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First published online 3 November 2004
doi: 10.1242/dev.01458

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Stabilization of ß-catenin in the mouse zygote leads to premature epithelial-mesenchymal transition in the epiblast

¹ Max-Planck Institute of Immunobiology, Stuebeweg 51, 79108 Freiburg, Germany
² Department of Pharmacology, Kyoto University Graduate School of Medicine, Yoshida-Konoé-cho, Sakyo-ku, Kyoto, 606-8501, Japan
³ The Jackson Laboratory, Bar Harbor, ME 04609-1500, USA

View larger version (49K): [in a new window]   Fig. 1. Mutant embryos develop to blastocysts but exhibit a phenotype at early gastrulation. Immunohistochemical detection of ß-catenin in wild-type (A) and mutant (B) ovaries revealed enhanced cytoplasmic localization of ß-catenin in mutant oocytes. (C) Genotyping of single oocytes from ß-catEx3flox/+;cre/+females by PCR demonstrates the efficient deletion of the floxed exon 3 of the ß-catenin gene by the oocyte-specific cre-recombinase; tail DNA from the same females was used as the control. (D) Western blot of cell lysates from mutant and wild-type (R1) ES cells shows the expression of the stabilized form of ß-catenin in the mutant cells. (E-H) Immunofluorescent detection of ß-catenin in wild-type (E), and mutant (F) blastocysts, and simultaneous detection for E-cadherin (green) and Oct4 (red) (G, wild type; H, mutant) revealed no differences between these embryos. (I-J) Phenotype of mutant (J,K) and wild-type (I) E6.5 embryos revealed that the epiblast of mutant embryos varied, being small or disorganized. Scale bar: 50 µm.

View larger version (145K): [in a new window]   Fig. 2. Expression of the stabilized form of ß-catenin leads to an ectodermal phenotype. (A,B) Histological sections of E6.5 wild-type and mutant embryos that were Hematoxylin and Eosin-stained. (C,D) Immunohistochemical analysis of sections from E5.5 embryos to detect ß-catenin. (E,F) Immunohistochemical analysis of sections from E6.5 embryos to detect ß-catenin. ß-catenin was localized at the cell membrane in wild-type embryos, whereas in the mutant epiblast, ß-catenin was enhanced and distributed throughout the cells. All embryos are oriented with the embryonic part to the left. EE, embryonic ectoderm. Arrow indicates thickened visceral endoderm in the mutant. Scale bar: 100 µm.

View larger version (54K): [in a new window]   Fig. 3. Nuclear function of ß-catenin in mutant epiblast. Males of the Wnt-reporter line, BAT-gal, were crossed to wild-type or ß-catEx3flox/ß-catEx3flox;cre/+ females to obtain embryos at E5.5 (A,B), E6.5 (C,D), and E7.5 (E,F). In wild type, ß-galactosidase-positive cells can first be detected on the posterior side of the proximal epiblast at E6.5 (C, arrow), and at E7.5 the ß-galactosidase expression domain extended throughout the posterior part of the embryo (E) (Maretto et al., 2003). In contrast, in mutant embryos, ß-galactosidase-positive cells were first found scattered throughout the epiblast at E5.5, and this positive staining became enhanced at E6.5 and E7.5, with no obvious pattern to the staining. Arrow: ß-galactosidase-positive cells in wild-type E6.5 embryos.

View larger version (90K): [in a new window]   Fig. 4. Expression of Wnt/ß-catenin target genes in the mutant epiblast. Immunohistochemical analysis of sections from wild-type (A) and mutant (B) E5.5 embryos revealed an enhanced cytoplasmic localization of ß-catenin in cells of the mutant epiblast. No difference between wild-type and mutant epiblast was observed for Oct4 (C,D), while some cells in the mutant epiblast expressed T-Brachyury protein (E,F). In situ hybridization analysis of the expression of Snai1 in wild-type (G) and mutant (H) embryos revealed expression in some cells of the mutant epiblast. All embryos are oriented with the embryonic part to the right. Scale bar: 100 µm.

View larger version (74K): [in a new window]   Fig. 5. Premature epithelial-mesenchymal transition in the epiblast of mutant embryos. Immunohistochemical analysis of E6.5 mutant embryo serial sections showed that some cells of the embryonic ectoderm (arrow) that were negative for E-cadherin (A) expressed T-Brachyury (B) and Lef1 (C), demonstrating their change from an epithelial to a mesenchymal cell fate. Arrow indicates cells that changed cell fate. Scale bar: 100 µm.

View larger version (56K): [in a new window]   Fig. 6. Embryonic patterning is changed as a result of stabilized ß-catenin expression. The expression of embryonic patterning genes was analyzed by whole-mount in situ hybridization of E6.5 wild-type and mutant embryos. In wild-type and mutant embryos Bmp4 had a similar expression pattern in the extra-embryonic ectoderm. The expression domain of Otx2 was less extensive in the mutant embryo epiblast in comparison to that of the wild-type embryo. No Nanog expression was found in the mutant epiblast. The mutant visceral endoderm (arrow) expressed no Hex. Arrow indicates mutant visceral endoderm negative for Hex; EE, embryonic ectoderm.

View larger version (111K): [in a new window]   Fig. 7. Mutant embryos exhibit a restricted differentiation potential. (A,B) Determination of ß-galactosidase expression in sections from E7.5 embryos that developed from aggregation chimeras between ROSA26 and mutant morulae revealed that mutant cells contributed mostly to extra-embryonic tissues and were only occasionally found in the embryonic ectoderm of the chimeras. In some cases the embryonic ectoderm cell layer was exclusively composed of ROSA26 cells (A,B). (C-E) Analysis of wild-type and mutant E6.5 embryos transplanted under the kidney capsule to induce teratomas. (C) Teratocarcinoma produced by transplantation of wild-type embryo. Several well differentiated tissues are visible, including bone, muscle, neural tissue and skin epithelium. (D) Adjacent section stained with antibody to ß-catenin. Higher magnification of area marked in panel C shows expression of ß-catenin in the membranes of the epithelial cells. (E) Histological section of tumor derived from transplanted mutant embryo. Cells similar to those present in extra-embryonic endoderm form small glandular structures (arrowhead). Hyalin substance corresponding to Reichert's membrane (arrow) fills extracellular spaces. Certain gland-like structures display substantial cellular and nuclear polymorphism (star). (F) Adjacent section stained with antibody to ß-catenin. There is intensive membrane and cytoplasmic staining. Cells exhibiting polymorphism (star) also show nuclear staining. Scale bar: 100 µm.