Fig. 5: Crypt-villus signaling gradients advance maturation from tuft-1 to tuft-2 states.
a Experimental setup to promote tuft cell maturation with villus-inspired medium. ChatBAC-eGFP reporter organoids were pretreated with IL-4 and IL-13 on the 3rd day after seeding in ENR (EGF, Noggin and R-spondin) medium. On the 4th day, factors were added to, or depleted from, the medium. Frequency of GFP(Chat)+ tuft-2 induction was tested at day 7 with flow cytometry. b Percentage of ChatBAC-eGFP+ tuft-2 cells in organoids treated with culture medium (ctrl, n = 11) or IL-4 and IL-13 in presence (#1, n = 4) or absence (#2, n = 7) of crypt or villus/autocrine-inspired signaling factors (#3, n = 6) (ANOVA P = 6.8 × 10−7, Tukey HSD test). c Representative flow cytometry analysis of fluorescent population frequencies in indicated reporter organoids treated with crypt (#1) or villus-inspired medium (#3) after IL-4 and IL-13 pretreatment. 5000 cells are shown per plot. d Percentage of mScarlet+ (left) and mScarlet+GFP+ (right) cells in indicated organoid reporter lines treated with crypt or villus-inspired medium (condition #1 vs #3, Fig. 5a, b) measured by flow cytometry (n = 7 (Nrep), 5 (Gng13) and 6 (Folr1) independent experiments, t-test). e Representative fluorescence image of ChatBAC-eGFP;NrepP2A-mScarlet-I organoids treated with crypt or villus-inspired medium regimen (condition #1 vs #3, Fig. 5a, b). White arrows: ChatBAC-eGFP+ cells. f Microscopic stills from live imaging experiments of ChatBAC-eGFP;NrepP2A-mScarlet-I organoids in villus-inspired medium (condition #3 of Fig. 5a, b). Left: confocal brightfield image of organoid at the start of imaging. Right: stills of fluorescence channels (mSarlet-I: red, eGFP: green) from timepoints preceding and following co-expression of fluorescent markers. Cell that shows co-expression is indicated with a white arrowhead throughout imaging timepoints (n = 2 independent experiments). g Average mScarlet-I signal over time in ChatBAC-eGFP;NrepP2A-mScarlet-I organoids treated with crypt or villus-inspired medium regimen (condition #1 vs #3, Fig. 5a, b). Shading in plot represents SEM. Number of cells comprising each graph are indicated. h Barplot showing fraction of mScarlet-I+ cells in ChatBAC-eGFP;NrepP2A-mScarlet-I organoids with stable/rising or declining fluorescence signal in crypt or villus medium with cytokines (IL4 + IL13). Related to g. Number of cells comprising each graph are indicated. i Experimental setup for tuft cell subtype-specific depletion with diphtheria toxin (DT). Organoids were treated as in condition #3 of Fig. 5a, b and DT was added with every medium change. j Percentage of mScarlet+ cells in indicated reporter lines and parental no knock-in control (ChatBAC-eGFP; no mScarlet-I or DTR) after differentiation during depletion with DT (n = 3 independent experiments, t-test). k Percentage of GFP+ cells in indicated reporter lines and parental no knock-in control (ChatBAC-eGFP; no mScarlet-I or DTR) after differentiation during depletion with DT (n = 3 independent experiments, ANOVA P = 0.04, Tukey HSD test). l RNA velocity-based trajectory inference with single cell transcriptomes from organoids treated with ENR, NR + IL4 + IL13 or villus-inspired medium superimposed on UMAP. Direction of arrows predict unidirectional differentiation from tuft-1 to tuft-2 transcriptomic states. m Schematic depicting linear model of intestinal tuft cell differentiation. Tuft-p: tuft precursors; IL4: interleukin-4; IL13: interleukin-13; ACh: acetylcholine; BMPs: bone morphogenetic proteins.
