Taken together, our findings suggest that β-catenin, acting independently of TCF/LEF factors, can reinforce the pluripotent status of mESCs and impair their efficient differentiation. The role of Wnt/β-catenin signaling in ESC pluripotency has been somewhat controversial. Long-term Wnt/β-catenin signaling promotes mesendodermal differentiation of ESCs (Bakre et al., 2007
; Lindsley et al., 2006
), whereas short-term treatment of ESCs with the GSK-3 inhibitor BIO or Wnt-3a enhances pluripotency of mouse and human ESCs (Sato et al., 2004
). Expression of Wnt ligands and receptors has been detected through in situ hybridization in the inner cell mass of mouse blastocysts, and active β-catenin has been detected in the nuclei of mouse blastocysts via immunofluorescence visualization (Kemp et al., 2005
; Xie et al., 2008
), suggesting that Wnt/β-catenin signaling plays a physiological role in the tissues from which ESCs are derived.
It has been reported that Wnt/β-catenin signaling can synergize with LIF/Stat3 signaling to sustain pluripotency in mESCs (Ogawa et al., 2006
). The role of LIF/Stat3 signaling in embryonic stem cell maintenance has been linked to the blockade of mesendodermal differentiation (Ying et al., 2003
). Our model, in which β-catenin directs mesendodermal differentiation through TCF-mediated signaling and promotes pluripotency through a TCF-independent mechanism, can help to explain the observed synergism between LIF and Wnt/β-catenin signaling.
Recently, it has been discovered that GSK-3 inhibitors (CHIR99021 or BIO) greatly enhance the ability to establish de novo mESC cell lines from C57BL/6 blastocysts (Gertsenstein et al., 2010
; Kiyonari et al., 2010
; Sato et al., 2009
). Previously, it had been very difficult to obtain germline-competent mESC lines from C57BL/6 mice via standard mESC culture conditions. De novo embryonic stem cell isolation from rats, which has been notoriously difficult, has also been facilitated with GSK-3 inhibitor supplementation (Buehr et al., 2008
; Li et al., 2009
). Taken together, and given the strong link between GSK-3 inhibition and β-catenin accumulation/activation, these studies strongly support a role for stabilized β-catenin in the positive regulation of pluripotency in mESCs. It is also important to note that the “ground state” of mESC pluripotency requires defined medium, which must be supplemented with an inhibitor of GSK-3 (Ying et al., 2008
Whereas it is widely assumed that most of the signaling functions of β-catenin rely on its interaction with TCFs, β-catenin can bind several other nuclear proteins to elicit biological effects (Le et al., 2008
). These alternative β-catenin signaling modes could explain some of our observations. We provide evidence suggesting that β-catenin’s interaction with the pluripotency regulator Oct-4 at least partially underlies its effects on sustaining pluripotency. Our qRT-PCR data reveal that not all Oct-4 target genes are upregulated under conditions that elevate active β-catenin levels. Thus, only a subset of Oct-4 target genes, including the key pluripotency regulator Nanog, is probably coregulated by β-catenin. The precise mechanism through which β-catenin selectively activates specific Oct-4 targets remains to be identified.
Of note, two recent studies endeavoring to elucidate the Oct-4 protein interaction network in mESCs failed to isolate β-catenin as a high-confidence Oct-4 interactor (Pardo et al., 2010
; van den Berg et al., 2010
). This is probably because both studies were performed in the absence of Wnt pathway stimulation, conditions under which the majority of cellular β-catenin would be membrane associated and spatially isolated from nuclear Oct-4.
We cannot discount the possibility that β-catenin knockdown may have influenced E-cadherin-mediated cell adhesion in our system (Nelson, 2008
). Reduced E-cadherin-mediated intercellular adhesion has recently been reported to reduce Nanog levels and to promote mESC differentiation (Chou et al., 2008
). Consistent with this, E-cadherin cell-cell contacts are required for the induction of pluripotency in iPSCs (Chen et al., 2010
). Thus, β-catenin’s junctional role in DKO mESCs could partly regulate the ability of the cells to exit the pluripotent state, independent of its nuclear functions.
Through western blot analyses, we were able to detect all four members of the TCF/LEF family in mESCs expressing GSK-3 or lacking it entirely. Based on our TCF reporter assays and TCF target gene analyses, our dominant-negative strategies with TCF1DN and TCF4DN appear to have efficiently blocked β-catenin/TCF/LEF activity contributed by all family members, which is not unexpected given that the DNA binding domains of the TCF family members are nearly identical (Arce et al., 2006
). The best-studied TCF/LEF family member in mESCs is TCF3, which acts as a transcriptional repressor of Nanog and possibly other genes involved in mESC self-renewal (Pereira et al., 2006
; Tam et al., 2008
; Yi et al., 2008
). TCF3 is atypical in that its repressive function appears to be retained in the presence of stabilized β-catenin (Pereira et al., 2006
). Thus, the highly elevated TCF activity detected in DKO cells is probably mediated by other TCF/LEF family members, suggesting that they are the primary effectors of Wnt/β-catenin signaling in mESCs. Stable expression of dominant-negative LEF1 (analogous to TCF1DN and TCF4DN used in our study) in wild-type mESCs does not appear to have a profound effect on the ability of the mESCs to self-renew in standard serum-containing or fully defined serum-free medium (Ying et al., 2008
). Thus, although mESCs harbor all TCF family members, they probably do not play an active role in sustaining pluripotency but may be poised to relay Wnt signals promoting differentiation.
Our experiments revealed that DKO cells are capable of proliferating indefinitely in serum-free medium, although they lose protein expression of Nanog and Sox2, suggesting that they are no longer pluripotent. Intriguingly, the DKO cells can be returned to a pluripotent state by switching back to medium with serum and LIF, suggesting that extrinsic factors are sufficient to “reprogram” them. The loss of Nanog and Sox2 proteins from DKO cells maintained in N2B27 medium was prevented, at least in a subset of cells, by blocking TCF activity through expression of TCF4DN. This suggests that TCF-mediated gene activation plays a prodifferentiation role. Indeed, sustained Wnt-β-catenin signaling has been shown to direct mESCs into mesendodermal progenitors, in part through TCF-mediated activation of brachyury
transcription (Bakre et al., 2007
Based on our collective data, we propose a model whereby β-catenin signaling plays a supportive role, in part through its interaction with Oct-4, in the maintenance of ground state pluripotency (). This role of β-catenin does not involve its transactivation of TCF/LEF-mediated transcription, which serves to promote mesendodermal differentiation. Given that β-catenin has been linked to the self-renewal of somatic stem cells and cancer-initiating cells (reviewed in Fodde and Brabletz, 2007
), our findings have important implications not only for embryonic stem cell biology but also for cancers arising from dysregulation of Wnt/β-catenin signaling.