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稳态的途径。该项目研究了Notch信号通路在肠干细胞调控中的作用，检测了由Lgr5、Olfm4、Bmi1和Lrig1标记的活跃和静止干细胞群。Notch是通过调节分泌谱系特异性转录因子Atoh1调控细胞命运的关键调节因子。我们最近的研究表明，Notch在肠干细胞的维持中也发挥着独特的作用，其机制不依赖于atoh1，这一发现可作为其应用的基本原理。我们已经确定，成年小鼠的慢性Notch抑制导致祖细胞增殖减少，隐窝基底柱状细胞（CBC）标记物Olfm4的表达显著降低。此外，这些研究表明，活跃循环的隐窝基柱状干细胞（CBC）是细胞Notch的直接靶点，Notch信号的破坏会导致CBC的丢失。该提议的主要假设是：Notch信号调节从ISC到命运祖细胞的转变，Notch信号的缺失导致干细胞库的耗尽和静止干细胞的激活来补充干细胞库。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），小鼠肠道类器官，重要的是，一个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发现相似。小鼠ISC细胞群的遗传操作和标记将利用激活或静止ISCs特有的Cre驱动程序。弯曲基因小鼠的菌株将允许在特定的ISC细胞群中缺失或激活Notch通路成分。RNA-Seq基因表达谱将用于鉴定缺口反应基因，计算模型将告知我们对祖细胞群体动态的理解。提出了三个具体目标：(1)验证CBC干细胞受Notch信号动态调节的假设；(2)验证Notch调节静止干细胞重新填充活跃循环干细胞和祖细胞库的能力；(3)验证Notch信号调控人体外肠类器官细胞分化和干细胞功能的假说。这些研究对于我们进一步了解不同肠道干细胞群的功能以及表征Notch信号在人类肠道上皮细胞稳态中的重要性具有重要意义。由于目前的人类疾病治疗都是针对Notch通路设计的，因此了解该通路在肠道中的功能是至关重要的，以避免由于该通路的系统性破坏而导致的肠道毒性。

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.