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.

项目摘要 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（GRNs）在其中发挥着重要作用。然而，分子 从干细胞到分化的整个途径的调节从未被任何组织所定义。的 拟南芥根具有简单的结构和明确的干细胞生态位，是研究植物根分化的一个易处理的模型。 细胞命运的转录调控。二十多年的工作已经概述了GRN， 内皮层（一种类似于哺乳动物上皮的组织）的增殖和特化。这个GRN是 足够详细地映射到现在的数学模型的动态。然而，关键问题仍然存在， 关于下游分化事件。这些包括什么调节控制内胚层命运稳定 差异化？这些调节因子与控制stem的转录事件有多密切 细胞增殖？直到最近，技术限制使得研究分子动力学非常困难 在整个器官或生物体的背景下，单个细胞类型的潜在发育。研究提出， 这里利用成像和转录谱的新进展来研究蛋白质和基因表达 细胞分辨率下的动力学这项建议的总体目标是扩大内胚层的拓扑结构， GRN和开始量化分化背后的动力学。为实现这一目标，提出了具体 目标包括在致敏遗传背景中进行正向遗传筛选，以发现新的调节因子。 内胚层同一性（目标1）。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，从而提供系统水平的洞察整个途径的调节， 干细胞向分化的内皮层分化（Aim 2）。为了通过实验量化已知的和新的 在分化的背景下，最先进的成像技术将被用来跟踪变化 在活根中，随着时间的推移，蛋白质积累以细胞分辨率增加（目标3）。总之，这些目标应 扩展内胚层GRN的架构，并开始定义信息如何通过它流动， 协调细胞分化事件。杜克的智力刺激和协作环境 大学，再加上个性化的指导和支持实验室的技术，提供了一个 资源丰富的环境中进行拟议的实验。这项研究计划将促进 遗传学、系统水平转录调控、多维数据分析和 延时显微镜，从而为长期研究计划提供基础， GRN是基本发展过程的基础。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械理解，从而产生 方法和概念的进步，通知组织再生。