Genetic and molecular dissection of hair-cell function

描述（申请人提供）：耳聋是最常见的遗传性疾病之一，每1000名儿童中就有一名患有耳聋。人类遗传学家在识别耳聋基因方面取得了显着进展，但在许多情况下，我们并不清楚这些基因的功能或突变引起的病理。此外，我们对听觉的理解落后于我们对视觉、味觉和触觉等其他感官的了解。毛细胞中直接介导机械传导的分子尚未被鉴定。为了深入了解机械转导的分子基础和耳聋基因的功能，我们将在模式脊椎动物生物斑马鱼中利用正向和反向遗传学。我们已经确定了总共24个基因所需的幼虫的听觉和前庭功能的大规模诱变筛选，并克隆了四个斑马鱼基因。所有四个基因的直系同源物都是人类或小鼠耳聋的原因，这表明我们的筛选和基因功能高度保守的相关性。我们将通过候选或定位克隆方法继续我们的克隆努力。此外，我们的反向遗传学研究已经证明是富有成效的，确定了一个潜在的候选基因的机械转导通道，NompC。作为在转导中的直接作用的进一步证据，我们将在斑马鱼毛细胞中定位NompC通道。一个特别的挑战将是表征和查明我们的突变体或吗啉注射动物的缺陷的性质。因此，我们将创建一个转基因钙指示线，这将使我们能够确定是否转导，突触传递，或听觉信号的中央处理在我们的听觉/前庭突变体的影响。这些实验的数据将有助于增加我们对耳聋基因生物学的理解，并可能为耳聋患者带来潜在的治疗方法。

DESCRIPTION (provided by applicant): Deafness is one of the most common hereditary diseases, affecting one out of 1000 children. Human geneticists have made remarkable progress in identifying genes responsible for deafness, yet in many cases, we do not clearly understand either the function these genes or the pathology caused by the mutations. Moreover, our understanding of the sense of hearing lags behind our knowledge of other senses such as vision, taste, and touch. The molecules that directly mediate mechanotransduction in hair cells have yet to be been identified. In order to gain insight into the molecular basis of mechanotransduction and the function of deafness genes, we will take advantage of both forward and reverse genetics in the model vertebrate organism, the zebrafish. We have identified a total of 24 genes required for larval auditory and vestibular function from large scale mutagenesis screens, and have cloned four zebrafish genes thus far. Orthologues of all four genes are responsible for deafness in either humans or mice, demonstrating the relevance of our screen and a high degree of conservation of gene function. We will continue our cloning efforts by candidate or positional cloning approaches. In addition, our reverse genetic studies have proven fruitful, identifying a potential candidate gene for the mechanotransduction channel, NompC. As further evidence for a direct role in transduction, we will localize the NompC channel in zebrafish hair cells. One particular challenge will be to characterize and pinpoint the nature of the defects in our mutants or morpholino-injected animals. We will therefore create a transgenic calcium indicator line that will enable us to determine whether transduction, synaptic transmission, or central processing of auditory signals is affected in our auditory/vestibular mutants. The data from these experiments will help to increase our understanding of the biology of deafness genes and may lead to potential therapies for deafness patients.