Defining the Notch niche for Intestinal Stem cells

PROJECT SUMMARY This proposal seeks to train a postdoctoral fellow in the experimental technologies and conceptual knowledge required to become a successful academic researcher in gastrointestinal stem cell biology. The research proposal aims to elucidate the mechanisms of Notch signaling in immature and adult intestinal stem cells that regulate both stem cell and Paneth cell maintenance. Notch is an essential intestinal stem cell (ISC) niche signal that promotes ISC self-renewal to maintain the stem cell pool. Notch also regulates epithelial cell fate choice, cellular renewal during homeostasis, and crypt repair after injury, yet the cellular and molecular mechanisms underlying these processes are poorly understood. Notch signaling requires cell-cell contact and neighboring Paneth cells are thought to be the Notch niche cells at homeostasis through the expression of Notch ligands DLL1 and DLL4 on their surface. However, ISCs begin relying on Notch signal during gestation, well before Paneth cell development, and tolerate Paneth cell loss in the mature intestine. How the ISC Notch niche is maintained when Paneth cells are absent remains unknown. Based on my preliminary analysis of ISCs after Paneth cell loss, I hypothesize that in the absence of Paneth cells, both adult and immature stem cells maintain Notch signaling by juxtacrine signaling due to ISC expression of the Notch ligands DLL1 and DLL4. This hypothesis will be tested in Aim 1 to define the Notch niche for ISCs throughout development. Moreover, functional Notch targets in ISCs as well as crosstalk between ISCs and their niche cells remain understudied. Our previous studies suggested that Paneth cell maintenance is regulated by an external, Notch-dependent signal. In Aim 2, I will test the hypothesis that ISCs provide a Notch- dependent survival signal to the Paneth cells. These aims will harness genetically engineered in vivo and ex vivo mouse models and employ advanced transcriptomics, cell imaging, and drug screening approaches. Altogether, this proposal will inform novel therapeutic approaches for regenerative medicine for the treatment of gastrointestinal diseases, especially those related to improper intestinal development or dysregulated Notch signaling. I designed these aims to rigorously test my hypotheses while generating new findings to support my transition to an independent investigator studying mechanisms underlying intestinal development, plasticity, and barrier repair. Furthermore, the execution of these aims will allow me to achieve my training goals in deepening my understanding of gastrointestinal physiology and stem cell biology, and in mastering state-of-the-art cellular and molecular techniques required to establish my independence.