Extended Data Fig. 1: Organoid monolayers capture key physiological features of the intestinal epithelium.

a, Organoid monolayers expressing Lgr5–eGFP-IRES-CreERT2 stained for GFP and F-actin (phalloidin). Representative image of 2 experiments. b, Organoid monolayers stained for Zonula occludens 1 (ZO-1). Top: Average intensity projections of the 10 most basal (Top left) or apical (Top right) planes of the monolayer. Bottom: lateral view of the monolayer. Representative image of 2 experiments. c, Snapshots of an extrusion event happening at the villus-like region. The extruding cell is indicated with the arrowhead in the orthogonal views (bottom row). The neighbours of the extruding cell are highlighted in colours in the top views of the monolayer (middle row). The lateral views correspond to the midplane indicated with a white dashed line at their corresponding top views (Top row). Representative of 9 extrusions from 3 experiments. d, Snapshots of a cell that exhibits retrograde flow due to division. The contour of the cell of interest is delineated in red. The track of this cell is delineated in yellow. The contour of the second daughter cell that appears after the division event is delineated in green. The first and last snapshots (0 min and 160 min) correspond to basal planes of the crypt (Z = 0 µm and 2 µm, respectively). Because division occurs apically, the second and third snapshots (48 min and 52 min), correspond to an apical plane where division is better observed (Z = 12 µm). 5 crypts from 3 experiments. e, Spontaneous formation of pressurized domes in the villus-like ___domain. Top left: medial view of an organoid monolayer expressing Lifeact–eGFP. The dashed orange line defines the regions where the monolayer has delaminated to form a pressurized dome (orange arrowheads in bottom panel). Top right: 3D traction map of the same crypt. Yellow vectors represent components tangential to the substrate and the colour map represents the component normal to the substrate. Horizontal cyan line indicates y-axis position of the lateral XZ view (bottom). Bottom: lateral view of the organoid monolayers. Yellow vectors represent tractions. Representative of 3 experiments. Scale vector, 300 Pa. All scale bars, 20 μm. Stiffness of all the gels, 5 kPa.