Fig. 4

WNT4 negatively regulates WNT/β-catenin signaling, and relies on ROR2 receptor to promote symmetric crypt fission. A RT-qPCR analysis of the expression changes of WNT/β-catenin target genes (Axin2, Myc, Ccnd1 and Ephb3) in mouse small intestinal organoids after knockdown (left) or overexpression (right) of Wnt4. B Colocalized expression of ROR2 (green) and WNT4 (red) in both normal mouse small intestinal tissue and organoid was detected by IF staining. Boxed regions are magnified and shown in the right column. Nuclei were counterstained with DAPI (blue). White arrows indicate colocalization. Scale bars, 100 μm (columns 1–4), 50 μm (column 5). C Lysate from the whole mouse small intestine was immunoprecipitated with anti-ROR2 or anti-IgG and analyzed by western blot with anti-WNT4 and anti-ROR2 antibodies. Yellow asterisk indicates the WNT4 band. D The symmetry ratio was calculated by dividing the length of the short daughter crypt by the length of the long daughter crypt on the basis of the 3D projections of mouse small intestinal organoids with overexpression of Wnt4 combined with or without knockdown of Ror2 (n = 20 for vector, n = 22 for Wnt4-OE, n = 24 for Wnt4-OE + si-Ctr, n = 21 for Wnt4-OE + si-Ror2). E The symmetry ratio was calculated by dividing the length of the short daughter crypt by the length of the long daughter crypt on the basis of the 3D projections of mouse small intestinal organoids with either knockdown of Wnt4 or knockdown of Ror2 (n = 19 for si-control, n = 21 for si-Wnt4, n = 20 for si-Ror2). All values are means ± SD. Statistical analyses were performed by unpaired Student’s t-test between two groups (A), and one-way ANOVA with Tukey’s multiple comparisons test (D, E). ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001