Elucidating the molecular mechanisms that shape the hair bundle

描述(由申请人提供)：要了解毛细胞的形态发生和运作，需要鉴定这些细胞中表达的基因和同源蛋白的功能。作为实现这一目标的第一步，我们使用DNA微阵列技术来鉴定成年斑马鱼毛细胞中存在的转录本。在从内耳的毛细胞中分离出纯净的毛细胞群体后，我们扩增和标记了RNA，并将得到的探针杂交到了寡核苷酸微阵列上。为了排除在上皮细胞中普遍表达的基因，我们对肝细胞进行了平行实验，并忽略了它们表达的基因。斑马鱼的毛细胞转录组包括与囊泡融合、转录调节和跨膜离子移动有关的基因；其中有一些基因的同源基因，当这些基因突变时，会导致人类和老鼠耳聋。更多的基因编码参与细胞骨架功能的蛋白质，因此可能有助于毛束的形成和维持。这些成分包括肌动蛋白结合蛋白和封帽蛋白以及鞭毛内运输系统的成分。最后，转录图谱包含许多功能未知的基因。利用适用于斑马鱼的分子生物学技术，我们已经开始研究这些基因的一些产物在毛束发育中所起的作用。公共卫生相关性毛细胞是内耳和侧线系统的机械感受器，负责一些水生脊椎动物的听力、平衡和水运动的检测。毛细胞有一根毛束，它支撑着机械转导装置。为了确定发束的蛋白质成分，并对遗传性耳聋有深入的了解，我们正在开发斑马鱼的功能基因组学方法。

DESCRIPTION (provided by applicant): An understanding of the morphogenesis and operation of hair cells requires identification of the genes expressed in these cells and of the functions of the cognate proteins. As an initial step towards this goal, we have used DNA-microarray technology to identify the transcripts present in hair cells of the adult zebrafish. After isolating pure populations of hair cells from the lagena of the inner ear, we amplified and labeled RNA and hybridized the resultant probes to oligonucleotide microarrays. To exclude genes expressed ubiquitously in epithelial cells, we conducted parallel experiments with hepatocytes and disregarded the genes that they expressed. The hair-cell transcriptome of the zebrafish includes genes involved in vesicle fusion, transcriptional regulation, and transmembrane ion movement; among these are homologs of genes that, when mutated, produce deafness in humans and mice. Additional genes encode proteins that are involved in cytoskeletal function, and might therefore contribute to the formation and maintenance of the hair bundle. These components include actin-binding and -capping proteins as well as constituents of the intraflagellar transport system. Finally, the transcriptional profile encompasses numerous genes of undetermined function. Using the molecular-biological techniques applicable to the zebrafish, we have begun to investigate the roles that some of these genes' products play in hair-bundle development. PUBLIC HEALTH RELEVANCE The hair cell is a mechanoreceptor of the inner ear and lateral-line system that is responsible for hearing, balance, and the detection of water movement by some aquatic vertebrates. The hair cell possesses a hair bundle, which holds the apparatus of mechanotransduction. To identify the protein constituents of the hair bundle and to give insights into hereditary deafness, we are developing a functional genomics approach in zebrafish.