Animals

Zebrafish (Danio rerio, AB strain) were reared and kept in the Karolinska Institutet (KI) Zebrafish Core Facility and the Institut Curie Animal Facility, according to standard protocols. Ethical permits used for zebrafish husbandry were Nr 5756/17 (to Eduardo Villablanca), 14049/19 (to KI Zebrafish Core Facility), and APAFiS #21197-2019062521156746-v2 (To Pedro Hernandez). Zebrafish embryos were collected by natural spawning of adults and were kept at 28 °C in E3 water. The zebrafish line TgBAC(cldn15la-GFP)pd1034, 36 referred to in the text as Tg(cldn15la:GFP), was provided by Prof. Michael Bagnat (Duke University Medical Center, USA); the line Tg(Tp1bglob:eGFP)um14, 45 referred in the manuscript as Tg(tp1:EGFP) was kindly provided by Prof. Olov Andersson (Department of Cell and Molecular Biology CMB, Karolinska Institutet, Sweden); and the line Tg(7xStat3-Hsv.Ul23:EGFP),48 referred in the manuscript as Tg(7xStat3:EGFP), was kindly provided by Prof. Francesco Argenton (Università degli Studi di Padova, Italy).

Wild-type mice (Mus musculus, C57/Bl6 strain) were purchased from Charles River Laboratories or Taconic and were kept in the Comparative Medicine facility from Karolinska University Hospital (AKM). The transgenic strain Lgr5-EGFP-IRES-creERT2,52 referred to in the text as Lgr5-GFP, was maintained in a C57/Bl6 background at the Comparative Medicine facility from Karolinska Institutet (KM-B). All mice used for experiments were 6–10 weeks old. Animals were handled according to protocols approved by the Stockholm Regional Ethics Committee (Nr 3227-2017 and 6778-2020). All experiments were performed following the national and institutional guidelines and regulations.

Isolation of fluorescent intestinal epithelial cells from zebrafish larvae

To acquire the intestinal epithelial population, approximately 100 of 6dpf TgBAC(cldn15la:GFP) zebrafish larvae were collected for the FACS experiment, then intestines were dissected and placed into PBS on ice with a maximum dissection time of 2 h. Intestine dissociation was performed using TrypLE Express (Gibco, Cat. No. 12605028) for 1 h at 37 °C, pipetting up and down every 10 min to support digestion. Digested samples were spun at 1500 g for 5 min at 4 °C and washed twice with PBS 1× before being resuspended together with PBS 1× and 10% FBS (fetal bovine serum). Filtered cells were immediately subjected to FACS at Institut Curie Flow Cytometry Platform using the Sony SH800 Cell Sorter. Dead cells were excluded from analysis using the combination of Calcein Blue (Invitrogen, Cat. No. 65-0855-39) and Propidium Iodide viability stains (Sigma-Aldrich, Cat. No. P4864). Non-transgenic and single transgenic controls (pools of 10 dissected guts) were prepared as above and used for gating and compensation. RNA isolation was done using on average 30,000 GFP+ or GFP− sorted cells with the Single-Cell RNA Purification Kit from Norgen Biotek Corp and reverse transcribed using Superscript IV Reverse Transcriptase (Life Technologies, Cat. No. 18090050) following the manufacturer’s instructions.

Whole-mount in situ hybridization in zebrafish larvae

Portions of the coding sequences for the ZFIN-annotated orthologs of the human IL10RA (Gene name: il10ra, Gene ID: 777651, ENSEMBL ID: ENSDARG00000100383) and IL10RB genes (Gene name: il10rb, Gene ID: 619391, ENSDARG00000078042) were amplified by PCR using the primers from Table S1, and subsequently cloned into a pCRII-TOPO vector (Invitrogen, Cat. No. 450640). Probe and control RNA sequences were synthesized in vitro by using T7 and SP6 RNA polymerases and subsequently purified by Lithium chloride (LiCl) precipitation. Whole-mount in situ hybridizations were performed in 3 dpf and 5 dpf zebrafish larvae as previously described30 with the following modifications. Permeabilization was done by treating fixed larvae with 10 μg/mL of Proteinase K (Qiagen, Cat. No. 19131) prepared in PBS supplemented with Tween-20 0.1% and DMSO 1% for 30 minutes at room temperature. Pre-hybridization was done at 60 °C for at least 3 h in Hybridization media (HM, Formamide 50%, Sodium citrate solution 5×, citric acid 9.2 mM, Heparin 50 μg/mL, RNAse-free yeast tRNA 500 μg/mL). Probe hybridization was done using 500 μL of HM containing 4 ng/mL of either probe or control RNA, and samples were incubated overnight at 60 °C. Anti-dig-AP antibody (Roche, Cat. No. 1109327493) was diluted 1:3000 in PBS supplemented with Tween-20 0.1%, sheep serum 2%, and bovine serum albumin (BSA) 2 mg/mL. Development of probe hybridizations was done by diluting NBT-BCIP stock solution (Roche, Cat. No. 11681451001) 1:50 in alkaline phosphatase buffer (Tris-HCl 100 mM pH 9.5, MgCl2, NaCl, Tween-20 0.1%). After 3 h of developing, samples were washed extensively with PBS plus Tween-20 0.1%, fixed in PFA 4%, washed again in PBS Tween-20 0.1%, and subsequently dehydrated in Methanol and stored at −80 °C for at least one night, to allow clearing of background signal. Larvae were rehydrated, transferred to Glycerol 85%, and imaged using a Nikon SMZ25 stereoscope equipped with a DS-Fi3 camera.

Generation of il10-Mut zebrafish lines

The zebrafish coding sequence for the ortholog of the human IL10 gene (Gene name: il10, Gene ID: 553957, ENSEMBL ID: ENSDARG00000078147)31 was targeted by CRISPR/Cas937 with specific sgRNAs (Table S1). For the maintenance of wild-type and il10 mutant stocks, zebrafish embryos coming from the incross of heterozygous il10 individuals were raised, and genotyping was performed once they reached adulthood by fin-clipping a portion of the tail fin. The il10uu1751 mutation created a restriction enzyme site for EcoNI, which is absent in wild-type fish. Carriers of the il10uu1751 mutation were screened by PCR followed by enzymatic digestion with EcoNI. Zebrafish carrying the il10uu1762 mutation were screened by PCR using combinations of primers in which the forward primer was specific for either the WT or mutant sequence. Primers used for genotyping are shown in Table S1.

Cloning of the zebrafish il10 and il17a/f3 genes and rescue experiments

The coding sequences for the zebrafish il10 and il17a/f3 genes were amplified by PCR using specific primers for both genes (Table S1) and then cloned in a pCR2.1-TOPO vector (Invitrogen, Cat. No. 450641). In vitro- transcribed mRNA was synthesized using the T7 mMessage mMachine kit (Invitrogen, Cat. No. AM1344), following the manufacturer’s guidelines. For injections, 1nL of 200 ng/μL of in vitro-transcribed mRNA was co-injected with 0.5% of Rhodamine-Dextran MW10000 (Invitrogen, Cat. No. D1824) in 1-cell stage wild-type or il10-Mut embryos. Embryos injected with 0.5% Rhodamine-dextran were used as control. At 2dpf, Rhodamine-positive embryos were selected and used for subsequent analysis.

Body-intestine isolation in zebrafish larva

Larvae from 3 dpf to 5 dpf were euthanized by overdosing them with MS-222 and intestines were isolated mechanically with the use of needles to pierce the tissue and extract the intestines. Around 10 intestines and their respective bodies were collected for analysis.

RNA extraction and qRT-PCR from zebrafish samples

For zebrafish samples, up to 10 tissue samples (i.e. whole larvae, intestines, or body carcasses) were pooled and collected in Trizol reagent (Invitrogen, Cat. no. 10296010), and tissues were homogenized by pipetting the samples repeatedly through 23G and 27G needles. Total RNA was extracted following Trizol manufacturer’s instructions. Synthesis of cDNA was performed using the iScript cDNA synthesis kit (BioRad, Cat. No. 1708841). Quantitative PCR was performed using iTaq Universal SYBR Green supermix (BioRad, Cat. No. 1725124), as previously reported.69 Primers used for zebrafish qRT-PCR analysis are found in Table S2.

Bleach and antibiotic treatments in zebrafish larvae

Zebrafish embryos between 24–28 hpf were treated with chlorine hypochlorite (0.04%) for 5 minutes and then washed twice with sterile E3 medium for 5 min. After washing, embryos were left in sterile E3 medium containing Ampicillin 100 μg/mL and Kanamycin 5 μg/mL and placed at 28 °C in isolated containers. Media was renewed every day in sterile conditions until the day of sample collection. For the quantification of bacterial loads, genomic DNA from 2 larvae per condition was extracted by (heat-basic), and PCR against 16S All Bacteria30 and zebrafish gDNA (for il23r gene) was performed. PCR products were run in a 2% agarose gel, and band intensities were measured in Fiji/ImageJ (NIH).

Alcian blue, neutral red stainings and quantifications in zebrafish larvae

Stainings for Neutral red (Sigma, Cat. No. N4638) and Alcian blue (Sigma, Cat. No. B8438) in 5dpf zebrafish larvae were performed as previously described.70 Alcian blue-stained larvae were additionally treated with H2O2 1.5%/KOH 0.5% to remove pigments before imaging. Larvae were mounted in a lateral position using 1% low-gelling point agarose (Sigma, Cat. No. A9414), and RGB images were acquired using a Nikon SMZ25 stereoscope equipped with a DS-Fi3 camera. Images were cropped to keep the mid-intestine section. Automatic unbiased analyses of the Alcian blue-stained area in the intestine of individual larvae were performed in Fiji/ImageJ software (NIH) using “Colour Deconvolution 1.7” and selecting the “Alcian blue & H” vector to identify the Alcian blue-stained area. For the automatic detection of the Neutral red-stained area, the function “Color Threshold” was used. The length of the alcian blue- and neutral red-stained regions were measured manually in randomized images.

Immunofluorescence staining in zebrafish larvae

Immunostaining was performed on whole larvae at 5 days post fertilization. Paraformaldehyde at 4% was used to fix zebrafish larvae overnight at 4 °C. The samples were then washed with distilled water. For whole-mount 2F11 immunostaining, fixed larvae were permeabilized with cold 100% acetone for 20 min at 4 °C before being washed three times with PBST (PBS supplemented with 0,5% Triton-X-100). Samples were further permeabilized with 1 mg/mL Collagenase from Clostridium histolyticum (Cat. No. C2139) for 2 h at room temperature. Samples were then washed with PBST and blocked with 10% of FBS/PBST at room temperature for more than 2 h. The primary mouse monoclonal 2F11 antibody (1:200; Abcam, Cat. No. ab71286) was diluted in blocking solution and incubated at 4 °C for more than 24 h. Following primary antibody incubation, the samples were washed with PBST solution and incubated for at least 2 h with secondary anti-mouse Alexa Fluor 488 antibody (1:500; Invitrogen, Cat. No. A11001) at room temperature in the dark. For whole-mount WGA staining, larvae were washed in distilled water for 1 h at room temperature, permeabilized with cold 100% acetone form 20 minutes at −20 °C, and washed with PBST. Larvae were incubated in Alexa Fluor 555-conjugated Wheat Germ Agglutinin (1:5000 from 5 mg/mL stock; Invitrogen, Cat. No. W32464) overnight at 4 °C and washed extensively with PBST. Imaging was performed using the Zeiss Axio Zoom.V16 stereoscope or the Zeiss LSM800 confocal microscope and analyzed with ImageJ software.

Chemical treatments in larval zebrafish

At 2 days post fertilization, pools of 10 larvae per milliliter were exposed to the Notch/γ-secretase inhibitor DAPT (50 μM; Tocris, Cat. No. 2634), the Jak2/Stat3 inhibitor Tyrphostin AG-490 (50 µM; Sigma-Aldrich, Cat. No. T3434), or the Notch activator Yhhu-3792 (Tocris, Cat. No. 6599), or. As a control for Notch activity induced by Yhhu-3792, RNA was extracted from treated larvae and the expression levels of the Notch downstream gene her6 were measured by qRT-PCR.

Fluorescent reporter imaging and analysis

The fluorescent reporters for Notch activity Tg(tp1:GFP) and Stat3 activity Tg(7xStat3:EGFP), in either wild-type or il10uu1751 genetic background, were imaged at 5dpf in an SMZ25 stereoscope equipped with a CoolLed laser set and a DS-Fi3 camera (Nikon), focusing on the intestinal region. Z-stacks were merged in Fiji/ImageJ software, and the number of GFP+ cells in the intestinal region was counted manually in Fiji/ImageJ.

Dissection, sectioning, and staining of adult zebrafish intestines

Zebrafish older than 1 year of age were euthanized with an overdose of MS-222. Intestines were collected and fixed in neutral buffered formalin (Sigma, Cat. No. HT-501128) overnight at room temperature, and subsequently transferred to ethanol 70% for at least 24 h. Tissues were dehydrated and embedded in paraffin, and 10 µm sections were collected and stained with alcian blue. Briefly, tissue sections were deparaffinized and rehydrated to PBS, and incubated in 3% alcian blue solution (Sigma) for 5 min. After staining, tissues were counterstained with nuclear fast red (Sigma, Cat. No. N3020). Images were acquired in a Nikon SMZ25 stereoscope and quantifications of goblet cells per villus area were performed using QuPath 0.2.3 (University of Edinburgh, UK).

Mouse intestinal organoid cultures

Mouse organoids were generated from crypts derived from the entire small intestine (SI). Briefly, SIs from WT mice were collected and flushed with PBS, cut opened longitudinally, and subsequently cut into five pieces of similar size. Tissue pieces were placed in PBS and were vigorously shaken to remove mucus. Tissue pieces were then transferred to cold PBS-EDTA 10 mM and incubated for 1 h in ice. After incubation, SI villi were removed by gentle scraping of the luminal side using two glass slides. Then SI crypts were scraped from the tissue pieces by applying stronger pressure with the glass slides and collected in recipient tubes filled with cold PBS. Collected crypts were centrifuged for 5 min at 300 × g and 4 °C, and then quantified using an upright microscope by placing 10 µL of crypt solution on a glass slide. The basic culture medium (ENR) contained advanced DMEM/F12, 1× penicillin/streptomycin, 1x Glutamax (Thermo Fisher Scientific), 10 mM HEPES (Thermo Fisher Scientific), 1x B27 supplement (Life Technologies, Cat. No.17504044), 1× N2 supplement (Life Technologies, Cat. No. 17504048), 1mM N-acetylcysteine (NAC, from Sigma, Cat. No. A9165) and was supplemented with 50 ng/mL of recombinant murine epidermal growth factor (EGF, from R&D, Cat. No. 2028-EG), 250 ng/mL recombinant murine R-Spondin (R&D, Cat. No. 3474-RS) and 100 ng/mL recombinant murine Noggin (Peprotech, Cat. No. 250-38). SI crypts were resuspended in 30–40% basic culture medium with 60–70% Matrigel (Corning, Cat. No. 356231) and 20 µl containing approximately 500 crypts were plated in a pre-warmed flat-bottom 48-well plate (Sarstedt, Cat. No. 83.3923). The plate was placed at 37 °C and allowed to solidify for 15 min before 200 µl of ENR medium (containing the different stimuli) was overlaid. The medium was replaced every 2 days with fresh medium and cultures were maintained at 37 °C in fully humidified chambers containing 5% CO2. During the first 2 days of culture, the ENR medium was supplemented with 10 µM of the ROCK inhibitor Y-27632 (Sigma, Cat. No. Y0503). For organoid in vitro stimulation, 25 ng/mL of recombinant murine IL-10 (Peprotech, Cat. No. 210-10) diluted in 0.1% bovine serum albumin (BSA, Sigma) was added in the ENR medium for the entire duration of the organoid cultures. Control organoids were supplemented with a similar volume of 0.1% BSA vehicle. Each condition was plated in technical triplicates. On day 4 of culture, crypt domains per IL-10-treated or vehicle-treated organoids were quantified in 2–3 wells/condition Each dot in the quantification plot represents one mouse and an average of 2–3 technical replicates.

Alcian blue stainings on mouse SI organoids

The Alcian blue protocol used for the staining of mouse SI organoids was adapted from the protocol used for whole-mount Alcian blue stainings in zebrafish. Organoids were collected after 4 days of culture, washed 5 times with cold PBS-BSA 0.1%, and replated in a new Matrigel stock in 24-well plates, to remove debris from the initial culture. After Matrigel gelification, organoids were fixed in PFA 4% for 45 minutes, washed twice with PBS-BSA 0.1%, and washed twice with acidic ethanol (70% Ethanol supplemented with 1% HCl). Organoids were stained with Alcian blue solution (Sigma, Cat. No. B8438) for 1 h at room temperature. After the incubation, stained organoids were washed extensively with acidic ethanol and were left overnight in acidic ethanol at 4 °C, to remove excessive staining from the Matrigel. Once the Matrigel became clear, organoids were washed with PBS-BSA 0.1% and imaged using the Nikon SMZ25 system.

RNA extraction from mouse organoids and qRT-PCR

After 4 days of culture, treated organoids were harvested and Matrigel was removed by consecutive washes with cold PBS-BSA 0.1%. Cleaned organoids were resuspended in RLT-plus buffer supplemented with 1% β-Mercaptoethanol, and RNA extraction was performed using the RNeasy extraction kit (Qiagen, Cat. No. 74136), following the manufacturer’s instructions. Similar to zebrafish samples, synthesis of cDNA was performed using the iScript cDNA synthesis kit (BioRad), and quantitative PCR was performed using iQ SYBR Green mix (BioRad). Primers used for mouse qRT-PCR analysis are found in Table S2.

Flow cytometry from organoid cell suspensions

For flow cytometry experiments, SI organoids were generated from crypts extracted from Lgr5-GFP mice. After 4 days of treatment, organoids were harvested and washed 5 times with cold PBS-BSA 0.1%. Organoids were disaggregated to single cells by incubating with TrypLE supplemented with DNase I for 5 min at 37 °C. Cells were incubated in Fc block (1:1000; Invitrogen, Cat. No. 14-0161-85) and Fixable Viability Dye eFluor 780 (1:1000; eBioscience, Cat. No. 65-0865-14) for 15 min at 4 °C, and subsequently stained with anti-Epcam PE-Cy7 (1:200; BioLegend, Cat. No. 118215), anti-CD24 BV421 (1:200; BioLegend, Cat. No. 101825) and WGA Alexa Fluor 555 (1:5000; Invitrogen) for 1 h at 4 °C. Flow cytometry was performed using an LSRFortessa flow cytometer (BD Biosciences, USA), and analysis was perfumed using FlowJo v10 (Treestar, USA).

Immunostaining of small intestine organoids

For immunofluorescence experiments, Matrigel-embedded isolated crypts extracted from small intestines of WT mice were plated in pre-warmed 8-well 15 µm glass-bottom chambers (Ibidi, Cat. No. 80826) and cultured as described above. On day 4 of culture, organoids were fixed in PBS-buffered Paraformaldehyde 4% for 45 minutes, permeabilized with Triton X-100 0.5% for 30 minutes and treated with Glycine 0.1 M for 1 h to block free aldehyde groups. Organoids were incubated in blocking solution (Normal goat serum 5%, Triton X-100 0.25%, BSA 0.1% in PBS) for 30 min before incubating with WGA Alexa Fluor 555 (1:5000, Invitrogen) and rabbit polyclonal anti-Lysozyme Ab-1 (1:200; Thermo Scientific, Cat. No. RB-372-A) overnight at 4 °C. After 3 washes with PBS-BSA 0.1%, organoids were incubated with goat anti-rabbit Alexa Fluor 647 (1:1000; Invitrogen, Cat. No. A21244) overnight at 4 °C. On the final day of staining, organoid nuclei were stained with DAPI (1 µg/mL, Molecular Probes, Cat. No. D1306) for 30 min at room temperature, washed, and kept in PBS until imaging within 0–3 days. Images from stained organoids were acquired in a Zeiss LSM800 confocal microscope (Zeiss, Germany) and analyzed using ImageJ.

Statistical analysis

Quantitative data were analyzed using GraphPad Prism v8.0 software (GraphPad, San Diego, CA, USA). Group comparisons were considered statistically significant when they reached a p-value below 0.05.