GoKawiilMice
All experimental procedures were conducted in accordance with guidelines approved by the Institutional Animal Care Committees at the University of California, San Francisco (UCSF) and the South Australian Health and Medical Research Institute (SAHMRI) and aligned with the NIH and NHMRC Guide for the Care and Use of Laboratory Animals, respectively. Mice of both sexes, aged 8–16 weeks, were used, and given ad libitum access to standard lab chow and sterile water. They were housed in a controlled environment under a 12-h light–dark cycle. TRPM5-GFP mice were used to visualize tuft cells (gift from R. F. Margolskee). Pou2f3−/− mice (Jackson Laboratory, strain 037040) were used to knock out tuft cells. For conditional knockout of Chat in intestinal tuft cells, VilCre mice (Jackson Laboratory, strain 021504) were crossed to Chatflox mice (gift from J. Chan). For gGRAB 5-HT3.0 sensor imaging, VilCre mice were crossed to the Rosa26gGRAB-5-HT3.0-P2A-jRGECO1a reporter line. GCaMP imaging in organoids used Tac1Cre mice (Jackson Laboratory, strain 021877) crossed with Polr2aGCaMP5g-IRES-tdTomato mice (gift from L. Jan, Jackson Laboratory, strain 024477). To inhibit serotonin release from EC cells, we expressed the tetanus toxin in EC cells using the Cre- and Flp-dependent tetanus toxin light-chain reporter mouse, RC::PFTox (gift from S. Dymecki). Specifically, ePetFlp hemizygous/RC::PFTox homozygous mice were crossed to Tac1Cre homozygous mice to produce Tac1Cre;ePetFlp;PFTox mice. For nerve fibre recordings, Scn10aCre mice (gift from W. Imlach, Jackson Laboratory, strain 036564) were crossed to the ChR2 reporter line (Rosa26lsl-ChR2; Jackson Laboratory, strain 012569). Genetically modified mice or control mice (littermates or age-matched mice) were randomly selected for all behavioural experiments.
Crypt cell isolation and organoid culture
Adult male Tac1Cre;Polr2aGCaMP5g-IRES-tdTomato, TRPM5-GFP and Pou2f3−/− mice were used to generate intestinal organoids as previously reported51. Approximately 8-cm pieces of the ileum were used to establish TRPM5-GFP organoids. For Tac1Cre;Polr2aGCaMP5g-IRES-tdTomato organoids, the upper jejunum was used to avoid ectopic expression of Tac1Cre in the lower intestine. Organoids were maintained and passaged every six days in organoid growth medium (advanced Dulbecco’s modified Eagle’s medium (DMEM)/F12 supplemented with penicillin–streptomycin, 10 mM HEPES, GlutaMAX, B27 (Thermo Fisher Scientific), 1 mM N-acetylcysteine (Sigma), 50 ng ml−1 mouse recombinant epidermal growth factor (Thermo Fisher Scientific), R-spondin 1 (10% final volume) and 100 ng ml−1 mouse Noggin (Peprotech)).
Cell lines
The R-spondin-1-expressing HEK293FT (ATCC) cells were maintained in DMEM, 20% fetal calf serum (FCS), 1% penicillin–streptomycin and 125 µg ml−1 zeocin (Thermo Fisher Scientific) at 37 °C, 5% CO 2 . Zeocin was removed after production of R-spondin 1 conditioned medium. HEK293T cells (ATCC) were grown in DMEM, 10% FCS and 1% penicillin–streptomycin at 37 °C, 5% CO 2 and transfected using Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer’s protocol. For biosensor experiments, 200 ng pDisplay-gGRAB ACh4h -IRES-mCherryCAAX or 200 ng pcDNA3-hM1R-P2A-GCaMP8m was transfected to HEK293FT cells in 24-well plates. For the 5-HT 3 biosensor experiment, 200 ng pcDNA3-5-HT 3A and 20 ng pcDNA3-mApple were co-transfected to HEK293FT cells in 24-well plates.
Induction of tuft cell hyperplasia
Tuft cell hyperplasia in organoids was induced by exposing organoids to 20 ng ml−1 IL-4 (R&D Systems) in the growth medium for two days (days 3–5), followed by one day of growth in IL-4-free medium. The biosensor experiments were then performed on day 6. To induce tuft cell hyperplasia in mice, the mice received intraperitoneal injections of 500 ng of IL-25 (R&D Systems) on days 0, 1, 2 and 3. Tissues were subsequently collected for imaging on day 5.
GCaMP imaging using intestinal organoids
Five days after passage, Tac1Cre;Polr2aGCaMP5g-IRES-tdTomato organoids were removed from Matrigel (Corning) and mechanically broken up with a 200-µl pipette. The organoid fragments were seeded onto Cell-Tak (Corning)-coated coverslips and placed in a recording chamber containing Ringer’s solution (140 mM NaCl, 5 mM KCl, 2 mM CaCl 2 , 2 mM MgCl 2 , 10 mM d-glucose and 10 mM HEPES-Na (pH 7.4)). EC cells were identified by tdTomato expression. GCaMP imaging was performed with an upright microscope equipped with a Grasshopper 3 (FLIR) camera and a Lambda LS light source (Sutter). Organoids were maintained under a constant laminar flow of Ringer’s solution applied by a pressure-driven microperfusion system (SmartSquirt, Automate Scientific). All pharmacological reagents were delivered by local perfusion. Acquired images were analysed with Fiji. Regions of interest (ROIs) were drawn around individual EC cells, and ∆F/F 0 was calculated.
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