MOLECULAR ANALYSIS OF EAR DEVELOPMENT

这项提议的长期目标是检验这样一个假设 在发育中的耳朵(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。一个 这一假说的推论是基因在再生过程中被激活 可能代表着以前未被识别的在发育中重要的基因。 这些假说将在雏鸡身上进行测试，在那里胚胎学 毛细胞的发育和再生的时间进程是明确的 已经得到了广泛的研究。巴拉尔德博士和拉斐尔博士将提供 在这两个重要领域的专业知识。这项建议的具体目的 目的：1.利用差异显示技术鉴定基因表达 并评估它们在耳朵发育中的作用。这种方法具有 已经产生了一个耳囊cdna，它在进化上编码了一种新的 保守的蛋白质。目的2.鉴定基底膜表达的基因 噪声(声损伤)后乳头状突与正常耳蜗组织RNA的比较 暴露在宽带噪声(110dBSPL，持续4小时)和 经过两天的恢复后，暴露在噪音中的动物。初步研究表明 已经确定了三个高度调控的基因，对于CDC42，一种小的GTP- 与肌动蛋白细胞骨架相互作用的结合蛋白；甲状旁腺 激素相关蛋白，一种即刻早期基因；以及 神经元钙/钙调蛋白调节的蛋白激酶II。 这些基因的mRNA和蛋白质的空间分布将是 耳蜗声损伤后及正常耳蜗组的原位检测 杂交和免疫组织化学。目标3.确定是否 在耳囊中识别的基因在毛细胞中表达 再生，并确定声学处理后基因是否被激活 创伤表现在耳朵发育的早期。这将会实现的 逆转录-聚合酶链式反应。目标4.开发干扰系统 利用禽类逆转录病毒表达这些基因的正常模式 将基因导入鸡胚胎的载体，以及逆转录病毒或 腺病毒载体将基因导入成年雏鸡耳蜗。这些 实验将评估这些基因的功能，在 在毛细胞发育和再生期间。这些研究的结果 研究应该提供对EAR所涉及的分子过程的洞察 开发，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.