Richard A. Baird - US grants

DESCRIPTION: 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.

DESCRIPTION (provided by applicant): We are requesting funds to support a recently constituted Inner Ear Core Consortium designed to enhance exiting research programs and to promote cooperative interactions among 18 scientists at Central institute for the Deaf (CID), Washington University Medical School (WUMS), and St. Louis University (SLU). This consortium consists of an administrative Core Center, three research-oriented core facilities (Digital Imaging Core Electron Microscopy Core, and Gene Expression Core), and one service-oriented core facility (Electronic Services Core). These core facilities are physically located in a new CID research building, recently completed and occupied, immediately adjacent to the south end of the WUMS campus. The scientists in the Inner Ear Consortium have research backgrounds in anatomy, biochemistry, cell biology, developmental biology, molecular biology, molecular genetics, and physiology. These scientists have primary appointments in the Fay and Carl Simons Center for Biology of Hearing and Deafness, one of two centers of excellence at CID, the Departments of Otolaryngology, Anatomy and Neurobiology, Anesthesiology, Molecular Biology and Pharmacology at WUMS, and the Department of Anatomy and Neurobiology at SLU. The research programs of these scientists address crucial issues in the development, function, and regeneration of sensory receptors and their neuronal innervation in the vertebrate inner ear. The Inner Ear Consortium maintains expensive common equipment, supports the common needs of inner ear scientists for digital imaging, ultrastructural analysis, and studies of gene expression. It encourages research collaborations that cross existing research programs by promoting interactions among (1) researchers who study the morphology and physiology of the normal, developing, and regenerating inner ear, (2) researchers who study the inner ear on systemic, cellular, and subcellular levels, and (3) researchers who study the auditory and vestibular systems. It promotes the use of mutant and transgenic animal models and trains inner ear scientists in new research techniques, fostering the transfer of these techniques among inner ear laboratories. The Inner Ear Consortium also provides limited assistance for pilot projects, especially those that advance existing scientific programs, involve multiple investigators, or promise to provide new techniques of general interest to inner ear scientists. Finally, it enhances the research training that consortium members provide to residents and medical students in the Department of Otolaryngology and graduate students in the Departments of Speech and Hearing Sciences at CID and the Division of Biology and Biological Sciences at WUMS.