their functional validation is unacceptably slow. Even in the case of genes with well-established links to hearing loss, there is often a lack of knowledge regarding underlying mechanisms of pathogenesis. There is an urgent need to fill this knowledge gap. Thus, our long-term goal is to illuminate the pathologies responsible for hearing loss and to develop novel treatment strategies. To accomplish this goal, we used CRISPR/Cas9 technology to generate a library of 50 zebrafish lines with mutations in established human hearing loss genes. Most (94%) human hearing loss genes have an orthologue in zebrafish, suggesting high functional conservation. Zebrafish are an ideal model organism to study hearing loss, given their external embryonic development, transparent body, accessible inner ear and the presence of lateral line neuromasts, which are functionally analogous to mechanoreceptors of the mammalian inner ear. We hypothesize that our mutant zebrafish lines will reveal distinct phenotypes and mechanisms corresponding to disrupted structure and function of the inner ear. The inner ear contains two complex sensory systems (auditory and vestibular) necessary for hearing and balance. These two systems are directly dependent on functional sensory epithelia, which are composed primarily of two cell types, the hair cells and the supporting cells. Sound waves are transduced into electrical signals by the hair bundles present in sensory hair cells in the inner ear. This process requires a specific ionic environment in the endolymph surrounding the hair cell stereocilia and the perilymph covering the hair cell body. This ionic homeostasis is maintained by ion channels and ion transporters. It is likely that many cases of hearing loss are caused by abnormalities in the hair cells themselves or in their environment. We will initially focus on two hearing loss genes, slc26a4 and stereocillin, as a ?training set? to elucidate their hearing loss phenotypes and mechanisms in zebrafish. Additionally, we will investigate 16 select mutants from our library for their roles in inner ear function. We will test our hypothesis by pursuing the following Specific Aims: 1) Elucidate the mechanisms and phenotypes underlying hearing loss in slc26a4 mutant zebrafish. 2) Determine the role of stereocillin in hair cell development and function in zebrafish. 3) Large-scale functional validation of candidate genes associated with non-syndromic hearing loss. Our research will provide novel insights into the cellular and molecular phenotypes of slc26A4 and stereocillin hearing loss mutants (Aims 1 & 2) and will enable us to expand our efforts by identifying additional hearing loss models within our knockout library (Aim 3). The results are expected to have a positive impact because insight into the pathology of genetic hearing loss will provide new targets for therapeutic interventions.
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