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Fig. 8. Eomesodermin (Eomes) homozygous mutant embryos are viable
through 6.0 dpc, but fail to differentiate the trophoblast lineage in vivo and
in culture. (A) Targeted disruption of Eomes gene. (Top panel)
Restriction maps of the mouse Eomes gene, the targeting construct and
the predicted structure of the targeted Eomes allele. The six exons
are shown as boxes, with the filled region indicating T-domain. In the
targeting construct, a 4.3 kb segment containing two-thirds of the T-domain
and the C-terminal half of Eomes was replaced with a PGK-neo cassette
(neo), which was flanked by a 3.4 kb of Eomes 5' and 2.1 kb of
Eomes 3' sequences (black boxes). An MC1-tk cassette (tk) was
used as negative selector. Arrows indicate the position of primers for
PCR-based genotyping. (Bottom panel) Representative Southern blot analysis of
tail DNA isolated from pups. The 5' probe recognizes 15.0 kb wild-type
and 10.7 kb targeted EcoRV fragments. (B,F) Immunolocalization of
Cdx2 (red) and Oct4 (blue) in 4.5 dpc embryos from
Eomes+/– intercrosses. (B)
Eomes+/–; (F) Eomes-/-. (C,G)
Immunolocalization of integrin 
7 (blue) and YOYO-1 (green) in 4.75 dpc
embryos from Eomes+/– intercrosses. Integrin
7 is expressed in the trophectoderm of the
Eomes+/– embryo (arrowheads in C), but is
undetectable in the Eomes-/- embryo (G). (D,H) Morphology
of embryos from Eomes+/– intercrosses at 6.0 dpc.
(D) Presumptive Eomes+/+ or
Eomes+/– embryo. (H) Presumptive
Eomes-/- embryo; arrowhead indicates an ICM-like
structure. (E,I) Trophoblast outgrowth assay. Blastocysts (3.5 dpc) from
Eomes+/– intercrosses were cultured individually in
tissue culture plates uncoated or pre-coated with ECM substrate for 96 hours.
(E) Outgrowth of an Eomes+/+ embryo. (I)
Eomes-/- embryo failed to attach, and remained as an
expanded blastocyst. Scale bars: 20 µm for C,F,G; 25µm for B,I; 40 µm
for D,E,H. GC, trophoblast giant cells. (J) Semi-quantitative RT-PCR analysis
for trophoblast markers in individual embryos from
Eomes+/– intercrosses. RNA was extracted from
individually cultured blastocysts and analyzed by RT-PCR. Culture conditions
are indicated genotype deduced from the absence or presence of an
Eomes-specific PCR product is indicated over each lane.
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