DEVELOPMENT AND STEM CELLS
RESEARCH ARTICLE 4159
Development 137, 4159-4169 (2010) doi:10.1242/dev.056630
© 2010. Published by The Company of Biologists Ltd
Initiation of trophectoderm lineage specification in mouse
embryos is independent of Cdx2
Guangming Wu1, Luca Gentile1, Takuya Fuchikami2, Julien Sutter1, Katherina Psathaki1, Telma C. Esteves1,
Marcos J. Araúzo-Bravo1, Claudia Ortmeier1, Gaby Verberk1, Kuniya Abe2 and Hans R. Schöler1,3,*
SUMMARY
The separation of the first two lineages – trophectoderm (TE) and inner cell mass (ICM) – is a crucial event in the development of
the early embryo. The ICM, which constitutes the pluripotent founder cell population, develops into the embryo proper, whereas
the TE, which comprises the surrounding outer layer, supports the development of the ICM before and after implantation. Cdx2,
the first transcription factor expressed specifically in the developing TE, is crucial for the differentiation of cells into the TE, as
lack of zygotic Cdx2 expression leads to a failure of embryos to hatch and implant into the uterus. However, speculation exists as
to whether maternal Cdx2 is required for initiation of TE lineage separation. Here, we show that effective elimination of both
maternal and zygotic Cdx2 transcripts by an RNA interference approach resulted in failure of embryo hatching and implantation,
but the developing blastocysts exhibited normal gross morphology, indicating that TE differentiation had been initiated.
Expression of keratin 8, a marker for differentiated TE, further confirmed the identity of the TE lineage in Cdx2-deficient
embryos. However, these embryos exhibited low mitochondrial activity and abnormal ultrastructure, indicating that Cdx2 plays a
key role in the regulation of TE function. Furthermore, we found that embryonic compaction does not act as a ‘switch’ regulator
to turn on Cdx2 expression. Our results clearly demonstrate that neither maternal nor zygotic Cdx2 transcripts direct the initiation
of ICM/TE lineage separation.
INTRODUCTION
The first visible cell lineage split in the development of the
mammalian embryo occurs during blastulation. The cells of the
inner part of the blastocyst, called the inner cell mass (ICM), are
pluripotent and eventually give rise to the embryo proper and to the
extra-embryonic tissues. By contrast, the cells of the outer layer
differentiate into an epithelium, called the trophectoderm (TE),
which subsequently develops into the placenta. To date, a full
understanding has been lacking of the molecular mechanisms that
underlie the fate decision for the initial totipotent cells of the
embryo to become either the TE or ICM lineage.
Cdx2 is a class I homeobox transcription factor that belongs to
the caudal-related homeobox gene family, which comprises
mammalian homologs of Drosophila caudal (James et al., 1994;
Suh et al., 1994). Cdx family members function as upstream
positive regulators of Hox genes (Lorentz et al., 1997), which are
purported to confer positional identity to cells along the
anteroposterior body axis and are sequentially activated in a
temporal and spatial manner (Davidson et al., 2003). Loss of Cdx
function has been shown to be associated with inactivation of Hox
gene expression (Davidson et al., 2003). Cdx1 and Cdx2, which are
regulated by p38 MAPK (Mapk14) (Houde et al., 2001), are
involved in defining the anteroposterior body axis
(Chawengsaksophak et al., 1997; Chawengsaksophak et al., 2004)
1
Department of Cell and Developmental Biology, Max Planck Institute for Molecular
Biomedicine, Röntgenstrasse 20, 48149 Münster, Germany. 2BioResource Center,
RIKEN Tsukuba Institute, Ibaraki 305-0074, Japan. 3University of Münster, Medical
Faculty, Domagkstr. 3, 48149 Münster, Germany.
*Author for correspondence ([email protected])
Accepted 11 October 2010
and in establishing anteroposterior patterning of the intestine
(Silberg et al., 2000). Dysregulation of Cdx2 expression has been
found in intestinal metaplasia and carcinomas (da Costa et al.,
1999; Bai et al., 2002; Eda et al., 2003). In colon cancer cells, for
example, oncogenic ras downregulates Cdx2 expression by
activating protein kinase C (PKC) pathway signaling and by
reducing activity of the Cdx2 promoter AP-1 site through changes
in the relative expression of c-June/c-Fos (Lorentz et al., 1999); by
contrast, in the embryo, ras-MAPK signaling activates Cdx2
expression (Lu et al., 2008). Most importantly, one study found that
although Cdx2 expression appears to be required for TE cell fate
specification in the early mouse embryo, Cdx2 zygotic mutants can
still initiate TE differentiation and blastulation (Strumpf et al.,
2005). Similarly, another study found that Cdx2-deficient embryos,
generated from Cdx2 short hairpin (sh) RNA-expressing somatic
cells using nuclear transfer, failed to implant into the uterus, just
like Cdx2–/– embryos (Meissner and Jaenisch, 2006). However,
neither study eliminated maternal Cdx2 expression. More recent
studies have found that Tead4 acts upstream of Cdx2 and is
essential for TE specification and blastocyst formation (Yagi et al.,
2007; Nishioka et al., 2008), although a subsequent study
contradicted these findings by reporting that maternal Cdx2 is
responsible for compaction and TE lineage initiation (Jedrusik et
al., 2010). Therefore, although it is well established that Cdx2 is
indispensable for the maintenance and proper functioning of the TE
lineage, its role in the initiation of TE lineage specification remains
obscure. It is therefore imperative to study the full effect of Cdx2
expression in early mammalian embryonic development by
eliminating both maternal and zygotic Cdx2 transcripts.
In the present study, we microinjected a robust Cdx2 small
interfering (si) RNA duplex into zygotes and metaphase II (MII)
oocytes and determined the effects of eliminating both maternal
and zygotic expression of Cdx2 on the initiation of ICM/TE lineage
DEVELOPMENT
KEY WORDS: Cdx2, Trophectoderm, Lineage specification, Preimplantation mouse embryos
4160 RESEARCH ARTICLE
MATERIALS AND METHODS
Embryo culture and microinjection of siCdx2 duplex
Fertilized oocytes were collected from the oviducts of primed B6C3F1
female mice after mating with CD1 male mice 18 hours post-hCG in M2
medium. Oocytes were cultured in KSOMAA (potassium simplex optimized
medium plus 19 natural amino acids) (Ho et al., 1995) at 37°C and 5% CO2
in air until microinjection. For MII oocyte microinjection, mature oocytes
were collected from the oviducts of primed B6C3F1 female mice 14 hours
post-hCG in M2 medium, injected with siCdx2, and then fertilized in vitro
in modified KSOM (Summers et al., 2000) with epididymal spermatozoa
from adult OG2 male mice.
We used the online tool BLOCK-iT RNAi Designer to select specific
target sequences for siRNA (https://rnaidesigner.invitrogen.com/
rnaiexpress/). The lyophilized siRNA duplexes (Invitrogen, Karlsruhe,
Germany) were resuspended in 1 ml DEPC-treated water according to the
manufacturer’s instructions and stored in single-use aliquots at –20°C. We
tested three regular oligonucleotides and three Stealth RNAi
oligonucleotides containing the coding region of Cdx2 (target sequence 5⬘GCAGTCCCTAGGAAGCCAA-3⬘) in zygotes and selected the most
effective duplex (siRNA3) for use in all our experiments. Unless otherwise
specified, a scrambled siRNA duplex was used as control (target sequence
5⬘-GCACCCGATAAGCGGTCAA-3⬘).
siRNAs were microinjected using an Eppendorf FemtoJet microinjector
and Narishige micromanipulators. Microinjection pipettes were pulled with
a Sutter P-97 pipette puller. siRNA solution (5 ml of 8 mM) was loaded into
the pipette and ~2 pl was injected into the cytoplasm of each oocyte. A
relatively consistent amount was carefully injected each time. Specifically,
from 73 separate experiments, 3894 of 4093 zygotes (95.1%) were
successfully injected with Cdx2 siRNA, in comparison with 3846 of 4065
zygotes (94.6%) successfully injected with scrambled siRNA.
In addition, from 18 separate experiments, 345 of 1202 MII oocytes
successfully injected with Cdx2 siRNA were fertilized in vitro, in
comparison with 207 of 743 MII oocytes successfully injected with
scrambled siRNA and fertilized in vitro and with 327 of 415 uninjected
MII oocytes that were fertilized in vitro. Unsuccessfully injected oocytes
or lysed oocytes were excluded from all subsequent experiments.
Following microinjection, oocytes were washed and cultured in
KSOMAA at 37°C and 5% CO2 in air and evaluated for cleavage twice
daily. The implantation capability of early-stage embryos was evaluated by
transfer of embryonic day (E) 3.5 mouse embryos into the uterus of a 2.5
days post-coitum (dpc) pseudopregnant CD1 female mouse.
Animal care was in accordance with the institutional guidelines of the
Max Planck Institute.
RNA extraction, cDNA synthesis and real-time reverse
transcription PCR
For real-time analysis of gene expression, embryos were harvested in RLT
buffer (Qiagen, Hilden, Germany) at different stages of development and
processed as previously described (Boiani et al., 2005). Briefly, total RNA
was extracted from individual blastocysts using the MicroRNeasy Kit
(Qiagen) and cDNA synthesis was performed with the High-Capacity
cDNA Archive Kit (Applied BioSystems, Darmstadt, Germany) according
to the manufacturer’s instructions.
Real-time PCR was carried out on the ABI PRISM 7900HT Sequence
Detection System (Applied BioSystems) using TaqMan probes (Applied
Biosystems) for the housekeeping gene Hprt1 (Mm00446968_m1) as a
control and the following genes of interest: Cdx2 (Mm00432449_m1),
Eomes (Mm01351984_m1), Efs (Mm00468700_m1), Atpaf1
(Mm00619286_g1),
Atp1b1
(Mm00437612_m1),
Akt3
(Mm00442194_m1),
Fgfr2
(Mm00438941_m1),
Rab13
(Mm00503311_m1),
Cdh1
(Mm00486906_m1),
Hand1
(Mm00433931_m1), Grn (Mm00433848_m1), Oct4 (Mm00658129_gH),
Gata6 (Mm00802636_m1), Sox17 (Mm00488363_m1). Oligos for Nanog
amplification were custom designed: PF, 5⬘-AACCAGTGGTTGAATACTAGCAATG-3⬘; PR, 5⬘-CTGCAATGGATGCTGGGATACT-3⬘; and
probe, 5⬘-6FAM-TTCAGAAGGGCTCAGCAC-MGB-3⬘. Three to six
biological replicates were used and each sample was run with three
technical replicates; negative controls lacked reverse transcriptase or
template. The cycle threshold (CT) values were collected using Applied
Biosystems SDS v2.0 software and transferred to a Microsoft Excel
spreadsheet for further relative quantification analysis using the DDCT
method (User Bulletin #2, ABI Prism 7700 Sequence Detection System,
1997). Some of the data were presented using the percentage of peak
method, which is an adaptation of the DDCT method (Wang et al., 2004).
Immunofluorescent staining of embryos
Immunocytochemical staining was performed as described previously with
minor modifications (Strumpf et al., 2005). Briefly, samples were fixed in
4% paraformaldehyde for 20 minutes, permeabilized with 0.1% Triton X100 (Sigma-Aldrich, St Louis, MO, USA) for 1 hour, and blocked with 3%
BSA in PBS for 1 hour. Control and Cdx2-deficient embryos were
processed and examined in parallel. Samples were incubated with the
following antibodies: monoclonal mouse anti-Cdx2 IgG (1:100; BioGenex,
San Ramon, CA, USA), rabbit polyclonal anti-Cdx2 [1:2000; gift from
Felix Beck (Victoria, Australia) and Alexey Tomilin, (Freiburg, Germany)],
rabbit anti-Nanog IgG (1:500; Cosmo Bio Company, Tokyo, Japan),
monoclonal mouse anti-Oct4 IgG (1:200; SC-5279; Santa Cruz Biotech.,
Santa Cruz, CA, USA), rabbit anti-Oct4 IgG [1:2500; generated in our
laboratory (Palmieri et al., 1994)], rat anti-E-cadherin IgG (1:100;
ECCD-2; Calbiochem, Darmstadt, Germany), Troma-1 (1:100; DSHB,
Iowa City, IA, USA) or rabbit anti-ISP1 IgG [1:100; kindly provided by
Dr D. Rancourt (O’Sullivan et al., 2001)], all at 4°C overnight, or with
rabbit anti-ZO-1 IgG (1:150; Zytomed, Berlin, Germany) at 37°C for
1 hour.
Embryo samples were then washed several times with PBS and stained
with Alexa 488-conjugated goat anti-rabbit IgG, Alexa 488-conjugated
goat anti-mouse IgG (Invitrogen), Cy3-conjugated goat anti-rat IgG, or
Cy3-conjugated goat anti-mouse IgG (Jackson Laboratory, Bar Harbor,
ME, USA) at 1:400 dilution for 1 hour at room temperature. After further
washes and counterstaining with 5 mM DRAQ5 (Biostatus, Shepshed, UK)
for 30 minutes, samples were examined under a laser-scanning confocal
microscope (UltraVIEW; PerkinElmer Life Sciences, Jügesheim,
Germany) with 488, 568 and 647 nm lasers. ECCD-2 immunostaining was
quantified with ImageJ 1.42 software (http://rsbweb.nih.gov/ij/
download.html). Troma-1 antibody and DRAQ5 staining were used to
determine the ICM:TE cell number ratio by comparing confocal images of
neighboring sections using a 5 mm scanning distance.
Transmission electron microscopy
Cells were fixed in 2.5% glutaraldehyde (Merck, Darmstadt, Germany) in
0.1 M sodium cacodylate buffer (pH 7.4), post-fixed in 1% aqueous osmium
tetroxide, dehydrated in ethanol and embedded in Epon 812 (Fluka, Buchs,
Switzerland). Ultrathin sections (50 nm) were prepared with an EM UC6
ultramicrotome (Leica Microsystems, Wetzlar, Germany), stained with 1%
uranyl acetate and then 3% lead citrate, and subsequently examined under a
Zeiss EM 109 electron microscope (Carl Zeiss, Oberkochen, Germany).
Measurement of mitochondrial membrane potential and ATP
content
Mitochondrial membrane potential was measured by staining with 5,5⬘,6,6⬘tetrachloro-1,1⬘,3,3⬘-tetraethylbenzimidazolyl carbocyanide iodide (JC-1;
Molecular Probes, Eugene, OR, USA) (Cossarizza et al., 1994). Cells were
incubated in KSOMAA containing 5.0 mg/ml JC-1 for 15 minutes at 37°C in
the dark. Cells were washed with KSOMAA, excited at 488 nm, and then
observed at either 510 nm (green mitochondria) or 590 nm (red-to-orange
mitochondria) with the confocal imaging system. The ATP content of each
embryo was measured as previously described (Boiani et al., 2004).
Induction and inhibition of embryonic compaction
To induce compaction, 4-cell stage embryos were transferred into
KSOMAA containing 200 mM DiC8 (Sigma-Aldrich) and incubated
overnight at 37°C. To prevent compaction, precompacted 8-cell stage
DEVELOPMENT
separation and cellular differentiation. As Cdx2 acts downstream
of Tead4, we also targeted Tead4 with siRNA to further confirm
these results. The findings of this study help to better define the
roles played by Cdx2 during early mammalian development.
Development 137 (24)
embryos were cultured in KSOMAA containing 200 mg/ml anti-E-cadherin
antibody (ECCD-1; Calbiochem). Embryo samples were collected after 18
and 24 hours of treatment and subjected to immunocytochemical analysis
for Cdx2 expression.
Measurement of DNA fragmentation by TUNEL assay
The degree of apoptosis in embryo samples was determined using terminal
deoxynucleotidyl transferase (TdT)-mediated dUTP-FITC nick end
labeling (TUNEL) with an in situ cell death detection kit (DeadEnd
Fluorimetric TUNEL System; Promega, Madison, WI, USA) according to
the manufacturer’s recommendations. Cells were then viewed/counted
under a confocal microscope.
Statistical analysis
The Jarque-Bera test was performed on the ICM:TE cell number ratio and
apoptotic cell numbers, revealing that the cell number did not follow a
normal, or Gaussian, distribution with a significance level 0.05.
Therefore, the Wilcoxon rank-sum test was performed as an appropriate
non-parametric statistical test with significance level 0.05 to compare
differences in cell number.
RESULTS
Maternal and zygotic expression of Cdx2
We examined Cdx2 mRNA and protein levels in developing early
mouse embryos by quantitative real-time PCR (qRT-PCR) and
immunocytochemistry. Maternal Cdx2 mRNA was readily
detectable in oocytes (CT value of 36.5 from pools of ten MII
oocytes) and 2-cell stage embryos, but levels were reduced by 73%
in 4-cell stage embryos and increased by 23-fold upon Cdx2
activation in 8- to 16-cell stage embryos (Fig. 1A). As previously
reported (Niwa, H. et al., 2005; Strumpf et al., 2005; Dietrich and
Hiiragi, 2007; Yagi et al., 2007), Cdx2 protein levels could only be
detected as a very weak nuclear signal in 8-cell stage embryos (Fig.
1C); however, a stronger Cdx2 protein signal was detected in some
blastomeres during the next cell cycle, gradually extending to all
outer blastomeres (see Fig. S1 in the supplementary material). This
expression profile provides evidence for the presence of maternal
Cdx2 transcripts in early mouse embryos and indicates that
activation of zygotic Cdx2 expression is concomitant with the first
lineage differentiation event during the asymmetric fourth division
after compaction in 8-cell stage mouse embryos (Johnson and
McConnell, 2004).
Effective reduction in maternal and zygotic Cdx2
by RNAi has profound effects on TE cell
differentiation, function and embryo
developmental potential
Of the six Cdx2-specific siRNA duplexes tested, siRNA3 was
selected for subsequent experiments owing to its unique sequence
and high efficiency (see Fig. S2A in the supplementary material);
siRNA3 is hereafter referred to as siCdx2. A scrambled siRNA
duplex (siControl) with the same composition as siCdx2 but no
specific target was used as control. A single injection of siCdx2
into MII oocytes or zygotes was found to reduce Cdx2 mRNA
levels by at least 95%, as determined by qRT-PCR (Fig. 1B).
Furthermore, siCdx2 injection also led to an efficient reduction in
Cdx2 protein to undetectable levels in each of the 8-cell (siControl,
n26; siCdx2, n28), morula (siControl, n26; siCdx2, n25) and
blastocyst stage (siControl, n71; siCdx2, n67) embryos, as
determined by three independent immunocytochemistry
experiments (Fig. 1C,D; see Fig. S2B in the supplementary
material) and a western blot with replicates (see Fig. S2C in the
supplementary material). The robust elimination of both maternal
and zygotic Cdx2 expression did not affect the rate of blastocyst
RESEARCH ARTICLE 4161
formation (see Table S1 in the supplementary material). As shown
in our time-lapse observations (see Fig. S2D in the supplementary
material), compaction and the onset of blastocyst formation were
unaffected in siCdx2-treated embryos. However, most likely owing
to a defect in tight junctions, Cdx2-deficient blastocysts could not
initially maintain the integrity of the blastocyst cavity, collapsed
three times and were delayed by ~7.5 hours in starting cavity
expansion, but were of similar size and gross morphology to
siControl embryos at 4.0 dpc. Then, after 5-8 pulses of collapse and
expansion, all Cdx2-deficient embryos eventually collapsed in the
zonae pellucidae between 5.5 and 6.0 dpc, rather than hatching like
control blastocysts.
To exclude the possibility that any residual maternal Cdx2
protein could have driven TE lineage separation and blastocyst
formation in siCdx2-treated embryos, we co-injected siCdx2 with
a rabbit polyclonal Cdx2 antibody at 100-fold higher concentration
than the effective concentration for immunostaining, but still
observed no phenotype prior to the blastocyst stage (see Table S2
in the supplementary material).
In addition, an increase in the ICM:TE cell number ratio was
found (0.58 versus 0.37; siControl, n40; siCdx2, n41;
P1.05⫻10–5), but the total number of cells in Cdx2-deficient E4.0
blastocysts was not different from that of control blastocysts (66.3
versus 66.9; P0.64; Fig. 1H). TUNEL analysis revealed the
presence of slightly more apoptotic cells within both the inner (5.4
versus 2.7; P7.35⫻10–5) and outer (2.9 versus 1.8; P3.48⫻10–4;
siControl, n19; siCdx2, n13) cells of Cdx2-deficient blastocysts
(Fig. 1I). However, transfer of Cdx2-deficient blastocysts into the
uterus of pseudopregnant foster mice did not lead to any
implantation (0 implanted out of 109 transferred in the siCdx2treated group versus 60 out of 108 in the siControl, from three
separate experiments), constituting another specific phenotype of
the zygotic Cdx2–/– embryos.
Gene expression analysis by qRT-PCR revealed that knockdown
of maternal Cdx2 led to a 77.5% reduction in the transcript levels
of the TE-associated gene Eomes in 8-cell stage embryos, whereas
a reduction in the Fgfr2 transcript and an increase in the transcripts
of the pluripotency gene Oct4 (Pou5f1 – Mouse Genome
Informatics) became significant only after blastulation (Fig. 2A,B;
Fig. 4; see Fig. S3A in the supplementary material). Notably,
Hand1, which is expressed in the TE and regulates trophoblast cell
differentiation into trophoblast giant cells (Cross et al., 1995), and
Grn, which is an important factor for blastocyst hatching, adhesion
and outgrowth (Qin et al., 2005), were overexpressed in Cdx2deficient blastocysts (Fig. 4).
To determine why the normally shaped Cdx2-deficient
blastocysts failed to hatch from the zonae pellucidae, we performed
gene expression studies with qRT-PCR or immunocytochemistry
on E4.0 Cdx2-deficient blastocysts. We found that Grn (an
autocrine growth factor) and ISP1 (a tryptase; Prss28 – Mouse
Genome Informatics), which have been reported to play crucial
roles in the hatching process (O’Sullivan et al., 2001; Qin et al.,
2005), were not reduced in Cdx2-deficient blastocysts (Fig. 4; see
Fig. S3B in the supplementary material). However, further
examination revealed the cause of this hatching failure.
Immunocytochemical analysis of embryos using the tight junctionassociated protein zona occludens 1 (ZO-1; Tjp1 – Mouse Genome
Informatics) showed that the apical zonular structure of the tight
junctions was discontinuous in siCdx2-treated embryos (Fig. 1F),
indicating compromised intercellular sealing and epithelial
integrity. This specific phenotype was previously observed in
experiments with mouse Cdx2–/– embryos (Strumpf et al., 2005).
DEVELOPMENT
Cdx2 in trophectoderm specification
Development 137 (24)
Fig. 1. Efficient reduction of Cdx2 mRNA and protein levels in different preimplantation stage mouse embryos by siCdx2 results in
phenotypes similar to Cdx2 knockout. (A)Relative Cdx2 mRNA expression levels as a percentage of peak expression in mouse embryos at different
preimplantation stages, as assessed by qRT-PCR. The maternal mRNA decreased at the 4-cell stage, followed by upregulation at the 8-cell stage due to
the zygotic activation of Cdx2. Embryo collection times (post-hCG): 2-cell stage, 40 hours; 4-cell stage, 52 hours; 8-cell stage, 62 hours; 16-cell stage,
72 hours; morula, 85 hours; early blastocyst, 96 hours; late blastocyst, 115 hours. (B) A comparison of Cdx2 mRNA levels in siControl- and siCdx2treated zygotes at different preimplantation stages demonstrated robust downregulation of Cdx2 by siCdx2 treatment. KD, knockdown. (C)Confocal
images of 8-cell stage embryos immunolabeled with anti-Cdx2 monoclonal antibody illustrate the reduction of Cdx2 protein (green) by siCdx2
treatment. Red, nuclear counterstaining with DRAQ5. (D)Confocal images of blastocyst stage embryos immunolabeled with anti-Cdx2 (green) and antiOct4 (red) antibodies. Following siCdx2 treatment, Cdx2 was eliminated, but Oct4 was ectopically expressed in the trophectoderm (TE). (E)DIC images
of E4.0 and E6.0 embryos. All siCdx2-treated embryos failed to hatch. Arrows indicate empty zonae pellucidae left by hatched control embryos. Scale
bar: 100mm. (F)Confocal images of ZO-1 (green) and E-cadherin (red) immunohistochemistry show normal E-cadherin distribution, which marks the
lateral-basal cell boundary, but disoriented cell polarity of the TE, as indicated by the presence of ZO-1 at both the apical and basal sides (arrowhead
and inset). (G)Defective tight junction (arrowhead) in the TE of a Cdx2-deficient blastocyst as shown by electron microscopy. Scale bar: 0.2 mM.
(H) Effect of Cdx2 depletion on TE and inner cell mass (ICM) cell numbers. The number of ICM cells was significantly increased (P<0.01), but the total
cell number remained unchanged (P0.64) upon Cdx2 depletion. (I)Quantitation of apoptotic cells shows an increase in apoptotic activity in a Cdx2deficient E4.0 blastocyst,. MII, metaphase II oocyte; ZP, zona pellucida. Error bars indicate standard deviation.
Moreover, we also observed the presence of the zonular form of
ZO-1 at both the apical and basal sides of the epithelium,
suggesting that some TE cells exhibited disorientation in cellular
apicobasal polarity. Examination of Cdx2-deficient blastocysts
under the electron microscope (Fig. 1G) showed that the tight
junction structure was actually missing, although ZO-1 protein was
DEVELOPMENT
4162 RESEARCH ARTICLE
Cdx2 in trophectoderm specification
RESEARCH ARTICLE 4163
Fig. 2. Significant effect of Cdx2 reduction on gene
expression at various developmental stages but no
blockage of expression of the differentiated TE marker
Krt8. (A)Relative gene expression levels as a percentage of
peak expression of Cdx2, Eomes, Fgfr2 and Oct4 in mouse
preimplantation embryos after siControl and siCdx2 injection
into zygotes. The downstream transcription factor Eomes was
reduced to a basal level. Fgfr2 reduction is shown at the early
and expanded blastocyst stages. Oct4 expression was increased
~2-fold in both the early and expanded blastocyst stages
(P<0.0001). Error bars indicate standard deviation. (B) Ectopic
expression of Nanog in the TE of Cdx2-deficient blastocysts
after microinjection of siCdx2 at the zygote stage. The
embryonic stem cell marker Nanog was ectopically expressed in
TE, as shown in this z-stack confocal image of an
immunostained siCdx2-treated blastocyst. (C)Sectional confocal
image showing the presence of Krt8 (as detected by the Troma1 antibody) in an interrupted pattern in Cdx2 knockdown
blastocysts.
Effect of Cdx2 knockdown on mitochondrial
activity
Upregulation of mitochondrial activity is a feature of TE
differentiation. The JC-1 assay was performed to explore the
impact of Cdx2 expression on mitochondrial function and revealed
a significant reduction in mitochondrial activity at the apical cortex
of 8-cell embryos and TE cells of blastocysts, indicating the
presence of a functionally defective TE (Fig. 3A). This finding was
corroborated by the underexpression of genes involved in both ATP
synthesis (Atpaf1) and consumption (Atp1b1) (Fig. 4A,B) in Cdx2deficient embryos, as determined by qRT-PCR. However, an
increase in cytoplasmic ATP content was found in blastocyst stage
embryos that had Cdx2 knockdown-induced mitochondrial
dysfunction, as compared with wild-type blastocysts (0.41 versus
0.17 pmol per embryo; Fig. 3B).
siCdx2 microinjection into MII oocytes produces a
phenotype identical to that of zygotes
Our results showed that microinjection of siCdx2 into MII oocytes
also led to an efficient reduction in Cdx2 mRNA expression levels
(Fig. 4A), with no protein detectable in blastocyst stage embryos
(siControl, n61; siCdx2, n52) by immunostaining. Changes in
the gene expression profile at E4.0 were similar to those observed
after injection of siCdx2 into zygotes (Fig. 4B), as determined by
qRT-PCR. Injection of siCdx2 into MII oocytes led to ectopic
expression of Oct4 and Nanog proteins in the outer cells of Cdx2deficient blastocysts (see Fig. S3C in the supplementary material),
comparable to that observed after siCdx2 injection into zygotes
(Fig. 1D; Fig. 2B). Injection of siCdx2 at an earlier time point
resulted in the same phenotype and gene expression changes as at
the zygote stage, further supporting the conclusion that the
initiation of compaction and separation of ICM/TE in Cdx2deficient embryos was not due to Cdx2 protein produced from
maternal mRNA before the mRNA was knocked down.
Embryonic compaction does not lead to activation
of Cdx2 expression
To evaluate the effect of embryonic compaction on the activation
of Cdx2 expression, we used two complementary approaches. First,
we induced compaction in 4-cell stage embryos with the natural
PKC agonist DiC8 (Pauken and Capco, 1999). Embryos at this
stage of normal development do not initiate compaction. However,
upon treatment with DiC8, 4-cell stage embryos rapidly initiated
DEVELOPMENT
detected by immunocytochemistry. However, the ultrastructure of
lateral-basal adherens junctions was present as in control embryos
(Fig. 1G). Immunocytochemical analysis of embryos revealed a
stronger intensity of E-cadherin, a major component of adherens
junctions, at the basolateral border of TE cells in Cdx2-deficient
embryos than in control embryos (Fig. 1F; P<0.01), without an
increase in mRNA levels (Fig. 4). Therefore, although adherens
junctions could compensate for the lack of tight junctions and
support delayed blastocoele formation and expansion in the
mutated embryos, they failed to maintain intercellular sealing for
the more demanding process of hatching.
Finally, to further confirm the identity of the outer cells, we
performed immunocytochemical analysis of blastocysts for keratin
8 (Krt8; also known as EndoA), a widely used marker for
differentiated TE, with the monoclonal antibody Troma-1 (Brulet et
al., 1980; Ralston and Rossant, 2008). We found that Krt8 was
present in the outer cells of Cdx2-deficient blastocysts, albeit with an
uneven distribution, clearly showing that establishment of the TE
lineage was independent of Cdx2 at the molecular level (Fig. 2C).
Our time-lapse analysis revealed that Cdx2-deficient embryos
compacted and initiated cavitation at the correct time and only failed
to hatch afterwards. In addition, the zonular form of ZO-1 was found
to be present at the apical sides of the outer cells and certain TEassociated genes (Hand1 and Grn) were actually overexpressed in
Cdx2-depleted embryos. Taking all these data together, we conclude
that elimination of both maternal and zygotic Cdx2 expression did
not block the initiation of ICM/TE lineage separation, but it did
impact the continuation of TE differentiation and function.
Fig. 3. Significant effect of Cdx2 knockdown on mitochondrial
activity. (A)Confocal images showing mitochondrial activity as
assessed by JC-1 staining at the 8-cell, morula and blastocyst stages.
siCdx2 treatment significantly reduced mitochondrial activity, as
indicated by the accumulation of the red aggregated form of JC-1 in
the mitochondria (appears yellow, as all mitochondria were stained
with the green form of JC-1). (B)Measurement of ATP content in
individual embryos, showing a significant increase in ATP from 0.17
pmol to 0.41 pmol in Cdx2-deficient blastocysts. Error bars indicate
standard deviation.
compaction, as seen by the flattening and adherence of
blastomeres, which became well compacted within 30 minutes
(Fig. 5A). Cdx2 expression could not be detected after 18 hours of
treatment by immunocytochemistry (Fig. 5B; DiC8, n21; Control,
n19). Using the second approach, we blocked the compaction of
8-cell stage embryos (Fig. 5C) with ECCD-1, an antibody directed
against the extracellular domain of E-cadherin (Yoshida-Noro et
al., 1984), and examined Cdx2 expression after 24 hours in 12- to
16-cell stage embryos (Fig. 5D; ECCD-1, n20; Control, n21).
Although there was no compaction, Cdx2 expression could still be
detected in 12-cell stage embryos. These results clearly
demonstrate that the compaction process does not lead to activation
of Cdx2 expression in mouse embryos.
Reduction in Tead4, which is upstream of Cdx2,
prevents the cavitation and separation of the ICM
and TE but does not prevent compaction
We tested the efficiency of four Tead4-specific siRNA duplexes to
knock down Tead4 mRNA expression levels by the same
microinjection approach that we used for Cdx2 knockdown.
Injected oocytes were cultured for 72 hours to reach the
morula/blastocyst stage and collected individually or in groups for
qRT-PCR. Two siRNA duplexes were found to result in a ~90%
knockdown efficiency of Tead4 (siTead4A and siTead4B; Fig. 6A).
Consistent with the zygotic Tead4 knockout phenotype (Yagi et al.,
Development 137 (24)
Fig. 4. Relative gene expression levels in E4.0 blastocysts after
microinjection of siCdx2 into MII oocytes and zygotes. Elimination
of Cdx2 RNA by microinjection of siCdx2 into mouse (A) MII oocytes
and (B) zygotes had similar effects on gene expression levels, as
assessed by qRT-PCR. Cdx2 knockdown reduced expression of genes
crucial in TE lineage differentiation, with overexpression of
pluripotency-related genes. Error bars indicate standard deviation.
2007; Nishioka et al., 2008), these two duplexes were found to
effectively prevent embryo cavitation and clear separation of the
ICM and TE (Fig. 6B), but had no effect upon compaction at 2.5
dpc (see Fig. S4 in the supplementary material), just as with Cdx2
knockdown embryos. Significant underexpression of TE marker
genes (Cdx2, Eomes, Fgfr2 and Atp1b1), but not pluripotencyrelated genes (Oct4, Nanog and Sox2), was observed by qRT-PCR
upon Tead4 knockdown (Fig. 6C).
DISCUSSION
In the present study, we defined the expression profile of Cdx2 in
mouse oocytes and embryos by the sensitive and specific technique
of qRT-PCR and systematically investigated the impact of
knocking down both maternal and zygotic mRNA levels by an
siRNA approach on the development of preimplantation embryos.
The consistency in gene expression profile and phenotype of the
siCdx2-treated embryos with those of the zygotic Cdx2-null
embryos (Strumpf et al., 2005), such as the failure of embryos to
hatch from the zonae pellucidae and implant into the uterus, the
underexpression of Eomes, and the ectopic expression of Oct4 and
Nanog in the TE, demonstrates the efficacy and specificity of our
siCdx2 treatment.
Developmental biologists have sought for years to understand
how mammalian embryos establish their developmental pattern and
initiate their first differentiation event – the separation of the TE
DEVELOPMENT
4164 RESEARCH ARTICLE
Fig. 5. Neither induction nor blockage of embryonic compaction
affects the activation of Cdx2 expression. (A)Premature
compaction induced within 30 minutes of treatment with the PKC
agonist DiC8 (right). Normal 4-cell stage mouse embryos are shown on
the left, with obvious, individual blastomeres. (B)Cdx2 expression (red)
could not be detected by immunocytochemistry 18 hours after DiC8
treatment. Nuclei are blue (DAPI) and Oct4 (a positive control for
transcriptional activity of the treated embryos) green. (C)Compaction
was inhibited by anti-E-cadherin (ECCD-1) antibody treatment for 24
hours (right). Normally compacted 14-cell stage embryos are shown on
the left. (D)Cdx2 was detected (arrowhead) in uncompacted 12-cell
stage embryos. Heavy cytoplasmic background (especially around the
cell surface) was due to cross-reaction of ECCD-1 (rat IgG monoclonal),
which was used at a very high concentration (200mg/ml) to block
embryo compaction, with the Cdx2 antibody (mouse IgG monoclonal).
from the ICM (Hiiragi et al., 2006). Cdx2, the first TE marker gene
to be expressed in mouse embryos, was reported in a study by
Strumpf and colleagues to be required for correct TE fate
specification (Strumpf et al., 2005). The zygotic Cdx2–/– embryos
failed to downregulate Oct4 and Nanog levels in the outer cells of
the blastocyst, resulting in loss of epithelial integrity and in a
subsequent failure to hatch and implant. However, these Cdx2-null
embryos were still capable of forming a TE, as evidenced by
cavitation and blastocyst formation, a finding that contradicted the
main conclusion of that study and the authors hypothesized that the
expression of maternal Cdx2 in these Cdx2–/– embryos might be
RESEARCH ARTICLE 4165
responsible for the initiation of TE lineage differentiation.
However, the same group later proposed that Cdx2 does not lead
the TE transcription factor hierarchy (Rossant and Tam, 2009), as
a loss-of-function Cdx2 mutation had no effect on the initiation of
blastocyst formation (Strumpf et al., 2005), and that Cdx2-mutant
cells were not excluded from the TE layer in chimeric blastocysts
(Ralston and Rossant, 2008). These cells, however, do not undergo
further trophoblast differentiation and thus are not functional
(Strumpf et al., 2005).
In the present study, we used an siRNA approach to robustly
knock down both maternal and zygotic Cdx2 transcripts, and are
the first group to provide unequivocal and direct evidence that
maternal Cdx2 is not a determinant of TE lineage commitment. In
these maternal and zygotic Cdx2-deficient embryos, compaction
and cavitation initiation were not interrupted, as clearly shown by
our time-lapse observations (see Fig. S2D in the supplementary
material), even though the morula-to-blastocyst transformation was
delayed as a consequence of a defect in ZO-1/tight junctions (Wang
et al., 2008). The outer cells of Cdx2-deficient blastocysts
expressed the TE marker Krt8, confirming their TE identity.
However, disorientation of the TE, as indicated by the presence of
linear ZO-1 at the base of the epithelium, also highlights the
importance of Cdx2 in cell polarization at the preimplantation
stages. An increased ICM:TE cell number ratio might reflect
compromised TE proliferation and function in regulating the
number of ICM cells. These embryos subsequently failed to hatch,
implant into the uterus or survive, indicating that TE lineage
separation is independent of Cdx2 but that TE function supporting
embryogenesis is completely dependent on Cdx2.
A recent report from M. Zernicka-Goetz’s group (Jedrusik et al.,
2010) has provided results that are contradictory to those of the
present study. In that study, the authors achieved a much lower
knockdown efficiency of maternal Cdx2 transcripts, of ~80-85%
before the 8-cell stage, with Cdx2 protein levels still detectable in
some of the treated embryos, but they reported a more severe
phenotype and claimed that the maternal pool of Cdx2 mRNA
played a crucial role in compaction and polarization at the 8- to 16cell stages of embryonic development. In our experiments, we
achieved a knockdown efficiency of maternal Cdx2 transcripts of
greater than 99.0%, without any detectable Cdx2 proteins, and yet
88.4% of these embryos compacted and developed to the blastocyst
stage, with the same morphology and developmental rate as those
injected with scrambled siRNA (87%) or non-injected embryos
(85.3%) (see Table S1 in the supplementary material). The failure
of such embryos to hatch and implant and the formation of
embryoid body (EB)-like structures on mouse embryonic
fibroblasts (MEFs) (Wu et al., 2010) were reported to be
phenotypes specific to zygotic Cdx2–/– embryos by Rossant’s group
(Strumpf et al., 2005), yet we did not observe any significant effect
on compaction and blastocyst formation.
In the study by M. Zernicka-Goetz’s group, heterogeneous
phenotypes were observed as embryos arrested at various stages of
development. Furthermore, their Cdx2-deficient embryos not only
totally lacked expression of TE-associated genes, such as Krt8,
Eomes, Ecad (Cdh1), aPKC (Prkcz), Par1 (Mark1) and Par3
(Pard3), but also that of the pluripotent marker gene Nanog, which
contradicts the findings of other publications, as a reciprocal
relationship between Nanog and Cdx2 has already been clearly
established (Chen et al., 2009) and overexpression of Nanog has
been observed in zygotic Cdx2 knockout experiments (Strumpf et
al., 2005) as well as in the present study. In support of our
observations, an independent group has reported that following
DEVELOPMENT
Cdx2 in trophectoderm specification
4166 RESEARCH ARTICLE
Development 137 (24)
injection of Cdx2-specific siRNA at the 1-cell stage, Cdx2-deficient
embryos do not exhibit developmental arrest prior to the blastocyst
stage and do overexpress the pluripotent gene Oct4 at the blastocyst
stage (Wang et al., 2010). In a broader sense, research on primates
has also indicated that CDX2 plays an essential role in functional
TE formation and that the ICM lineage of CDX2-depleted embryos
supports the isolation of functional embryonic stem cells
(Sritanaudomchai et al., 2009), which is consistent with our
observations in the mouse.
In the present study, we observed an increase in mitochondrial
activity concomitant with TE differentiation. This is consistent with
the involvement of Na+/K+-ATPase in mouse embryos preparing
for cavitation and production of nascent blastocoele fluid (Wiley,
1984) and with the stage-specific translocation of active
mitochondria during early porcine embryo development (Sun et al.,
2001). Interestingly, TE cells have been reported to produce and
consume most of the ATP of the blastocyst (Houghton, 2006).
Functional analysis with JC-1 staining showed reduced activity in
the mitochondria of Cdx2-deficient embryos. Further gene
expression analysis demonstrated that the reduction in
mitochondrial activity was mediated by the underexpression of
genes involved in both ATP synthesis and consumption. Krt8 has
been shown to modulate the shape, distribution and function of
hepatocyte mitochondria and to protect cells from apoptosis (Tao
et al., 2009), suggesting that some of the phenotypes of Cdx2deficient embryos might be mediated by the reduced and uneven
Krt8 expression pattern that we observed. Reduction in Krt8
expression has also been observed in zygotic Cdx2–/– embryos
(Ralston and Rossant, 2008). Of note, an increase in ATP content
was consistently found in Cdx2-deficient embryos, in duplicate
experiments with triplicate tests, which was probably due to
underexpression of the ATP-dependent Na+ pump of the TE, a
main consumer of ATP at this developmental stage (Houghton,
2006). These data indicate that deficiency in Cdx2 has profound
effects on mitochondrial activity and that Cdx2 is indeed required
for proper functioning of the TE. Further study is needed to
determine precisely how Cdx2 regulates mitochondrial activity and
ATP levels in the embryo.
Observations from classical embryonic experiments have
suggested that ICM/TE lineage separation is initiated by
asymmetric cell divisions that act to establish the inner and outer
cell populations following compaction and polarization in 8-cell
stage mouse embryos (Johnson and Rossant, 1981). During
compaction, changes at the cellular level include enhanced cell
adhesion between blastomeres resulting in the formation of a
smooth ball of blastomeres, and the establishment of cell
polarity, as demonstrated by the formation of apical and
basolateral surfaces on the blastomeres (Hyafil et al., 1980; Butz
and Larue, 1995; Ohsugi et al., 1996). At the next cell division
after compaction, the developmental fate of the embryo becomes
restricted, as outer blastomeres become destined to form the first
epithelial layer, the TE, and inner blastomeres are pluripotent
and form the ICM (Tarkowski and Wroblewska, 1967; Johnson
and Rossant, 1981). Par3, Par6 and aPKC are components of an
apical polarity complex that has been shown to influence
TE/ICM fate choice (Plusa et al., 2005). In contrast to the two
DEVELOPMENT
Fig. 6. Effective knockdown of Tead4 in preimplantation mouse embryos by microinjection of siRNA into zygotes reproduces the
Tead4–/– embryo phenotype. (A)Tead4 knockdown efficiency (%) determined by qRT-PCR in a single E4.5 mouse blastocyst in comparison to the
scrambled siRNA control. Error bars indicate standard deviation. (B) Failure of blastulation 72 hours (E3.5) after microinjection of siTead4A and
siTead4B into mouse zygotes. In this particular experiment, 25 zygotes per group were successfully injected. Note that the phenotype is consistent
and highly reproducible within the siTead4A and siTead4B groups (except for one embryo in each group that was blocked at the 2-cell stage), but
was variable in groups with lower Tead4 knockdown efficiency. (C)Tead4 knockdown reduced the expression of genes crucial in TE lineage
differentiation but did not reduce the expression of pluripotency-related genes. Shown are qRT-PCR results from pools of six embryos, with
biological and technical triplicates.
Fig. 7. Postulated TE differentiation regulatory network. Cdx2 is
not required for the TE lineage specification but it is critical for further
differentiation of TE. Interactions among the processes of compaction
and cellular polarization could affect the distribution of Cdx2 along the
inside/outside axis and influence tight junction formation and
localization. These interactions could therefore affect ICM/TE lineage
allocation. SCMC, subcortical maternal complex.
studies by M. Zernicka-Goetz’s group (Jedrusik et al., 2008;
Jedrusik et al., 2010), other studies have shown that Cdx2
appears to act downstream of the first lineage decision in the
early mouse embryo – i.e. following cell polarization – to
promote TE cell fate determination in a cell-autonomous manner,
suggesting that processes influencing lineage allocation or
morphogenesis might regulate Cdx2 expression along the
inside/outside axis of the embryo (Ralston and Rossant, 2008).
However, it is not yet clear whether compaction induces Cdx2
expression. It is known that compaction can be induced
prematurely in 4-cell stage embryos by PKC activators (Winkel
et al., 1990; Ohsugi et al., 1993; Pauken and Capco, 1999). With
this approach, we showed that induction of compaction does not
activate Cdx2 expression. Moreover, blocking compaction with
ECCD-1, a monoclonal antibody that specifically neutralizes Ecadherin-mediated adhesion (Johnson et al., 1986) and induces
disorientation of cell polarity in mouse early embryos (Fleming
et al., 1989), does not prevent Cdx2 expression. These results
suggest that Cdx2 expression and compaction are two parallel
processes in regulating TE differentiation. This conclusion is
consistent with a very recent report from Rossant’s group that
demonstrated that the epithelial integrity mediated by E-cadherin
is not required for Cdx2 expression (Stephenson et al., 2010).
We next sought to confirm that even though we effectively
knocked down maternal Cdx2 transcripts and attempted to block
the function of any residual proteins with a Cdx2 antibody, we still
could not confer an essential role for maternal Cdx2 in embryo
compaction and initiation of the TE lineage, contrary to M.
Zernicka-Goetz’s group (Jedrusik et al., 2010). Here, we add
another piece of evidence to support our conclusions. Tead4 has
been reported by two studies to act upstream of Cdx2 and to be
essential for TE specification (Yagi et al., 2007; Nishioka et al.,
2008). In these two studies, zygotic Tead4–/– embryos were found
to be devoid of the TE lineage, and expression of Tead4 was
activated at the 2-cell stage and was postulated to subsequently
trigger differentiation of totipotent blastomeres into trophoblast by
the differential activation of the protein through the Hippo
signaling pathway (Nishioka et al., 2008; Nishioka et al., 2009). In
the present study, we attempted to knock down Tead4 by the same
RESEARCH ARTICLE 4167
siRNA approach that we used to knock down Cdx2. After
specifically and efficiently knocking down Tead4 transcripts, we
found that none of the treated embryos was capable of initiating
cavitation, consistent with the results of the two previous Tead4
knockout studies. In these Tead4–/– embryos, the maternal pool of
Cdx2, which was not at all affected, did not sustain the embryos
through the early stages of development to support initiation of TE
separation in the positive-feedback loop manner proposed by M.
Zernicka-Goetz’s group. Our Tead4 knockdown results provided
another solid piece of evidence to support the validity of our Cdx2
knockdown approach and confirmed that maternal Cdx2 does not
lead the cascade that regulates TE lineage specification.
Notably, Nishioka et al. (Nishioka et al., 2008) performed a
trophoblast outgrowth assay and showed that all the TE-specific
genes examined, including Cdx2, Eomes, Fgfr2 (Arman et al.,
1998), integrin alpha 7 (Itga7) (Klaffky et al., 2001), placental
cadherin (Cdh3) (Niwa, T. et al., 2005) and the giant-cell-specific
genes Hand1 (Riley et al., 1998) and Prl3d1 (Faria et al., 1991),
were not expressed in Tead4–/– embryos corresponding to the late
blastocyst stage. Similarly, expression of Hand1 and Prl3d1 was
detected in Cdx2+/+ and Cdx2+/– embryos but not in Cdx2–/–
embryos after 72 hours of culture of 3.5 dpc embryos (Strumpf et
al., 2005). On the contrary, another Tead4 knockout study (Yagi et
al., 2007) concluded that Tead4 was required for expression of
some, but not all, TE-specific genes, as expression of Fgfr2 was
not affected in Tead4–/– embryos. Our qRT-PCR analysis (Fig. 6C)
showed significant downregulation of TE-associated genes (Cdx2,
Eomes, Atp1b1 and Fgfr2) and significant overexpression of
Hand1. These results, together with previous observations that
Tead4–/– embryos weakly and briefly express Cdx2 between the 8and 18-cell stages, and that they appear to have normal adherens
junctions, cell polarities and JNK and p38 MAPK signaling
(Nishioka et al., 2008), lead us to propose the existence of
additional, Cdx2- and Tead4-independent mechanisms in TE
lineage specification, which might include the subcortical maternal
complex (SCMC) and its component Floped (also known as
Moep19 or Ooep) in the mouse oocyte cortex (Herr et al., 2008; Li
et al., 2008). Although the SCMC does not appear to be sufficient
for establishing the TE lineage, the persistence of the SCMC in the
outer cells of E3.5 Tead4-null embryos indicates that it might be
present in early mouse embryos as an intrinsic pre-patterning factor
related to blastomere polarity and TE specification (Li et al., 2008).
As illustrated in Fig. 7, based on the results of this study we
confirm the findings of recent studies and clarify conflicting studies
by concluding that although Cdx2 plays a crucial role in the
maintenance of TE differentiation and function, neither maternal
nor zygotic Cdx2 expression is essential for initiating the
segregation of the ICM and TE lineages in the early stages of
mouse embryonic development.
Acknowledgements
We thank Jeanine Mueller-Keuker, Margit Preusser and Susanne Koelsch for
assistance in preparing the manuscript and Dr D. Rancourt for providing the
ISP1 antibody. The content of this manuscript is solely the responsibility of the
authors and does not necessarily represent the official views of the Eunice
Kennedy Shriver National Institute of Child Health & Human Development or
the National Institutes of Health. This research was supported by the Max
Planck Society and grant NIH R01HD059946-01 from the Eunice Kennedy
Shriver National Institute of Child Health & Human Development. This work
was also supported in part by DFG grant Germ Cell Potential DFG FOR 1041
SCHO 340/7-1. Deposited in PMC for release after 12 months.
Competing interests statement
The authors declare no competing financial interests.
DEVELOPMENT
Cdx2 in trophectoderm specification
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.056630/-/DC1
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