Notch Pathway Regulation of Intestinal Epithelial Cell Homeostasis

项目摘要 这个修订后的新R 01应用程序的重点是调节肠道上皮细胞稳态的途径。 该项目研究Notch信号通路在调节肠道干细胞中的作用， 检查由Lgr 5、Olfm 4、Bmi 1和Lrig 1标记的活性和静止干细胞群体。 Notch是公认的通过调节分泌谱系来调节细胞命运的关键调节因子- 特异性转录因子Atoh 1。我们最近的研究表明，Notch也发挥着独特的作用， 在维持肠干细胞的Atoh 1独立的机制，这一发现服务于 作为本申请的基本原理。我们已经确定，慢性Notch抑制， 成年小鼠导致祖细胞增殖减少， 隐窝基底柱状细胞（CBC）标记Olfm 4。此外，这些研究表明， 活跃循环的隐窝基底柱状干细胞（CBC）是直接的细胞Notch靶点， Notch信号转导的缺失导致CBC的丢失。该提案的主要假设是： 信号传导调节从ISC到命运祖细胞的转变，Notch信号传导的缺失导致 干细胞库的耗尽和静止干细胞的活化以补充库。为了验证这一 假设研究将检查完整的小鼠模型（遗传和药理学），小鼠肠 类器官，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中的发现相似。遗传操作 并且小鼠中ISC细胞群的标记将利用对活性或特异性的Cre驱动子。 静止ISC。基因缺失小鼠的品系将允许Notch途径的缺失或激活 特定ISC细胞群中的组分。RNA-Seq基因表达谱将用于鉴定 Notch-responsive基因和计算模型将为我们理解祖细胞 种群动态提出了三个具体目标：（1）测试CBC干细胞的假设， 受Notch信号传导的动态调节;（2）检验Notch调节细胞凋亡的能力的假设。 静止干细胞以重新填充活跃的循环干细胞和祖细胞库;（3）测试 Notch信号转导调控体外人细胞分化和干细胞功能假说 衍生的肠类器官这些研究对于我们进一步了解 不同的肠道干细胞群体，并表征Notch信号传导的重要性， 人肠上皮细胞稳态。由于目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， 避免可能由该途径的系统性破坏引起的肠毒性。

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.