Molecular Genetics of Otolith Formation in the Zebrafish

描述(申请人提供)：这项建议的总体目标是阐明控制耳石和耳锥形成的分子机制，耳石和耳锥是脊椎动物内耳中的生物矿物质，将线性加速传递到感觉毛细胞。耳石和耳石的蛋白质被认为在它们的形成中起着关键作用。然而，由于这些蛋白质中的大多数都是未知的，这一假设的检验受到了阻碍。因此，这项建议的具体目标是：第一，确定斑马鱼耳石的大约24种组成蛋白质的序列；第二，研究这些蛋白质的表达模式；第三，测试这些蛋白质是否对耳石的形成是必要的。在人类中，耳锥缺陷与常见的前庭缺陷有关，如良性阵发性位置性眩晕和可能的Pendred综合征。然而，人类迷宫的不可达性要求建立一个动物模型来研究耳锥的生理学和病理学。斑马鱼耳石是哺乳动物耳锥的进化直系物，基于它们共同的位置、功能、组成和一般结构，因此是一个有效的模式系统。此外，控制耳石和耳锥形成的机制很可能是相似的，这是基于斑马鱼和哺乳动物内耳结构之间发现的高度分子保守。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，易于观察和操纵基因表达，可与小鼠相媲美的基因组学基础设施，以及其透明和外部发育的胚胎的可及性。蛋白质纯化与质谱学测序、cDNA分子克隆、原位杂交与定量RT-PCR、吗啡注射与突变挽救相结合，将为今后哺乳动物耳石病的研究提供有效的候选基因和机制。

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.