2969DEVELOPMENT AND DISEASE RESEARCH ARTICLE

INTRODUCTIONDuring early embryogenesis, gastrulation results in the formation ofthree definitive germ layers and establishment of the embryonicbody plan (Tam and Loebel, 2007). At this stage, undifferentiatedepiblast cells undergo an epithelial to mesenchymal transition(EMT) and migrate through a structure called the primitive streak(PS) to generate the mesoderm and endoderm. Distinct regions ofthe PS induce different subpopulation of mesoderm and endodermprogenitors (Tam and Loebel, 2007). The anterior PS has thepotential to form the definitive endoderm and anterior mesoderm,including hepatic endoderm and cardiac mesoderm. The middleregion of PS forms the lateral plate mesoderm; and the mostposterior region of PS develops extra-embryonic mesodermprogenitors, which give rise to hematopoietic and vascular cells ofblood islands. Mesoderm and endoderm generation depends onsuccessful completion of the EMT and migration away from the PS.The EMT program is the process by which polarized epithelial cellsare converted into individually motile cells; it occurs not only duringearly embryogenesis, but also during tumor progression (Thiery,2002). The important markers of EMT lose epithelial polarities andadherence junctions following the downregulation of E-cadherin,which is an important cell-adhesion molecule of the apical-basalpolarity and intercellular adhesion. Although several signalingpathways involved in the EMT processes have been identified

(Thiery, 2002), elucidation of the molecular mechanisms that triggerand promote EMT is important for a better understanding ofembryogenesis and tumorigenesis.

The canonical Wnt signaling functions are well established infundamental biological processes (Moon et al., 2004; Tam andLoebel, 2007). In early embryogenesis, Wnt/β-catenin signalinghas pivotal roles in the formation of the PS, mesoderm andendoderm (Lickert et al., 2002; Tam and Loebel, 2007), and thestabilization of β-catenin leads to premature EMT in mouseembryo (Kemler et al., 2004). Wnt binds to its receptor Frizzledand co-receptor Lrp5/6, and increases the level of cytoplasmic andnuclear β-catenin, followed by inhibition of the GSK3-mediateddegradation pathway. Upon inhibition of GSK3 activity, stabilizedβ-catenin translocates into the nucleus, where it serves as a co-activator of the Lef/Tcf family of DNA-binding proteins to formactive transcriptional complexes for specific target genes (Moonet al., 2004). There is accumulating evidence that the Wnt/β-catenin signaling pathway also has an important role in stem cellmaintenance and regulation of the cell fate decision in severalstem cell systems, including hematopoietic, epidermal andintestinal stem cells (Moon et al., 2004).

Embryonic stem (ES) cells possess the remarkable propertyof indefinite self-renewal and pluripotency, the ability todifferentiate to all cell types of an organism; they also provide anexcellent model system for studying cell fate determination inearly mammalian development (Keller, 2005). Multiple signalingpathways, such as those involving growth factors, transcriptionalregulators and epigenetic modifiers, have crucial roles inregulating the balance between ES-cell self-renewal and lineagecommitment (Keller, 2005). It is very important to elucidatemolecular mechanisms of the self-renewal and lineagecommitment for efficient production of functional cells requiredfor use of hES cells in transplantation therapy and drug discovery.Like other stem cell systems, canonical Wnt/β-catenin signalingis implicated in mouse ES (mES) cell self-renewal anddifferentiation, depending on the context (Gadue et al., 2006; Hao

Defining early lineage specification of human embryonicstem cells by the orchestrated balance of canonical Wnt/β-catenin, Activin/Nodal and BMP signalingTomoyuki Sumi1, Norihiro Tsuneyoshi1,2, Norio Nakatsuji2,3 and Hirofumi Suemori1,*

The canonical Wnt/β-catenin signaling has remarkably diverse roles in embryonic development, stem cell self-renewal and cancerprogression. Here, we show that stabilized expression of β-catenin perturbed human embryonic stem (hES)-cell self-renewal, suchthat up to 80% of the hES cells developed into the primitive streak (PS)/mesoderm progenitors, reminiscent of early mammalianembryogenesis. The formation of the PS/mesoderm progenitors essentially depended on the cooperative action of β-catenintogether with Activin/Nodal and BMP signaling pathways. Intriguingly, blockade of BMP signaling completely abolished mesodermgeneration, and induced a cell fate change towards the anterior PS progenitors. The PI3-kinase/Akt, but not MAPK, signalingpathway had a crucial role in the anterior PS specification, at least in part, by enhancing β-catenin stability. In addition,Activin/Nodal and Wnt/β-catenin signaling synergistically induced the generation and specification of the anterior PS/endoderm.Taken together, our findings clearly demonstrate that the orchestrated balance of Activin/Nodal and BMP signaling defines the cellfate of the nascent PS induced by canonical Wnt/β-catenin signaling in hES cells.

KEY WORDS: Primitive streak, Mesoderm, Endoderm, Stem cells, β-Catenin, Wnt

Development 135, 2969-2979 (2008) doi:10.1242/dev.021121

1Laboratory of Embryonic Stem Cell Research, Stem Cell Research Center, Institutefor Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan. 2Department of Development and Differentiation,Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin,Sakyo-ku, Kyoto 606-8507, Japan. 3Institute for Integrated Cell-Material Sciences(iCeMS), Kyoto University, 69 Konoe-cho, Yoshida, Sakyo-ku, Kyoto 606-8507,Japan.

*Author for correspondence (e-mail: hsuemori@frontier.kyoto-u.ac.jp)

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et al., 2006; Lindsley et al., 2006), but precise roles of thissignaling in human ES (hES) cells remains controversial (Dravidet al., 2005; Sato et al., 2004).

We report here that the activation of canonical Wnt/β-cateninsignaling in hES cells by conditional activation of stabilized β-catenin disrupted hES-cell self-renewal. Rather, the canonicalWnt/β-catenin and BMP signaling pathway in hES cells hassignificant roles in establishing the posterior PS/mesodermprogenitors, whereas attenuation of BMP signaling changes the cellfate to the anterior PS/endoderm progenitors. In addition, Activinand Wnt/β-catenin signaling pathways synergistically function ininducing undifferentiated hES cells to differentiate into the anteriorPS/endoderm progenitors. This is the first in vitro model system thatconsistently recapitulates the human early embryogenesis and thatenables us to analyze molecular events during the process of earlyembryogenesis from the epiblast to the PS formation, followed bylineage specification into the mesoderm and endoderm. Moreimportantly, our findings will also be relevant to directeddifferentiation of specific tissue and cells from hES cells.

MATERIALS AND METHODSActivation of β-catenin signaling in hES cellsThe �Nβ-cateninER construct, in which the N-terminal 90 amino acids weredeleted, was generated by in-frame insertion into the expression vectorcontaining the hormone-binding domain of a mutant estrogen receptor(Littlewood et al., 1995; Sumi et al., 2007). Cell lines expressing ΔNβ-cateninER were obtained by transfection of hES cell lines KhES-1 andKhES-3 with ΔNβ-cateninER expression plasmids, followed by puromysinselection as described previously (Sumi et al., 2007). To activate ΔNβ-cateninER, hES cells stably expressing ΔNβ-cateninER were treated with4OHT (100 nM), as indicated.

Maintenance and differentiation of hES cellsThe hES cell line HES-3 was purchased from ES Cell International. The hEScell lines KhES-1, KhES-3 and HES-3 were maintained as describedpreviously (Suemori et al., 2006). For differentiation of hES cells, the cellswere dissociated into small clumps and cultured on plates coated withmatrigel (BD Biosciences, San Jose, CA) in DMEM/F12 with N2 and B27supplements (Invitrogen, Carlsbad, CA). The next day, the medium waschanged to N2B27 medium with or without 4OHT (100 nM, Sigma, StLouis, MO) in the presence or absence of 250 ng/ml Noggin-Fc chimera(R&D Systems Minneapolis, MN) except as otherwise indicated, andrenewed daily thereafter. For Activin-induced differentiation, hES cells werecultured in RPMI supplemented with 2% FBS in the presence or absence of100 ng/ml Activin A (R&D Systems), 100 ng/ml DKK1 (R&D Systems) or100 nM 4OHT for 3 days, and then analyzed as described below. SB431542(Sigma), U0126 and LY294002 (Promega, Madison, WI) were used at afinal concentration of 10 μM. GSK-3 inhibitor-IX and -X (BIO and BIO-Acetoxime, Calbiochem, La Jolla, CA) were used at a final concentration of2 to 10 μM. For endothelial cell differentiation, cells cultured with 4OHTfor 3 days were trypsinized and plated on collagen I-coated dishes, and thencultured in a StemPro34 serum free medium (Invitrogen) with 20 ng/mlVEGF165 (R&D Systems) for an additional 6 days. For endoderm andcardiac differentiation, β-catenin-activated cells cultured with or withoutNoggin (250 ng/ml) for 4 days were trypsinized and plated on collagen I-coated dishes, and then cultured in a N2B27 medium without 4OHT in thepresence of 10 ng/ml BMP4 (R&D Systems) and 5 ng/ml FGF2 for anadditional 4 days. All data shown are representative results obtained from atleast two independent clones of two different human ES cell lines.

Quantitative and semi-quantitative PCR analysisTotal RNA was isolated from cells using RNeasy Micro Kit (QIAGEN,Valencia, VA) and reverse-transcribed using Omniscript RT Kit (QIAGEN)according to the manufacturer’s protocol. For semi-quantitative PCR, PCRreactions were optimized to allow for semi-quantitative comparisons withinthe log phase of amplification. Real-time RT-PCR analysis was performed

on an ABI Prism 7500 Real-time PCR system using the PowerSYBR GreenPCR Master Mix (Applied Biosystems, Foster City, CA). The expressionvalue of each gene was normalized against the amount of GAPDH andcalculated by the ΔΔCt method. The expression level of each gene in thecontrol sample (vehicle or undifferentiated ES cells) was defined as 1.0. Thenormalized expression values for all control and treated samples wereaveraged, and average fold-change was determined. Details of the primersused for the semi-quantitative and quantitative PCR can be provided onrequest.

Western blot and immunofluorescence analysisCell lysates were prepared and subjected to sodium dodecyl sulfate-polyacrylamide gel elctrophoresis (SDS-PAGE), followed by westernblotting as described previously (Sumi et al., 2007). Forimmunofluorescence analysis, cells plated on the culture slides were fixedwith 3.7% formaldehyde and permeabilized with 0.25% Triton X-100. Afterblocking, the cells were incubated with primary antibodies, followed byincubation with secondary antibodies. Alexa Fluor 488- or 555-conjugatedsecondary antibodies were purchased from Invitrogen. Cells were mountedonto glass slides with Vectashield (Vector Laboratories, Burlingame, CA),and then analyzed using a BX61 fluorescence microscope (Olympus, CenterValley, PA). Antibodies against the following proteins were used: VEGFR2/KDR (clone#89115), SOX17 and CXCR4 (clone#44717) (R&DSystems); E-cadherin, Smad2/3, GSK3β and β-catenin (BD Biosciences);N-cadherin (GC-4, Sigma); ZO-1 (Invitrogen); Nanog (ReproCELL);HNF3β, Oct3/4, Brachyury and ERα (Santa Cruz Biotechnology, SantaCruz, CA); SSEA3 and SSEA4 (Developmental Hybridoma Bank); TRA-1-60 and TRA-1-81 (Chemicon); Phospho-Smad1 (Ser463/465), Smad1,phospho-Smad2 (Ser465/467), phospho-MAPK (Thr202/Tyr204), MAPK,phospho-Akt (Ser473), Akt and phospho-GSK3α/β (Ser21/9) (CellSignaling, Danvers, MA).

FACS analysisSingle cell suspensions were fixed with 1% formaldehyde for 30 minutes at4°C, and incubated first with primary antibody and then with Alexa Fluor488-, 555-conjugated secondary antibody (Invitrogen). FACS analysis wasperformed using a FACSCalibur Flow Cytometer (Becton Dickinson).

Karyotype analysis of hES cellsChromosome spreads were prepared as described elsewhere (Suemori et al.,2006). Briefly, hES cells were incubated in ES medium with KaryoMAXColcemid Solution (Invitrogen; 0.1 μg/ml of colcemid) for 2 hours,trypsinized, incubated in 0.075 M KCl for 10 minutes and fixed in Carnoy’sfixative.

RESULTSTemporal emergence of the primitive streak andmesoderm induced by stabilized β-cateninTo examine the role of canonical Wnt/β-catenin signaling in hEScell growth and differentiation, we generated stable hES cell linesconstitutively expressing a fusion protein of a stabilized β-catenin,a mutant ligand-binding domain of the estrogen receptor (ER)(Littlewood et al., 1995). The stabilized β-catenin was produced bydeletion of the N-terminal 90 amino acids, including GSK3phosphorylation sites (Barth et al., 1997). This fusion protein (ΔNβ-cateninER) can be conditionally activated in response to the ERagonist 4-hydroxy-tamoxifen (4OHT), thus enabling consistent andreversible activation of β-catenin. We obtained several independenthES cell clones with stable expression of the ΔNβ-cateninER fusionproteins, following the transfection of this expression plasmid andpuromycin selection. Results presented here were obtained usingone or two clones, but similar results were obtained with furtherindependent clones from two different hES cell lines: KhES-1 andKhES-3. These transgenic clones had normal karyotype andexpressed cell-surface markers for hES cells, including SSEA3,SSEA4, TRA-1-60, TRA-1-81 and pluripotent markers POU5F1,

RESEARCH ARTICLE Development 135 (17)

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SOX2 and NANOG comparable with the parental hES cells (see Fig.S1A-D in the supplementary material). No obvious effect of 4OHTon the undifferentiated state of parental hES cells was observed (seeFig. S1B,D in the supplementary material). When ΔNβ-cateninERcells were cultured in chemically defined medium (Yao et al., 2006)without 4OHT, they formed tightly contacted compact colonies withinvisible cell-cell boundaries (Fig. 1A). Thus, ΔNβ-cateninER cellsmaintained an undifferentiated state in culture without 4OHT. In thepresence of 4OHT, however, the morphology of the cells began todifferentiate with dissociation of the cell-cell junctions and inductionof cell scattering within 1 to 2 days, resembling the EMT (Thiery,2002). By day 3, β-catenin-activated cells exhibited a similarmorphology (Fig. 1A), and proliferated well during the entire

process, but some cells underwent apoptotic-like cell death betweendays 4 and 5, and detached from the substrata (data not shown). Theundifferentiated hES cells (vehicle) displayed epithelial-like apical-basal polarity with a component of tight junctions, ZO-1, and formedadherence junctions composed of E-cadherin (Fig. 1B,C). Bycontrast, mesenchymal marker N-cadherin was expressed only at theperiphery of ES cell colonies, suggesting spontaneous differentiationof a subpopulation of ES cells (Ullmann et al., 2007). After 3 daysof β-catenin activation, cells had disorganized ZO-1 localization andmarkedly decreased expression of E-cadherin, and displayed amesenchymal phenotype, including dissociated adherence junctionsand a gradual change from E-cadherin to N-cadherin expression(Fig. 1B,C). These results indicate that conditional β-catenin

2971RESEARCH ARTICLEWnt, Activin and BMP signaling specify human ES cell fate

Fig. 1. Temporal dynamics of the primitive streak and mesoderm progenitors in β-catenin-activated hES cells. (A) The ΔNβ-cateninER cellswere cultured in defined medium with or without 4OHT (days 0-3), and the representative morphology at the indicated time periods was shown.Upper or lower panels shows low or high magnification images, respectively. Scale bars: 100 μm. (B) Immunostaining analysis of the day-3 ΔNβ-cateninER-activated cells. Cells were cultured with or without 4OHT for 3 days, and subjected to immunostaining with indicated antibodies, andnuclei were counterstained with diaminopimelic acid (DAPI). Scale bar: 100 μm. (C) The ΔNβ-cateninER cells were cultured with (days 1-3) orwithout (day 0) 4OHT for the indicated time periods, and subjected to immunostaining with anti-E-cadherin and anti-N-cadherin antibodies. Nucleiwere counterstained with DAPI. Scale bar: 100 μm. (D) Quantitative real-time PCR (qPCR) analysis on RNA isolated from undifferentiated ΔNβ-cateninER cells (day 0) and β-catenin-activated cells harvested at daily intervals (days 1-5) was performed using specific primers for the genesindicated. (E) Immunoblot analysis of cell lysates from ΔNβ-cateninER cells. Cells were cultured for the indicated time periods with 4OHT, and celllysates were subjected to SDS-PAGE and probed with specific antibodies as indicated. D

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activation in hES cells does not support their self-renewal, butresults in enhanced differentiation via EMT induction within a fewdays.

To further characterize the properties of β-catenin-activated cells,we examined the expression profiles of genes related to pluripotencyand differentiation. Cells activated by β-catenin had a rapidlyreduced expression of pluripotent markers NANOG, SOX2 andPOU5F1 within 1 day of activation, which progressively decreasedto undetectable levels by day 3 (Fig. 1D). By contrast, the expressionof the PS/nascent mesoderm markers T (Brachyury), GSC andMIXL1 (Tam and Loebel, 2007) transiently peaked at day 1 ofactivation, and gradually decreased by day 5. Expression of an earlymarker of ventral mesoderm (KDR) and pre-cardiac mesoderm(NKX2-5), and of a mesoderm/endoderm marker (CXCR4)(D’Amour et al., 2005; Yasunaga et al., 2005) increased in an orderly

manner following the induction of T, MIXL1 and GSC. The BMP,Nodal and FGF signaling pathways are important for the emergenceof the PS, mesoderm and endoderm in mouse (Tam and Loebel,2007). Gene expression analysis showed the progressive inductionof BMP4 and FGF8, and a temporal induction of NODAL by β-catenin activation, suggesting the involvement of these factors in thedevelopment of the PS/mesoderm in β-catenin-induced hES celldifferentiation (Fig. 1D). By contrast, the trophectoderm markerCGA was not expressed (Fig. 3A). The ectoderm marker PAX6 wasdominated by β-catenin activation (Fig. 3A), as the canonical Wntsignaling suppresses neural differentiation (Aubert et al., 2002).Immunoblot and immunofluorescence analysis confirmeddownregulation of Oct4 and Nanog, and induction of Brachyury andKDR protein (Fig. 1B,E). Thus, these temporal gene expressionpatterns in differentiating hES cells recapitulate the emergence ofthe PS, and mark the period of the transition towards mesoderm inearly mammalian development.

To evaluate the relative proportion of mesoderm progenitorsinduced by β-catenin activation, we analyzed the expression ofCXCR4 and KDR as mesoderm markers by fluorescence-activatedcell sorting (FACS) analysis. Weak levels of KDR expression weredetected in ~50% of undifferentiated ES cells, while CXCR4 wasnot expressed (Fig. 2A). A representative 5-day kinetic experimentindicated that the KDR+/CXCR4+ cells were immediately detectedafter 2 days of β-catenin activation. After 3 days of activation, morethan 80% of cells became KDR+/CXCR4+ double positive, and thenthis proportion declined between 4 and 5 days. To examine whetherthe KDR+/CXCR4+ cells have the potential to differentiate towardsmesoderm derivatives, β-catenin-activated cells at day 3 werecultured under conditions that induce the endothelial cell lineagewith VEGF. These cells had endothelial cell-like morphology andprominent induction of endothelial cell markers, including CD34,CDH5 (VE-cadherin), PECAM, TEK (Tie-2) and VWF (vonWillebrand factor) (Fig. 2B,C). When these endothelial-like cellswere cultured on Matrigel with VEGF, they formed a meshwork ofcells, resembling the capillary-like structures formed by matureendothelial cells (Fig. 2D). These results demonstrate that hES-derived mesoderm progenitors induced by β-catenin have a potentialto differentiate into an endothelial cell lineage.

Antagonism of BMP signaling changes themesoderm progenitor fate towards the anteriorPSTo examine whether BMP and Activin/Nodal signaling are involvedin the β-catenin-mediated formation of PS/mesoderm progenitors,the BMP and Activin/Nodal signaling pathways were attenuated byNoggin or SB431542 (SB), which inhibit BMP or Activin/Nodalreceptors ALK4/5/7, respectively (Fig. 3A). In the presence ofNoggin, β-catenin-induced expression of the mesoderm markersKDR, FOXF1 and VENTX, and of the PS marker MIXL1, which isalso expressed in the posterior PS/mesoderm (Robb et al., 2000),was markedly diminished, whereas Noggin consistently enhancedthe expression of T and GSC (Fig. 3A; see Fig. S2 in thesupplementary material). By contrast, exposure of cells to SB in thepresence of 4OHT prevented the induction of differentiationmarkers, except for T and trophectoderm marker CGA. Thesefindings demonstrate the requirement of both BMP andActivin/Nodal signaling pathway for the β-catenin-inducedmesoderm formation in differentiating hES cells. Intriguingly, incontrast to the abolished mesoderm induction by Noggin, BMPsignaling blockade induced the expression of the anteriorPS/endoderm markers FOXA1, FOXA2, CER1, SHH and SOX17

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Fig. 2. Characterization of β-catenin-activated hES cells. (A) TheΔNβ-cateninER cells cultured with or without 4OHT for the indicatedtime periods were subjected to immunostaining with anti-KDR andanti-CXCR4 antibodies, and analyzed by FACS. The gated regiondenoted the cells scored as antigen-positive (the percent cells scoredpositive is shown in each panel). ES, unstimulated ΔNβ-cateninER cells.(B) Developmental potentials of β-catenin-activated cells towardsmesoderm lineage. RT-PCR analysis of endothelial cell-specific markersat different time points: the ΔNβ-cateninER cells were cultured with4OHT for 3 days (day 3), and further cultured with vascular endothelialgrowth factor (VEGF) for additional 3 days (day 6). The transgenic celllines derived from different ES cell lines are shown (K1βcatER derivedfrom KhES-1 cells, K3βcatER derived from KhES-3 cells). (C) Morphologyof endothelial-like cells derived from β-catenin-activated cells (day 6).(D) Capillary tube-like networks formed on matrigel for 24 hours afterculturing endothelial-like cells derived from β-catenin-activated cells.Scale bar: 100 μm.

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(Fig. 3A) (D’Amour et al., 2005; Kanai-Azuma et al., 2002; Kuboet al., 2004; Yasunaga et al., 2005). The visceral endoderm markerSOX7, ectoderm marker PAX6 and trophectoderm marker CGA werenot induced in Noggin-treated cells, indicating that they didnot differentiate into the visceral endoderm, ectoderm andtrophectoderm lineages. Immunofluorescence analysis indicatedthat more than 70% of FOXA2-positive cells were observed onlyfollowing combined Noggin and β-catenin activation, and they wereco-expressed with Brachyury and SOX17 (Fig. 3B).

The anterior PS/endoderm populations develop definitiveendoderm and anterior mesoderm derivatives, including hepatic,pancreatic and cardiac lineage (Tam and Loebel, 2007). To examinewhether β-catenin-activated cells treated with Noggin have a similardifferentiation potential with cells in the anterior PS, β-catenin-activated cells with or without Noggin at day 3 were harvested andre-cultured with FGF2 and BMP4 for 4 days. These factors have acrucial role in the differentiation of definitive endoderm andmesoderm progenitors (Gouon-Evans et al., 2006; Mima et al.,1995; Zhang and Bradley, 1996). The expression of early hepaticand pancreatic endoderm markers (AFP and PDX1), cardiac markers[NKX2-5 and α myosin heavy chain (MYH6)] was prominentlyinduced only in the Noggin-treated cells (Fig. 3C). These results

indicate that β-catenin-activated cells treated with Noggin possessthe characteristic of anterior PS/endoderm progenitors similar to thatof mouse ES cells and embryo (Gadue et al., 2006).

To determine the optimal requirement of β-catenin and Nogginfor generation of mesoderm and the anterior PS progenitors, cellswere cultured with these factors for various time periods and thenanalyzed for gene expression. Induction of T, GSC and FOXA2, andinhibition of mesoderm (KDR) was dependent on the Noggin dose(see Fig. S2A in the supplementary material), and not observed inthe absence of 4OHT, indicating the necessity for both β-cateninactivation and BMP signaling inhibition in the cell fate changetoward the anterior PS progenitors. The expression of T and GSCand mesoderm marker FOXF1 was upregulated within 1 day of β-catenin activation, and reached peak levels by continuous 3 days ofactivation. The addition of Noggin repressed expression of theFOXF1 gene, whereas the expression of the T, GSC and FOXA2genes was induced to maximal levels 3 days after β-cateninactivation (see Fig. S2B in the supplementary material). Thus,transient activation of β-catenin in hES cells was sufficient to inducemesoderm progenitors, but continuous activation was required formaximal induction of mesoderm markers. In contrast to therequirement for β-catenin, the last 2 days of Noggin treatment were

2973RESEARCH ARTICLEWnt, Activin and BMP signaling specify human ES cell fate

Fig. 3. BMP signaling blockade changes cell fate frommesoderm to the anterior PS/endoderm. (A) The qPCRanalysis on RNA isolated at the day 3 from unstimulated hEScells (vehicle: v) and β-catenin-activated cells (OHT) with orwithout Noggin (Nog; 250 ng/ml) or SB431542 (SB) wasperformed using specific primers for the genes indicated.(B) Expression of the anterior PS/endoderm markers in cellsactivated by β-catenin with Noggin. The ΔNβ-cateninER cellswere cultured with 4OHT in the presence or absence Nogginfor 3 days, and subjected to immunostaining with T, FOXA2and SOX17. Nuclei were counterstained with DAPI. Scalebar: 100 μm. (C) Developmental potentials of β-catenin-activated cells toward definitive endoderm and cardiaclineage. The day 3 β-catenin-activated cells with or withoutNoggin were cultured with BMP4 and FGF2 for additional4 days, and analyzed by RT-PCR. The transgenic cell linesderived from different ES cell lines are shown (K1βcatERderived from KhES-1 cells, K3βcatER derived from KhES-3cells). ES, unstimulated ΔNβ-cateninER cells.

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sufficient to inhibit FOXF1 and to induce FOXA2, althoughcontinuous treatment of Noggin needed for maximal induction of Tand GSC (see Fig. S2C in the supplementary material).

Involvement of PI3-kinase and MAPK signalingpathways in β-catenin-induced mesoderm andendoderm differentiationTo understand the molecular mechanisms of the PS specification viaβ-catenin and/or BMP antagonism, we investigated the downstreamtargets of Activin/Nodal and BMP signaling that might affect cellfate specification. SMAD transcriptional factors have a central rolein the TGFβ signaling pathway, and graded Nodal/SMAD signalinggoverns cell fate decisions in the PS (Vincent et al., 2003).Immunoblot analysis revealed that the expression of Oct4 proteinwas dramatically reduced in both 4OHT- and Noggin-treated cells,while Brachyury expression was induced in those cells (Fig. 4A).Neither Activin nor BMP4 alone induced the expression ofBrachyury protein in these culture conditions, indicating therequirement of cooperative actions with β-catenin signaling.Consistent with a previous report that Activin/Nodal signalingsupports hES-cell self-renewal (James et al., 2005), an active(phosphorylated) form of SMAD2 protein (P-SMAD2), but not

SMAD1, was observed in the undifferentiated hES cells (vehicle) aswell as in Activin A-treated cells. Activated SMAD2 in β-cateninactivated cells with or without Noggin, however, was reducedcompared with that in a vehicle control. Although Activin/Nodalsignaling is essential for generation of the PS, mesoderm andendoderm, persistent activation of SMAD2 results in maintenanceof the pluripotent state (James et al., 2005). Thus, the reducedSMAD2 signaling activity might be necessary to form the PS,mesoderm and endoderm progenitors in hES cell differentiation. Bycontrast, SMAD1 (P-SMAD1) was activated in 4OHT-treated cellsas well as in BMP4-treated cells, whereas it was completely blockedby Noggin (Fig. 4A). To examine the role of Activin/Nodal signalingin the differentiation of the anterior PS progenitors induced by β-catenin and BMP signaling blockade, ES cells were treated with SBduring hES cell differentiation. Inhibition of Activin/Nodalsignaling completely suppressed FOXA2 protein expressioninduced by β-catenin and Noggin (Fig. 4B). These findings, togetherwith the data shown in Fig. 3A, indicate that Activin/Nodal signalingis essential for the formation and specification of the nascent PS inβ-catenin-mediated hES cell differentiation.

In addition to the SMADs pathway, accumulating evidenceindicates that SMAD-independent pathways are involved in TGFβsignaling (Derynck and Zhang, 2003). BMP signaling has anantagonistic role in the canonical Wnt/β-catenin signaling pathwaythrough inhibition of the PI3-kinase/Akt signaling pathway byPTEN (He et al., 2004; Kobielak et al., 2007). Consistent with therequirement for Akt signaling to maintain ES cell pluripotency(Watanabe et al., 2006), an active form of Akt (P-Akt) was observedin undifferentiated hES cells (vehicle), and later downregulated inβ-catenin activated cells (Fig. 4C). There was a slight increase inthe inactive form of GSK3β (P-GSK3β), which is phosphorylatedby Akt, in β-catenin activated cells, regardless of the reduced activeform of Akt, suggesting the involvement of an Akt-independentregulatory pathway (Etienne-Manneville and Hall, 2003). Bycontrast, inhibition of BMP signaling enhanced phosphorylation ofboth Akt and GSK3β. Immunofluorescence analysis showed thattotal β-catenin was localized at the cell membrane in the absence of4OHT, whereas ΔNβ-cateninER was diffusively distributed withincells, indicating that the ΔNβ-cateninER protein was kept in aninactive form in the absence of 4OHT (Fig. 4D). When cells weretreated with 4OHT, ΔNβ-cateninER was concentrated in the nucleieven in the presence or absence of Noggin (Fig. 4D). By contrast,total β-catenin was localized at the cell membrane and slightly inthe nucleus by 4OHT treatment in comparison with vehicle controlcells. Conversely, in Noggin-treated cells, β-catenin waspreferentially accumulated in the cytoplasm and nuclei comparedwith cell treated with 4OHT alone (Fig. 4D). These data suggestthat inhibition of BMP signaling by Noggin might enhance thestability of endogenous β-catenin through the Akt/GSK3β signalingpathway during the PS specification. The cadherin familymodulates nucleo-cytoplasmic localization, stability andtransactivation of β-catenin (Moon et al., 2004). An increasedcytoplasmic pool of β-catenin due to disorganized interactions withcadherin family members might thereby enhance β-catenin-mediated transactivation. Indeed, expression of T transcript andprotein, which is a direct downstream target of β-catenin(Yamaguchi et al., 1999), and GSC, a direct target of T (Messengeret al., 2005), was enhanced in Noggin-treated cells compared withcells treated with 4OHT alone (Fig. 3A; see Fig. S2A-C in thesupplementary material). By contrast, Erk1/2 phosphorylation wasenhanced in β-catenin-activated cells, but was even moreprominent in BMP-antagonized cells (Fig. 4C).

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Fig. 4. Involvement of Activin/Nodal, PI3-kinase and MAPKsignaling pathways in β-catenin-induced mesoderm and theanterior PS differentiation. (A,C) Immunoblot analysis of total celllysates from ΔNβ-cateninER cells. Cells were cultured for 3 days with orwithout 4OHT, Noggin (Nog; 250 ng/ml), Activin A (25 ng/ml) or BMP4(10 ng/ml). Cell lysates were subjected to SDS-PAGE, and probed withspecific antibodies as indicated. (B) The ΔNβ-cateninER cells werecultured with 4OHT and Noggin in the presence or absence ofSB431542 (SB) for 3 days, and immunostained with anti-N-cadherinand FOXA2 antibodies. Scale bar: 100 μm. (D) Localization of β-cateninin ΔNβ-cateninER cells. Cells were cultured with or without 4OHT andNoggin, and subjected to immunostaining with anti-β-catenin (total)and anti-ER antibodies (to recognize exogenous ΔNβ-cateninER). Nucleiwere counterstained with DAPI. Scale bar: 50 μm.

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It has been shown that inhibition of BMP and FGF/MAPKsignaling pathways potentiates the induction of endoderm inXenopus and zebrafish (Poulain et al., 2006; Sasai et al., 1996). Toexplore the possible role of the PI3-kinase and MAPK signalingpathways in cell fate specification induced by β-catenin and BMPantagonism, cells were cultured in the presence of MEK1/2 (U0126)or PI3-kinase (LY294002) inhibitors. Consistent with an earlierfinding that Erk2 is essential for mesoderm induction (Yao et al.,2003), inhibition of MEK1/2 completely abolished the induction ofmesoderm progenitors (FOXF1), whereas inhibition of PI3-kinasehad no effect on mesoderm induction (Fig. 5A). Interestingly, theexpression of FOXA2 was slightly, but consistently, upregulated bythe inhibition of MEK1/2, compared with cells treated with 4OHTalone, suggesting that uncommitted progenitors change their cellfate toward the anterior PS progenitors following MEK1/2 signalingblockade (Fig. 5A). By contrast, FOXA2 expression induced by theantagonism of BMP signaling was completely blocked by theinhibition of PI3-kinase, but not MEK1/2, signaling.Immunofluorescence analysis of FOXA2 protein expressionconfirmed the quantitative RT-PCR results (Fig. 5B). Thus, thesefindings demonstrate that the PI3-kinase, but not MEK1/2, pathway,is essential for changing the differentiation of β-catenin-mediatedhES cells from mesoderm to the anterior PS progenitors.

Generation of mesoderm and the anterior PS fromgenetically unmanipulated ES cellsOur findings suggest that mesoderm and the anterior PS progenitorscan be developed from hES cells by a combination of BMP signalinginhibition and β-catenin activation. The 6-bromoindirubicin-3�-oxime (BIO), a GSK3 inhibitor, sufficiently activates canonical

Wnt/β-catenin signaling (Sato et al., 2004). We examined whethermesoderm and the anterior PS progenitors could be derived fromgenetically unmanipulated hES cells using BIO. At a lowerconcentration of BIO (2 μM), hES cells formed an undifferentiatedcompact colony and maintained their undifferentiated state (Fig.6A,B), as described previously (Sato et al., 2004). By contrast,treatment with a 2.5-fold higher concentration of BIO (5 μM)stimulated the dissociation of cell-cell adhesion and mesoderminduction with a concurrent loss of pluripotent markers (Fig. 6A,B).Immunofluorescence analysis showed that β-catenin was mainlylocalized at the cell membrane and cytoplasm by the lowerconcentrations of BIO, whereas the higher concentrations of BIOprominently enhanced nuclear accumulation of β-catenin (Fig. 6E).These cells, however, had lower proliferation rates than cellsactivated by ΔNβ-cateninER (data not shown). It might be due to thefact that BIO targets various protein kinases, such as Cdk family andMAP kinases (Meijer et al., 2003), and/or that GSK3β has a role inchromosomal alignment during mitosis (Tighe et al., 2007). Thus,the biphasic effect of the canonical Wnt/β-catenin signaling on thedifferentiation and maintenance of pluripotency was observed.Moreover, the combination of Noggin and BIO induced expressionof FOXA2 and repressed mesoderm markers in hES cells (Fig.6C,D). Similar data were obtained in another hES cell line, HES-3and KhES-1 cells (Fig. 6E-G; see Fig. S3A in the supplementarymaterial).

We further examined whether the canonical Wnt/β-catenin isinvolved in the specification of mesendoderm/endoderm induced byActivin during hES cell differentiation, because it has beendemonstrated the synergistic interaction of the canonical Wnt andNodal/Activin signaling pathway in mesoderm and endodermspecification in mouse embryo and ES cell system (Gadue et al.,2006; Tam and Loebel, 2007). Consistent with a previous report(D’Amour et al., 2005), expression of mesendoderm markers (T andGSC) and definitive endoderm markers (CER1 and FOXA2) wasinduced in the presence of a high concentration of Activin A (Fig.7A). By contrast, expression of these mesendoderm/endodermmarkers was markedly diminished when Wnt signaling wasinhibited by the addition of DKK1 (Glinka et al., 1998). Conversely,activation of ΔNβ-cateninER by 4OHT with Activin enhancedexpression of mesendoderm/endoderm markers rather than Activinalone (Fig. 7B). Immunoblot analysis showed that phosphorylationof SMAD2 and expression of FOXA2 protein were enhanced incells by β-catenin activation with Activin (Fig. 7C). Taken together,these data indicate that mesendoderm/endoderm specification ofhES cells in the culture was defined by the cooperative interactionof Wnt/β-catenin and Activin signaling pathway.

DISCUSSIONGenetic evidence from a wide variety of vertebrate speciesdemonstrates that the canonical Wnt/β-catenin signaling hascrucial roles in diverse developmental processes duringembryonic patterning, such as the PS, mesoderm and axisformation, and is also involved in the formation of definitiveendoderm progenitors (Lickert et al., 2002; Tam and Loebel,2007). In the present study, we clearly demonstrated thecooperative interactions of the canonical Wnt/β-catenin,Activin/Nodal and BMP signaling pathways for the induction andspecification of the PS, mesoderm and endoderm during hES celldifferentiation. The stabilization of β-catenin in hES cells issufficient to induce the posterior PS and mesoderm formationthrough the induction of Activin/Nodal and BMP signaling.Importantly, Activin/Nodal and Wnt/β-catenin signaling were

2975RESEARCH ARTICLEWnt, Activin and BMP signaling specify human ES cell fate

Fig. 5. Inhibition of PI3-kinase or MAP-kinase signaling abolishesthe anterior PS or mesoderm induction, respectively. The ΔNβ-cateninER cells were cultured with 4OHT and Noggin (Nog) in thepresence or absence of U0126 (U) and LY294002 (LY) for 3 days, andthen analyzed by qPCR (A), and stained with specific antibodies asindicated (B). v, vehicle control. Scale bar: 100 μm.

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synergistically required for the generation and specification of theanterior PS/endoderm. Moreover, we found that blockade of BMPor MAPK, but not PI3-kinase, signaling completely abolishedmesoderm induction, and instead changed the cell fate toward theanterior PS/endoderm progenitors, indicating that BMP andMAPK signaling have an antagonistic role in the formation of theanterior PS/endoderm progenitors. Taken together, our findingsindicate that balance of BMP and Activin/Nodal signaling withthe canonical Wnt/β-catenin signaling specifies the cell fate of thenascent PS into the mesoderm or endoderm progenitors (Fig. 7D).Our conclusion is supported by reports using mouse ES cellsystem (Murry and Keller, 2008; Nostro et al., 2008).

BMP signaling negatively regulates endoderm formation inXenopus (Sasai et al., 1996), but the molecular mechanismsresponsible for this inhibition are unclear. There are several linesof evidence for antagonistic interactions between BMP andWnt/β-catenin signaling. In Xenopus, Brachyury (Xbra), which isa direct target of Wnt signaling, associates with SMAD1 inresponse to BMP4, and inhibits goosecoid induction andanteriorization of Xbra in the posterior-ventral region of theembryo (Messenger et al., 2005). In mice, BMP signalingsuppresses the expression of Lef1, and consequently attenuatesthe transcriptional activity of β-catenin/Lef1 (Jamora et al., 2003).In addition, PTEN decreases the stability of β-catenin under the

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Fig. 6. Generation of mesoderm and the anterior PS from genetically unmanipulated hES cell lines, KhES-3 and HES-3 cells. (A) Phase-contrast microscopy of KhES-3 cells, treated with BIO at different concentrations for 3 days. Scale bar: 100 μm. (B,C) The qPCR analysis on RNAisolated after 3 days from untreated KhES-3 cells (vehicle, v) or BIO-treated cells (BIO, 5 μM) with or without Noggin (Nog; 250 ng/ml) for 3 dayswas performed using specific primers for the genes indicated. (D) Expression of N-cadherin and FOXA2 in BIO-treated KhES-3 cells. The hES cellswere cultured with BIO (5 μM) in the presence or absence Noggin for 3 days, and stained with specific antibodies as indicated. Scale bar: 100 μm.(E) Phase-contrast microscopy and localization of β-catenin in BIO-Acetoxime-treated HES-3 cells. Cells were cultured for 3 days with BIO-Acetoximethat exhibits greater selectivity for GSK3 than for BIO (left panels, phase-contrast images), and subjected to immunostaining with anti-β-cateninantibody (right panels). Scale bars: 100 μm. (F) qPCR analysis of RNA isolated from HES-3 cells, with or without BIO-Acetoxime (0-10 μM) for 3days, was performed as described in B. (G) Expression of N-cadherin and FOXA2 in BIO-Acetoxime-treated HES-3 cells. The HES-3 cells werecultured with BIO-Acetoxime (10 μM) in the presence or absence Noggin for 3 days, and subjected to immunostaining with anti-N-cadherin andanti-FOXA2 antibodies. Scale bar: 100 μm.DEVELO

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control of BMP signaling via the inhibition of PI3-kinase/Aktsignaling and subsequent activation of GSK3β (He et al., 2004;Kobielak et al., 2007). Consistent with these notions, PI3-kinasesignaling was essential for the generation of endodermprogenitors, at least in part, through the Akt-mediated modulationof cytoplasmic free β-catenin levels. As enhanced activation ofWnt/β-catenin signaling is necessary for the specification ofanterior endoderm progenitors in mES cells (Zamparini et al.,2006), our data suggest that modulation of cytoplasmic free-β-catenin levels, associated with BMP-induced inhibition of thePI3-kinase/Akt pathway, provides a molecular link between BMPand Wnt signaling pathways for the cell fate specification of thenascent PS in hES cell differentiation.

Previous reports have shown that the canonical Wnt/β-cateninsignaling pathway supports self-renewal and pluripotency of bothmouse and human ES cells (Hao et al., 2006; Sato et al., 2004).Although these reports seem to contradict our findings, there is someevidence that Wnt signaling plays a role in the lineage specificationof ES cells, depending on their context (Dravid et al., 2005; Gadueet al., 2006; Hao et al., 2006; Lindsley et al., 2006). Importantly,pluripotent epiblast stem cells (EpiSCs) have been recentlyestablished from mouse post-implantation epiblasts, and appear tohave features similar to those of hES cells (Brons et al., 2007; Tesaret al., 2007). These reports suggest that the distinct properties ofhES-cell self-renewal and differentiation might be due to theirepiblast origin. In agreement with this, in mouse embryo the epiblastdifferentiates prematurely into mesoderm cells when stabilized β-catenin is constitutively expressed (Kemler et al., 2004). Thepossibility that the Wnt signaling pathway has different functions atvarious developmental stages by cooperating with distinct partnersand/or regulating distinct downstream targets might affect theinterpretation of the effect of Wnt signaling on self-renewal anddifferentiation of mES and hES cells.

Alternatively, there might be a threshold of Wnt/β-catenin activityinvolved in the biphasic property of Wnt signaling. Blockade ofGSK3β with higher concentrations of BIO prominently inducednuclear translocation of β-catenin and mesoderm differentiation ofunmanipulated hES cells, whereas the lower concentrations of BIOseemed to support their self-renewal (Fig. 6; see Fig. S3A in thesupplementary material). A similar activity-dependent effect of β-catenin on self-renewal or differentiation was obtained by titratingthe 4OHT concentration (see Fig. S3B in the supplementarymaterial). At the lower concentrations of 4OHT, which anticipatesmodest activation of β-catenin, hES cells were seeminglymaintained in a self-renewal state, despite the weak induction ofmesoderm markers, whereas at the higher concentration of 4OHTthe undifferentiated stem cell state of the hES cells was abolished.These findings are consistent with a model in which small changesin the cellular levels of crucial transcriptional factors, such as Oct3/4and PU.1, define the lineage commitment of stem cells (Gurdon andBourillot, 2001; Laslo et al., 2006; Niwa et al., 2000). Thus, wepropose that the canonical Wnt/β-catenin signaling in hES cells hasbiphasic roles in controlling self-renewal and differentiation,depending on a specific threshold of β-catenin activity, althoughdistinct properties of the individual hES cell lines reflect differencesin their susceptibility to BIO (Fig. 6; see Fig. S3 in thesupplementary material).

In summary, we have demonstrated that the canonical Wnt/β-catenin signaling pathway in differentiating hES cells has significantroles in establishing the PS/mesendoderm and mesodermprogenitors, which recapitulates the global developmental programduring the early embryogenesis. More importantly, we show that thenascent PS populations changed their cell fate to the anteriorPS/endoderm or posterior PS/mesoderm progenitors followingmodulation of the Activin/Nodal and BMP signaling pathways.Thus, the reciprocal balance of Activin/Nodal and BMP signaling

2977RESEARCH ARTICLEWnt, Activin and BMP signaling specify human ES cell fate

Fig. 7. Synergistic interaction ofActivin and canonical Wnt/β-catenin signaling pathways inthe anterior PS/endodermspecification during hES celldifferentiation. (A) qPCR analysison RNA isolated from HES-3 cells,with or without Activin A (100ng/ml) or DKK1 (100 ng/ml) for3 days, was performed (v, vehiclecontrol; A, Activin A treated).(B,C) The ΔNβ-cateninER cells werecultured with Activin A (100 ng/ml)in the presence or absence of 4OHTfor 3 days, and then analyzed byqPCR (B), or cell lysates weresubjected to SDS-PAGE and probedwith specific antibodies as indicated(C) (OHT, 4OHT treated).(D) Proposed model for earlylineage determination of hES celldifferentiation by the canonicalWnt/β-catenin, Activin and BMPsignaling pathways (see Discussion).

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pathways have crucial roles in the cell fate specification of the naivePS/mesendoderm, which is induced by activation of the canonicalWnt/β-catenin signaling in hES cell differentiation (Fig. 7D).Because precise regulation of cell lineages is indispensable forefficient production of functional cells from hES cells, our findingswould be valuable for devising methods for such functional cellproduction. Future studies, such as genome-wide epigenetic andgene expression analysis, will further enhance our understanding ofhow lineage specification of hES cells is determined.

We thank Dr Pierre Chambon (IGBMC, France) for the kind gift of thepCreERT2 plasmid. This work was supported by the Grant-in-Aid for YoungScientists (B) and the National BioResource Project of the Ministry of Education,Culture, Sports, Science, and Technology (MEXT), and by the Japan Society forthe Promotion of Science.

Supplementary materialSupplementary material for this article is available athttp://dev.biologists.org/cgi/content/full/135/17/2969/DC1

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