Early embryonic death in mice lacking the beta-catenin-binding protein Duplin

Abstract

The Wnt signaling pathway plays a pivotal role in vertebrate early development and morphogenesis. Duplin (axis duplication inhibitor) interacts with beta-catenin and prevents its binding to Tcf, thereby inhibiting downstream Wnt signaling. Here we show that Duplin is expressed predominantly from early- to mid-stage mouse embryogenesis, and we describe the generation of mice deficient in Duplin. Duplin(-/-) embryos manifest growth retardation from embryonic day 5.5 (E5.5) and developmental arrest accompanied by massive apoptosis at E7.5. The mutant embryos develop into an egg cylinder but do not form a primitive streak or mesoderm. Expression of beta-catenin target genes, including those for T (brachyury), Axin2, and cyclin D1, was not increased in Duplin(-/-) embryos, suggesting that the developmental defect is not simply attributable to upregulation of Wnt signaling caused by the lack of this inhibitor. These results suggest that Duplin plays an indispensable role, likely by a mechanism independent of inhibition of Wnt signaling, in mouse embryonic growth and differentiation at an early developmental stage.

FIG. 1.

Duplin expression during mouse development. (A) Immunoblot analysis of Duplin expression in wild-type ES cells (equivalent to E3.5), embryos (E8.5, E12.5, and E16.5), and newborn animals. The expression of α-tubulin was examined as a loading control. (B) Whole-mount in situ hybridization of wild-type embryos at E7.75, E8.5, and E10.5 with a Duplin antisense riboprobe. The result of a control experiment with a sense riboprobe is shown for an E7.75 embryo. Scale bars, 100 μm.

FIG. 2.

Gene targeting of the mouse Duplin locus. (A) Schematic representation of the wild-type Duplin allele, the targeting vector, and the mutant allele after homologous recombination. A 13-kb genomic fragment including all exons of Duplin was replaced by a loxP-neo cassette. Exons and the probe used for hybridization are denoted by open and filled boxes, respectively. Restriction sites: B, BamHI; B2, BglII; E5, EcoRV; H3, HindIII; P, PstI; S, SalI; X, XbaI. (B) Southern blot analysis with the probe shown in panel A of tail genomic DNA from the offspring of heterozygote crosses after digestion with BglII and EcoRV. The 5.4- and 8.0-kb bands corresponding to the wild-type and mutant alleles, respectively, are indicated.

FIG. 3.

Histopathology of Duplin^−/− embryos. The development of Duplin^+/+ (A, D, G, and J) and Duplin^−/− (B, E, H, and K) embryos is shown at E5.5, E6.5, E7.5, and E8.5, respectively. Higher-magnification views of the Duplin^−/− embryos are also shown (C, F, I, and L). Arrowheads in panel I indicate apoptotic cells with condensed nuclei. Abbreviations: ec, ectoderm; me, mesoderm; pen, parietal endoderm; ven, visceral endoderm; xec, extraembryonic ectoderm. Scale bars, 100 μm.

FIG. 4.

Pronounced apoptosis in early Duplin^−/− embryos. Sections of Duplin^+/+ (A and C) and Duplin^−/− (B and D) embryos at E6.0 (A and B) and E7.5 (C and D) were subjected to the TUNEL assay. Scale bars, 100 μm.

FIG. 5.

Reduced expression of the products of β-catenin target genes in Duplin^−/− embryos. Sections of Duplin^+/+ (A, D, G, J, M, and P) and Duplin^−/− (B, E, H, K, N, and Q) embryos at E7.5 were subjected to immunohistochemistry with antibodies to β-catenin (A and B), to brachyury (D and E), to Axin2 (G and H), to cyclin D1 (J and K), to cyclin D2 (M and N), or to cyclin D3 (P and Q). Higher-magnification views of the Duplin^−/− embryo sections are also shown (C, F, I, L, O, and R). Scale bars, 100 μm.

FIG. 6.

Defective outgrowth of Duplin^−/− blastocysts in vitro. (A to J) Blastocysts from heterozygote intercrosses were cultured for 6 days. The development of Duplin^+/+ (A, C, E, G, and I) and Duplin^−/− (B, D, F, H, and J) blastocysts was examined at E5.5 (A and B), E6.5 (C and D), E7.5 (E and F), E8.5 (G and H), and E9.5 (I and J). (K to N) Outgrown Duplin^+/+ (K and M) and Duplin^−/− (L and N) blastocysts at E9.5 were subjected to immunofluorescence analysis with antibodiesto β-catenin (K and L) or to cyclin D1 (M and N). Abbreviations: icm, inner cell mass; tr, trophoectoderm; zp, zona pellucida. Scale bars, 100 μm.