Fig. 3. 5-HT generated by enteric serotonergic neurons contributes to ISC self-renewal.

a Lgr5 in situ hybridization was performed in SI and colon tissues from Tph1- and Tph2-KO mice. Typical image were shown in left panel, and 100 fields from five mice were observed and shown in right panel. Scale bars, 100 μm. b, c Lgr5^GFP;Tph1^−/− mice and Lgr5^GFP;Tph2^−/− mice were sacrificed on P60 and intestine tissues were collected for immunofluorescence staining by indicated antibodies. Typical images of small intestine (b) and large intestine (c) are shown. 100 fields from five mice were observed and calculated. Scale bars, 50 μm. d Tph1 and Tph2 KO mice were treated with 10 Gy’s radiation, and sacrificed at indicated time points. Intestinal tissues were collected for H&E staining (upper panel) and numbers of intact crypts were shown in lower panel (means ± SD). 20 fields from five mice were taken for each group. Scale bars, 100 μm. e Fluorescence staining of intestinal tissue from 2-month-old Lgr5^GFP, Lgr5^GFP;Tph1^DTR and Lgr5^GFP;Tph2^DTR mice 6 days after the first diphtheria toxin (DT) treatment. 100 ng/mouse DT treatments were performed on day 0 and day 3. 100 fields from five mice were observed. Scale bars, 50 μm. f LR^lacZ;Tph1^DTR and LR^lacZ;Tph2^DTR mice were treated with 100 ng/mouse DT every 3 days, and used for lineage tracing analysis, and intestinal tissues were stained for lacZ observation. 6 mice were used. *P < 0.05, **P < 0.01, ***P < 0.001 by unpaired one-tailed Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001 by unpaired one-tailed Student’s t-test; ns, not significant. At least three independent repeats were performed for each experiment and the representative results were shown.