Preliminary in vivo and in vitro studies have demonstrated that hair cell regeneration in the bullfrog vestibular otolith organs following gentamicin ototoxicity is accomplished via both mitotic and non-mitotic mechanisms. Using morphological, immunocytochemical, dye-labeling, and cell ablation techniques in organotypic cultures, we will determine which mitotic and non-mitotic mechanisms underlie hair cell regeneration and document how these mechanisms interact to bring about hair cell recovery after damage to existing hair cells. We will incubate organotypic cultures for varying periods in aphidicolin, a blocker of mitotic division, to compare the in vitro dynamics of mitotic and non-mitotic hair cell regeneration (Project 1). Using electron microscopy, we will compare the cell morphology of mature and immature hair cells on an ultrastructural level. We will intracellularly label, using Lucifer Yellow and Texas Red-conjugated dextrans, selected cells in living organs and follow, in organ culture, migration patterns and morphological changes in these cells after gentamicin treatment (Project 2). These studies will reveal if marginal cells migrate into the sensory macula or undamaged hair cells migrate into damaged macular regions. They will also determine if supporting cells (SCs) de-differentiate and undergo one or more rounds of mitotic division or transdifferentiate into hair cells (Hcs) without undergoing mitotic division. Using histological and immunocytochemical techniques, we will compare the intracellular distributions of mitochondria, cytoskeletal, and calcium-binding proteins in the HCs and SCs (Project 3). These studies will determine if damaged HCs repair their hair bundles and if SCs undergo morphological and immunocytochemical changes after damage to existing hair cells. They will also provide developmental markers for proliferating and transdifferentiating cells. Using photoinactivation or laser ablation to ablate individual HCs and SCs, we will also determine how much damage is required to initiate proliferation or hair cell regeneration, how long each process takes to produce mature hair cells, and how each process produces multiple hair cell phenotypes (Project 4). The proposed studies, by documenting the morphological, immunocytochemical, and ultrastructural changes that occur in HCs and SCs after damage to existing hair cess, will shed light on the intercellular and intracellular mechanisms that initiate and regulate hair cell regeneration and, ultimately, suggest new means to stimulate the regeneration of auditory and vestibular hair cells in higher vertebrates.
