The biophysical and molecular mechanisms of reliability in development

描述(申请人提供)：早期果蝇胚胎的分割是通过一系列以特定模式精确表达的基因来完成的。定义这些模式边界的边界是在发育非常早的时候建立的，具有单个细胞直径的显著空间精度，这是胚胎发育过程中发育精度和可靠性的最早证据。边界形成的确切分子或生物物理机制及其准确性尚不清楚。一般而言，精确的形态梯度作为转录输入来定位边界，而协作性或补偿等机制被认为有助于锐化和维持边界。在这里，我们建议通过采用遗传学实验、精确测量和数学建模相结合的方法，对果蝇早期胚胎中的双类形态原梯度及其靶基因进行定量检验。我们建立了一种在单个分子水平上对整个胚胎中多个基因的mRNA进行定量的新方法，为确定同一胚胎中mRNAs和蛋白质的绝对数量提供了一种方法。我们将使用这种方法结合表达荧光标记的双标记物的胚胎的实时成像来解决以下问题：1.如何实现精确和稳定的转录输入？2.输入因素的协同性如何激活和锐化边界？3.系统对基因剂量变化的反应是什么？允许胚胎补偿对这种输入变化的反应的机制是什么？

DESCRIPTION (provided by applicant): Segmentation of the early Drosophila embryo is accomplished through a cascade of genes that are precisely expressed in specific patterns. The boundaries that define the borders of these patterns are established very early during development with the remarkable spatial accuracy of a single cell diameter, representing the earliest evidence of developmental precision and reliability during embryogenesis. The exact molecular or biophysical mechanisms underlying the formation of boundaries and their accuracy are unknown. In general, precise morphogen gradients serve as transcriptional inputs that position a boundary, and mechanisms such as cooperativity or compensation are thought to help sharpen and maintain the boundary. Here we propose to put these hypotheses to a quantitative test by employing a combination of genetic experiments, precise measurements and mathematical modeling of the Bicoid morphogen gradient and its target genes in early Drosophila embryos. We have developed a new method to quantify mRNA of multiple genes at the single molecule level in whole embryos, which provides an approach to determine absolute numbers of both mRNAs and proteins in the same embryo. We will use this method in combination with live imaging of embryos expressing fluorescently tagged Bicoid to address the following questions: 1. how is a precise and stable transcriptional input achieved? 2. How does cooperativity of input factors activate and sharpen a boundary? 3. What are the responses of the system to gene dosage changes and what are the mechanisms that allow the embryo to compensate the response to such input changes?