Factor Mediating Ureteric Migration and Morphogenesis

描述（由申请人提供）：肾收集系统通过输尿管芽（UB）的分支形态发生而产生，我们能够通过在后肾间充质细胞条件培养基存在下培养分离的UB在体外复制该过程。我们在前一个资助期内从该条件培养基中纯化蛋白质，证明了多种可溶性因子调节UB分支。在这里，我们认为，这些生长因子的能力，以产生明确的区别UB提示和秸秆是至关重要的正常收集系统的发展。因此，我们试图确定特定生长因子产生茎尖和茎杆的细胞和分子机制（SA 1）。由于我们已经发现，不同的生长因子集提供了非常独特的模式孤立UB形态-“细分支（提示和茎）”，“茎为中心”和“提示为中心”的表型，我们将使用这些明显不同的条件，以确定分子和机制，导致提示和茎规格。在拟议的研究中，我们计划将我们已经确定的生长因子与细胞表面信号传导（通过GFR α 1和脂筏）和细胞内信号传导途径（通过Rho/ROCK）以及细胞内蛋白分选途径（特别是涉及ER分子伴侣的那些）连接起来，这是适当表达尖端和茎特异性基因产物所必需的。这些联系的逻辑主要基于上一个供资期间完成的工作，在申请中详细阐述。(SA1技术：分离的UB培养物、密度梯度分级、转染、mRNA的选择性下调、激光捕获显微切割、微阵列）。此外，我们已经确定了至少一个剩余的有效活性UB分支在体外，并旨在纯化和表征它。（SA 2技术：柱层析，质谱，蛋白质表达）。总之，拟议的研究应该提供一个更完整的图片调节UB分支的可溶性因子，以及它们如何在细胞表面和细胞内产生收集系统发育过程中的提示和柄。这些试验是以大量初步数据为基础的，直接来自上一个供资期间完成的工作。我们在完成该项目所需的技术方面拥有成熟的专业知识。

DESCRIPTION (provided by applicant): The kidney collecting system arises through branching morphogenesis of the ureteric bud (UB), a process that we have been able to replicate in vitro by culturing the isolated UB in the presence of a metanephric mesenchyme cell conditioned medium. Our purification of proteins from this conditioned medium during the previous funding period has demonstrated that multiple soluble factors regulate UB branching. Here we argue that the ability of these growth factors to generate clear distinctions between UB tips and stalks is critical for normal collecting system development. We therefore seek to identify the cellular and molecular mechanisms by which particular growth factors work to generate tips and stalks (SA1). Since we have found that different sets of growth factors give very distinct patterns of isolated UB morphogenesis--"fine branching (tips and stalks)," "stalk-centric" and "tip-centric" phenotypes-we will use these clearly different conditions to identify molecules and mechanisms that lead to tip and stalk specification. In the proposed studies, we plan to connect the growth factors we have identified to cell surface signaling (via GFRalpha1 and lipid rafts) and intracellular signaling pathways (via Rho/ROCK), as well as intracellular protein sorting pathways (especially those involving ER chaperones), necessary for appropriate expression of tip and stalk-specific gene products. The logic of these connections, based largely on work completed during the previous funding period, is developed in detail in the application. (SA1 techniques: isolated UB culture, density gradient fractionation, transfections, selective down regulation of mRNA, laser capture microdissection, microarrays). In addition, we have identified at least one remaining potent activity for UB branching in vitro and aim to purify and characterize it. (SA2 techniques: column chromatography, mass spectrometry, protein expression). Together, the proposed studies should provide a much more complete picture of the soluble factors that regulate UB branching and how they act at the cell surface and intracellularly to generate tips and stalks during collecting system development. The experiments are based on considerable preliminary data and follow directly from work completed during the previous funding period. We have proven expertise in the techniques necessary to complete the project.