Fig 2: OPGhigh M cells cluster more in cecal patches than in Peyer’s patches.a Whole-mount immunostaining of the FAE of Peyer’s patches (left) and cecal patches (right) for OPG (green) and Spi-B (red). Nuclei were stained with DAPI (blue); scale bars: 50 µm. b Scatter plots of the fluorescence intensities of OPG versus Spi-B. Red dots represent cells stained with anti-Spi-B antibody that was conjugated with HyLyte Fluor 555 and anti-OPG antibody that was conjugated with HyLyte Fluor 647. Blue dots represent background fluorescence intensity of randomly selected non-stained cells. Fluorescence intensities were measured for at least 3000 cells from five FAEs of three mice. c Frequencies of OPGhigh M cells in Peyer’s patches and cecal patches were quantified. *p < 0.05, ***p < 0.005. Student’s t-test, n = 5 FAE from three animals. The source data underlying panels b and c are provided as a Source Data file.

Fig 3: Upon RANKL stimulation enterocytes and microfold cells originate from a common progenitor cell population. Transcription factors SPIB and SOX drive microfold cell differentiation while ONECUT2 promotes enterocyte cell differentiation by inducing transcriptional activation of Hnf4g.

Fig 4: ONECUT2 regulates M cell number and mucosal immunity in vivo. (A) Immunohistochemistry analysis for GP2 and SPIB protein expression in Peyer's patches of small intestine sections from mice treated with PBS control or CSRM617 for 14 days. Nuclear counterstaining with hematoxylin (bright field) or DAPI (fluorescence) is shown. Scale bars are annotated. Dotted rectangles show amplified areas. Yellow arrow heads show GP2+ cells and red arrow heads mark autofluorescence signal. Quantification of SPIB+ cells from n = 6 Peyer's patches sections from mice treated as in (A). (B) RT-qPCR analysis showing relative mRNA expression of M cell markers Spib, Gp2 and Tnfaip2 in small intestine sections from mice treated as described in Supplementary Figure S3D. Unpaired t test P <0.05 is represented by (*) and P <0.01 by (**). (C) Relative mRNA expression of ONECUT2-targeted genes Klf5, Klf6, Hoxb8, Foxn3, Tfcp2l1 and Hnf4g detected in tissue described in (B). Unpaired t test P <0.05 is represented by (*), P <0.01 by (**) and P <0.001 by (***). (D) Secretory IgA (SIgA) levels in faecal samples from mice treated as described in (B). Unpaired t test P <0.05 is represented by (*). (E) Relative mRNA expression of markers from IgA+ B cells Ccr9 and Itgb7 detected in tissue from (B).

Fig 5: M-cell differentiation and activation are enhanced in the absence of OPG.a The numbers of M cells labeled with Tnfaip2 (green) and GP2 (red) are prominently increased in the follicle-associated epithelium (FAE) of Payer’s patches from Opg-/- mice. Each indicated channel is shown separately in gray scale. Scale bars: 200 µm. b The numbers of Tnfaip2-positive or GP2-positive M cells in the FAE of Peyer’s patches from WT and Opg-/- mice were quantified. Values are presented as the mean ± standard deviation. ***p < 0.005 (Student’s t-test, n = 4 of WT and 9 of Opg-/- mice). c The expression levels of M cell-associated genes in the FAE of Peyer’s patches were normalized to Gapdh expression and are presented relative to the expression in the villus epithelium (VE). Values are presented as the mean ± standard deviation. **p < 0.01, ***p < 0.005 (Student’s t-test, n = 4 animals). d Whole-mount staining images of GP2 (red), Spi-B (green), and DAPI (blue) in cecal FAE. Scale bars: 100 µm. e The numbers of Tnfaip2-positive or GP2-positive M cells in the FAE of cecal patches from WT and Opg-/- mice were quantified. Values are presented as the mean ± standard deviation. *p < 0.05 (Student’s t-test, n = 4 of WT and 9 of Opg-/- mice). f The uptake of latex beads into Peyer’s patches and cecal patches was measured. Data from three independent experiments are presented as the mean ± standard deviation. ***p < 0.005. n.s. not significant (Student’s t-test, n = 7 animals). The source data underlying panels b, c, e and f are provided as a Source Data file.