(A) Graph showing the expression level in arbitrary units (a.u.) of Ovol1 and Cldn7 from RNA-sequencing of individual Rreb1+/+ and Rreb1-/- embryos. Each point represents a single embryo. ***p<0.001, unpaired t-test. Bars represent median and IQR. (B) Confocal optical sections showing transverse cryosections through immunostained embryos. Sb, 50 μm. In the Rreb1-/- shown in the lower panel, SNAIL is expressed laterally on either side of the posterior epiblast rather than at the posterior pole. Thus, it is unclear whether this expression demarcates a primitive streak-like structure in this case (PS?). (G) Arrowheads indicate ectopic SNAIL expression in epiblast cells. White lines demarcate a region containing a large cluster of epiblast cells ectopically expressing SNAIL, which exhibit more punctate Beta-CATENIN localization than in surrounding SNAIL-negative epiblast cells. Pr, proximal; Ds, distal; A, anterior; P, posterior; L, left; R, right; Meso, mesoderm; Endo, endoderm; Epi, epiblast; PS, primitive streak. (C) Confocal optical sections of transverse cryosection of immunostained E7.5 Rreb1-/- embryo. Arrowhead marks ectopic cells, in the upper panel, SOX2 + cells leaving the epiblast and traversing the outer endoderm layer and in the lower panel, GATA6 +mesoderm cells traversing the epiblast. Arrow marks SOX2 +debris on the outside of the embryo which may represent dead cells. Sb, 25 μm. (D) Images highlighting a chain of cells apparently exiting the epiblast and traversing the outer endoderm layer. Chain of cells is artificially colored in orange in lower panel. (E) Confocal sagittal optical section (upper panel) and maximum intensity project (MIP) (lower panel) of an immunostained E7.5 chimeric embryo containing Rreb1-/- ESCs. Arrowheads indicate ectopic SOX2 + cells. Sb, 50 μm. (F) Graph showing the proportion of SOX2HI cells localized inside the Epi, at the Epi-VE interface, mesoderm or amniotic cavity in E7.5 Rreb1-/- chimeric embryos. Data shown as the percentage of the total SOX2HI cells analyzed per embryo in each location. Each point represents scoring for an individual embryo. Total number of cells per location is shown above each bar. For all box plots, top and bottom edges of boxes represent third and first quartiles, respectively (interquartile range, IQR). Middle lines mark the median. Whiskers extend to 1.5 * IQR. (G) Confocal MIPs of immunostained E8.5 (Sb, 100 μm) and 9.5 (Sb, 200 μm) chimeric embryos containing Rreb1-/- ESCs. Arrowheads indicate ectopic SOX2 + cells. (H) Confocal sagittal optical section of a pre-gastrulation E6.0 chimeric embryo containing Rreb1-/- ESCs. Arrowheads mark ectopic SOX2 + cells. mCherry marks ESC progeny. Sb, 25 μm. Boxes show regions displayed at higher magnification. Brackets mark primitive streak. (I) Quantification of SOX2 protein levels in arbitrary units (a.u.) in normal anterior (aEpi) and posterior (pEpi) Epi cells and SOX2 high (SOX2HI) cells in E7.5 Rreb1-/- chimeric embryos. Data shown relative to mean SOX2 levels within typical aEpi cells. Each point represents a measurement from an individual nucleus (n = 696 cells, ***p<0.0001). A, anterior; P, posterior; L, left; R, right; Pr, proximal; Ds, distal; Epi, epiblast; Endo, endoderm; Meso; mesoderm; PS, primitive streak; NE, neurectoderm; Am, amnion; Al, allantois; ExE, extraembryonic ectoderm.