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. •N-terminally truncated β-catenin isoforms are produced in mESCs upon inducible knockout•β-Catenin is fully deleted upon CRISPR/Cas9 whole-gene knockout•Wnt/β-catenin prevents differentiation without affecting pluripotency genes•β-Catenin/TCF/LEF functions are required to prevent spontaneous differentiation In this article, Cosma and colleagues show that previously published inducible β-catenin knockout models produce N-terminally truncated isoforms in mouse embryonic stem cells (mESCs). Complete β-catenin knockout was obtained by CRISPR/Cas9 whole-gene deletion. With the newly generate knockout ESC model it was observed that β-catenin/TCF/LEF functions are required to prevent spontaneous differentiation of mESCs.