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First published online 23 March 2005
doi: 10.1242/dev.01801

Development 132, 2093-2102 (2005)
Published by The Company of Biologists 2005
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Cdx2 is required for correct cell fate specification and differentiation of trophectoderm in the mouse blastocyst

Dan Strumpf1, Chai-An Mao1,*, Yojiro Yamanaka1, Amy Ralston1, Kallayanee Chawengsaksophak1, Felix Beck2 and Janet Rossant1,3,{dagger}

1 Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto M5G 1X5, Ontario, Canada
2 Department of Biochemistry, University of Leicester, Leicester LE1 7RH, UK
3 Department of Molecular and Medical Genetics, University of Toronto, Toronto M5S 1A8, Ontario, Canada



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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 {alpha}7 (blue) and YOYO-1 (green) in 4.75 dpc embryos from Eomes+/– intercrosses. Integrin {alpha}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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  		Fig. 1. Cdx2 is expressed in cells of the TE lineage. (A) DIC image of late morula stage embryo (~16 cell). (B) Immunofluorescent localization of Cdx2 in embryo shown in A. Monoclonal anti-Cdx2 antibody detects high levels of Cdx2 in nuclei of outer cells (arrow) and lower cytoplasmic levels (arrowhead). (C) Polyclonal anti-Cdx2 (CNL) antibody detects similar expression pattern, including cytoplasmic staining (arrowhead) at late morula (~25 cell) stage. (D) Oct4 and Cdx2 protein in same embryo shown in C. Cdx2 (red) is restricted to outer cells, but Oct4 (green) is still detectable in all outer cells. Oct4 and Cdx2 colocalize in outer cells (e.g. black arrowhead). (E-I) Composite DIC confocal images of polyclonal anti-Cdx2 staining of (E) 3.25 dpc early blastocyst, (F) 3.5 dpc blastocyst, (G) 4.5 dpc implanting blastocyst, (H) 4.75 dpc and (I) 5.5 dpc. Arrow indicates nuclear staining, white arrowhead indicates cytoplasmic. Red arrowheads demarcate polar TE. Bracket indicates ExE. (J) DIC image of Cdx2-/- morula. (K) Monoclonal anti-Cdx2 staining of embryo shown in J. (L) DIC image of Cdx2-/- blastocyst. (M) Monoclonal anti-Cdx2 staining of embryo shown in L. (N) DIC image of wild-type blastocyst. (O) Monoclonal anti-Cdx2 staining of embryo shown in N. Scale bar: 15 µm in A-G; 30 µm in H,I; 20 µm J-O.

 


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  		Fig. 2. Cultured Cdx2-/- embryos fail to form an expanded blastocyst. Bright-field images of Cdx2+/+ embryos (A-D) and Cdx2-/- embryos (E-I). Eight-cell embryos from Cdx2+/– intercrosses were individually cultured, monitored for 72 hours, and images recorded at (A,E) 6 hours; (B,F) 24 hours; (G) 30 hours; (C,H) 48 hours; and (D,I) 72 hours. Scale bars: 25 µm.

 


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  		Fig. 3. Epithelial integrity of the TE is not maintained in Cdx2 mutant embryos. (A) Immunolocalization of ZO-1{alpha}+ in Cdx2+/– early blastocyst. (B) ZO-1{alpha} + in Cdx2-/- early blastocyst. (C) Immunolocalization of E-cadherin in wild type embryo shown in A. (D) E-cadherin in Cdx2-/- embryo shown in B. (E) ZO-1{alpha} + in Cdx2+/– late blastocyst. (F) ZO-1{alpha} + in extreme example of a Cdx2-/- late blastocyst. (G) E-cadherin in Cdx2+/– late blastocyst. (H) E-cadherin in typical Cdx2-/- late blastocyst. (A-F) Projections of multiple optical sections. (G,H) Single sections.

 


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  		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 {alpha}7 (blue) in embryo shown in A. Oct4 levels are reduced in the PE (arrows). Integrin {alpha}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 {alpha}7 (blue) in embryo shown in D. Integrin {alpha}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.

 


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  		Fig. 5. Oct4 and Nanog are expressed in outer cells of Cdx2-/- blastocysts. (A,B) Fluorescent in situ hybridization of Oct4 (red) and Cdx2 (blue), and YOYO-1 nuclear dye (green). (A) Cdx2+/– or Cdx2+/+ blastocyst. (B) Cdx2-/- blastocyst. Each panel is a composite image of single confocal optical sections. (C,D) Fluorescent whole-mount in situ hybridization of Nanog (red) and YOYO-1 (green). Each panel is a composite projected image of two confocal optical sections. (C) Cdx2+/+ blastocyst. (D) Cdx2-/- blastocyst. Scale bars: 20 µm.

 


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  		Fig. 6. Nanog is not downregulated in the outer cells of Cdx2-/- embryos. (A) Immunolocalization of Nanog in Cdx2+/+ morula. (B) Nanog in Cdx2-/- morula, littermate of embryo shown in A. Nanog is detected in inner and outer cells in non-mutant and mutant morulae. (C) Nanog in Cdx2+/+ in early blastocyst. Low levels of Nanog can be detected in occasional TE nuclei (arrowhead), but is largely downregulated in the TE by this stage. (D) Nanog in Cdx2-/- early blastocyst. Nanog expression persists in TE cells in Cdx2 mutants. All panels are projections of multiple optical sections.

 


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  		Fig. 7. Cultured Cdx2-/- embryos exhibit excessive cell death at the expanded blastocyst stage. (A-B') Composite images of single confocal optical sections superimposed on DIC images. Zona pellucida-free eight-cell stage embryos from Cdx2+/– intercrosses were cultured for 48 hours followed by TUNEL analysis. (A) Cdx2+/–; TUNEL-positive nuclei were detected in the ICM. (B,B') Cdx2-/- blastocyst; TUNEL-positive nuclei were observed in the ICM and TE. (B') Optical section taken at a different level of the embryo in B. Scale bars: 25 µm. (C) The number of TUNEL-positive nuclei in the ICM and TE of Cdx2+/+ or Cdx2+/– embryos (n=16) and Cdx2-/- embryos (n=7) was scored from confocal optical sections of individual embryos following TUNEL staining. The values plotted represent the mean number of TUNEL-positive nuclei (±s.e.m.). The increase in the number of TUNEL positive nuclei in the ICM and TE in Cdx2-/- embryos compared with Cdx2+/– or Cdx2+/+ embryos was significant at P<0.001 using Student's t-test analysis.

 

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© The Company of Biologists Ltd 2005