Date of Submission

Spring 2016

Academic Programs and Concentrations

Biology

Project Advisor 1

Michael Tibbetts

Abstract/Artist's Statement

In humans, much of permanent hearing loss and balance issues are caused by the irreversible death of inner ear mechanosensory hair cells. Hair cells act as sensory receptors for both the auditory and vestibular systems and are housed in the inner ear structure of the fluid filled cochlea, and in lateral line neuromasts of fish. Where mammals have lost the ability to renew dead hair cells, many non-mammalian vertebrates have retained their capacity for regeneration through the mantle support cells that surround hair cells in neuromasts. The large protocadherin protein Fat2 is involved in many developmental systems regulating cellular polarity and adhesion, a property crucial to hair cell function, in addition to displaying heightened activity during hair cell regeneration. This makes Fat2 an intriguing element to investigate, and this present study sought to elucidate its role in hair cell development and regeneration in the zebrafish Daneo rerio. Fat2 was found to localize in the periphery of neuromasts and is a key factor in both hair cell development and regeneration. To determine Fat2 localization in zebrafish, 7 day old fish underwent immunofluorescence staining using monoclonal antibodies against Fat2. Once its presence in neuromasts during hair cell development and regeneration was confirmed, morpholino knockdowns were performed on zebrafish embryos to observe the effects of Fat2 loss. Larvae were allowed to develop five days before hair cell staining and counting, with the experimental regeneration group subjected to CuSO4-induced hair cell ablation 24 hours before staining and counting. Immunofluorescent staining was imaged with confocal microscopy, and Fat2 was found to cluster near the body surface in neuromast mantle support cells and the inner ear. This technique has not yet been explored in this context, and the results implicate Fat2 in hair cell regulation and maintenance as well as demonstrate that Fat2 can be successfully detected and imaged by immunofluorescence. Loss of Fat2 in developing zebrafish led to delayed growth and deafness, and hair cell counts revealed significantly reduced numbers per neuromast in morphants for both developmental and regenerative assays. Interestingly, hair cell number per neuromast displayed a marked decrease anterior-posteriorly along the lateral line in development but not regeneration. Given the results obtained it’s clear Fat2 plays a major role in hair cell development and regeneration. However, what’s uncertain is how it functions in these systems. Future investigation in the effects of Fat2 loss could elucidate if they arise from hindered organismal or neuromast development, and whether they’re caused by disruption of proper cellular polarity and adhesion.

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