Extended Data Fig. 3: Vinculin is recruited towards the tight junctions in a tension-dependent manner during blastocyst expansion.

a, b, Immunostaining of wild-type embryos at the late blastocyst stage (E4.25), showing co-localization of ZO1 (green) with ppMRLC (red) (a) and MYH9 (red) (b). Line scans are shown on the right. c, d, Immunostaining of wild-type embryos at the late blastocyst stage (E4.25), showing co-localization of vinculin (red) with occludin (green) (c) and non-localization with α-catenin (green) (d). Line scans are shown on the right. A and B mark the apical and basal sides of trophectoderm cell–cell junctions. Scale bars, 5 μm. e, Left, vinculin is more enriched at the mural trophectoderm junctions than at the polar trophectoderm junctions in E4.25 blastocysts (box plot shown at top right, n = 61 cells from 11 embryos), which correlates with higher cortical tension in mural trophectoderm cells compared to those of polar trophectoderm cells (bottom right, n = 167 cells from 25 embryos). Scale bars, 20 μm. f, Top, immunostaining of blastocysts shows stronger signals of vinculin at trophectoderm junctions of reduced blastocysts (1/4 and 1/2 blastocysts) compared to whole embryos at E3.75, but equally strong signals in all blastocysts at E4.25. Scale bars, 10 μm. Bottom, box plot of vinculin signal intensity in various blastocysts at E3.75 (black, n = 58 cells from 15 embryos) and E4.25 (red, n = 54 cells from 17 embryos). An overall higher intensity of vinculin at E4.25 across all blastocysts is consistent with the trend in trophectoderm cortical tension (Fig. 1e). g, Immunostaining of vinculin in Bb(−)-treated and control embryos at the E4.25 stage. Bottom, box plot of vinculin signal intensity in control (n = 52 cells from 11 embryos) versus Bb(−)-treated blastocysts (n = 28 cells from 5 embryos). Scale bar, 20 μm. h, Schematic depicting tension-dependent vinculin recruitment at tight junctions, possibly through actin binding, as trophectoderm cells are increasingly stretched during cavity expansion. Mann–Whitney U test. All box plots show median, 25th and 75th percentiles, and whiskers extending to maximum and minimum data points. In a-d, representative examples from three independent experiments are shown.