Canonical Wnt pathway controls mESC self-renewal through inhibition of spontaneous differentiation via β-catenin/TCF/LEF functions

The Wnt/β-catenin signaling pathway is a key regulator of embryonic stem cell (ESC) self-renewal and differentiation. Constitutive activation of this pathway has been shown to increase mouse ESC (mESC) self-renewal and pluripotency gene expression. In this study, we generated a novel β-catenin knockout model in mESCs to delete putatively functional N-terminally truncated isoforms observed in previous knockout models. We showed that aberrant N-terminally truncated isoforms are not functional in mESCs. In the generated knockout line, we observed that canonical Wnt signaling is not active, as β-catenin ablation does not alter mESC transcriptional profile in serum/LIF culture conditions. In addition, we observed that Wnt signaling activation represses mESC spontaneous differentiation in a β-catenin-dependent manner. Finally, β-catenin (ΔC) isoforms can rescue β-catenin knockout self-renewal defects in mESCs cultured in serum-free medium and, albeit transcriptionally silent, cooperate with TCF1 and LEF1 to inhibit mESC spontaneous differentiation in a GSK3-dependent manner.The authors would like to thank the Genomics Unit at the CRG for assistance with the mRNA-seq (library prep and sequencing), and CRG and the Bristol University flow-cytometry facilities. This work was supported by the European Union's Horizon2020 Research and Innovation Programme (CellViewer No 686637 to M.P.C.), Ministerio de Ciencia e Innovación, grant BFU2017-86760-P (AEI/FEDER, UE), and an AGAUR grant from Secretaria d’Universitats i Recerca del Departament d’Empresa I Coneixement de la Generalitat de Catalunya (2017 SGR 689 to M.P.C.)