Fig. 6: Positive feedback of Wnt signaling on BMP signaling contributes to the villus length control.

a Immunofluorescence staining of Ki67 in Villin^CreERT2; Apc^flox/+mice at 16 dpi. Quantification of the villus length and epithelial cell number from the proximal to the distal small intestine and Ki67⁺ cells in the jejunum. N = 3 mice/group. Scale bars, 200 µm. The white arrows indicate the apex or the base of the villus. b RT-qPCR of Axin2, Ki67, Bmp2, Bmpr1a, Pmp22, and Slc34a2 in intestinal organoids derived from control and Apc heterozygous (hez) mice. N = 3 independent experiments. c RT-qPCR of Axin2, Ki67, Id1, and Id2 in intestinal organoids cultured in E and ER medium. E, EGF; R, R-spondin,. N = 3 independent experiments. d Immunofluorescence staining and quantification of p-Smad1/5 in the proximal region of the small intestine between control and Apc hez mice, with error bars representing the standard deviation (SD), visualized as shaded regions in the plots. N = 3 mice/group. Scale bars, 200 µm. e A schematic diagram of a two-stage cell lineage model regulated by Wnt and BMP signaling, modeled using the Hill equation. Wnt signaling exhibits a feedback regulation on BMP signaling. Conversely, BMP signaling inhibits Wnt signaling (Created in BioRender. Liu (2025) https://BioRender.com/2tio9zc). f The mathematical model shows BMP activity, cell removal rate, and villus length under three conditions: (i) normal; (ii) Wnt activation without BMP regulation (γ_1 = 0); (iii) Wnt activation with BMP regulation (γ_1). The colormap represents progenitor cell proportion along the crypt-villus axis, with a dashed line marking the crypt-villus boundary. g Immunofluorescence staining of Ki67 in the proximal small intestine of the indicated mice. Mice were injected with tamoxifen for five days and then sacrificed 2 days later. N = 3 mice/group. Scale bars, 200 µm. The white arrows indicate the apex of the villus. For a, villus length and cell number were analyzed by two-way ANOVA with Tukey’s multiple comparison test; Ki67⁺ cells were analyzed by unpaired t-test with Welch-correction (two-sided). b, c Analyzed by unpaired t-test with Welch-correction (two-sided); g was analyzed by one-way ANOVA with Tukey’s multiple comparison test. Data represent mean ± SD.