Hair Cell Regeneration: Molecular Regulation

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Auditory hair cells are mechanoreceptors in the inner ear that transduce sound waves into neural signals. In humans, hair cell loss is irreversible and leads to profound, permanent hearing deficits. In contrast, mature birds regenerate hair cells in the chicken auditory epithelium (basilar papilla, or BP). Despite this ability, little is known about the identity, cell lineage, or molecular regulation of avian hair cell progenitors. Four Specific Aims are proposed to address these issues. In Aim I, the dynamics of progenitor cell division are examined using in vivo and in vitro approaches. First, using two nucleotide analogs, I will determine if progenitor cells divide more than once after a single lesion and after two lesions separated by a recovery period. Second, I will perform cell lineage analysis in primary BP cultures to assess the number of hair cell progenitors that have stem-cell like behavior (i.e., form colonies containing hair cells) versus those that divide only once or twice prior to forming hair cells and/or supporting cells. These experiments will help define the types of progenitor cells present in the mature avian BP, and if the presence of stem cells is confirmed, they will provide a foundation for experiments in Aim II. In Aim II, two methods are proposed to generate purified cultures of hair cell progenitors: clonal expansion and viral-mediated selection of cells with mitotic potential. Progenitor cells derived by each method are examined for 1) their response to known regulators of supporting cell division and 2) their ability to regenerate cells with hair-cell and supporting-cell phenotypes. Experiments in Aim III will test if systematic variations in morphology exist among quiescent and dividing supporting cells in the intact BP. Otocysts are infected with a retrovirus encoding GFP in ovo, and chicks are allowed to mature to post-hatch. GFP-labeled supporting cells in the BP of control chicks and of chicks exposed to gentamicin are compared to determine if 1) distinct morphological subsets exist among quiescent supporting cells and 2) if dividing cells exhibit morphological features that distinguish them from growth arrested cells. In Aim IV, I will test the function of a potential regulator of progenitor cell cycling - the homeobox-like transcription factor, cProxl - in cultures enriched for hair cell progenitors. Cultured cells are infected with a retrovirus encoding full-length or dominant-negative Proxl, and the effects of cProxl misexpression or inhibition on cell division are assessed. I hypothesize that cProx 1 activity is necessary and sufficient to stimulate cells to withdraw from the cell cycle. I hope the information generated in these experiments will be useful toward promoting hair cell regeneration in mammalian species.