Fig. 2. Inactivation of Wnt-β-catenin signaling derails on-time implantation. (A) Implantation in mice receiving empty or Dkk1 ADV on days 5 and 6 of pregnancy. Implantation sites (IS) were visualized by the blue dye method. Numbers within the bar indicate the number of mice with IS/total number of mice examined. (B,C) Representative photomicrograph of uteri with (B, left) and without (B, right) IS (blue bands) and (C) unimplanted morphologically normal blastocysts recovered from those without blue reaction. (D) Implantation in mice receiving vehicle, PKF115-584 or CGP049090 (each 10 mg/kg body weight) on day 5. Numbers within the bar indicate the number of mice with IS/total number of mice examined. (E) In situ hybridization showing comparable expression of amphiregulin (Areg) and Hoxa10 in day-4 uteri of mice receiving empty or Dkk1 ADV. (F-I) Overexpression of Dkk1 via Dkk1 ADV exerts no effects on the cellular level of total β-catenin (F), but, remarkably, attenuates nuclear stabilization of active dephosphorylated β-catenin (G) and c-Myc expression (H) in blastocyst trophectoderm (Tr) when examined at midnight of day 4 (day 4.5). By contrast, Nanog, an inner cell mass (ICM) marker gene, is expressed normally in ICM cells of blastocysts recovered from pregnant females receiving either Dkk1 ADV or empty vectors on day 4.5 (I). Representative immunofluorescence staining images depict Cy3-labeled antigens in red, SYTO-13-labeled nuclei in green, and the merge in yellow. Scale bars: 50 µm in C,F-I; 200 µm in E.