Utilizing Small Molecule Screens to Delineate Embryonic Signaling Mechanisms

描述(由申请人提供)：化学筛选为识别能够调节胚胎发育和控制生物过程的小分子提供了一种有价值的方法。通过从特定的分析中鉴定出化合物，就有可能确定建立胚胎分化途径的时间诱导事件。此外，与基因筛选相比，化学筛选的优势包括识别可能具有在多个生物体中发挥作用的分子。整个生物体化学筛选还可以通过消除高通量筛选中经常发现的有毒和非特定化合物来快速验证化合物。该提案中概述的目标结合了实验胚胎学、分子生物学和高通量筛选技术，以确定影响调控斑马鱼发育的最早分子事件的化合物。模式生物长期以来一直被用作药物发现的工具，但整个生物筛选尚未在高通量化学遗传学应用中得到广泛应用。这在一定程度上是因为生物体所需的化合物数量相对较多，以及通常通过肉眼观察来分析化合物效果的繁琐性质。尽管如此，由于人们对系统生物学的兴趣与日俱增，以及对生物途径的复杂性的认识，人们对以高通量筛选形式开发基于生物体的表型分析的热情日益高涨。在这项申请中，我们建议利用匹兹堡大学现有斑马鱼和药物发现项目的优势和资源，开发和实施在斑马鱼中进行化学筛选的工具和程序。药物发现中心拥有广泛的高通量筛选能力，包括多模式平板阅读器、ArrayScanll自动图像采集和分析仪器以及自动液体处理器。我们从这项提案中概述的分析中确定的化合物，以及我们适用于斑马鱼研究的筛选技术，将成为科学界的重要工具。

DESCRIPTION (provided by applicant): Chemical screens provide a valuable approach for identifying small molecules that can regulate embryogenesis and control biological processes. With the identification of chemical compounds from specific assays, it is possible to define the temporal inductive events that establish embryonic differentiation pathways. Furthermore, an advantage of chemical screens over genetic screens includes the identification of molecules that may possess the ability to function in multiple organisms. Whole organism chemical screens also allow for rapid validation of chemical compounds by eliminating toxic and non-specific compounds often found in high-throughput screening. The Aims outlined in this proposal combine experimental embryology, molecular biology, and high throughput screening technology to identify chemical compounds that influence the earliest molecular events regulating zebrafish development. Model organisms have long served as a tool for drug discovery, but whole organism screens have not yet found wide application in high-throughput chemical genetics applications. This has been partly because of the relatively greater amounts of compounds required for organisms and the laborious nature of assaying compound effect, which is typically done by visual observation. Nonetheless, because of the growing interest in systems biology and the recognition of the complexities of biological pathways, there is increasing enthusiasm for exploiting organism-based phenotypic assays in an high throughput screening format. In this application, we propose to leverage the strengths and resources of our existing zebrafish and drug discovery programs at the University of Pittsburgh to develop and implement tools and procedures for conducting chemical screens in zebrafish. The drug discovery center has extensive high throughput screening capabilities, including multimode plate readers, an ArrayScanll automated image acquisition and analysis instrument, and automated liquid handlers. The chemical compounds we identify from the assays outlined in this proposal, and the screen technology we adapt to zebrafish research will become important tools for the scientific community.