CELLULAR COMMUNICATION IN MORPHOGENESIS

这是一个识别和描述反应途径的建议 在果蝇细胞中，缺氧是由缺氧引起的。期间 在上一个资助期，Krasnow博士开发了一个果蝇模型系统， 用于研究气管的发育，气管是一种上皮衍生组织， 为幼虫细胞输送氧气。他发现有两个线索 决定了气管结构的最终模式， 有树干和树枝第一个是发育遗传程序， 指定了典型的主枝和次分支模式， 第二个线索是氧气的缺乏，这影响了终端分支的密度。 这第二步是本建议的主要重点。 在过去的资助期间，Krasnow博士提出了一些重要的建议， 关于缺氧细胞如何产生吸引信号的发现 气管的末端分支。关键信号似乎是果蝇 成纤维细胞生长因子（FGF）的同系物，称为无分支。的合成 FGF在缺氧的细胞中增加。FGF水平的上升反过来 吸引表达FGF受体（命名为 喘不过气来）。他还分离出了果蝇中两个亚基的同源物， 缺氧诱导因子I（tango和类似物）以及 调节HIF-1蛋白的稳定性。这些蛋白质是从 哺乳动物系统需要低氧反应。在基因筛查中，博士 Krasnow还发现了一个潜在的反应基因，命名为cropped。他展示 Cropped蛋白定位于细胞核以应对缺氧。 这项建议的第一个目的是确定五种缺氧反应是否 tango、similar、dVHL、cropped、branchless等基因都是同一个基因的成员， 或不同的途径。由于它们是通过不同的方式识别的（尽管 它们都对缺氧有反应）它们不一定代表一个单一的 和对缺氧的统一反应。Cropped和HIF-1都是 转录因子克拉斯诺医生将首先确定 转录调节HIF-1，反之亦然。这将通过使 一个基因的突变克隆和检测另一个基因的表达。第二 问题将是，如果作物和HIF-1诱导无分支的FGF， 可能是缺氧状态的输出信号。修剪的或HIF-1的突变克隆 （探戈）将被诱导，并将确定无分支表达。的 这两个实验的基本原理是，如果一个人缺乏功能， 一个基因损害另一个基因的表达，它们是同一个基因的成员。 反应途径（并建立其上位关系）。另一个计划 目的是探讨HIF-1和Branchless与一氧化氮的关系。 （NO）介导的反应，另一种缺氧反应途径最近在 帕特·奥法雷尔的实验室 第二个目标是进行全面的基因筛选， 缺氧含有8000个果蝇cDNA的微阵列芯片将用 从不同来源（包括组织培养细胞， 不同发育阶段和不同组织的动物）， 暴露在缺氧环境中微阵列芯片也将用于 鉴定由HIF-1（和Cropped）调控的基因。三个标准将用于 找到感兴趣的基因：首先，具有相同的诱导谱的基因， 已知的HIF-1靶点; 2，在野生型动物中诱导而不是在HIF-1中诱导的基因 突变动物;三，由HIF-1过度表达诱导的基因。 最后一个目的是利用遗传学方法鉴定其他缺氧反应基因。 屏幕所提出的F1筛选涉及产生突变的F1基因的有丝分裂克隆。 染色体臂的幼虫和检查能力的突变克隆吸引 末端气管分支无法吸引异性的变异克隆体 对缺氧没有反应一个完善的FLP/FRT的变体 一个系统，允许检查克隆在活幼虫建议，这 目的.

This is a proposal to identify and characterize response pathways in Drosophila cells that are triggered by lack of oxygen (hypoxia). During the previous funding period, Dr. Krasnow has developed a Drosophila model system for studying the development of trachea, an epithelial derived tissue that delivers oxygen to larval cells. He has found that there are two cues that dictate the final pattern of the tracheal structure, which resembles a tree with a trunk and branches. First is the developmental genetic program that specifies the stereotypical primary and secondary branch patterns, and the second cue is lack of oxygen that influences the density of terminal branches. It is this second step that is the main focus of this proposal. During the past funding period, Dr. Krasnow has made a number of important discoveries regarding how cells deprived of oxygen produce signals to attract the terminal branches of trachea. The key signal appears to be the Drosophila homolog of Fibroblast Growth Factor (FGF), called Branchless. The synthesis of FGF increases in cells deprived of oxygen. The rise in FGF levels in turn attracts terminal tracheal branches which express the FGF receptor (named Breathless). He has also isolated Drosophila homologs of the two subunits of Hypoxia Inducible Factor I (tango and similar) as well as a factor (VHL) that regulates HIF-1 protein stability. These are proteins known from the work in mammalian systems to be needed for hypoxic response. In a genetic screen, Dr. Krasnow has also identified a potential response gene, named cropped. He shows that Cropped protein localizes to the nucleus in response to hypoxia. The first aim of this proposal is to determine if the five hypoxic response genes -- tango, similar, dVHL, cropped, branchless -- are members of the same or different pathways. Since they were identified by different means (although they all respond to hypoxia) they don't necessarily have to represent a single and unified response to the lack of oxygen. Cropped and HIF-1 are both transcription factors. Dr. Krasnow will first determine if Cropped transcriptionally regulates HIF-1 or vice versa. This will be done by making mutant clones of one gene and examining the expression of the other. The second question will be to ask if Cropped and HIF-1 induces Branchless FGF, the probable output signal of hypoxic conditions. Mutant clones of cropped or HIF-1 (tango) will be induced and Branchless expression will be determined. The rationale for both of these experiments is that if the lack of function in one gene compromises the expression of another gene, they are members of the same response pathway (and establishes their epistatic relationships). Another plan is to determine the relationship of HIF-1 and Branchless to nitric oxide (NO)-mediated response, another hypoxic response pathway recently described in Pat O'Farrel's lab. The second aim is to carry out comprehensive screens for genes that respond to hypoxia. Microarray chips with 8000 Drosophila cDNAs will be screened with probe cDNAs prepared from different sources (including tissue culture cells, animals of different developmental stages, and different tissues) that have been exposed to hypoxic conditions. Microarray chips will also be used to identify genes regulated by HIF-1 (and Cropped). Three criteria will be used to find genes of interest: first, genes with the same induction profile as the known HIF-1 target; two, genes induced in wildtype animals but not in HIF-1 mutant animals; three, genes induced by the over expression of HIF-1. The last aim is to identify additional hypoxia response genes using a genetic screen. The proposed F1 screen involves generating mitotic clones of mutated chromosome arms in larvae and examining the ability of mutant clones to attract terminal tracheal branches. Mutant clones that fail to attract are presumably unable to respond to hypoxia. A variation of the well established FLP/FRT system that allows examination of clones in living larvae is suggested for this purpose.