Fig. 4: Macropinocytosis regulates tissue crowding during epithelial development.

a Representative images of multiciliated cells (MCCs, marked with acetylated tubulin ab) in ST47 embryo after treatment with DMSO or Roscovitine. Scale bar 300 μm. b Quantification of MCC number in embryo treated for 24 h with either DMSO or Roscovitine (n = 4 exp. and 50 DMSO and 51 Roscovitine embryos; two-sided unpaired t-test, p-value = 1.42e-014). c Representative image of macropinocytic events (arrowheads) occurring next to MCCs in the epithelium of a TubA1A-membrane RFP transgenic embryo labeling multiciliated cells (red) and injected with Lifeact-FP (cyan). Dashed lines represent rosette-like structure around MCCs. Scale bar 30 μm. d Representative SEM image showing a macropinocytotic event (arrowhead) adjacent to an MCC. Scale bar 5 μm. e Quantification of the number of events (MP) per cell per hour for cells adjacent or not adjacent to MCCs (n = 4 exp. 191 macropinocytotic events; two-sided paired t-test, p-value = 0.0034). f, h, j Images of a simulated epithelium with multiciliated cells (asterisk). Cells are color-coded for effective pressure (f), tension (h) or shear stress (j). Dashed lines represent rosette-like structure around MCCs. g, i, k Percentage of non-MCC neighbors and MCC neighbors that are in the highest decile of effective pressure (g) and shear stress (k) or in the lowest decile of tension (i) (n = 10 simulations; two-sided paired t-test; effective pressure p-value = 0.0004, tension p-value = 0.0021, shear stress p-value = 5.4e-05). l Quantification of the fold difference in macropinocytotic events comparing 1 h before and 1 h after treatment with DMSO (n = 9 exp.), 5 μM LY294002 (n = 4 exp.; p-value = 8.0e-06), or 1 mM Amiloride (n = 7 exp.; p-value = 0.0002); two-sided unpaired t-tests. m Representative images of MCCs (marked with acetylated tubulin ab; red/white) in ST46 embryos stained with phalloidin (cyan) after treatment with DMSO, LY294002 or Amiloride. Scale bar 300 μm. n Quantification of the number of MCCs after treatment with DMSO (n = 115 embryos), 5 μM LY294002 (n = 81 embryos; p-value = 0.0457) or 1 mM Amiloride (n = 49 embryos; p-value < 0.0001); two-sided unpaired t-tests. Source data are provided as a Source Data file.