Synchronization of blastocyst activation with uterine receptivity is essential to normal implantation. While a wide range of signaling molecules helps in specifying uterine receptivity for implantation (Wang and Dey, 2006
), there is limited information regarding the signaling network that governs blastocyst activation (Hamatani et al., 2004b
; Paria et al., 1998
; Wang et al., 2003
). Our present findings using multiple approaches demonstrate an essential role of nuclear Wnt-β-catenin signaling in ensuring blastocyst competency to implantation.
Although recent evidence suggests that preimplantation embryos possess the machinery of Wnt-β-catenin signaling (Hamatani et al., 2004a
; Kemp et al., 2005
; Wang et al., 2004
), it remained unknown whether this signaling is critical for the development of preimplantation embryos to blastocysts and subsequent implantation. Studies with genomic β-catenin null mice showed that β-catenin null mutant embryos from heterozygous crossings developed to blastocysts and implanted normally in heterozygous mother, but showed first sign of abnormal development of embryonic ectoderm on day 7.5 of pregnancy (Haegel et al., 1995
; Huelsken et al., 2000
). However, the contribution of nuclear β-catenin signaling in early embryo development and blastocyst implantation cannot be assessed in this mouse model, since β-catenin null mutant blastocysts contain a large amount of maternally derived β-catenin, and cannot be distinguished from littermate heterozygous and wild-type blastocysts by immunostaining, even on days 5 and 6 in culture (Haegel et al., 1995
A recent study using conditional elimination of β-catenin in oocytes provides evidence that zygotes, even with depletion of both maternal and zygotic β-catenin, form blastocysts in culture, suggesting that β-catenin does not play a critical role during preimplantation embryo development (De Vries et al., 2004
). However, a potential role of β-catenin in blastocyst function during implantation is predicted by this study. For example, although oocytes with conditional deletion of β-catenin develop into blastocysts, female mice with maternal β-catenin depletion produce reduced number of pups when crossbred with wild-type males in comparison to those of wild-type to wild-type mating. However, this reduction in pup numbers is rescued in females with conditional deletion of both β-catenin and E-cadherin in oocytes (De Vries et al., 2004
). Considering diverse roles of β-catenin in cellular functions, including its association with E-cadherin in adherens junctional complexes and functioning as an intermediate in canonical Wnt pathways, this study suspected that paternal derived β-catenin in blastocysts with maternal β-catenin depletion is primarily incorporated into adherens junctions, causing insufficiency for nuclear Wnt signaling and thereby leading to loss of blastocysts during the periimplantation period. In contrast, simultaneous depletion of β-catenin and E-cadherin restores nuclear β-catenin signaling in blastocysts, because in the presence of less E-cadherin more β-catenin is available for nuclear Wnt signaling (De Vries et al., 2004
). Our present investigation using the strategy of DKK1-mediated functional inhibition of nuclear β-catenin signaling and small molecule inhibitors of Wnt signaling provides direct evidence that canonical Wnt-β-catenin signaling is unlikely required for preimplantation embryo development, but is essential for normal blastocyst functions during implantation. Our reciprocal embryo transfer experiments also reveal that silencing of Wnt-β-catenin pathway does not interfere with uterine receptivity, but primarily blocks blastocysts’ competency to implant, highlighting the necessity of nuclear β-catenin signaling in blastocyst activation for implantation.
The significance of this pathway in blastocysts is further evidenced from our findings in delayed implantation models, showing that the activity of nuclear β-catenin signaling distinguishes blastocyst dormancy and activation. Coincident with blastocyst activation, Wnt antagonist DKK1 is downregulated, while canonical ligand Wnt3a is induced at higher levels, leading to intracellular accumulation of dephospho β-catenin in blastocyst Tr cells. Interestingly, DKK2 is upregulated in activated blastocysts, perhaps functioning as a negative or positive feedback regulator of β-catenin signaling depending on the presence or absence of cell-surface Kremen 2 receptors (Mao and Niehrs, 2003
). In addition, sFRP1 remaining only
in the ICM of activated blastocysts may help maintain the pluripotency of ICM cells by suppressing Wnt signaling during blastocyst activation. This is consistent with early observations that inhibition of endogenous Wnt signals in mouse embryonic stem cells prevents the cells from differentiating into mesoderm (Lindsley et al., 2006
); whereas constitutive expression of active β-catenin protein in early embryos leads to premature epithelial-mesenchymal transition in the embryonic ectoderm layer of early postimplantation embryos (Kemler et al., 2004
). This enhanced β-catenin signaling, particularly in Tr cells, is physiologically relevant to blastocyst functions, since our gain-of-function experiments demonstrate that Wnt3a is able to induce PPARδ expression in the Tr and confer blastocyst competency for implantation in cooperation with GW501516, a selective PPARδ agonist.
In parallel to activation of canonical Wnt signaling, the RhoA signaling, a potential mediator of noncanonical Wnt pathway (Veeman et al., 2003
), was attenuated in Tr cells with blastocyst activation. Since Rho proteins are required for maintenance of adherens junctions (Braga et al., 1997
; Sahai and Marshall, 2002
), this downregulation of RhoA GTPase protein and activity perhaps causes cytoskeletal reorganization and disassembly of adherens junction, thus destabilizing the leading edge of epithelial Tr cells conducive to blastocyst-uterine attachment. However, the molecular basis of divergence of Wnt signaling during blastocyst activation remains unknown.
Dvl proteins function as intermediate transducers balancing the transduction of Wnt-Fzd receptor downstream to β-catenin dependent vs independent pathways (Capelluto et al., 2002
; Itoh et al., 2005
; Torres and Nelson, 2000
). For example, nuclear translocation of vesicular Dvl proteins triggers the accumulation and stabilization of β-catenin, whereas actin-binding Dvl trafficking to the plasma membrane results in RhoA activation, affecting cell shape and morphology. In this respect, our findings of increased cytoplasmic accumulation of Dvl1 and Dvl3, and nuclear translocation of Dvl1 in the implantation-competent Tr are well correlated with activation of β-catenin signaling and attenuation of RhoA signaling during blastocyst activation, supporting the concept that Dvl controls the diversification of Wnt pathways. However, our observation of accumulation of Dvl1 & Dvl3 proteins in the Tr of implantation-competent blastocysts is contradictory to a recent report showing enrichment of Dvl3 in ICM cells of implanting blastocysts, although this study states similar activation of canonical Wnt pathway without showing detail cell-type distribution of active β-catenin (Na et al., 2007
). To confirm our findings in blastocysts, we also examined the activation of the Wnt-β-catenin signaling in Tr-derived TS cells in culture. The results showing localization of Dvl1 primarily in the nucleus and of Dvl2 and Dvl3 in both the cytoplasm and nucleus along with nuclear stabilization of active β-catenin in response to Wnt3a uphold our initial findings in delayed and activated blastocysts.
Another intriguing finding is the internalization and nuclear import of Wnt family of receptors in Tr cells during blastocyst activation. The significance and underlying mechanism of this phenomenon during Wnt signaling transduction remain mostly unknown. Recent evidence shows that endocytosis and nuclear import of Fzd2 receptor transduce Wingless signaling critical for synapse development in Drosophila
(Mathew et al., 2005
). It was also worthy of noting that reduced cell-surface Kremen 2 receptors with increased levels of DKK2 in Tr cells perhaps further enhance nuclear β-catenin signaling during blastocyst activation. Therefore, it is possible that the internalization of Wnt receptors in response to Wnt ligands involves the transduction of Wnt downstream signaling rather than the simple inactivation of receptors. Nonetheless, our present study illustrates the physiological significance of canonical vs noncanonical Wnt pathways in blastocyst functions during implantation.