Ectopic γ-catenin expression partially mimics the effects of stabilized β-catenin on embryonic stem cell differentiation.

β-catenin, an adherens junction component and key Wnt pathway effector, regulates numerous developmental processes and supports embryonic stem cell (ESC) pluripotency in specific contexts. The β-catenin homologue γ-catenin (also known as Plakoglobin) is a constituent of desmosomes and adherens junctions and may participate in Wnt signaling in certain situations. Here, we use β-catenin((+/+)) and β-catenin((-/-)) mouse embryonic stem cells (mESCs) to investigate the role of γ-catenin in Wnt signaling and mESC differentiation. Although γ-catenin protein is markedly stabilized upon inhibition or ablation of GSK-3 in wild-type (WT) mESCs, efficient silencing of its expression in these cells does not affect β-catenin/TCF target gene activation after Wnt pathway stimulation. Nonetheless, knocking down γ-catenin expression in WT mESCs appears to promote their exit from pluripotency in short-term differentiation assays. In β-catenin((-/-)) mESCs, GSK-3 inhibition does not detectably alter cytosolic γ-catenin levels and does not activate TCF target genes. Intriguingly, β-catenin/TCF target genes are induced in β-catenin((-/-)) mESCs overexpressing stabilized γ-catenin and the ability of these genes to be activated upon GSK-3 inhibition is partially restored when wild-type γ-catenin is overexpressed in these cells. This suggests that a critical threshold level of total catenin expression must be attained before there is sufficient signaling-competent γ-catenin available to respond to GSK-3 inhibition and to regulate target genes as a consequence. WT mESCs stably overexpressing γ-catenin exhibit robust Wnt pathway activation and display a block in tri-lineage differentiation that largely mimics that observed upon overexpression of β-catenin. However, β-catenin overexpression appears to be more effective than γ-catenin overexpression in sustaining the retention of markers of naïve pluripotency in cells that have been subjected to differentiation-inducing conditions. Collectively, our study reveals a function for γ-catenin in the regulation of mESC differentiation and has implications for human cancers in which γ-catenin is mutated and/or aberrantly expressed

A Single TCF Transcription Factor, Regardless of Its Activation Capacity, Is Sufficient for Effective Trilineage Differentiation of ESCs

Co-expression and cross-regulation of the four TCF/LEFs render their redundant and unique functions ambiguous. Here, we describe quadruple-knockout (QKO) mouse ESCs lacking all full-length TCF/LEFs and cell lines rescued with TCF7 or TCF7L1. QKO cells self-renew, despite gene expression patterns that differ significantly from WT, and display delayed, neurectoderm-biased, embryoid body (EB) differentiation. QKO EBs have no contracting cardiomyocytes and differentiate poorly into mesendoderm but readily generate neuronal cells. QKO cells and TCF7L1-rescued cells cannot efficiently activate TCF reporters, whereas TCF7-rescued cells exhibit significant reporter responsiveness. Surprisingly, despite dramatically different transactivation capacities, re-expression of TCF7L1 or TCF7 in QKO cells restores their tri-lineage differentiation ability, with similar lineage marker expression patterns and beating cardiomyocyte frequencies observed in EBs. Both factors also similarly affect the transcriptome of QKO cells. Our data reveal that a single TCF, regardless of its activation capacity, is sufficient for effective trilineage differentiation of ESCs

Stabilized γ-catenin suppresses neuronal differentiation in embryoid body assays and reduces overall tri-lineage differentiation efficiency.

<p>(A and B) Embryoid bodies were generated using control, β-catS33A, and γ-catS28A mESCs, and assayed for neuronal differentiation after 10 days. (A) Only WT EBs displayed immunofluorescent staining for the neuronal marker, β-III-tubulin. (B) The transcript levels of the neuronal markers <i>β-III-tubulin</i>, <i>Nestin</i>, <i>Map2</i>, <i>Tyrosine hydroxylase</i>, the endoderm marker <i>α-Fetoprotein</i>, and the mesoderm marker <i>Cardiac Troponin</i>, were reduced in EBs derived from mESCs overexpressing β-catS33A or γ-catS28A, as assessed by qRT-PCR analyses. Bars represent means and error bars indicate s.e.m. (n = 2). Size bar  = 200 µm.</p

Quantitative RT-PCR Primers.

<p>Quantitative RT-PCR Primers.</p

Overexpressed stabilized γ-catenin is less efficient than overexpressed stabilized β-catenin in sustaining retention of markers of ground state pluripotency in WT mESCs induced to differentiate.

<p>The indicated mESC cell lines were maintained for 72 hours using conditions identical to those described in Fig. 5A, namely, incubation in ES, EB or N2B27 media. Six markers of embryonic stem cells: <i>Esrrb</i>, <i>Klf4</i>, <i>Nanog</i>, <i>Pecam-1</i>, <i>Rex1</i> and <i>Stella</i> and two markers of EpiSCs: <i>FGF5</i> and <i>Otx2</i>, were assessed by qRT-PCR analyses. Data are presented in 8 individual graphs, where the transcript level for each gene is shown relative to that detected in wild-type cells maintained for 72 hours in standard ES medium (mean value arbitrarily set at 1). Bars represent the mean values of three experimental replicates and error bars indicate s.e.m. Asterisks indicate statistically significant differences (p<0.05) between the indicated means as determined by ANOVA analyses with Tukey's multiple comparison post-tests, which were applied to the gene expression datasets from the EB and N2B27 experiments.</p

Ectopic expression of γ-catenin in β-cat^(−/−) cells rescues TCF target gene activation upon GSK-3 inhibition.

<p>(A) Cytosolic γ-catenin is not stabilized after GSK-3 inhibition in β-cat^(−/−) mESCs. (B) Established β-catenin/TCF target genes (<i>Axin2, Brachyury and Cdx1</i>) are not induced by GSK-3 inhibition in β-cat^(−/−) mESCs, indicating that γ-catenin does not substitute for β-catenin in this context. Bars represent means and error bars indicate s.e.m. (n = 3) (C) Myc-tagged β-catenin, and variants of γ-catenin, were stably expressed under the control of the CAG promoter in β-cat^(−/−) mESCs, and clones were isolated, expanded and screened for transgene expression by western blotting. (D) Ectopic, stable expression of γ-catenin partially rescues the transactivation of β-catenin/TCF target genes (<i>Axin2</i>, <i>Brachyury</i> and <i>Cdx1</i>) in β-cat^(−/−) mESCs after GSK-3 inhibition. Bars represent means and error bars indicate s.e.m. (n = 3). Asterisks indicate p<0.05 in unpaired t-test analyses between indicated groups.</p

Cytosolic γ-catenin is stabilized in response to GSK-3 inhibition/ablation in wild-type mESCs.

<p>(A) Robust cytosolic stabilization of β-catenin and γ-catenin in mESCs after treatment with CHIR99021 (15 µM for ∼24 hours), and in mESCs lacking both isoforms (α and β) of GSK-3 (DKO), as assessed by western blotting of hypotonic lysates. Increases in β-catenin and γ-catenin levels were not clearly observed using whole cell lysates for western blotting purposes after the same treatment. (B) β-catenin and γ-catenin stabilization in mESCs after Wnt pathway activation using Wnt3a-conditioned medium (∼24 hours). (C) β-catenin and γ-catenin stabilization in DKO mESCs is reversed by re-expression of either wild-type GSK-3α or GSK-3β, but not their kinase-dead forms (K148A and K85A, respectively).</p

Ectopic expression of γ-cateninS28A in mESCs activates the Wnt pathway without altering cytosolic β-catenin levels.

<p>(A) mESCs were stably transfected with a Myc-tagged S28A variant of γ-catenin. Clones were isolated, expanded and screened for γ-catS28A expression by western blotting of hypotonic lysates using a tag-specific antibody. (B) Alkaline phosphatase staining of WT, γ-catS28A-expressing and β-catS33A-expressing mESC colonies, visualized using bright-field microscopy. Bar  = 400 µm. (C) Western blot analyses of nuclear and cytosolic extracts prepared from WT and γ-catS28A cell lines, as indicated. Elevated levels of γ-cat protein were detected in both cytoplasmic and nuclear lysates of γ-catS28A-overexpressing cell lines. (D) TCF reporter activity of mESCs stably expressing γ-catS28A. (E) Induction of the β-catenin/TCF target genes, <i>Axin2, Brachyury</i> and <i>Cdx1</i>, but not <i>c-Myc</i>, in mESCs stably expressing γ-catS28A or β-catS33A. (F) Expression of stabilized γ-catenin in WT mESCs does not result in detectable levels of cytosolic β-catenin as determined by western blot analysis of hypotonic lysates. Cytosolic levels of β-catenin in GSK-3 DKO mESCs or WT mESCs treated with CHIR99021 (24 h, 15 µM) are provided as positive controls. For (D) and (E), Bars represent means and error bars indicate s.e.m. (n = 3).</p

Ectopic γ-catenin expression delays, whereas endogenous γ-catenin knockdown accelerates, loss of pluripotency in differentiating mESCs.

<p>(A and B) The indicated mESC lines were seeded into standard ES medium or media conducive to differentiation, either N2B27 (lacking serum and LIF), or DMEM with 5% FBS and lacking LIF (EB medium), and assayed after 72 hours for β-galactosidase activity and the expression of pluripotency markers (Oct-4, Nanog and Sox2) by western blot analysis. (A) mESC lines expressing either β-catS33A or γ-catS28A exhibited a highly compact colony morphology and retained high levels of alkaline phosphatase staining after 72 hours of differentiation. Bar  = 400 µm. (B) These lines also retained the expression of Nanog and Sox2, whereas the control line (WT-Control) lost expression of these markers as assessed by western blotting. (C) Bulk EBs were generated using γ-catenin wild-type and knockdown lines by culturing 2×10⁶ cells on low attachment dishes with 5% FBS and lacking LIF. Western blot analysis using whole cell extracts taken on days 0, 3 and 7 revealed that wild-type and control (NegSH) lines retained the expression of Oct-4 and Nanog more effectively than γ-catenin knockdown lines. (D) mESCs stably expressing β-catS33A or γ-catS28A were seeded into N2B27 medium with LIF, and cultured as loosely adherent spheres, with passaging every 3–4 days over prolonged culture. The expression of Oct-4 and Nanog protein was readily detected in western blots of whole cell lysates from these stable cell lines.</p