Microfluidic Temperature Steps Robustness of Embryonic Development

描述(申请人提供)：项目总结。这项提案描述了一项多学科研究计划，旨在开发、验证和传播微流控技术，使活的果蝇胚胎的发育能够通过温度步骤在空间和时间上进行控制。果蝇胚胎的发育过程是强大的-即使在不同的环境条件下，如温度，它也能准确地工作。虽然许多单独的分子在果蝇发育中的功能是已知的，但这些分子如何共同作用使发育网络变得强大还不清楚。新的微流控技术可以不同地控制活胚胎不同部位的温度，这可能成为确定导致这种发育健壮性的机制的有力工具。具体目标1侧重于开发新的微流控技术，该技术将利用层流在胚胎周围产生一个急剧的温度台阶，其中胚胎的一部分将在一个层流的较暖温度下发育，而胚胎的另一部分将在第二个层流的较低温度下发育。胚胎表面的温度分布将使用数值模拟和共聚焦显微镜进行定量表征。对暴露在温度阶跃下的胚胎进行实时成像在识别动态过程中至关重要，例如蛋白质浓度随时间的变化。特定目标2采用在特定目标1中开发的技术来挖掘和双光子显微镜，以便在温度步骤中实时成像胚胎发育。关于具体目标3的拟议研究将通过回答关于分子一级和核分裂一级的稳健性机制的四个重要问题，来验证在具体目标1和具体目标2中开发的微流控技术。相关性：了解发育对于了解由发育和分化途径的缺陷和错误(如癌症和衰老)引起的人类疾病和状况至关重要。研究模式生物的发育，特别是果蝇黑腹果蝇，可以更好地理解人类的发育，因为生物发育的基本机制的许多部分都是相似的。该方案中开发的微流控技术将有助于理解果蝇发育网络无差错运行的机制，该技术将被推广到测试和理解其他模式生物发育中的差错

DESCRIPTION (provided by applicant): Project summary. This proposal describes a multi-disciplinary research program that aims to develop, validate and disseminate microfluidic technology to allow development of a live Drosophila embryo to be controlled in space and time using temperature steps. The process of Drosophila embryo development is robust - it works precisely even under varying environmental conditions such as temperature. While the function of many individual molecules present in Drosophila development is known, it is unknown how these molecules work together to make developmental network robust. New microfluidic technology that can differentially control temperature around different parts a living embryo could become a powerful tool in determining the mechanisms responsible for this robustness of development. Specific Aim 1 focuses on development of new microfluidic technology that will use laminar flow to create a sharp temperature step around the embryo, where one part of the embryo will develop at the warmer temperature of one laminar stream, and the other part of the embryo will develop at the cooler temperature of the second laminar stream. The temperature profile at the surface of the embryo will be quantitatively characterized using numerical simulations and confocal microscopy. Real-time imaging of an embryo being exposed to temperature step is critical in identifying dynamic processes such as changes in protein concentration as a function of time. Specific Aim 2 adapts technology developed in Specific Aim 1 to DIG and 2-photon microscopy in order to image embryonic development in the temperature step in real time. Proposed research in Specific Aim 3 will validate the microfluidic technology developed in Specific Aim 1 and Specific Aim 2 by answering four important questions concerning the mechanism of robustness, both at the molecular level and at the level of nuclear divisions. Relevance: Understanding development is essential for understanding of human diseases and conditions caused by defects and errors in developmental and differentiation pathways (such as cancer and aging). Studying development in model organisms, in particular, the fruit fly Drosophila melanogaster leads to a better understanding of human development, since many parts of the basic machinery of development are similar among organisms. The microfluidic technology developed in this proposal will enable understanding of the mechanism that provides error-free operation of developmental network in the fruit fly Drosophila melanogaster, and this technology will be extendable to testing and understanding errors in development of other model organisms