Extended Data Fig. 9: Increased cortical tension leads to reduced cavity expansion rate and size, whereas ECCD1 treatment preserves tight junctions.

a, Volume expansion rate in embryos treated with 20 μM lysophosphatidic acid (LPA, n = 16 embryos), and 0.5 nM calyculin A (CalyA, n = 21 embryos) compared to wild type (n = 38). LPA and calyculin A are known to activate cortical contractility. b, Maximum cavity diameter reached for wild type (n = 14 embryos), LPA (n = 16 embryos) and calyculin A (n = 25 embryos). c, Total cell number for wild type (n = 30 embryos) and embryos treated with LPA (n = 15 embryos) and calyculin A (n = 11 embryos) is similar, which indicates that the reduced expansion rate and cavity size is not due to a reduced cell-proliferation rate. Box plots show median line, 25th and 75th percentiles, and whiskers extending to maximum and minimum data points. Mann–Whitney U test. d, e, Tight junctions remain sealed and functional, despite impaired functions of adherens junctions. d, Top, immunostaining of E5.0 embryos treated with 50 μg ml⁻¹ of ECCD1 (representative example from three independent experiments). A and B indicate the apical and basal sides of cell–cell junctions, respectively. Bottom, corresponding line scans show that tight junctions appear intact, as shown by the decoupling of signals of ZO1 and occludin from E-cadherin. A.U., arbitrary units. Scale bar, 5 μm. e, Top, tight-junction leakage assay shows that an ECCD1-treated embryo loaded with fluozinc (100 μM) displayed no increase in fluorescence intensity within the cavity when ZnCl₂ (200 μM) was added. Bottom, percentage of blastocysts showing no difference in permeability (green) in ECCD1-treated embryos compared to control embryos. N, number of embryos. Scale bar, 20 μm.