Notch Signaling Regulates Generation of Progenitors from Intestinal Stem Cells

描述（由申请人提供）：本NRSA F30申请要求Alexis Carulli女士在胃肠道生理学领域获得奖学金支持，重点研究肠上皮细胞稳态和对损伤和修复的反应。该研究项目调查了Notch信号通路在调节肠干细胞（ISCs）中的作用。我们最近的研究表明，Notch对ISC维持以及下游细胞命运决定都具有关键功能。我们已经表明，成年小鼠中的慢性Notch抑制导致分泌细胞类型的数量增加，细胞增殖减少，以及ISC标志物Olfm 4的显著减少。我的初步数据表明，Olfm 4反应是Notch 1（N1）特异性的，这表明N1是ISC的主要Notch受体。此外，我已经表明，急性Notch抑制导致Olfm 4的瞬时敲低、分泌细胞增加和细胞增殖的长期持续增加，这与慢性Notch抑制所见的抑制形成鲜明对比。很明显，Notch信号在ISC中起着重要作用，但具体的细胞反应尚不清楚。为了进一步了解Notch-ISC调控的潜在细胞动力学，本提案将测试以下假设：Notch信号传导途径调节从ISC到命运祖细胞的转变，并且Notch信号传导的丧失导致干细胞池的耗尽和静止干细胞的激活以补充池。为了检验这一假设，将采用Notch抑制的遗传和药理学小鼠模型。ISC细胞群的遗传操作和标记将利用对活性或静止细胞特异性的Cre驱动子。 ISCs。突变基因小鼠的品系将允许特定ISC细胞群中Notch途径组分的缺失。提出了三个具体目标：（1）测试Notch 1受体信号传导需要维持ISCs和N1和N2受体信号传导需要调节细胞命运肠上皮细胞谱系的假设。(2)检验Notch抑制导致的ISC丧失导致静止干细胞活化以重新填充活跃的循环干细胞和祖细胞库的假设。(3)开发数学模型以确定Notch信号传导是否调节不对称干细胞分裂与祖细胞的对称形成。这些目标将为我提供哺乳动物生理学、分子生物学和数学建模方面的全面培训，推动我作为一名物理学家和科学家的职业生涯。

DESCRIPTION (provided by applicant): This NRSA F30 application requests fellowship support for Ms. Alexis Carulli in the field of gastrointestinal physiology, focusing on the study o intestinal epithelial cell homeostasis and responses to damage and repair. The research project investigates the role of the Notch signaling pathway for regulation of intestinal stem cells (ISCs) Our recent studies have shown that Notch has critical functions for both ISC maintenance as well as downstream cell fate decisions. We have shown that chronic Notch inhibition in adult mice results in increased numbers of secretory cell types, decreased cellular proliferation, and a marked decrease in the ISC marker Olfm4. My preliminary data suggests that the Olfm4 response is Notch1(N1)- specific, suggesting that N1 is the predominate Notch receptor for the ISC. Furthermore, I have shown that acute Notch inhibition leads to transient knockdown of Olfm4, increased secretory cells, and a long-lasting increase in cellular proliferation, a stark contrast to the suppression seen with chronic Notch inhibition. It is clear that Notch signaling plays an important role in the ISC, but the specific cellular responses are unknown. To further understand the underlying cellular dynamics with Notch-ISC regulation, this proposal will test the following hypothesis: The Notch signaling pathway 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 both genetic and pharmacologic mouse models of Notch inhibition will be employed. Genetic manipulation and marking of ISC cell populations will take advantage of Cre drivers specific for active or quiescent ISCs. Strains of floxed-gene mice will allow deletion of Notch pathway components in specific ISC cell populations. Three specific aims are proposed: (1) Test the hypothesis that Notch1 receptor signaling is required to maintain ISCs and that both N1 and N2 receptor signaling is required for regulation of cell fate to enterocyte lineages. (2) Test the hypothesis that ISC loss by Notch inhibition leads to activation of a quiescent stem cell to repopulate the active cycling stem and progenitor pool. (3) Develop a mathematical model to determine if Notch signaling regulates asymmetric stem cell division vs. symmetric formation of progenitor cells. United, these aims will provide comprehensive training in mammalian physiology, molecular biology, and mathematical modeling, advancing my career as a physician-scientist.