Bridging the gap between transcriptional activation and cell fate specification in the Drosophila visual system

细胞命运的指定是通过严格调控关键转录因子的表达来实现的 发展中的层次、时间和地点。这些决定因素的表达受以下因素控制 促进剂和促进剂。这项建议集中在两种情况下，基因表达的水平和时间 在果蝇的视觉系统中，对细胞命运的规定尤为重要。许多突变都有 已被证明会导致异常的基因调控和细胞命运，从而更好地理解这些 这些过程与理解疾病的遗传基础高度相关。 视觉系统中随机基因激活的实时成像：在目标1中，我将检查随机的 果蝇视网膜中细胞命运的规范，否则等同的R7光感受器前体 两种命运中对色觉很重要的一种特殊概率。这一决定由 无刺转录因子的随机、细胞内源性表达。我将研究两个可能的来源 无刺随机性：启动子水平转录激活的可变性，以及 染色质可及性。为了量化潜在的转录动态，我将成像基因 使用基于MS2和PP7的实时转录成像系统实时表达。MS2实时成像 最近对果蝇胚胎基因调控的研究进行了革命性的研究，提供了一个新的水平 定量测量。这种方法与CRISPR/Cas9对无脊椎动物的直接标记相结合 轨迹将使我能够在这个细胞命运决定中确定随机性的起源。 影响随机命运的因素：在目标2中，我将直接测试两个预测差异有多大的模型 影响无骨气表达概率的因素。为了直接测试转录起始与转录启动的作用。 染色质状态，我将使用CRISPR/Cas9修改无刺基本启动子，将其替换为 已被证明在果蝇中或多或少强健地启动表达的特征启动子 胚胎，例如通过招募暂停的RNA聚合酶II。这将使我能够测试 随机命运决策中的推动者。如果随机结果不受此类更改的影响，我将测试 局部染色质状态通过局部、有针对性地改变染色质状态来评估对 产生的命运的随机比率。这一方法将得到转录的实时成像的补充， 将为评估具体修改的数量影响提供另一种手段。 研究转录时间和水平在神经母细胞命运中的时间转换中的作用：目标3 我将研究延髓神经母细胞中五种不同转录因子之间的一系列时间转换 在苍蝇大脑中产生神经多样性。尽管我们知道它们之间存在交叉监管 时间转录因子，为这些转变计时的机制仍不清楚。我会测试一下 个体因素表达的水平和变异性对每个时间点的时间或输出的影响 窗户。MS2成像与荧光蛋白标签结合使用将能够直接比较 随着序列中下一个因子转录的启动，一个时间因子的表达水平。这 将提供对管理时间窗口及其转换的定时的机制的洞察。一个 然后将使用基于CRISPR的方法来修改单个转录因子的激活量 在时间序列中测试转录水平或可变性是否在控制持续时间中起作用 时间窗口。 了解基因调控如何影响细胞命运将有助于更好地理解细胞命运是如何 坚定不移地坚持下去。最终，我希望这将导致新的编程策略或 有目的地对细胞命运进行重新编程。K99过渡奖将允许我获得额外的 对定量成像方法进行培训，并为这种新方法的工具开发提供时间 研究了随机命运规则和时间命运规范。 这项提议的培训阶段将在我的导师克劳德·德斯普兰博士的实验室进行。我可以的 想不到更好的地方来研究关于果蝇视觉发育的令人兴奋和引人入胜的问题 系统，或用于工具开发和与有洞察力的同事协作。纽约大学纽约大学研究中心 发育遗传学、生物成像设备以及邻近的基因组学和系统中心 生物学为拟议的工作提供了必要的设施和设备。我完全享受到了 研究生院和博士后学术研究的挑战和兴奋。这些经历， 并有机会与其他人分享它们，鼓励我以以下目标规划我的职业生涯 建立独立的学术研究实验室。

Cell fate specification occurs through the tightly regulated expression of key transcription factors at precise levels, times, and places during development. The expression of these determinants is controlled by enhancers and promoters. This proposal focuses on two cases where the level and timing of gene expression are especially important for cell fate specification in the Drosophila visual system. A number of mutations have been shown to cause abnormal gene regulation and cell fate, making a better understanding of these processes highly relevant to understanding the genetic basis of disease. Live imaging of stochastic gene activation in the visual system: In Aim 1, I will examine the stochastic specification of cell fate in the Drosophila retina, where otherwise equivalent R7 photoreceptor precursors have a particular probability of taking one of two fates important for color vision. This decision is controlled by the stochastic, cell-intrinsic expression of the transcription factor Spineless. I will examine two possible sources of spineless stochasticity: variability in transcriptional activation at the level of the promoter, and variation in chromatin accessibility. In order to quantify the underlying transcriptional dynamics, I will image gene expression in real time using the MS2 and PP7-based live transcriptional imaging systems. MS2 live imaging has recently revolutionized the study of gene regulation in the Drosophila embryo by providing a new level of quantitative measurement. This approach combined with direct tagging by CRISPR/Cas9 of the spineless locus will allow me to determine the origin of stochasticity in this cell fate decision. Factors that influence stochastic fate: In Aim 2, I will directly test two models that predict how different factors influence the probability of spineless expression. To directly test the role of transcriptional initiation vs. chromatin state, I will modify the spineless basal promoter using CRISPR/Cas9 by replacing it with characterized promoters that have been shown to initiate expression more or less robustly in the Drosophila embryo, for instance through recruitment of paused RNA Polymerase II. This will allow me to test the role of the promoter in stochastic fate decisions. If stochastic outcomes are unaffected by such changes, I will test the role of local chromatin state via local, targeted changes in chromatin state to evaluate the effect on the stochastic ratio of fates produced. This approach will be complemented by live imaging of transcription, which will provide an additional means of assessing the quantitative effects of specific modifications. Studying the role of transcriptional timing and levels in temporal transitions in neuroblast fate: Aim 3 will examine a series of temporal transitions between five different transcription factors in medulla neuroblasts that generate neural diversity in the fly brain. Although we know that there is cross regulation among these temporal transcription factors, the mechanisms that time these transitions are still unknown. I will test the influence of the level and variability of expression of individual factors on the timing or output of each temporal window. MS2 imaging used in combination with fluorescent protein tags will enable direct comparison between expression levels of one temporal factor with the initiation of transcription of the next factor in the series. This will provide insight into mechanisms that govern the timing the temporal windows and their transitions. A CRISPR-based approach will then be used to modify the amount of activation of individual transcription factors in the temporal series to test whether transcriptional levels or variability play a role in controlling the duration of the temporal window. Understanding how gene regulation influences cell fate will lead to a better understanding of how cell fate is determined and maintained. Ultimately, I hope that this will lead to new strategies for programming or reprograming cell fates in a purposeful way. A K99 Transition Award would allow me to receive additional training in quantitative imaging methods and provide time for tool development for this novel approach to studying stochastic fate regulation and temporal fate specification. The training phase of this proposal will be performed in the laboratory of my mentor Dr. Claude Desplan. I can think of no better place to study exciting and engaging questions in the development of the Drosophila visual system, or for tool development and collaboration with insightful colleagues. The NYU Center for Developmental Genetics, Biological Imaging Facility, and neighboring Center for Genomics and Systems Biology provide the facilities and equipment necessary for the proposed work. I have thoroughly enjoyed the challenges and excitement of academic research in graduate school and as a postdoc. These experiences, and the opportunity to share them with others, have encouraged me to plan my career with the goal of establishing an independent academic research laboratory.