Notch Pathway Regulation of Intestinal Epithelial Cell Homeostasis

描述(由申请人提供)：本修订后的新R01申请侧重于调节肠上皮细胞动态平衡的途径。该项目研究TH Notch信号通路在肠道干细胞调控中的作用，检测由Lgr5、Olfm4、Bmi1和Lrig1标记的活跃和静止的干细胞群体。Noch通过调节分泌谱系特异的转录因子Atoh1，被认为是细胞命运的关键调节因子。我们最近的研究表明，Notch在维持肠道干细胞的Atoh1非依赖性机制中也发挥着明显的作用，这一发现是这一应用的基本原理。我们已经证实，在成年小鼠中，长期抑制Notch会导致祖细胞增殖减少，并显著减少隐窝基底柱状细胞(CBC)标记Olfm4的表达。此外，这些研究表明，主动循环的隐窝基底部的柱状干细胞(CBC)是一个直接的细胞Notch靶点，而Notch信号的中断会导致CBCs的丢失。这一提议的压倒一切的假设是：Notch信号调节从ISC到命中注定的祖细胞的转变，而Notch信号的丢失会导致干细胞库的耗尽，并激活静止的干细胞来补充干细胞池。为了验证这一假设，研究将检查完整的小鼠模型(遗传和药物)、小鼠肠道器官，以及重要的是，一个新的人类器官模型。该项目将首次测试Notch对人类肠道的调节是否与在小鼠身上的发现一致。对小鼠ISC细胞群体的遗传操作和标记将利用针对活跃或静止ISCs的Cre驱动因素。FLOXED基因小鼠的品系将允许特定ISC细胞群体中Notch途径组件的缺失或激活。RNA-Seq基因表达谱将用于识别Notch反应基因，计算建模将使我们了解祖细胞群体动力学。提出了三个具体目标：(1)检验CBC干细胞受Notch信号动态调节的假说；(2)检验Notch调节静止干细胞重新填充活跃的周期干细胞库和祖细胞库的能力的假说；(3)检验Notch信号调节人体外来源的肠道器官中细胞分化和干细胞功能的假说。这些研究对于进一步了解不同肠道干细胞群的功能以及研究Notch信号对人类肠上皮细胞内环境平衡的重要性具有重要意义。由于目前的人类疾病治疗正在设计以Notch途径为靶点，了解该途径在肠道中的功能至关重要，以避免该途径的全身干扰可能导致的肠道毒性。

DESCRIPTION (provided by applicant): This revised new R01 application focuses on pathways regulating intestinal epithelial cell homeostasis. The project investigates the role of th 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 differentiatin 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 population 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.