Birds are the only warm-blooded vertebrates known to regenerate sensory hair cells (HCs) of the inner ear in maturity. Recent studies have demonstrated that birds utilize two mechanisms to generate new HCs: renewed cell division of progenitor cells, the non-sensory supporting cells (SCs), and direct transdifferentiation of SCs into HCs, without intervening mitosis. Studies of avian HC regeneration offer an important opportunity to elucidate cellular and molecular mechanisms governing these SC responses to HC loss. Such studies are critical preludes for directing analyses of SC behavior in mammalian HC-epithelia toward determining why HC regeneration is not completed in mammals. We will focus on the role of two molecules, Atoh1 and Notch, which are critical for mammalian HC development. Aim 1 will address signals regulating the SC decision to directly transdifferentiate after HC damage in mature chicken BPs by testing the hypotheses that Notch activation antagonizes transdifferentiation while Atoh1 activation promotes it. We will examine damaged BPs to determine if temporospatial expression patterns of genes in the Notch pathway support the proposed role for Notch. We will activate or inhibit Notch, its ligands, and its effectors and determine if direct transdifferentiation becomes altered in response. We will also test the hypothesis that Atoh1 function is necessary and sufficient for direct transdifferentiation. In Aim 2, we will address signals regulating the SC decision to divide after HC damage in avian BPs by testing the hypothesis that Atoh1 and Notch both inhibit SC division. We will determine if overexpression of Atoh1 leads to cell-autonomous inhibition of SC division and if inhibition of Atoh1 promotes division. We will determine which genes in the Notch pathway are altered in dividing SCs and in cells surrounding them. We will determine if manipulation of Notch function significantly alters rates of SC division. In Aim 3, we will assess the extent to which Atoh1 expression and direct transdifferentiation occur spontaneously in SCs of the adult mouse utricle after HC damage. We will examine how expression of genes in the Notch pathway is altered in adult mouse utricles after HC loss. We will also test the hypotheses that inhibition of Notch signaling in adult mouse utricles promotes direct transdifferentiation and SC division. [unreadable] [unreadable] [unreadable]