Fig. 6: An organoid-based platform for functional characterization of tuft cell properties.
a Microscopic images of ChatBAC-eGFP+ cell in organoid showing typical tuft cell morphology (observed in 3 independent experiments). Left: overview confocal brightfield image overlayed with GFP fluorescence channel. Right: zoom-in of indicated region. White dotted line outlines ChatBAC-eGFP+ tuft cell. b Dynamic protrusions in Chat+ tuft-2 cells that can span several neighboring cells. Merge of fluorescence channels is shown: green, eGFP; red, nuclei visualized with H2B-mScarlet-I. Three stills of indicated timepoints are shown. White triangles with letters indicate protrusions. c. Protrusion lengths over time. Triangles with letters indicate single protrusion tracks corresponding to protrusions shown in microscopy images of (b). d Relationship between maximum protrusion length and protrusion lifetime. e Microscopic stills from live imaging of NrepP2A-mScarlet-I-nls organoids stained with anti-CD24, visualizing membrane protrusions of Nrep+ tuft-1 cells. Merge of fluorescence channels is shown: cyan, anti-CD24; red, NrepP2A-mScarlet-I-nls. White triangles with letters indicate protrusions. Three different examples of Nrep+ cells with dynamic protrusions are shown. f Relative expression level of tuft-specific GPCRs within indicated cell types, as extracted from the mouse in vivo integrated single-cell dataset (Fig. 1d) and organoids (Fig. 3n). g Experimental setup for live imaging experiments to monitor tuft cell activation. Tuft-subtype reporter organoids expressing the Tq-Ca-FLITS calcium biosensor (left, representative image of 10 examined organoids) are re-plated to form 2D monolayers that can be apically stimulated and are compatible with high temporal resolution imaging (right). h Fluorescent image of 2D monolayer of reporter organoid with CellMask deep red incubation to label cell boundaries (membranes, white) and mScarlet-I reporter signal to label tuft identity. Image is prior to stimulation with cis-epoxysuccinic acid (cESA) (n = 3 independent experiments). i Fluorescent intensity fluctuations of Tq-Ca-FLITS biosensor within the field-of-view cell monolayer as shown in (d), following stimulation with cESA at t = 0. Time (seconds) post cESA exposure is indicated at the top of each panel. White outline indicates Nrep+ tuft cell. j Single-cell traces of Tq-Ca-FLITS intensity fluctuations within monolayer of (e). Traces are colored by mScarlet-I signal measured in the corresponding cell. k Responsiveness of tuft-reporter positive cells following stimulation with 1.5 mM cESA. Per bar, each point represents a separate experiment and is colored for the medium used (number of cells comprising each bar and p-values are indicated, t-test). l As in panel d, but now monolayer of ChatBAC-eGFP reporter organoid prior exposure to propionate (n = 2 independent experiments). m As in panel e, but Tq-Ca-FLITS intensity fluctuation post propionate exposure. Time since propionate addition is indicated at the top of each panel. Arrow indicates ChatBAC-eGFP+ tuft cell. n Single-cell traces of Tq-Ca-FLITS intensity fluctuations within monolayer of panel i. Traces are colored by GFP signal measured in the corresponding cell. o Responsiveness of non-tuft cells (mScarlet-I− GFP−) in the proximity of the ChatBAC-eGFP+ cell shown in (h). Number of cells comprising each bar are indicated at the top of each bar. Results of one representative experiment are shown.
