a regulatory network involving β‐catenin, e‐cadherin, pi3k ... publications/2015huan… ·...

A Regulatory Network Involving b-Catenin,E-Cadherin, PI3K/Akt, and Slug BalancesSelf-Renewal and Differentiation of HumanPluripotent Stem Cells in Response to WntSignaling

TYNG-SHYAN HUANG,a LI LI,a LILIAN MOALIM-NOUR,a DEYONG JIA,a JIAN BAI,a,b ZEMIN YAO,a,c

STEFFANY A. L. BENNETT,a,c DANIEL FIGEYS,a,c LISHENG WANGa,c,d

Key Words. Wnt • b-Catenin • E-cadherin • Slug • Human embryonic stem cell • Self-renewal •

Differentiation

ABSTRACT

The mechanisms underlying disparate roles of the canonical Wnt signaling pathway in maintain-ing self-renewal or inducing differentiation and lineage specification in embryonic stem cells(ESCs) are not clear. In this study, we provide the first demonstration that self-renewal versusdifferentiation of human ESCs (hESCs) in response to Wnt signaling is predominantly determinedby a two-layer regulatory circuit involving b-catenin, E-cadherin, PI3K/Akt, and Slug in a time-dependent manner. Short-term upregulation of b-catenin does not lead to the activation of T-cell factor (TCF)-eGFP Wnt reporter in hESCs. Instead, it enhances E-cadherin expression on thecell membrane, thereby enhancing hESC self-renewal through E-cadherin-associated PI3K/Aktsignaling. Conversely, long-term Wnt activation or loss of E-cadherin intracellular b-catenin bind-ing domain induces TCF-eGFP activity and promotes hESC differentiation through b-catenin-induced upregulation of Slug. Enhanced expression of Slug leads to a further reduction of E-cadherin that serves as a b-catenin “sink” sequestering free cytoplasmic b-catenin. The forma-tion of such a framework reinforces hESCs to switch from a state of temporal self-renewal asso-ciated with short-term Wnt/b-catenin activation to definitive differentiation. STEM CELLS

2015;33:1419–1433

INTRODUCTION

Canonical Wnt/b-catenin signaling has beensuggested to facilitate the self-renewal ofmouse and human embryonic stem cells (ESCs)[1–8]. It has also been shown to enhancereprogramming of somatic cells to pluripotentstem cells [9, 10]. However, the specific role ofWnt/b-catenin in ESCs remains unclear. Anumber of studies have yielded opposingresults in that Wnt signaling stimulates ESCdifferentiation and lineage specification[11–16]. How does one reconcile these seem-ingly disparate outcomes of one signalingpathway in ESCs?

The canonical Wnt signaling pathway isactivated when a Wnt ligand protein binds toits receptors Frizzled and Lrp5/6, which leadsto the inhibition of the GSK3b (Ser-Thr kinaseglycogen synthase kinase 3b)-mediated degra-dation of b-catenin. This allows the cytoplas-mic b-catenin accumulate and translocate intothe nucleus where it interacts with the lymph-

oid enhancer factor and T-cell factor (LEF/TCF)transcriptional complexes [17–19]. Subse-quently, this interaction induces or suppressesthe expression of many specific downstreamtarget genes, such as pluripotency gene Sox2,Nanog, Oct3/4, mesoderm gene Brachyury, celladhesion molecule E-cadherin, and E-cadherinsuppressors Snail and Slug [1, 20, 21]. Thegene expression profiles and subsequent bio-logical functions, however, seem to be celltype- and/or signaling network dependent[19].

In addition to its pivotal role as a down-stream effector of canonical Wnt signaling, b-catenin is also an essential structure adaptor.It was first discovered as a component of amammalian cell adhesion complex linking tothe cytoplasmic tail of E-cadherin to mediatecell-cell adhesion [22]. E-cadherin is a trans-membrane glycoprotein that mediates calcium-dependent, homophilic cell-cell adhesion in allepithelial tissues including ESCs [18]. With theexception of being a supplementary marker for

aDepartment of Biochemistry,Microbiology andImmunology, Faculty ofMedicine; and cOttawaInstitute of Systems Biology,University of Ottawa, Ottawa,Ontario, Canada; bShenyangPharmaceutical University,Shenyang, Liaoning, People’sRepublic of China;dRegenerative MedicineProgram, Ottawa HospitalResearch Institute, Ottawa,Ontario, Canada

Correspondence: Lisheng Wang,Ph.D., M.D., Department ofBiochemistry, Microbiology andImmunology, Faculty ofMedicine, University of Ottawa,451 Smyth Road, Ottawa, ONK1H 8M5 Canada.Telephone: 613-562-5624;Fax: 613-562-5452; e-mail:[email protected]

Received April 9, 2014;accepted for publicationNovember 28, 2014; firstpublished online in STEM CELLS

EXPRESS December 23, 2014.

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http://dx.doi.org/10.1002/stem.1944

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EMBRYONIC STEM CELLS/INDUCED

PLURIPOTENT STEM CELLS

undifferentiated hESCs [23], the active role of E-cadherin inESC self-renewal was not recognized until recently. A plausibleexplanation may be from a clear demonstration in 1996 thatE-cadherin was dispensable for mouse ESC maintenance [24].However, a recent study has shown that the loss of E-cadherin results in rapid differentiation of mouse ESCs, sug-gesting a supporting role of E-cadherin in mouse ESC self-renewal [25]. In addition, several groups including ours havereported that E-cadherin contributes to hESC self-renewal[26–30] and reprogramming of adult cells into pluripotentstem cells [31–34]. Furthermore, a recent study showedprominent E-cadherin expression in na€ıve hESC colonies thatwas associated with enhanced single-cell survival [35]. None-theless, the function and mechanism underlying cell-cell adhe-sion and Wnt signaling pathway in ESCs are not yet clearlyunderstood [36].

We here demonstrate that crosstalk between b-catenin,E-cadherin intracellular b-catenin binding domain, PI3K/Akt, andE-cadherin suppressor Slug is key to Wnt/b-catenin signaling inhESCs. The apparently disparate effects of Wnt/b-catenin signalin self-renewal and differentiation of hESCs are time-dependentand regulated by a two-layer circuit. In the first layer, short-termWnt/b-catenin activation augments E-cadherin-mediatedcell-cell adhesion that associates with PI3K/Akt signaling andenhances hESC self-renewal. Conversely, the second layer ischaracterized by long-term Wnt/b-catenin activation that pro-motes hESC differentiation definitively due to the increased freecytoplasmic b-catenin exceeding the binding capacity ofE-cadherin and translocating to nuclei to functionally interactwith TCF. Upregulation of Slug but not Snail gene expressionafter prolonged Wnt/b-catenin activation serves as a turningpoint that switches hESCs from temporal self-renewal to defini-tive differentiation. A time lag between the upregulation ofE-cadherin and its repressor Slug transcripts during the switchfrom short- to long-term Wnt activation points to an intricatenetwork underlying the seemingly opposing functions of Wnt sig-naling in ESCs. Our results highlight a delicate time-dependentbalance within Wnt signaling and will lead to more effectivemanipulation of human pluripotent stem cells for potential appli-cations. Integration of the aforementioned regulatory circuitwith Wnt/b-catenin and other signaling pathways may also pro-vide broad implications for stem cell and cancer research.

MATERIALS AND METHODS

hESCs Culture, GSK3 Inhibitors, Wnt3a Treatments,and Clonogenic Assays

H1 and H9 hESC lines were obtained from Wicell and CA1 cellline was a gift from Dr. Nagy (Mount Sinai hospital, Toronto,Canada). All cell lines were approved for use by the localethics board and the Stem Cell Oversight Committee of theCanadian Institutes of Health Research. Undifferentiated hESCwere maintained under feeder-free conditions in mouseembryonic fibroblast-conditioned medium (MEFCM) supple-mented with 12 ng/ml human recombinant basic fibroblastgrowth factor (bFGF, BD) as described previously [27, 28].Chemical defined medium (CDM) supplemented with 1xB27,1xITS-G, 1xNEAA, and 40 ng/ml bFGF [37] was used in themajority of experiments. The dosages of GSK3 inhibitor 6-bromoindirubin-30-oxime BIO (Calbiochem, Darmstadt, Ger-

many, http://www.emdmillipore.com/CA/en) used, after serialdilution, in MEFCM and CDM were 2.5 and 0.83 lM, respec-tively; CHIR99021 (Tocris Bioscience, Bristol, UK, http://www.tocris.com), 3–6 lM; Wnt3a (R&D Systems, Minneapolis, MN,http://www.rndsystems.com), 100–200 ng/ml; PI3K inhibitorLY294002 (Cell signaling, Beverly, MA, http://www.cellsignal.com), 5 lM; Akt activator II SC79 (EMD Millipore, Etobicoke,Canada, http://www.emdmillipore.com/CA/en) [38], 4–6 lg/ml; Akt inhibitor VIII (Cayman Chemical, Ann Arbor, MI,https://www.caymanchem.com), 3–6 lM; IWP2 (CaymanChemical), 2 lM. Clonogenic and self-renewal assays wereperformed as described previously [27]. See Supporting Infor-mation for detailed procedures.

Plasmids Transfection and siRNA Knockdown

Plasmid hE-cadherin/Db-catenin-pcDNA3 (EcadDb), obtainedfrom Addgene (Cambridge, MA, https://www.addgene.org)(#45772), was a kind gift from Dr. Barry Gumbiner [39]. Thismutant does not contain a b-catenin binding region due todeletion of the last 35 amino acids of the E-cadherin cytoplas-mic domain, as defined by Stappert and Kemler [40]. PlasmidpcDNA3-bS33Y Beta-catenin (bS33Y, Addgene #19286) [41]contains a tyrosine to serine missense mutation at codon 33(S33Y, presumptive GSK3b phosphorylation site), leading tocellular accumulation and an ability to activate TCF transcrip-tion [41]. Other plasmids obtained from Addgene include hE-cadherin-pcDNA3 (#45769) [39], pLKO-siSlug3 (#10905) [42],and pLKO-scrambled (#1864) [43]. See Supporting Informationfor siRNA targeted Slug and E-cadherin knockdown.

Generation of Tet-On Stable hESC Cell Lines

Stable E-cadherin-Tet-On and vector-Tet-On hESCs were gener-ated from H9 and H1 hESCs as reported previously [28]. StableEcadDb-Tet-On and bS33Y-Tet-On hESCs were generated usingLenti-X Tet-On advanced inducible expression system (Clon-tech, Mountain View, CA, http://www.clontech.com) accordingto the manufacturer’s instructions [28]. EcadDb and bS33Ygenes were amplified by PCR from hEcadDb-pcDNA3 andpcDNA3-bS33Y Beta-catenin, respectively. The amplified frag-ments were cloned into pLVX-Tight-Puro vector and verified byDNA sequencing (see Supporting Information for details). Sta-ble transduced hESC colonies were selected and maintained aspreviously described [28]. To upregulate E-cadherin expression,the selected colonies were treated with 1 lg/ml of doxycy-cline. To express bS33Y and EcadDb, the hESCs were treatedwith 2 lg/ml of doxycycline unless otherwise specified.

Generation of Transgenic Wnt Reporter 7xTCF-eGFPhESCs

Four Wnt reporter hESC sublines (7xTCF-eGFP-EcadDb-Tet-On,7xTCF-eGFP-bS33Y-Tet-On, 7xTCF-eGFP-vector-Tet-On, and7xTCF-eGFP) were generated by transduction of the estab-lished EcadDb-, bS33Y-, and vector-Tet-On or wild-type hESCswith a 73TCF-eGFP//SV40-PuroR lentiviral vector containingseven Tcf/Lef-binding sites and a puromycin resistance gene(Addgene #24305, a kind gift from Dr. Roel Nusse [44]). Lenti-virus was produced by transient transfection of 293T cells asdescribed previously [45]. Transduced hESCs were selectedwith puromycin (2 lg/ml) for 14 days. Wnt reporter Tet-On-hESC lines were maintained on drug-resistant DR4 MEF feedercells in hESC medium supplemented with bFGF (4 ng/ml),

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puromycin (1 lg/ml), and G418 (100 lg/ml). All Wnt reporterlines were passaged on Matrigel in MEFCM supplementedwith bFGF (12 ng/ml) and puromycin (1 lg/ml) or G418 (100lg/ml for Tet-On lines) for two passages prior to the Wntactivation assay. The expression levels of TCF-eGFP weredetermined by flow cytometry.

Flow Cytometry

To preserve membrane E-cadherin expression, hESCs were dis-sociated with Cell Dissociation Buffer (Life Technologies, Wal-tham, MA, http://www.lifetechnologies.com) for 14 minutes,filtered through a 40-lm cell strainer (BD), and immunola-beled with either Alexa-Flour647 mouse anti-human E-cad-herin antibody or isotype control (BD Biosciences,Mississauga, Canada, http://www.bdbiosciences.com) for 120minutes on ice. Live cells identified by 7-AAD exclusion wereanalyzed for E-cadherin and TCF-eGFP expression on a CyAnADP 9 Analyzer (Beckman Coulter, Mississauga, Canada,https://www.beckmancoulter.com). Detailed procedures aredescribed in Supporting Information.

Real-Time Quantitative PCR and Western Blot

Total RNA extraction, quantitative PCR (Q-PCR), and Westernblot were performed as described previously [27]. Forwardand reverse primers used in Q-PCR, antibodies used in West-ern blotting, and additional procedures are described in Sup-porting Information.

Immunofluorescence Staining, FluorescenceMicroscopy, and Image Analysis

Experimental methods have been described previously [27,28] and in Supporting Information.

Other Experimental Procedures

See Supporting Information.

Statistical Analysis

Results are expressed as mean6 SD. Statistical significancewas determined using a Student’s t test, ANOVA, or chi-square wherever appropriate. Results were considered signifi-cant when p< .05 or <.01.

RESULTS

Short-Term GSK3 Inhibition Temporally PromotesE-Cadherin Expression and hESC Self-Renewal WhereasLong-Term Inhibition Generates Opposing Results

Inhibition of GSK3 has been reported to either promote self-renewal or induce differentiation of hESCs [2, 14, 46]. Wespeculated that the dual role of GSK3 inhibition might betime-dependent and controlled by undefined mechanisms.Consistent with our hypothesis, brief inhibition of GSK3 withBIO for 6 hours caused hESC colonies to change from thinand flat to thick and compact morphology with sharp bounda-ries and hard-to-distinguish individual cells (characteristics ofundifferentiated colonies) when observed 24 hours later afterthe removal of BIO (Fig. 1A). Notably, gene and proteinexpression levels of E-cadherin, Nanog, and Oct3/4 were ele-vated (Supporting Information Fig. S1A, S1B, and Fig. 1B–1E),and E-cadherin protein on the cell membrane was enriched

(Fig. 1B, 1C, immunocytochemistry; Fig. 1F, 1G, flow cytome-try) after short-term BIO treatment. Using clonogenic assays,a crucial method to assess ESC self-renewal by reseeding anequal number of single hESCs in each group, we observed anapproximately eightfold increase in total clonogenic capacityafter short-term BIO treatment (3-fold increase in clonogenicassays 3 2.6-fold increase in total cell number after removalof BIO and continual culture for 4 days, Fig. 1H, 1I). It can beconcluded that short-term BIO treatment enhances E-cadherinexpression on the cell membrane and promotes hESC self-renewal.

Next, to address whether prolonged inhibition of GSK3suppresses E-cadherin expression and promotes differentia-tion, we examined hESCs after treatment with BIO for 6 hours(short-term) and 4 days (long-term), respectively. In contrastto short-term BIO treatment, long-term BIO treatment mark-edly reduced Oct3/4 and E-cadherin protein levels (Fig. 1D–1G), and significantly downregulated pluripotency transcriptsNanog, Oct3/4, and Sox2 (Fig. 1J).

Consistent with the above observations, Mixl1 and Bra-

chyury transcripts (primitive streak/mesoderm markers) weresignificantly upregulated after 4 days BIO treatment whileremaining unchanged after 6 hours treatment (Fig. 1K). West-ern blot confirmed that long-term BIO treatment resulted indifferentiation of hESCs, as indicated by a significant downreg-ulation of Nanog and E-cadherin, and upregulation of Bra-chyury protein levels (Fig. 1L). Specifically, Wnt reporter TCF-eGFP was not expressed after treatment with BIO for 6 hoursbut significantly upregulated after 4 days (Fig. 1M–1O). Thus,the opposing results in response to short- and long-term BIOtreatment indicate a time-dependent manner of Wnt signalingpathway in the regulation of hESC self-renewal versus differ-entiation. Similar results were also obtained by activating Wntsignaling pathway using GSK3 inhibitor CHIR99021 (SupportingInformation Fig. S2). Collectively, these data illustrate thatshort-term GSK3 inhibition enhances E-cadherin expression onthe cell membrane and hESC self-renewal whereas long-terminhibition results in opposite outcomes.

Dual Function of b-Catenin in Canonical Wnt SignalingPathway Is Responsible for hESC Self-Renewal andDifferentiation

Effects of GSK3 inhibition could be complicated by the diversecellular activities of GSK3 (downstream of BIO andCHIR99021), which involves transcription, translation, cellcycle, metabolism, antiapoptosis, and signal transduction [47].To exclude possible nonspecific activity of GSK3, we usedrecombinant protein Wnt3a and obtained similar results (Sup-porting Information Figs. S3, S4). As b-catenin is well recog-nized as the pivotal downstream effector of the Wntsignaling, we decided to examine b-catenin to further studythe discrepancy of Wnt signaling pathway on hESC self-renewal and differentiation. To controllable over-expression ofb-catenin in hESCs, we generated a stable hESC line using atetracycline-inducible lentiviral expression vector thatexpresses a nondegradable b-catenin mutant bS33Y [41]. Aprevious study has demonstrated that this mutant is function-ally identical to b-catenin, but undegradable due to the sub-stitution of a specific residue that is normally phosphorylatedby GSK3b, and therefore is able to activate TCF transcription[41].

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Figure 1.



Notably, after short-term bS33Y expression, the increased lev-els of b-catenin and Nanog proteins (data not shown), and anenhanced self-renewal capacity (clonogenic assays), wereobserved (Fig. 2A). These results further confirm that b-cateninsignaling is involved in hESC self-renewal. Conversely, long-term(4–6 days) expression of bS33Y led to an increase in total cyto-plasmic b-catenin protein and a loss of Nanog expression in adoxycycline dosage-dependent manner (Fig. 2B, 2C). Significantly,after long-term expression of bS33Y with a high dosage of doxy-cycline, approximately 30% of colonies exhibited lower levels ofNanog and some cells completely lost Nanog expression togetherwith accumulated b-catenin in the nuclei (Fig. 2D, right panel),indicative of a differentiation property of hESCs due to the pro-longed Wnt/b-catenin activation. Consistently, after long-termexpression of bS33Y, clonogenic assays revealed a dose-dependent impairment in hESC self-renewal (Fig. 2E), and West-ern blotting showed diminished Nanog expression (Fig. 2F). Spe-cifically, following an increase in TCF-eGFP activity, E-cadherinexpression on the cell membrane was decreased in a doxycyclinedosage-dependent manner (Fig. 2G–2J). Together, these findingsdemonstrate that depending on the duration of Wnt activation,b-catenin has dual function in the control of hESC self-renewalversus differentiation through yet to be determined mechanisms.

Short-Term Upregulation of b-CateninEnhances hESC Self-Renewal ThroughE-Cadherin-Associated PI3K/Akt Activation

To investigate how b-catenin enhances hESC self-renewal, weexamined the relationship between b-catenin and the adhesionmolecule E-cadherin. Several groups, including ours, have recentlyshown that E-cadherin plays an essential role in the maintenanceof hESCs [26–30], and in the reprogramming of somatic cells[31–34]. Our previous study also suggested that inadequate for-mation of E-cadherin-mediated cell-cell contacts in hESCs ham-pered clonogenic capacity and self-renewal of hESCs [27, 28].Since Wnt signaling has been shown to drive an epithelial-to-mesenchymal transition [48], one may speculate that even tran-sient Wnt activation would repress E-cadherin in hESCs.

To address this issue, we simultaneously assessed Wntreporter TCF-eGFP activity and E-cadherin expression in hESCsusing flow cytometry. Significantly, while the levels of E-cadherin transcript and protein were increased, and E-cadherin on the cell membrane was enriched (both percent-age and median fluorescence intensity) after treatment withBIO, CHIR99021, or Wnt3a for 6 hours (Figs. 1, 4D-scrambled1 CHIR99021, Supporting Information Figs. S1–S4),the TCF-eGFP expression was not elevated. This suggests that

short-term Wnt activation in hESCs leads to limited functionalinteraction of b-catenin and TCF in the nucleus. In contrast,long-term Wnt activation resulted in a significant increase inTCF-eGFP reporter activity, which was inversely correlatedwith the E-cadherin downregulation (Fig. 1, Supporting Infor-mation Figs. S2, S4). It is apparent that short-term upregula-tion of b-catenin in hESCs mainly promotes E-cadherinexpression on the cell membrane (as an intracellular adaptorprotein for E-cadherin) but not Wnt signaling in the nucleus.

To further elaborate whether E-cadherin is involved inhESC self-renewal, we subsequently over-expressed E-cadherinin hESCs using a tetracycline-inducible lentiviral vector [28],and observed an increase in the expression of Nanog andOct3/4 transcripts, formation of thick compact colonies withmore than a fivefold increase in cell density, and enrichedjunctional E-cadherin expression at cell-cell contacts (Fig. 3A–3C). We then asked whether loss of E-cadherin results in adecline of hESC self-renewal. Indeed, siRNA targeted E-cadherin knockdown significantly inhibited Nanog and Oct3/4

expression and impaired clonogenic capacity (Fig. 3D, 3E).Finally, we demonstrated that E-cadherin knockdown alsoabrogated short-term GSK3 inhibition-induced upregulation ofNanog and Oct3/4 expression (Supporting Information Fig.S5). Collectively, the above results suggest that short-termWnt/b-catenin activation enhances hESC self-renewal throughthe augmentation of E-cadherin expression.

To examine the underlying mechanism between E-cadherin expression and hESC self-renewal, we tested theeffect of E-cadherin-targeted knockdown on PI3K/Akt activa-tion. It has been documented that PI3K/Akt signaling enhan-ces the expression of pluripotency genes and antagonizeshESC differentiation induced by over-activation of Erk andWnt signaling [14, 49]. Significantly, E-cadherin knockdown inhESCs resulted in a reduction of Akt activation via decreasedAkt phosphorylation on Ser473, indicative of reduced PI3K/Aktsignaling (Fig. 4A). Conversely, E-cadherin upregulation led toan increase in PI3K/Akt activation (Fig. 4B) and clonogeniccapacity in hESCs (Fig. 4C), while inhibition of PI3K/Akt abro-gated the enhanced clonogenic capacity (Fig. 4C) and elevatedNanog and Oct3/4 expression induced by E-cadherin upregula-tion (Supporting Information Fig. S5). Specifically, short-termGSK3 inhibition-induced upregulation of E-cadherin protein,Akt phosphorylation, and Nanog and Oct3/4 transcripts wasabolished by E-cadherin knockdown, but restored by a specificAkt activator SC79 (Fig. 4D, Supporting Information Fig. S5).Furthermore, similar to E-cadherin knockdown, Akt suppres-sion blocked short-term GSK3 inhibition-induced upregulationof Nanog and Oct3/4 transcripts (Supporting Information Fig.

Figure 1. Short-term GSK3 inhibition temporally promotes E-cadherin expression and human embryonic stem cell (hESC) self-renewalwhereas long-term inhibition generates opposite results. (A–L): hESCs were treated with either vehicle (Veh) or BIO for 6 hours. Twenty-four hours after removal of BIO, hESCs exhibit thick and compact colonies (A, bright-field images) that express higher levels of E-cadherin (E-cad), Nanog, and Oct3/4 (B and D, immunofluorescence staining; C and E, quantitative image analysis of fluorescence inten-sity, n> 100 from three independent experiments for each group; F and G, flow cytometry). They also show high cloning efficiency (H,clonogenic assays, n 5 3). After removal of BIO and continual culture for 4 days, BIO treatment leads to a high cell yield (I). In contrast,long-term BIO treatment leads to opposing results (D–G; J, Q-PCR; L, Western blot), significantly enhancing the expression of mesodermmarker Mixl1 and Brachyury (K, Q-PCR, HNF3b: endoderm, Pax6: ectoderm). Nuclei were counterstained with DAPI (D, blue). Scalebars5 100 mm (A), 10 mm (B), and 20 mm (D). (M–O): Representative flow cytometric histogram (M), percentage of live TCF-eGFP-positive cells (pregated on 7-AAD-negative live cells), and MFI after treatment with BIO for 6 hours or 4 days. All data are mean6 SD.**, p< .01 compared to vehicle control. Abbreviations: GFP, green fluorescent protein; MFI, median fluorescence intensity; TCF, T-cellfactor.



Figure 2. Dual function of b-catenin in canonical Wnt signaling pathway is responsible for hESC self-renewal and differentiation. (A):bS33Y-Tet-On hESCs were treated with doxycycline (DOX, 2 mg/ml) for 24 hours, followed by clonogenic assays (mean6 SD, n 5 3, **,p< .01). (B–D): bS33Y-Tet-On hESCs were treated with different dosages of DOX for 4–6 days, followed by immunofluorescence stainingof b-catenin and Nanog (B), and quantification of mean pixel intensity of Nanog (C, n >100 cells/group). The nuclear translocation of b-catenin was observed in 1,000 ng/ml of DOX group (D). Nuclei were counterstained with DAPI (blue). White arrows indicate cytoplasmicb-catenin. Scale bars5 10 mm (insets in D: 1 mm). (E, F): Clonogenic assays (E, mean6 SD, n 5 3, *, p< .05; **, p< .01) and Westernblot analysis (F) for the same cells as described in (B)–(D). (G): TCF-eGFP reporter activity remains invisible in the absence of DOX for4–6 days but visible in the nuclear region in a DOX dose-dependent manner, scale bars5 20 mm. (H, I): Flow cytometric analysis of7xTCF-eGFP hESCs as described in (G), pregated on single 7-AAD-negative live cells. The TCF-eGFP reporter activity was measured by thepercentage of GFP-positive cells after treatment with different concentrations of DOX for 4–6 days (H). The expression of GFP-positivecells is normalized to DOX-untreated group that was arbitrarily set at 1 (I). (J): Flow cytometric analysis of the cells in (H) and (I) for E-cadherin expression (E-cad1) on the cell membrane indicates an inverse association with TCF-eGFP activity as shown in (G)–(I), pregatedon single 7-AAD-negative live cells. Data in (I) and (J) are means6 SD, n 5 3, two independent experiments; **, p< .01 compared toDOX-untreated group. Abbreviations: GFP, green fluorescent protein; hESC, human embryonic stem cell; TCF, T-cell factor.

S5). Taken together, these data demonstrate that short-termWnt/b-catenin activation enhances hESCs self-renewal throughE-cadherin upregulation that is associated with PI3K/Aktactivation.

E-Cadherin Sequesters b-Catenin to SuppressWnt-Induced hESC Differentiation

While E-cadherin has recently been revealed as an importantcomponent in hESC self-renewal [26–30], its regulatory role inWnt/b-catenin signaling in ESCs remains undefined. It is knownthat cytoplasmic b-catenin can either be phosphorylated byGSK3b which led to its degradation, or bound by E-cadherinintracellular domain through direct protein-protein interactionsat the cell-cell junctions. In either fate, accumulation of freecytoplasmic b-catenin will be minimized, thus preventing trans-location of b-catenin into the nucleus to initiate expression oftargeted downstream genes through interaction with the LEF/TCF complex [18, 50]. The lack of nuclear shift of b-catenin andTCF-eGFP reporter activity in undifferentiated hESCs (Fig. 2B,2D, 2G, 2H, DOX-untreated group) prompted us to proposethat, in addition to association with PI3K/Akt activation to regu-late hESC self-renewal and differentiation, E-cadherin also influ-ences Wnt signaling by sequestering b-catenin.

To determine whether disruption of interaction betweenE-cadherin and b-catenin impairs hESC self-renewal, we trans-fected hESCs with a truncated E-cadherin that lacks the

b-catenin-binding domain (EcadDb [39]) in combination withother genes. Stappert and Kemler [40] have shown thatEcadDb mutant allows more cytoplasmic b-catenin to enterthe nucleus and interact with LEF/TCF complex. Particularly,ectopic coexpression of bS33Y and EcadDb, but not coexpres-sion of bS33Y and wild-type E-cadherin, led to a significantdownregulation of pluripotency genes Nanog, Oct3/4, andSox2 (Fig. 5A). However, this effect was rescued by coexpres-sion of bS33Y with wild-type E-cadherin instead of EcadDb(Fig. 5A), demonstrating that the repressed expression of plu-ripotency genes after Wnt activation relies on the b-catenin-binding ability of E-cadherin. Importantly, the Brachyury tran-script was also significantly upregulated in comparison to con-trols after short-term ectopic coexpression of bS33Y andEcadDb (Fig. 5B). These results suggest that mutating theb-catenin binding domain of E-cadherin abrogates self-renewalenhanced by short-term Wnt activation (as shown in Fig. 1)and sensitizes hESCs to Wnt-induced differentiation.

To further confirm the above observation, we generated astable hESC line using a tetracycline-inducible lentiviral expres-sion vector to control EcadDb expression using EcadDb plas-mid [39]. It is known that accumulation of b-catenin that isspecifically unphosphorylated at GSK3b sites is critical forb-catenin/TCF-mediated transcription [51]. We therefore useda monoclonal antibody to detect the transcriptionally activeform of b-catenin (active b-catenin), which recognizes the

Figure 3. b-Catenin enhances human embryonic stem cell (hESC) self-renewal through upregulation of E-cadherin. (A–C): Inducibleover-expression of E-cadherin (E-cad) in hESCs enhances E-cadherin expression in cell-cell junctions (A, immunofluorescence, left panel:enlarged images; DOX: 3 days), and increases the expression of E-cadherin, Oct3/4, and Nanog (B, Q-PCR, DOX: 24 hours) and cell den-sity (C, bright-field image and cell counting; low panel: enlarged images; DOX: 3 days). Scale bars5 10 mm (A), 20 mm (low panel of C),and 100 mm (upper panel of C); DOX, 2 mg/ml. (D, E): Reduced expression levels of E-cadherin, Oct3/4, and Nanog (D, Q-PCR) anddiminished clonogenic capacity (E, clonogenic assays, n 5 3, **, p< .01) in hESC 24 hours after E-cadherin siRNA knockdown. H1, H9, orCA1 hESC lines were used in this figure.



signaling form of b-catenin specifically unphosphorylated atS37 and T41 [51, 52]. In comparison to untreated control, weobserved an increase in nuclear-localized active b-cateninwhen EcadDb expression was induced by the addition of dox-ycycline alone for 4 days (Fig 5C–5E), indicative of a state ofWnt/b-catenin signaling activation. Importantly, DOX-inducedEcadDb over-expression plus Wnt3a stimulation led to a fur-ther increase in the nuclear accumulation of active b-cateninand high-level expressions of Brachyury and Mixl1 (Fig. 5C–5F). This suggests that hESCs expressing EcadDb without b-catenin-binding domain are more sensitive to Wnt/b-cateninactivation, resulting in early differentiation.

To quantify the extent by which hESC differentiation isdependent on functional interactions between E-cadherin andb-catenin, we cultured EcadDb-Tet-On hESCs in the presenceor absence of Wnt3a for 4 days, then performed clonogenicassays and measured the numbers and intensity of alkalinephosphatase-positive (AP1) colonies. Again, after addition ofboth Wnt3a (activation of Wnt signaling) and DOX (inductionof EcadDb expression), differentiation was significantlyenhanced based on the loss of AP staining and gain of Bra-

chyury and Mixl1 expression (Fig. 5F–5H, AP1 colonies:Wnt3a alone vs. DOX1Wnt3a, 43%6 5% vs. 11%6 6%). Assuch, in addition to its association with PI3K/Akt activation, E-cadherin serves as b-catenin sink to delay hESC differentiationin response to prolonged Wnt activation.

We finally asked whether introducing EcadDb into hESCsenhances functional interaction of b-catenin and TCF if E-cadherin truly acts to sequester b-catenin away from cyto-plasm and TCF. Indeed, after being treated with DOX alonefor 4 days, both percentage of TCF-eGFP-positive cells andmedian fluorescence intensity were increased up to five- tosevenfold in comparison to DOX-untreated cells (Fig. 6A [fluo-rescence microscopy], 6B–6D [flow cytometry]), demonstrat-ing that E-cadherin is capable of minimizing nuclear b-catenin-TCF signaling. Consistently, EcadDb hESCs were also more sen-sitive to Wnt3a treatment, exhibiting an early TCF activationand greater TCF-eGFP reporter activity than control groups(Fig. 6A–6D). Collectively, it can be concluded that E-cadherinsequesters b-catenin to suppress its nuclear localization andfunctional interaction with TCF.

Interestingly, hESC differentiation progressed even afterremoving BIO at day 4 and cultivating in conventional condi-tions supporting self-renewal, indicating that hESC differentia-tion induced by prolonged Wnt/b-catenin activation isirreversible. This prompted us to examine the regulatory cir-cuits associated with Wnt and E-cadherin that switch hESCsfrom self-renewal to definite differentiation.

b-Catenin-Induced Slug Upregulation Reinforces hESCDifferentiation by Downregulating E-Cadherin andUpregulating Brachyury

Nuclear transactivation of b-catenin has been shown to upregu-late expression of E-cadherin suppressor genes Snail and Slug inother cell types to initiate epithelial-mesenchymal transition [1,21, 53]. While b-catenin/TCF transcription-dependent and -independent in the control of ESC self-renewal have been wellstudied, little is known about the crosstalk between Wnt/b-cat-enin, Snail/Slug, and E-cadherin in ESC fate decision. Becauseinteraction between E-cadherin and b-catenin at cell-cell junc-tions favors hESC self-renewal (Figs. 3–6), we propose the

Figure 4. E-cadherin associates with PI3K/Akt activation inresponse to short-term Wnt/b-catenin signaling-enhanced humanembryonic stem cell (hESC) self-renewal. (A): Decreased phospho-rylated AKT (pAKTS473) in hESCs after E-cadherin knockdown for48 hours (Western blot). (B): Increased phosphorylated AKT(pAKTS473) after E-cadherin upregulation. E-cadherin-Tet-On hESCswere treated with DOX (2 mg/ml) for 48 hours, followed by West-ern blot analysis. (C): Inhibition of PI3/Akt with LY294002 abro-gates the enhanced clonogenic capacity induced by E-cadherinupregulation. E-cadherin-Tet-On hESCs were treated with or with-out DOX (2 mg/ml) in the presence or absence of LY294002 (5mM) for 24 hours, followed by clonogenic assays (n 5 3). (D): E-cadherin knockdown abolishes short-term GSK3 inhibition-inducedupregulation of E-cadherin and phosphorylated Akt, while specificAkt activator SC79 counteracts the effect of E-cadherin knock-down. After siRNA knockdown for 44 hours, hESCs (H9 and H1lines) were treated with either CHIR99021 (6 mM) or vehicle(DMSO) for 6 hours, followed by Akt inhibitor VIII (6 mM), or Aktactivator II SC79 (6 mg/ml), or vehicle for an additional 30minutes. All data in this figure are mean6 SD and were gener-ated from H1 and H9 lines. **, p< .01.



Figure 5.



balance between self-renewal and differentiation in responseto Wnt/b-catenin signaling is coordinated by a two-layer circuit:(a) interaction between E-cadherin and b-catenin at cell mem-brane, and (b) crosstalk among E-cadherin, Snail/Slug, and Wnt/b-catenin pathway as determined below.

We first asked to what extent Snail or Slug was involved inWnt/b-catenin signaling in the control of hESC self-renewal anddifferentiation. We found that Slug but not Snail transcript wasincreased significantly in a timely manner after 1 day BIO treat-ment and further upregulated in both transcript and proteinlevels after 4 days BIO treatment (Fig. 7A, 7B). Despite the ini-tial increase of Snail protein in hESCs after short-term Wnt acti-vation, unexpectedly, neither Snail protein nor its transcript wassignificantly augmented in comparison to Slug after long-termWnt activation (Fig. 7A, 7B), suggesting that Slug is predomi-nantly involved in the Wnt/b-catenin pathway in hESCs.

We next examined how a crosstalk between E-cadherinand b-catenin influences Slug expression in hESCs. Ectopiccoexpression of bS33Y and EcadDb (which lacks the b-cateninbinding domain), but not coexpression of bS33Y and wild-typeE-cadherin, led to upregulation of Slug expression (Fig. 7C)and downregulation of pluripotency genes Nanog, Oct3/4,and Sox2 in hESCs (Fig. 5A). This suggests that loss of a b-catenin-binding domain in E-cadherin promotes Slug expres-sion and impairs hESC self-renewal. Consistently, ectopicexpression of EcadDb mutant in response to short-termWnt3a treatment further augmented Slug expression in hESCs(Fig. 7D). Comparable results were observed in a Tet-On sta-ble hESC cell line expressing EcadDb after short-term Wnt3atreatment as well (data not shown). These findings demon-strate that E-cadherin and b-catenin work together to regu-late Slug expression after Wnt activation in hESCs.

We then asked whether Slug plays a potent role in thecontrol of hESC self-renewal and differentiation in response toWnt signaling. To address this, we carried out transient knock-down of Slug using pLKO-siSlug (Fig. 7E) [42]. Suppression ofSlug followed by long-term BIO treatment for 4 days resultedin a significant decrease of differentiation marker Brachyury

(Fig. 7F, 7G), and an increase in clonogenic capacity in com-parison to scrambled control (Fig. 7H), indicating that Sluginhibition has an adverse effect on hESC differentiation afterupregulation of Wnt signaling. Taken together, the above dataprovide the first evidence that loss of E-cadherin b-catenin-binding domain or long-term Wnt/b-catenin activation upre-gulates Slug expression, which reinforces hESC differentiation.

DISCUSSION

The disparate effects of Wnt/b-catenin signaling pathway onESC self-renewal, differentiation, and lineage commitment arenot fully understood. In this study, we demonstrate that theseemingly controversial biological roles of Wnt pathway inhESCs are largely time-dependent and regulated by a two-layer circuit. Short-term activation of Wnt/b-catenin signalingtemporally enhances hESC self-renewal via upregulating E-cadherin expression at cell-cell junctions (the first layer), lead-ing to PI3K/Akt activation. Enhanced E-cadherin expressionalso serves as a “sink” to reduce the accumulation of freecytoplasmic b-catenin in favor of hESC self-renewal. Con-versely, long-term activation of Wnt/b-catenin signalingincreases free cytoplasmic b-catenin that exceeds the bindingcapacity of E-cadherin, leading to translocation of b-catenininto the nucleus and upregulation of E-cadherin suppressorSlug expression. The upregulated Slug, but not Snail, in turninhibits E-cadherin expression and reinforces the accumulationof b-catenin (the second layer), which leads to definitive dif-ferentiation of hESCs (working model, Fig. 7I).

The potential role of cell-cell adhesion molecules in theregulation of Wnt/b-catenin signaling pathway in ESCs hasbeen speculated but not well documented, mainly due to theearly demonstration of E-cadherin as an unnecessary compo-nent in mouse ESC self-renewal [24]. As a result, the majorityof studies regarding Wnt/b-catenin signaling in ESCs arefocused on its nuclear regulation via TCF transcriptional fac-tors [1, 36, 54] or TCF-independent functions through interac-tion with Oct3/4 [13, 55]. Current opposing observationsregarding endogenous Wnt signaling in hESC self-renewal ver-sus differentiation further complicate this debate [8, 56],although we did not observe significant changes after inhibi-tion of endogenous Wnt ligands’ secretion with IWP2 (Sup-porting Information Fig. S2). Recent findings on the importantrole of E-cadherin in ESCs [25–30, 35] prompted us to investi-gate whether and how E-cadherin and its associated mole-cules regulate Wnt/b-catenin signaling pathway in ESCs.

Human ESCs provide a unique system to address thisquestion because their self-renewal is highly dependent on E-cadherin expression. Interestingly, a recent report has sug-gested that retention of stabilized b-catenin in the cytoplasmmaintains self-renewal of pluripotent stem cells [57]. Here, wedemonstrate that temporal hESC self-renewal in response toshort-term b-catenin activation is dependent on E-cadherin

Figure 5. E-cadherin sequesters b-catenin to suppress Wnt-induced hESC differentiation. (A): Q-PCR analysis of hESCs cotransfectedwith bS33Y plasmids and either wild-type E-cadherin (WT-Ecad) or EcadDb for 24 hours. pcDNA: control vector. (B): In comparison tocontrol groups, co-overexpression of EcadDb and bS33Y for 24 hours significantly increases Brachyury expression (Q-PCR). (C): EcadDb-Tet-On hESCs were treated with or without doxycycline (DOX1, 2 mg/ml) or vehicle in the presence or absence of Wnt3a protein (100ng/ml) for 4–6 days and analyzed by double-immunostaining. Nuclei were counterstained with DAPI (blue). Scale bar5 20 mm. (D): Sub-cellular localization analysis of the expression of active b-catenin and Brachyury as shown in (C). Plots of the intensity profile of activeb-catenin (red), Brachyury (green), and DAPI (blue) over a linear section of a whole cell (white lines in C) are representative of >100cells/group analyzed. Data are expressed as AU versus length in microns. The arrowheads indicate the cell-cell contact areas; the yellowarea in the middle of each plot marks the nuclear region; mean6 SD in red on each plot represent the mean pixel intensity of thenuclear active b-catenin from >100 cells/group. (E): Quantification of the ratio of active b-catenin nuclear staining/DAPI intensity forthe indicated groups in (C). Data are mean6 SD, four replicates from H9 line, **, p< .01 compared to DOX2Wnt3a2 group. (F, G):Doxycycline-induced EcadDb over-expression promotes hESC differentiation in response to Wnt3a treatment as revealed by significantupregulation of Brachyury and Mixl1 (F, Q-PCR; G, Western blot). (H): Alkaline phosphatase (AP) staining and quantitative analyses. Atday 4 of treatments as indicated, cells were dissociated and reseeded. AP-positive colonies were counted when >50% of the cells withinthe colony were positive for AP staining. The percentages of AP1 colonies were quantitatively assessed and are shown at the bottom.The dashed line indicates the boundary of the colony. Scale bar5 100 mm. The results represent three replicates6 SD from H1 and H9lines. *, p<.05; **, p<.01. Abbreviations: AU, arbitrary unit; hESC, human embryonic stem cell.



upregulation, which associates with PI3K/Akt activation (Figs.3, 4). Considerable studies have shown that PI3K/Akt pathwayis specifically responsible for ESC self-renewal [14, 49, 58, 59].Our findings provide the first evidence that links E-cadherin-mediated cell-cell adhesion to PI3K/Akt activation in hESCself-renewal.

In addition, we also provide the first demonstration thatWnt signaling-induced hESC differentiation can be suppressedby cell-cell adhesion molecule E-cadherin. One underlyingmechanism is via E-cadherin intracellular domain that seques-ters free cytoplasmic b-catenin (Fig. 5). It is possible thatother mechanisms may also come into play given the wide-array of E-cadherin and Wnt functions and cell type-dependencies, which warrants further investigation in humanpluripotent stem cells. For instance, in mouse ESCs, there isnegligible transcriptional activation of b-catenin during self-renewal. b-Catenin forms a complex with Oct3/4 and E-

cadherin in the membrane and it is this complex that isinvolved in regulating mESC self-renewal and differentiation[60, 61]. In cancer cells, E-cadherin enhances the interactionbetween caveolin-1 and b-catenin near the cell membranethereby reducing b-catenin and TCF/LEF-dependent transcrip-tion [62]. E-Cadherin-mediated cell-cell adhesion may alsoenhance the turnover of cytoplasmic b-catenin by promotingthe activity of a b-catenin phospho-destruction complexlocated near adhesion junctions [63]. Conversely, unligated E-cadherin initiates Wnt signaling since stabilization of E-cadherin-mediated cell-cell junctions (i.e., formation of E-cadherin-p120-catenin complex) inhibits the assembly of theWnt signalosome [63].

One unexpected finding in our study is the involvement ofE-cadherin suppressor Slug but not Snail in reinforcing theswitch of hESCs from self-renewal to differentiation inresponse to Wnt/b-catenin signaling. Although Snail protein

Figure 6. E-cadherin sequesters b-catenin and suppresses the functional interaction of b-catenin and TCF in hESCs. Analysis of 7xTCF-eGFP-EcadDb-Tet-On hESCs after treatment with doxycycline (DOX1, 2 mg/ml) or vehicle in the presence or absence of Wnt3a protein(100 ng/ml) for the indicated time points. The cells were cultured on Matrigel-coated plates. Media, DOX, and Wnt3a were changeddaily. (A): TCF-eGFP (green) expression pattern of hESCs in the absence or presence of DOX and/or Wnt3a for 6 hours or 4 days. Scalebar5 50 mm. (B–D): Representative flow cytometric plots (B), fold changes of TCF-eGFP-positive cells over DOX2Wnt3a2 control (C) andMFI (D) in the absence or presence DOX and/or Wnt3a for 4 days, pregated on single 7-AAD-negative live cells. Mean6 SD from threeindependent experiments. *, p< .05; **, p< .01. Abbreviations: GFP, green fluorescent protein; hESC, human embryonic stem cell; MFI,median fluorescence intensity; TCF, T-cell factor.



Figure 7. b-Catenin-induced Slug upregulation reinforces human embryonic stem cell (hESC) differentiation by downregulating E-cadherin and upregulating Brachyury. (A, B): Long-term but not short-term BIO treatment enhances Slug expression in hESCs (A, Q-PCR;B, Western blot). (C): hESCs cotransfected with EcadDb and bS33Y, but not with vectors (pcDNA) or with wild-type E-cadherin (WT-Ecad)and bS33Y, enhances Slug expression (Q-PCR). (D): Over-expression of EcadDb together with short-term Wnt3a treatment (100 ng/ml)promotes Slug expression in hESCs (Q-PCR). (E): hESCs infected with pLKO-siSlug plasmid (siSlug) for 2 days reduce Slug expression(Western blot). Scrambled: pLKO-puro vector with a scrambled sequence that does not target any mRNA. (F–H): Slug knockdown (pre-siSlug) followed by BIO treatment for 4 days results in a decrease of differentiation marker Brachyury (F, Q-PCR; G, Western blot) andclonogenic capacity (H, clonogenic assays, n 5 3) in hESCs. All data are mean6 SD, *, p<.05; **, p< .01. (I): The proposed workingmodel (detailed in Supporting Information Fig. S7).

was temporarily increased after short-term Wnt/b-cateninactivation (Fig. 7B), hESCs retained self-renewal propertieswith increased clonogenicity (Fig. 1H). In contrast, Slug wasupregulated after 1 day Wnt/b-catenin activation and furtherincreased thereafter (Fig. 7A, 7B). A similar trend was alsoobserved when EcadDb mutant (lack of b-catenin bindingcapacity) was over-expressed after short-term activation ofWnt signaling pathway (Fig. 7C, 7D). Furthermore, transientknockdown of Slug was capable of abrogating long-term Wntactivation-induced high level Brachyury expression, low levelE-cadherin expression, and poor clonogenic capacity (Fig. 7F–7H). These data suggest that increasing Slug expression repre-sents a turning point in the fate decision of hESC self-renewalversus differentiation. TCF1, TCF3, and TCF4 binding sites inSlug promoter have recently been revealed by Chip-seq inseveral human cancer cell lines (ENCODE Consortium). There-fore, it is possible that E-cadherin indirectly suppresses Slugexpression by sequestering b-catenin and reducing its effecton TCFs/Slug.

Although the direct connection between Slug and Bra-chyury is unclear, Brachyury has been shown to bind to the E-cadherin promoter, and over-expression of Brachyury resultedin downregulation of E-cadherin and upregulation of Slug inepithelial tumor cells [64]. It is possible that high levels ofBrachyury and Slug expression after long-term Wnt activationfurther suppress E-cadherin expression, thereby skewing thebalance from self-renewal to differentiation.

Our proposed working model (Fig. 7I), which includesWnt/b-catenin, E-cadherin, PI3K/Akt, and Slug, provides amechanistic insight underlying the dynamic regulationbetween self-renewal and differentiation in hESCs. To date,several signaling pathways have been shown to significantlyinfluence ESC fate, including PI3K, Wnt, Activin A/Smad2,3,and Raf/Mek/Erk pathways [65]. Recently, Singh et al. pro-posed the existence of a signaling network crosstalk in hESCs,which involves PI3K/Akt suppressing Erk and Wnt signaling toallow Smad2/3 to activate a specific subset of target genesrequired for self-renewal [14]. Our results further expand thecurrent working model by revealing a new regulatory networkcomprising of b-catenin, E-cadherin, PI3K, and Slug in hESCs.Since b-catenin collaborates with Smad2/3 to induce mesen-doderm gene expression [14], it is possible that E-cadherincould indirectly affect Smad2/3 pathway through reducingcytoplasmic-free b-catenin. As Mek/Erk signaling has been

shown to be downregulated by E-cadherin-dependent PI3K/Akt pathway in differentiating intestinal epithelial cells [66], E-cadherin-associated PI3K activation may also contribute to theinhibition of Erk pathway to reduce hESC differentiation.

CONCLUSIONS

In summary, this study provides direct evidence, for the firsttime, that integrates b-catenin, the cell-cell adhesion moleculeE-cadherin, PI3K/Akt, and Slug into the current signaling net-work in the control of cell fate of pluripotent stem cells. Ourwork shows how E-cadherin and Slug actively tunes Wnt sig-naling pathway and also provides a unique tool to controllablypromote hESC self-renewal by short-term Wnt activation. Inaddition, these findings may shed light on the dual-role ofcanonical Wnt signaling in developmental and cancer biology.

ACKNOWLEDGMENTS

We apologize to all those colleagues whose important workcould not be cited owing to space limitations. We thankAndrew Sulaiman and Sarah Ooi for thorough reading of themanuscript. This work is supported by an operating grantfrom the Canadian Institutes of Health Research (CIHR) MOP-111224 and Heart and Stroke Foundation of Ontario NA7186to L.W.; a Canada Research Chair in Proteomics and SystemsBiology and a NSERC strategic grant to D.F. (STPGP/396508-2010); CIHR operating grant (MOP 62826) to S.A.L.B.

AUTHOR CONTRIBUTIONS

T.-S.H. and L.L.: conception and design, collection and assem-bly of data, data analysis and interpretation, and manuscriptwriting; L.M.-N.: collection and assembly of data and dataanalysis and interpretation; D.J. and J.B.: collection of data;Z.Y., S.A.L.B., and D.F.: provision of study material; L.W.: con-ception and design, data interpretation, manuscript writing,financial support, and final approval of manuscript. T.-S.H.,L.L., and L.M.-N. contributed equally to this study.

DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

The authors indicate no potential conflicts of interest.

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