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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/βcatenin, 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 embryonic development, stem cell 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 primitive streak (PS)/mesoderm progenitors, reminiscent of early mammalian
embryogenesis. 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/endoderm.
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.
INTRODUCTION
During early embryogenesis, gastrulation results in the formation of
three definitive germ layers and establishment of the embryonic
body plan (Tam and Loebel, 2007). At this stage, undifferentiated
epiblast 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 of
the PS induce different subpopulation of mesoderm and endoderm
progenitors (Tam and Loebel, 2007). The anterior PS has the
potential to form the definitive endoderm and anterior mesoderm,
including hepatic endoderm and cardiac mesoderm. The middle
region of PS forms the lateral plate mesoderm; and the most
posterior region of PS develops extra-embryonic mesoderm
progenitors, which give rise to hematopoietic and vascular cells of
blood islands. Mesoderm and endoderm generation depends on
successful completion of the EMT and migration away from the PS.
The EMT program is the process by which polarized epithelial cells
are converted into individually motile cells; it occurs not only during
early embryogenesis, but also during tumor progression (Thiery,
2002). The important markers of EMT lose epithelial polarities and
adherence junctions following the downregulation of E-cadherin,
which is an important cell-adhesion molecule of the apical-basal
polarity and intercellular adhesion. Although several signaling
pathways involved in the EMT processes have been identified
1
Laboratory of Embryonic Stem Cell Research, Stem Cell Research Center, Institute
for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyoku, 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: [email protected])
Accepted 3 July 2008
(Thiery, 2002), elucidation of the molecular mechanisms that trigger
and promote EMT is important for a better understanding of
embryogenesis and tumorigenesis.
The canonical Wnt signaling functions are well established in
fundamental biological processes (Moon et al., 2004; Tam and
Loebel, 2007). In early embryogenesis, Wnt/β-catenin signaling
has pivotal roles in the formation of the PS, mesoderm and
endoderm (Lickert et al., 2002; Tam and Loebel, 2007), and the
stabilization of β-catenin leads to premature EMT in mouse
embryo (Kemler et al., 2004). Wnt binds to its receptor Frizzled
and co-receptor Lrp5/6, and increases the level of cytoplasmic and
nuclear β-catenin, followed by inhibition of the GSK3-mediated
degradation pathway. Upon inhibition of GSK3 activity, stabilized
β-catenin translocates into the nucleus, where it serves as a coactivator of the Lef/Tcf family of DNA-binding proteins to form
active transcriptional complexes for specific target genes (Moon
et al., 2004). There is accumulating evidence that the Wnt/βcatenin signaling pathway also has an important role in stem cell
maintenance and regulation of the cell fate decision in several
stem cell systems, including hematopoietic, epidermal and
intestinal stem cells (Moon et al., 2004).
Embryonic stem (ES) cells possess the remarkable property
of indefinite self-renewal and pluripotency, the ability to
differentiate to all cell types of an organism; they also provide an
excellent model system for studying cell fate determination in
early mammalian development (Keller, 2005). Multiple signaling
pathways, such as those involving growth factors, transcriptional
regulators and epigenetic modifiers, have crucial roles in
regulating the balance between ES-cell self-renewal and lineage
commitment (Keller, 2005). It is very important to elucidate
molecular mechanisms of the self-renewal and lineage
commitment for efficient production of functional cells required
for use of hES cells in transplantation therapy and drug discovery.
Like other stem cell systems, canonical Wnt/β-catenin signaling
is implicated in mouse ES (mES) cell self-renewal and
differentiation, depending on the context (Gadue et al., 2006; Hao
DEVELOPMENT
KEY WORDS: Primitive streak, Mesoderm, Endoderm, Stem cells, β-Catenin, Wnt
2970 RESEARCH ARTICLE
MATERIALS AND METHODS
Activation of β-catenin signaling in hES cells
The ⌬Nβ-cateninER construct, in which the N-terminal 90 amino acids were
deleted, was generated by in-frame insertion into the expression vector
containing the hormone-binding domain of a mutant estrogen receptor
(Littlewood et al., 1995; Sumi et al., 2007). Cell lines expressing ΔNβcateninER were obtained by transfection of hES cell lines KhES-1 and
KhES-3 with ΔNβ-cateninER expression plasmids, followed by puromysin
selection as described previously (Sumi et al., 2007). To activate ΔNβcateninER, hES cells stably expressing ΔNβ-cateninER were treated with
4OHT (100 nM), as indicated.
Maintenance and differentiation of hES cells
The hES cell line HES-3 was purchased from ES Cell International. The hES
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
were dissociated into small clumps and cultured on plates coated with
matrigel (BD Biosciences, San Jose, CA) in DMEM/F12 with N2 and B27
supplements (Invitrogen, Carlsbad, CA). The next day, the medium was
changed to N2B27 medium with or without 4OHT (100 nM, Sigma, St
Louis, MO) in the presence or absence of 250 ng/ml Noggin-Fc chimera
(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
100 ng/ml Activin A (R&D Systems), 100 ng/ml DKK1 (R&D Systems) or
100 nM 4OHT for 3 days, and then analyzed as described below. SB431542
(Sigma), U0126 and LY294002 (Promega, Madison, WI) were used at a
final concentration of 10 μM. GSK-3 inhibitor-IX and -X (BIO and BIOAcetoxime, Calbiochem, La Jolla, CA) were used at a final concentration of
2 to 10 μM. For endothelial cell differentiation, cells cultured with 4OHT
for 3 days were trypsinized and plated on collagen I-coated dishes, and then
cultured in a StemPro34 serum free medium (Invitrogen) with 20 ng/ml
VEGF165 (R&D Systems) for an additional 6 days. For endoderm and
cardiac differentiation, β-catenin-activated cells cultured with or without
Noggin (250 ng/ml) for 4 days were trypsinized and plated on collagen Icoated dishes, and then cultured in a N2B27 medium without 4OHT in the
presence of 10 ng/ml BMP4 (R&D Systems) and 5 ng/ml FGF2 for an
additional 4 days. All data shown are representative results obtained from at
least two independent clones of two different human ES cell lines.
Quantitative and semi-quantitative PCR analysis
Total 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, PCR
reactions were optimized to allow for semi-quantitative comparisons within
the log phase of amplification. Real-time RT-PCR analysis was performed
on an ABI Prism 7500 Real-time PCR system using the PowerSYBR Green
PCR Master Mix (Applied Biosystems, Foster City, CA). The expression
value of each gene was normalized against the amount of GAPDH and
calculated by the ΔΔCt method. The expression level of each gene in the
control sample (vehicle or undifferentiated ES cells) was defined as 1.0. The
normalized expression values for all control and treated samples were
averaged, and average fold-change was determined. Details of the primers
used for the semi-quantitative and quantitative PCR can be provided on
request.
Western blot and immunofluorescence analysis
Cell lysates were prepared and subjected to sodium dodecyl sulfatepolyacrylamide gel elctrophoresis (SDS-PAGE), followed by western
blotting as described previously (Sumi et al., 2007). For
immunofluorescence analysis, cells plated on the culture slides were fixed
with 3.7% formaldehyde and permeabilized with 0.25% Triton X-100. After
blocking, the cells were incubated with primary antibodies, followed by
incubation with secondary antibodies. Alexa Fluor 488- or 555-conjugated
secondary antibodies were purchased from Invitrogen. Cells were mounted
onto glass slides with Vectashield (Vector Laboratories, Burlingame, CA),
and then analyzed using a BX61 fluorescence microscope (Olympus, Center
Valley, PA). Antibodies against the following proteins were used: VEGF
R2/KDR (clone#89115), SOX17 and CXCR4 (clone#44717) (R&D
Systems); 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, Santa
Cruz, CA); SSEA3 and SSEA4 (Developmental Hybridoma Bank); TRA1-60 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) (Cell
Signaling, Danvers, MA).
FACS analysis
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
488-, 555-conjugated secondary antibody (Invitrogen). FACS analysis was
performed using a FACSCalibur Flow Cytometer (Becton Dickinson).
Karyotype analysis of hES cells
Chromosome spreads were prepared as described elsewhere (Suemori et al.,
2006). Briefly, hES cells were incubated in ES medium with KaryoMAX
Colcemid 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’s
fixative.
RESULTS
Temporal emergence of the primitive streak and
mesoderm induced by stabilized β-catenin
To examine the role of canonical Wnt/β-catenin signaling in hES
cell growth and differentiation, we generated stable hES cell lines
constitutively 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 by
deletion of the N-terminal 90 amino acids, including GSK3
phosphorylation sites (Barth et al., 1997). This fusion protein (ΔNβcateninER) can be conditionally activated in response to the ER
agonist 4-hydroxy-tamoxifen (4OHT), thus enabling consistent and
reversible activation of β-catenin. We obtained several independent
hES cell clones with stable expression of the ΔNβ-cateninER fusion
proteins, following the transfection of this expression plasmid and
puromycin selection. Results presented here were obtained using
one or two clones, but similar results were obtained with further
independent clones from two different hES cell lines: KhES-1 and
KhES-3. These transgenic clones had normal karyotype and
expressed cell-surface markers for hES cells, including SSEA3,
SSEA4, TRA-1-60, TRA-1-81 and pluripotent markers POU5F1,
DEVELOPMENT
et al., 2006; Lindsley et al., 2006), but precise roles of this
signaling in human ES (hES) cells remains controversial (Dravid
et al., 2005; Sato et al., 2004).
We report here that the activation of canonical Wnt/β-catenin
signaling in hES cells by conditional activation of stabilized βcatenin disrupted hES-cell self-renewal. Rather, the canonical
Wnt/β-catenin and BMP signaling pathway in hES cells has
significant roles in establishing the posterior PS/mesoderm
progenitors, whereas attenuation of BMP signaling changes the cell
fate to the anterior PS/endoderm progenitors. In addition, Activin
and Wnt/β-catenin signaling pathways synergistically function in
inducing undifferentiated hES cells to differentiate into the anterior
PS/endoderm progenitors. This is the first in vitro model system that
consistently recapitulates the human early embryogenesis and that
enables us to analyze molecular events during the process of early
embryogenesis from the epiblast to the PS formation, followed by
lineage specification into the mesoderm and endoderm. More
importantly, our findings will also be relevant to directed
differentiation of specific tissue and cells from hES cells.
Development 135 (17)
SOX2 and NANOG comparable with the parental hES cells (see Fig.
S1A-D in the supplementary material). No obvious effect of 4OHT
on the undifferentiated state of parental hES cells was observed (see
Fig. S1B,D in the supplementary material). When ΔNβ-cateninER
cells were cultured in chemically defined medium (Yao et al., 2006)
without 4OHT, they formed tightly contacted compact colonies with
invisible cell-cell boundaries (Fig. 1A). Thus, ΔNβ-cateninER cells
maintained an undifferentiated state in culture without 4OHT. In the
presence of 4OHT, however, the morphology of the cells began to
differentiate with dissociation of the cell-cell junctions and induction
of cell scattering within 1 to 2 days, resembling the EMT (Thiery,
2002). By day 3, β-catenin-activated cells exhibited a similar
morphology (Fig. 1A), and proliferated well during the entire
RESEARCH ARTICLE 2971
process, but some cells underwent apoptotic-like cell death between
days 4 and 5, and detached from the substrata (data not shown). The
undifferentiated hES cells (vehicle) displayed epithelial-like apicalbasal polarity with a component of tight junctions, ZO-1, and formed
adherence junctions composed of E-cadherin (Fig. 1B,C). By
contrast, mesenchymal marker N-cadherin was expressed only at the
periphery of ES cell colonies, suggesting spontaneous differentiation
of a subpopulation of ES cells (Ullmann et al., 2007). After 3 days
of β-catenin activation, cells had disorganized ZO-1 localization and
markedly decreased expression of E-cadherin, and displayed a
mesenchymal phenotype, including dissociated adherence junctions
and a gradual change from E-cadherin to N-cadherin expression
(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 ΔNβcateninER-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 ΔNβcateninER 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
Wnt, Activin and BMP signaling specify human ES cell fate
Fig. 2. Characterization of β-catenin-activated hES cells. (A) The
ΔNβ-cateninER cells cultured with or without 4OHT for the indicated
time periods were subjected to immunostaining with anti-KDR and
anti-CXCR4 antibodies, and analyzed by FACS. The gated region
denoted the cells scored as antigen-positive (the percent cells scored
positive is shown in each panel). ES, unstimulated ΔNβ-cateninER cells.
(B) Developmental potentials of β-catenin-activated cells towards
mesoderm lineage. RT-PCR analysis of endothelial cell-specific markers
at different time points: the ΔNβ-cateninER cells were cultured with
4OHT for 3 days (day 3), and further cultured with vascular endothelial
growth factor (VEGF) for additional 3 days (day 6). The transgenic cell
lines derived from different ES cell lines are shown (K1βcatER derived
from KhES-1 cells, K3βcatER derived from KhES-3 cells). (C) Morphology
of endothelial-like cells derived from β-catenin-activated cells (day 6).
(D) Capillary tube-like networks formed on matrigel for 24 hours after
culturing endothelial-like cells derived from β-catenin-activated cells.
Scale bar: 100 μm.
activation in hES cells does not support their self-renewal, but
results in enhanced differentiation via EMT induction within a few
days.
To further characterize the properties of β-catenin-activated cells,
we examined the expression profiles of genes related to pluripotency
and differentiation. Cells activated by β-catenin had a rapidly
reduced expression of pluripotent markers NANOG, SOX2 and
POU5F1 within 1 day of activation, which progressively decreased
to undetectable levels by day 3 (Fig. 1D). By contrast, the expression
of the PS/nascent mesoderm markers T (Brachyury), GSC and
MIXL1 (Tam and Loebel, 2007) transiently peaked at day 1 of
activation, and gradually decreased by day 5. Expression of an early
marker 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
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 βcatenin 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 ectoderm 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
cultured under conditions that induce the endothelial cell lineage
with VEGF. These cells had endothelial cell-like morphology and
prominent induction of endothelial cell markers, including CD34,
CDH5 (VE-cadherin), PECAM, TEK (Tie-2) and VWF (von
Willebrand factor) (Fig. 2B,C). When these endothelial-like cells
were cultured on Matrigel with VEGF, they formed a meshwork of
cells, resembling the capillary-like structures formed by mature
endothelial cells (Fig. 2D). These results demonstrate that hESderived mesoderm progenitors induced by β-catenin have a potential
to differentiate into an endothelial cell lineage.
Antagonism of BMP signaling changes the
mesoderm progenitor fate towards the anterior
PS
To examine whether BMP and Activin/Nodal signaling are involved
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
receptors ALK4/5/7, respectively (Fig. 3A). In the presence of
Noggin, β-catenin-induced expression of the mesoderm markers
KDR, FOXF1 and VENTX, and of the PS marker MIXL1, which is
also expressed in the posterior PS/mesoderm (Robb et al., 2000),
was markedly diminished, whereas Noggin consistently enhanced
the expression of T and GSC (Fig. 3A; see Fig. S2 in the
supplementary material). By contrast, exposure of cells to SB in the
presence of 4OHT prevented the induction of differentiation
markers, except for T and trophectoderm marker CGA. These
findings demonstrate the requirement of both BMP and
Activin/Nodal signaling pathway for the β-catenin-induced
mesoderm formation in differentiating hES cells. Intriguingly, in
contrast to the abolished mesoderm induction by Noggin, BMP
signaling blockade induced the expression of the anterior
PS/endoderm markers FOXA1, FOXA2, CER1, SHH and SOX17
DEVELOPMENT
2972 RESEARCH ARTICLE
Wnt, Activin and BMP signaling specify human ES cell fate
RESEARCH ARTICLE 2973
(Fig. 3A) (D’Amour et al., 2005; Kanai-Azuma et al., 2002; Kubo
et al., 2004; Yasunaga et al., 2005). The visceral endoderm marker
SOX7, ectoderm marker PAX6 and trophectoderm marker CGA were
not induced in Noggin-treated cells, indicating that they did
not differentiate into the visceral endoderm, ectoderm and
trophectoderm lineages. Immunofluorescence analysis indicated
that more than 70% of FOXA2-positive cells were observed only
following combined Noggin and β-catenin activation, and they were
co-expressed with Brachyury and SOX17 (Fig. 3B).
The anterior PS/endoderm populations develop definitive
endoderm and anterior mesoderm derivatives, including hepatic,
pancreatic and cardiac lineage (Tam and Loebel, 2007). To examine
whether β-catenin-activated cells treated with Noggin have a similar
differentiation potential with cells in the anterior PS, β-cateninactivated cells with or without Noggin at day 3 were harvested and
re-cultured with FGF2 and BMP4 for 4 days. These factors have a
crucial role in the differentiation of definitive endoderm and
mesoderm progenitors (Gouon-Evans et al., 2006; Mima et al.,
1995; Zhang and Bradley, 1996). The expression of early hepatic
and pancreatic endoderm markers (AFP and PDX1), cardiac markers
[NKX2-5 and α myosin heavy chain (MYH6)] was prominently
induced only in the Noggin-treated cells (Fig. 3C). These results
indicate that β-catenin-activated cells treated with Noggin possess
the characteristic of anterior PS/endoderm progenitors similar to that
of mouse ES cells and embryo (Gadue et al., 2006).
To determine the optimal requirement of β-catenin and Noggin
for generation of mesoderm and the anterior PS progenitors, cells
were cultured with these factors for various time periods and then
analyzed for gene expression. Induction of T, GSC and FOXA2, and
inhibition of mesoderm (KDR) was dependent on the Noggin dose
(see Fig. S2A in the supplementary material), and not observed in
the absence of 4OHT, indicating the necessity for both β-catenin
activation and BMP signaling inhibition in the cell fate change
toward the anterior PS progenitors. The expression of T and GSC
and mesoderm marker FOXF1 was upregulated within 1 day of βcatenin activation, and reached peak levels by continuous 3 days of
activation. The addition of Noggin repressed expression of the
FOXF1 gene, whereas the expression of the T, GSC and FOXA2
genes was induced to maximal levels 3 days after β-catenin
activation (see Fig. S2B in the supplementary material). Thus,
transient activation of β-catenin in hES cells was sufficient to induce
mesoderm progenitors, but continuous activation was required for
maximal induction of mesoderm markers. In contrast to the
requirement for β-catenin, the last 2 days of Noggin treatment were
DEVELOPMENT
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-cateninactivated 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. 4. Involvement of Activin/Nodal, PI3-kinase and MAPK
signaling pathways in β-catenin-induced mesoderm and the
anterior PS differentiation. (A,C) Immunoblot analysis of total cell
lysates from ΔNβ-cateninER cells. Cells were cultured for 3 days with or
without 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 with
specific antibodies as indicated. (B) The ΔNβ-cateninER cells were
cultured with 4OHT and Noggin in the presence or absence of
SB431542 (SB) for 3 days, and immunostained with anti-N-cadherin
and FOXA2 antibodies. Scale bar: 100 μm. (D) Localization of β-catenin
in ΔNβ-cateninER cells. Cells were cultured with or without 4OHT and
Noggin, and subjected to immunostaining with anti-β-catenin (total)
and anti-ER antibodies (to recognize exogenous ΔNβ-cateninER). Nuclei
were counterstained with DAPI. Scale bar: 50 μm.
sufficient to inhibit FOXF1 and to induce FOXA2, although
continuous treatment of Noggin needed for maximal induction of T
and GSC (see Fig. S2C in the supplementary material).
Involvement of PI3-kinase and MAPK signaling
pathways in β-catenin-induced mesoderm and
endoderm differentiation
To understand the molecular mechanisms of the PS specification via
β-catenin and/or BMP antagonism, we investigated the downstream
targets of Activin/Nodal and BMP signaling that might affect cell
fate specification. SMAD transcriptional factors have a central role
in the TGFβ signaling pathway, and graded Nodal/SMAD signaling
governs cell fate decisions in the PS (Vincent et al., 2003).
Immunoblot analysis revealed that the expression of Oct4 protein
was 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 of
Brachyury protein in these culture conditions, indicating the
requirement of cooperative actions with β-catenin signaling.
Consistent with a previous report that Activin/Nodal signaling
supports hES-cell self-renewal (James et al., 2005), an active
(phosphorylated) form of SMAD2 protein (P-SMAD2), but not
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 βcatenin 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
PTEN (He et al., 2004; Kobielak et al., 2007). Consistent with the
requirement for Akt signaling to maintain ES cell pluripotency
(Watanabe et al., 2006), an active form of Akt (P-Akt) was observed
in undifferentiated hES cells (vehicle), and later downregulated in
β-catenin activated cells (Fig. 4C). There was a slight increase in
the inactive form of GSK3β (P-GSK3β), which is phosphorylated
by Akt, in β-catenin activated cells, regardless of the reduced active
form of Akt, suggesting the involvement of an Akt-independent
regulatory pathway (Etienne-Manneville and Hall, 2003). By
contrast, inhibition of BMP signaling enhanced phosphorylation of
both Akt and GSK3β. Immunofluorescence analysis showed that
total β-catenin was localized at the cell membrane in the absence of
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
treated with 4OHT, ΔNβ-cateninER was concentrated in the nuclei
even in the presence or absence of Noggin (Fig. 4D). By contrast,
total β-catenin was localized at the cell membrane and slightly in
the nucleus by 4OHT treatment in comparison with vehicle control
cells. Conversely, in Noggin-treated cells, β-catenin was
preferentially accumulated in the cytoplasm and nuclei compared
with cell treated with 4OHT alone (Fig. 4D). These data suggest
that inhibition of BMP signaling by Noggin might enhance the
stability of endogenous β-catenin through the Akt/GSK3β signaling
pathway during the PS specification. The cadherin family
modulates nucleo-cytoplasmic localization, stability and
transactivation of β-catenin (Moon et al., 2004). An increased
cytoplasmic pool of β-catenin due to disorganized interactions with
cadherin family members might thereby enhance β-cateninmediated transactivation. Indeed, expression of T transcript and
protein, which is a direct downstream target of β-catenin
(Yamaguchi et al., 1999), and GSC, a direct target of T (Messenger
et al., 2005), was enhanced in Noggin-treated cells compared with
cells treated with 4OHT alone (Fig. 3A; see Fig. S2A-C in the
supplementary material). By contrast, Erk1/2 phosphorylation was
enhanced in β-catenin-activated cells, but was even more
prominent in BMP-antagonized cells (Fig. 4C).
DEVELOPMENT
2974 RESEARCH ARTICLE
Fig. 5. Inhibition of PI3-kinase or MAP-kinase signaling abolishes
the anterior PS or mesoderm induction, respectively. The ΔNβcateninER cells were cultured with 4OHT and Noggin (Nog) in the
presence or absence of U0126 (U) and LY294002 (LY) for 3 days, and
then analyzed by qPCR (A), and stained with specific antibodies as
indicated (B). v, vehicle control. Scale bar: 100 μm.
It has been shown that inhibition of BMP and FGF/MAPK
signaling pathways potentiates the induction of endoderm in
Xenopus and zebrafish (Poulain et al., 2006; Sasai et al., 1996). To
explore the possible role of the PI3-kinase and MAPK signaling
pathways in cell fate specification induced by β-catenin and BMP
antagonism, cells were cultured in the presence of MEK1/2 (U0126)
or PI3-kinase (LY294002) inhibitors. Consistent with an earlier
finding that Erk2 is essential for mesoderm induction (Yao et al.,
2003), inhibition of MEK1/2 completely abolished the induction of
mesoderm progenitors (FOXF1), whereas inhibition of PI3-kinase
had no effect on mesoderm induction (Fig. 5A). Interestingly, the
expression of FOXA2 was slightly, but consistently, upregulated by
the inhibition of MEK1/2, compared with cells treated with 4OHT
alone, suggesting that uncommitted progenitors change their cell
fate toward the anterior PS progenitors following MEK1/2 signaling
blockade (Fig. 5A). By contrast, FOXA2 expression induced by the
antagonism of BMP signaling was completely blocked by the
inhibition of PI3-kinase, but not MEK1/2, signaling.
Immunofluorescence analysis of FOXA2 protein expression
confirmed the quantitative RT-PCR results (Fig. 5B). Thus, these
findings demonstrate that the PI3-kinase, but not MEK1/2, pathway,
is essential for changing the differentiation of β-catenin-mediated
hES cells from mesoderm to the anterior PS progenitors.
Generation of mesoderm and the anterior PS from
genetically unmanipulated ES cells
Our findings suggest that mesoderm and the anterior PS progenitors
can be developed from hES cells by a combination of BMP signaling
inhibition and β-catenin activation. The 6-bromoindirubicin-3⬘oxime (BIO), a GSK3 inhibitor, sufficiently activates canonical
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
material).
We further examined whether the canonical Wnt/β-catenin is
involved in the specification of mesendoderm/endoderm induced by
Activin during hES cell differentiation, because it has been
demonstrated the synergistic interaction of the canonical Wnt and
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
(D’Amour et al., 2005), expression of mesendoderm markers (T and
GSC) and definitive endoderm markers (CER1 and FOXA2) was
induced in the presence of a high concentration of Activin A (Fig.
7A). By contrast, expression of these mesendoderm/endoderm
markers was markedly diminished when Wnt signaling was
inhibited by the addition of DKK1 (Glinka et al., 1998). Conversely,
activation of ΔNβ-cateninER by 4OHT with Activin enhanced
expression of mesendoderm/endoderm markers rather than Activin
alone (Fig. 7B). Immunoblot analysis showed that phosphorylation
of SMAD2 and expression of FOXA2 protein were enhanced in
cells by β-catenin activation with Activin (Fig. 7C). Taken together,
these data indicate that mesendoderm/endoderm specification of
hES cells in the culture was defined by the cooperative interaction
of Wnt/β-catenin and Activin signaling pathway.
DISCUSSION
Genetic evidence from a wide variety of vertebrate species
demonstrates that the canonical Wnt/β-catenin signaling has
crucial roles in diverse developmental processes during
embryonic patterning, such as the PS, mesoderm and axis
formation, and is also involved in the formation of definitive
endoderm progenitors (Lickert et al., 2002; Tam and Loebel,
2007). In the present study, we clearly demonstrated the
cooperative interactions of the canonical Wnt/β-catenin,
Activin/Nodal and BMP signaling pathways for the induction and
specification of the PS, mesoderm and endoderm during hES cell
differentiation. The stabilization of β-catenin in hES cells is
sufficient to induce the posterior PS and mesoderm formation
through the induction of Activin/Nodal and BMP signaling.
Importantly, Activin/Nodal and Wnt/β-catenin signaling were
DEVELOPMENT
Wnt, Activin and BMP signaling specify human ES cell fate
2976 RESEARCH ARTICLE
Development 135 (17)
synergistically required for the generation and specification of the
anterior PS/endoderm. Moreover, we found that blockade of BMP
or MAPK, but not PI3-kinase, signaling completely abolished
mesoderm induction, and instead changed the cell fate toward the
anterior PS/endoderm progenitors, indicating that BMP and
MAPK signaling have an antagonistic role in the formation of the
anterior PS/endoderm progenitors. Taken together, our findings
indicate that balance of BMP and Activin/Nodal signaling with
the canonical Wnt/β-catenin signaling specifies the cell fate of the
nascent PS into the mesoderm or endoderm progenitors (Fig. 7D).
Our conclusion is supported by reports using mouse ES cell
system (Murry and Keller, 2008; Nostro et al., 2008).
BMP signaling negatively regulates endoderm formation in
Xenopus (Sasai et al., 1996), but the molecular mechanisms
responsible for this inhibition are unclear. There are several lines
of evidence for antagonistic interactions between BMP and
Wnt/β-catenin signaling. In Xenopus, Brachyury (Xbra), which is
a direct target of Wnt signaling, associates with SMAD1 in
response to BMP4, and inhibits goosecoid induction and
anteriorization of Xbra in the posterior-ventral region of the
embryo (Messenger et al., 2005). In mice, BMP signaling
suppresses the expression of Lef1, and consequently attenuates
the transcriptional activity of β-catenin/Lef1 (Jamora et al., 2003).
In addition, PTEN decreases the stability of β-catenin under the
DEVELOPMENT
Fig. 6. Generation of mesoderm and the anterior PS from genetically unmanipulated hES cell lines, KhES-3 and HES-3 cells. (A) Phasecontrast 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.
Wnt, Activin and BMP signaling specify human ES cell fate
RESEARCH ARTICLE 2977
control of BMP signaling via the inhibition of PI3-kinase/Akt
signaling and subsequent activation of GSK3β (He et al., 2004;
Kobielak et al., 2007). Consistent with these notions, PI3-kinase
signaling was essential for the generation of endoderm
progenitors, at least in part, through the Akt-mediated modulation
of cytoplasmic free β-catenin levels. As enhanced activation of
Wnt/β-catenin signaling is necessary for the specification of
anterior endoderm progenitors in mES cells (Zamparini et al.,
2006), our data suggest that modulation of cytoplasmic free-βcatenin levels, associated with BMP-induced inhibition of the
PI3-kinase/Akt pathway, provides a molecular link between BMP
and Wnt signaling pathways for the cell fate specification of the
nascent PS in hES cell differentiation.
Previous reports have shown that the canonical Wnt/β-catenin
signaling pathway supports self-renewal and pluripotency of both
mouse and human ES cells (Hao et al., 2006; Sato et al., 2004).
Although these reports seem to contradict our findings, there is some
evidence that Wnt signaling plays a role in the lineage specification
of ES cells, depending on their context (Dravid et al., 2005; Gadue
et al., 2006; Hao et al., 2006; Lindsley et al., 2006). Importantly,
pluripotent epiblast stem cells (EpiSCs) have been recently
established from mouse post-implantation epiblasts, and appear to
have features similar to those of hES cells (Brons et al., 2007; Tesar
et al., 2007). These reports suggest that the distinct properties of
hES-cell self-renewal and differentiation might be due to their
epiblast origin. In agreement with this, in mouse embryo the epiblast
differentiates prematurely into mesoderm cells when stabilized βcatenin is constitutively expressed (Kemler et al., 2004). The
possibility that the Wnt signaling pathway has different functions at
various developmental stages by cooperating with distinct partners
and/or regulating distinct downstream targets might affect the
interpretation of the effect of Wnt signaling on self-renewal and
differentiation of mES and hES cells.
Alternatively, there might be a threshold of Wnt/β-catenin activity
involved in the biphasic property of Wnt signaling. Blockade of
GSK3β with higher concentrations of BIO prominently induced
nuclear translocation of β-catenin and mesoderm differentiation of
unmanipulated hES cells, whereas the lower concentrations of BIO
seemed to support their self-renewal (Fig. 6; see Fig. S3A in the
supplementary material). A similar activity-dependent effect of βcatenin on self-renewal or differentiation was obtained by titrating
the 4OHT concentration (see Fig. S3B in the supplementary
material). At the lower concentrations of 4OHT, which anticipates
modest activation of β-catenin, hES cells were seemingly
maintained in a self-renewal state, despite the weak induction of
mesoderm markers, whereas at the higher concentration of 4OHT
the undifferentiated stem cell state of the hES cells was abolished.
These findings are consistent with a model in which small changes
in the cellular levels of crucial transcriptional factors, such as Oct3/4
and PU.1, define the lineage commitment of stem cells (Gurdon and
Bourillot, 2001; Laslo et al., 2006; Niwa et al., 2000). Thus, we
propose that the canonical Wnt/β-catenin signaling in hES cells has
biphasic roles in controlling self-renewal and differentiation,
depending on a specific threshold of β-catenin activity, although
distinct properties of the individual hES cell lines reflect differences
in their susceptibility to BIO (Fig. 6; see Fig. S3 in the
supplementary material).
In summary, we have demonstrated that the canonical Wnt/βcatenin signaling pathway in differentiating hES cells has significant
roles in establishing the PS/mesendoderm and mesoderm
progenitors, which recapitulates the global developmental program
during the early embryogenesis. More importantly, we show that the
nascent PS populations changed their cell fate to the anterior
PS/endoderm or posterior PS/mesoderm progenitors following
modulation of the Activin/Nodal and BMP signaling pathways.
Thus, the reciprocal balance of Activin/Nodal and BMP signaling
DEVELOPMENT
Fig. 7. Synergistic interaction of
Activin and canonical Wnt/βcatenin 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).
pathways have crucial roles in the cell fate specification of the naive
PS/mesendoderm, which is induced by activation of the canonical
Wnt/β-catenin signaling in hES cell differentiation (Fig. 7D).
Because precise regulation of cell lineages is indispensable for
efficient production of functional cells from hES cells, our findings
would be valuable for devising methods for such functional cell
production. Future studies, such as genome-wide epigenetic and
gene expression analysis, will further enhance our understanding of
how lineage specification of hES cells is determined.
We thank Dr Pierre Chambon (IGBMC, France) for the kind gift of the
pCreERT2 plasmid. This work was supported by the Grant-in-Aid for Young
Scientists (B) and the National BioResource Project of the Ministry of Education,
Culture, Sports, Science, and Technology (MEXT), and by the Japan Society for
the Promotion of Science.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/17/2969/DC1
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DEVELOPMENT
Wnt, Activin and BMP signaling specify human ES cell fate