Role of the Archipelago gene in Drosophila tracheal morphogenesis

描述（由申请人提供）：我们的长期目标是利用果蝇黑腹果蝇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，我们关注的是archipelago （ago）基因的一种新功能，我们根据其在苍蝇眼睛中的抗增殖作用确定了该基因，其人类同源基因Fbw7随后被证明可以抑制多种组织类型中的肿瘤发生。ago基因编码一个F-box蛋白（ago），该蛋白通过直接结合蛋白酶体降解蛋白，并将其招募到SCF e3泛素连接酶中进行多泛素化。我们之前已经确定了两个Ago靶点，细胞周期调节因子Cyclin E和原致癌转录因子dMyc，这两个靶点都是Fbw7的靶点。然而，我们对ago/Fbw7靶点的全部知识的缺乏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/Fbw7缺失的生物学后果的尝试。在目前的提案中，我们提出了大量未发表的工作，证明了前连接酶在苍蝇胚胎气管系统有丝分裂后形态发生中的新作用，气管系统是一个分支的上皮管网络，类似于人类血管系统，通过发育中的胚胎输送氧气。我们假设ago通过一个新的靶点在这一途径中起作用：无气管蛋白（Trh），它是缺氧诱导因子(HIF)-1 α转录因子家族的一员，可激活成纤维细胞生长因子（FGF）途径，FGF是苍蝇气管发育和哺乳动物血管和肺发育的已知调节剂。在Specific Aim 1中，我们试图验证我们的初步数据提出的一个假设，即Ago与气管细胞中的Trh结合，并将其靶向降解。我们发现ago可能在这一过程中与dVHL基因一起冗余作用，该基因编码HIF-1 α泛素连接酶，已知参与气管发育，在Specific Aim 2中，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，成熟组织中ago的失活能够非细胞自主地诱导气管末端异位分支，这一过程通常由氧可用性控制。因此，我们假设ago在抑制气管分支的稳态机制中起重要作用。我们在Specific Aim 3的目标是定义这一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 In the current proposal, we present a substantial body of unpublished work that demonstrates a novel role for the ago ligase in post-mitotic morphogenesis of the fly embryonic tracheal system, a branched network of epithelial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.