Extended Data Fig. 8: Keratins regulate blastocyst morphogenesis.
From: Keratins are asymmetrically inherited fate determinants in the mammalian embryo
a, Punctate desmosome structures labelled using immunofluorescence for desmoplakin (Dsp) colocalize with K8 along the trophectoderm cell–cell junctions of the blastocyst. Data are from three independent experiments. b, Analysis of apical surface curvature in control and K8/K18-knockdown blastocysts. Individual cells within the intact embryo were computationally-segmented in 3D. Single cells (blue) are selected for apical surface analysis. Middle panels show rendering of the apical surfaces (orange) of the selected cells. The right panels show fitting of the cell apical surface to a sphere for calculation of radius of apical surface curvature. Data are from three independent experiments. c, K8/K18-knockdown blastocysts display morphogenetic defects, revealed by smaller blastocyst volume, higher junctional tortuosity, and trophectoderm cells with lower radius of apical surface curvature. **P = 0.004 for blastocyst volume; ***P < 0.0001 for junctional tortuosity; ***P < 0.0001 for surface curvature; unpaired, two-tailed Mann–Whitney U-test. d, 2D confocal planes of live control and K8/K18-knockdown blastocysts, microinjected with fluorescent nanoparticles (yellow). Data are from three independent experiments. e, Images show nanoparticles within single trophectoderm cells, in control and K8/K18-knockdown embryos. Middle panels show representative trajectories of nanoparticle movement. Graph shows their mean squared displacement (MSD) over lag time. Thicker lines represent the mean of individual curves. The graph has two phases revealing different cytoskeletal properties: a time-independent (short lag times) and a time-dependent (long lag times) phase. These phases are associated with elasticity and viscosity, respectively40. Differences in MSD during the time-independent phase reveal higher elasticity, indicative of lower cytoplasmic stiffness, in the K8/K18-knockdown cells. f, Co-injection of keratin rescue constructs with K8/K18 siRNAs can restore blastocyst morphology to control conditions. Unpaired, two-tailed Student’s t-test for blastocyst volume and surface curvature; unpaired, two-tailed Mann–Whitney U-test for junction tortuosity; NS, not significant. Scale bars, 10 μm.
