The long term objective of this proposal is to test the hypothesis that genes expressed in the developing ear (otocyst and associated neuronal structures) will be re-expressed during hair cell regeneration. A corollary of this hypothesis is that genes activated during regeneration may represent previously unidentified genes important in development. These hypotheses will be tested in the chick, where the embryological development is well defined and the time course of hair cell regeneration has been studied extensively. Drs. Barald and Raphael will provide expertise in these two important areas.
The specific aims of this proposal are;
Aim 1. To use differential display to identify genes expressed in the otocyst and to assess their role in ear development. This approach has already produced an otocyst cDNA that encodes a novel evolutionarily conserved protein.
Aim 2. To identify genes expressed int he basilar papilla after noise (acoustic trauma) by comparing RNA from normal cochleas exposed to broad band noise (110 dB SPL for 4 hours), and cochleas of noise-exposed animals after two days of recovery. Preliminary studies have already identified three highly regulated genes, for CDC42, a small GTP- binding protein that interacts with the actin cytoskeleton; parathyroid hormone-related protein, an immediate early gene; and the delta subunit of neuronal calcium/calmodulin-regulated protein kinase II. The temporal and spatial distribution of the mRNA and proteins for these genes will be determined in normal cochleas and after acoustic trauma by in situ hybridization and immunohistochemistry.
Aim 3. To determine whether the genes identified in the otocyst are expressed during hair cell regeneration, and to determine whether genes activated after acoustic trauma are expressed in early ear development. This will be accomplished by reverse transcriptase-PCR.
Aim 4. To develop systems for perturbing the normal patterns of expression of these genes by using avian retroviral vectors to introduce genes into chick embryos and either retroviral or adenovirus vectors to introduce genes into adult chick cochleas. These experiments will assess the function of these genes, both during development and during hair cell regeneration. The results of these studies should provide insight into the molecular processes involved in ear development, and determine whether these processes are reactivated during hair cell regeneration.
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