Fig. 4. Reduced expression of trophectoderm markers in Cdx2^-/- blastocysts. (A) DIC image of Cdx2^+/+ 4.5 dpc blastocyst. Mural TE (mTE), polar TE (pTE), inner cell mass (ICM) and primitive endoderm (PE, arrowheads) indicated. (B) Immunolocalization of Oct4 (red), integrin 7 (blue) in embryo shown in A. Oct4 levels are reduced in the PE (arrows). Integrin 7 is specifically expressed in the trophectoderm (arrowheads in B,C). (C) Composite image of B and YOYO-1 (green, nuclear staining) (B and C are single optical sections). (D) DIC image of Cdx2^-/- 4.5 dpc, a littermate of the embryo shown in A, encased in its zona pellucida (arrowhead). (E) Oct4 (red) and integrin 7 (blue) in embryo shown in D. Integrin 7 expression is undetectable, while Oct4 expression is found in almost all cells (compare E with F). (F) YOYO-1 (green) staining in embryo shown in D,E. Many nuclei are fragmented (e.g. arrowhead) (E,F are projected image composed of 10 confocal optical sections). Scale bars: 20 µm. (G-I) Trophoblast outgrowth formation assay. Blastocysts (3.5 dpc) from Cdx2^+/– intercrosses were individually cultured in tissue culture plates uncoated or pre-coated with ECM substrate. (G) Outgrowth of a Cdx2^+/– embryo. (H,I) Cdx2^-/- embryos failed to attach, and formed a rounded mass of cells devoid of a typical blastocoel. Parietal endoderm cells were detected in some Cdx2^-/- embryos cultures (K; arrowheads). Scale bars: 50µm. GC, trophoblast giant cells. (J) Semi-quantitative RT-PCR analysis for trophoblast markers in individual embryos from Cdx2^+/– intercrosses. RNA was extracted from individually cultured blastocysts and analyzed by RT-PCR. Culture conditions are indicated (top): non-cultured 3.5 dpc blastocyst (Blastocyst), 1 day in KSOM (+1d KSOM), additional 48 hours culture in presence of serum (+2d culture). Presumptive genotype is indicated over each lane: wt/het, Cdx2^+/+ or Cdx2^+/– embryo; null, Cdx2^-/- embryo; C, control TS cell-derived RNA.