Drosophila deafness genes: analysis of chordotonal organ functions

描述(申请人提供)：这项建议的长期目标是了解约翰斯顿器官(JO)的发育和功能，这是遗传模式生物果蝇中的听觉器官。果蝇JO与哺乳动物内耳同源，因为它们的特性都依赖于无机盐家族高度保守的转录因子的活性。小鼠无性同源基因1(Math1)和苍蝇无性基因在互惠转基因抢救实验中可以相互替代功能。此外，人类无调性同系物1(Atoh1)可以介导药物性耳聋哺乳动物听觉毛细胞的再生。因此，苍蝇JO代表了一种强大的听觉基因发现资源。Jo在几个方面不同于其他脉络膜器官，这是它专门用于听力的。在幼虫发育的早期阶段，当JO发生许多关键事件时，包括前体细胞的不对称分裂，感觉器官细胞谱系的指定，以及正确分化所必需的细胞形状变化，JO在蛹内被遮蔽，非常容易被解剖。我们的总体策略是设计出更好地可视化正在发展的JO的方法，并将这些方法作为系统表达微阵列分析的基础，用于基因发现。首先，我们将描述几个JO基因在这些早期蛹阶段中的作用。为了确定这些基因的医学相关性，我们将筛选它们的人类同源基因与分离耳聋的家系之间的关联。其次，我们将培养解剖的触角盘，以动态地显示体内的细胞谱系和标记表达，而不是模糊的蚕蛹。我们将用几个标记来确定文化发展的保真度，并定义这些阶段的显著事件。第三，我们将利用这种方法的较高吞吐量，在这些关键阶段从野生型和突变型触角圆盘中提取足够的RNA，以便使用微阵列分析比较基因表达。我们计划最初使用CUT突变体作为范例。切割的转录因子是正常JO发育所必需的，哺乳动物的同源基因CDP/CUX1在内耳表达。因此，我们希望我们的实验结果将使我们能够识别在这些关键阶段发挥作用的CUT的靶基因。总体而言，这些研究将为未来哺乳动物内耳的发育和功能生物学研究提供信息，并加速我们对人类听觉障碍的理解。

DESCRIPTION (provided by applicant): The long term objectives of this proposal are to understand the development and function of Johnston's organ (JO), which is the auditory organ in the genetic model organism, the fruit fly Drosophila. The Drosophila JO is homologous to the mammalian inner ear because they both rely for their specification on the activity of highly conserved transcription factors of the atonal family. The mouse atonal homolog 1 (Math1) and the fly atonal genes can substitute for each other's function in reciprocal transgenic rescue experiments. Furthermore, the human atonal homolog 1 (Atoh1) can mediate regeneration of auditory hair cells in pharmacologically deafened mammals. Thus, the fly JO represents a powerful gene discovery resource for hearing. JO differs from other chordotonal organs in several ways that specialize it for hearing. At early pupal stages when many critical events of JO development occur, including asymmetric divisions of precursor cells, specification of sense organ cell lineages, and cell shape changes essential for correct differentiation, the JO is obscured within the puparium and very fragile to dissect. Our general strategy is to devise methods to better visualize the developing JO, and to use these methods as the basis for systematic expression microarray analysis for gene discovery. First we will characterize the roles of several JO genes at these early pupal stages. To characterize the medical relevance of these genes, we will screen for associations of their human homologs with families segregating deafness. Second, we will culture dissected antennal disks to image cell lineages and marker expression dynamically in vivo, away from the obscuring puparium. We will determine the fidelity of development in culture with several markers, and define the salient events at these stages. Third, we will exploit the higher throughput of this approach to recover sufficient RNA from wild-type and mutant antennal disks at these critical stages to compare gene expression using microarray analysis. We plan to use cut mutants initially as a paradigm. The cut transcription factor is required for normal JO development, and the mammalian homolog, CDP/Cux1 is expressed in the inner ear. Thus, we expect that the results of our experiments will permit us to identify target genes of cut that act at these critical stages. Overall, these studies will inform future research on the developmental and functional biology of the mammalian inner ear, and accelerate our understanding of human auditory disorders.