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以特定模式精确表达的基因级联完成的。定义这些模式的边界的边界在发育过程中很早就建立了，具有单细胞直径的显著空间准确性，代表了胚胎发生过程中发育精确性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，并且诸如协同性或补偿的机制被认为有助于锐化和维持边界。在这里，我们建议把这些假设进行定量测试，采用遗传实验，精确测量和数学建模的Bicoid形态梯度和其靶基因在早期果蝇胚胎的组合。我们已经开发了一种新的方法来量化多个基因的mRNA在单分子水平上在整个胚胎，这提供了一种方法来确定mRNA和蛋白质在同一胚胎的绝对数量。我们将使用这种方法与表达荧光标记的Bicoid的胚胎的实时成像相结合，以解决以下问题：1。如何实现精确和稳定的转录输入？2.输入要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？胚胎补偿这种输入变化的机制是什么？

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?