Validation of the CBF:H2B-Venus reporter construct in embryos. Schematic of electroporation of a pCX:NICD and pCX:mCherry construct into the ve of an E6.5 embryo (A). Electroporation of pCX:mCherry into a CBF:H2B-Venus transgenic embryo and subsequent culture shows expression of mCherry but no reporter activation in the ve (B). Electroporation of pCX:NICD and pCX:mCherry into a CBF:H2B-Venus transgenic embryo and subsequent culture shows expression of mCherry as well as reporter activation in the ve (C, and D, white arrowheads). Of note, the ve does not normally express the nuclear-localized Venus reporter, though cytoplasmic fluorescence which is background fluorescence likely due to fixation of samples is sometimes observed (see Figures 4B and D). mCherry is not localized in the epiblast (epi), these 3D projections depict reporter expression on the embryos surface in the ve layer (see panels B3 and B1). Cells appear yellow due to strong Venus expression in epiblast (epi), this bleeds through from the underlying epiblast layer in 3D projections. By contrast, 2D data (see panels B2 and B3) reveal that there are no green (Venus-positive) cells in ve which comprises only mCherry-positive cells.