MOLECULAR MECHANISMS IN DEVELOPMENT

这里提出的研究的长期目标是了解组织的信号机制 以及发育过程中的图案组织。我们研究细胞如何发出信号并对图案提示做出反应，聚焦 在形象盘和气管细胞形成的长丝状径向延伸(细胞线)上。我们的工作已经完成 结果表明，这些结构存在于远距离细胞信号传递部位，从细胞接收信号延伸而来 信号，似乎与产生信号的细胞直接接触。这些观察结果表明 包括直接接触和调节释放和摄取的选择性信号模型。五个具体 我们提出的目标旨在帮助我们了解这些细胞延伸的结构和功能 为我们提出的细胞-细胞信号传递模型提供新的证据。 目的特异细胞素1的动力学和结构。光学显微镜和电子显微镜的改进方法 将被用来对细胞素进行成像。更好的成像将提供解决有关 细胞突起的生长、稳定和分布。 目的研究Vav在细胞线状丝状伪足的产生和功能中的作用.我们 最近发现，果蝇Rho gef，Vav，当在S2和S2中表达时，可以诱导长丝足 CI-8细胞。我们将在培养细胞中表征这些丝状足细胞的动态和功能，我们将 分析Vav在形象盘发育过程中的作用。我们将进行RNAi干扰筛选以 确定促进或抑制丝状足形成的功能，并将研究这些功能的作用 在苍蝇发育过程中。 特异的AIM3A系统来模拟丝状伪足介导的信号传递.我们已经开发出一种分析方法 S2细胞中的HH信号可能依赖于细胞间的接触。我们将与这项测试一起努力 确定信号是否需要接触，并询问通过丝状突起进行接触的细胞 可以发信号。 目的：探讨HSPGs在细胞素分布中的作用。我们将确定是否 细胞素可以延伸到缺乏HSPG的细胞上。这些实验将测试是否抑制了 形态原在缺乏HSPG的区域的移动与细胞素的缺乏相关。 特异的AIM 5细胞素功能。我们将在具有异位的想象盘中映射细胞素 信令中心或在其A/P信令中心降低DPP水平的信令中心，以便检查 信号转导与细胞质定位的关系。我们还将监控磁盘中的DPP信号，这些磁盘 细胞素已经被破坏，我们将跟踪信号蛋白的运动，以确定 细胞素起到了信号传输的管道作用。

The long-term goal of the research proposed here is to understand the signaling mechanisms that organize and pattern tissues during development. We study how cells signal and respond to patterning cues, focusing on the long filopodial extensions (cytonemes) that imaginal disc and tracheal cells make. Our work has shown that these structures are present at sites of long distance cell signaling, extending from cells receiving signals and appearing to make direct contact with cells producing the signals. These observations suggest a model for selective signaling involving direct contact and regulated release and uptake. The five Specific Aims we propose are designed to help us understand the structure and function of these cell extensions and to provide new evidence relevant to our proposed model for cell-cell signaling. SPECIFIC AIM 1 Cytoneme dynamics and structure. Improved methods for light and EM microscopy will be used to image cytonemes. Better imaging will provide means to address basic questions about the growth, stability and distribution of the cell extensions. SPECIFIC AIM 2 The role of Vav in the production and function of cytoneme-like filopodia. We recently discovered that the Drosophila Rho GEF, Vav, can induce long filopodia when expressed in S2 and CI-8 cells. We will characterize the dynamics and function of these filopodia in cultured cells, and we will analyze the role of vav during imaginal disc development. We will carry out an RNAi intereference screen to identify functions that enhance or suppress filopodia formation and will examine the role of these functions during fly development. SPECIFIC AIM 3 A system to model filopodia-mediated signaling. We have developed an assay of Hh signaling in S2 cells that may be dependent upon cell-cell contact. We will work with this assay to establish if contact is required for signaling and will ask if cells that make contact via filopodial extensions can signal. SPECIFIC AIM 4 The role of HSPGs in the distribution of cytonemes. We will determine whether cytonemes can extend over cells that lack HSPGs. These experiments will test whether inhibition of morphogen movement through regions lacking HSPGs correlates with the absence of cytonemes. SPECIFIC AIM 5 Cytoneme function. We will map cytonemes in imaginal discs that have ectopic signaling centers or that have reduced levels of Dpp at their A/P signaling center in order to examine the relationship between signaling and cytoneme orientation. We will also monitor Dpp signaling in discs whose cytonemes have been disrupted and we will track the movement of signaling proteins to determine if cytonemes function as conduits for signal traffic.