Initiation of trophectoderm lineage ... - Development · DEVELOPMENT AND STEM CELLS RESEARCH...

4159DEVELOPMENT AND STEM CELLS RESEARCH ARTICLE

INTRODUCTIONThe first visible cell lineage split in the development of themammalian embryo occurs during blastulation. The cells of theinner part of the blastocyst, called the inner cell mass (ICM), arepluripotent and eventually give rise to the embryo proper and to theextra-embryonic tissues. By contrast, the cells of the outer layerdifferentiate into an epithelium, called the trophectoderm (TE),which subsequently develops into the placenta. To date, a fullunderstanding has been lacking of the molecular mechanisms thatunderlie the fate decision for the initial totipotent cells of theembryo to become either the TE or ICM lineage.

Cdx2 is a class I homeobox transcription factor that belongs tothe caudal-related homeobox gene family, which comprisesmammalian homologs of Drosophila caudal (James et al., 1994;Suh et al., 1994). Cdx family members function as upstreampositive regulators of Hox genes (Lorentz et al., 1997), which arepurported to confer positional identity to cells along theanteroposterior body axis and are sequentially activated in atemporal and spatial manner (Davidson et al., 2003). Loss of Cdxfunction has been shown to be associated with inactivation of Hoxgene expression (Davidson et al., 2003). Cdx1 and Cdx2, which areregulated by p38 MAPK (Mapk14) (Houde et al., 2001), areinvolved in defining the anteroposterior body axis(Chawengsaksophak et al., 1997; Chawengsaksophak et al., 2004)

and in establishing anteroposterior patterning of the intestine(Silberg et al., 2000). Dysregulation of Cdx2 expression has beenfound in intestinal metaplasia and carcinomas (da Costa et al.,1999; Bai et al., 2002; Eda et al., 2003). In colon cancer cells, forexample, oncogenic ras downregulates Cdx2 expression byactivating protein kinase C (PKC) pathway signaling and byreducing activity of the Cdx2 promoter AP-1 site through changesin the relative expression of c-June/c-Fos (Lorentz et al., 1999); bycontrast, in the embryo, ras-MAPK signaling activates Cdx2expression (Lu et al., 2008). Most importantly, one study found thatalthough Cdx2 expression appears to be required for TE cell fatespecification in the early mouse embryo, Cdx2 zygotic mutants canstill 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 somaticcells using nuclear transfer, failed to implant into the uterus, justlike Cdx2–/– embryos (Meissner and Jaenisch, 2006). However,neither study eliminated maternal Cdx2 expression. More recentstudies have found that Tead4 acts upstream of Cdx2 and isessential for TE specification and blastocyst formation (Yagi et al.,2007; Nishioka et al., 2008), although a subsequent studycontradicted these findings by reporting that maternal Cdx2 isresponsible for compaction and TE lineage initiation (Jedrusik etal., 2010). Therefore, although it is well established that Cdx2 isindispensable for the maintenance and proper functioning of the TElineage, its role in the initiation of TE lineage specification remainsobscure. It is therefore imperative to study the full effect of Cdx2expression in early mammalian embryonic development byeliminating both maternal and zygotic Cdx2 transcripts.

In the present study, we microinjected a robust Cdx2 smallinterfering (si) RNA duplex into zygotes and metaphase II (MII)oocytes and determined the effects of eliminating both maternaland zygotic expression of Cdx2 on the initiation of ICM/TE lineage

Development 137, 4159-4169 (2010) doi:10.1242/dev.056630© 2010. Published by The Company of Biologists Ltd

1Department of Cell and Developmental Biology, Max Planck Institute for MolecularBiomedicine, Röntgenstrasse 20, 48149 Münster, Germany. 2BioResource Center,RIKEN Tsukuba Institute, Ibaraki 305-0074, Japan. 3University of Münster, MedicalFaculty, Domagkstr. 3, 48149 Münster, Germany.

*Author for correspondence ([email protected])

Accepted 11 October 2010

SUMMARYThe separation of the first two lineages – trophectoderm (TE) and inner cell mass (ICM) – is a crucial event in the development ofthe early embryo. The ICM, which constitutes the pluripotent founder cell population, develops into the embryo proper, whereasthe 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, aslack of zygotic Cdx2 expression leads to a failure of embryos to hatch and implant into the uterus. However, speculation exists asto whether maternal Cdx2 is required for initiation of TE lineage separation. Here, we show that effective elimination of bothmaternal 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-deficientembryos. However, these embryos exhibited low mitochondrial activity and abnormal ultrastructure, indicating that Cdx2 plays akey role in the regulation of TE function. Furthermore, we found that embryonic compaction does not act as a ‘switch’ regulatorto turn on Cdx2 expression. Our results clearly demonstrate that neither maternal nor zygotic Cdx2 transcripts direct the initiationof ICM/TE lineage separation.

KEY WORDS: Cdx2, Trophectoderm, Lineage specification, Preimplantation mouse embryos

Initiation of trophectoderm lineage specification in mouseembryos is independent of Cdx2Guangming 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,*

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separation and cellular differentiation. As Cdx2 acts downstreamof Tead4, we also targeted Tead4 with siRNA to further confirmthese results. The findings of this study help to better define theroles played by Cdx2 during early mammalian development.

MATERIALS AND METHODSEmbryo culture and microinjection of siCdx2 duplexFertilized oocytes were collected from the oviducts of primed B6C3F1female mice after mating with CD1 male mice 18 hours post-hCG in M2medium. Oocytes were cultured in KSOMAA (potassium simplex optimizedmedium plus 19 natural amino acids) (Ho et al., 1995) at 37°C and 5% CO2

in air until microinjection. For MII oocyte microinjection, mature oocyteswere collected from the oviducts of primed B6C3F1 female mice 14 hourspost-hCG in M2 medium, injected with siCdx2, and then fertilized in vitroin modified KSOM (Summers et al., 2000) with epididymal spermatozoafrom adult OG2 male mice.

We used the online tool BLOCK-iT RNAi Designer to select specifictarget 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 themanufacturer’s instructions and stored in single-use aliquots at –20°C. Wetested three regular oligonucleotides and three Stealth RNAioligonucleotides containing the coding region of Cdx2 (target sequence 5�-GCAGTCCCTAGGAAGCCAA-3�) in zygotes and selected the mosteffective duplex (siRNA3) for use in all our experiments. Unless otherwisespecified, a scrambled siRNA duplex was used as control (target sequence5�-GCACCCGATAAGCGGTCAA-3�).

siRNAs were microinjected using an Eppendorf FemtoJet microinjectorand Narishige micromanipulators. Microinjection pipettes were pulled witha Sutter P-97 pipette puller. siRNA solution (5 ml of 8 mM) was loaded intothe pipette and ~2 pl was injected into the cytoplasm of each oocyte. Arelatively consistent amount was carefully injected each time. Specifically,from 73 separate experiments, 3894 of 4093 zygotes (95.1%) weresuccessfully injected with Cdx2 siRNA, in comparison with 3846 of 4065zygotes (94.6%) successfully injected with scrambled siRNA.

In addition, from 18 separate experiments, 345 of 1202 MII oocytessuccessfully injected with Cdx2 siRNA were fertilized in vitro, incomparison with 207 of 743 MII oocytes successfully injected withscrambled siRNA and fertilized in vitro and with 327 of 415 uninjectedMII oocytes that were fertilized in vitro. Unsuccessfully injected oocytesor lysed oocytes were excluded from all subsequent experiments.

Following microinjection, oocytes were washed and cultured inKSOMAA at 37°C and 5% CO2 in air and evaluated for cleavage twicedaily. The implantation capability of early-stage embryos was evaluated bytransfer of embryonic day (E) 3.5 mouse embryos into the uterus of a 2.5days post-coitum (dpc) pseudopregnant CD1 female mouse.

Animal care was in accordance with the institutional guidelines of theMax Planck Institute.

RNA extraction, cDNA synthesis and real-time reversetranscription PCRFor real-time analysis of gene expression, embryos were harvested in RLTbuffer (Qiagen, Hilden, Germany) at different stages of development andprocessed as previously described (Boiani et al., 2005). Briefly, total RNAwas extracted from individual blastocysts using the MicroRNeasy Kit(Qiagen) and cDNA synthesis was performed with the High-CapacitycDNA Archive Kit (Applied BioSystems, Darmstadt, Germany) accordingto the manufacturer’s instructions.

Real-time PCR was carried out on the ABI PRISM 7900HT SequenceDetection System (Applied BioSystems) using TaqMan probes (AppliedBiosystems) for the housekeeping gene Hprt1 (Mm00446968_m1) as acontrol 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�-AACCAGTGGTTGAATA -CTAGCAATG-3�; PR, 5�-CTGCAATGGATGCTGGGATACT-3�; andprobe, 5�-6FAM-TTCAGAAGGGCTCAGCAC-MGB-3�. Three to sixbiological replicates were used and each sample was run with threetechnical replicates; negative controls lacked reverse transcriptase ortemplate. The cycle threshold (CT) values were collected using AppliedBiosystems SDS v2.0 software and transferred to a Microsoft Excelspreadsheet 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 peakmethod, which is an adaptation of the DDCT method (Wang et al., 2004).

Immunofluorescent staining of embryosImmunocytochemical staining was performed as described previously withminor modifications (Strumpf et al., 2005). Briefly, samples were fixed in4% paraformaldehyde for 20 minutes, permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, St Louis, MO, USA) for 1 hour, and blocked with 3%BSA in PBS for 1 hour. Control and Cdx2-deficient embryos wereprocessed and examined in parallel. Samples were incubated with thefollowing antibodies: monoclonal mouse anti-Cdx2 IgG (1:100; BioGenex,San Ramon, CA, USA), rabbit polyclonal anti-Cdx2 [1:2000; gift fromFelix 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 ourlaboratory (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 byDr D. Rancourt (O’Sullivan et al., 2001)], all at 4°C overnight, or withrabbit anti-ZO-1 IgG (1:150; Zytomed, Berlin, Germany) at 37°C for1 hour.

Embryo samples were then washed several times with PBS and stainedwith Alexa 488-conjugated goat anti-rabbit IgG, Alexa 488-conjugatedgoat anti-mouse IgG (Invitrogen), Cy3-conjugated goat anti-rat IgG, orCy3-conjugated goat anti-mouse IgG (Jackson Laboratory, Bar Harbor,ME, USA) at 1:400 dilution for 1 hour at room temperature. After furtherwashes and counterstaining with 5 mM DRAQ5 (Biostatus, Shepshed, UK)for 30 minutes, samples were examined under a laser-scanning confocalmicroscope (UltraVIEW; PerkinElmer Life Sciences, Jügesheim,Germany) with 488, 568 and 647 nm lasers. ECCD-2 immunostaining wasquantified with ImageJ 1.42 software (http://rsbweb.nih.gov/ij/download.html). Troma-1 antibody and DRAQ5 staining were used todetermine the ICM:TE cell number ratio by comparing confocal images ofneighboring sections using a 5 mm scanning distance.

Transmission electron microscopyCells were fixed in 2.5% glutaraldehyde (Merck, Darmstadt, Germany) in0.1 M sodium cacodylate buffer (pH 7.4), post-fixed in 1% aqueous osmiumtetroxide, dehydrated in ethanol and embedded in Epon 812 (Fluka, Buchs,Switzerland). Ultrathin sections (50 nm) were prepared with an EM UC6ultramicrotome (Leica Microsystems, Wetzlar, Germany), stained with 1%uranyl acetate and then 3% lead citrate, and subsequently examined under aZeiss EM 109 electron microscope (Carl Zeiss, Oberkochen, Germany).

Measurement of mitochondrial membrane potential and ATPcontentMitochondrial 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 wereincubated in KSOMAA containing 5.0 mg/ml JC-1 for 15 minutes at 37°C inthe dark. Cells were washed with KSOMAA, excited at 488 nm, and thenobserved at either 510 nm (green mitochondria) or 590 nm (red-to-orangemitochondria) with the confocal imaging system. The ATP content of eachembryo was measured as previously described (Boiani et al., 2004).

Induction and inhibition of embryonic compactionTo induce compaction, 4-cell stage embryos were transferred intoKSOMAA containing 200 mM DiC8 (Sigma-Aldrich) and incubatedovernight at 37°C. To prevent compaction, precompacted 8-cell stage

RESEARCH ARTICLE Development 137 (24)

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embryos were cultured in KSOMAA containing 200 mg/ml anti-E-cadherinantibody (ECCD-1; Calbiochem). Embryo samples were collected after 18and 24 hours of treatment and subjected to immunocytochemical analysisfor Cdx2 expression.

Measurement of DNA fragmentation by TUNEL assayThe degree of apoptosis in embryo samples was determined using terminaldeoxynucleotidyl transferase (TdT)-mediated dUTP-FITC nick endlabeling (TUNEL) with an in situ cell death detection kit (DeadEndFluorimetric TUNEL System; Promega, Madison, WI, USA) according tothe manufacturer’s recommendations. Cells were then viewed/countedunder a confocal microscope.

Statistical analysisThe Jarque-Bera test was performed on the ICM:TE cell number ratio andapoptotic cell numbers, revealing that the cell number did not follow anormal, or Gaussian, distribution with a significance level 0.05.Therefore, the Wilcoxon rank-sum test was performed as an appropriatenon-parametric statistical test with significance level 0.05 to comparedifferences in cell number.

RESULTSMaternal and zygotic expression of Cdx2We examined Cdx2 mRNA and protein levels in developing earlymouse embryos by quantitative real-time PCR (qRT-PCR) andimmunocytochemistry. Maternal Cdx2 mRNA was readilydetectable in oocytes (CT value of 36.5 from pools of ten MIIoocytes) and 2-cell stage embryos, but levels were reduced by 73%in 4-cell stage embryos and increased by 23-fold upon Cdx2activation in 8- to 16-cell stage embryos (Fig. 1A). As previouslyreported (Niwa, H. et al., 2005; Strumpf et al., 2005; Dietrich andHiiragi, 2007; Yagi et al., 2007), Cdx2 protein levels could only bedetected as a very weak nuclear signal in 8-cell stage embryos (Fig.1C); however, a stronger Cdx2 protein signal was detected in someblastomeres during the next cell cycle, gradually extending to allouter blastomeres (see Fig. S1 in the supplementary material). Thisexpression profile provides evidence for the presence of maternalCdx2 transcripts in early mouse embryos and indicates thatactivation of zygotic Cdx2 expression is concomitant with the firstlineage differentiation event during the asymmetric fourth divisionafter compaction in 8-cell stage mouse embryos (Johnson andMcConnell, 2004).

Effective reduction in maternal and zygotic Cdx2by RNAi has profound effects on TE celldifferentiation, function and embryodevelopmental potentialOf the six Cdx2-specific siRNA duplexes tested, siRNA3 wasselected for subsequent experiments owing to its unique sequenceand high efficiency (see Fig. S2A in the supplementary material);siRNA3 is hereafter referred to as siCdx2. A scrambled siRNAduplex (siControl) with the same composition as siCdx2 but nospecific target was used as control. A single injection of siCdx2into MII oocytes or zygotes was found to reduce Cdx2 mRNAlevels by at least 95%, as determined by qRT-PCR (Fig. 1B).Furthermore, siCdx2 injection also led to an efficient reduction inCdx2 protein to undetectable levels in each of the 8-cell (siControl,n26; siCdx2, n28), morula (siControl, n26; siCdx2, n25) andblastocyst stage (siControl, n71; siCdx2, n67) embryos, asdetermined by three independent immunocytochemistryexperiments (Fig. 1C,D; see Fig. S2B in the supplementarymaterial) and a western blot with replicates (see Fig. S2C in thesupplementary material). The robust elimination of both maternaland zygotic Cdx2 expression did not affect the rate of blastocyst

formation (see Table S1 in the supplementary material). As shownin our time-lapse observations (see Fig. S2D in the supplementarymaterial), compaction and the onset of blastocyst formation wereunaffected in siCdx2-treated embryos. However, most likely owingto a defect in tight junctions, Cdx2-deficient blastocysts could notinitially maintain the integrity of the blastocyst cavity, collapsedthree times and were delayed by ~7.5 hours in starting cavityexpansion, but were of similar size and gross morphology tosiControl embryos at 4.0 dpc. Then, after 5-8 pulses of collapse andexpansion, all Cdx2-deficient embryos eventually collapsed in thezonae pellucidae between 5.5 and 6.0 dpc, rather than hatching likecontrol blastocysts.

To exclude the possibility that any residual maternal Cdx2protein could have driven TE lineage separation and blastocystformation in siCdx2-treated embryos, we co-injected siCdx2 witha rabbit polyclonal Cdx2 antibody at 100-fold higher concentrationthan the effective concentration for immunostaining, but stillobserved no phenotype prior to the blastocyst stage (see Table S2in the supplementary material).

In addition, an increase in the ICM:TE cell number ratio wasfound (0.58 versus 0.37; siControl, n40; siCdx2, n41;P1.05�10–5), but the total number of cells in Cdx2-deficient E4.0blastocysts was not different from that of control blastocysts (66.3versus 66.9; P0.64; Fig. 1H). TUNEL analysis revealed thepresence of slightly more apoptotic cells within both the inner (5.4versus 2.7; P7.35�10–5) and outer (2.9 versus 1.8; P3.48�10–4;siControl, n19; siCdx2, n13) cells of Cdx2-deficient blastocysts(Fig. 1I). However, transfer of Cdx2-deficient blastocysts into theuterus of pseudopregnant foster mice did not lead to anyimplantation (0 implanted out of 109 transferred in the siCdx2-treated group versus 60 out of 108 in the siControl, from threeseparate experiments), constituting another specific phenotype ofthe zygotic Cdx2–/– embryos.

Gene expression analysis by qRT-PCR revealed that knockdownof maternal Cdx2 led to a 77.5% reduction in the transcript levelsof the TE-associated gene Eomes in 8-cell stage embryos, whereasa reduction in the Fgfr2 transcript and an increase in the transcriptsof the pluripotency gene Oct4 (Pou5f1 – Mouse GenomeInformatics) 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 celldifferentiation into trophoblast giant cells (Cross et al., 1995), andGrn, which is an important factor for blastocyst hatching, adhesionand outgrowth (Qin et al., 2005), were overexpressed in Cdx2-deficient blastocysts (Fig. 4).

To determine why the normally shaped Cdx2-deficientblastocysts failed to hatch from the zonae pellucidae, we performedgene expression studies with qRT-PCR or immunocytochemistryon E4.0 Cdx2-deficient blastocysts. We found that Grn (anautocrine growth factor) and ISP1 (a tryptase; Prss28 – MouseGenome Informatics), which have been reported to play crucialroles in the hatching process (O’Sullivan et al., 2001; Qin et al.,2005), were not reduced in Cdx2-deficient blastocysts (Fig. 4; seeFig. S3B in the supplementary material). However, furtherexamination revealed the cause of this hatching failure.Immunocytochemical analysis of embryos using the tight junction-associated protein zona occludens 1 (ZO-1; Tjp1 – Mouse GenomeInformatics) showed that the apical zonular structure of the tightjunctions was discontinuous in siCdx2-treated embryos (Fig. 1F),indicating compromised intercellular sealing and epithelialintegrity. This specific phenotype was previously observed inexperiments with mouse Cdx2–/– embryos (Strumpf et al., 2005).

4161RESEARCH ARTICLECdx2 in trophectoderm specification

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Moreover, we also observed the presence of the zonular form ofZO-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 blastocystsunder the electron microscope (Fig. 1G) showed that the tightjunction structure was actually missing, although ZO-1 protein was


Fig. 1. Efficient reduction of Cdx2 mRNA and protein levels in different preimplantation stage mouse embryos by siCdx2 results inphenotypes similar to Cdx2 knockout. (A)Relative Cdx2 mRNA expression levels as a percentage of peak expression in mouse embryos at differentpreimplantation stages, as assessed by qRT-PCR. The maternal mRNA decreased at the 4-cell stage, followed by upregulation at the 8-cell stage due tothe 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 siCdx2-treated zygotes at different preimplantation stages demonstrated robust downregulation of Cdx2 by siCdx2 treatment. KD, knockdown. (C)Confocalimages of 8-cell stage embryos immunolabeled with anti-Cdx2 monoclonal antibody illustrate the reduction of Cdx2 protein (green) by siCdx2treatment. Red, nuclear counterstaining with DRAQ5. (D)Confocal images of blastocyst stage embryos immunolabeled with anti-Cdx2 (green) and anti-Oct4 (red) antibodies. Following siCdx2 treatment, Cdx2 was eliminated, but Oct4 was ectopically expressed in the trophectoderm (TE). (E)DIC imagesof E4.0 and E6.0 embryos. All siCdx2-treated embryos failed to hatch. Arrows indicate empty zonae pellucidae left by hatched control embryos. Scalebar: 100mm. (F)Confocal images of ZO-1 (green) and E-cadherin (red) immunohistochemistry show normal E-cadherin distribution, which marks thelateral-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 (arrowheadand 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 totalcell number remained unchanged (P0.64) upon Cdx2 depletion. (I)Quantitation of apoptotic cells shows an increase in apoptotic activity in a Cdx2-deficient E4.0 blastocyst,. MII, metaphase II oocyte; ZP, zona pellucida. Error bars indicate standard deviation.

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detected by immunocytochemistry. However, the ultrastructure oflateral-basal adherens junctions was present as in control embryos(Fig. 1G). Immunocytochemical analysis of embryos revealed astronger intensity of E-cadherin, a major component of adherensjunctions, at the basolateral border of TE cells in Cdx2-deficientembryos than in control embryos (Fig. 1F; P<0.01), without anincrease in mRNA levels (Fig. 4). Therefore, although adherensjunctions could compensate for the lack of tight junctions andsupport delayed blastocoele formation and expansion in themutated embryos, they failed to maintain intercellular sealing forthe more demanding process of hatching.

Finally, to further confirm the identity of the outer cells, weperformed immunocytochemical analysis of blastocysts for keratin8 (Krt8; also known as EndoA), a widely used marker fordifferentiated TE, with the monoclonal antibody Troma-1 (Brulet etal., 1980; Ralston and Rossant, 2008). We found that Krt8 waspresent in the outer cells of Cdx2-deficient blastocysts, albeit with anuneven distribution, clearly showing that establishment of the TElineage was independent of Cdx2 at the molecular level (Fig. 2C).Our time-lapse analysis revealed that Cdx2-deficient embryoscompacted and initiated cavitation at the correct time and only failedto hatch afterwards. In addition, the zonular form of ZO-1 was foundto be present at the apical sides of the outer cells and certain TE-associated genes (Hand1 and Grn) were actually overexpressed inCdx2-depleted embryos. Taking all these data together, we concludethat elimination of both maternal and zygotic Cdx2 expression didnot block the initiation of ICM/TE lineage separation, but it didimpact the continuation of TE differentiation and function.

Effect of Cdx2 knockdown on mitochondrialactivityUpregulation of mitochondrial activity is a feature of TEdifferentiation. The JC-1 assay was performed to explore theimpact of Cdx2 expression on mitochondrial function and revealeda significant reduction in mitochondrial activity at the apical cortexof 8-cell embryos and TE cells of blastocysts, indicating the

presence of a functionally defective TE (Fig. 3A). This finding wascorroborated by the underexpression of genes involved in both ATPsynthesis (Atpaf1) and consumption (Atp1b1) (Fig. 4A,B) in Cdx2-deficient embryos, as determined by qRT-PCR. However, anincrease in cytoplasmic ATP content was found in blastocyst stageembryos that had Cdx2 knockdown-induced mitochondrialdysfunction, as compared with wild-type blastocysts (0.41 versus0.17 pmol per embryo; Fig. 3B).

siCdx2 microinjection into MII oocytes produces aphenotype identical to that of zygotesOur results showed that microinjection of siCdx2 into MII oocytesalso led to an efficient reduction in Cdx2 mRNA expression levels(Fig. 4A), with no protein detectable in blastocyst stage embryos(siControl, n61; siCdx2, n52) by immunostaining. Changes inthe gene expression profile at E4.0 were similar to those observedafter injection of siCdx2 into zygotes (Fig. 4B), as determined byqRT-PCR. Injection of siCdx2 into MII oocytes led to ectopicexpression of Oct4 and Nanog proteins in the outer cells of Cdx2-deficient 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 pointresulted in the same phenotype and gene expression changes as atthe zygote stage, further supporting the conclusion that theinitiation of compaction and separation of ICM/TE in Cdx2-deficient embryos was not due to Cdx2 protein produced frommaternal mRNA before the mRNA was knocked down.

Embryonic compaction does not lead to activationof Cdx2 expressionTo evaluate the effect of embryonic compaction on the activationof Cdx2 expression, we used two complementary approaches. First,we induced compaction in 4-cell stage embryos with the naturalPKC agonist DiC8 (Pauken and Capco, 1999). Embryos at thisstage of normal development do not initiate compaction. However,upon treatment with DiC8, 4-cell stage embryos rapidly initiated


Fig. 2. Significant effect of Cdx2 reduction on geneexpression at various developmental stages but noblockage of expression of the differentiated TE markerKrt8. (A)Relative gene expression levels as a percentage ofpeak expression of Cdx2, Eomes, Fgfr2 and Oct4 in mousepreimplantation embryos after siControl and siCdx2 injectioninto zygotes. The downstream transcription factor Eomes wasreduced to a basal level. Fgfr2 reduction is shown at the earlyand 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) Ectopicexpression of Nanog in the TE of Cdx2-deficient blastocystsafter microinjection of siCdx2 at the zygote stage. Theembryonic stem cell marker Nanog was ectopically expressed inTE, as shown in this z-stack confocal image of animmunostained siCdx2-treated blastocyst. (C)Sectional confocalimage showing the presence of Krt8 (as detected by the Troma-1 antibody) in an interrupted pattern in Cdx2 knockdownblastocysts.

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compaction, as seen by the flattening and adherence ofblastomeres, which became well compacted within 30 minutes(Fig. 5A). Cdx2 expression could not be detected after 18 hours oftreatment by immunocytochemistry (Fig. 5B; DiC8, n21; Control,n19). Using the second approach, we blocked the compaction of8-cell stage embryos (Fig. 5C) with ECCD-1, an antibody directedagainst the extracellular domain of E-cadherin (Yoshida-Noro etal., 1984), and examined Cdx2 expression after 24 hours in 12- to16-cell stage embryos (Fig. 5D; ECCD-1, n20; Control, n21).Although there was no compaction, Cdx2 expression could still bedetected in 12-cell stage embryos. These results clearlydemonstrate that the compaction process does not lead to activationof Cdx2 expression in mouse embryos.

Reduction in Tead4, which is upstream of Cdx2,prevents the cavitation and separation of the ICMand TE but does not prevent compactionWe tested the efficiency of four Tead4-specific siRNA duplexes toknock down Tead4 mRNA expression levels by the samemicroinjection approach that we used for Cdx2 knockdown.Injected oocytes were cultured for 72 hours to reach themorula/blastocyst stage and collected individually or in groups forqRT-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.,

2007; Nishioka et al., 2008), these two duplexes were found toeffectively prevent embryo cavitation and clear separation of theICM and TE (Fig. 6B), but had no effect upon compaction at 2.5dpc (see Fig. S4 in the supplementary material), just as with Cdx2knockdown embryos. Significant underexpression of TE markergenes (Cdx2, Eomes, Fgfr2 and Atp1b1), but not pluripotency-related genes (Oct4, Nanog and Sox2), was observed by qRT-PCRupon Tead4 knockdown (Fig. 6C).

DISCUSSIONIn the present study, we defined the expression profile of Cdx2 inmouse oocytes and embryos by the sensitive and specific techniqueof qRT-PCR and systematically investigated the impact ofknocking down both maternal and zygotic mRNA levels by ansiRNA approach on the development of preimplantation embryos.The consistency in gene expression profile and phenotype of thesiCdx2-treated embryos with those of the zygotic Cdx2-nullembryos (Strumpf et al., 2005), such as the failure of embryos tohatch from the zonae pellucidae and implant into the uterus, theunderexpression of Eomes, and the ectopic expression of Oct4 andNanog in the TE, demonstrates the efficacy and specificity of oursiCdx2 treatment.

Developmental biologists have sought for years to understandhow mammalian embryos establish their developmental pattern andinitiate their first differentiation event – the separation of the TE


Fig. 3. Significant effect of Cdx2 knockdown on mitochondrialactivity. (A)Confocal images showing mitochondrial activity asassessed by JC-1 staining at the 8-cell, morula and blastocyst stages.siCdx2 treatment significantly reduced mitochondrial activity, asindicated by the accumulation of the red aggregated form of JC-1 inthe mitochondria (appears yellow, as all mitochondria were stainedwith the green form of JC-1). (B)Measurement of ATP content inindividual embryos, showing a significant increase in ATP from 0.17pmol to 0.41 pmol in Cdx2-deficient blastocysts. Error bars indicatestandard deviation.

Fig. 4. Relative gene expression levels in E4.0 blastocysts aftermicroinjection of siCdx2 into MII oocytes and zygotes. Eliminationof Cdx2 RNA by microinjection of siCdx2 into mouse (A) MII oocytesand (B) zygotes had similar effects on gene expression levels, asassessed by qRT-PCR. Cdx2 knockdown reduced expression of genescrucial in TE lineage differentiation, with overexpression ofpluripotency-related genes. Error bars indicate standard deviation.

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from the ICM (Hiiragi et al., 2006). Cdx2, the first TE marker geneto be expressed in mouse embryos, was reported in a study byStrumpf and colleagues to be required for correct TE fatespecification (Strumpf et al., 2005). The zygotic Cdx2–/– embryosfailed to downregulate Oct4 and Nanog levels in the outer cells ofthe blastocyst, resulting in loss of epithelial integrity and in asubsequent failure to hatch and implant. However, these Cdx2-nullembryos were still capable of forming a TE, as evidenced bycavitation and blastocyst formation, a finding that contradicted themain conclusion of that study and the authors hypothesized that theexpression of maternal Cdx2 in these Cdx2–/– embryos might be

responsible for the initiation of TE lineage differentiation.However, the same group later proposed that Cdx2 does not leadthe TE transcription factor hierarchy (Rossant and Tam, 2009), asa loss-of-function Cdx2 mutation had no effect on the initiation ofblastocyst formation (Strumpf et al., 2005), and that Cdx2-mutantcells were not excluded from the TE layer in chimeric blastocysts(Ralston and Rossant, 2008). These cells, however, do not undergofurther trophoblast differentiation and thus are not functional(Strumpf et al., 2005).

In the present study, we used an siRNA approach to robustlyknock down both maternal and zygotic Cdx2 transcripts, and arethe first group to provide unequivocal and direct evidence thatmaternal Cdx2 is not a determinant of TE lineage commitment. Inthese maternal and zygotic Cdx2-deficient embryos, compactionand cavitation initiation were not interrupted, as clearly shown byour time-lapse observations (see Fig. S2D in the supplementarymaterial), even though the morula-to-blastocyst transformation wasdelayed as a consequence of a defect in ZO-1/tight junctions (Wanget al., 2008). The outer cells of Cdx2-deficient blastocystsexpressed the TE marker Krt8, confirming their TE identity.However, disorientation of the TE, as indicated by the presence oflinear ZO-1 at the base of the epithelium, also highlights theimportance of Cdx2 in cell polarization at the preimplantationstages. An increased ICM:TE cell number ratio might reflectcompromised TE proliferation and function in regulating thenumber of ICM cells. These embryos subsequently failed to hatch,implant into the uterus or survive, indicating that TE lineageseparation is independent of Cdx2 but that TE function supportingembryogenesis 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 thepresent study. In that study, the authors achieved a much lowerknockdown efficiency of maternal Cdx2 transcripts, of ~80-85%before the 8-cell stage, with Cdx2 protein levels still detectable insome of the treated embryos, but they reported a more severephenotype and claimed that the maternal pool of Cdx2 mRNAplayed a crucial role in compaction and polarization at the 8- to 16-cell stages of embryonic development. In our experiments, weachieved a knockdown efficiency of maternal Cdx2 transcripts ofgreater than 99.0%, without any detectable Cdx2 proteins, and yet88.4% of these embryos compacted and developed to the blastocyststage, with the same morphology and developmental rate as thoseinjected with scrambled siRNA (87%) or non-injected embryos(85.3%) (see Table S1 in the supplementary material). The failureof such embryos to hatch and implant and the formation ofembryoid body (EB)-like structures on mouse embryonicfibroblasts (MEFs) (Wu et al., 2010) were reported to bephenotypes specific to zygotic Cdx2–/– embryos by Rossant’s group(Strumpf et al., 2005), yet we did not observe any significant effecton compaction and blastocyst formation.

In the study by M. Zernicka-Goetz’s group, heterogeneousphenotypes were observed as embryos arrested at various stages ofdevelopment. Furthermore, their Cdx2-deficient embryos not onlytotally 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, whichcontradicts the findings of other publications, as a reciprocalrelationship between Nanog and Cdx2 has already been clearlyestablished (Chen et al., 2009) and overexpression of Nanog hasbeen observed in zygotic Cdx2 knockout experiments (Strumpf etal., 2005) as well as in the present study. In support of ourobservations, an independent group has reported that following


Fig. 5. Neither induction nor blockage of embryonic compactionaffects the activation of Cdx2 expression. (A)Prematurecompaction induced within 30 minutes of treatment with the PKCagonist DiC8 (right). Normal 4-cell stage mouse embryos are shown onthe left, with obvious, individual blastomeres. (B)Cdx2 expression (red)could not be detected by immunocytochemistry 18 hours after DiC8treatment. Nuclei are blue (DAPI) and Oct4 (a positive control fortranscriptional activity of the treated embryos) green. (C)Compactionwas inhibited by anti-E-cadherin (ECCD-1) antibody treatment for 24hours (right). Normally compacted 14-cell stage embryos are shown onthe left. (D)Cdx2 was detected (arrowhead) in uncompacted 12-cellstage embryos. Heavy cytoplasmic background (especially around thecell surface) was due to cross-reaction of ECCD-1 (rat IgG monoclonal),which was used at a very high concentration (200mg/ml) to blockembryo compaction, with the Cdx2 antibody (mouse IgG monoclonal).DEVELO

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injection of Cdx2-specific siRNA at the 1-cell stage, Cdx2-deficientembryos do not exhibit developmental arrest prior to the blastocyststage and do overexpress the pluripotent gene Oct4 at the blastocyststage (Wang et al., 2010). In a broader sense, research on primateshas also indicated that CDX2 plays an essential role in functionalTE formation and that the ICM lineage of CDX2-depleted embryossupports the isolation of functional embryonic stem cells(Sritanaudomchai et al., 2009), which is consistent with ourobservations in the mouse.

In the present study, we observed an increase in mitochondrialactivity concomitant with TE differentiation. This is consistent withthe involvement of Na+/K+-ATPase in mouse embryos preparingfor cavitation and production of nascent blastocoele fluid (Wiley,1984) and with the stage-specific translocation of activemitochondria during early porcine embryo development (Sun et al.,2001). Interestingly, TE cells have been reported to produce andconsume most of the ATP of the blastocyst (Houghton, 2006).Functional analysis with JC-1 staining showed reduced activity inthe mitochondria of Cdx2-deficient embryos. Further geneexpression analysis demonstrated that the reduction inmitochondrial activity was mediated by the underexpression ofgenes involved in both ATP synthesis and consumption. Krt8 hasbeen shown to modulate the shape, distribution and function ofhepatocyte mitochondria and to protect cells from apoptosis (Taoet al., 2009), suggesting that some of the phenotypes of Cdx2-deficient embryos might be mediated by the reduced and unevenKrt8 expression pattern that we observed. Reduction in Krt8expression has also been observed in zygotic Cdx2–/– embryos

(Ralston and Rossant, 2008). Of note, an increase in ATP contentwas consistently found in Cdx2-deficient embryos, in duplicateexperiments with triplicate tests, which was probably due tounderexpression of the ATP-dependent Na+ pump of the TE, amain consumer of ATP at this developmental stage (Houghton,2006). These data indicate that deficiency in Cdx2 has profoundeffects on mitochondrial activity and that Cdx2 is indeed requiredfor proper functioning of the TE. Further study is needed todetermine precisely how Cdx2 regulates mitochondrial activity andATP levels in the embryo.

Observations from classical embryonic experiments havesuggested that ICM/TE lineage separation is initiated byasymmetric cell divisions that act to establish the inner and outercell populations following compaction and polarization in 8-cellstage mouse embryos (Johnson and Rossant, 1981). Duringcompaction, changes at the cellular level include enhanced celladhesion between blastomeres resulting in the formation of asmooth ball of blastomeres, and the establishment of cellpolarity, as demonstrated by the formation of apical andbasolateral surfaces on the blastomeres (Hyafil et al., 1980; Butzand Larue, 1995; Ohsugi et al., 1996). At the next cell divisionafter compaction, the developmental fate of the embryo becomesrestricted, as outer blastomeres become destined to form the firstepithelial layer, the TE, and inner blastomeres are pluripotentand form the ICM (Tarkowski and Wroblewska, 1967; Johnsonand Rossant, 1981). Par3, Par6 and aPKC are components of anapical polarity complex that has been shown to influenceTE/ICM fate choice (Plusa et al., 2005). In contrast to the two


Fig. 6. Effective knockdown of Tead4 in preimplantation mouse embryos by microinjection of siRNA into zygotes reproduces theTead4–/– embryo phenotype. (A)Tead4 knockdown efficiency (%) determined by qRT-PCR in a single E4.5 mouse blastocyst in comparison to thescrambled siRNA control. Error bars indicate standard deviation. (B) Failure of blastulation 72 hours (E3.5) after microinjection of siTead4A andsiTead4B into mouse zygotes. In this particular experiment, 25 zygotes per group were successfully injected. Note that the phenotype is consistentand highly reproducible within the siTead4A and siTead4B groups (except for one embryo in each group that was blocked at the 2-cell stage), butwas variable in groups with lower Tead4 knockdown efficiency. (C)Tead4 knockdown reduced the expression of genes crucial in TE lineagedifferentiation but did not reduce the expression of pluripotency-related genes. Shown are qRT-PCR results from pools of six embryos, withbiological and technical triplicates.

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studies by M. Zernicka-Goetz’s group (Jedrusik et al., 2008;Jedrusik et al., 2010), other studies have shown that Cdx2appears to act downstream of the first lineage decision in theearly mouse embryo – i.e. following cell polarization – topromote TE cell fate determination in a cell-autonomous manner,suggesting that processes influencing lineage allocation ormorphogenesis might regulate Cdx2 expression along theinside/outside axis of the embryo (Ralston and Rossant, 2008).However, it is not yet clear whether compaction induces Cdx2expression. It is known that compaction can be inducedprematurely in 4-cell stage embryos by PKC activators (Winkelet al., 1990; Ohsugi et al., 1993; Pauken and Capco, 1999). Withthis approach, we showed that induction of compaction does notactivate Cdx2 expression. Moreover, blocking compaction withECCD-1, a monoclonal antibody that specifically neutralizes E-cadherin-mediated adhesion (Johnson et al., 1986) and inducesdisorientation of cell polarity in mouse early embryos (Fleminget al., 1989), does not prevent Cdx2 expression. These resultssuggest that Cdx2 expression and compaction are two parallelprocesses in regulating TE differentiation. This conclusion isconsistent with a very recent report from Rossant’s group thatdemonstrated that the epithelial integrity mediated by E-cadherinis not required for Cdx2 expression (Stephenson et al., 2010).

We next sought to confirm that even though we effectivelyknocked down maternal Cdx2 transcripts and attempted to blockthe function of any residual proteins with a Cdx2 antibody, we stillcould not confer an essential role for maternal Cdx2 in embryocompaction and initiation of the TE lineage, contrary to M.Zernicka-Goetz’s group (Jedrusik et al., 2010). Here, we addanother piece of evidence to support our conclusions. Tead4 hasbeen reported by two studies to act upstream of Cdx2 and to beessential for TE specification (Yagi et al., 2007; Nishioka et al.,2008). In these two studies, zygotic Tead4–/– embryos were foundto be devoid of the TE lineage, and expression of Tead4 wasactivated at the 2-cell stage and was postulated to subsequentlytrigger differentiation of totipotent blastomeres into trophoblast bythe differential activation of the protein through the Hipposignaling pathway (Nishioka et al., 2008; Nishioka et al., 2009). Inthe present study, we attempted to knock down Tead4 by the same

siRNA approach that we used to knock down Cdx2. Afterspecifically and efficiently knocking down Tead4 transcripts, wefound that none of the treated embryos was capable of initiatingcavitation, consistent with the results of the two previous Tead4knockout studies. In these Tead4–/– embryos, the maternal pool ofCdx2, which was not at all affected, did not sustain the embryosthrough the early stages of development to support initiation of TEseparation in the positive-feedback loop manner proposed by M.Zernicka-Goetz’s group. Our Tead4 knockdown results providedanother solid piece of evidence to support the validity of our Cdx2knockdown approach and confirmed that maternal Cdx2 does notlead the cascade that regulates TE lineage specification.

Notably, Nishioka et al. (Nishioka et al., 2008) performed atrophoblast outgrowth assay and showed that all the TE-specificgenes examined, including Cdx2, Eomes, Fgfr2 (Arman et al.,1998), integrin alpha 7 (Itga7) (Klaffky et al., 2001), placentalcadherin (Cdh3) (Niwa, T. et al., 2005) and the giant-cell-specificgenes Hand1 (Riley et al., 1998) and Prl3d1 (Faria et al., 1991),were not expressed in Tead4–/– embryos corresponding to the lateblastocyst stage. Similarly, expression of Hand1 and Prl3d1 wasdetected in Cdx2+/+ and Cdx2+/– embryos but not in Cdx2–/–

embryos after 72 hours of culture of 3.5 dpc embryos (Strumpf etal., 2005). On the contrary, another Tead4 knockout study (Yagi etal., 2007) concluded that Tead4 was required for expression ofsome, but not all, TE-specific genes, as expression of Fgfr2 wasnot 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 ofHand1. These results, together with previous observations thatTead4–/– embryos weakly and briefly express Cdx2 between the 8-and 18-cell stages, and that they appear to have normal adherensjunctions, cell polarities and JNK and p38 MAPK signaling(Nishioka et al., 2008), lead us to propose the existence ofadditional, Cdx2- and Tead4-independent mechanisms in TElineage specification, which might include the subcortical maternalcomplex (SCMC) and its component Floped (also known asMoep19 or Ooep) in the mouse oocyte cortex (Herr et al., 2008; Liet al., 2008). Although the SCMC does not appear to be sufficientfor establishing the TE lineage, the persistence of the SCMC in theouter cells of E3.5 Tead4-null embryos indicates that it might bepresent in early mouse embryos as an intrinsic pre-patterning factorrelated to blastomere polarity and TE specification (Li et al., 2008).

As illustrated in Fig. 7, based on the results of this study weconfirm the findings of recent studies and clarify conflicting studiesby concluding that although Cdx2 plays a crucial role in themaintenance of TE differentiation and function, neither maternalnor zygotic Cdx2 expression is essential for initiating thesegregation of the ICM and TE lineages in the early stages ofmouse embryonic development.

AcknowledgementsWe thank Jeanine Mueller-Keuker, Margit Preusser and Susanne Koelsch forassistance in preparing the manuscript and Dr D. Rancourt for providing theISP1 antibody. The content of this manuscript is solely the responsibility of theauthors and does not necessarily represent the official views of the EuniceKennedy Shriver National Institute of Child Health & Human Development orthe National Institutes of Health. This research was supported by the MaxPlanck Society and grant NIH R01HD059946-01 from the Eunice KennedyShriver National Institute of Child Health & Human Development. This workwas also supported in part by DFG grant Germ Cell Potential DFG FOR 1041SCHO 340/7-1. Deposited in PMC for release after 12 months.

Competing interests statementThe authors declare no competing financial interests.


Fig. 7. Postulated TE differentiation regulatory network. Cdx2 isnot required for the TE lineage specification but it is critical for furtherdifferentiation of TE. Interactions among the processes of compactionand cellular polarization could affect the distribution of Cdx2 along theinside/outside axis and influence tight junction formation andlocalization. These interactions could therefore affect ICM/TE lineageallocation. SCMC, subcortical maternal complex.

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Supplementary materialSupplementary material for this article is available athttp://dev.biologists.org/lookup/suppl/doi:10.1242/dev.056630/-/DC1

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Initiation of trophectoderm lineage ... - Development · DEVELOPMENT AND STEM CELLS RESEARCH...

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