Molecular Genetics of Otolith Formation in the Zebrafish

斑马鱼耳石形成的分子遗传学

• 批准号: 7086292
• 负责人: Richard Kollmar
• 金额: $ 28.8万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7086292
• 关键词: biological models; developmental genetics; ear hair cell; gene expression; in situ hybridization; labyrinth; mass spectrometry; molecular cloning; molecular genetics; otocyst /otolith; polymerase chain reaction; protein purification; protein quantitation /detection; protein sequence; protein structure function; proteomics; zebrafish

DESCRIPTION (provided by applicant): The general goal of this proposal is to elucidate the molecular mechanisms that govern the formation of otoliths and otoconia, the biominerals in vertebrate inner ears that convey linear accelerations to the sensory hair cells. The proteins of otoliths and otoconia are thought to play a critical role in their formation. However, testing of this hypothesis is hampered by the fact that most of these proteins are not known. The specific aims of this proposal are, therefore, first to determine the sequence of the about two dozen constituent proteins of zebrafish otoliths; second, to examine the proteins' expression patterns; and third, to test whether these proteins are necessary for otolith formation. In humans, otoconial defects are associated with common vestibular deficits, such as benign paroxysmal positional vertigo and possibly Pendred syndrome. The inaccessibility of the human labyrinth, however, calls for an animal model to study the physiology and pathology of otoconia. Zebrafish otoliths are evolutionary orthologs of mammalian otoconia, based on their shared location, function, composition, and general structure, and thus a valid model system. Furthermore, the mechanisms that govern otolith and otoconia formation are likely to be similar, based on the high degree of molecular conservation found between inner-ear structures of zebrafish and mammals. The zebrafish offers numerous experimental advantages for studies of otolith formation, such as simple husbandry, easy observation and manipulation of gene expression, a genomics infrastructure rivaling that of the mouse, and the accessibility of its transparent and externally-developing embryos. The combination of protein purification and sequencing by mass-spectrometry, molecular cloning of cDNAs, in situ hybridization and quantitative RT-PCR, morpholino injections and mutant rescue will lead to an efficient identification of candidate genes and mechanisms for future studies of mammalian otoconia.

描述（由申请人提供）：该提案的一般目标是阐明控制耳石和耳细胞症的分子机制，这是脊椎动物内耳中的生物矿物质，这些内耳中的生物膜将线性加速输送到感官毛细胞。耳石和耳尾症的蛋白质被认为在其形成中起着至关重要的作用。但是，这些假设的检验受到了以下事实的障碍：这些蛋白质中的大多数尚不清楚。因此，该提案的具体目的是首先确定斑马鱼耳石的大约二十个成分蛋白的序列。其次，检查蛋白质的表达模式；第三，要测试这些蛋白是否是耳石形成所必需的。在人类中，耳尾部缺陷与常见的前庭缺陷有关，例如良性阵发性位置眩晕和可能的pendred综合征。然而，人类迷宫的无法访问呼吁动物模型研究奥托托西病的生理学和病理学。斑马鱼耳石是基于其共享位置，功能，组成和一般结构，因此是有效的模型系统的哺乳动物耳鼻虫的进化直系同源物。此外，根据斑马鱼和哺乳动物内耳结构之间发现的高度分子保护，控制耳石和耳鼻喉科的机制可能是相似的。斑马鱼为耳石形成的研究提供了许多实验优势，例如简单的饲养，简单的观察和对基因表达的操纵，一种与小鼠相抗衡的基因组学基础设施以及其透明和外部发展的胚胎的可及性。蛋白质纯化和测序通过质谱，cDNA的分子克隆，原位杂交和定量RT-PCR，morpholino注射和突变救援的结合将导致对哺乳动物豆科可将来研究的候选基因和机制有效鉴定。