Fig. 2. 5-HT drives self-renewal of ISCs.

a The content of the indicated neurotransmitters in SPF and GF colon tissues were detected by mass spectrum, and normalized to the levels in SPF mice. SPF or GF mice (n = 3) were used for detection. GABA, γ-aminobutyric acid; 5-HT, 5-hydroxytryptamine; DA, dopamine; 3-MT, 3-methoxytyramine; 5-HIAA, 5-hydroxyindoleacetic acid; Ach, acetylcholine. b Two-month-old Lgr5^GFP mice were treated with indicated neurotransmitters or neurotransmitter antagonists. Three days later, crypts of SI (left) and colon (right) were collected, and Lgr5^GFP+ ISCs were detected by FACS. Ratios of Lgr5^GFP+ ISCs in total crypt cells were shown as scatter diagram. Riluzole, glutamic acid antagonist; Primidone, GABA antagonist; pCPA, tryptophan hydroxylase inhibitor; Chlorpromazine, DA antagonist; Atractylodin, ethanolamine antagonist. Lgr5^GFP mice (n = 8) for each group. c Two-month old mice were treated with 5-HT inhibitor pCPA for 36 h and 72 h, and representative H&E images of SI and colon were shown in left panel. 100 fields from five mice were observed and shown in right panel. 150 mg/kg pCPA was intraperitoneally injected. d Lgr5 in situ hybridization was performed in SI and colon tissues from pCPA-treated mice. Typical images were shown in left panel, and statistical results of 100 fields from five mice were shown in right panel. e pCPA-treated mice were treated with 10 Gy’s radiation, and sacrificed at indicated time points. Intestinal tissues were collected for H&E staining (upper panel). Numbers of intact crypts were shown in lower panel (means ± SD). Five 2-month-old mice were used and 20 fields were taken for each group. 150 mg/kg pCPA was intraperitoneally injected. f Sert KO SI and colon crypts were collected for ISC detection by FACS. Lgr5^GFP; Sert^+/+ (Sert^+/+) or Lgr5^GFP; Sert^−/− (Sert^−/−) mice (n = 8) were used and the ratios of Lgr5^GFP+ ISCs were shown. g LR^lacZ mice were crossed with Sert^−/− mice, followed by administration of TAM for lineage tracing analysis through intestinal whole-mount staining for β-gal. 200 μL TAM in sunflower oil (10 μg/μL) was intraperitoneally injected. 7 mice were sacrificed at indicated time points and typical sections of P65 were shown. h LR^lacZ;Sert^−/− mice were used for lineage tracing analysis, and intestinal tissues were stained for lacZ. n = 7 mice per group. For c–e, scale bars, 100 μm. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant; statistics was performed by unpaired one-tailed Student’s t-test. At least three independent repeats were performed for each experiment and the representative results were shown.