| The organ of Corti is an organized mosaic of nonsensory supporting cells and sensory hair cells the latter are vulnerable to damage from ototoxic drugs, loud noise and sound, aging, or infection. Once lost, hair cells are spontaneously regenerated in non-mammalian species, but auditory hair cell loss in mammals results in permanent hearing loss because these cells are not replaced. Avian hair cell regeneration by direct and indirect transdifferentiation is known to rely on the reinitiation of Notch signaling and subsequent transcription of the basic-helix-loop-helix (bHLH) gene Atoh1 in areas of hair cell loss. In mammals, Atoh1 is necessary and sufficient for hair cell development and can also force mature supporting cells to change fate to a sensory hair cell if over-expressed. Atoh1 is antagonized in cochlear development by the Notch-dependent bHLH genes Hes1 and Hes5, which direct cells to a supporting cell fate.This dissertation includes molecular descriptions of Notch signaling proteins in the organ of Corti in late development, maturity, and trauma. The studies were aimed at elucidating whether mammals preserve the ability to reinitiate Notch signaling following trauma and to learn as many details of the response as possible. Immunohistochemistry and Western blotting of cochlear proteins demonstrate low level Notch signaling in the normal mature epithelium, and an up-regulation in signaling that occurs within twenty-four hours of ototoxic drug administration. The response is characterized by increases in Jagged1 in the pillar cells, Notch1 in supporting cell nuclei, and Hes1 and Hes5 in the nuclei and cytoplasm of supporting cells which is reflective of their developmental locations. No Atoh1, Jagged2 or Delta1 were observed at any time following a lesion. Signaling peaks at two weeks, and diminishes to below baseline levels by two months. This observation about the up-regulation of Atoh1-repressors presents an opportunity and ideal timeframe for therapeutic Notch knockdown following hair cell loss. To develop a future tool to examine Notch knockdown in vivo, we engineered a recombinant adenoviral vector which expresses a dominant-negative version of the Notch signaling binding protein, Mastermind-like (MAML). This vector, Ad.dnMAML1-GFP , presents an improvement over the traditional means of Notch knockdown by offering stable expression of a specific transcription-deficient Notch substrate fused with a fluorescent reporter. |
