Characterizing RasGRP1 as a critical regulator of the chemosensory tuft cell lineage in the intestine

About This Grant

PROJECT SUMMARY Tuft cells are rare chemosensory cells, which reside in the epithelial lining of the small intestine (SI) where they serve as key intestinal sentinels by detecting luminal cues and triggering type II immune responses to infections. Specifically, tuft cells signal to and activate type 2 innate lymphoid cells (ILC2s) to mount an immune response and enhance tuft cell production from undifferentiated epithelial cells through a feed forward mechanism. Recent work has demonstrated that fully differentiated tuft cells can dedifferentiation and regenerate all intestinal lineages under damage conditions, highlighting a new facet of epithelial regeneration; however, the molecular signals that balance tuft cell specification and epithelial plasticity remain incompletely understood. Previous work from the Roose lab has identified the Ras Guanine Nucleotide Exchange Factor, RasGRP1, as a suppressor of proliferative EGFR/Ras signals in colorectal cancer. My foundational studies, using a novel multi-antibody spectral flow panel capable, show that mice deficient for Rasgrp1 (Rasgrp1-/-) have significantly fewer tuft cells and clear helminth infections less efficiently than wild-type controls. Single-cell RNA sequencing of sorted tuft cells reveals a reduction in immature tuft cell populations in Rasgrp1-/- mice, which also show increased proliferative activity compared to wild-type controls. Strikingly, I recently discovered that intestinal stem cell (ISC) ablation in Rasgrp1-/- mice results in lethality, suggesting that RasGRP1 is required for the dedifferentiation process that normally restores the ISC pool after injury. These findings support my central hypothesis that RasGRP1 coordinates both forward differentiation and dedifferentiation programs in response to environmental cues. In Aim 1, I will test whether RasGRP1 promotes commitment to the tuft cell lineage by combining transgenic overexpression models, multiplexed spectral flow cytometry, and organoid-based pharmacological assays to mechanistically dissect how RasGRP1 modulates progenitor proliferation and tuft cell generation during homeostasis and infection. I will also determine whether RasGRP1-driven tuft cell hyperplasia spontaneously activates the tuft–ILC2 circuit and alters local immune composition. In Aim 2, I will define whether RasGRP1 enables progenitor-to-ISC dedifferentiation during epithelial injury. I will map RasGRP1 expression patterns in homeostasis and post-injury, use ISC ablation and conditional knockout models to test whether RasGRP1 loss impairs ISC recovery. I will apply innovative real-time lineage tracing and live imaging of organoids to directly visualize RasGRP1+ cells regenerating ISCs. Together, these integrated approaches will reveal how RasGRP1 governs epithelial plasticity and the balance between differentiation and regeneration. Insights gained from this proposal will have broad implications for understanding intestinal homeostasis, type II immunity, and epithelial repair. Furthermore, I anticipate these findings to have implications for future therapeutic strategies to modulate tuft cell proportions in diseases characterized by disrupted epithelial homeostasis or impaired immune responses, such as chronic infections, inflammatory disorders, obesity, or cancer.