DEVELOPMENT AND STEM CELLSRESEARCH ARTICLE
During early mammalian development, two extra-embryonic
lineages, trophectoderm (TE) and primitive endoderm (PrE), are
specified and segregated away from the pluripotent epiblast (EPI).
Extra-embryonic tissues serve to pattern the early embryo, as well
as providing the essential maternal-fetal connection required to
sustain the embryo after implantation.
Stem cell lines that retain properties of each of the three lineages,
TE, PrE and EPI, of the implanting (late) blastocyst can be derived
and propagated in culture. Embryonic stem (ES) cells represent the
EPI (Evans and Kaufman, 1981; Martin, 1981), whereas extra-
embryonic endoderm (XEN) cells represent the PrE (Kunath et al.,
2005), and trophoblast stem (TS) cells represent the TE (Tanaka et
al., 1998). These embryo-derived cell types serve as models for
investigating the mechanisms that regulate lineage specification,
commitment and maintenance. Lineage choice is governed by the
expression of key lineage-specific transcription factors that regulate
downstream signaling pathways (reviewed by Arnold and
Robertson, 2009; Niwa, 2007; Rossant and Tam, 2009), such that
ES cells can be converted into TS-like (Niwa et al., 2005) or XEN-
like (Shimosato et al., 2007) cells simply by misexpression of
appropriate transcription factors.
In the mouse embryo, commitment to the PrE lineage occurs
prior to overt differentiation of the PrE. This model is supported by
the salt-and-pepper distribution of PrE markers in cells within the
inner cell mass (ICM) of early blastocyst stage embryos (Chazaud
et al., 2006; Plusa et al., 2008). A number of transcription factors,
including members of the GATA, SOX and HNF families, are
expressed by the PrE and its derivatives, as well as by ES cells that
are directed to differentiate into extra-embryonic endoderm (ExEn).
Loss- and gain-of-function studies have demonstrated that these
transcription factors play key roles in ExEn lineage specification,
maintenance and differentiation. FGF/MAPK signaling has been
shown to regulate Gata6 expression, and so may function upstream
of these transcription factors (Arman et al., 1998; Chazaud et al.,
2006; Feldman et al., 1995; Goldin and Papaioannou, 2003;
Nichols et al., 2009; Yamanaka et al., 2010).
Although the mechanisms of PrE specification have been
relatively well studied, little is known about the signaling pathways
regulating ExEn lineage expansion and differentiation towards PrE
derivatives: visceral endoderm (VE) and parietal endoderm (PE).
Higher vertebrates have two platelet-derived growth factor
receptors, PDGFR and PDGFR, which form homo- and
heterodimers, and at least four PDGF ligands (reviewed by Andrae
et al., 2008; Hoch and Soriano, 2003). Owing to this complexity,
the effects of PDGF signaling on early development, especially
early lineage specification and expansion, have not been
comprehensively investigated. We have identified PDGFR as a
marker of the PrE lineage of the blastocyst and its ExEn
derivatives, in both embryos and in ex vivo paradigms of the ExEn
lineage. From investigating the relationship between PDGFR and
the PrE lineage-determining transcription factors, we propose a
model whereby initiation of Pdgfra expression requires GATA6
and its maintenance requires GATA4 and GATA6.
Development 137, 3361-3372 (2010) doi:10.1242/dev.050864
© 2010. Published by The Company of Biologists Ltd
1Developmental Biology Program, Sloan-Kettering Institute, 1275 York Avenue,
Box 371, New York, NY 10065, USA. 2Institut Pasteur, CNRS URA2578,
Mouse Functional Genetics, 75015 Paris, France.
*Author for correspondence (firstname.lastname@example.org)
Accepted 10 August 2010
The inner cell mass (ICM) of the implanting mammalian blastocyst comprises two lineages: the pluripotent epiblast (EPI) and
primitive endoderm (PrE). We have identified platelet-derived growth factor receptor alpha (PDGFR) as an early marker of the
PrE lineage and its derivatives in both mouse embryos and ex vivo paradigms of extra-embryonic endoderm (ExEn). By combining
live imaging of embryos and embryo-derived stem cells expressing a histone H2B-GFP fusion reporter under the control of Pdgfra
regulatory elements with the analysis of lineage-specific markers, we found that Pdgfra expression coincides with that of GATA6,
the earliest expressed transcriptional regulator of the PrE lineage. We show that GATA6 is required for the activation of Pdgfra
expression. Using pharmacological inhibition and genetic inactivation we addressed the role of the PDGF pathway in the PrE
lineage. Our results demonstrate that PDGF signaling is essential for the establishment, and plays a role in the proliferation, of
XEN cells, which are isolated from mouse blastocyst stage embryos and represent the PrE lineage. Implanting Pdgfra mutant
blastocysts exhibited a reduced number of PrE cells, an effect that was exacerbated by delaying implantation. Surprisingly, we
also noted an increase in the number of EPI cells in implantation-delayed Pdgfra-null mutants. Taken together, our data suggest a
role for PDGF signaling in the expansion of the ExEn lineage. Our observations also uncover a possible role for the PrE in
regulating the size of the pluripotent EPI compartment.
KEY WORDS: Blastocyst, Mouse embryo, Epiblast, Primitive endoderm, Extra-embryonic endoderm, Embryonic stem (ES) cell, XEN cell,
PDGF signaling, GATA
A role for PDGF signaling in expansion of the extra-
embryonic endoderm lineage of the mouse blastocyst
Jérôme Artus1, Jean-Jacques Panthier2and Anna-Katerina Hadjantonakis1,*
Using pharmacological inhibition and genetic inactivation we
addressed the role of PDGF receptor signaling in the ExEn. Our
results suggest that the PDGF pathway exerts a mitogenic effect on
XEN cells, and that this activity is mediated intracellularly by
MEK and PKC signaling. We also noted that implanting Pdgfra
mutant blastocysts exhibit a reduction in the number of PrE cells,
an effect exacerbated by delaying implantation, suggesting a role
for PDGF signaling in expansion/maintenance of the PrE. In
addition, implantation-delayed mutant blastocysts exhibited an
increase in EPI cell number, uncovering a previously unrecognized
role for the PrE in regulating the size of the EPI compartment.
MATERIALS AND METHODS
Embryo collection and in vitro culture
Mice were maintained on a mixed genetic background (129/B6/ICR).
Embryos were obtained from ICR or PdgfraH2B-GFP/+females mated with
PdgfraH2B-GFP/+males (Hamilton et al., 2003). Blastocysts were recovered
in M2 media (Chemicon) and cultured for 1-3 days in ES cell media on
0.1% gelatin-coated chambered coverglass slides (Lab-Tek) at 37°C in 5%
CO2. Decidua were dissected from uteri in D-MEM/F-12 (Gibco)
containing 5% newborn calf serum. Postimplantation embryos were
processed for sectioning within decidua. Diapause was induced following
intraperitoneal injection of 10 g tamoxifen (Sigma) and 2-3 mg
progesterone (Abraxis) at E2.5. Embryos were recovered 2-3 days later.
ES/XEN cell culture
ES cells were maintained on mitomycin C-treated primary murine
embryonic fibroblasts (MEFs) in recombinant leukemia inhibitory factor
(LIF) (Mereau et al., 1993) under standard conditions (Nagy et al., 2003).
PdgfraH2B-GFP/+XEN cells were routinely cultured on gelatin coated-dishes
in ES cell media in the absence of LIF and feeders and passaged every 2
days at a 1 in 5 dilution.
Inactivation of Pdgfra in XEN cells
Deletion of the floxed allele in PdgfraH2B-GFP/fl, ROSA26CreERT2/+XEN cell
line was induced either by addition of 4-hydroxytamoxifen (4-OHT,
Sigma) or infection with a self-excising Cre-expressing retrovirus (Silver
and Livingston, 2001) (following the protocol of L. Le Cam, Institut de
Rechercher en Cancérologie de Montpellier, France).
ES/XEN cell line isolation
Blastocysts were collected from matings using KitW-lacZ/+(Bernex et al.,
1996), PdgfraH2B-GFP/+(Hamilton et al., 2003), Pdgfrafl/fl(Tallquist and
Soriano, 2003) and ROSA26CreERT2/CreERT2(Cheng et al., 2010) alleles.
Blastocysts were cultured individually for 5 days in 4-well plates on MEFs
in ES cell media containing LIF. ICMs were dissociated in 0.25% trypsin-
EDTA, and passaged into 24-well dishes containing MEFs. Media were
changed every 2 days. Cells were passaged 7-10 days later into fresh 24-
well plates until confluency, then cultured in the absence of MEFs and
genotyped by PCR.
ES cell differentiation assays
ES cells were maintained on gelatin in the absence of MEFs prior to
differentiation. PdgfraH2B-GFP/+cells (Hamilton et al., 2003) were plated at
2?105cells per 35-mm dish containing gelatin-coated glass coverslips.
Next day, ES cell media were replaced with LIF-free media containing
10% FCS and 1 M trans-retinoic acid (Sigma). Media were changed daily.
Transfections of plasmids pCMV5-Flag (gift of M. E. Donohoe, Cornell
University, NY, USA), pCMV-Tag2 Gata4 and pCMV-Tag2 Gata6 (gift of
Y. Hayashi, Nagoya University, Japan) were carried out using
Lipofectamine 2000 (Invitrogen). Embryoid bodies were generated by
plating 1?106cells onto a 10-cm Petri dish (VWR) with culture in LIF-
free medium containing 10% FBS. Media were changed every 2 days.
XEN cells were plated at 1?104-105cells per well of a 24-well plate
(Falcon). Media were changed daily and counts were performed in triplicate.
Inhibitor compounds used were: Gleevec (gift of P. Besmer, Sloan-Kettering
Institute, NY, USA) and bisindolylmaleimide (BIS), LY294002 and U0126
(all from Cell Signaling). Recombinant human PDGF-AA (R&D Systems)
and mouse SCF proteins (R&D Systems) were added at 10 ng/ml and 20
ng/ml, respectively, to N2B27 medium (Ying and Smith, 2003).
Blastocysts and cells cultured on coverslips were immunostained as
previously described (Artus et al., 2005). Primary antibodies used were:
CDX2 (Biogenex), FOXA2 (1:400, Abcam), GATA4 (1:300, Santa Cruz),
GATA6 (1:100, R&D Systems), Ki67 (1:400, Vector Laboratories), NANOG
(1:700, Cosmo Bio), OCT4 (1:200, Santa Cruz), PDGFR (1:100,
eBioscience), SOX7 (1:100, R&D Systems) and SOX17 (1:400, R&D
Systems). Alexa Fluor-conjugated secondary antibodies (Invitrogen) were
used at 1:200. DNA was counterstained with Hoechst 33342 (1:200,
Invitrogen). Blastocysts were genotyped after analysis by PCR (Artus et al.,
2005) using the primers detailed in Fig. 5B and Table S1 in the
supplementary material. Coverslips were mounted in Vectashield (Vector
Laboratories). Decidua-containing embryos were fixed overnight in 4%
paraformaldehyde (PFA) at 4°C, embedded in PBS containing 4% agarose
and 5% sucrose and sectioned at 200 m using a vibrating microtome
(VT1000S, Leica). F-actin was visualized with Alexa Fluor-phalloidin
(1:500, Invitrogen). Embryoid bodies were fixed overnight in 4% PFA at
4°C, cryoprotected in PBS containing 30% sucrose, embedded in O.C.T.
compound (Tissue-Tek) and sectioned at 12 m using a cryostat (CM3050S,
Image acquisition and processing
Laser-scanning confocal data were acquired on a Zeiss LSM510 META.
Fluorescence was excited with a 405 nm diode laser (Hoechst), a 488 nm
argon laser (GFP) or a 543 nm HeNe laser (Alexa Fluor 546, 568). Images
were acquired using Plan Apochromat 20?/NA0.75, Plan Neofluar
40?/NA1.3 or Plan Apochromat 63?/NA1.4 objectives. Optical sections
ranged from 0.38-4 m. Raw data were processed using Zeiss AIM (Carl
Zeiss Microsystems), IMARIS 6.3.1 (Bitplane), Photoshop CS2 and
Illustrator CS2 (Adobe).
RT-PCR and qPCR
Total RNA was extracted using TRIzol (Invitrogen), reverse transcribed
using the Superscript III First-Strand Synthesis Kit (Invitrogen) and 50 ng
of the RNA used as template for PCR amplification. Quantitative (q) PCR
was performed using SYBR Green (Roche) on a LightCycler 480 real-time
PCR instrument and analyzed with LightCycler 480 software (version
220.127.116.11). For sequences of primers and cycling conditions, see Table S1
in the supplementary material.
Pdgfra is expressed in the PrE and its derivatives
We have identified Pdgfra as one of the earliest markers of the PrE
lineage in the mouse blastocyst (Plusa et al., 2008). To determine
whether Pdgfra is expressed in later PrE derivatives, we investigated
the expression of the PdgfraH2B-GFPreporter in explanted blastocysts.
Under the conditions used, embryos hatched from their zona pellucida
after 1 day of culture, then attached to the dish and formed
outgrowths. The TE spread onto the bottom of the dish and eventually
differentiated into trophoblast giant cells (TGCs). Concomitantly, the
ICM expanded to produce an outgrowth (Fig. 1Aa-c). Nuclear-
localized GFP was detected within the ICM after 1 to 3 days in
culture (Fig. 1Aa?-c?). After 2 days in culture, weak GFP signal was
also detected in cells located on the bottom of the dish outside of the
outgrowth. These weakly GFP-expressing cells had large nuclei or
were bi-nucleated, and were likely to be TGCs (Fig. 1Ab?,c?).
We next confirmed that, within ICM outgrowths, GFP colocalized
with PrE-specific markers, including the transcription factors GATA4
(Fig. 1Ad) and GATA6 (Fig. 1Ae). Of the GFP-positive cells, 91%
were GATA4 positive (out of 291 GFP-positive cells, 264 were
GATA4 positive; n2 embryos) and 56% were GATA6 positive (out
Development 137 (20)
of 383 GFP-positive cells, 215 were GATA6 positive; n4 embryos).
Although the majority of GFP-expressing cells were also GATA4
positive, we noted the presence of a minor population of GFP-
positive cells that did not express GATA4. These cells could
represent later PrE derivatives or, alternatively, mesoderm
derivatives, which have been reported to express PDGFR (see Fig.
S1A2 in the supplementary material) (Orr-Urtreger and Lonai, 1992).
Notably, GFP reporter expression did not colocalize with either EPI-
or TE-specific transcription factors (Fig. 1Af,g).
Next, to determine whether Pdgfra is expressed in XEN cells,
which represent the PrE lineage, we established XEN cell lines
from the PdgfraH2B-GFPstrain. Live imaging has revealed that even
though they are clonal, XEN cells exhibit heterogeneity in their
morphology (Kunath et al., 2005). The PdgfraH2B-GFPreporter was
expressed in all cells at equivalent levels (see Fig. 4E), suggesting
that Pdgfra is homogeneously expressed in XEN cells irrespective
of morphological transitions.
We next determined that Pdgfra expression was maintained within
the PE and VE, the two PrE derivative tissues, encompassing the
period preceding and during gastrulation, from embryonic day (E)
5.5 to 7.5 (Fig. 1B and see Fig. S1 in the supplementary material).
PdgfraH2B-GFPreporter expression was downregulated at ~E6.5 in
VE cells overlying the epiblast, coincident with the onset of
gastrulation (Fig. 1Bb), such that a gradient of GFP signal was
detected along the proximal-distal axis (see Fig. S1B,B2 in the
supplementary material), as confirmed by PDGFR localization (see
Fig. S1A1 in the supplementary material). At E7.5, GFP was
visualized in the endoderm layer of the visceral yolk sac, confirming
that Pdgfra expression is maintained in this ExEn derivative (Fig.
1Be,f). These observations indicate that Pdgfra is dynamically
expressed in vivo in the PrE and its derivatives.
Pdgfra expression in ex vivo models of ExEn
We next determined whether Pdgfra expression serves as an ExEn
marker in various ES cell models of ExEn formation. These
paradigms have proved invaluable for studying diverse aspects of
ExEn biology (Coucouvanis and Martin, 1999; Fujikura et al.,
2002; Rula et al., 2007; Shimosato et al., 2007; Yang et al., 2007).
We used the PdgfraH2B-GFPreporter (Hamilton et al., 2003; Plusa
et al., 2008) to visualize Pdgfra expression in these assays.
Retinoic acid (RA) promotes the differentiation of ES cells into
various cell types, including ExEn derivatives (Soprano et al.,
2007). PdgfraH2B-GFP/+ES cells propagated in the presence of LIF,
and thus grown in an undifferentiated state, expressed neither
PDGFR nor the GFP reporter (Fig. 2Aa). However, a 4-day
treatment with 1 M RA induced the expression of both
endogenous PDGFR protein and the GFP reporter (Fig. 2Ab).
GFP was detected at low levels 2 days after RA addition (see Fig.
S2Ac,h in the supplementary material) and had increased by 3 days
(see Fig. S2Ad,i in the supplementary material). GFP-positive cells
expressed GATA4 (Fig. 2Ac) and GATA6 (Fig. 2Ad), suggesting
that they had acquired an ExEn identity. Their identity was
confirmed using additional markers, including SOX17, SOX7 and
FOXA2 (Fig. 2B). Immunodetection of AFP as a VE marker
(Dziadek and Adamson, 1978) and SPARC as a PE marker
(Holland et al., 1987; Mason et al., 1986) revealed that RA
treatment induced a heterogeneous population containing both VE-
and PE-like cells (see Fig. S3 in the supplementary material).
Ectopic transient expression of GATA4 or GATA6 in ES cells is
sufficient to induce ExEn differentiation (Fujikura et al., 2002;
Shimosato et al., 2007). Ectopic expression of either GATA4 or
GATA6 was sufficient to induce the GFP reporter (Fig. 2Ae,f),
which was detected from 15 hours post-transfection (see Fig.
PDGF signaling promotes expansion of ExEn
Fig. 1. Pdgfra is expressed in the
primitive endoderm (PrE) and its extra-
embryonic endoderm (ExEn) derivatives.
(A)Pdgfra expression during mouse
blastocyst outgrowth. (a-c)Single bright-field
(bf) optical sections of PdgfraH2B-GFP/+
blastocyst cultured in vitro for 3 days.
(a? ?-c? ?) GFP (green) is expressed in the inner
cell mass (ICM; asterisk). (b?,c?) GFP is weakly
detected in trophoblast giant cells (TGCs;
arrowheads). (d,e)After 3 days in culture,
GFP colocalizes with PrE markers GATA4 (d,
red) and GATA6 (e, red). (f,g)Mutually
exclusive expression of GFP, OCT4 (f, red) and
CDX2 (g, red) after 3 days culture. (a’-c’,d-g)
Three-dimensional projections of z-stacks.
(B)(a-d? ?) Pdgfra expression in the PrE
derivatives parietal endoderm (PE, arrows)
and visceral endoderm (VE, arrowheads) at
E5.5 (a), E6.5 (b) and E7.5 (c,d). (c,c?) Extra-
embryonic region of an E7.5 embryo. (a-c?)
Three-dimensional projections of z-stacks.
(d,d?) Magnified view of the boxed region
from c and c?. (e,f)GFP is detected in the
endoderm of the visceral yolk sac at E9.5.
(e)Orthogonal views of z-stack of an E9.5
yolk sac. (f)Three-dimensional projection of e.
White arrowhead, VE; yellow arrowhead,
mesoderm derivatives of the yolk sac. Blue,
Hoechst; green, GFP; red, F-actin. Scale bars:
S2Ba,f in the supplementary material). These data demonstrate that
activation of the PdgfraH2B-GFP/+reporter occurred concomitant
with ExEn specification.
When cultured without LIF under non-adherent conditions, ES
cells form aggregates called embryoid bodies (EBs), which
recapitulate several developmental events (Coucouvanis and
Martin, 1995; Martin et al., 1977). These include the formation of
an outer layer of cells that resembles VE (Coucouvanis and Martin,
1995; Coucouvanis and Martin, 1999). Upon EB formation at 5
days of differentiation, we noted that the outer layer of cells
expressed GATA6, but did not express the PdgfraH2B-GFPreporter
(Fig. 2Ag). Our observation that Pdgfra is downregulated in the
Development 137 (20)
Fig. 2. Pdgfra expression in ex vivo models of PrE formation is regulated by GATA6. (A)(a) PdgfraH2B-GFP/+mouse ES cells propagated in the
presence of LIF do not express PDGFR protein or GFP reporter. (b-d)Upon retinoic acid (RA) treatment, both nuclear-localized GFP and
endogenous PDGFR protein are detected (b). GFP-positive cells express GATA4 (c) and GATA6 (d). (e,f)Expression of GFP and GATA4 is detected
48 hours after ectopic expression of GATA4 (e) or GATA6 (f). Few GFP-positive cells do not express GATA4 (arrowheads) upon GATA6
misexpression. (g)Section through an embryoid body at 5 days of differentiation. Cells in the outer layer express GATA6 but not the GFP reporter.
(a,b)Single optical section. (c-g)Three-dimensional projections of the z-stacks. Blue, Hoechst; green, GFP; red, PDGFR, GATA4 or GATA6.
(B)Requirement for GATA6, but not GATA4, for Pdgfra expression upon RA treatment. (a-c)Wild-type (a), Gata4–/–(b) and Gata6–/–(c) ES cells
cultured in the presence of LIF express the pluripotency markers OCT4 and NANOG. (d-f)Upon RA treatment, ES cell differentiation is visualized by
loss of Oct4 and Nanog expression. (g-u)ExEn formation induced by RA treatment is visualized by co-expression of PDGFR, GATA4, GATA6,
SOX17, SOX7 and FOXA2. Like wild-type ES cells (left column), Gata4–/–ES cells differentiate into ExEn (middle column). In the absence of Gata6,
ES cells fail to differentiate into ExEn derivatives (right column). (a-u)Single optical sections. Blue, Hoechst; green, NANOG (a-f) or PDGFR (g-u);
red, OCT4 (a-f), GATA4 (g-i), GATA6 (j-l), SOX17 (m-o), SOX7 (p-r) or FOXA2 (s-u). Scale bars: 20m.
distal VE at E6.5 might suggest that the outer layer of cells in these
EBs resembles the distal portion of the VE that overlies the
epiblast. To further explore the identity of the surface endoderm
layer of EBs, we investigated the presence of other ExEn markers
at 3, 5 and 7 days of EB formation (see Fig. S4 in the
supplementary material). No ExEn markers were detected after 3
days of EB formation. However, by 5 days, robust localization of
GATA6, FOXA2 and SOX17 and weak localization of GATA4 and
HNF4 were observed in the outer layer of cells. After 7 days, all
ExEn markers analyzed were detected in the outer layer of cells
and their distribution appeared relatively homogeneous, except for
HNF4, which was distributed in patches. Interestingly, SOX7,
which is detected in the proximal VE overlying the extra-
embryonic ectoderm at E6.5-7.5 (Kanai-Azuma et al., 2002), was
not detected in cells in the outer layer of EBs, supporting the notion
that the outer layer of EBs resembles the distal VE overlying the
epiblast. Collectively, these observations reveal that Pdgfra is
expressed in ExEn cell types formed upon ES cell differentiation.
By contrast, Pdgfra is not expressed in the ExEn cells that
constitute the outer layer of EBs, suggesting that these cells are
likely to represent a subdomain of the VE.
GATA6 regulates Pdgfra expression
GATA4 and GATA6 are considered to be the key regulators of
ExEn identity based on the finding that their misexpression is
sufficient to direct ES cells towards an ExEn fate (Shimosato et
al., 2007), as confirmed using the PdgfraH2B-GFPreporter (Fig.
2Ae,f). However, the respective roles of these two transcription
factors in the regulation of Pdgfra expression has remained
unclear. Previous studies have shown that GATA binding sites
within cis-regulatory sequences upstream of the Pdgfra gene are
crucial for expression and can be bound by GATA4 (Wang and
Song, 1996). Having previously demonstrated that in the mouse
embryo, PDGFR and GATA6 are the earliest known markers of
the PrE lineage and that their expression precedes that of GATA4
(Plusa et al., 2008), we suggest a model whereby GATA6 is
involved in the activation of Pdgfra expression, and GATA4 and
GATA6 are involved in its maintenance. In support of such a
model, we noted that 48 hours after GATA6 misexpression in ES
cells, a small percentage of GFP-positive cells did not express
GATA4 (Fig. 2Af, arrowheads).
We analyzed the kinetics of expression of the endogenous
Gata4, Gata6 and Pdgfra genes 24 hours after forced expression
of the genes encoding to GATA transcription factors. Upon Gata4
misexpression, a small percentage of GFP-positive cells did not
express detectable levels of GATA4 (see Fig. S5A in the
supplementary material). However, all GFP-positive cells
expressed GATA6 (see Fig. S5B in the supplementary material).
By contrast, upon Gata6 misexpression, all GFP-positive cells
expressed GATA6 (see Fig. S5D in the supplementary material),
but most did not express GATA4 (see Fig. S5C in the
supplementary material, white arrowheads). These observations
suggest that activation of Pdgfra expression is preceded by, and
might require, GATA6.
Next, we reasoned that if GATA6 is an important regulator of
Pdgfra expression then, in the absence of GATA6, Pdgfra should
not be expressed. We investigated Pdgfra expression upon RA
treatment of Gata4 (Soudais et al., 1995) or Gata6 (Morrisey et al.,
1998) mutant ES cells. It has been shown that differentiation of ES
cells into ExEn induced by RA treatment requires Gata6 but not
Gata4 (Capo-Chichi et al., 2005). As expected, RA treatment of
wild-type, Gata4- and Gata6-deficient ES cells induced their
differentiation, as indicated by the absence of detectable levels of
OCT4 (POU5F1 – Mouse Genome Informatics) and NANOG
proteins (Fig. 2Bd-f). Wild-type RA-treated ES cells acquired an
ExEn identity (Fig. 2B). In Gata4-deficient differentiated cells
(Fig. 2Bh), PDGFR colocalized with GATA6 (Fig. 2Bk), SOX17
(Fig. 2Bn), SOX7 (Fig. 2Bq) and FOXA2 (Fig. 2Bt). By contrast,
Gata6-deficient ES cells (Fig. 2Bl) failed to differentiate into ExEn
and expressed neither Pdgfra nor PrE markers (Fig. 2B). These
data led us to propose that Gata6 expression is required for Pdgfra
expression, both for its initial activation and for its maintenance in
the early mouse embryo and in cell-based assays of PrE formation
(see Fig. 7).
Inhibition of RTK, MEK1/2 or PKC signaling affects
ExEn cell proliferation
Several observations suggest that receptor tyrosine kinase (RTK)
signaling mediated by MAPK is necessary for the formation of the
PrE. The most compelling data come from the analysis of GRB2
adaptor molecule-deficient embryos and EBs in which PrE fails to
form (Chazaud et al., 2006; Cheng et al., 1998). To investigate a
potential role for PDGF signaling in the PrE, we adopted a
pharmacological approach using small-molecule compounds to
inhibit signal transduction. Gleevec (also know as Imatinib)
occupies the active site of tyrosine kinases, leading to a decrease
in their activity (Carroll et al., 1997). Gleevec inhibits several
kinases, including PDGFR, KIT and ABL proteins. Addition of
Gleevec to embryos affected blastocyst outgrowth, especially the
TE and its derivatives, and so could not be used to investigate PrE
formation (data not shown). Several explanations could account for
the deleterious effects of Gleevec in early embryos. Pdgfra is
expressed in TGCs (Fig. 1Ab?-c?). Additionally, the KIT receptor
is also expressed in TGCs and a role for KIT signaling in the TE
lineage is supported by KIT ligand promotion of TE outgrowth
(Mitsunari et al., 1999).
To address the role of RTK signaling in the PrE, we analyzed
the effect of Gleevec on XEN cells. We first assayed the effect
of an increasing dose of Gleevec on the proliferation of wild-
type XEN cells (Fig. 3A). We estimated the doubling time to be
21 (control), 20 (1 M), 25 (5 M) and 31 (10 M) hours. We
then determined whether the effect of Gleevec on XEN cell
proliferation is also cell density-dependent (Fig. 3B). At 10?104
cells per well, 10 M Gleevec increased cell doubling time by
1.4-fold, whereas at 5?104and 1?104cells per well the
doubling time was increased by 2.7- and 4.2-fold, respectively.
Thus, Gleevec treatment affects XEN cell proliferation in a dose-
and cell density-dependent manner.
We next investigated whether the reduction in cell
proliferation was due to cell death, cell cycle exit, or both.
Immunodetection of the active, cleaved form of caspase 3 in
cells treated with Gleevec did not reveal an increase in apoptosis
(data not shown). However, we observed a decrease in the
percentage of Ki67-positive cells: 53% of cells treated for 48
hours with 10 M Gleevec as compared with 85% of untreated
cells (Fig. 3C). These data suggest that, in response to Gleevec,
XEN cells exit the cell cycle. We determined the expression of
genes encoding cell cycle regulators of the G1 to S transition,
including cyclins and the CDK inhibitors of the CIP/KIP and
INK4 families. We noted that, in contrast to ES cells, which lack
a G1 phase and do not express any CDK inhibitors, XEN cells
expressed p21Cip1(Cdkn1a) and low levels of p15INK4b(Cdkn2b),
p16INK4a(Cdkn2a), p18INK4c(Cdkn2c) and p19ARF(Cdkn2a) (Fig.
3D,E). Upon 24 and 48 hours of Gleevec treatment, we did not
PDGF signaling promotes expansion of ExEn
observe a significant upregulation in the levels of CDK
inhibitors that could explain, at a transcriptional level, the
inhibition of XEN cell proliferation in response to Gleevec.
Upon ligand-mediated receptor activation, RTKs signal through
various second messengers (Andrae et al., 2008). To determine the
second messenger(s) through which RTKs exert their mitogenic
effect, we employed a pharmacological approach using U0126
[MEK1/2 (MAP2K1/2) inhibitor], LY294002 (PI3K inhibitor) and
BIS (PKC inhibitor) (Fig. 3F). We confirmed the specificity and
lack of cytotoxicity of the compounds (see Fig. S6 in the
supplementary material). Inhibition of MEK1/2 using U0126 at 10
M led to a decrease in cell proliferation (Fig. 3G). Inhibition of
PI3K activity by LY294002 produced a moderate effect on cell
proliferation (Fig. 3H), whereas PKC inhibition using 10 M BIS
led to a dramatic decrease in proliferation (Fig. 3I). Based on these
observations, we propose that in the PrE, RTK activity promotes
cell cycle progression through MAPK and PKC signaling (see Fig.
7). Since Gleevec treatment inhibits several kinases, including
PDGFR, KIT and ABL, these data did not allow us to determine
the respective role of each RTK in XEN cells.
Development 137 (20)
Fig. 3. Inhibition of RTK activity, MEK1/2 or PKC signaling affects XEN cell proliferation. (A)Dose-dependent effect of Gleevec. Gleevec
concentrations: 0 (black), 1 (yellow), 5 (orange) and 10 (red)M. (B)Cell density-dependent effect of Gleevec. Cells were plated at 1?104(red),
5?104(orange) and 10?104(black) cells/well (24-well plate) in the presence of 10M Gleevec (solid lines; dotted lines indicate controls).
(C)Reduction in the number of Ki67-positive cells after 48 hours of Gleevec treatment. Blue, Hoechst; green, Ki67. Scale bar: 50m. (D,E)RT-PCR
(D) and qPCR (E) analyses of cell cycle regulator expression in mouse embryonic fibroblast (MEF), ES and extra-embryonic endoderm (XEN) cells in
the presence and absence of Gleevec. (F)Schematic representation of signal transduction pathways activated upon ligand binding to RTK; the
inhibitors used to block their activities are indicated. (G-I)Proliferation curves depicting the effect of 1-10M U0126 (G), 5-20M LY294002 (H)
and 1-10M BIS (I). Cells were split at 5?104cells/well (24-well plate). Error bars indicate s.e.m.
The PDGF pathway is essential for XEN cell
establishment and is involved in proliferation
Expression analyses suggest that both Pdgfra and Kit, but not Abl1,
genes are expressed in PrE and in XEN cells (Brown et al., 2010;
Kunath et al., 2005). To investigate the functional requirement for
PDGFR and KIT in XEN cells, we determined whether we could
isolate Kit- or Pdgfra-deficient cells. Seven KitW-lacZ/W-lacZXEN cell
lines were isolated from blastocysts recovered from KitW-lacZ/+
heterozygous intercrosses (Bernex et al., 1996), indicating that Kit
is not required for the establishment or maintenance of XEN cells
(Fig. 4A). Furthermore, KitW-lacZ/W-lacZXEN cells proliferated at a
similar rate to wild-type XEN cells in serum and serum-free culture
conditions (data not shown). To determine whether we could
recapitulate the mitogenic effect of Gleevec in the absence of KIT
signaling, Kit-deficient XEN cells were cultured in the presence of
Gleevec (Fig. 4B). Their proliferation was comparable to that of
wild-type XEN cells.
By contrast, no homozygous PdgfraH2B-GFP/H2B-GFPXEN cells
were recovered (29.9 with 1 degree of freedom, P0.0016) (Fig.
4C). To further validate a role for the PDGF pathway in XEN cell
proliferation we tested the effect of PDGF-AA on wild-type XEN
cells cultured in serum-free conditions and noted that it enhanced
proliferation (Fig. 4D). By contrast, addition of recombinant kit
ligand (KITL; SCF) had no effect.
The failure to isolate Pdgfra-deficient XEN cell lines suggested
that PDGF signaling is required for XEN cell establishment, but
prevented us from determining whether it is required for the
maintenance of XEN cells in culture. To investigate the
requirement for PDGF signaling in established XEN cell lines, we
derived XEN cells in which Pdgfra could be conditionally
inactivated. This cell line carried two Pdgfra alleles, the
PdgfraH2B-GFPnull allele and a conditional mutant Pdgfraflallele
that is hypomorphic in the unexcised configuration (Tallquist and
Soriano, 2003), as well as a ubiquitous inducible Cre-ERT2
transgene (Cheng et al., 2010) (Fig. 5A). Induction of Cre activity
by addition of 4-OHT resulted in mosaic deletion of the Pdgfrafl
allele (Fig. 5C). In addition, infection with a self-excising Cre-
expressing retrovirus (Silver and Livingston, 2001) also induced
mosaic deletion of the Pdgfraflallele in cultured XEN cells (Fig.
5D). To overcome the mosaic genotype (PdgfraH2B-GFP/flversus
PdgfraH2B-GFP/+), cells were subcloned after 4-OHT treatment.
PdgfraH2B-GFP/+XEN cell lines proliferated in the absence of
MEFs, and the rate of cell division progressively increased when
cells were maintained for extended passages (Fig. 5E). Treatment
with 5 M Gleevec affected proliferation even in the absence of
Pdgfra, suggesting that other RTKs inhibited by Gleevec might
adapt to the loss of PDGF signaling. However, in serum-free
conditions, Pdgfra-deficient XEN cells failed to proliferate (Fig.
5F). This phenotype could not be rescued by addition of FGF4 or
SCF (data not shown). These data suggest that PDGF signaling is
crucial for the propagation of ExEn cells in vitro, and that this
effect is masked in serum-containing culture conditions.
The PDGF pathway plays a role in PrE lineage
expansion around the time of implantation
Our observations suggesting a role for the PDGF pathway in
XEN cells cultured in vitro contrast with the apparent absence of
a detectable phenotype in preimplantation Pdgfra-null mutant
PDGF signaling promotes expansion of ExEn
Fig. 4. PDGFR , but not KIT,
signaling is required for XEN cell
establishment. (A)Isolation of Kit-
deficient XEN cell lines. (B)Dose-
dependent effect of Gleevec in the
absence of Kit. Kit-deficient XEN cells
were plated at 5?104cells/well (24-
well plate) and treated with Gleevec
at final concentrations of 0 (black), 1
(yellow), 5 (orange) and 10 (red) M.
(C)Failure to isolate Pdgfra-deficient
XEN cell lines. (D)Effect of the
addition of recombinant PDGF-AA
(green) or SCF (purple) on XEN cells,
compared with control (black). Wild-
type XEN cells plated at 5?104
cells/well (24-well plate); 10 ng/ml
PDGF-AA or 20 ng/ml SCF was
added to the serum-free N2B27 cell
culture medium. Error bars indicate
s.e.m. (E)Four-dimensional time-
lapse imaging of PdgfraH2B-GFP/+XEN
cells showing homogeneous levels of
fluorescence during the acquisition
period. Nuclear GFP staining allows
cell tracking. Insets show the boxed
region (dashed line) at higher
magnification. z-stacks were acquired
every 15 minutes for a total of 15
hours. Green, GFP; red, tracked
nucleus; purple, dragon tail.
embryos (Hamilton et al., 2003; Soriano, 1997). We therefore
determined the respective composition of PrE, EPI and TE
lineages in implanting wild-type and mutant blastocysts (E4.5)
(Fig. 6A,D; see Movies 1 and 2 in the supplementary material).
Although mutant embryos had the same mean TE cell number
(68.7±17.3 TE cells versus 74.4±14.1 for heterozygotes and
72.6±9.7 for wild type) or EPI cell number (17.3±6.6 EPI cells
versus 17.7±6.1 for heterozygotes and 17.5±5.0 for wild type),
they exhibited a significant reduction in the number of PrE cells
(16.2±6.8 PrE cells versus 25.8±5.6 for heterozygotes and
25.3±5.3 for wild type) (Fig. 6D). These findings suggest that
the PDGF pathway might be important in the maintenance of the
Artificially delaying implantation through the induction of a
period of diapause has been used to demonstrate the role of LIF
receptor signaling in the maintenance of the EPI lineage despite the
absence of a phenotype at the blastocyst stage (Li et al., 1995;
Nichols et al., 2001; Ware et al., 1995). Diapause preserves the
general topology of the three lineages of the blastocyst, but
promotes an expansion in cell number (Fig. 6B-F; see Movie 3 in
the supplementary material). In PdgfraH2B-GFP/+3-day diapause
blastocysts, the PrE layer was maintained at the interface between
the blastocoel and the EPI, and expressed the PdgfraH2B-GFP
reporter as well as GATA4 (Fig. 6Ca) and SOX17 (Fig. 6Cb). The
EPI lineage of these implantation-delayed blastocysts was
encapsulated by TE and PrE, and expressed OCT4 (Fig. 6Cc) and
low levels of NANOG (Fig. 6Cd) (Batlle-Morera et al., 2008).
Implantation-delayed blastocyst stage PdgfraH2B-GFP/H2B-GFPmutant
embryos also contained GATA4 and SOX17-positive PrE cells with
robust nuclear-localized GFP signal that were in contact with the
blastocoel (Fig. 6Ce-h). However, the number of PrE cells was
reduced (7.5±5.1 PrE cells versus 23.2±6.6 for heterozygotes and
25.1±6.7 for wild type) (Fig. 6F), such that in the most affected
embryos the PrE layer was present as a small patch of cells that did
not line the entire interface between the EPI and blastocoel (Fig.
6Cg,h and see Movie 4 in the supplementary material). These data
suggest that PDGF signaling is required for maintenance of the PrE
lineage in implantation-delayed blastocysts (Fig. 7).
PrE limits the size of the EPI compartment
Interestingly, although the absence of Pdgfra resulted in a reduction
of the number of PrE cells, the number of EPI cells was increased
in PdgfraH2B-GFP/H2B-GFPmutants (30.3±7.9 EPI cells versus
22.8±6.2 for heterozygotes and 20.1±4.8 for wild type) (Fig. 6F).
This imbalance could result from an alteration in cell fate
specification of the ICM, whereby the wild-type balance is skewed
towards EPI. Alternatively, if cell fate specification is unaffected,
reciprocal tissue interactions might be essential earlier than
previously believed, with the PrE layer functioning to regulate the
size of the EPI. To distinguish between these possibilities, we
analyzed the distribution of EPI and PrE cells in 2-day diapause
embryos (Fig. 6E). Although we observed a reduction in the
number of PrE cells in mutant embryos (10.5±4.7 PrE cells versus
16.1±3.5 for heterozygotes and 16.8±2.3 for wild type), the number
of EPI cells did not deviate between mutant (14.9±4.4 cells),
heterozygous (13.5±4.1 cells) and wild-type (14.4±4.4 cells)
embryos. We therefore conclude that between 2 and 3 days of
diapause, the EPI compartment exhibits inappropriate expansion in
Development 137 (20)
Fig. 5. Conditional
inactivation of Pdgfra in
XEN cells impairs their
proliferation. (A)Strategy for
of Pdgfra alleles and
genotyping primers used
detection of floxed and
deleted alleles upon addition
of 4-OHT for the indicated
periods of time. (D)PCR
detection of floxed and
deleted alleles after infection
with Cre-expressing retrovirus.
(E,F)Proliferation curves of
PdgfraH2B-GFP/+(red) XEN cell
lines depict (E) the effect of
5M Gleevec and (F) the
failure of PdgfraH2B-GFP/+XEN
cells to proliferate in N2B27
medium. Error bars indicate
the absence of Pdgfra or a PrE layer. Based on this observation, we
suggest that either PDGF signaling or PrE tissue might be required
for regulating the size of the EPI.
In this study, we have characterized the expression, regulation and
function of Pdgfra in the PrE and its derivatives. We previously
reported that in early preimplantation stage mouse embryos, Pdgfra
is initially expressed in a subset of blastomeres at the 16-cell stage
and is then progressively restricted to the nascent PrE layer in the
blastocyst stage embryo prior to implantation (Plusa et al., 2008).
Here we show that Pdgfra expression is maintained after
implantation in the two PrE derivatives, the PE and VE, eventually
being downregulated in the distal VE, which overlies the epiblast.
This localization correlates with our recent findings for other genes
and transgenes that are initially expressed throughout the VE at
early postimplantation stages, but later downregulated in the distal
VE [which is also referred to as embryonic visceral endoderm
(emVE) (Mesnard et al., 2006)] overlying the epiblast by E6.5,
including Afp (Dziadek and Adamson, 1978; Kwon et al., 2006),
Hnf4a (Duncan et al., 1994; Kwon et al., 2008) and Ttr (Kwon and
Hadjantonakis, 2009; Makover et al., 1989).
We investigated the expression of Pdgfra in various cellular
models of ExEn formation including ES cell differentiation and
XEN cells. Collectively, our data suggest that GATA6 is a key
regulator of Pdgfra (Fig. 7). This model is supported by our
observation that Pdgfra is not expressed upon RA treatment of
Gata6-deficient ES cells, whereas it is expressed in RA-treated
Gata4-deficient ES cells. This model validates our in vivo studies
in which we reported the onset of Gata6 expression at the ~8-cell
stage, Pdgfra at the ~8- to 16-cell stage and Gata4 at the ~64-cell
stage (Plusa et al., 2008).
Our studies have uncovered a role for PDGF signaling in the
proliferation of ExEn cells, acting via MEK and PKC (Fig. 7). Our
failure to establish Pdgfra-deficient XEN cell lines, as well as the
decreased proliferative capacity of conditionally Pdgfra-deficient
XEN cells, support such a function.
Intriguingly, Pdgfra-deficient ES cells were able to differentiate
into PrE, suggesting that the PDGF pathway is not required in the
determination of PrE identity (see Fig. S7 in the supplementary
material). An unresolved question concerns the upstream signal(s)
that regulate MAPK activity within the PrE lineage. Several studies
have reported an essential role for FGF signaling in PrE formation
(Arman et al., 1998; Feldman et al., 1995; Goldin and
Papaioannou, 2003; Nichols et al., 2009; Yamanaka et al., 2010).
However, XEN cells do not appear to require FGF signaling, as
they can be maintained in serum-free conditions. Furthermore,
exogenous addition of FGF4 protein does not elicit any detectable
change in XEN cell proliferation or morphology (our unpublished
data). By contrast, FGF4 is required to derive and maintain TS cells
in an undifferentiated state (Tanaka et al., 1998). In conclusion, our
data support a model whereby PDGF pathway signaling via MEK
PDGF signaling promotes expansion of ExEn
Fig. 6. Implantation-delayed blastocysts lacking PDGFR exhibit defects in the PrE and pluripotent epiblast (EPI) lineages.
(Aa-Ch) PdgfraH2B-GFP/+(top row) and PdgfraH2B-GFP/H2B-GFP(bottom row) mouse embryos at E4.5 (A), 2 days (B) and 3 days (C) after tamoxifen
injection. Blue, Hoechst; green, GFP; red, GATA4 (a,e), SOX17 (b,f), OCT4 (c,g) and NANOG (d,h). Scale bar: 20m. (D-F)Distribution of PrE (blue),
EPI (red) and trophectoderm (TE; green) cells in Pdgfra+/+(WT), PdgfraH2B-GFP/+(HET) and PdgfraH2B-GFP/H2B-GFP(HOM) embryos at E4.5 (D), 2 days (E)
and 3 days (F) after tamoxifen injection. Gray, ICM. **, P<0.007; ***, P<0.0001. Error bars indicate s.d.
and PKC plays a key role in XEN cell propagation. However, the
exact and respective roles of MEK and PKC remain unclear, and
we cannot exclude cross-talk, as has been reported in various cell
systems (Lee et al., 2006; Okazaki et al., 2000; Robin et al., 2004).
Future studies will be required to address the specific and
combinatorial roles of these signal transducers.
Genetic inactivation of members of the PDGF pathway does not
affect the PrE in embryos (reviewed by Andrae et al., 2008; Hoch
and Soriano, 2003), arguing in favor of a model whereby the PDGF
pathway is not required for the acquisition of PrE identity. Several
explanations could account for this apparent disparity. First, the
presence of maternal stores of protein or mRNA accumulated in the
oocyte could compensate for the absence of zygotic expression.
However, Pdgfra transcripts are only detected from the 8-cell stage
onwards by RT-PCR (Plusa et al., 2008), and we failed to detect
GFP reporter expression in PdgfraH2B-GFP/+oocytes (see Fig. S8 in
the supplementary material) or in embryos prior to the 16-cell stage
(Plusa et al., 2008). Alternatively, PDGFR, the second member of
the PDGF receptor family, could be expressed during this period.
Pdgfrb expression has not been reported during the pre/peri-
implantation period in wild-type embryos (Hamatani et al., 2004;
Wang et al., 2004). However, Pdgfra and Pdgfrb double-mutant
embryos can be recovered at postimplantation stages (P. Soriano,
personal communication). Lastly, signaling through other RTKs
could compensate for the absence of PDGF signaling. Candidates
include KIT, owing to its expression in XEN cells (Brown et al.,
2010; Kunath et al., 2005). However, our data suggest that KIT
signaling is not essential in XEN cells, as demonstrated by our
isolation of Kit-deficient XEN cell lines, as well as the absence of
a mitogenic effect when wild-type XEN cells were cultured in the
presence of SCF. Nevertheless, we cannot exclude the possibility
that, in vivo in Pdgfra mutant embryos, KIT signaling or signaling
through another RTK might compensate for the absence of
The role that we report for PDGF signaling in the establishment
of the XEN cells and in the regulation of their proliferation might
reveal a hitherto unsuspected requirement for this pathway in the
maintenance of an exclusively in vitro cell type that has no
identical in vivo counterpart. XEN cells were only isolated recently,
and to date they are not well characterized. XEN cells might indeed
represent the PrE lineage in aspects of their general morphological
characteristics, gene expression and developmental potential
(Kunath et al., 2005), but they might not be identical to any cell
type in the embryo. It is therefore possible, as with the LIF
signaling pathway in mouse ES cells (Li et al., 1995; Smith et al.,
1988; Ware et al., 1995; Williams et al., 1988), that XEN cell
signaling requirements are distinct from those of the PrE lineage in
However, our studies have revealed that E4.5 Pdgfra-deficient
embryos exhibit a reduction in the number of PrE cells, a situation
that is exacerbated when embryos are implantation delayed. We
suggest that PDGF signaling functions in PrE lineage expansion
and maintenance. In addition, because we observed an expansion
of the EPI compartment in implantation-delayed embryos lacking
Pdgfra, we propose that the PrE regulates the size of the EPI
compartment. Thus, a reduction in the number of PrE cells results
in a reciprocal expansion of the EPI. This hypothesis is supported
by a previous report demonstrating that the efficiency of ES cell
isolation from the ICM is increased upon PrE removal (Brook and
Gardner, 1997). Indeed, these observations warrant further
investigation as they suggest that juxtaposition of, and therefore
reciprocal signaling between, PrE and EPI tissues might serve to
regulate compartment size within the peri-implantation mammalian
We thank Alexandra Joyner and Philippe Soriano for the ROSA26CreERT2and
Pdgfra mouse strains, respectively; Michael Parmacek, Philippe Soriano and
David Wilson for ES cell lines; Mary Donohoe, Yoshitaka Hayashi and Daniel
Silver for plasmids; Peter Besmer for Gleevec; Florence Bernex and Geneviève
Aubin-Houzelstein for KitW-lacZhusbandry; Laurent Le Cam for the retroviral
infection protocol; Kemar Brown and Ann Foley for advice on qPCR;
Ferdinando Rossi and Yasemin Yozgat for advice on immunoblotting; Ann
Foley, Tilo Kunath and Jennifer Nichols for valuable discussions; and Florence
Bernex, Mary Donohoe and Ann Foley for critical reading and comments on
the manuscript. Work in A.-K.H.’s laboratory is supported by the National
Institutes of Health (RO1-HD052115 and RO1-DK084391) and NYSTEM.
Deposited in PMC for release after 12 months.
Competing interests statement
The authors declare no competing financial interests.
Supplementary material for this article is available at
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