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Background: Intestinal tuft cells have only been recently investigated thoroughly due to their roles in type 2 immunity and chemosensory function. This rare population of intestinal epithelium is implicated in extensively interacting with other cell types and involved in nutrient absorption. Doublecortin-like kinase 1 (DCLK1) is widely used for tuft cell identification in the mouse intestine. However, the effects of tuft cell deletion on mouse intestines have not been fully understood. Here, we investigated the effects of tuft cell deletion on the epithelial cell lineages and overall impact on mouse intestine. Methods: Tuft cell deletion was induced in Dclk1-IRES-GFP-CreERT2/+; Rosa-DTA (Dclk1-DTA) mice by a single dose of tamoxifen injection. Cre-negative DTA/+ mice and DTA-negative Dclk1-CreERT2 mice of littermates received tamoxifen injection at same time as controls. Mouse bodyweight was monitored daily, and intestinal tissues were sampled 2, 4, or 7 days after the tamoxifen injection. Immunostaining of intestinal tissue sections was performed for lysozyme (LYZ), trefoil factor 3 (TFF3), cluster of differentiation 3 (CD3), DCLK1, mast cell protease 1 (MCPT1), Ly6G, and F4/80. Image analysis was conducted using Qupath. Results: Dclk1-DTA mice showed a significant decrease in bodyweight to 11% of their original weight on day 4 after tamoxifen injection. They showed significantly shortened small intestinal length compared to control mice, however, colonic length showed no significant difference. Immunofluorescent staining of induced Dclk1-DTA mouse tissues revealed that DCLK1+ tuft cells were decreased on day 2, almost completely deleted on day 4, and recovered on day 7. Overall brush border structures defined by villin and SGLT1 staining were intact. Correlated with the tuft cell reduction, mislocalized Paneth-like cells, identified with LYZ+ granules, were observed in the middle or upper part of villi with increasing frequency on day 4 and decreasing on day 7. To confirm goblet/Paneth intermediate cells, multiplexed immunofluorescent (MxIF) staining was employed with LYZ and TFF3. Double-positive (LYZ+/TFF3+) cells were significantly increased in the small intestine of tuft cell-depleted mice on day 4. Intraepithelial lymphocytes, mucosal neutrophils, and F4/80+ macrophages/dendritic cells did not show significant changes after the tuft cell deletion. This indicates that there is no inflammatory reactions following tuft cell deletion. Conclusion: Acute ablation of intestinal tuft cell may cause nutrient malabsorption and changes in mucosal defense response in vivo due to the alteration of cell differentiation lineage and the decrease in absorptive area. Our findings support the importance of tuft cell function in nutrient sensing and mucosal defense mechanism. NIH R01DK128190 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.