Extended Data Fig. 1. Cell and tissue quantification.

A, Example of selecting crypt and villus region for quantification of single-cell apical and basal size in bulged organoid. Upper panel, from left to right: Z-projection of ZO-1 and Lyz staining in bulged organoid, selected apical segmentation with Lyz signal in 3D and ZO-1 signal (white) projected in 3D apical side in villus from xy view, in villus from yz view, in crypt from xy view and in crypt from yz view. Lower panel, from left to right: selected basal segmentation with Lyz signal in 3D and ZO-1 signal (white) project in 3D basal side in villus from xy view, outline of villus basal segmentation from xy view, crypt basal segmentation from xy view, outline of crypt basal segmentation from xy view and yz view. Colour of heatmap indicates the size of single-cell apical or basal membranes. B-C’. Tissue compaction along crypt-villus axis in the development of intestinal organoid and in vivo tissue. B. Representative images of bulged and budded organoids with CD44 and DAPI staining. B’. Plot of cell density in crypt (red dashed double arrowhead line) and villus (blue dashed double arrowhead line) tissue as indicated in B. Two-tailed t-test for bulged crypt (n=22) and bulged villus (n=22), p < 10 ^-5; for budded crypt (n=19) and budded villus (n=19), P < 10 ^-11. C. Representative images of immunostaining on the section of mouse intestine at the age of P1 and P11. C’. Plot of cell density in crypt (red dashed double arrowhead line) and villus (blue dashed double arrowhead line) regions as indicated in C. Two-tailed t-test for P1 crypt (n=32) and P1 villus (n=31), p < 10 ^-10; for P11 crypt (n=40) and P11 villus (n=44), P < 10 ^-23. Experiments and imaging analysis in A, and experiments in B-C were repeated at least three times independently with similar results. Scale bars, 20 μm. Violin plot lines in B’ and C’ denote quartile for each group.