Abstract
Canonical Wnt signaling plays a rate-limiting role in regulating self-renewal and differentiation in mouse embryonic stem cells (ESCs). We have previously shown that mutation in the Apc (adenomatous polyposis coli) tumor suppressor gene constitutively activates Wnt signaling in ESCs and inhibits their capacity to differentiate towards ecto-, meso-, and endodermal lineages. However, the underlying molecular and cellular mechanisms through which Wnt regulates lineage differentiation in mouse ESCs remain to date largely unknown. To this aim, we have derived and studied the gene expression profiles of several Apc-mutant ESC lines encoding for different levels of Wnt signaling activation. We found that down-regulation of Tcf3, a member of the Tcf/Lef family and a key player in the control of self-renewal and pluripotency, represents a specific and primary response to Wnt activation in ESCs. Accordingly, rescuing Tcf3 expression partially restored the neural defects observed in Apc-mutant ESCs, suggesting that Tcf3 down-regulation is a necessary step towards Wnt-mediated suppression of neural differentiation. We found that Tcf3 down-regulation in the context of constitutively active Wnt signaling does not result from promoter DNA methylation but is likely to be caused by a plethora of mechanisms at both the RNA and protein level as shown by the observed decrease in activating histone marks (H3K4me3 and H3-acetylation) and the upregulation of miR-211, a novel Wnt-regulated microRNA that targets Tcf3 and attenuates early neural differentiation in mouse ESCs. Our data show for the first time that Wnt signaling down-regulates Tcf3 expression, possibly at both the transcriptional and post-transcriptional levels, and thus highlight a novel mechanism through which Wnt signaling inhibits neuro-ectodermal lineage differentiation in mouse embryonic stem cells.
Highlights
Embryonic stem cells (ESCs) are in vitro cultured cells derived from the preimplantation-stage embryo, which possess unconfined capacity for self-renewal and multi-lineage differentiation towards different embryonic germ layers
The future successes of regenerative medicine largely rely on our knowledge of, and our capacity to manipulate, the cellular and molecular mechanisms governing stem cell differentiation
Using a set of Apc-mutant ESCs harbouring different levels of Wnt signaling, we found that, among others, down-regulation of Tcf3, a key member of the pluripotency circuit, as well as induction of a novel Wnt-regulated microRNA, miR-211, represent two important downstream effects through which Wnt signaling inhibits neural differentiation in mouse ESCs
Summary
Embryonic stem cells (ESCs) are in vitro cultured cells derived from the preimplantation-stage embryo, which possess unconfined capacity for self-renewal and multi-lineage differentiation towards different embryonic germ layers. Pluripotency and self-renewal are two essential features of ESCs, which make them a very robust and suitable model for stem cell research, and a promising source for regenerative medicine. Wnt/b-catenin signaling has been shown to play a major role in maintaining self-renewal as well as in regulating ESCs differentiation [1,2,3,4,5,6]. The canonical Wnt/b-catenin signaling pathway is controlled by post-translational modifications of b-catenin leading to its differential protein stability and sub-cellular localization. In the absence of active Wnt signaling, b-catenin is negatively regulated by the socalled ‘‘destruction complex’’, consisting of the Apc and Axin scaffolding proteins and the glycogen synthase and casein kinases (GSK and CK1), resulting in proteolytic degradation and low levels of cytoplasmic b-catenin. Ligand-mediated Wnt signaling activation leads to nuclear translocation of b-catenin where it binds to members of the Tcf/Lef family of transcriptional factors modulating the expression of a broad spectrum of downstream target genes [7,8,9]
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