The biophysical and molecular mechanisms of reliability in development

描述（由申请人提供）：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早期建立的，具有单细胞直径的显著空间精度，代表了胚胎发生期间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。一般来说，精确的形态梯度作为定位边界的转录输入，而协作或补偿等机制被认为有助于锐化和维持边界。在这里，我们提出通过遗传学实验、精确测量和早期果蝇胚胎中Bicoid形态发生梯度及其靶基因的数学建模相结合的方法，对这些假设进行定量检验。我们开发了一种在全胚胎单分子水平上定量多基因mRNA的新方法，为确定同一胚胎中mRNA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与表达荧光标记Bicoid的胚胎的实时成像相结合来解决以下问题：一个精确而稳定的转录输入是如何实现的？2. 输入要素的协同性是如何激活和锐化边界的？3. 系统对基因剂量变化的反应是什么？允许胚胎补偿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?