MOLECULAR ANALYSIS OF EAR DEVELOPMENT

本提案的长期目标是检验以下假设： 在发育中的耳（耳囊和相关神经元）中表达的基因 结构）将在毛细胞再生过程中重新表达。 一 这一假说的一个推论是，在再生过程中激活的基因 可能代表了以前未发现的在发育中重要的基因。 这些假设将在小鸡身上进行测试，其中胚胎学 毛细胞再生的时间过程 已经被广泛研究过了。 巴拉德博士和拉斐尔将提供 在这两个重要领域的专业知识。 本提案的具体目标 目标：1。 为了利用差异显示技术来鉴定表达于 并评估它们在耳发育中的作用。 这种方法有 已经产生了一个耳囊cDNA， 保守蛋白 目标二。 为了鉴定基底动脉中表达的基因， 通过比较正常耳蜗的RNA， 暴露于宽带噪声（110 dB SPL持续4小时）， 暴露于噪音的动物经过两天的恢复。 初步研究已经 已经确定了三个高度调控的基因，CDC 42，一个小的GTP- 与肌动蛋白细胞骨架相互作用的结合蛋白;甲状旁腺 一种即时早期基因，即与乳腺癌相关的蛋白质; 神经元钙/钙调素调节蛋白激酶II。 时间和 这些基因的mRNA和蛋白质的空间分布将是 在正常耳蜗和声损伤后， 杂交和免疫组织化学。 目标3。 以确定是否 在耳囊中鉴定的基因在毛细胞中表达， 再生，并确定是否基因激活后，声学 创伤在早期耳部发育中表现出来。 这将是完成 通过逆转录酶-PCR。 目标4。 为了开发一个系统， 通过使用禽类逆转录病毒， 将基因导入鸡胚的载体和逆转录病毒或 腺病毒载体将基因导入成年鸡耳蜗。 这些 实验将评估这些基因的功能， 发育和毛细胞再生过程中。 的结果予以 研究应该提供深入的分子过程参与耳 发展，并确定这些进程是否在 毛细胞再生

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.