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First published online 11 March 2009
doi: 10.1242/dev.033951
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1 Department of Surgery and Laboratory For Regenerative Medicine, West Forvie
Building, Robinson Way, University of Cambridge, Cambridge CB2 0SZ, UK.
2 CR-UK Viral Oncology Group, Wolfson Institute for Biomedical Research,
University College London, London WC1E 6BT, UK.
3 Laboratoire de transfert de gènes dans le foie: applications
thérapeutiques. Inserm U804, Université Paris XI, 94276 Le
Kremlin Bicêtre, France.
* Author for correspondence (e-mail: lv225{at}cam.ac.uk)
Accepted 16 February 2009
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Nanog, hESCs, Activin, Nodal, Smad2/3, Neuroectoderm, Endoderm, Mesendoderm, Extraembryonic, Mouse, Human
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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) and bone
morphogenetic protein (Ying et al.,
2003) to maintain pluripotency, whereas their human counterparts
rely on Activin/Nodal (James et al.,
2005; Vallier et al.,
2004) and fibroblast growth factors
(Xu et al., 2005). However, in
vitro and in vivo studies have established that a core transcription factor
circuit involving Oct4, Sox2 and Nanog is necessary for pluripotency in both
species. Indeed, constitutive expression of Nanog is sufficient to prevent
differentiation of mouse and human ESCs
(Chambers et al., 2003;
Darr et al., 2006), and loss of
function confirms that Nanog is necessary to block primitive endoderm
differentiation (Chambers et al.,
2003; Hyslop et al.,
2005; Mitsui et al.,
2003). These apparent contradictory observations underline the
lack of knowledge concerning the mechanisms linking extracellular signalling
and the core transcriptional network (including Nanog), especially in
hESCs.
Here, we show that Smad2/3, the downstream effectors of Activin/Nodal
signalling, bind and directly control the activity of the Nanog gene in hESCs.
Accordingly, inhibition of Activin/Nodal signalling resulted in a loss of
Nanog expression while inducing differentiation toward neuroectoderm.
Knockdown of Nanog expression mimicked this effect, which is strictly
dependent on FGF signalling. Conversely, constitutive expression of Nanog was
sufficient to maintain the pluripotent status of hESCs in the absence of
Activin/Nodal signalling, by specifically blocking neuroectoderm
differentiation. In addition, our biochemical analyses showed that Nanog
interacts directly with Smad2/3 proteins, the direct effectors of
Activin/Nodal signalling, to limit their transcriptional activity, which is
crucial for the cell fate choice between pluripotency and differentiation in
hESCs. Importantly, similar results were obtained using pluripotent stem cells
derived from the epiblast layer of pre-gastrula stage mouse embryos (mEpiSCs)
demonstrating that these mechanisms are evolutionarily conserved, consistent
with the proposed homology between hESCs and EpiSCs
(Brons et al., 2007;
Tesar et al., 2007). Taken
together, these results demonstrate that Activin/Nodal signalling blocks
neuroectoderm differentiation of pluripotent cells by maintaining Nanog
expression, and they also provide the basis for a model explaining for the
first time how Activin/Nodal signalling can maintain the pluripotency of hESCs
without inducing differentiation towards endoderm.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). For
embryoid body (EB) formation and differentiation, hESC colonies were grown in
non-adherent conditions as described
(Vallier et al., 2004). For
differentiation into extraembryonic tissues, hESCs and EpiSCs were grown in
CDM with 10 ng/ml BMP4 for 7 days. For neuroectoderm differentiation, hESCs
and EpiSCs were grown in CDM with 10 µM SB431542 and 12 ng/ml FGF2 for 7
days. For mesendoderm differentiation, hESCs were grown in CDM with 5 ng/ml
Activin but without FGF2 for 3 days, and then for 4 additional days in the
presence of 100 ng/ml Activin, 10 ng/ml BMP4 and 20 ng/ml FGF2 (our
unpublished results).
Microarray analysis
Microarray analyses were performed as described by Brons et al.
(Brons et al., 2007). All
hybridisations employed are publicly available in MIAME format from the
ArrayExpress microarray repository (European Bioinformatics Institute;
http://www.ebi.ac.uk/arrayexpress)
under Accession Number E-MEXP-1741.
RT-PCR and real-time PCR
Total RNAs were extracted using the RNeasy Mini Kit (Qiagen). For each
sample, 0.6 µg of total RNA was reverse transcribed using Superscript II
(Invitrogen). RT-PCR was performed as described
(Vallier et al., 2004).
Real-time PCR reaction mixtures were prepared as described (SensiMiX protocol
Quantace), denatured at 94°C for 5 minutes, cycled at 94°C for 30
seconds, 60°C for 30 seconds and 72°C for 30 seconds for 40 cycles,
then subjected to a final extension step at 72°C for 10 minutes. Primer
sequences have been described elsewhere
(Vallier et al., 2004;
Brons et al., 2007). RT-PCR
reactions were performed using a Stratagene Mx3005P in triplicate and
normalised to porphobilinogen deaminase (PBGD) in the same run. Data represent
the mean of three independent experiments and error bars indicate standard
deviation.
Nuclear extracts and Smad/Nanog co-immunoprecipitations
The nuclear extract was prepared as described
(Dyer and Herzog, 1995). The
final protein concentration was 2 mg/ml in HEMG110 buffer (Hepes pH 7.6, 0.5
mM EDTA, 5 mM MgCl2, 110 mM KCl, 20% glycerol, 0.2 mM DTT, Roche
Complete cocktail, 0.5 mM PMSF). In each experiment, 0.5-1.0 mg nuclear
protein and 3 µl antibody [
Smad2/3 (Cell Signalling), rabbit IgG
(Sigma)] were used and incubated for 3 hours at 4°C. Protein G beads (10
µl, Roche) were added and samples incubated for an additional hour. Beads
were washed five times with HEMG110 and proteins eluted with LDS-loading
buffer at 70°C. PAGE and western blotting was performed with the
Invitrogen NuPAGE system. Primary antibodies [Nanog (R&D systems),
Sox2 (Abcam)] were probed with secondary HRP antibodies (Sigma) and
detected using the LumilitePlus kit (Roche). The HA and FLAG
immunoprecipitations (IPs) were essentially performed as described above,
using Roche and Sigma M2 affinity matrixes. The Flag-HA-hNanog construct is
driven by the CAG promoter and was transfected by Lipofectamine 2000
(Invitrogen). Cells were harvested after 42 hours.
Chromatin immunoprecipitation (ChIP)
ChIP was carried out as previously described by Forsberg and colleagues
(Okabe et al., 1996), using
antibody directed against Smad2 (Abcam) or Nanog (R&D Systems). Enrichment
was measured by quantitative real-time PCR using SYBR green (SensiMix
Quantace). Results were normalised against control region H located in the
3' untranslated region of the Nanog gene
(Fig. 1D) and are expressed as
±s.d. from three experiments. Previous studies have described the
location of the Smad2/3-binding regions in the promoter of Lefty
(Besser, 2004) and of Smad7
(Denissova et al., 2000).
Mutation of Smad2/3-binding sites
Potential Smad-binding sites in the Nanog6 promoter construct were
mutated using the Quick Change II Mutagenesis Kit (Stratagene) following the
manufacturer's instructions. Constructs were sequenced to confirm the presence
of the desired mutation and to check the integrity of the promoter sequence.
The sequence of the Smad2/3-(1) binding site was mutated from AGAC to GGCC
(-310 to -307) and the sequence of the Smad2/3-(2) binding site from AGAC to
GGCC (-302 to -299).
Generation of hESCs with stable knockdown of Nanog
Five shRNA-Nanog expression vectors (Sigma, SHGLY-NM_024865) and one
shRNA-non-targeting expression vector (Sigma, SC001) were stably transfected
into H9 hESC lines in CDM supplemented with Activin and FGF2. After selection,
60 puro-resistant colonies were picked (10 colonies for each shRNA-Nanog and
10 colonies for the shRNA-Non-Targeting control) and each sub-line was
screened for the expression of Nanog using immunostaining analyses
(Fig. 3B). Importantly, absence
of Nanog protein was not observed in any of the 10 sub-lines expressing the
shRNA-non-targeting control. Knockdown of Nanog expression was confirmed by
real-time PCR in 12 shRNA-Nanog-hESCs sub-lines with two randomly chosen
shRNA-non-targeting-hESCs used as controls
(Fig. 3A).
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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),
and induced a marked increase in early markers of neuroectoderm
differentiation, including GBX2, HOXA1, OLIG3 and SIX1
(Fig. 1A; see also Table S1 in
the supplementary material). More surprisingly, the expression of
NANOG was strongly downregulated in the absence of Activin/Nodal
signalling, whereas the expression of OCT4 (POU5F1 - Human
Gene Nomenclature Database) and SOX2 did not change as substantially
(Fig. 1A). These observations
were confirmed by real-time PCR analyses showing that the expression of
NANOG was downregulated by 75% after 24 hours of SB431542 treatment,
similar to the decrease in expression of NODAL and LEFTYA
(see Fig. S1A in the supplementary material). By contrast, the decrease in
OCT4 expression occurred more progressively, while the expression of
SOX2 remained relatively constant (Fig. S1A in the supplementary
material). Interestingly, normal levels of NANOG, NODAL and Lefty
transcripts could be re-established in hESCs by adding Activin after SB431542
treatment (see Fig. S1B in the supplementary material). Similar results were
obtained either in the presence or absence of cycloheximide, indicating that
reactivation of NANOG transcription by Activin/Nodal signalling did
not require de novo protein synthesis. Consistent with the marked
downregulation of NANOG expression resulting from the inhibition of
Smad2/3-mediated TGFβ signalling, overexpression of SMAD3 in hESCs
resulted in a doubling of NANOG transcription. Conversely, expression
of a dominant-negative form of SMAD3 resulted in a 10-fold decrease in
NANOG transcription (see Fig. S1C in the supplementary material).
Finally, similar results were obtained in mEpiSCs (Fig. S1D,E in the
supplementary material). Taken together, these results show that Activin/Nodal
signalling is necessary for the expression of NANOG in hESCs, and that this
requirement is evolutionarily conserved in pluripotent cells derived from the
epiblast of post-implantation mouse embryos.
Smad2/3 proteins directly control Nanog expression in hESCs
To distinguish between the hypotheses of direct transcriptional regulation
of Nanog by Smad2/3 and action through an unknown intermediate, we
looked for functional SMAD2/3-binding sites in the human NANOG
promoter. We first determined that a 379-bp region located upstream of the
NANOG ATG was sufficient to recapitulate the transcriptional activity
induced by Activin/Nodal signalling (Fig.
1B). Interestingly, this region contains binding sites for OCT4,
SOX2 and Nanog (Boyer et al.,
2005), and also two consensus Smad2/3-binding sites [S2/3-(1) and
S2/3-(2), see Fig. S1F in the supplementary material]. Mutation of these
Smad2/3-binding sites revealed that the site nearest to the Nanog-binding
site, S2/3-(2), was crucial for the transcriptional activity induced by
Activin/Nodal signalling (Fig.
1C, see also Fig. S1G in the supplementary material), suggesting
that this Smad2/3-binding site is functional. Interestingly, sequence
alignment of the human NANOG promoter region to mouse, dog and cow
equivalents revealed that the mouse promoter does not contain similar
Smad2/3-binding sites (Fig. S1F in the supplementary material). This provides
a further indication that the location of binding sites for highly
evolutionarily conserved transcription factors can vary between humans and
mice (Odom et al., 2007).
Finally, chromatin immunoprecipitation (ChIP) assays were performed to
identify genomic regions bound by SMAD2/3 in the NANOG promoter
(Fig. 1D). These analyses
showed that Smad2/3 binds the same genomic region (containing the putative
Smad2/3-binding sites) that was identified using luciferase assays. Taken
together, these results reinforce the recent study by Thomson and colleagues
(Xu et al., 2008), which
showed that NANOG expression is directly controlled by SMAD2/3 in hESCs. The
similar dependence of Nanog transcription on Smad2/3 in hESCs and
mEpiSCs suggests that humans and mice share this direct link between
extracellular growth factors and the core controlling transcriptional network
despite their distinct genomic organisation.
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)
expressing SOX1, PAX6, SOX2, HOXA1, GBX2 and NCAM
(Fig. 2A,B; Fig. S2E in the
supplementary material; our unpublished results). By contrast, NANOG-hESCs
grown under the same conditions did not undergo neuroectoderm differentiation,
as shown by the expression of the pluripotency markers OCT4 and
TRA-1-60, and by the absence of NCAM, SOX1, PAX6, HOXA1 and
GBX2 expression (Fig.
2A-C; see also Fig. S2E in the supplementary material). In
addition, FGF5 was not detected in NANOG-hESCs grown in the absence
of Activin/Nodal signalling, thereby excluding the possibility that NANOG
could block the differentiation of hESCs at a primitive ectoderm-like stage in
these culture conditions (Fig. S2E in the supplementary material)
(Darr et al., 2006).
Interestingly, NANOG-overexpressing cells could be grown for prolonged periods
in the presence of SB431542 while maintaining the expression of pluripotency
markers and the absence of SOX1 and PAX6
(Fig. 2A). These findings show
that NANOG overexpression was sufficient to substitute for Activin/Nodal
signalling in the maintenance of hESC pluripotency. Similar results were
obtained with Nanog-overexpressing mEpiSCs (see Fig. S3A,B in the
supplementary material). These results were also confirmed by growing hESCs as
embryoid bodies (EBs), showing that NANOG inhibited neural development
regardless of the culture conditions used to induce differentiation (Fig.
S4A-C in the supplementary material). Finally, overexpression of OCT4 or SOX2
in hESCs was unable to prevent the neuroectoderm differentiation of hESCs
grown in the presence of SB431542 (data not shown), demonstrating that the
ability to inhibit hESC differentiation was unique to Nanog and was not shared
by other components of the core pluripotency transcriptional circuit. Taken
together, these results indicate that among the effectors of pluripotency
downstream of Activin/Nodal signalling, Nanog expression is sufficient to
block neuroectoderm differentiation in both hESCs and mEpiSCs.
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) led
us to determine whether this effect was related to NANOG. Accordingly, we
knocked down NANOG in hESCs by stably expressing a short hairpin RNA (shRNA)
directed against NANOG transcripts (see Materials and methods).
Immunostaining and real-time PCR analyses showed that NANOG expression was
reduced by 90% (Fig. 3A-C),
which represents a decrease in NANOG expression similar to that induced by the
inhibition of Activin/Nodal signalling in hESCs (see
Fig. 1B). OCT4
expression was also decreased by 60%, whereas SOX2 expression was
maintained at normal hESC levels (Fig.
3C). Importantly, shNanog-hESCs were able to proliferate almost
indefinitely in CDM supplemented with Activin and FGF2 while maintaining a low
level of OCT4 expression (Fig.
3D,E), confirming that Nanog is not necessary for self renewal
(Chambers et al., 2007). The
analysis of markers for extraembryonic tissues and for each of the three
primary germ layers showed that the decrease in NANOG expression did not
induce increases in primitive endoderm (SOX7, GATA6), trophectoderm
(HAND1, eomesodermin) or mesendoderm (brachyury, eomesodermin,
MIXL1, SOX17) markers (Fig.
3C). Notably, these experiments were performed in CDM containing
Activin and FGF2, which does not possess any BMP-like activity
(Vallier et al., 2005) that
could interfere with the outcome of Nanog knockdown or could induce
extraembryonic tissue differentiation. In striking contrast, NANOG knockdown
increased the expression of neuroectoderm markers (SOX1, SIX1, GBX2, OLIG3,
HOXA1; see Fig. 3C-E), showing
that NANOG plays a key role in preventing neuroectoderm differentiation of
hESCs. Importantly, NANOG knockdown in hESCs resulted in increases in
neuroectoderm marker expression (GBX2, HOXA1 and SOX1) that were similar to
those observed following pharmacological inhibition of Activin/Nodal
signalling (Fig. 2A; Fig. S2E
in the supplementary material). Taken together, these observations indicate
that NANOG is responsible for inhibiting the neuroectoderm differentiation of
hESCs, and that it mediates the effects of Activin/Nodal signalling in
achieving this important element of pluripotency.
Nanog blocks the expression of neuroectoderm markers induced by FGF2 through mechanisms independent of BMP signalling
FGF signalling has been shown to be necessary for inducing neuroectoderm
specification in amphibian and chick embryos
(Stern, 2005), whereas BMP
signalling is known to inhibit the same differentiation
(Munoz-Sanjuan and Brivanlou,
2002). In principle, Nanog could block neuroectoderm
differentiation by interfering with an FGF inductive effect or by enhancing a
BMP inhibitory effect. To distinguish between these two possibilities,
wild-type hESCs and Nanog-overexpressing hESCs were grown in CDM supplemented
with SB431542 and FGF2, with SB431542 and SU5402 (a chemical inhibitor of FGF
receptors), with SB431542, FGF2 and BMP4, or with SB431542, FGF2 and Noggin
(an inhibitor of BMPs; see Fig.
2D). The inhibition of FGF signalling resulted in a decrease in
GBX2, SOX2, SOX1, PAX6, OLIG3 and HOXA1 expression in
wild-type cells grown in the presence of SB431542, confirming that FGF
signalling is necessary for the neuroectoderm specification of hESCs
(Fig. 2D). Importantly,
addition of BMP4 completely inhibited the expression of neuroectoderm markers
(Fig. 2D, data not shown),
confirming the inhibitory effect of BMP signalling on neuroectoderm
specification. However, the presence of BMP4 did not maintain pluripotency (as
shown by the decrease in OCT4 expression, see
Fig. 2D), but instead drove the
differentiation of wild-type hESCs and NANOG-hESCs into extraembryonic tissues
(see below). These results demonstrate that BMP signalling is capable of
blocking the neuroectoderm specification of hESCs but that this occurs only by
promoting differentiation along the extraembryonic pathway. Moreover, the
inhibition of BMPs using Noggin did not induce the expression of neuroectoderm
markers in NANOG-hESCs (Fig.
2D) grown in the absence of Activin/Nodal signalling. These
observations exclude the possibility that Nanog can block neuroectoderm
differentiation through mechanisms involving BMP signalling. We then
determined whether the induction of neuroectoderm markers induced by the
knockdown of NANOG in hESCs was dependent on FGF signalling by growing
shNanog-hESCs in CDM containing Activin and SU5402. The inhibition of FGF
signalling strongly decreased the expression of SOX2, GBX2, SOX1, PAX6,
HOXA1 and OLIG3 (Fig.
3E), confirming that FGF signalling is necessary for the
expression of these neuroectoderm markers. However, the inhibition of FGF
signalling did not restore pluripotency markers
(Fig. 3E), confirming that FGF
signalling is also involved in mechanisms controlling the pluripotency of
hESCs. Taken together, these results demonstrate that Nanog is necessary to
block the expression of neuroectoderm markers induced by FGF signalling, which
is also necessary for hESC self renewal.
Constitutive expression of Nanog is unable to prevent extraembryonic differentiation
Genetic studies in the mouse have shown that the function of Nanog in mESCs
and in the pre-implantation embryo is to block extraembryonic endoderm
differentiation (Mitsui et al.,
2003). To examine whether this function was conserved in hESCs, we
analysed the effect of NANOG overexpression on extraembryonic differentiation
induced by BMP4 (Xu et al.,
2002) (our unpublished results). After 7 days of culture in the
presence of BMP4, NANOG-hESCs adopted a homogeneous, broad cellular morphology
typical of extraembryonic differentiation induced in these conditions (data
not shown). BMP4-treated NANOG-hESCs became OCT4 negative
(Fig. 4A,B; see also Fig. S5A-C
in the supplementary material), indicating that Nanog was not sufficient to
maintain pluripotency in the presence of BMP4. These observations were
confirmed by FACS analyses determining the proportion of undifferentiated
hESCs in culture before and after BMP4 treatment. In control conditions, 90%
of both wild-type and NANOG-hESCs were positive for the pluripotency marker
TRA-1-60 (see Fig. S5B in the supplementary material), whereas after 7 days of
BMP4 treatment only 25-35% of either wild-type or NANOG-hESCs remained
positive for TRA-1-60 (Fig. S5B in the supplementary material). In addition,
expression of markers for primitive endoderm (SOX7, GATA4, GATA6, AFP,
H19) and trophectoderm (CDX2, EOMES, HAND1, hCG)
could be detected in Nanog-expressing cells, as well as in wild-type cells
(Fig. 4B, see also Fig. S5C in
the supplementary material). Immunostaining analyses showed that NANOG and
these markers (CDX2, EOMES, GATA4, GATA6) were co-expressed in the same cells
(Fig. 4A), further
demonstrating that Nanog did not block extraembryonic differentiation.
Importantly, NANOG-hESCs also differentiated into cells expressing
extraembryonic markers when induced to form EBs in a medium containing FBS
(see Fig. S6A,B in the supplementary material). The extraembryonic outcome was
thus independent of the culture system used to induce the differentiation of
NANOG-hESCs. Finally, similar results were obtained with Nanog-mEpiSCs grown
in CDM in the presence of BMP4 (see Fig. S5D,E in the supplementary material).
Taken together, these results show that Nanog functions specifically to
prevent neuroectoderm differentiation, rather than acting as a general blocker
of differentiation.
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90%
of cells) express SOX17, CXCR4, MIXL1 and GSC
(Fig. 5A-C; data not shown).
Gene expression profiling analysis of hESCs cultured under these conditions
confirms that they express a large number of known definitive endoderm
markers, including GSC, LIM1, GATA4, GATA6 and FOXA2,
whereas the expression of extraembryonic tissue markers (SOX7, AFP)
and neuroectoderm markers (SOX1, SOX2) cannot be detected (data not
shown). hESCs constitutively expressing NANOG that were grown in these
conditions maintained the expression of OCT4 at levels similar to those in
pluripotent cells while showing a limited induction of expression of endoderm
markers (GSC, MIXL1, SOX17; see Fig.
5A,B). In addition, FACS analysis revealed that 90% of the
wild-type cells expressed the definitive endoderm marker CXCR4 after
differentiation compared with only 30% of the NANOG-expressing cells,
suggesting that constitutive expression of NANOG could deter endoderm
differentiation (Fig. 5C).
However, the expression of SOX2 was strongly diminished in NANOG-hESCs subject
to this protocol, whereas expression of the mesendoderm markers brachyury and
eomesodermin was induced (Fig.
5B). Immunostaining analyses confirmed that NANOG protein was
systematically co-expressed in the same cells as brachyury
(Fig. 5A), indicating that
NANOG did not prevent the onset of expression of mesendoderm markers but
suggesting that it was able to interfere with further progression to
definitive endoderm. This interpretation is not contradicted by the
maintenance of OCT4 expression in NANOG-hESCs, as Oct4 (like Nanog) is also
expressed in the mesendoderm of gastrulating mouse embryos
(Hart et al., 2004).
Furthermore, permissiveness for mesendoderm but not endoderm differentiation
in NANOG-hESCs is also reinforced by the induction of the mesendoderm marker
PDGFR and by the relatively low number of NANOG cells expressing CXCR4
(Fig. 5C). Finally, similar
results were observed when Nanog-mEpiSCs were grown in endoderm-inducing
culture conditions (see Fig. S7A,B). Taken together, these results indicate
that rather than preventing mesendoderm specification in hESCs and mEpiSCs,
Nanog limits the progression of mesendoderm progenitors towards definitive
endoderm cells.
Nanog binds Smad2/3 in hESCs and modulates activity of the Activin/Nodal signalling pathway
Studies in amphibians and in mice have shown that high activity of
Activin/Nodal signalling is necessary to specify the endoderm germ layer
(Dunn et al., 2004). In
addition, BMP signalling has been shown to be essential for mesendoderm
specification (Davis et al.,
2004; Fujiwara et al.,
2002). Therefore, the effect of Nanog on mesendoderm progression
could involve modulating the activity of these signalling pathways. To address
this hypothesis, NANOG-overexpressing cells were grown in culture conditions
inductive for endoderm differentiation in the presence of increasing doses of
Activin, BMP or FGF2 (Fig. 5D).
A high dose of Activin, BMP or FGF was not sufficient to restore normal levels
of endoderm markers (SOX17, GSC) in NANOG-hESCs, while expression of
the mesendoderm marker brachyury was maintained in all these conditions
(Fig. 5D). These observations
show that an increase in extracellular factors cannot bypass the inhibitory
effect of Nanog, and thus that Nanog could interfere directly with
intracellular components of the Activin or BMP pathways. Interestingly, Nanog
has been shown to interact directly with Smad1 to modulate mouse ESC
differentiation (Suzuki et al.,
2006). However, we have been unable to detect any direct
interaction between Smad1 and Nanog in hESCs (data not shown), and we have
shown that Nanog overexpression does not block the inductive effects of BMP
extraembryonic differentiation. Therefore Nanog is unlikely to effectively
regulate hESC or mEpiSC differentiation through direct interaction with Smad1,
as has been reported for mESCs (Suzuki et
al., 2006). However, Nanog protein itself shares homology with the
common (C-) Smad4 protein (Hart et al.,
2004) and thus Nanog might interact with Smad2/3. To test this
hypothesis, the SBE4 luciferase reporter
(Jonk et al., 1998) for
Activin/Nodal-induced, Smad2/3-mediated signalling was transfected into hESCs
(with or without SMAD2/3 or/and NANOG expression vectors) in the presence of
increasing doses of Activin. This analysis showed that overexpression of NANOG
decreased (but did not abolish) the transcriptional activity associated with
increasing doses of Activin and with increased levels of SMAD2 (data not
shown) or SMAD3 (Fig. 5E). This
result suggests that NANOG overexpression is able to modulate the
Activin/Nodal signalling pathway in hESCs. Interestingly, a similar
Nanog-mediated modulation of Smad3-induced transcriptional activity was
observed in mEpiSCs (see Fig. S8A in the supplementary material). Together,
these observations suggest the existence of a negative-feedback loop in which
Activin/Nodal-induced Nanog limits the transcriptional activity induced by
Activin/Nodal signalling. To understand the mechanism of this interaction, we
performed co-immunoprecipitation of NANOG or SMAD2/3, followed by western blot
analyses. These revealed that the protein complexes containing NANOG also
contained SMAD2/3 proteins (Fig.
3F). Importantly, these experiments were performed on nuclear
extracts, and addition of SB431542 to the medium blocked the Smad2/3-Nanog
interaction. This suggests that Nanog-complexed Smad2/3 must be phosphorylated
and thus located in the nucleus, where it could affect the transcription of
target genes. Similar Smad2/3-Nanog interactions were observed in mEpiSCs (see
Fig. S8B in the supplementary material), suggesting that they are
evolutionarily conserved. Finally, ChIP analyses showed that NANOG and SMAD2/3
proteins were capable of binding to the same genomic region in four different
target genes of Activin/Nodal signalling, including LEFTYA, SMAD7,
SnoN (SKIL - Human Gene Nomenclature Database) and
NANOG itself (Fig. 1D,
see also Fig. S1H in the supplementary material, data not shown), suggesting
that the protein complexes containing NANOG and SMAD2/3 could regulate the
promoters of target genes in hESCs. Interestingly, constitutive expression of
NANOG during mesendoderm differentiation of hESCs limits the upregulation of
known Smad2/3 target genes (LEFTYA and NODAL) to levels
equivalent to those in hESCs (Fig.
5B). Consequently, the negative-feedback loop involving
Activin/Nodal-mediated Nanog expression could act by limiting, but not
entirely blocking, the transcriptional activity of Smad2/3.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). Their
work showed that suppression of Nanog expression was not sufficient to induce
full differentiation of mESCs, and suggested that Nanog acts to limit the
probability of differentiation into multiple lineages induced by extracellular
signals. Our results showing that Nanog blocks neuroectoderm differentiation
in hESCs and mEpiSCs indicate a more lineage-specific role for Nanog than that
in mESCs. Indeed, Nanog overexpression blocked neither mesendoderm
differentiation nor BMP-induced extraembryonic differentiation in hESCs and
mEpiSCs. Interestingly, the neuroectoderm markers analysed in our study have
been shown to be direct target genes of Nanog in hESCs (SIX1, GBX2, PAX6,
HOXA1, OLIG3) (Boyer et al.,
2005), suggesting that Nanog could block the activity of
transcription factors downstream of FGF signalling (as it does with Smad2/3
for Activin/Nodal signalling). Together, these observations suggest that Nanog
acts in hESCs by blocking differentiation induced by signalling pathways that
are also necessary for hESC pluripotency and self renewal (i.e. Activin/Nodal
and FGF).
Therefore, the generalised function of Nanog in pluripotent stem cells is
to safeguard pluripotency against the differentiation-inducing effects of
essential extracellular signals. Importantly, however, Nanog does not protect
hESCs, mEpiSCs and mESCs against the same differentiative events. Indeed,
gain- and loss-of-function studies have shown that Nanog primarily blocks
primitive endoderm differentiation of mESCs in vitro
(Hamazaki et al., 2004) and of
mouse inner cell mass in vivo (Mitsui et
al., 2003). Conversely, Nanog blocks neuroectoderm and definitive
endoderm differentiation of hESCs and of mEpiSCs. In vivo studies reinforce
our results, as mouse embryos mutant for Nanog fail to develop beyond the late
epiblast stage (Hamazaki et al.,
2004), when Nanog expression in the epiblast becomes dependent on
Nodal signalling (Mesnard et al.,
2006; Mitsui et al.,
2003). In addition, absence of Nodal expression and consequently
of Nanog expression in post-implantation mouse embryos results in
neuralisation of the epiblast, suggesting that Nanog acts in vivo to prevent
neuroectoderm differentiation of the mouse epiblast before gastrulation
(Camus et al., 2006), thus
reinforcing our findings on its role in hESCs and mEpiSCs. The apparent
similarity in the role of Nanog in hESCs, mEpiSCs and the pluripotent epiblast
(and the dissimilarity of its role in mESCs) reinforces the hypothesis that
hESCs and mESCs represent distinct stages of early mammalian development
(Brons et al., 2007;
Tesar et al., 2007). In this
hypothesis, hESCs closely resemble pluripotent cells from post-implantation
stages in vivo, in contrast to mESCs, which closely resemble pluripotent cells
from the inner cell mass (Nichols et al.,
2001). This hypothesis also implies that the functions of
pluripotency factors change progressively during early mammalian development.
Before implantation, the core pluripotency transcription factor circuit blocks
formation of the extraembryonic lineages, whereas after implantation it blocks
formation of the primary germ layers.
In this context, a recent study by Smith and colleagues
(Ying et al., 2008) showed
that general repression of differentiation signals by small molecules results
in a `ground state of pluripotency' in mouse ESCs. This model does not appear
to apply to hESCs and mEpiSCs, which strictly depend on Activin/Nodal
signalling to maintain the expression of Nanog and thereby to maintain their
pluripotent state. Therefore, two or more distinguishable pluripotent states
relying on different growth factors but on similar core transcriptional
networks appear to exist in vitro and in vivo during embryonic development.
Interestingly, we recently observed that human induced pluripotent stem cells
rely, like hESCs, on Activin/Nodal signalling to maintain their pluripotent
state (our unpublished results). Therefore, understanding the function of
pluripotency factors in each of these pluripotent states will be crucial to
achieving control over the differentiation of human pluripotent cells, whether
derived from mammalian embryos or by inducing pluripotency in cells of somatic
origin (Takahashi et al.,
2007; Yu et al.,
2007).
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/8/1339/DC1
|
Footnotes |
|---|
Present address: Department of Craniofacial Development, King's College
London, London SE1 9RT, UK 
Present address: Hospital for Sick Children, Toronto Medical Discovery
Tower, Toronto, Ontario M5G 1L7, Canada
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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