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First published online 16 January 2008
doi: 10.1242/dev.015339

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Inactivation of nuclear Wnt-β-catenin signaling limits blastocyst competency for implantation

¹ Departments of Pediatrics, Vanderbilt University Medical Center, Nashville, TN, USA.
² Departments of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, TN, USA.
³ Departments of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN, USA.
⁴ Departments of Pharmacology, Vanderbilt University Medical Center, Nashville, TN, USA.
⁵ Department of Medicine, Stanford University School of Medicine, Center for Clinical Sciences Research 3100, 269 Campus Drive, Stanford, CA 94305, USA.

View larger version (50K): [in this window] [in a new window]   Fig. 1. Consequence of silencing nuclear β-catenin for mouse preimplantation embryo development. (A,B) Immunofluorescence localization of total (A) and dephosphorylated (active, B) β-catenin in mouse preimplantation embryos. (C-F) Recombinant Dkk1 protein (5 µg/ml) and PKF115-584 (0.1 µM) block nuclear import of active dephosphorylated β-catenin and Cdx2 expression in preimplantation embryos, without interfering with the cellular level of total β-catenin and the development of 2-cell embryos to blastocysts in culture. Two-cell embryos recovered by flushing day-2 pregnant oviducts were cultured in groups of 5-10 in 25 µl of M16 medium under silicon oil in an atmosphere of 5% CO2 and 95% air at 37°C for 72 hours and the number of blastocysts that developed was recorded. Experiments were repeated 3-5 times. The numbers above the bar in C indicate the number of blastocysts developed per the number of cultured 2-cell embryos. Cy3-labeled active β-catenin in red, SYTO-13-labeled nuclei in green, and the merge in yellow. ICM, inner cell mass; Tr, trophectoderm; veh: vehicle; PKF, PKF115-584. Scale bars: 50 µm.

View larger version (51K): [in this window] [in a new window]   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.

View larger version (60K): [in this window] [in a new window]   Fig. 3. Wnt pathways in dormant and activated mouse blastocysts. (A) Differential patterns of phosphorylated (inactive) and dephosphorylated (active) β-catenin during blastocyst activation. Whereas total β-catenin distribution was comparable in dormant (Dor) and activated (Act) blastocysts, phosphorylated β-catenin was primarily detected in the inner cell mass (ICM) and active β-catenin mostly in the trophectoderm (Tr) of activated blastocysts. (B) Wnt3a was induced in activated blastocyst Tr, whereas Wnt4 and Wnt5a were detected at high levels in the same cell-types in both dormant and activated blastocysts. (C) Dynamic expression of Wnt antagonists Dkk1, Dkk2 and Sfrp1 in delayed-implanting blastocysts. Whereas Dkk1 was downregulated, Dkk2 was induced in the Tr with blastocyst activation. By contrast, Sfrp1 expression was restricted to the ICM of activated blastocysts. (D) Expression of Wnt receptor subtypes Fzd2, Fzd4, Lrp5, Lrp6, Kremen1 and Kremen2 in dormant and activated blastocysts. An intriguing observation is the internalization and nuclear import of Wnt receptors in activated blastocysts. (E) Expression of Dvl1-3 proteins in delayed-implanting blastocysts. It is notable that Dvl1 and Dvl3 increasingly accumulated in the cytoplasm with visible nuclear localization of Dvl1 in the Tr during blastocyst activation. (F) c-Myc was induced in Tr cells of activated blastocysts. (G) Downregulation of total and GTP-bound (active) RhoA GTPase in the Tr during blastocyst activation. Representative immunofluorescence staining images depict Cy3-labeled antigens in red, SYTO-13-labeled nuclei in green, and merge in yellow. Scale bars: 50 µm.

View larger version (46K): [in this window] [in a new window]   Fig. 4. Nuclear β-catenin signaling in TS cells in culture. Western blotting analysis of Wnt-family components in TS cells. (A) Time-dependent accumulation and nuclear translocation of active β-catenin in response to recombinant Wnt3a protein (50 ng/ml) in differentiating TS cells. (B) Wnt3a-induced c-Myc and Ppar expression in differentiating TS cells. (C) Dkk1 (1 µg/ml) and PKF115-584 (1 µM) blocked Wnt3a-induced cytoplasmic accumulation of active dephosphorylated β-catenin in TS cells by 2 hours of co-treatments. Notably, even basal levels of nuclear β-catenin disappeared following PKF115-584 treatment. (D) Similar treatment of PKF115-584 (1 µM) downregulated Wnt3a-induced c-Myc and Ppar expression in differentiating TS cells. (E) Dvl1-3 proteins in TS cells. Whereas Dvl1 was only detected in the nucleus, Dvl2 and Dvl 3 were detected in both the cytoplasm and nucleus in response to Wnt3a. (F) Wnt receptors Fzd2, Fzd4, Lrp5, Lrp6, Kremen1 and Kremen2 in TS cells. Wnt3a facilitated nuclear import of Wnt receptor subtypes in differentiating TS cells, mimicking the finding in blastocysts during activation. C, M and N indicate cytoplasmic, membrane and nuclear protein extraction, respectively.

View larger version (51K): [in this window] [in a new window]   Fig. 5. Wnt-β-catenin signaling synergizes with that of Ppar to confer blastocyst competency for implantation. (A,B) Overexpressing levels of Dkk1 (A) or PKF115-584 (B) blocked activation of dormant blastocysts for implantation in response to E2 (3 ng/mouse). Numbers within the bar indicate the number of mice with implantation sites (IS)/total number of mice examined. (C,D) Representative photomicrographs of uteri without blue bands (C, right) and morphologically dormant blastocysts (D) recovered from mice treated with PKF115-584. (E,F) Recombinant Wnt3a protein (200 ng/ml) induced nuclear stabilization of active dephosphorylated β-catenin and Ppar expression in dormant blastocysts in culture. Co-treatment of Wnt3a with Dkk1 (1 µg/ml) or PKF115-584 (1 µM) antagonized Wnt3a-induced β-catenin stabilization. Cy3-labeled antigens in red, SYTO-13-labeled nuclei in green, and merge in yellow. (G,H) Wnt3a and/or GW501516 conferred blastocyst implantation competency. Dormant blastocysts were cultured in the presence of vehicle, Wnt3a (200 ng/ml) and/or GW501516 (a selective Ppar agonist, 1 µM) for 24 hours before transfer into pseudopregnant delayed recipients. Numbers within the bar in G indicate the number of recipients with IS/total number of mice examined, and those in H indicate the number of IS/total number of blastocysts transferred; *P<0.05, Student's t-test. Scale bars: 50 µm.