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Fig. 2. Homozygous Ncad k.i. mutants fail to form an intact trophectoderm. (A-B'') Double immunolabeling of E- and N-cad protein. E-cad is localized at the cell-cell contact sites in wt (A) and heterozygous Ncad k.i. E4.5 blastocysts (A'), but is absent in homozygous Ncad k.i. embryos (A''). N-cad immunolabeling is observed in heterozygous (B') and homozygous mutants (B''). No N-cad staining above background is observed in wt embryos (B). Co-localization of E- and N-cad at cell-cell contact sites of both ICM and TE is seen in heterozygous preimplantation embryos (A',B'). Additional punctate staining of N-cad is seen on the apical membrane of TE cells (B', arrow). (C-E''') Time-lapse analysis of the preimplantation development of Ncad k.i. embryos. Recordings were started at the 8-cell stage (C-C'') and continued for 24 hours. Representative pictures of recordings are shown. At the 8-cell stage, wt (C), heterozygous (C') and homozygous (C'') embryos were indistinguishable, and all developed normally to compacted morulae (D-D''). (E-E''') Representative pictures of in vitro-cultured E4.5 embryos. Wt (E) and Ecad^+/Ncad (E') embryos formed expanded blastocysts, whereas homozygous Ecad^Ncad/Ncad mutants showed loosening of cell-cell contacts and formed either no cavity (E'') or only small cavity-like cysts (E'''). (F) RT-PCR analysis of single E3.5 blastocysts: wt (1), heterozygous (2) and homozygous k.i. (3) embryos with primers for Ecad and Ncad mRNA as indicated. Scale bar: 25 µm.

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