Extended Data Fig. 4: Apical polarization in PrE cells is required for directed migration and sorting.
From: Coupling of cell shape, matrix and tissue dynamics ensures embryonic patterning robustness
a. Heatmap indicating gene expression of extracellular matrix-related components in EPI and PrE cells from E3.5 blastocysts analysed by single-cell qPCR (22 cells from 3 embryos at E3.5) from ref. 13. b. Boxplot indicating differential expression of Prkci among EPI and PrE cells in the ICM cells at different stages from ref. 13, n = 36, 11, 11, 4, 4 cells from 6, 3, 3, 1, 1 embryos for the different groups, respectively. c. Immunofluorescence images of E3.5 and E4.0 isolated ICMs. Quantification of laminin deposition around EPI and PrE cells in E3.5 and E4.0 isolated ICMs. n = 18, 27, 37, 42 cells for the different groups, analysed from 7,7 embryos at stages E3.5 and E4.0 respectively. Two-sided Mann–Whitney U-test, p = 2.54e−07 for stage E3.5, p = 1.46e−13 for stage E4.0. d. Time-lapse imaging of ICMs isolated from E3.5 blastocysts expressing PdgfraH2B-GFP and R26-H2B-mCherry, treated with aPKC inhibitor Gö6983. White arrowhead marks a PrE cell unable to maintain its surface position. Time is indicated as hh:mm, t = 00:00 corresponds to start of live-imaging at stage E3.5 + 3 hours, following completion of immunosurgery. e. Single-cell tracking and analysis of distance of fluorescence-labelled EPI and PrE cells from the cavity surface in chimeric blastocysts treated with the aPKC inhibitor Gö6983. Individual cell position curves were smoothed using a rolling average. Grey dotted lines mark the average position of ICM–TE interface, and ICM–cavity interface set at d = 0. n = 7 EPI cells, 19 PrE cells from 7 embryos. f. Representative images of EPI (top) and PrE (bottom) cell shape and its quantification in mosaic-labelled E3.75 blastocysts upon inhibition of aPKC with Gö6983. White asterisks, EPI cells of interest. White arrowheads, PrE cells. Two-sided Mann–Whitney U-test, p = 0.6, n = 48, 80 measurements for EPI and PrE cells from 7 blastocysts, respectively. g. Box and scatter-plot for total ICM cell number in WT, Prkci+/+;Prkcz−/−, and Prkci+/−;Prkcz−/− blastocysts at E4.5 stage. n = 25,17 and 14 blastocysts for the experimental groups, respectively. Two-sided independent-samples t-test, p = 2.23e−08 for Prkci+/+;Prkcz−/−, and p = 1.25e−06 for Prkci+/−;Prkcz−/− blastocysts. Scale bars, 20 μm. ns, non-significant, ***p ≤ 0.001.
