Stochastic gene expression in differentiation and development

本研究的主要目的是利用秀丽隐杆线虫和哺乳动物细胞系作为实验模型系统，探索随机基因表达在发育和细胞分化过程中的作用。在本提案的第一个程序中，我描述了一种单分子mRNA检测技术，该技术可以精确测量发育和分化途径中内源性基因的随机表达。我们将使用这种技术来确定秀丽隐杆线虫内胚层规范途径的表达波动。接下来，我们将使用该技术来更好地理解不完全外显现象。许多秀丽隐杆线虫突变体表现出不完全外显现象，其中只有一定比例的基因相同突变体表现出有缺陷的突变表型，而其余的生物体通常与野生型难以区分。我们将验证这样的假设，即这些突变允许控制发育的基因表达程序中的随机事件影响生物体的最终发育结果。最后，我们将使用mRNA计数方法来确定哺乳动物细胞命运决定的随机成分，使用糖皮质激素途径和造血祖细胞作为实验模型系统。第二个项目侧重于阐明microrna在控制基因表达波动中的作用。在过去的十年中，microrna在包括发育和分化在内的多种生物过程中发挥着至关重要的作用。项目2将探讨microRNAs控制基因表达波动的潜在作用。首先，我们将通过实验确定单个细胞中microRNA的浓度与其靶标之间的关系。其次，我们将验证一个假设，即为了抑制mRNA波动的翻译扩增，可以使用mirna选择性地降低可以显著波动的基因的翻译率。最后，我们将探讨microrna介导的反馈网络是否可以降低靶基因表达的波动。

The overarching goal of this proposal is to the explore the role of stochastic gene expression during development and cell differentiation using the nematode worm Caenorhabditis elegans and mammalian cell lines as experimental model systems. In the first program of this proposal I describe a single molecule mRNA detection technique that allows accurate measurements of stochastic expression of endogenous genes in developmental and differentiation pathways. We will use this technique to determine expression fluctuations in the C. elegans endodermal specification pathway. Next, we will use this technology to better understand the phenomenon of incomplete penetrance. Many C. elegans mutants exhibit the phenomenon of incomplete penetrance, where only a certain fraction of genetically identical mutants exhibit the defective mutant phenotype, while the remaining organisms are often indistinguishable from the wild-type. We will test the hypothesis that these mutants allow stochastic events in the gene expression program controlling development to influence the organism's ultimate developmental outcome. Finally we will use the mRNA counting method to determine the stochastic component of mammalian cell fate decisions using the glucocorticoid pathway and hemapoietic progenitor cells as experimental model systems. The second program focuses on elucidating the roles of microRNAs in controlling gene expression fluctuations. During the last decade it has become clear that microRNAs play crucial roles in diverse biological processes including development and differentiation. Program 2 will explore the potential roles for microRNAs controlling gene expression fluctuations. First, we will experimentally determine the relationship between the concentration of a microRNA and its target in a single cell. Second, we will test the hypothesis that in order to dampen translational amplification of mRNA fluctuations, miRNAs may be employed to selectively lower the translation rate for genes that can fluctuate significantly. Finally, we will explore if microRNA-mediated feedback networks can lower fluctuations in target gene expression.