Notch Signaling Regulates Generation of Progenitors from Intestinal Stem Cells

描述（由申请人提供）：该申请申请为Alexis Carulli女士在胃肠生理学领域提供奖学金支持，重点研究肠上皮细胞稳态和对损伤和修复的反应。该研究项目探讨了Notch信号通路在肠干细胞（ISCs）调控中的作用。我们最近的研究表明，Notch在ISC维持和下游细胞命运决定中都具有关键功能。我们已经证明，成年小鼠的慢性Notch抑制导致分泌细胞类型数量增加，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抑制导致Olfm4的短暂敲低，分泌细胞增加，细胞增殖长期增加，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，本研究将验证以下假设：Notch信号通路调节从ISC到命运祖细胞的转变，Notch信号通路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，将采用Notch抑制的遗传和药理学小鼠模型。ISC细胞群体的遗传操作和标记将利用活跃或静止的Cre驱动因子

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.