676 Evidence for Histamine-Mediated Potentiation of TRPV4 and TRPA1 Signalin in Submucosal Neurons of IBS Patients and Mouse DRG Neurons

Background: To date no discrete genetic signature has been defined for isolated Dclk1+ tuft cells within the small intestine. Furthermore, recent reports on the functional significance of Dclk1+ cells in the small intestine have been inconsistent. These cells have been proposed to be fully differentiated cells, reserve stem cells, and tumor stem cells. Here we present a discrete "candidate" gene signature of Dclk1+ cells obtained from Dclk1-CreER;Rosa26-YFP mice, which will be a valuable animal model for future work seeking to uncover the latent abilities and functional role of Dclk1 expressing tuft cells and their subsets. Methods: The Dclk1-CreER;Rosa26-YFP compound mice were injected with tamoxifen to induce the expression of YFP driven by Dclk1 promoter. We FACS-sorted Dclk1+ cells from the small intestinal epithelium of Dclk1-CreER;Rosa26-YFP mice. We determined the mRNA expression levels of Dclk1 and other stem cell associated markers (Bmi1 and Lgr5), and pluripotency factors in the isolated Dclk1+ and Dclk1cells. To analyze the expression of key markers of quiescence survival and longevity, mRNA expression of cell cycle regulators and survival factors were analyzed. To explore the clonogenic capacity of the otherwise quiescent cells, we investigated self-renewal capacity in the Dclk1+ and Dclk1population. Results: Following tamoxifen administration, intestines were collected for YFP and Dclk1 staining, and we found that YFP co-localized with Dclk1 expression. Analysis of sorted YFP+ cells demonstrated marked enrichment (~6000 fold) for Dclk1 mRNA compared with YFPcells, confirming successful isolation of Dclk1+ cells. The Dclk1+ population was enriched (~6 fold) for the quiescent putative stem cell marker Bmi1, whereas the Dclk1fraction was enriched ~174 fold for Lgr5. We observed a relatively higher expression of pluripotency genes (~5-80 fold) as well as a significant increase in pro-survival genes (~4-9 fold) and quiescence regulatory markers (~8-24 fold) in the Dclk1+ cells compared to Dclk1cells. A 14-fold increase in self-renewal capability was observed in in vitro isolated Dclk1+ cells by clonogenic assay. The unique genetic profile presented in this study suggests that Dclk1+ cells may be relatively quiescent under normal homeostatic conditions but maintain their pluripotency and metabolic machinery required for survival/longevity through the expression of Cdkn1A, Cdkn1B, Oct4, Sox2, Nanog, and Klf4, Wif1, RelA, Akt and AMPK, and vital regulation of Raptor, Rictor, p53 and Survivin. Conclusion: Taken as a whole, these results provide additional support for the hypothesis that while the majority of Dclk1+ cells are committed tuft cells, they also maintain the molecular capability for stemness in reserve, and could play a role in the maintenance of the stem cell niche in an injury repair environment.