Drosophila deafness genes: analysis of chordotonal organ functions

描述（由申请人提供）：本提案的长期目标是了解遗传模式生物果蝇的听觉器官庄士敦器官（JO）的发育和功能。果蝇的JO与哺乳动物的内耳是同源的，因为它们都依赖于高度保守的atonal家族转录因子的活性。小鼠无调性同源基因1 （Math1）与果蝇无调性基因在互惠转基因拯救实验中可以相互替代。此外，人类无民族同源物1 （Atoh1）可以介导药理学耳聋哺乳动物听觉毛细胞的再生。因此，果蝇JO代表了一个强大的听力基因发现资源。JO与其他脊索器官的不同之处在于它专门用于听觉。在早期蛹期，JO发育的许多关键事件发生，包括前体细胞的不对称分裂，感觉器官细胞系的规范，以及正确分化所必需的细胞形状变化，JO在蛹内被掩盖，非常脆弱，难以解剖。我们的总体策略是设计出更好地可视化发展中的JO的方法，并将这些方法作为基因发现的系统表达微阵列分析的基础。首先，我们将描述几个JO基因在这些早期蛹期的作用。为了描述这些基因的医学相关性，我们将筛选它们的人类同源物与家族分离性耳聋的关联。其次，我们将培养解剖的触角盘，在远离模糊的蛹的情况下，在体内动态成像细胞系和标记表达。我们将用几个标记来确定文化发展的保真度，并定义这些阶段的突出事件。第三，我们将利用这种方法的更高通量，在这些关键阶段从野生型和突变型天线盘中恢复足够的RNA，使用微阵列分析来比较基因表达。我们计划首先使用切割突变体作为范例。剪切转录因子是正常JO发育所必需的，其在哺乳动物中的同源物CDP/Cux1在内耳中表达。因此，我们期望我们的实验结果将使我们能够确定在这些关键阶段起作用的靶基因。总的来说，这些研究将为哺乳动物内耳的发育和功能生物学的未来研究提供信息，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.