Determining how noisy gene expression controls stochastic fate choices during development

发育期间的细胞命运规格通常被认为是由以下因素驱动的高度可再现的过程： 细胞谱系和信号传导。细胞多样性也可以由细胞内固有的分子噪音（即变异性）引起。 随机细胞命运指定过程中的基因表达，其中细胞随机选择两种或更多种 命运与谱系和信号机制相比，对基因表达中的噪声如何影响基因表达的知之甚少。 在发育过程中决定命运。 本计画旨在探讨果蝇视觉系统中细胞命运的随机特化 黑腹菌蝇眼包含一个简单的、随机的、二元的命运选择。眼睛是由两个随机拼接而成的 颜色检测光感受器亚型，由不同的光检测视紫红质蛋白的表达定义。 这种二元决定是由转录因子Spineless（Ss）控制的，其以随机的方式表达。 R7光感受器的子集（65%）。基因spineless（ss）的随机表达在一个 通过两个增强子元件（“早期”和“晚期”增强子）和一个增强子元件， 转录抑制子，Klu. ss的早期表达是嘈杂的，在基因水平上产生变异， 前体R7细胞之间的表达。通过操纵DNA元件（增强子和沉默子）和Klu 转录因子，SS早期表达的强度可以被调节。值得注意的是，这些早期的操纵 表达引起终末分化的R7细胞中Ss的开/关比率的变化。基于这些 研究发现，假设R7祖细胞（即早期）中ss表达的可变水平设定了R7祖细胞的表达水平。 在终末分化的R7细胞中的开/关表达频率（即晚期）。该项目旨在解决 促进早期β表达的变异性以及这种变异性的后果。 基因表达噪音的主要来源来自转录过程。转录是 固有的随机性，并在基因之间的振幅，频率和持续时间不同的突发中发生， 细胞三色RNA荧光原位杂交将用于监测单个细胞中的转录 为了确定Klu和不同的DNA元件如何控制固定组织中早期ss表达的变异（目的 1）。为了从机制上理解转录如何影响早期表达变异，本项目 目的是通过使用MS 2/MCP系统和实时成像来可视化早期ss表达（目的1）。最后，一个链接 早期表达和终末细胞命运之间的关系将通过真实的监测内源性ss表达来确定。 从前体细胞阶段到终末分化的时间（Aim 2）。该项目将在 约翰霍普金斯大学小罗伯特J.约翰斯顿的实验室。申请人将接受额外的培训， 普林斯顿大学和巴斯德研究所的合作者（托马斯格雷戈博士）。该项目的结果 将阐明在视网膜发育过程中控制ss表达的机制，特别是， 在后生动物发育过程中，分子变异性驱动随机细胞命运决定

Cell fate specification during development is often thought of as a highly reproducible process driven by cell lineage and signaling. Cellular diversity can also arise from the inherent molecular noise (i.e. variability) in gene expression during stochastic cell fate specification, where a cell randomly chooses between two or more fates. Compared to lineage and signaling mechanisms, very little is known about how noise in gene expression drives fate decisions during development. This project aims to address stochastic cell fate specification in the visual system of Drosophila melanogaster. The fly eye contains a simple, stochastic, binary fate choice. The eye is a random mosaic of two color-detecting photoreceptor subtypes, defined by expression of different light-detecting Rhodopsin proteins. This binary decision is controlled by the transcription factor Spineless (Ss), which is expressed in a random subset (65%) of R7 photoreceptors. Stochastic expression of the gene spineless (ss) is controlled in a temporal manner throughout development by two enhancer elements (“early” and “late” enhancers), and a transcriptional repressor, Klu. Early expression of ss is noisy, producing variability in the levels of gene expression between precursor R7 cells. By manipulating DNA elements (enhancers and silencers) and the Klu transcription factor, the strength of ss expression early can be tuned. Remarkably, these manipulations of early expression cause changes in the on/off ratio of Ss in terminally differentiated R7 cells. Based on these findings, it is hypothesized that variable levels of ss expression amongst R7 progenitors (i.e. early) sets the on/off expression frequency in terminally differentiated R7 cells (i.e. late). This project aims to address what promotes early ss expression variability as well as the consequences of this variability. A major source of gene expression noise arises from the process of transcription. Transcription is inherently stochastic, and occurs in bursts that vary in amplitude, frequency, and duration between genes and cells. Three-color RNA fluorescence in situ hybridization will be used to monitor transcription in individual cells to determine how Klu and different DNA elements control variation in in early ss expression in fixed tissue (Aim 1). To gain a mechanistic understanding of how transcriptional affects early expression variability, this project aims to visualize early ss expression by using the MS2/MCP system and live imaging (Aim 1). Finally, a link between early expression and terminal cell fate will be made by monitoring endogenous ss expression in real time from the precursor cell stage through terminal differentiation (Aim 2). This project will be carried out at Johns Hopkins University in the lab of Robert J. Johnston Jr. The applicant will receive additional training from collaborators at Princeton University and the Institut Pasteur (Dr. Thomas Gregor). The results of this project will elucidate the mechanisms controlling expression of ss during retina development, in particular, how molecular variability drives a stochastic cell fate decision during metazoan development