Fig. 2: Acquisition of the apical domain decreases surface tension and is sufficient for retaining PrE cells at the fluid interface.
From: Coupling of cell shape, matrix and tissue dynamics ensures embryonic patterning robustness
a, Schematic representation and time-lapse images of a mosaic-labelled ICM isolated from an R26-creER;mTmG blastocyst. Time is indicated as h:min, t = 00:00 marks stage E3.5 + 3 h. Yellow arrowheads denote cell shape changes in a surface cell. b, Representative immunofluorescence images of E3.5 ICMs highlighting cell shape among EPI and PrE at the ICM–fluid interface. Analysis of surface cell aspect ratio from E3.5 isolated ICMs to compare EPI and PrE cell shape. n = 53 and 38 EPI and PrE cells from 16 isolated ICMs. Two-sided Mann–Whitney U-test, P = 6.44 × 10−3. c, Immunofluorescence image of an E3.5 isolated ICM showing the distribution of ppMRLC and Actin in EPI (top) and PrE (bottom) cells on the ICM surface, and quantification of normalized ppMRLC distribution at the outer cell cortex. n = 29 EPI and 42 PrE cells from 10 ICMs. Two-sided Mann–Whitney U-test, P = 2.18 × 10−9. d, Micropipette aspiration of E3.5 ICMs expressing PdgfraH2B-GFP (green) and membrane tdTomato (mT, magenta) and scatter-plot of measured surface tension of outer cells versus logarithm of PdgfraH2B-GFP fluorescence intensity of the cell. White arrowhead marks the site of cell aspiration and the white dotted line indicates cell surface contour. n = 40 cells from 24 ICMs. Black dotted line denotes linear regression with slope −432 ± 69 pN μm−1, Pearson’s R = −0.71 and P = 2.8 × 10−7. Interfacial tension is calculated using the Young–Laplace equation where γcm indicates cell–medium interfacial tension, Pc, aspiration pressure, Rp, radius of pipette and Rc, curvature radius of cell surface. e, Left, schematic for the experimental strategy using chimeric ICMs to test the functional role of apical polarity in cell positioning. Right, immunofluorescence images of 2× chimeric ICMs composed of cells from WT + WT combination (top) and WT + Prkci+/−Prkcz−/− combination (bottom). IS, immunosurgery. f, Analysis of the surface retention of WT versus Prkci+/−Prkcz−/− cells in chimeric ICMs from e. The plot indicates the proportion of all surface cells that are mT+ for WT + WT and WT + Prkci+/−Prkcz−/− combinations. n = 10 and 22 ICMs for the two groups, respectively. Two-sided Mann–Whitney U-test, P = 1.08 × 10−3. Scale bars, 20 μm. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.
