Fig. 3: Cell sorting involves active directed migration of PrE cells towards the surface via actin-mediated protrusions.
From: Coupling of cell shape, matrix and tissue dynamics ensures embryonic patterning robustness
a, Experimental strategy to generate mosaic-labelled cells in large blastocysts to visualize EPI/PrE cell dynamics. b, Time-lapse images of a representative EPI cell (top) and PrE cell (bottom) expressing PdgfraH2B-GFP and membrane tdTomato from mosaic-labelled blastocysts. White dotted lines denote cavity surface; white asterisk indicates EPI cell of interest; white arrowheads denote membrane protrusions in PrE cells. Time is indicated as h:min, t = 00:00 corresponds to start of live-imaging at stage E3.5 + 3 h. c, Distance of fluorescence-labelled EPI and PrE cells from the cavity surface. Cell position curves were smoothed using a rolling average. Grey dotted lines, average position of ICM–trophectoderm (TE) interface and the ICM–cavity interface. n = 14 EPI cells and 31 PrE cells from 13 embryos. d, Representative images (left) and circularity quantification (right) of EPI (top) and PrE (bottom) cell shapes in mosaic E3.75 blastocysts. White asterisks denote EPI cells; white arrowheads indicate PrE cell protrusions. Two-sided Mann–Whitney U-test, P = 7.9 × 10−22. n = 68 and 84 measurements from 14 embryos for EPI and PrE, respectively. e, Schematic and polar histogram of direction of PrE cell protrusions in the ICM with respect to the cavity. n = 113 measurements from 12 embryos. Two-sided Mann–Whitney U-test compared with Extended Data Fig. 3c, P = 1.31 × 10−6. f, Scatter-plot of angle of protrusions in PrE cells versus distance of the cell from the cavity. Dotted line, linear regression. Pearson’s R = 0.179, P = 0.058. n = 113 measurements from 12 embryos. g, Representative images of control and latrunculin B (LatB)-treated E4.0 isolated ICMs (left) and quantification of sorting score (right). n = 20 and 11 ICMs for control and LatB-treated ICMs, respectively. Mann–Whitney U-test, P = 6.16 × 10−6. h, Representative images of control and CK-666-treated E4.0 isolated ICMs and quantification of sorting score. n = 20 and 12 ICMs for control and CK-666-treated ICMs, respectively. Mann–Whitney U-test, P = 3.69 × 10−4. i, Immunofluorescence images (left) and quantification of number of ectopic PrE cells (right) of Rac1+/+, Rac1+/− and Rac1−/− E4.5 blastocysts White arrowhead denotes ectopic PrE cell. n = 9, 17 and 16 blastocysts, respectively. Mann–Whitney U-test, P = 0.133 (Rac1+/+ versus Rac1+/−), P = 0.001 (Rac1+/+ and Rac1−/−). Scale bars, 20 μm. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.
