REGULATION OF INNER EAR DEVELOPMENT AND REGENERATION

本提案的一个主要目标是调查两个关键的功能， 调节分子的类别-生长因子及其受体，以及 同源结构域蛋白-在内耳的发展，并在 受损的感觉毛细胞的再生。此外，我们将致力于 涉及开发用于细胞类型特异性分子标记的探针。两个PCR- 将采用基于技术的方法来鉴定在特定细胞中表达的基因。 内耳发育和再生中的细胞亚群。一 第一种调查方法是全球范围的，而第二种调查方法仅限于 酪氨酸激酶和同源结构域蛋白。代表转录本的cDNA 在特定的发展阶段，在特定的 内耳亚区，或仅在损伤后再生期间， 成年人的器官将被鉴定和克隆。DNA序列分析将 以确定编码蛋白质与蛋白质之间的关系 在可用的数据库中。看起来有趣的cDNA将更充分地 其特征在于采用原位杂交定位转录物 在发育和受伤的内耳中。这些研究报告将 主要用于鸡。然而，如果结果证明， 将在小鼠中重复研究，以表征可能的 差异，这可能是更大的能力，再生的 与哺乳动物相比，鸟类的毛细胞。对于最有用的细胞类型 特异性标记物，其特征在于针对 将开发编码蛋白质。描述性数据表明， 这些研究中表征的基因产物可能是 开发，功能的研究将通过检查 减少或消除表达对发育的影响 基因产物。将采用两种方法。一种方法 将使用反义寡核苷酸来降低 目的基因的转录本。第二 方法将涉及检查内耳的发育， 在75 kDa神经营养因子受体中具有基因靶向突变的小鼠。

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.