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Transcription factor AP-2{gamma} is essential in the extra-embryonic lineages for early postimplantation development

Heidi J. Auman1, Timothy Nottoli1,*, Olga Lakiza1,{dagger}, Quinton Winger2, Stephanie Donaldson1 and Trevor Williams1,2,{ddagger}

1 Department of Molecular, Cellular and Developmental Biology, Yale University, 266 Whitney Avenue, New Haven, CT 06511, USA
2 Departments of Craniofacial Biology, and Cellular and Structural Biology, BRB151, Campus Box C286, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Denver, CO 80262, USA
* Present address: Gene Targeting Service, Section of Comparative Medicine, Yale University School of Medicine, PO Box 208016, New Haven, CT 06520-8016, USA
{dagger} Present address: Northwestern University Medical School, Children’s Memorial Hospital, 2300 Children’s Plaza, MC 204, Chicago, IL 60614, USA



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  		Fig. 1. Targeted disruption of Tcfap2c (the gene for AP-2{gamma}) and representative genotype analysis of 7.5 d.p.c. embryos from an Tcfap2c+/– intercross. (A) Derivation of an Tcfap2c-null allele. The wild-type Tcfap2c gene (top), targeting vector (middle) and recombinant locus (bottom). Exons 6 and 7 (black boxes), and restriction enzyme sites (B, BamHI; C, ClaI; H, HindIII; P, PstI; N, NcoI) are shown along with the neomycin resistance marker (neo) and two copies of the HSV thymidine kinase gene (tk). The positions of the primer pairs used for ES colony screening by PCR amplification are shown beneath, as are the positions of the 5' and 3' probes used for Southern blot analysis (checked boxes). The position of the primers used for identifying the wild-type allele in subsequent mice are shown towards the top of the figure (Gamwt5 and Xgamma3'). (B) DNA samples were subjected to PCR analysis using a mixture of three primers (see Materials and Methods). Amplification of the wild-type allele produces a fragment of 190 bp (upper band), while amplification of the targeted allele results in a 140 bp product (lower band). Abbreviations: WT, wild type; KO, size of Tcfap2c–/– allele PCR product.

 


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  		Fig. 2. Analyses of gross morphology (A) and expression of Tcfap2c (B) in embryos from Tcfap2c+/– intercrosses. (A, parts a, d) Wild type late allantoic bud stage embryo recovered at 7.5 d.p.c., covered by parietal endoderm/Reichert’s membrane and associated trophoblast cells (part a) and with Reichert’s membrane dissected away (part d). (A, parts b,c,e,f) Tcfap2c–/– embryos recovered at 7.5 d.p.c. (b,c,e) and 10.5 d.p.c. (A, part f). (A, part b) Tcfap2c–/– mutant with Reichert’s membrane intact. Embryos are oriented with mesometrial pole to the top and are of the same magnification. (B) RNA in situ hybridization with an AP-2{gamma} antisense probe on sagittal sections of 7.5 d.p.c. embryos from Tcfap2c+/– intercrosses. Mesometrial pole is towards the top. (B, parts a-d) Darkfield images. (B, parts e,f) Brightfield images. AP-2{gamma} transcripts localize to trophoblast derivatives in wild-type conceptuses (a,c,e) but not in Tcfap2c–/– conceptuses (b,d,f). AP-2{gamma} transcripts are present in the antimesometrial decidua of both wild type (a,c,e) and mutant (b,d,f) conceptuses. Abbreviations: –/–, Tcfap2c–/–; gc, giant cell; em, embryonic region; ex, extra-embryonic region; epc, ectoplacental cone; hf, headfold; exe, extra-embryonic ectoderm; ee, embryonic ectoderm; de, deciduum.

 


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  		Fig. 3. AP-2{gamma} protein localization from 2.5-7.5 d.p.c. (A-G) Sagittal sections of 5.5-7.5 d.p.c. embryos. The mesometrial pole is positioned towards the top, and the antimesometrial pole is to the bottom. AP-2{gamma} protein is localized to wild-type extra-embryonic tissues and is present in the trophoblast giant cells (gc), extra-embryonic ectoderm (exe), ectoplacental cone (epc) and parietal endoderm (pe). No AP-2{gamma} protein was detected in 7.5 d.p.c. Tcfap2c–/– mutants (E,G). (H-M) Whole-mount analysis of morulae and blastocysts. (H,I) Fluorescent and (J,M) DAB stained immunodetection of AP-2{gamma} protein. (K,L) DNA counterstaining with Hoechst. AP-2{gamma} protein is present in all cells of the morula (H) and in all cells of both wild-type (I,J) and Tcfap2c–/– (M) blastocysts.

 


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  		Fig. 4. Histological sections of normal (A,C) and Tcfap2c–/– (B,D-H) embryos at 7.5 d.p.c. Conceptuses were sectioned longitudinally along the antimesometrial-mesometrial (AM-M) axis of the deciduum, resulting in sagittal sections of the embryos in A,C-E,G,H. The Tcfap2c–/– mutant shown in B,F is misoriented with respect to the AM-M axis, resulting in a cross-section of the embryo. The mesometrial pole is located towards the top. Individual embryos are described in the text. The line in A,B represents the AM-M axis. The line in G represents the abnormal angle of the dorsoventral axis. Scale bars: 100 µm. Abbreviations: WT, wild type; ch, chorion; am, amnion; epc, ectoplacental cone; exe, extra-embryonic ectoderm; ee, embryonic ectoderm; gc, giant cell; pe, parietal endoderm; ve, visceral endoderm; de, deciduum; me, mesoderm; AM, antimesometrial pole; M, mesometrial pole; D, dorsal; V, ventral.

 


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  		Fig. 5. In situ hybridization analysis of T (A,B), Hnf3b (C,D) and Otx2 (E,F) expression in 7.5 d.p.c. wild-type (A,C,E) and littermate Tcfap2c–/– embryos (B,D,F). Embryos are shown at the same magnification. Mesometrial pole is towards the top, anterior is towards the left and posterior is to the right.

 


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  		Fig. 6. In vitro outgrowths after 7 days in culture of wild-type (A) and Tcfap2c–/– (B-D) embryos collected at 3.5 d.p.c. from Tcfap2c+/– intercrosses. Outgrowths are shown at the same magnification. ICM, inner cell mass derivative; gc, giant cell; WT, wild type.

 


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  		Fig. 7. RNA in situ hybridization analyses of trophoblast markers in 7.5 d.p.c. embryos from Tcfap2c+/– intercrosses. (A) Detection of transcripts for Pl1 (A, parts a-d), Ada (A, parts e-h) and Mash2 (A, parts i-l) in longitudinal sections of wild-type (A, parts a,e,i) and Tcfap2c mutant (A, parts c,g,k) conceptuses. (A, parts b,d,f,h,j,l) The equivalent brightfield images. The mesometrial pole is oriented towards the top, and the antimesometrial pole is towards the bottom. (B) Whole-mount detection of transcripts for Fgfr2 (B, parts a,b), Bmp4 (B, parts c,d), Cdx2 (B, parts e-h) and Eomes (B, parts i-l) in wild type (B, parts a,c,e,i) and Tcfap2c–/– (B, parts b,d,f-h,j-l) embryos. Mesometrial pole is towards the top. Embryos are at the same magnification. Abbreviations: WT, wild type; –/–, Tcfap2c–/–; gc, giant cell; epc, ectoplacental cone; ch, chorion; exe, extra-embryonic ectoderm; al, allantoic bud; ps, primitive streak. Scale bars: 100 µm.

 


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  		Fig. 8. Chimeras derived from ROSA26-tagged Tcfap2c–/– ES cells (A-C) and from injection of ROSA26-tagged wild-type ES cells into blastocysts from Tcfap2c+/– intercrosses (D,E). (A) Adult chimeras generated by injection of Tcfap2c–/– ES cells into wild-type blastocysts. (B,C) X-gal-stained chimeras at 10.5 d.p.c. and 12.5 d.p.c., respectively, generated by aggregation of ROSA26-tagged Tcfap2c–/– ES cells with wild-type tetraploid embryos. (D,E) X-gal-stained littermate chimeras at 10.5 d.p.c. generated by injection of wild-type ROSA26-tagged ES cells into an Tcfap2c+/– blastocyst (D) and an Tcfap2c–/– blastocyst (E). Scale bars: 600 µm in B-E.

 

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© The Company of Biologists Ltd 2002