Figure 2.
TA cells control R-spondin production in the crypt niche. (A) qRT-PCR of Rspo1, Rspo2, and Rspo3 mRNA levels in whole intestinal tissue from URI(+/+)Int and URI(Δ/Δ)Int mice following 6 d of tamoxifen treatment (n = 6, 4). (B) WB in intestinal tissue from URI(+/+)Int and URI(Δ/Δ) Int mice following 6 d of tamoxifen treatment. Membranes are blotted with the indicated antibodies. (C) Representative in situ hybridization (ISH) pictures for R-spondin 1 in intestinal tissue from URI(+/+)Int-Lgr5-EGFP and URI(Δ/Δ)Int-Lgr5-EGFP mice following 6 d of tamoxifen treatment. Black dashed lines are for crypts; green dashed lines highlight stromal cells. 1 = upper part of the crypt; 2 = bottom part of the crypt; 3 = stromal cells. (D) Representative co-IF for lysozyme and R-spondin 3 in URI(+/+)Int and URI(Δ/Δ)Int mice following 6 d of tamoxifen treatment. (E) Gating strategy for flow cytometry to sort Lgr5− high side-scattering/forward scattering (high size/high granularity), Lgr5low (TA), and Lgr5high (ISC) cells. (F) qRT-PCR of Uri mRNA levels in sorted Lgr5− high side-scattering/forward scattering (high size/high granularity) and Lgr5low (TA) cells from isolated crypts from URI(+/+)Int-Lgr5-EGFP mice (n = 4, 4, 3, 3). (G) qRT-PCR of Defa4 mRNA levels in sorted Lgr5− high side-scattering/forward scattering (high size/high granularity) and Lgr5high (ISCs) from isolated crypts from URI(+/+)Int-Lgr5-EGFP (n = 3). (H) qRT-PCR of Rspo1, Rspo2, and Rspo3 mRNA levels in sorted Lgr5− high side-scattering/forward scattering cells (high size/high granularity) from URI(+/+)Int-Lgr5-EGFP mice. (I) qRT-PCR of Rspo1, Rspo2, and Rspo3 mRNA levels in sorted Lgr5− high side-scattering/forward scattering cells (high size/high granularity) from URI(+/+)Int-Lgr5-EGFP and from URI(Δ/Δ)Int-Lgr5-EGFP. (J) qRT-PCR of ISC regulator genes (Dll4, BMP2, BMP4, EGF, and TGFβ) expressed by Paneth cells isolated from URI(+/+)Int-Lgr5-EGFP and URI(Δ/Δ)Int-Lgr5-EGFP mice (n = 4, 3). (K) Representative co-IF for lysozyme and R-spondin 1 in URI(+/+)Int-Lgr5-EGFP and URI(Δ/Δ)Int-Lgr5-EGFP mice following 6 d of tamoxifen treatment. (L) Quantification of R-spondin 1 intensity in K. (M) Scheme of organoid differentiation protocol. Isolated crypts were culture in ENR-CV for 4–6 d to enrich for ISCs. Stemmed organoids were then harvested, washed, and embedded in fresh Matrigel and culture in ENR-CD for 4–6 d to induce Paneth cell differentiation. (N) Morphology of organoids culture in ENR-CV (control) or ENR-CD (Paneth cell differentiation) for 4–6 d. (O) qRT-PCR depicting stem cell markers (Lgr5) and Paneth cell markers (Cryptdin, Defa4) in organoids cultured in ENR-CV or ENR-CD conditions. (P) qRT-PCR showing R-spondins mRNA levels in organoids cultured in ENR-CV or ENR-CD conditions. Data represent mean ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; Student’s t test and one-way ANOVA. Scale bars represent 10 µm in C; 20 µm in K; 100 µm in D; and 150 µm in N. WB are representative of at least three independent experiments. ISH, IF, and IHC are representative of at least three independent mice. Source data are available for this figure: SourceData F2.

TA cells control R-spondin production in the crypt niche. (A) qRT-PCR of Rspo1, Rspo2, and Rspo3 mRNA levels in whole intestinal tissue from URI(+/+)Int and URI(Δ/Δ)Int mice following 6 d of tamoxifen treatment (n = 6, 4). (B) WB in intestinal tissue from URI(+/+)Int and URI(Δ/Δ) Int mice following 6 d of tamoxifen treatment. Membranes are blotted with the indicated antibodies. (C) Representative in situ hybridization (ISH) pictures for R-spondin 1 in intestinal tissue from URI(+/+)Int-Lgr5-EGFP and URI(Δ/Δ)Int-Lgr5-EGFP mice following 6 d of tamoxifen treatment. Black dashed lines are for crypts; green dashed lines highlight stromal cells. 1 = upper part of the crypt; 2 = bottom part of the crypt; 3 = stromal cells. (D) Representative co-IF for lysozyme and R-spondin 3 in URI(+/+)Int and URI(Δ/Δ)Int mice following 6 d of tamoxifen treatment. (E) Gating strategy for flow cytometry to sort Lgr5− high side-scattering/forward scattering (high size/high granularity), Lgr5low (TA), and Lgr5high (ISC) cells. (F) qRT-PCR of Uri mRNA levels in sorted Lgr5− high side-scattering/forward scattering (high size/high granularity) and Lgr5low (TA) cells from isolated crypts from URI(+/+)Int-Lgr5-EGFP mice (n = 4, 4, 3, 3). (G) qRT-PCR of Defa4 mRNA levels in sorted Lgr5− high side-scattering/forward scattering (high size/high granularity) and Lgr5high (ISCs) from isolated crypts from URI(+/+)Int-Lgr5-EGFP (n = 3). (H) qRT-PCR of Rspo1, Rspo2, and Rspo3 mRNA levels in sorted Lgr5− high side-scattering/forward scattering cells (high size/high granularity) from URI(+/+)Int-Lgr5-EGFP mice. (I) qRT-PCR of Rspo1, Rspo2, and Rspo3 mRNA levels in sorted Lgr5− high side-scattering/forward scattering cells (high size/high granularity) from URI(+/+)Int-Lgr5-EGFP and from URI(Δ/Δ)Int-Lgr5-EGFP. (J) qRT-PCR of ISC regulator genes (Dll4, BMP2, BMP4, EGF, and TGFβ) expressed by Paneth cells isolated from URI(+/+)Int-Lgr5-EGFP and URI(Δ/Δ)Int-Lgr5-EGFP mice (n = 4, 3). (K) Representative co-IF for lysozyme and R-spondin 1 in URI(+/+)Int-Lgr5-EGFP and URI(Δ/Δ)Int-Lgr5-EGFP mice following 6 d of tamoxifen treatment. (L) Quantification of R-spondin 1 intensity in K. (M) Scheme of organoid differentiation protocol. Isolated crypts were culture in ENR-CV for 4–6 d to enrich for ISCs. Stemmed organoids were then harvested, washed, and embedded in fresh Matrigel and culture in ENR-CD for 4–6 d to induce Paneth cell differentiation. (N) Morphology of organoids culture in ENR-CV (control) or ENR-CD (Paneth cell differentiation) for 4–6 d. (O) qRT-PCR depicting stem cell markers (Lgr5) and Paneth cell markers (Cryptdin, Defa4) in organoids cultured in ENR-CV or ENR-CD conditions. (P) qRT-PCR showing R-spondins mRNA levels in organoids cultured in ENR-CV or ENR-CD conditions. Data represent mean ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; Student’s t test and one-way ANOVA. Scale bars represent 10 µm in C; 20 µm in K; 100 µm in D; and 150 µm in N. WB are representative of at least three independent experiments. ISH, IF, and IHC are representative of at least three independent mice. Source data are available for this figure: SourceData F2.

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