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

弥合果蝇视觉系统中转录激活和细胞命运规范之间的差距

• 批准号: 9341328
• 负责人: Michael William Perry
• 金额: $ 13.58万
• 依托单位: NEW YORK UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2019-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9341328
• 关键词: Active Sites; Adult; Affect; Alpha Cell; Atherosclerosis; Award; Biological; Brain; CRISPR/Cas technology; Cells; Chromatin; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Color Visions; Complement; Complex; DNA; Development; Disease; Doctor of Philosophy; Drosophila genus; Embryo; Engineering; Enhancers; Ensure; Epigenetic Process; Equipment; Eye diseases; Feedback; Frequencies; Gene Abnormality; Gene Activation; Gene Components; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Transcription; Genomics; Goals; Hemophilia A; Image; Imaging Device; Individual; Laboratories; Laboratory Research; Lead; Light; Measurement; Mentors; Modeling; Modification; Mutation; Nucleic Acid Regulatory Sequences; Osteoporosis; Outcome; Output; Phase; Photoreceptors; Physiology; Play; Polydactyly; Postdoctoral Fellow; Probability; Process; Proteins; RNA Polymerase II; Recruitment Activity; Regulation; Regulator Genes; Research; Retina; Role; Schools; Series; Source; System; Systems Biology; Testing; Time; To specify; Training; Transcription Initiation; Transcriptional Activation; Transcriptional Regulation; Variant; Visual system structure; Work; base; blue cone monochromacy; career; cell fate specification; cell type; developmental genetics; experience; fly; human disease; imaging modality; imaging system; insight; mutant; neuroblast; neurogenesis; novel strategies; predictive modeling; promoter; quantitative imaging; relating to nervous system; response; time use; tool development; transcription factor

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.

细胞命运的特化是通过严格调控关键转录因子的精确表达来实现的。 开发过程中的级别、时间和地点。这些决定因素的表达由 增强子和启动子。这项建议集中在两种情况下，基因表达的水平和时间， 对果蝇视觉系统中的细胞命运规范尤为重要。一些突变 已被证明会导致异常的基因调控和细胞命运，使人们更好地了解这些 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，我将检查随机基因激活的情况。 果蝇视网膜中细胞命运的特化，在那里，在其他方面等同的R7感光细胞前体具有 一个特定的概率采取两个命运之一重要的颜色视觉。这一决定由 转录因子Spineless的随机细胞内在表达。我将研究两个可能的来源， 无脊椎随机性：在启动子水平上转录激活的变异性， 染色质可及性为了量化潜在的转录动力学，我将对基因进行成像， 使用基于MS 2和PP 7的活转录成像系统在真实的时间中检测表达。MS 2实时成像 最近彻底改变了果蝇胚胎基因调控的研究， 定量测量这种方法结合了CRISPR/Cas9对无脊椎动物的直接标记， 基因座将允许我确定细胞命运决定中随机性的起源。 影响随机命运的因素：在目标2中，我将直接测试两个模型， 影响懦弱表达的可能性。为了直接测试转录起始相对于转录起始的作用， 在染色质状态下，我将使用CRISPR/Cas9修饰无刺基底启动子，将其替换为 这些启动子在果蝇中或多或少地启动表达 胚胎，例如通过暂停的RNA聚合酶II的募集。这将使我能够测试的作用， 随机命运决定中的启动子。如果随机结果不受这些变化的影响，我将测试 局部染色质状态的作用，通过染色质状态的局部、靶向变化来评估对 随机产生的命运比率。这种方法将得到转录实时成像的补充， 将提供评估特定修改的定量效果的额外手段。 研究转录时间和水平在成神经细胞命运时间转换中的作用：目的3 将研究在髓质成神经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。虽然我们知道这些之间存在交叉调节， 时间转录因子，这些转换的时间机制仍然未知。我将测试 个体因素表达的水平和可变性对每个时间点的时间或输出的影响 窗口与荧光蛋白标签组合使用的MS 2成像将能够直接比较 一个时间因子的表达水平与该系列中下一个因子的转录起始有关。这 将提供洞察机制，管理时间窗口及其过渡的时间。一 然后，基于CRISPR的方法将用于修改单个转录因子的激活量。 在时间序列，以测试是否转录水平或变异性发挥作用，在控制的持续时间， 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发生变化的。 确定和维护。最终，我希望这将导致新的规划战略， 有目的地改变细胞的命运K99过渡奖将使我能够获得额外的 定量成像方法的培训，并为这种新方法的工具开发提供时间， 研究随机命运调节和时间命运规范。 本提案的培训阶段将在我的导师Claude Desplan博士的实验室进行。我可以 我想没有比这更好的地方来研究果蝇视觉发育中令人兴奋和引人入胜的问题了 系统，或用于工具开发和与有洞察力的同事协作。纽约大学研究中心 发育遗传学，生物成像设施，以及邻近的基因组学和系统中心 生物学提供拟议工作所需的设施和设备。我已经彻底享受了 在研究生院和博士后的学术研究的挑战和兴奋。这些经历， 以及与他人分享的机会，鼓励我规划我的职业生涯， 建立独立的学术研究实验室。