Extended Data Fig. 5: Extracellular matrix deposited in the ICM guides PrE cells towards the cavity surface.
From: Coupling of cell shape, matrix and tissue dynamics ensures embryonic patterning robustness
a. Immunofluorescence maximum intensity projection of an isolated ICM at stage E3.5. b. Maximum intensity projections of laminin and active integrinβ1 (9EG7) in PrE cells in the ICM in 3× blastocysts at stage E3.75. Manders’ coefficients for colocalised fractions are: Laminin overlapping Itgβ1 = 0.185, and Itgβ1 overlapping Laminin = 0.891. c. Immunofluorescence images of an inner PrE cell at the onset of migratory activity. Yellow arrowhead, site of protrusion; yellow dotted line, cell boundary. d. Maximum intensity projection of a 3× blastocyst at stage E3.75 to visualize ECM distribution in the ICM. e. Immunofluorescence images and radial laminin fluorescence intensity in 3× isolated ICMs at E3.75 stage, plotted as mean±SD. Yellow dotted arrow, line segments along which fluorescence intensity was measured. Intensity profiles were smoothed using a rolling average, and lines of the same colour correspond to measurements from the same embryo. Data from n = 8 embryos. f. Quantification of nuclear GATA6/GATA4 fluorescence intensities and laminin accumulation around cells located at the ICM surface or inside the ICM from 3× blastocysts at stages E3.5, E3.75 and E4.5. GATA6/GATA4 measurements from 366, 493, and 350 cells from n = 9,13,10 3× blastocysts for stages E3.5, E3.75 and E4.5 respectively. Laminin measurements from 140, 216, 199 cells from n = 9,13,10 3× blastocysts for stages E3.5, E3.75 and E4.5 respectively. g. Immunofluorescence images of non-migratory and migratory PrE cells in 3× blastocysts at stages E3.5 and E3.75, respectively. Quantification of local membrane curvature and laminin distribution along the PrE cell contour. Yellow dotted lines, cell contour traced starting at the yellow circle clockwise until the flat arrowhead. Line profiles were smoothed using a rolling average. n = 19, 17 cells from 15, 14 embryos at E3.5 and E3.75 stages, respectively. h. Orientation of PrE cell membrane regions with highest average curvature and highest average laminin distribution along the cell contour from (g) at stages E3.5 and E3.75, respectively. The cell contour was binned into 6 length intervals, and the curvature and laminin intensity were averaged for each bin. n = 19, 17 cells from 15, 14 embryos at E3.5 and E3.75 stages, respectively. i. Immunofluorescence and brightfield images of control and coated PMMA microbeads incubated with E-cadherin-Fc chimeric protein and laminin protein. j. PrE cell proportion in the ICM of blastocysts with implanted beads at stage E4.5. Data from n = 5,4 embryos for the two groups respectively, two-sided independent-samples t-test, p = 0.46. Scale bars, 20 μm. ns, non-significant, *** p ≤ 0.001.
