Notch Signaling Regulates Generation of Progenitors from Intestinal Stem Cells

描述(由申请人提供)：本NRSA F30申请为Alexis Carulli女士申请胃肠生理领域的奖学金支持，重点是肠上皮细胞动态平衡以及损伤和修复反应的研究。该研究项目研究了Notch信号通路在肠道干细胞(ISCs)调控中的作用。我们最近的研究表明，Notch对ISC的维持以及下游细胞的命运决定都具有关键功能。我们已经证明，长期抑制成年小鼠的Notch会导致分泌细胞类型增加，细胞增殖减少，ISC标记物Olfm4显著减少。我的初步数据表明，Olfm4的反应是Notch1(N1)特异性的，这表明N1是ISC的主要Notch受体。此外，我已经证明，急性Notch抑制会导致Olfm4的瞬时下调，分泌细胞增加，细胞增殖长期增加，这与慢性Notch抑制形成鲜明对比。很明显，Notch信号在ISC中起着重要的作用，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，该提议将检验以下假设：Notch信号通路调节从ISC到命中祖细胞的转变，而Notch信号的丢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。为了验证这一假设，我们将采用Notch抑制的遗传学和药理学小鼠模型。ISC细胞群体的遗传操作和标记将利用针对活动或静止的CRE驱动程序 ISCS.FLOXED基因小鼠的品系将允许删除特定ISC细胞群中的Notch途径组件。提出了三个具体的目标：(1)验证Notch1受体信号是维持ISCs所必需的假设，以及N1和N2受体信号都是调节细胞向肠细胞系的命运所必需的假设。(2)验证通过Notch抑制导致ISC丢失导致静止干细胞激活以重新填充活跃的循环干和祖细胞池的假设。(3)建立一个数学模型来确定Notch信号是否调节干细胞的不对称分裂和祖细胞的对称形成。联合起来，这些目标将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，促进我作为一名内科科学家的职业生涯。

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.