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？ 与公共卫生相关：胚胎发育是一个非常可靠的过程。它对遗传和环境扰动具有很强的抵抗力，并且从一个胚胎到下一个胚胎具有高度的重复性。另一方面，最微小的错误，在分子水平上，可能会产生最严重的后果，如发育或出生缺陷，而发育信号通路的错误可能导致癌症的形成。因此，了解构成发育可靠性的机制是至关重要的。发育系统是否具有纠错机制，或者发育过程在每个阶段都非常精确和可重复吗？这些问题的答案是分子性质的，需要一种定量的方法，为我们提供管理发育的分子过程的精确测量。

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? PUBLIC HEALTH RELEVANCE: Embryonic development is an extraordinary reliable process. It is robust against genetic and environmental perturbations, and it is highly reproducible from one embryo to the next. On the other hand, the tiniest error, down at the molecular level, can have the severest consequences, such as developmental or birth defects, and errors in developmental signaling pathways can lead to the formation of cancer. Hence, it is crucial to understand the mechanisms that underlie developmental reliability. Do developmental systems possess mechanisms for error correction, or are developmental processes very precise and reproducible at each stage? The answers to these questions are of molecular nature, and demand a quantitative approach that provides us with precise measurements of the molecular processes that govern development.