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First published online 30 July 2008
doi: 10.1242/dev.021121
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1 Laboratory of Embryonic Stem Cell Research, Stem Cell Research Center,
Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho,
Shogoin, Sakyo-ku, Kyoto 606-8507, Japan.
2 Department of Development and Differentiation, Institute for Frontier Medical
Sciences, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto
606-8507, Japan.
3 Institute for Integrated Cell-Material Sciences (iCeMS), Kyoto University, 69
Konoe-cho, Yoshida, Sakyo-ku, Kyoto 606-8507, Japan.
* Author for correspondence (e-mail: hsuemori{at}frontier.kyoto-u.ac.jp)
Accepted 3 July 2008
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Primitive streak, Mesoderm, Endoderm, Stem cells, β-Catenin, Wnt
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). 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
(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
co-activator 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/β-catenin 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 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 β-catenin 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.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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 Nβ-cateninER were obtained by transfection of hES cell
lines KhES-1 and KhES-3 with Nβ-cateninER expression plasmids,
followed by puromysin selection as described previously
(Sumi et al., 2007). To
activate Nβ-cateninER, hES cells stably expressing
Nβ-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 BIO-Acetoxime, 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 I-coated 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 Ct 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
sulfate-polyacrylamide 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 ER
(Santa Cruz Biotechnology, Santa Cruz, CA);
SSEA3 and SSEA4 (Developmental Hybridoma Bank); TRA-1-60 and TRA-1-81
(Chemicon); Phospho-Smad1 (Ser463/465), Smad1, phospho-Smad2 (Ser465/467),
phospho-MAPK (Thr202/Tyr204), MAPK, phospho-Akt (Ser473), Akt and
phospho-GSK3/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.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). 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 (Nβ-cateninER) 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
Nβ-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, 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
Nβ-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,
Nβ-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 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
apical-basal 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 activation in hES cells does not
support their self-renewal, but results in enhanced differentiation via EMT
induction within a few days.
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)
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 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 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 hES-derived 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 (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).
|
). To examine whether β-catenin-activated cells treated
with Noggin have a similar differentiation potential with cells in the
anterior PS, β-catenin-activated 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
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 β-catenin 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 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).
|
).
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 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 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 Nβ-cateninER was diffusively distributed within
cells, indicating that the Nβ-cateninER protein was kept in an
inactive form in the absence of 4OHT (Fig.
4D). When cells were treated with 4OHT, Nβ-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-catenin-mediated
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).
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.
|
). 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
Nβ-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 Nβ-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 |
|---|
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
; 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
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).
|
), 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 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-β-catenin 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.
|
;
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 β-catenin 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 β-catenin
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/β-catenin 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 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.
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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ACKNOWLEDGMENTS |
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