Fig. 3.
Aberrant trophectoderm specification in embryos lacking maternal and zygotic expression of Cdx2. (A) Expression of trophectoderm (TE) specific cytokeratins, recognised by TROMA-1 antibody, is reduced in Cdx2 MZ-KO (N=6) and M-KO (N=7) embryos in comparison to both heterozygous (N=4) and homozygous (N=4) controls allowed to develop for the same time period (4.5 days post-fertilisation). Fluorescent signal intensity was quantified using ImageJ and the pipeline was automated using a macro. Image stacks in the appropriate channels were convolved with a Gaussian blur (σ=2) to reduce the effects of detector noise, then thresholded using Li׳s Minimum Cross Entropy thresholding method. This was applied to areas that include all of the labelled membrane while excluding as much background as possible. The mean intensity value of thresholded areas in the stack was calculated. (B) Representative embryos from the quantitative analysis depicted in A. (C) Ectopic expression of Nanog (white arrows) in outside cells of Cdx2 maternal-zygotic null (N=29) but not in control (Cdx2loxP) embryos. (D) Positive Cdx2 antibody control showing immunofluorscent detection of Cdx2 in trophoblast stem (TS) cells (top panel), and negative antibody control in E3.5 blastocyst (bottom panel) showing Cdx2 restriction to outer (TE) cells and omission from the ICM (indicated with dashed line).