Architecture and Dynamics of a Gene Regulatory Network Controlling Cell Fate

控制细胞命运的基因调控网络的结构和动力学

• 批准号: 9908461
• 负责人: Rachel Maczis Shahan
• 金额: $ 6.49万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-02-01 至 2022-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9908461
• 关键词: Arabidopsis; Architecture; Cell Differentiation process; Cell Fate Control; Cell Maintenance; Cell Maturation; Cell Proliferation; Cells; Complex; Coupled; Data Analyses; Development; Developmental Process; Ectopic Expression; Embryonic Development; Endoderm Cell; Environment; Epithelial; Epithelium; Event; Expression Profiling; Foundations; Future; Gene Expression; Gene Expression Profiling; Genes; Genetic; Genetic Screening; Genetic Transcription; Goals; Image; Imaging Techniques; Individual; Knowledge; Lead; Lighting; Logic; Maps; Mentors; Methodology; Microscopy; Modeling; Molecular; Organ; Organism; Overlapping Genes; Pathway interactions; Pattern; Peptides; Plant Roots; Play; Process; Proteins; Regulation; Regulator Genes; Reporter; Research; Resolution; Resources; Role; Signal Transduction; Signaling Molecule; Structure; Study models; System; Technology; Testing; Time; TimeLine; Tissues; Training; Transcriptional Regulation; Universities; Work; Zebrafish; cancer cell; cell type; collaborative environment; design; differential expression; experimental study; forward genetics; genetic approach; insight; mathematical model; molecular dynamics; multidimensional data; mutant; non-Native; novel; organ regeneration; pluripotency; programs; protein expression; single-cell RNA sequencing; stem cell differentiation; stem cell niche; stem cell proliferation; stem cells; tissue regeneration; transcription factor

Project Summary Cell fate acquisition is a fundamental developmental process in all multicellular organisms and a growing body of evidence indicates that gene regulatory networks (GRNs) play an important role. However, the molecular regulation of the entire pathway from stem cell to differentiation has never been defined for any tissue. The Arabidopsis root, with its simple structure and defined stem cell niche, is a tractable model for studying the transcriptional regulation of cell fate. Over two decades of work have outlined the GRN that orchestrates cell proliferation and specification of the endodermis, a tissue analogous to the mammalian epithelium. This GRN is mapped in sufficient detail to now mathematically model its dynamics. However, crucial questions remain regarding downstream differentiation events. These include what regulators control endodermal fate stabilization and differentiation? And how closely are such regulators connected to the transcriptional events controlling stem cell proliferation? Until recently, technological constraints made it very difficult to study the molecular dynamics underlying development of a single cell type in the context of an entire organ or organism. The research proposed here utilizes new advances in imaging and transcriptional profiling to study protein and gene expression dynamics at cellular resolution. The overall goal of this proposal is to expand the topology of the endodermal GRN and begin to quantify the dynamics underlying differentiation. To achieve this goal, the proposed specific aims include conducting a forward genetic screen in a sensitized genetic background to uncover novel regulators of endodermal identity (Aim 1). In parallel, single cell RNA-sequencing experiments will define gene cascades underlying cell maturation events, thus providing systems-level insight into regulation of the entire pathway from stem cell to differentiated endodermis (Aim 2). To experimentally quantify the dynamics of known and novel regulators in the context of differentiation, state-of-the-art imaging techniques will be employed to track changes in protein accumulation over time at a cellular resolution in living roots (Aim 3). Together, these aims should expand the architecture of the endodermal GRN and begin to define how information flows through it to orchestrate cell differentiation events. The intellectually stimulating and collaborative environment of Duke University, coupled with individualized mentoring and enabling technology in the sponsoring lab, provide a resource-rich environment in which to conduct the proposed experiments. This research plan will facilitate advanced training in genetics, systems-level transcriptional regulation, multi-dimensional data analysis, and time-lapse microscopy, thereby providing the foundation for a long-term research program to dissect and model the GRNs underlying fundamental developmental processes. Insights gained from this work will deepen our mechanistic understanding of how stem cell progeny traverse the pathway to differentiation, thereby producing methodological and conceptual advances to inform tissue regeneration.

项目概要 细胞命运获得是所有多细胞生物和生长体的基本发育过程 大量证据表明基因调控网络（GRN）发挥着重要作用。然而，分子 对于任何组织，从干细胞到分化的整个途径的调节从未被定义。这 拟南芥根结构简单，干细胞生态位明确，是研究植物根系的易处理模型。 细胞命运的转录调控。二十多年的工作已经概述了协调细胞的 GRN 内皮层（类似于哺乳动物上皮的组织）的增殖和规范。这个GRN是 绘制得足够详细，现在可以对其动态进行数学建模。然而，关键问题仍然存在 关于下游分化事件。其中包括控制内胚层命运稳定的调节因子 和差异化？这些调节因子与控制茎的转录事件的联系有多紧密 细胞增殖？直到最近，技术限制使得研究分子动力学变得非常困难 单个细胞类型在整个器官或生物体中的潜在发育。研究提出 这里利用成像和转录分析的新进展来研究蛋白质和基因表达 细胞分辨率的动力学。该提案的总体目标是扩展内胚层的拓扑结构 GRN 并开始量化差异化背后的动态。为实现这一目标，建议具体 目标包括在敏感的遗传背景下进行正向遗传筛选，以发现新的调节因子 内胚层特性（目标 1）。与此同时，单细胞 RNA 测序实验将定义基因级联 潜在的细胞成熟事件，从而提供对整个途径的调节的系统级洞察 干细胞分化为内皮层（目标 2）。通过实验量化已知和新颖的动态 在差异化背景下，监管机构将采用最先进的成像技术来跟踪变化 活根中蛋白质随着时间的推移以细胞分辨率积累（目标 3）。这些目标应该共同实现 扩展内胚层 GRN 的架构并开始定义信息如何流经它 协调细胞分化事件。杜克大学的智力刺激和协作环境 大学加上赞助实验室的个性化指导和支持技术，提供了 资源丰富的环境可以进行所提出的实验。该研究计划将有助于 遗传学、系统级转录调控、多维数据分析等方面的高级培训 延时显微镜，从而为长期研究计划的解剖和建模提供基础 GRNs 是基本发育过程的基础。从这项工作中获得的见解将加深我们的 对干细胞后代如何穿越分化途径的机制理解，从而产生 为组织再生提供信息的方法和概念进展。