Defining Atoh1 function and regulation in avian supporting cells during auditory hair cell regeneration

Sensorineural hearing loss is irreversible in all mammals, including humans, since neither hair cells nor neurons are regenerated. In contrast to mammals, non-mammalian vertebrates replace hair cells after damage. In the avian basilar papilla, hair cell injury activates neighboring supporting cells to undergo direct transdifferentiation or mitotic division, both of which contribute to regeneration of hair cells. The molecules that enable hair cell regeneration in birds are not well understood. This dissertation presents a series of experiments to evaluate whether Atoh1, a transcription factor required for hair cell development, is sufficient for hair cell differentiation in avian basilar papilla during regeneration after aminoglycoside damage and to determine if the bone morphogenetic factor BMP4, a protein required for development of auditory epithelia, inhibits Atoh1 mRNA expression and subsequent hair cell differentiation after damage. In the first study, I tracked the activity of the Atoh1 enhancer in cultured basilar papillae to determine if it is an accurate predictor of hair cell fate, and I forced expression of mouse Atoh1 in supporting cells to test the hypothesis that higher levels of Atoh1 push supporting cells to divide or transdifferentiate. This first study determined that about half of supporting cells with Atoh1 enhancer activity do not differentiate into hair cells, but relief from notch-mediated lateral inhibition or forced overexpression of Atoh1 significantly increase the likelihood that a supporting cell will differentiate as a hair cell or proliferate. In the second study, I used in situ hybridization to determine that Bmp4 mRNA is expressed in hair cells in mature chicken basilar papilla. BMP4 receptors are transcribed in supporting cells and hair cells, while inhibitor of DNA binding (Id) mRNA, a downstream effector of BMP4, is enriched in supporting cells in control tissues. Upon hair cell loss, Bmp4 mRNA expression is lost, while Atoh1 mRNA is upregulated in supporting cells. Concurrently, downstream Id effectors and receptors to BMP4 are upregulated in the area of damage. Given the observation that Bmp4 and Atoh1 have opposing expression patterns after hair cell loss, damaged basilar papillae were cultured with BMP4 protein or its inhibitor noggin after hair cell loss to determine if BMP4 antagonizes Atoh1 expression and subsequent hair cell differentiation. BMP4 eliminated Atoh1 transcripts along the length of the basilar papilla, while noggin increased Atoh1 expression. Further, BMP4 application significantly decreased the number of regenerated hair cells, while noggin application significantly increased them. These findings suggest BMP4 antagonizes hair cell regeneration by reducing Atoh1 transcripts in supporting cells, preventing them from dividing or transdifferentiating. My results are consistent with other investigators’ observations that Atoh1 is necessary for hair cell differentiation during development but additional factors such as notch ligands and BMP4 limit ATOH1’s expression. These are important considerations as investigators examine the potential for ATOH1 to stimulate auditory hair cell regeneration in humans.