Fig. 5

Different cell types that produce Wnt ligands in the small intestinal epithelium. A Heatmap with scaled expression of genes involved in the Wnt (top) and Notch (bottom) signaling pathways. Expression levels are color-coded, with yellow indicating high expression and purple indicating low expression. Dll1/4, Delta-like canonical Notch ligand 1/4; Hes1, Hes family basic helix-loop-helix (bHLH) transcription factor 1; Notch1, Notch homolog protein 1; Rnf43, ring finger protein 43; Tnfrsf19, tumor necrosis factor receptor superfamily, member 19. B Top, violin plot showing the expression levels of Wnt3 in different cell clusters of Defa6-tdTom cells. Dots represent individual cells. Bottom, heatmap showing the expression of indicated Wnt ligands in Tcf4 wt Defa-tdTom cells. In cells with homozygous Tcf4 cKO, downregulation of Wnt ligands was detected. C Immunohistochemical analysis of the crypt compartment after genetic knockout of the Wls gene, 8 days after recombination, using indicated Cre recombinase-expressing mouse strains. Left and center, images showing the absence of Olfm4 and reduced PCNA staining observed only after inhibition of Wnt ligand secretion in all epithelial and subepithelial cells. Right, additional panel of Muc2 and tdTomato staining highlighting the mucus-producing goblet cells in the intestinal crypts and showing the effects of Paneth cell depletion using diphtheria toxin A (DTA). Bottom, documentation of recombination in Defa6⁺ and Pdgfrα⁺ cells using tdTomato reporter protein fluorescence. The scale bars correspond to 0.15 mm. Wls, Wntless. D Growth of intestinal organoids from crypts containing Tcf7l2 wt, Tcf7l2 cKO, or Wls cKO in Defa6-tdTom cells (upper panel) or Wls cKO in all cells induced by administration of 4-hydroxytamoxifen (4-OHT) in culture medium (bottom panel). E Representative fluorescence images of Tcf7l2 wt and cKO organoids showing Olfm4-positive crypt compartments and Defa6-tdTom cells. The scale bars correspond to 0.1 mm