REGULATION OF INNER EAR DEVELOPMENT AND REGENERATION

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A major goal of this proposal is to investigate the function of two key classes of regulatory molecules - growth factors and their receptors, and homeodomain proteins - in the development of the inner ear, and in the regeneration of damaged sensory hair cells. Also, effort will be devoted to developing probes for cell-type specific molecular markers. Two PCR- based techniques will be employed to identify genes expressed in specific cell subpopulations in the developing and regenerating inner ear. One survey technique is global in scope, while the second will be limited to tyrosine kinases and homeodomain proteins. cDNAs representing transcripts expressed only at a particular stage of development, in a particular subregion of the inner ear, or only during regeneration after injury of the adult organ, will be identified and cloned. DNA sequence analysis will be performed to determine the relationship of encoded proteins to proteins in available databases. cDNAs which appear interesting will be more fully characterized employing in situ hybridization to localize transcripts within the developing and injured inner ear. These studies will be performed primarily in chickens. Where the results warrant, however, studies will be repeated in mouse, in order to characterize possible differences which may account for the greater capacity for regeneration of hair cells in birds as compared to mammals. For the most useful cell-type specific markers characterized as cDNAs monoclonal antibodies against the encoded proteins will be developed. Where descriptive data suggests that gene products characterized in these studies may be key regulators of development, studies of function will be performed by examining the consequences for development of reducing or eliminating expression of the gene products in question. Two approaches will be employed. One approach will employ anti-sense oligonucleotides to reduce the levels of transcripts for the genes of interest, in explant cultures. The second approach will involve examination of the development of the inner ear in mice with gene targeted mutations in the 75 kDa neurotrophin receptor.