Role of the Archipelago gene in Drosophila tracheal morphogenesis

描述（由申请人提供）：我们的长期目标是使用果蝇Drosophila melanogaster作为模型系统来鉴定限制细胞增殖的基因，并研究这些基因在发育组织中的功能。在这里，我们专注于群岛（ago）基因的一种新功能，我们根据其在蝇眼中的抗增殖作用进行了鉴定，其人类直系同源物Fbw 7随后被证明可以抑制各种组织类型的肿瘤发生。ago基因编码F-box蛋白（Ago），其通过直接结合蛋白质进行蛋白酶体降解，并将其募集到SCF E3-泛素连接酶中进行聚泛素化。我们先前已经鉴定了两个Ago靶标，细胞周期调节因子细胞周期蛋白E和原癌基因转录因子dMyc，这两者也被Fbw 7靶向。然而，我们缺乏对ago/Fbw 7靶基因的完整库的了解，限制了我们对该基因在发育中的作用的理解，并阻碍了预测人类ago/Fbw 7缺失的生物学后果的尝试。在目前的建议中，我们提出了大量未发表的工作，证明了一个新的作用，为前连接酶在有丝分裂后形态发生的苍蝇胚胎气管系统，一个分支网络的上皮管类似于人类的血管输送氧气通过发育中的胚胎。我们假设ago通过一个新的靶点在该途径中起作用：无气管蛋白（Trh），一种缺氧诱导因子（HIF）-1 α转录因子家族的成员，激活成纤维细胞生长因子（FGF）途径，一种已知的果蝇气管发育和哺乳动物血管和肺发育的调节因子。在具体目标1中，我们试图测试我们的初步数据所提出的假设，即Ago结合气管细胞中的Trh，并靶向其降解。我们已经发现，ago可能在这个过程中与dVHL基因，它编码一个已知参与气管发育的HIF-1 α泛素连接酶，并在具体目标2，我们建议测试ago和dVHL之间的遗传和功能相互作用。我们还发现，在成熟组织中，ago的失活能够非细胞自主地诱导异位末端气管分支，这是一个通常由氧供应控制的过程。因此，我们推测，前在抑制气管分支的稳态机制中起着重要作用。我们在《特定目标3》中的目标是定义这种作用并阐明其分子基础。

DESCRIPTION (provided by applicant): It is our long-term goal to use the fruit fly Drosophila melanogaster as a model system to identify genes that restrict cell proliferation, and to study the functions of these genes in developing tissues. Here we focus on a novel function of the archipelago (ago) gene, which we identified based on its anti-proliferative role in the fly eye, and whose human ortholog Fbw7 was subsequently shown to suppress tumorigenesis in a variety of tissue types. The ago gene encodes an F-box protein (Ago) that targets proteins for proteasomal degradation by binding directly to them, and recruiting them into an SCF E3-ubiquitin ligase for poly- ubiquitination. We have previously identified two Ago targets, the cell cycle regulator Cyclin E, and the proto- oncogenic transcription factor dMyc, both of which are also targeted by Fbw7. However, our lack of knowledge of the full repertoire of ago/Fbw7 targets limits our understanding of the role of the gene in development, and hinders attempts to predict the biological consequences of ago/Fbw7 loss in humans. In the current proposal, we present a substantial body of unpublished work that demonstrates a novel role for the ago ligase in post-mitotic morphogenesis of the fly embryonic tracheal system, a branched network of epithelial tubes similar to the human vasculature which duct oxygen through the developing embryo. We hypothesize that ago acts in this pathway via a novel target: the Trachealess protein (Trh), a member of the Hypoxia Inducible Factor (HIF)-1 alpha family of transcription factors that activates the Fibroblast Growth Factor (FGF) pathway, a known regulator of tracheal development in flies and vascular & lung development in mammals. In Specific Aim 1, we seek to test a hypothesis suggested by our preliminary data that Ago binds Trh in tracheal cells, and targets it for degradation. We have found that ago may act redundantly in this process with the dVHL gene, which encodes a HIF-1 alpha ubiquitin-ligase known to be involved in tracheal development, and in Specific Aim 2, we propose to test genetic and functional interactions between ago and dVHL. We also find that inactivation of ago in mature tissues is able to non-cell autonomously induce ectopic terminal tracheal branching, a process that is normally controlled by oxygen availability. Thus, we hypothesize that ago plays an important role in the homeostatic mechanisms that restrain tracheal branching. Our goal in Specific Aim 3 is to define this role and elucidate its molecular basis.