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doi: 10.1242/10.1242/dev.00355

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Dp1 is required for extra-embryonic development

Matthew J. Kohn1, Roderick T. Bronson2, Ed Harlow3, Nicholas J. Dyson3 and Lili Yamasaki1,*

1 Department of Biological Sciences, Columbia University, New York, NY 10027 USA
2 Tufts University School of Veterinary Medicine, North Grafton, MA 01536 USA
3 Laboratory of Molecular Oncology, MGH Cancer Center, Charlestown, MA 02129, USA



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  		Fig. 1. Inactivation of the Dp1 locus. (A)The Dp1 locus is shown with the indicated coding exons and restriction enzyme sites (E, EcoRI; X, Xba; N, EcoNI). A star denotes a polymorphic 129Sv EcoNI site not seen in the C57BL/6 strain. The targeting vector and mutant Dp1 allele are shown, in which part or all of exons 4-9 (hatched boxes) is deleted with the insertion of the neoR gene in the opposite reading frame. A 5' external probe and a 3' internal probe (below the wild-type allele) used for Southern analysis and six primers (arrowheads) used for genomic PCR are shown. (B) Southern analysis with the 5' external probe and EcoNI digestion was performed on F2 animals to visualize an 8.5 kb wild-type C57BL/6 band and a 6.6 kb mutant 129Sv band. Only wild-type and Dp1+/- F2 animals are recovered. (C) Genomic PCR on laser-captured embryos from microdissected decidua is shown.

 


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  		Fig. 2. Dp1-deficient embryos die despite expressing DP2. (A) Dp1+/+ and Dp1-/- embryos at E10.5 are shown for comparison. Note the small size and decreased number of somites in this Dp1-/- embryo, the most-developed of the E10.5 mutants recovered. (B) Dp1+/- and Dp1-/- embryos at E9.5 are shown for comparison. Again, note the small size and developmental delay (absence of turning, unfused neural folds) of the Dp1-/- embryo. Scales bars to the right of A and B: 1 mm. (C) Western blots of manually dissected E9.5 embryo lysates are shown with an anti-DP1 monoclonal antibody (top panel), an anti-DP2 monoclonal antibody (second panel), an anti-PCNA antibody (third panel) and an anti-actin antibody (bottom panel). Lanes marked A-F are E9.5 embryos recovered from a single Dp1+/- pregnant female. A positive control E9.5 wild-type embryo is shown in the lane marked 9.5. Genotyping of yolk sacs established the genotypes shown at the bottom of the panels. (D) A developmental timecourse is shown of DP1 and DP2 protein expression in manually dissected embryos from E8.5 to E12.5 by western blotting.

 


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  		Fig. 3. Expression of DP1 in extra-embryonic and embryonic tissues. Conceptuses were stained immunohistochemically with an anti-DP1 monoclonal antibody (A,C,E,F) or mouse IgG (B,D,G,H). (A,B) Serial sections of a wild-type embryo at E7.5. (C-H) Serial sections of a wild-type embryo at E8.5. The regions in C,D indicated by a star and triangle are shown at higher magnification in E-H. Expression of DP1 occurs in the embryo (e), in the ectoplacental cone (epc) and in the chorion (ch). Additionally, despite the background seen with mouse IgG, clear nuclear expression of DP1 is visible in trophoblast giant cells (tg).

 


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  		Fig. 4. In situ analysis of Dp1-deficient conceptuses at E8.5. Decidua containing a wild-type (A,D,G,J) and two Dp1-deficient (B-C,E-F,H-I,K-L) conceptuses were analyzed after serial sectioning. The genotypes were established for these embryos using laser-capture microdissection and then genomic PCR. (A-C) Hematoxylin and Eosin staining. (D-F) Immunohistochemical staining with TROMA1, which detects abundant trophectoderm-derived tissue in the wild-type conceptuses. (G-L) BrdU incorporation after a 30 minute in vivo labeling. (G-I) Low magnification; (J-L) high magnification. The embryo is the serpentine-like structure at the distal pole of each conceptus. The star denotes the position of the ectoplacental cone (EPC) and secondary trophoblastic giant (TG) cells at the proximal pole of the conceptus. The arrowheads denote the positions of trophoblast giant cells at the distal pole of the conceptus. Note the intensely BrdU-positive EPC and trophoblast giant cells surrounding the wild-type, but not the mutant embryos.

 


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  		Fig. 5. In situ analysis of Dp1-deficient conceptuses at E7.5. Decidua containing a wild-type (A,E,I) and three abnormal, presumptive Dp1 mutant (B-D,F-H,J-L) conceptuses were analyzed after serial sectioning. (A-D) Visualized with Hematoxylin and Eosin staining. (E-H) Immunohistochemical staining with TROMA1 to detect trophectoderm-derived tissue. (I-L) BrdU incorporation after a 30 minute in vivo labeling. The star indicates the position of the ectoplacental cone (EPC) and secondary trophoblastic giant (TG) cells at the proximal pole of the conceptus. The arrowhead indicates the position of a trophoblast giant cell at the distal pole of the wild-type conceptus.

 


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  		Fig. 6. In situ analysis of Dp1-deficient conceptuses at E6.5. Decidua containing a wild-type (A,D,G) and two abnormal, presumptive Dp1 mutants (B,C,E,F,H,I) conceptuses were analyzed after serial sectioning. (A-C) Hematoxylin and Eosin staining. (D-F) Immunohistochemical staining with TROMA1 to detect trophectoderm-derived tissue. (G-I) BrdU incorporation after a 2 hour in vivo labeling. The star indicates the position of the ectoplacental cone (EPC) and secondary trophoblastic giant (TG) cells at the proximal pole of the conceptus. The arrowheads indicate the positions of a trophoblast giant cell.

 

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