Fig. 2

Venus and GATA6 expression in blastocyst-stage embryos. a Live imaging of Gata6 ^H2B-Venus/+ heterozygous blastocysts for 17 h from E3.5-E4.25. Venus is expressed both in the ICM and TE. Schematic diagram of E3.5 stage blastocyst depicts ‘salt-and-pepper’ distribution of cells fated to PrE (GATA6+, blue) and Epi (NANOG+, red), while TE is indicated as green. By E4.5, PrE and Epi cells have sorted to the exterior versus interior of the ICM, respectively. b Co-localization of Venus (green) with endogenous immunolabeled GATA6 protein (blue) in fixed Gata6 ^H2B-Venus/+ embryos. Nuclei are stained with Hoechst (grey). For (a) and (b), images are 5 μm projections of whole z-stacks. c Box plots showing the level of Venus expression in each blastocyst lineage (TE, Trophectoderm; EPI, Epiblast; PRE, Primitive Endoderm) in Gata6 ^H2B-Venus/+ heterozygous blastocysts at E3.5 and E4.5. Each dot represents the average level of Venus expression (as logarithm) for each embryo and lineage (n = 4 embryos at E3.5; n = 3 embryos at E4.5). PrE cells expressed significantly higher levels of Venus than EPI cells at both stages (ANOVA, with post-hoc Tukey’s range test; p < 0.01). d Scatter plots showing the expression of Venus and endogenous GATA6 (as logarithms) in ICM cells of all embryos at each stage (blue: PrE; red: EPI cells). Expression levels of Venus show a positive and significant correlation with those of GATA6 PrE cells (blue) at both E3.5 and E4.5. A weak correlation between Venus and GATA6 levels was observed in the EPI of E3.5 but not E4.5 embryos. Pearson’s correlation coefficient and p values are shown in the graphs next to the corresponding group. ** = p < 0.01 Scale bars = 20 μm