Fig 2: Notch activation in Defa4Cre-expressing cells leads to loss of secretory cell differentiation and increased expression of the stem cell marker Olfm4. Histologic and immunofluorescent staining of the small intestine from Defa4Cre;Rosa26EYFP and Defa4Cre;Rosa26NICD-ires-nGFP mice. (A) PAS/Alcian blue staining. Dashed line denotes wild-type crypt in jejunum of Defa4Cre;Rosa26NICD-ires-nGFP intestine. (B) Immunofluorescent staining of GFP or NICD (nGFP+) with cell type–specific markers, lysozyme, MUC2, chromogranin A, and villin. (C) Immunofluorescent staining of GFP or NICD (nGFP+) with the stem cell marker Olfm4. Scale bars: 50 μm.

Fig 3: Conserved numbers but impaired functionality of stem cells in Cd44?ie mice.a IHC staining of OLFM4. Scale bar: 50 µm. b Average number of OLFM4-positive stem cells per crypt. Error bars, ±SE. N = number of mice, n = number of crypts. c SI organoids observed 5 days after Cd44 deletion and controls. Scale bar: 200 µm. d Budded vs. round organoid numbers after deletion of Cd44 and passaging (P) every 5 days. n = number of organoids. Error bars, ±SE. e Representative images of f. Scale bar: 200 µm. f Relative organoid number along passages in Cd44fl/fl;VillinCreERT2 organoid cultured with or without 4-OHT treatment. N = 3 mice. Scale bar: 500 µm. g Organoid vs. cystic organoid numbers after deletion of Cd44 and treatment with Wnt3a CM, control CM or CHIR. N = 3 experiments. Error bars, ±SE. h Representative images of g CHIR and Wnt3a conditions. Scale bar: 200 µm. Student’s t test; *p < 0.05, **p < 0.01, ns = not significant. 4-OHT 4-hydroxytamoxifen. The dotted line in g indicates that the CHIR and Wnt3a conditions are two independent but related experiments.

Fig 4: PPARd and PPARa function redundantly to enhance stem cell activity in a HFD(A) Schematic of intestinal Ppard conditional deletion with Villin-CreERT2 (Ppard-iKO), including timeline of tamoxifen (T) injections for long-term deletion, diet, and tissue collection.(B and C) Quantification of organoids per crypt from WT (Ppardfl/fl) or Ppard-iKO mice in (A) (B), accompanied by representative bright-field images (C). LT-iKO indicates deletion of intestinal Ppard prior to establishment on the diet. Scale bar, 200 µm.(D) qRT-PCR of transcripts amplified from crypts collected from WT (Ppardfl/fl) or Ppard-iKO mice on chow or HFD. Transcripts were normalized to ß-actin.(E) Immunoblot of flow-sorted ISCs (Lgr5-EGFPhi) and progenitors (Lgr5-EGFPlow) from WT and Ppard-iKO (Ppardfl/fl; Villin-CreERT2; Lgr5-eGFP-IRES-CreERT2) mice on a control diet or HFD. Villin-CreERT2; Lgr5-eGFP-IRES-CreERT2 animals were used as WT controls.(F) Quantification of organoids per crypt from mice injected daily for 30 days with the PPARa agonist WY-14643 (4 mg/kg) or vehicle.(G) Schematic of the intestinal Ppard conditional allele with Villin-CreERT2 crossed to Ppara-null (Ppard/a-iKO), including timeline of T injections, diet, and tissue collection. Villin-CreERT2 transgenic mice were used as WT controls.(H) Quantification of OLFM4+ cells per jejunal crypt. Each data point represents quantification of 20+ crypts from one animal (n = 6).(I) Representative images of OLFM4+ immunohistochemistry (IHC) from (H). Scale bar, 20 µm.(J) Immunoblot blot of flow-sorted ISCs (Lgr5-EGFPhi) and progenitors (Lgr5-EGFPlow) from WT and Ppard/a-iKO mice on a control diet or HFD. Ppard/a-iKO mice carried Lgr5-eGFP-IRES-CreERT2 for isolation of ISC and progenitor populations. Villin-CreERT2; Lgr5-eGFP-IRES-CreERT2 animals were used as WT controls.(K) Quantification of organoids per crypt from mice in (G).To compare differences in diet, control WT mice were fed chow in (B)–(D) and a purified control diet in (E) and (H)–(K). In (B), (D), (F), and (K), each data point represents the average of 2–3 technical replicates of one animal. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.0001, one-way ANOVA.

Fig 5: The Effect of Androgen on ISC ProliferationFor counting of BrdU+ cells, 10 crypts were randomly chosen for investigation.(A) For detection of BrdU+ cells per crypt in the ORX model, IHC was performed with primary antibody to BrdU. Scale bar, 100 μm.(B) For detection of BrdU+ cells per crypt in the inhibition of AR model, IHC was performed with primary antibody to BrdU. Scale bar, 100 μm.(C) For detection of BrdU+ cells per crypt in the OVX model, IHC was performed with primary antibody to BrdU. Scale bar, 100 μm.(D) For analysis of active β-CATENIN and total β-CATENIN expression, western blotting was performed.(E) For detection of intestinal stem cells in the ORX model, IHC was performed with primary antibody to Olfm4+. Scale bar, 50 μm.(F) For analysis of relative mRNA levels of Bmp4, quantitative real-time PCR was performed in the ORX, OVX, and inhibition of AR models.(G) The phosphorylation of Smad1/5 was assessed by western blot analysis.Data, indicated as mean ± SD, correspond to three independent experiments (A, C, n = 6; B, n = 5; D, G, n = 2; E, F, n = 4 mice/group/experiment). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.