(A) Single optical sections depicting Zfp281 protein expression in pre- and post-implantation. At pre-implantation, nuclear-localized Zfp281 protein is observed in epiblast (Epi), primitive endoderm (PrE) and trophectoderm (TE) cells, as quantified in (B). Zfp281 expression is specific to epiblast at post-implantation (E5.75), quantified in (B). Immunohistochemistry of Zfp281KO embryo at E5.75 shows that the protein is not expressed, confirming the mutant as a protein null. It also reveals VE-specific background. High-magnification insets (top-right) show protein distribution in regions highlighted, white dashed lines delimit the VE layer. At onset of gastrulation (E6.5), Zfp281 is expressed in all epiblast-derived cells. (B) Quantification of nuclear levels of Zfp281 using MINS software at mid- (E3.5) and late (E4.25–4.5) blastocyst stage, revealing protein expression in all three cell types. n = 5 embryos (308 cells) for mid-blastocyst stage and three embryos (448 cells) for late blastocyst stage. See Figure 1—figure supplement 1 for immunohistochemistry of additional stages. (C) Quantification of nuclear levels of Zfp281 in VE and Epi cells in WT and Zfp281KO embryos at E5.75 using Imaris software. n = 6 embryos for the WT (20 cells per genotype) and n = 3 embryos for Zfp281KO. The expression of Zfp281 in the VE was ruled out through quantitative fluorescence level comparisons of wild-type (WT) and Zfp281KO embryos, which lack Zfp281 protein. (D) At E6.5, the A-P axis is established and WT embryos initiate gastrulation at their posterior, while Zfp281KO embryos display a thickened visceral endoderm epithelium (black arrowhead) and no A-P polarity. Insets (top-right) depict thickened VE layer (delineated by black dashed lines) in Zfp281KO embryo compared to the WT. (E) Zfp281KO embryos die around E8.0 and exhibit aberrant gross morphology at E7.75 when compared to WT with either cells of the epiblast layer undergoing apoptosis and/or constriction at the embryonic/extra-embryonic junction (white arrowheads). A = Anterior, p=Posterior, Pr = proximal, D = Distal, BF = brightfield, Scale bars represent 50 µm. Statistical significance was calculated on the average level of corrected fluorescence per embryo using Student T-test.