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DEVELOPMENT AND DISEASE RESEARCH ARTICLE 2969

Development 135, 2969-2979 (2008) doi:10.1242/dev.021121 Defining early lineage specification of human embryonic stem cells by the orchestrated balance of canonical Wnt/β- catenin, Activin/Nodal and BMP signaling Tomoyuki Sumi1, Norihiro Tsuneyoshi1,2, Norio Nakatsuji2,3 and Hirofumi Suemori1,*

The canonical Wnt/β-catenin signaling has remarkably diverse roles in , self-renewal and cancer progression. Here, we show that stabilized expression of β-catenin perturbed human embryonic stem (hES)-cell self-renewal, such that up to 80% of the hES cells developed into the (PS)/ progenitors, reminiscent of early . The formation of the PS/mesoderm progenitors essentially depended on the cooperative action of β-catenin together with Activin/Nodal and BMP signaling pathways. Intriguingly, blockade of BMP signaling completely abolished mesoderm generation, and induced a cell fate change towards the anterior PS progenitors. The PI3-kinase/Akt, but not MAPK, signaling pathway 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/. Taken together, our findings clearly demonstrate that the orchestrated balance of Activin/Nodal and BMP signaling defines the cell fate 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

INTRODUCTION (Thiery, 2002), elucidation of the molecular mechanisms that trigger During early embryogenesis, results in the formation of and promote EMT is important for a better understanding of three definitive germ layers and establishment of the embryonic embryogenesis and tumorigenesis. body plan (Tam and Loebel, 2007). At this stage, undifferentiated The canonical Wnt signaling functions are well established in cells undergo an epithelial to mesenchymal transition fundamental biological processes (Moon et al., 2004; Tam and (EMT) and migrate through a structure called the primitive streak Loebel, 2007). In early embryogenesis, Wnt/β-catenin signaling (PS) to generate the mesoderm and endoderm. Distinct regions of has pivotal roles in the formation of the PS, mesoderm and the PS induce different subpopulation of mesoderm and endoderm endoderm (Lickert et al., 2002; Tam and Loebel, 2007), and the progenitors (Tam and Loebel, 2007). The anterior PS has the stabilization of β-catenin leads to premature EMT in mouse potential to form the definitive endoderm and anterior mesoderm, (Kemler et al., 2004). Wnt binds to its receptor Frizzled including hepatic endoderm and cardiac mesoderm. The middle and co-receptor Lrp5/6, and increases the level of cytoplasmic and region of PS forms the ; and the most nuclear β-catenin, followed by inhibition of the GSK3-mediated posterior region of PS develops extra-embryonic mesoderm degradation pathway. Upon inhibition of GSK3 activity, stabilized progenitors, which give rise to hematopoietic and vascular cells of β-catenin translocates into the nucleus, where it serves as a co- blood islands. Mesoderm and endoderm generation depends on activator of the Lef/Tcf family of DNA-binding to form successful completion of the EMT and migration away from the PS. active transcriptional complexes for specific target genes (Moon The EMT program is the process by which polarized epithelial cells et al., 2004). There is accumulating evidence that the Wnt/β- are converted into individually motile cells; it occurs not only during catenin signaling pathway also has an important role in stem cell early embryogenesis, but also during tumor progression (Thiery, maintenance and regulation of the cell fate decision in several 2002). The important markers of EMT lose epithelial polarities and stem cell systems, including hematopoietic, epidermal and adherence junctions following the downregulation of E-cadherin, intestinal stem cells (Moon et al., 2004). which is an important cell-adhesion molecule of the apical-basal Embryonic stem (ES) cells possess the remarkable property polarity and intercellular adhesion. Although several signaling of indefinite self-renewal and pluripotency, the ability to pathways involved in the EMT processes have been identified differentiate to all cell types of an organism; they also provide an excellent model system for studying in early mammalian development (Keller, 2005). Multiple signaling pathways, such as those involving growth factors, transcriptional regulators and epigenetic modifiers, have crucial roles in 1Laboratory of Research, Stem Cell Research Center, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo- regulating the balance between ES-cell self-renewal and lineage ku, Kyoto 606-8507, Japan. 2Department of Development and Differentiation, commitment (Keller, 2005). It is very important to elucidate Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin, molecular mechanisms of the self-renewal and lineage Sakyo-ku, Kyoto 606-8507, Japan. 3Institute for Integrated Cell-Material Sciences (iCeMS), Kyoto University, 69 Konoe-cho, Yoshida, Sakyo-ku, Kyoto 606-8507, commitment for efficient production of functional cells required Japan. for use of hES cells in transplantation therapy and drug discovery. Like other stem cell systems, canonical Wnt/β-catenin signaling *Author for correspondence (e-mail: [email protected]) is implicated in mouse ES (mES) cell self-renewal and

Accepted 3 July 2008 differentiation, depending on the context (Gadue et al., 2006; Hao DEVELOPMENT 2970 RESEARCH ARTICLE Development 135 (17) et al., 2006; Lindsley et al., 2006), but precise roles of this on an ABI Prism 7500 Real-time PCR system using the PowerSYBR Green signaling in human ES (hES) cells remains controversial (Dravid PCR Master Mix (Applied Biosystems, Foster City, CA). The expression et al., 2005; Sato et al., 2004). value of each gene was normalized against the amount of GAPDH and We report here that the activation of canonical Wnt/β-catenin calculated by the ΔΔCt method. The expression level of each gene in the signaling in hES cells by conditional activation of stabilized β- control sample (vehicle or undifferentiated ES cells) was defined as 1.0. The catenin disrupted hES-cell self-renewal. Rather, the canonical normalized expression values for all control and treated samples were β averaged, and average fold-change was determined. Details of the primers Wnt/ -catenin and BMP signaling pathway in hES cells has used for the semi-quantitative and quantitative PCR can be provided on significant roles in establishing the posterior PS/mesoderm request. progenitors, whereas attenuation of BMP signaling changes the cell fate to the anterior PS/endoderm progenitors. In addition, Activin Western blot and immunofluorescence analysis and Wnt/β-catenin signaling pathways synergistically function in Cell lysates were prepared and subjected to sodium dodecyl sulfate- inducing undifferentiated hES cells to differentiate into the anterior polyacrylamide gel elctrophoresis (SDS-PAGE), followed by western PS/endoderm progenitors. This is the first in vitro model system that blotting as described previously (Sumi et al., 2007). For immunofluorescence analysis, cells plated on the culture slides were fixed consistently recapitulates the human early embryogenesis and that with 3.7% formaldehyde and permeabilized with 0.25% Triton X-100. After enables us to analyze molecular events during the process of early blocking, the cells were incubated with primary antibodies, followed by embryogenesis from the epiblast to the PS formation, followed by incubation with secondary antibodies. Alexa Fluor 488- or 555-conjugated lineage specification into the mesoderm and endoderm. More secondary antibodies were purchased from Invitrogen. Cells were mounted importantly, our findings will also be relevant to directed onto glass slides with Vectashield (Vector Laboratories, Burlingame, CA), differentiation of specific tissue and cells from hES cells. and then analyzed using a BX61 (Olympus, Center Valley, PA). Antibodies against the following proteins were used: VEGF R2/KDR (clone#89115), SOX17 and CXCR4 (clone#44717) (R&D MATERIALS AND METHODS Systems); E-cadherin, Smad2/3, GSK3β and β-catenin (BD Biosciences); Activation of β-catenin signaling in hES cells N-cadherin (GC-4, Sigma); ZO-1 (Invitrogen); Nanog (ReproCELL); The ⌬Nβ-cateninER construct, in which the N-terminal 90 amino acids were HNF3β, Oct3/4, and ERα (Santa Cruz Biotechnology, Santa deleted, was generated by in-frame insertion into the expression vector Cruz, CA); SSEA3 and SSEA4 (Developmental Hybridoma Bank); TRA- containing the hormone-binding domain of a mutant estrogen receptor 1-60 and TRA-1-81 (Chemicon); Phospho-Smad1 (Ser463/465), Smad1, (Littlewood et al., 1995; Sumi et al., 2007). Cell lines expressing ΔNβ- phospho-Smad2 (Ser465/467), phospho-MAPK (Thr202/Tyr204), MAPK, cateninER were obtained by transfection of hES cell lines KhES-1 and phospho-Akt (Ser473), Akt and phospho-GSK3α/β (Ser21/9) (Cell KhES-3 with ΔNβ-cateninER expression plasmids, followed by puromysin Signaling, Danvers, MA). selection as described previously (Sumi et al., 2007). To activate ΔNβ- cateninER, hES cells stably expressing ΔNβ-cateninER were treated with FACS analysis 4OHT (100 nM), as indicated. Single cell suspensions were fixed with 1% formaldehyde for 30 minutes at 4°C, and incubated first with primary antibody and then with Alexa Fluor Maintenance and differentiation of hES cells 488-, 555-conjugated secondary antibody (Invitrogen). FACS analysis was The hES cell line HES-3 was purchased from ES Cell International. The hES performed using a FACSCalibur Flow Cytometer (Becton Dickinson). cell lines KhES-1, KhES-3 and HES-3 were maintained as described previously (Suemori et al., 2006). For differentiation of hES cells, the cells Karyotype analysis of hES cells were dissociated into small clumps and cultured on plates coated with Chromosome spreads were prepared as described elsewhere (Suemori et al., matrigel (BD Biosciences, San Jose, CA) in DMEM/F12 with N2 and B27 2006). Briefly, hES cells were incubated in ES medium with KaryoMAX μ supplements (Invitrogen, Carlsbad, CA). The next day, the medium was Colcemid Solution (Invitrogen; 0.1 g/ml of colcemid) for 2 hours, changed to N2B27 medium with or without 4OHT (100 nM, Sigma, St trypsinized, incubated in 0.075 M KCl for 10 minutes and fixed in Carnoy’s Louis, MO) in the presence or absence of 250 ng/ml -Fc chimera fixative. (R&D Systems Minneapolis, MN) except as otherwise indicated, and renewed daily thereafter. For Activin-induced differentiation, hES cells were cultured in RPMI supplemented with 2% FBS in the presence or absence of RESULTS 100 ng/ml Activin A (R&D Systems), 100 ng/ml DKK1 (R&D Systems) or Temporal emergence of the primitive streak and 100 nM 4OHT for 3 days, and then analyzed as described below. SB431542 mesoderm induced by stabilized β-catenin (Sigma), U0126 and LY294002 (Promega, Madison, WI) were used at a To examine the role of canonical Wnt/β-catenin signaling in hES final concentration of 10 μM. GSK-3 inhibitor-IX and -X (BIO and BIO- cell growth and differentiation, we generated stable hES cell lines Acetoxime, Calbiochem, La Jolla, CA) were used at a final concentration of constitutively expressing a fusion of a stabilized β-catenin, 2 to 10 μM. For endothelial cell differentiation, cells cultured with 4OHT a mutant ligand-binding domain of the estrogen receptor (ER) for 3 days were trypsinized and plated on collagen I-coated dishes, and then (Littlewood et al., 1995). The stabilized β-catenin was produced by cultured in a StemPro34 serum free medium (Invitrogen) with 20 ng/ml deletion of the N-terminal 90 amino acids, including GSK3 VEGF165 (R&D Systems) for an additional 6 days. For endoderm and Δ β cardiac differentiation, β-catenin-activated cells cultured with or without phosphorylation sites (Barth et al., 1997). This fusion protein ( N - Noggin (250 ng/ml) for 4 days were trypsinized and plated on collagen I- cateninER) can be conditionally activated in response to the ER coated dishes, and then cultured in a N2B27 medium without 4OHT in the agonist 4-hydroxy-tamoxifen (4OHT), thus enabling consistent and presence of 10 ng/ml BMP4 (R&D Systems) and 5 ng/ml FGF2 for an reversible activation of β-catenin. We obtained several independent additional 4 days. All data shown are representative results obtained from at hES cell clones with stable expression of the ΔNβ-cateninER fusion least two independent clones of two different human ES cell lines. proteins, following the transfection of this expression plasmid and puromycin selection. Results presented here were obtained using Quantitative and semi-quantitative PCR analysis Total RNA was isolated from cells using RNeasy Micro Kit (QIAGEN, one or two clones, but similar results were obtained with further Valencia, VA) and reverse-transcribed using Omniscript RT Kit (QIAGEN) independent clones from two different hES cell lines: KhES-1 and according to the manufacturer’s protocol. For semi-quantitative PCR, PCR KhES-3. These transgenic clones had normal karyotype and reactions were optimized to allow for semi-quantitative comparisons within expressed cell-surface markers for hES cells, including SSEA3,

the log phase of amplification. Real-time RT-PCR analysis was performed SSEA4, TRA-1-60, TRA-1-81 and pluripotent markers POU5F1, DEVELOPMENT Wnt, Activin and BMP signaling specify human ES cell fate RESEARCH ARTICLE 2971

SOX2 and NANOG comparable with the parental hES cells (see Fig. process, but some cells underwent apoptotic-like cell death between S1A-D in the supplementary material). No obvious effect of 4OHT days 4 and 5, and detached from the substrata (data not shown). The on the undifferentiated state of parental hES cells was observed (see undifferentiated hES cells (vehicle) displayed epithelial-like apical- Fig. S1B,D in the supplementary material). When ΔNβ-cateninER basal polarity with a component of tight junctions, ZO-1, and formed cells were cultured in chemically defined medium (Yao et al., 2006) adherence junctions composed of E-cadherin (Fig. 1B,C). By without 4OHT, they formed tightly contacted compact colonies with contrast, mesenchymal marker N-cadherin was expressed only at the invisible cell-cell boundaries (Fig. 1A). Thus, ΔNβ-cateninER cells periphery of ES cell colonies, suggesting spontaneous differentiation maintained an undifferentiated state in culture without 4OHT. In the of a subpopulation of ES cells (Ullmann et al., 2007). After 3 days presence of 4OHT, however, the morphology of the cells began to of β-catenin activation, cells had disorganized ZO-1 localization and differentiate with dissociation of the cell-cell junctions and induction markedly decreased expression of E-cadherin, and displayed a of cell scattering within 1 to 2 days, resembling the EMT (Thiery, mesenchymal phenotype, including dissociated adherence junctions 2002). By day 3, β-catenin-activated cells exhibited a similar and a gradual change from E-cadherin to N-cadherin expression morphology (Fig. 1A), and proliferated well during the entire (Fig. 1B,C). These results indicate that conditional β-catenin

Fig. 1. Temporal dynamics of the primitive streak and mesoderm progenitors in β-catenin-activated hES cells. (A) The ΔNβ-cateninER cells were 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, and nuclei were counterstained with diaminopimelic acid (DAPI). Scale bar: 100 μm. (C) The ΔNβ-cateninER cells were cultured with (days 1-3) or without (day 0) 4OHT for the indicated time periods, and subjected to immunostaining with anti-E-cadherin and anti-N-cadherin antibodies. Nuclei were 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 genes indicated. (E) Immunoblot analysis of cell lysates from ΔNβ-cateninER cells. Cells were cultured for the indicated time periods with 4OHT, and cell

lysates were subjected to SDS-PAGE and probed with specific antibodies as indicated. DEVELOPMENT 2972 RESEARCH ARTICLE Development 135 (17)

manner following the induction of T, MIXL1 and GSC. The BMP, Nodal and FGF signaling pathways are important for the emergence of the PS, mesoderm and endoderm in mouse (Tam and Loebel, 2007). Gene expression analysis showed the progressive induction of BMP4 and FGF8, and a temporal induction of NODAL by β- catenin activation, suggesting the involvement of these factors in the development of the PS/mesoderm in β-catenin-induced hES cell differentiation (Fig. 1D). By contrast, the trophectoderm marker CGA was not expressed (Fig. 3A). The marker PAX6 was dominated by β-catenin activation (Fig. 3A), as the canonical Wnt signaling suppresses neural differentiation (Aubert et al., 2002). Immunoblot and immunofluorescence analysis confirmed downregulation of Oct4 and Nanog, and induction of Brachyury and KDR protein (Fig. 1B,E). Thus, these temporal gene expression patterns in differentiating hES cells recapitulate the emergence of the PS, and mark the period of the transition towards mesoderm in early mammalian development. To evaluate the relative proportion of mesoderm progenitors induced by β-catenin activation, we analyzed the expression of CXCR4 and KDR as mesoderm markers by fluorescence-activated cell sorting (FACS) analysis. Weak levels of KDR expression were detected in ~50% of undifferentiated ES cells, while CXCR4 was not expressed (Fig. 2A). A representative 5-day kinetic experiment indicated that the KDR+/CXCR4+ cells were immediately detected after 2 days of β-catenin activation. After 3 days of activation, more than 80% of cells became KDR+/CXCR4+ double positive, and then this proportion declined between 4 and 5 days. To examine whether the KDR+/CXCR4+ cells have the potential to differentiate towards mesoderm derivatives, β-catenin-activated cells at day 3 were Fig. 2. Characterization of β-catenin-activated hES cells. (A) The cultured under conditions that induce the endothelial cell lineage ΔNβ-cateninER cells cultured with or without 4OHT for the indicated with VEGF. These cells had endothelial cell-like morphology and time periods were subjected to immunostaining with anti-KDR and prominent induction of endothelial cell markers, including CD34, anti-CXCR4 antibodies, and analyzed by FACS. The gated region CDH5 (VE-cadherin), PECAM, TEK (Tie-2) and VWF (von denoted the cells scored as antigen-positive (the percent cells scored Willebrand factor) (Fig. 2B,C). When these endothelial-like cells Δ β positive is shown in each panel). ES, unstimulated N -cateninER cells. were cultured on Matrigel with VEGF, they formed a meshwork of (B) Developmental potentials of β-catenin-activated cells towards cells, resembling the capillary-like structures formed by mature mesoderm lineage. RT-PCR analysis of endothelial cell-specific markers at different time points: the ΔNβ-cateninER cells were cultured with endothelial cells (Fig. 2D). These results demonstrate that hES- β 4OHT for 3 days (day 3), and further cultured with vascular endothelial derived mesoderm progenitors induced by -catenin have a potential growth factor (VEGF) for additional 3 days (day 6). The transgenic cell to differentiate into an endothelial cell lineage. lines derived from different ES cell lines are shown (K1βcatER derived from KhES-1 cells, K3βcatER derived from KhES-3 cells). (C) Morphology Antagonism of BMP signaling changes the of endothelial-like cells derived from β-catenin-activated cells (day 6). mesoderm progenitor fate towards the anterior (D) Capillary tube-like networks formed on matrigel for 24 hours after PS culturing endothelial-like cells derived from β-catenin-activated cells. To examine whether BMP and Activin/Nodal signaling are involved μ Scale bar: 100 m. in the β-catenin-mediated formation of PS/mesoderm progenitors, the BMP and Activin/Nodal signaling pathways were attenuated by Noggin or SB431542 (SB), which inhibit BMP or Activin/Nodal activation in hES cells does not support their self-renewal, but receptors ALK4/5/7, respectively (Fig. 3A). In the presence of results in enhanced differentiation via EMT induction within a few Noggin, β-catenin-induced expression of the mesoderm markers days. KDR, FOXF1 and VENTX, and of the PS marker MIXL1, which is To further characterize the properties of β-catenin-activated cells, also expressed in the posterior PS/mesoderm (Robb et al., 2000), we examined the expression profiles of genes related to pluripotency was markedly diminished, whereas Noggin consistently enhanced and differentiation. Cells activated by β-catenin had a rapidly the expression of T and GSC (Fig. 3A; see Fig. S2 in the reduced expression of pluripotent markers NANOG, SOX2 and supplementary material). By contrast, exposure of cells to SB in the POU5F1 within 1 day of activation, which progressively decreased presence of 4OHT prevented the induction of differentiation to undetectable levels by day 3 (Fig. 1D). By contrast, the expression markers, except for T and trophectoderm marker CGA. These of the PS/nascent mesoderm markers T (Brachyury), GSC and findings demonstrate the requirement of both BMP and MIXL1 (Tam and Loebel, 2007) transiently peaked at day 1 of Activin/ for the β-catenin-induced activation, and gradually decreased by day 5. Expression of an early mesoderm formation in differentiating hES cells. Intriguingly, in marker of ventral mesoderm (KDR) and pre-cardiac mesoderm contrast to the abolished mesoderm induction by Noggin, BMP (NKX2-5), and of a mesoderm/endoderm marker (CXCR4) signaling blockade induced the expression of the anterior

(D’Amour et al., 2005; Yasunaga et al., 2005) increased in an orderly PS/endoderm markers FOXA1, FOXA2, CER1, SHH and SOX17 DEVELOPMENT Wnt, Activin and BMP signaling specify human ES cell fate RESEARCH ARTICLE 2973

Fig. 3. BMP signaling blockade changes cell fate from mesoderm to the anterior PS/endoderm. (A) The qPCR analysis on RNA isolated at the day 3 from unstimulated hES cells (vehicle: v) and β-catenin-activated cells (OHT) with or without Noggin (Nog; 250 ng/ml) or SB431542 (SB) was performed using specific primers for the genes indicated. (B) Expression of the anterior PS/endoderm markers in cells activated by β-catenin with Noggin. The ΔNβ-cateninER cells were cultured with 4OHT in the presence or absence Noggin for 3 days, and subjected to immunostaining with T, FOXA2 and SOX17. Nuclei were counterstained with DAPI. Scale bar: 100 μm. (C) Developmental potentials of β-catenin- activated cells toward definitive endoderm and cardiac lineage. The day 3 β-catenin-activated cells with or without Noggin were cultured with BMP4 and FGF2 for additional 4 days, and analyzed by RT-PCR. The transgenic cell lines derived from different ES cell lines are shown (K1βcatER derived from KhES-1 cells, K3βcatER derived from KhES-3 cells). ES, unstimulated ΔNβ-cateninER cells.

(Fig. 3A) (D’Amour et al., 2005; Kanai-Azuma et al., 2002; Kubo indicate that β-catenin-activated cells treated with Noggin possess et al., 2004; Yasunaga et al., 2005). The visceral endoderm marker the characteristic of anterior PS/endoderm progenitors similar to that SOX7, ectoderm marker PAX6 and trophectoderm marker CGA were of mouse ES cells and embryo (Gadue et al., 2006). not induced in Noggin-treated cells, indicating that they did To determine the optimal requirement of β-catenin and Noggin not differentiate into the visceral endoderm, ectoderm and for generation of mesoderm and the anterior PS progenitors, cells trophectoderm lineages. Immunofluorescence analysis indicated were cultured with these factors for various time periods and then that more than 70% of FOXA2-positive cells were observed only analyzed for gene expression. Induction of T, GSC and FOXA2, and following combined Noggin and β-catenin activation, and they were inhibition of mesoderm (KDR) was dependent on the Noggin dose co-expressed with Brachyury and SOX17 (Fig. 3B). (see Fig. S2A in the supplementary material), and not observed in The anterior PS/endoderm populations develop definitive the absence of 4OHT, indicating the necessity for both β-catenin endoderm and anterior mesoderm derivatives, including hepatic, activation and BMP signaling inhibition in the cell fate change pancreatic and cardiac lineage (Tam and Loebel, 2007). To examine toward the anterior PS progenitors. The expression of T and GSC whether β-catenin-activated cells treated with Noggin have a similar and mesoderm marker FOXF1 was upregulated within 1 day of β- differentiation potential with cells in the anterior PS, β-catenin- catenin activation, and reached peak levels by continuous 3 days of activated cells with or without Noggin at day 3 were harvested and activation. The addition of Noggin repressed expression of the re-cultured with FGF2 and BMP4 for 4 days. These factors have a FOXF1 gene, whereas the expression of the T, GSC and FOXA2 crucial role in the differentiation of definitive endoderm and genes was induced to maximal levels 3 days after β-catenin mesoderm progenitors (Gouon-Evans et al., 2006; Mima et al., activation (see Fig. S2B in the supplementary material). Thus, 1995; Zhang and Bradley, 1996). The expression of early hepatic transient activation of β-catenin in hES cells was sufficient to induce and pancreatic endoderm markers (AFP and PDX1), cardiac markers mesoderm progenitors, but continuous activation was required for [NKX2-5 and α myosin heavy chain (MYH6)] was prominently maximal induction of mesoderm markers. In contrast to the β induced only in the Noggin-treated cells (Fig. 3C). These results requirement for -catenin, the last 2 days of Noggin treatment were DEVELOPMENT 2974 RESEARCH ARTICLE Development 135 (17)

SMAD1, was observed in the undifferentiated hES cells (vehicle) as well as in Activin A-treated cells. Activated SMAD2 in β-catenin activated cells with or without Noggin, however, was reduced compared with that in a vehicle control. Although Activin/Nodal signaling is essential for generation of the PS, mesoderm and endoderm, persistent activation of SMAD2 results in maintenance of the pluripotent state (James et al., 2005). Thus, the reduced SMAD2 signaling activity might be necessary to form the PS, mesoderm and endoderm progenitors in hES cell differentiation. By contrast, SMAD1 (P-SMAD1) was activated in 4OHT-treated cells as well as in BMP4-treated cells, whereas it was completely blocked by Noggin (Fig. 4A). To examine the role of Activin/Nodal signaling in the differentiation of the anterior PS progenitors induced by β- catenin and BMP signaling blockade, ES cells were treated with SB during hES cell differentiation. Inhibition of Activin/Nodal signaling completely suppressed FOXA2 protein expression induced by β-catenin and Noggin (Fig. 4B). These findings, together with the data shown in Fig. 3A, indicate that Activin/Nodal signaling is essential for the formation and specification of the nascent PS in β-catenin-mediated hES cell differentiation. In addition to the SMADs pathway, accumulating evidence indicates that SMAD-independent pathways are involved in TGFβ signaling (Derynck and Zhang, 2003). BMP signaling has an antagonistic role in the canonical Wnt/β-catenin signaling pathway through inhibition of the PI3-kinase/Akt signaling pathway by Fig. 4. Involvement of Activin/Nodal, PI3-kinase and MAPK PTEN (He et al., 2004; Kobielak et al., 2007). Consistent with the signaling pathways in β-catenin-induced mesoderm and the requirement for Akt signaling to maintain ES cell pluripotency anterior PS differentiation. (A,C) Immunoblot analysis of total cell Δ β (Watanabe et al., 2006), an active form of Akt (P-Akt) was observed lysates from N -cateninER cells. Cells were cultured for 3 days with or in undifferentiated hES cells (vehicle), and later downregulated in without 4OHT, Noggin (Nog; 250 ng/ml), Activin A (25 ng/ml) or BMP4 β-catenin activated cells (Fig. 4C). There was a slight increase in (10 ng/ml). Cell lysates were subjected to SDS-PAGE, and probed with β β specific antibodies as indicated. (B) The ΔNβ-cateninER cells were the inactive form of GSK3 (P-GSK3 ), which is phosphorylated cultured with 4OHT and Noggin in the presence or absence of by Akt, in β-catenin activated cells, regardless of the reduced active SB431542 (SB) for 3 days, and immunostained with anti-N-cadherin form of Akt, suggesting the involvement of an Akt-independent and FOXA2 antibodies. Scale bar: 100 μm. (D) Localization of β-catenin regulatory pathway (Etienne-Manneville and Hall, 2003). By in ΔNβ-cateninER cells. Cells were cultured with or without 4OHT and contrast, inhibition of BMP signaling enhanced phosphorylation of Noggin, and subjected to immunostaining with anti-β-catenin (total) both Akt and GSK3β. Immunofluorescence analysis showed that and anti-ER antibodies (to recognize exogenous ΔNβ-cateninER). Nuclei total β-catenin was localized at the cell membrane in the absence of μ were counterstained with DAPI. Scale bar: 50 m. 4OHT, whereas ΔNβ-cateninER was diffusively distributed within cells, indicating that the ΔNβ-cateninER protein was kept in an inactive form in the absence of 4OHT (Fig. 4D). When cells were sufficient to inhibit FOXF1 and to induce FOXA2, although treated with 4OHT, ΔNβ-cateninER was concentrated in the nuclei continuous treatment of Noggin needed for maximal induction of T even in the presence or absence of Noggin (Fig. 4D). By contrast, and GSC (see Fig. S2C in the supplementary material). total β-catenin was localized at the cell membrane and slightly in the nucleus by 4OHT treatment in comparison with vehicle control Involvement of PI3-kinase and MAPK signaling cells. Conversely, in Noggin-treated cells, β-catenin was pathways in β-catenin-induced mesoderm and preferentially accumulated in the cytoplasm and nuclei compared endoderm differentiation with cell treated with 4OHT alone (Fig. 4D). These data suggest To understand the molecular mechanisms of the PS specification via that inhibition of BMP signaling by Noggin might enhance the β-catenin and/or BMP antagonism, we investigated the downstream stability of endogenous β-catenin through the Akt/GSK3β signaling targets of Activin/Nodal and BMP signaling that might affect cell pathway during the PS specification. The cadherin family fate specification. SMAD transcriptional factors have a central role modulates nucleo-cytoplasmic localization, stability and in the TGFβ signaling pathway, and graded Nodal/SMAD signaling transactivation of β-catenin (Moon et al., 2004). An increased governs cell fate decisions in the PS (Vincent et al., 2003). cytoplasmic pool of β-catenin due to disorganized interactions with Immunoblot analysis revealed that the expression of Oct4 protein cadherin family members might thereby enhance β-catenin- was dramatically reduced in both 4OHT- and Noggin-treated cells, mediated transactivation. Indeed, expression of T transcript and while Brachyury expression was induced in those cells (Fig. 4A). protein, which is a direct downstream target of β-catenin Neither Activin nor BMP4 alone induced the expression of (Yamaguchi et al., 1999), and GSC, a direct target of T (Messenger Brachyury protein in these culture conditions, indicating the et al., 2005), was enhanced in Noggin-treated cells compared with requirement of cooperative actions with β-catenin signaling. cells treated with 4OHT alone (Fig. 3A; see Fig. S2A-C in the Consistent with a previous report that Activin/Nodal signaling supplementary material). By contrast, Erk1/2 phosphorylation was supports hES-cell self-renewal (James et al., 2005), an active enhanced in β-catenin-activated cells, but was even more

(phosphorylated) form of SMAD2 protein (P-SMAD2), but not prominent in BMP-antagonized cells (Fig. 4C). DEVELOPMENT Wnt, Activin and BMP signaling specify human ES cell fate RESEARCH ARTICLE 2975

Wnt/β-catenin signaling (Sato et al., 2004). We examined whether mesoderm and the anterior PS progenitors could be derived from genetically unmanipulated hES cells using BIO. At a lower concentration of BIO (2 μM), hES cells formed an undifferentiated compact 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 mesoderm induction with a concurrent loss of pluripotent markers (Fig. 6A,B). Immunofluorescence analysis showed that β-catenin was mainly localized at the cell membrane and cytoplasm by the lower concentrations of BIO, whereas the higher concentrations of BIO prominently enhanced nuclear accumulation of β-catenin (Fig. 6E). These cells, however, had lower proliferation rates than cells activated by ΔNβ-cateninER (data not shown). It might be due to the fact that BIO targets various protein kinases, such as Cdk family and MAP kinases (Meijer et al., 2003), and/or that GSK3β has a role in chromosomal alignment during mitosis (Tighe et al., 2007). Thus, the biphasic effect of the canonical Wnt/β-catenin signaling on the differentiation and maintenance of pluripotency was observed. Moreover, the combination of Noggin and BIO induced expression of FOXA2 and repressed mesoderm markers in hES cells (Fig. 6C,D). Similar data were obtained in another hES cell line, HES-3 and KhES-1 cells (Fig. 6E-G; see Fig. S3A in the supplementary Fig. 5. Inhibition of PI3-kinase or MAP-kinase signaling abolishes material). the anterior PS or mesoderm induction, respectively. The ΔNβ- We further examined whether the canonical Wnt/β-catenin is cateninER cells were cultured with 4OHT and Noggin (Nog) in the involved in the specification of mesendoderm/endoderm induced by presence or absence of U0126 (U) and LY294002 (LY) for 3 days, and Activin during hES cell differentiation, because it has been then analyzed by qPCR (A), and stained with specific antibodies as demonstrated the synergistic interaction of the canonical Wnt and indicated (B). v, vehicle control. Scale bar: 100 μm. Nodal/Activin signaling pathway in mesoderm and endoderm specification in mouse embryo and ES cell system (Gadue et al., 2006; Tam and Loebel, 2007). Consistent with a previous report It has been shown that inhibition of BMP and FGF/MAPK (D’Amour et al., 2005), expression of mesendoderm markers (T and signaling pathways potentiates the induction of endoderm in GSC) and definitive endoderm markers (CER1 and FOXA2) was Xenopus and zebrafish (Poulain et al., 2006; Sasai et al., 1996). To induced in the presence of a high concentration of Activin A (Fig. explore the possible role of the PI3-kinase and MAPK signaling 7A). By contrast, expression of these mesendoderm/endoderm pathways in cell fate specification induced by β-catenin and BMP markers was markedly diminished when Wnt signaling was antagonism, cells were cultured in the presence of MEK1/2 (U0126) inhibited by the addition of DKK1 (Glinka et al., 1998). Conversely, or PI3-kinase (LY294002) inhibitors. Consistent with an earlier activation of ΔNβ-cateninER by 4OHT with Activin enhanced finding that Erk2 is essential for mesoderm induction (Yao et al., expression of mesendoderm/endoderm markers rather than Activin 2003), inhibition of MEK1/2 completely abolished the induction of alone (Fig. 7B). Immunoblot analysis showed that phosphorylation mesoderm progenitors (FOXF1), whereas inhibition of PI3-kinase of SMAD2 and expression of FOXA2 protein were enhanced in had no effect on mesoderm induction (Fig. 5A). Interestingly, the cells by β-catenin activation with Activin (Fig. 7C). Taken together, expression of FOXA2 was slightly, but consistently, upregulated by these data indicate that mesendoderm/endoderm specification of the inhibition of MEK1/2, compared with cells treated with 4OHT hES cells in the culture was defined by the cooperative interaction alone, suggesting that uncommitted progenitors change their cell of Wnt/β-catenin and Activin signaling pathway. fate toward the anterior PS progenitors following MEK1/2 signaling blockade (Fig. 5A). By contrast, FOXA2 expression induced by the DISCUSSION antagonism of BMP signaling was completely blocked by the Genetic evidence from a wide variety of vertebrate species inhibition of PI3-kinase, but not MEK1/2, signaling. demonstrates that the canonical Wnt/β-catenin signaling has Immunofluorescence analysis of FOXA2 protein expression crucial roles in diverse developmental processes during confirmed the quantitative RT-PCR results (Fig. 5B). Thus, these embryonic patterning, such as the PS, mesoderm and axis findings demonstrate that the PI3-kinase, but not MEK1/2, pathway, formation, and is also involved in the formation of definitive is essential for changing the differentiation of β-catenin-mediated endoderm progenitors (Lickert et al., 2002; Tam and Loebel, hES cells from mesoderm to the anterior PS progenitors. 2007). In the present study, we clearly demonstrated the cooperative interactions of the canonical Wnt/β-catenin, Generation of mesoderm and the anterior PS from Activin/Nodal and BMP signaling pathways for the induction and genetically unmanipulated ES cells specification of the PS, mesoderm and endoderm during hES cell Our findings suggest that mesoderm and the anterior PS progenitors differentiation. The stabilization of β-catenin in hES cells is can be developed from hES cells by a combination of BMP signaling sufficient to induce the posterior PS and mesoderm formation inhibition and β-catenin activation. The 6-bromoindirubicin-3Ј- through the induction of Activin/Nodal and BMP signaling. β oxime (BIO), a GSK3 inhibitor, sufficiently activates canonical Importantly, Activin/Nodal and Wnt/ -catenin signaling were DEVELOPMENT 2976 RESEARCH ARTICLE Development 135 (17)

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 RNA isolated 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 days was performed using specific primers for the genes indicated. (D) Expression of N-cadherin and FOXA2 in BIO-treated KhES-3 cells. The hES cells were 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-Acetoxime that exhibits greater selectivity for GSK3 than for BIO (left panels, phase-contrast images), and subjected to immunostaining with anti-β-catenin antibody (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 3 days, was performed as described in B. (G) Expression of N-cadherin and FOXA2 in BIO-Acetoxime-treated HES-3 cells. The HES-3 cells were cultured with BIO-Acetoxime (10 μM) in the presence or absence Noggin for 3 days, and subjected to immunostaining with anti-N-cadherin and anti-FOXA2 antibodies. Scale bar: 100 μm.

synergistically required for the generation and specification of the BMP signaling negatively regulates endoderm formation in anterior PS/endoderm. Moreover, we found that blockade of BMP Xenopus (Sasai et al., 1996), but the molecular mechanisms or MAPK, but not PI3-kinase, signaling completely abolished responsible for this inhibition are unclear. There are several lines mesoderm induction, and instead changed the cell fate toward the of evidence for antagonistic interactions between BMP and anterior PS/endoderm progenitors, indicating that BMP and Wnt/β-catenin signaling. In Xenopus, Brachyury (), which is MAPK signaling have an antagonistic role in the formation of the a direct target of Wnt signaling, associates with SMAD1 in anterior PS/endoderm progenitors. Taken together, our findings response to BMP4, and inhibits goosecoid induction and indicate that balance of BMP and Activin/Nodal signaling with anteriorization of Xbra in the posterior-ventral region of the the canonical Wnt/β-catenin signaling specifies the cell fate of the embryo (Messenger et al., 2005). In mice, BMP signaling nascent PS into the mesoderm or endoderm progenitors (Fig. 7D). suppresses the expression of Lef1, and consequently attenuates Our conclusion is supported by reports using mouse ES cell the transcriptional activity of β-catenin/Lef1 (Jamora et al., 2003). β system (Murry and Keller, 2008; Nostro et al., 2008). In addition, PTEN decreases the stability of -catenin under the DEVELOPMENT Wnt, Activin and BMP signaling specify human ES cell fate RESEARCH ARTICLE 2977

Fig. 7. Synergistic interaction of Activin and canonical Wnt/β- catenin signaling pathways in the anterior PS/endoderm specification during hES cell differentiation. (A) qPCR analysis on RNA isolated from HES-3 cells, with or without Activin A (100 ng/ml) or DKK1 (100 ng/ml) for 3 days, was performed (v, vehicle control; A, Activin A treated). (B,C) The ΔNβ-cateninER cells were cultured with Activin A (100 ng/ml) in the presence or absence of 4OHT for 3 days, and then analyzed by qPCR (B), or cell lysates were subjected to SDS-PAGE and probed with specific antibodies as indicated (C) (OHT, 4OHT treated). (D) Proposed model for early lineage determination of hES cell differentiation by the canonical Wnt/β-catenin, Activin and BMP signaling pathways (see Discussion).

control of BMP signaling via the inhibition of PI3-kinase/Akt Alternatively, there might be a threshold of Wnt/β-catenin activity signaling and subsequent activation of GSK3β (He et al., 2004; involved in the biphasic property of Wnt signaling. Blockade of Kobielak et al., 2007). Consistent with these notions, PI3-kinase GSK3β with higher concentrations of BIO prominently induced signaling was essential for the generation of endoderm nuclear translocation of β-catenin and mesoderm differentiation of progenitors, at least in part, through the Akt-mediated modulation unmanipulated hES cells, whereas the lower concentrations of BIO of cytoplasmic free β-catenin levels. As enhanced activation of seemed to support their self-renewal (Fig. 6; see Fig. S3A in the Wnt/β-catenin signaling is necessary for the specification of supplementary material). A similar activity-dependent effect of β- anterior endoderm progenitors in mES cells (Zamparini et al., catenin on self-renewal or differentiation was obtained by titrating 2006), our data suggest that modulation of cytoplasmic free-β- the 4OHT concentration (see Fig. S3B in the supplementary catenin levels, associated with BMP-induced inhibition of the material). At the lower concentrations of 4OHT, which anticipates PI3-kinase/Akt pathway, provides a molecular link between BMP modest activation of β-catenin, hES cells were seemingly and Wnt signaling pathways for the cell fate specification of the maintained in a self-renewal state, despite the weak induction of nascent PS in hES cell differentiation. mesoderm markers, whereas at the higher concentration of 4OHT Previous reports have shown that the canonical Wnt/β-catenin the undifferentiated stem cell state of the hES cells was abolished. signaling pathway supports self-renewal and pluripotency of both These findings are consistent with a model in which small changes mouse and human ES cells (Hao et al., 2006; Sato et al., 2004). in the cellular levels of crucial transcriptional factors, such as Oct3/4 Although these reports seem to contradict our findings, there is some and PU.1, define the lineage commitment of stem cells (Gurdon and evidence that Wnt signaling plays a role in the lineage specification Bourillot, 2001; Laslo et al., 2006; Niwa et al., 2000). Thus, we of ES cells, depending on their context (Dravid et al., 2005; Gadue propose that the canonical Wnt/β-catenin signaling in hES cells has et al., 2006; Hao et al., 2006; Lindsley et al., 2006). Importantly, biphasic roles in controlling self-renewal and differentiation, pluripotent epiblast stem cells (EpiSCs) have been recently depending on a specific threshold of β-catenin activity, although established from mouse post-implantation , and appear to distinct properties of the individual hES cell lines reflect differences have features similar to those of hES cells (Brons et al., 2007; Tesar in their susceptibility to BIO (Fig. 6; see Fig. S3 in the et al., 2007). These reports suggest that the distinct properties of supplementary material). hES-cell self-renewal and differentiation might be due to their In summary, we have demonstrated that the canonical Wnt/β- epiblast origin. In agreement with this, in mouse embryo the epiblast catenin signaling pathway in differentiating hES cells has significant differentiates prematurely into mesoderm cells when stabilized β- roles in establishing the PS/mesendoderm and mesoderm catenin is constitutively expressed (Kemler et al., 2004). The progenitors, which recapitulates the global developmental program possibility that the has different functions at during the early embryogenesis. More importantly, we show that the various developmental stages by cooperating with distinct partners nascent PS populations changed their cell fate to the anterior and/or regulating distinct downstream targets might affect the PS/endoderm or posterior PS/mesoderm progenitors following interpretation of the effect of Wnt signaling on self-renewal and modulation of the Activin/Nodal and BMP signaling pathways.

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