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Fig. 5. Development of Ncad-GFP k.i. embryos in the absence of maternal E-cad. Representative pictures of time-lapse recordings showing embryos without maternal E-cad but expressing paternal E-cad (Ecad^-/+) (A,B) or expressing N-cad-GFP from the k.i. allele (Ecad^-/Ncad-GFP) (A',B'). Both embryos compact in a similar fashion around E3.0 (A,A'). By E4.0, embryos expressing paternal E-cad develop to early blastocyst (B), whereas embryos expressing paternal N-cad-GFP from the k.i. allele begin to deteriorate shortly after compaction (B'). Immunolabeling shows expression and membrane localization of E-cad from the paternal allele in Ecad^-/+ embryos (C) and no E-cad expression in embryos carrying a paternal Ncad-GFP k.i. allele (C'). The same embryos were co-stained with a polyclonal anti-ezrin antibody (D,D'). Expression of N-cad-GFP was confirmed by immunostaining with a monoclonal anti-GFP antibody (E'). Only low levels of background staining are seen in embryos with a paternal E-cad allele (E). Embryos shown in E and E' were co-stained for ZO-1 (F,F'). (G-H') Oct4 and Cdx2 are expressed in Ncad-GFP k.i. embryos in the absence of maternal E-cad. 3D-reconstruction pictures from multiple optical sections show nuclear localization of Oct4 mostly in the ICM of Ecad^-/+ early blastocyst (G) and inner cells of Ecad^-/Ncad-GFP morulae (G'). Cdx2 is expressed exclusively in outer cells in both Ecad^-/+ (H) and Ecad^-/Ncad-GFP (H') embryos. Co-expression of Oct4 and Cdx2 is still observed in trophectodermal cells of E3.5 embryos. Arrows show ICM, arrowheads show TE. Scale bar: 25 µm.

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