Microfluidic Temperature Steps to Understand Robustness of Embryonic Development

项目摘要。该提案描述了一个多学科的研究计划，旨在开发， 验证和传播微流控技术，使果蝇胚胎的发育成为可能。 使用温度步骤在空间和时间上进行控制。果蝇胚胎发育的过程是 坚固耐用-即使在温度等变化的环境条件下也能精确工作。而 果蝇发育中存在的许多单个分子的功能是已知的，但不知道这些分子是如何作用的。 分子一起工作，使发育网络健壮。新的微流体技术， 有区别地控制不同部位的温度，活胚胎可能成为一种强有力的工具， 确定负责这种发展稳健性的机制。具体目标1重点关注 开发新的微流体技术，该技术将使用层流来产生急剧的温度阶跃 在胚胎周围，胚胎的一部分将在一个层流的温暖温度下发育 第二层流的温度较低，胚胎的另一部分将在第二层流的温度较低时发育。 源源不断的胚胎表面的温度分布将通过以下方法定量表征： 数值模拟和共焦显微镜。实时成像胚胎暴露于 温度步骤在确定动态过程中是至关重要的，例如蛋白质浓度的变化， 时间的功能。Special Aim 2将Special Aim 1中开发的技术应用于DIG和2-光子 显微镜，以便在温度步骤中真实的时间成像胚胎发育。提出 《特定目标3》中的研究将验证《特定目标1》和《特定目标2》中开发的微流体技术 目的2通过回答关于稳健性机制的四个重要问题，无论是在分子水平， 在核武器的层面上。 相关性：了解发展对于了解人类疾病和状况至关重要 由发育和分化途径中的缺陷和错误（如癌症和衰老）引起。 研究模式生物的发育，特别是果蝇（Drosophila melanogaster）， 更好地了解人类发展，因为发展的基本机制的许多部分 在生物体中是相似的。该提案中开发的微流体技术将使人们能够理解 在果蝇中提供发育网络无差错运行的机制 黑腹，这项技术将可扩展到测试和理解错误的发展， 其他模式生物

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 1focuses 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 1to 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 1and 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