Notch Pathway Regulation of Intestinal Epithelial Cell Homeostasis

肠上皮细胞稳态的Notch通路调节

• 批准号: 8631158
• 负责人: LINDA C. SAMUELSON
• 金额: $ 32.98万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-18 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8631158
• 关键词: Adult; Apoptosis; Biological Assay; Biological Models; Cell Count; Cell Differentiation process; Cell Fate Control; Cell Lineage; Cell Proliferation; Cell physiology; Cells; Chronic; Columnar Cell; Computer Simulation; Data; Endocrine; Enterocytes; Epithelial Cells; Gene Expression Profiling; Genes; Genetic; Genetic Models; Growth; Homeostasis; Human; Immunohistochemistry; In Vitro; Intestines; Kinetics; Laboratory Research; Maintenance; Malignant Neoplasms; Measures; Modeling; Mus; Notch Signaling Pathway; Organoids; Pathway interactions; Play; Population; Population Dynamics; Process; Radiation; Radiation Injuries; Receptor Signaling; Regulation; Reverse Transcriptase Polymerase Chain Reaction; Role; Secretory Cell; Signal Pathway; Signal Transduction; Source; Stem cells; System; Testing; Time; Tissues; Toxic effect; Villus; base; cell type; design; genetic manipulation; human disease; human stem cells; intestinal epithelium; mathematical model; mouse model; notch protein; progenitor; public health relevance; repaired; response; stem; stem cell fate; stem cell population; transcription factor; transcriptome sequencing

Project Summary This revised new R01 application focuses on pathways regulating intestinal epithelial cell homeostasis. The project investigates the role of the Notch signaling pathway for regulation of intestinal stem cells, examining both active and quiescent stem cell populations marked by Lgr5, Olfm4, Bmi1, and Lrig1. Notch is well established as a critical regulator of cell fate via regulation of the secretory lineage- specific transcription factor Atoh1. Our recent studies have shown that Notch also plays a distinct role in the maintenance of intestinal stem cells in an Atoh1-independent mechanism and this finding serves as the underlying rationale for this application. We have established that chronic Notch inhibition in adult mice results in decreased progenitor cell proliferation and a marked decrease in expression of the crypt base columnar cell (CBC) marker Olfm4. Furthermore, these studies demonstrated that the actively cycling crypt base columnar stem cell (CBC) is a direct cellular Notch target and that disruption of Notch signaling results in loss of CBCs. The overriding hypothesis for this proposal is: Notch signaling regulates the transition from ISC to fated progenitor cells and loss of Notch signaling leads to depletion of the stem cell pool and activation of quiescent stem cells to replenish the pool. To test this hypothesis studies will examine intact mouse models (genetic and pharmacologic), mouse intestinal organoids and, importantly, a new human organoid model. This project will, for the first time, test whether Notch regulation of human intestine parallels the findings in the mouse. Genetic manipulation and marking of ISC cell populations in mouse will take advantage of Cre drivers specific for active or quiescent ISCs. Strains of floxed-gene mice will allow deletion or activation of Notch pathway components in specific ISC cell populations. RNA-Seq gene expression profiling will be used to identify Notch-responsive genes and computational modeling will inform our understanding of progenitor cell population dynamics. Three specific aims are proposed: (1) Test the hypothesis that CBC stem cells are dynamically regulated by Notch signaling; (2) Test the hypothesis that Notch regulates the ability of quiescent stem cells to repopulate the active cycling stem and progenitor cell pool; (3) Test the hypothesis that Notch signaling regulates cellular differentiation and stem cell function in human in vitro derived intestinal organoids. These studies are important to further our understanding of the function of different intestinal stem cell populations and to characterize the importance of Notch signaling for human intestinal epithelial cell homeostasis. Since current human disease therapies are under design to target the Notch pathway, it is crucial to understand the function of this pathway in the intestine to avoid the intestinal toxicity that can result from systemic disruption of this pathway.

项目概要 此次修订后的新 R01 应用重点关注调节肠上皮细胞稳态的途径。 该项目研究了 Notch 信号通路在调节肠道干细胞中的作用， 检查由 Lgr5、Olfm4、Bmi1 和 Lrig1 标记的活性和静止干细胞群。 Notch 已被广泛认为是通过调节分泌谱系来调节细胞命运的关键因子。 特异性转录因子Atoh1。我们最近的研究表明，Notch 也发挥着独特的作用 以 Atoh1 独立机制维持肠道干细胞，这一发现有助于 作为此应用程序的基本原理。我们已经确定，慢性Notch抑制 成年小鼠导致祖细胞增殖减少，并且 隐窝基底柱状细胞 (CBC) 标记 Olfm4。此外，这些研究表明 活跃循环的隐窝基底柱状干细胞 (CBC) 是一个直接的细胞 Notch 目标，并且这种破坏 Notch 信号传导导致 CBC 丢失。该提案最重要的假设是：Notch 信号传导调节从 ISC 到命运祖细胞的转变，Notch 信号传导的丧失会导致 干细胞库的耗尽和静止干细胞的激活以补充干细胞库。为了测试这个 假设研究将检查完整的小鼠模型（遗传和药理学）、小鼠肠道 类器官，更重要的是，一种新的人类类器官模型。该项目将首次测试 Notch 对人类肠道的调节是否与小鼠的研究结果相似。基因操纵 小鼠 ISC 细胞群的标记将利用特定于活性或 静态 ISC。 floxed基因小鼠品系将允许Notch通路缺失或激活 特定 ISC 细胞群中的成分。 RNA-Seq 基因表达谱将用于鉴定 Notch响应基因和计算模型将有助于我们了解祖细胞 人口动态。提出了三个具体目标：（1）检验 CBC 干细胞的假设 由Notch信号动态调节； (2)检验Notch调节能力的假设 静止干细胞重新填充活跃循环干细胞和祖细胞池； (3) 测试 Notch 信号传导在体外调节人类细胞分化和干细胞功能的假设 衍生的肠道类器官。这些研究对于加深我们对功能的理解具有重要意义。 不同的肠道干细胞群并表征Notch信号传导的重要性 人肠上皮细胞稳态。由于当前的人类疾病疗法正在设计中 为了靶向 Notch 通路，了解该通路在肠道中的功能至关重要 避免该途径的系统性破坏可能导致的肠道毒性。