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. 2013 Mar 4;8(3):e57428. doi: 10.1371/journal.pone.0057428

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© 2013 Tanaka et al

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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In Q-PCR analyses, E7.5 Dullard ^−/− embryos (Du^−/−) showed significant down-regulation of WNT/β-catenin-dependent downstream genes: Brachyury (P<0.0002, Student’s t-test), Axin2 (P<0.008), Lef1 (P<0.01) and Ifitm1 (P<0.03), compared with that in Dullard ^+/− embryos (Du^+/−). Dullard ^+/−; Wnt3 ^+/− embryos (Du^+/−; Wnt3^+/−) showed significant down-regulation of WNT/β-catenin- dependent downstream genes: Brachyury (P<0.02), Axin2 (P<0.01) and Lef1 (P<0.002) compared with that in Du^+/−, and also showed significant up-regulation of Brachyury (P<0.008), Axin2 (P<0.02), Lef1 (P<0.03) and Ifitm1 (P<0.001) compared with that in Du^−/−. Expression of BMP downstream target genes (Id1 and Msx2) was unchanged. Du^−/− showed significant up-regulation of Wnt3 (P<0.0008) and Dkk1 (P<0.01) compared with that in Du^+/−. Du^+/−; Wnt3^+/− showed significant down-regulation of Wnt3 and Dkk1 compared with that in Du^+/− [Wnt3 (P<0.02) and Dkk1 (P<0.05)] and Du^−/− [Wnt3 (P<0.0005) and Dkk1 (P<0.001)], respectively. mRNA expression levels were normalized to those of Gapdh (internal control), and the levels in Dullard ^+/− embryos (≈ control) were set to 1. Arbp, internal control.