A Resource for the Transcriptional Signature of Bona Fide ......ResearchArticle A Resource for the...

Research ArticleA Resource for the Transcriptional Signature ofBona Fide Trophoblast Stem Cells and Analysis of TheirEmbryonic Persistence

Georg Kuales1 Matthias Weiss1 Oliver Sedelmeier1

Dietmar Pfeifer2 and Sebastian J Arnold134

1Renal Department Centre for Clinical Research University Medical Centre Breisacher Strasse 66 79106 Freiburg Germany2Department of Hematology Oncology and Stem Cell Transplantation University Medical Centre Freiburg Germany3Institute of Experimental and Clinical Pharmacology and Toxicology University of Freiburg Freiburg Germany4BIOSS Centre for Biological Signalling Studies Albert Ludwigs University Freiburg Germany

Correspondence should be addressed to Sebastian J Arnold sebastianarnolduniklinik-freiburgde

Received 25 May 2015 Accepted 22 July 2015

Academic Editor Valerie Kouskoff

Copyright copy 2015 Georg Kuales et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Trophoblast stem cells (TSCs) represent the multipotent progenitors that give rise to the different cells of the embryonic portionof the placenta Here we analysed the expression of key TSC transcription factors Cdx2 Eomes and Elf5 in the early developingplacenta of mouse embryos and in cultured TSCs and reveal surprising heterogeneity in protein levels We analysed persistence ofTSCs in the early placenta and find that TSCs remain in the chorionic hinge until E95 and are lost shortly afterwards To define thetranscriptional signature of bona fide TSCs we used inducible gain- and loss-of-function alleles of Eomes or Cdx2 and 119864119900119898119890119904GFPto manipulate and monitor the core maintenance factors of TSCs followed by genome-wide expression profiling Combinatorialanalysis of resulting expression profiles allowed for defining novel TSC marker genes that might functionally contribute to themaintenance of the TSC state Analyses by qRT-PCR and in situ hybridisation validated novel TSC- and chorion-specific markergenes such as BokMtd Cldn26 Duox2 Duoxa2 Nr0b1 and Sox21 Thus these expression data provide a valuable resource forthe transcriptional signature of bona fide and early differentiating TSCs and may contribute to an increased understanding of thetranscriptional circuitries that maintain andor establish stemness of TSCs

1 Introduction

Trophectoderm (TE) and inner cell mass (ICM) are the firstcell lineages that are specified at the 16- to 32-cell (morula)stage around embryonic day 3 (E30) of mouse development(reviewed in [1 2]) Mutual negative feedback inhibition ofthe two key transcription factors Cdx2 and Oct4 establishesthe lineages of Cdx2-expressing TE and Oct4-positive ICMcells [3ndash6] Multiple signals including cell polarity cell-cell contacts positional information and Hippo signallingconverge on the phosphorylation state of Yes-associatedprotein (YAP) YAP forms a transcriptional complex withTEAD4 to activate Cdx2 transcription in predominantlyouter peripheral cells of the early embryo thus priming

the TE programme [4ndash13] Additional layers of regulationensure lineage restricted Cdx2 and Oct4 expression such asNotch-signalling which cooperates with TEAD4 to directlyregulate Cdx2 expression [14] Similarly the transcriptionfactor AP2120574 promotes Cdx2 expression and downregulatesHippo signalling [15] GATA3 acts in parallel with CDX2and downstream of TEAD4 to induce an overlapping set oftarget genes in the trophoblast lineage [16] Within the polarTEwhich is defined by its vicinity to the ICMat the blastocyststage (E35) a population of trophoblast stem cells (TSCs)is established and maintained by ICM-derived Fgf- andNodal-signals to generate the main cellular source for for-mation of the embryonic part of the placenta [13 17ndash20]Following initial specification of TE fate by Cdx2-functions

Hindawi Publishing CorporationStem Cells InternationalVolume 2015 Article ID 218518 13 pageshttpdxdoiorg1011552015218518

2 Stem Cells International

additional transcriptional regulators including Eomes [4]and Elf5 [21] initiate expression in the TE lineage or becomespecifically restricted to TE cells such as AP2120574 [22 23]Following implantation around E45 the polar TE givesrise to the extraembryonic ectoderm (ExE) which containsTSCs and the ectoplacental cone (EPC) which mediates theembryonic invasion into the decidual wall then connects theembryo to the maternal uterus TSCs retain their stem cellcharacteristics namely self-renewal capacity and multipo-tency only in the polar TE and the ExE while cells of themural TE lack supportive signalling from the ICM and thusdifferentiate to primary trophoblast giant cells (TGCs) [17]TSC maintenance is controlled by transcriptional circuitriesinvolving Elf5 [24 25] Ets2 [26] andAP2120574 [23 27] which actin positive feed forward loops tomaintainCdx2 andorEomesexpression After E75 the core set of TSC marker genesincluding Cdx2 Eomes Elf5 and Esrr120573 remain expressed inthe chorion in addition to other embryonic and extraembry-onic expression domains [24 28ndash30]

TSCs can be routinely isolated and cultured from E35blastocysts and E65 ExE explants under defined condi-tions [17] Assessing the potential to isolate cells with TSCcharacter beyond E65 Uy and colleagues have shown thatthe frequency to obtain TSC clones increases until the 4-somite pair stage (simE80 to E825) but then rapidly drops andno TSCs can be isolated following the 11-somite pair stagearound E85 to E875 [31] The T-box transcription factorEomes was previously used to mark TSCs through differentdevelopmental stages and expression of an EomesGFP BACtransgene was detected until E145 in the outer peripheryof the murine placenta [32] To date it was not clearlyshown until which embryonic stage cells with TSC characterare maintained in the TSC niche which is functionallydefined by high levels of Fgf and Tgf120573 signals [17 33] Inaddition toEomes [34] andCdx2 [35] also other transcriptionfactors share essential functions for TSC self-renewal andmultipotency including Elf5 [24] Esrr120573 [29] Ets2 [26] AP2120574[23 27 36] and Sox2 [37] A recent report demonstrated thatthe functional loss of the histone demethylase Lsd1 in TSCsresults in premature migration of TSCs from their nichedemonstrating additional requirements of proper epigeneticregulation for the propagation of TSCs [38 39]

In addition to their functional importance during TSCmaintenance Cdx2 [3 11] Eomes [3] Elf5 [25] Tead4 [11]AP2120574 [23] andGata3 [16] also evoke lineage conversion frommouse embryonic stem (mES) cells to the TE lineage whenoverexpressed (reviewed in [40]) While the transcriptionalprogramme involving Cdx2 Eomes Elf5 AP2120574 and Ets2is key for the maintenance of the trophoblast stemnessstate genetic deletions of each of these transcription fac-tors generate remarkable different embryonic phenotypesCdx2-deficiency results in implantation defects [41] Eomes-deficient embryos show early postimplantation [34] and thedeletions of Elf5 [24] Ets2 [26 42 43] or AP2120574 [36] lead topostgastrulation lethality This diversity in loss-of-functionphenotypes might indicate differential requirements of thesefactors for the stemness maintaining regulatory circuitryAlternatively different states of TSCs might exist with dif-ferential developmental potential and distinct requirements

of transcriptional regulation in analogy to different statesof pluripotency such as that found in naıve or primedpluripotent stem cells [39 44]

In the current study we have analysed the endoge-nous expression of key stemness-maintaining factors Cdx2Eomes and Elf5 in the chorion of gastrulation stage embryosto characterise TSCs by overlapping marker gene expressionWe traced Eomes expression through later gastrulation stagesand demonstrate that it is lost around E95 in the regionemanating from the chorionic hinge Surprisingly TSCmark-ers consistently show heterogenous partially nonoverlappingexpression in different areas of the late TSC niche poten-tially indicating different states of TSCs during placentaldevelopmentThe analysis of TSCs cultured under stemness-maintaining conditions additionally revealed heterogeneousTSC marker expression in vitro underscoring our findingsfrom embryonic analyses in the chorion To determine thetranscriptional state that describes undifferentiated bona fideTSCs and to monitor transcriptional changes during earlyTSC differentiation we used genetic tools to manipulateEomes and Cdx2 expression in TSC cultures followed bygenome wide transcriptional profiling First we generatedTSCs that harbour an 119864119900119898119890119904GFP reporter allele therebymarking bona fide TSCs that were enriched by fluorescenceactivated cell sorting (FACS) and forced towards differentia-tion by removal of stemness maintaining conditions Secondwe employed TSCs that allow for the inducible deletion ofEomes gene function and third we used inducible expressionof key TSC transcriptional regulators Cdx2 and Eomes inmouse ES (mES) cells for the identification of downstreamtarget genes Resulting differential expression profiles allowfor a detailed description of the TSC signature and thechanges during early differentiation which might be directlyor indirectly regulated by a combination of Fgf- and Tgf120573-signalling and the key regulatory factors of TSCs Cdx2 andEomesWe used resulting expression profiles to identify novelcandidate TSC marker genes at stemness state To validatethis approach a handful of differentially expressed geneswere selected and tested by qRT-PCRs of bona fide TSCsand during early differentiation Additionally expression ofnovel candidates was analysed in the TSC compartment ofpostimplantation embryos by in situhybridisation analysis Insummary this study characterises and describes the expres-sion signature of TSCs within the embryo and in culturedTSCs

2 Materials and Methods

21 Cell Culture Genetically modified TSCs were isolatedfromE35 blastocysts of animals carrying alleles for119864119900119898119890119904GFP

[45]119864119900119898119890119904CA [47] and R26CreER [58] according to standardprotocols [17] TSCs cultured in stemnessmaintaining condi-tions (SMC) were kept in 70 mouse embryonic fibroblast-(MEF-) conditioned TSC-medium (MCM) supplementedwith hrFGF4 and heparin (F4H) on MEFs [17] For dif-ferentiation conditions (DC) TSCs were cultured in TSC-medium without MCM and F4H on gelatinized plates with-out feeder cells [17] Cre-mediated excision of the conditional119864119900119898119890119904

CA allele was induced by administration of 1120583gmL

Stem Cells International 3

4-hydroxytamoxifen (Sigma Aldrich H7904) to the culturemedium for up to 3 days

To generate mES cells that inducibly express Cdx2 orEomes in response to doxycycline administration we per-formed inducible cassette exchange (ICE) using A2loxcremES cells [59] and the p2lox-V5 vector system using gatewaycloning (Invitrogen) [48] Expression was induced by admin-istration of 1120583gmL doxycycline (Fargon 137087) to the EScell culture medium composed of DMEM (Gibco 11960)15 ES cell-qualified FBS (Gibco 16141) 2mM L-glutamine(Gibco 25030) 50UmL penicillin50 120583gmL streptomycin(Gibco 15070) 01mM nonessential amino acids (Gibco11140) 1mM sodium pyruvate (Gibco 11360) 01mM 120573-mercaptoethanol (Sigma Aldrich M7522) and 1000UmLleukemia inhibitory factor (Merck Millipore ESG1107)

22 Flow Cytometry Single cell suspensions of 119864119900119898119890119904GFP

TSCs cultured in SMC were FACS-purified for GFPhigh cellsusing aMoFlo Legacy cell sorter (BeckmanCoulter) Selectedcells were reseeded in SMC without MEF feeder cells for 24 hbefore preparation of the RNA

23 Immunofluorescence Staining of E75ndashE145 Tissue Sec-tions Whole decidua (E65ndashE85) or placenta (E105ndashE145)was isolated from 119864119900119898119890119904GFP pregnant females on ice washedin PBS and fixed in 4 PFA at 4∘C for 1 h (E65 E75)or 3 h (E85ndashE145) Fixed tissues were rinsed in PBS andsuccessively transferred to 15 and 30 sucrose dissolvedin PBS until samples no longer floated Tissues were sub-sequently incubated in embedding medium (75 fish skingelatin 15 sucrose) for 1 h at 37∘C and snap frozen inliquid nitrogen Frozen blocks were cut at 8 120583m sectionson a cryostat (CM3050s Leica) and sections mounted onSuperFrost Plus slides (R Langenbrinck 03-0060) Washingsteps blocking incubation with primary and secondaryantibodies and DAPI staining were performed as describedfor immunofluorescence on TSCs

24 Immunofluorescence Staining on TSCs TSC cultureswere grown on coverslips coated with 01 gelatin brieflyrinsed in PBS and fixed in 4 PFA for 30min on ice Fixedcells were washed with PBS containing 01 Tween-20 (PBS-T) blocked for 1 h in PBS-T with 5 bovine serum albumin(BSA-T) and incubated with primary antibodies (Supple-mentary Table S1 available online at httpdxdoiorg1011552015218518) in 5 BSA-T over night at 4∘C After washing offthe primary antibodies samples were incubated for 45minat RT with conjugated secondary antibodies (SupplementaryTable S1) diluted in 5 BSA-T Cells were counterstained for10min at RT with DAPI diluted 1 1000 in PBS and mountedwith Fluoromount-G Fluorescent images were captured on aZeiss Axiovert 200M equipped with a 20x Plan-Apochromatobjective Displayed images were eventually merged andbrightness adjusted using Adobe Photoshop CS5 software

25 RNA Preparation for Microarray and Quantitative RT-PCR Total RNA was purified from TSC and ES cell culturedishes using the RNeasy Mini Kit (QIAGEN 74104) using350 120583L RLT lysis buffer per 6 cm culture dish On-column

DNaseI digestionwas applied andRNAeluted in 50 120583LRNasefree water yielding concentrations of 179ndash1686 ng120583L RNA

26 Microarray and Analysis of Microarray Data To identifygenes overrepresented in TSCs gene expression datasetsobtained from the microarray were sorted for highest nega-tive (119864119900119898119890119904GFP TSC differentiation and 119864119900119898119890119904CA) or positive(induced Eomes or Cdx2 expression) fold changes Resultinglists from induced Eomes deletion or induced Cdx2 orEomes expression were additionally filtered for genes thatwere regulated at least 15-fold when compared to respectivecontrols Only genes with 119901 values le 005 were consideredfor further analyses Finallymedian expression values ofmEScell- and TSC-based experimental subsets were calculated foreach gene Gene lists were compared by Venn diagrams usingthe Manteia data mining system (httpmanteiaigbmcfr)

27 Validation by Quantitative RT-PCR and Statistical Anal-yses For reverse transcription the QIAGEN QuantiTectReverse Transcription Kit (205311) was used according to theprotocol with an input of 10120583gRNAper sampleQuantitativePCR was carried out in triplicate from three independentexperiments using the Roche Light Cycler 480 (SN 1126)detection system and the Light Cycler 480 DNA SYBR GreenI Master kit (Roche 04707516001) and the primers listed inSupplementary Table S2 36B4 and 120573cat served as referencegenes mRNA expression levels over time were calculatedrelative to day 0 which was set to 1 Statistical analyses wereperformed using a two-sided Studentrsquos 119905-test and the standarderror of themeanwas calculated for each dataset consisting ofbiological triplicates in technical triplicates each individually

28 Validation by In Situ Hybridization Deciduae contain-ing embryos were dissected and fixed in 4 PFAin PBSdehydrated through ethanol series and embedded in paraffinbefore 8 120583M sections were prepared on a Leica RM2165microtome In situ hybridization on paraffin sectionswas per-formed according to standard protocols [60] using BokMtd-Cldn26- Cyp26a1- Duox2- Duoxa2- Eomes- Nr0b1- andSox21-specific probes Eosin counterstaining was performedaccording to standard protocols [60]

29 Animals All mice were housed in the pathogen-freebarrier facility of the University Medical Centre Freiburg inaccordance with the institutional guidelines and approval bythe regional board

3 Results

31 Bona Fide TSCs Are Marked by Coexpression of EomesCdx2 and Elf5 and Are Lost from the Chorionic Hingearound E95 To investigate the spatial and temporal dis-tribution of TSCs during placentogenesis we performeddetailed immunofluorescence (IF) staining using antibod-ies specific for EOMES CDX2 and ELF5 in combinationwith a previously described 119864119900119898119890119904GFP reporter allele [45]EOMES protein and the GFP-reporter can be detected in theTSC compartment of the extraembryonic ectoderm (ExE)


in the primitive streak (PS) and in the visceral endoderm(VE) during early gastrulation at E65 (Figures 1(a)ndash1(a10158401015840))[45] One day later at E75 Eomes expression is refined to asubregion of the chorion which we refer to as the chorionichinge (ChH) and expression is gradually reduced in theremainder of the chorion (Figure 1(b)) CDX2 colocalizeswith EOMES within the chorion at E75 but unlike EOMESthe staining intensity shows no gradual changes and isuniform throughout the entire chorion (Figure 1(b1015840)) CDX2is additionally found in the extraembryonic mesoderm of theallantois (Figure 1(b1015840)) Finally we stained for expression ofElf5 which acts in the transcriptional circuitry thatmaintainsTSC stemness by positive feed-forward regulation of Eomesand Cdx2 expression [25] Strongest ELF5 staining wasdetected in the ChH and the distal portion of the ectoplacen-tal cone (EPC) facing the ectoplacental cavity while reducedlevels are found throughout the remainder of the chorion andthe proximal EPC (Figure 1(c)) At E85 EOMES expressingcells are remaining within the tip of the ChH which isdetached from the surrounding tissues while the central partof the chorion has started to fuse with the allantois and theEPC (Figures 1(d)ndash1(d10158401015840)) EOMES-positive cells are almostentirely lost around E95 from the forming placenta (Figures1(e) and 1(e1015840)) with only little EOMES staining in a regionemanating from the former ChH (Figure 1(e1015840)) FollowingE105 placental EOMES staining could not be detected apartfrom single EOMES positive cells that most likely representplacental natural killer cells (Figures 1(e) and 1(f)) [46]In summary these detailed marker analyses suggest thatEOMES-positive TSCs remain in the chorionic hinge as thefunctional stem cell niche of TSCs until E95 and are notfound at later time points

32 TSCs in Culture ShowHeterogeneous Staining for StemnessFactors and Can Be Identified by High Levels of the 119864119900119898119890119904119866119865119875Reporter Expression TSCs can be cultured under conditionsof continuous stimulation with Fgf- and Tgf120573- growth factorsfor extended periods of time and multiple passages [17 33]However TSCs in vitro exhibit an eminent tendency towardsspontaneous differentiation and in stemness-maintainingconditions about 5ndash10 of TSCs differentiate towards thetrophoblast giant cell fate indicated early by an increase innuclear and total cell size TSC cultures thus intrinsicallycontain heterogeneous cell types to various degrees To studythe degree of heterogeneity in cultured TSCs we first inves-tigated the coexpression of TSC markers using antibodiesagainst CDX2 EOMES and ELF5 in TSCs cultured onfeeder layers of mouse embryonic fibroblasts (MEF) in thepresence of MEF-conditioned TSC-medium (MCM) humanrecombinant FGF4 (hrFGF4) and heparin (F4H) (Figures2(a)ndash2(c))Double-IF staining for EOMES andCDX2 showeda surprising degree of mutual staining Predominantly cellsat the periphery of colonies showed strong CDX2 stainingwhile only very weak EOMES signal could be observedThese CDX2 positive cells exhibited a markedly enlargednucleus in comparison to CDX2 and EOMES double positivecells indicating that they may have differentiated from TSCsthat normally present as cells with small nuclei and cellbodies Colabelling for EOMES and CDX2 (Figure 1(a)) as

well as EOMES and ELF5 (Figure 1(b)) and CDX2 and ELF5(Figure 1(c)) was very consistently found in small cells likelyrepresenting bona fide TSCs In larger more differentiatedcells the CDX2 signal is only partially overlapping withEOMES and ELF5 (Figures 1(a) and 1(c)) while ELF5- andEOMES-staining are predominantly overlapping in all celltypes (Figure 1(b))

To allow for the analysis of TSCs that harbour a stemness-indicating reporter gene we generated TSCs from E35blastocysts carrying the 119864119900119898119890119904GFP knock-in allele [45]To characterize resulting 119864119900119898119890119904GFP TSCs we comparedGFP-reporter expression with 120572-EOMES antibody staining119864119900119898119890119904

GFP reporter and EOMES protein expression widelyoverlap and highest levels of EOMES protein staining werereflected by strongest reporter activity (Figure 2(d)) As anexception cells undergoing mitosis loose nuclear EOMESstaining while cytoplasmic GFP expression remains clearlydetectable Highest levels of GFP-reporter expression werefound in populations of small cells that morphologicallyqualify for bona fide TSCs In summary our marker analysisin cultured TSCs demonstrates that only the combination ofmultiple stemness markers allows for unambiguous identifi-cation of bona fide TSCs However the generation of TSCscarrying the119864119900119898119890119904GFP allele is a suitable tool for the isolationof pure populations of undifferentiated TSCs (GFPhigh) thatare characterised by high-level expression of Eomes

33 Variable Experimental Setups Delineate the Transcrip-tional Characteristics of Bona Fide TSCs 119864119900119898119890119904GFP TSCsallow for the enrichment of GFPhigh cells representing fullyundifferentiated and thus bona fide TSCsThese should serveas a suitable source to delineate the transcriptional signatureof TSC stemness Eomes maintains TSCs in an undifferenti-ated state and the genetic deletion of Eomes prohibits TSCmaintenance and induces differentiation [34 41] In reversethe expression of Eomes or Cdx2 (and other factors) in mEScells initiates the transcriptional programme that induceslineage conversion towards the TSC lineage [3] To define thetranscriptional signature of TSCs in their bona fide stemnessstate downstreamofEomes andCdx2 we thus employed threeexperimental settings followed by genomewide expressionanalyses (Figure 3) First we enriched 119864119900119898119890119904GFP TSCs forGFPhigh cells and compared cells under stemnessmaintainingculture conditions with cells that were differentiated by with-drawal ofMCM F4H andMEFs by gene expression profilingat daily intervals over a period of three days (Figure 3(a)) Inthe second approach we inducedCre-mediated gene deletionin TSCs that homozygously carry an Eomes conditionalallele in combination with a tamoxifen- (Tx-) inducible Cre-estrogen receptor fusion (119864119900119898119890119904CACA R26CreERT) [47] andmonitored gene expression at 24 h intervals for three days(Figure 3(b)) The recombination efficiency was monitoredby PCR (Figure 3(b1015840)) and EOMES levels by Western blotshowing that EOMES protein was almost absent for 24 h afterTx-administration (Figure 3(b10158401015840)) Third to identify genesthat are positively regulated by Eomes or Cdx2 we gener-ated mES cells with inducible overexpression of both tran-scription factors mES cells with inducible expression were


Eomes Merge

E65

E75

E85

E95

E10

5

ChH

ChH

EPC

ExE

(a)

(b)

(d)

(e)

(f)

(c)

PS

VE

EPC

Ch

Al

Ch

EPC

ChH ChH

Pl

Pl

pTGC

NK

EPCav

Cdx2Eomes

Eomes

Eomes

Elf5

Eomes

EomesGFP

lowast

lowast

lowast

100120583m

100120583m

500120583m

500120583m

100120583m

500120583m

e998400

d998400

d998400998400

f998400

(a998400) (a998400998400)

(b998400)

(d998400) (d998400998400)

(e998400)

(f998400)

Figure 1 EOMES colocalizes with TSC markers CDX2 and ELF5 in the E75 chorionic hinge and expression is lost around E95Immunofluorescence staining shows nuclear EOMES and 119864119900119898119890119904GFP reporter expression in (andasha10158401015840) the visceral endoderm (VE) the primitivestreak (PS) and the extraembryonic ectoderm (ExE) at E65 (b) within the chorion (Ch) and the chorionic hinge at E75 and (dndashd10158401015840) in thechorion at E85 Asterisks in (a1015840) indicates unspecific signals in extraembryonic tissues (b and b1015840) TSCmarkers EOMES and CDX2 colocalizein cells of the chorion at E75 EOMES staining is most prominent in the chorionic hinge and shows a gradient along the chorion whileCDX2 does not show graded reduction along the chorion (c) ELF5 similarly localizes to the chorion with strongest staining in the chorionichinge and additionally expands into the ectoplacental cone (EPC) adjacent to the ectoplacental cavity (EPCav) (e e1015840) Faint EOMES stainingcan be detected at E95 in the region emanating from the chorionic hinge and (f f1015840) staining is entirely lost at E105 (e) EOMES can beadditionally found in parietal trophoblast giant cells (pTGC) and natural killer cells (NK) at E95 Al allantois EPC ectoplacental coneEPCav ectoplacental cavity ExE extraembryonic ectoderm Ch chorion ChH chorionic hinge Pl placenta PS primitive streak pTGCparietal trophoblast giant cells VE visceral endoderm NK NK cells


Eomes Cdx2 DAPIMerge

100120583m

(a)

(b)

(c)

(d)

Eomes Elf5 DAPIMerge

100120583m

Cdx2 Elf5 DAPIMerge

100120583m

DAPIMerge Eomes EomesGFP

100120583m

Figure 2 TSCs in culture exhibit heterogeneous patterns of EOMES CDX2 and ELF5 staining which is recapitulated by 119864119900119898119890119904GFP reporterexpression Cultured TSCs are colabelled with antibodies against EOMES CDX2 and ELF5 or the 119864119900119898119890119904GFP knock-in reporter allele (a)EOMES staining and CDX2 staining largely overlap in TSCs of small size which morphologically match undifferentiated TSCs (solid boxedregion) but staining diverges in enlarged cells (dashed boxed region) (b) ELF5 and EOMES immunostaining widely labels the same cellsand accordingly (c) ELF5 and CDX2 do not colocalize in enlarged cells ((c) dashed boxed region) (d) EOMES protein levels and 119864119900119898119890119904GFPreporter expression closely correlate and highest fluorescence intensities are found in cells with strongest EOMES staining

generated by site-specific introduction of cDNAs includingan N-terminal fusion to a V5-tag [48] into the engineereddoxycycline-inducible Hprt locus of p2lox mES cells usinginducible cassette exchange (ICE) [49] Resulting mES cellsallow for temporally regulated and moderate overexpressionlevels of Eomes orCdx2 inmES cells asmonitored byWesternblot (Figures 3(c) and 3(c1015840)) Expression profiles of mEScells were performed before doxycycline induction and at 24and 48 h following induced expression to identify the earlyinduced genes downstream of Eomes or Cdx2 that initiatelineage conversion of mES cells towards the TE lineage

All three experimental settings were subsequently used forgene array based transcriptional profiling (see SupplementaryTable S3)

34 Comparative Expression Profiling Reveals the Transcrip-tional Signature of TSCs To reveal the transcriptional sig-nature that constitutes andor maintains a stable TSC statedownstream of the key transcription factors Eomes andCdx2 we performed a combined analysis of our independentgene array datasets and identified those genes that showeddifferential expression in multiple experiments (Figure 4 and


(b)

(c)

Forced differentiation

FACS

MEF feeder layer

0d 1d 2d 3dTSCs EomesGFP FACSEomesGFP

lowast lowast lowast lowast

minus1d

Induced expression

ESCsMEF depletion

0d 1d 2dlowast lowast lowast

Induced LOFMEF depletion

0d 1d 2d 3dlowast lowast lowast lowast

TSCs EomesCACA R26CreERT

(b998400998400)(b998400)

(c998400)

NullCA+

+ + +minus minus minus

Eomes

CA+

Eomes

CAC

A

R26

CreER

T

R26

CreER

TEo

mes

CA+

3d Tx

10075

10075

0d 1d 2d 3d 4dEomesCACA R26CreERT

+Tx

0d 1d 2d 3d 4d +Tx

120572 Eomes

120572 Eomes

EomesCA+ R26CreERT

(a)

p2lox-Eomes-V536h Dox120572 V5

10075

+minus

p2lox-Cdx2-V5

50

37

+minus 36h Dox120572 V5

FACSEomesGFP differentiation

Induced Eomes LOF

Induced expression EomesCdx2

Figure 3 Experimental approaches for the detection and manipulation of the core TSC maintenance factors Eomes and Cdx2 followedby gene expression profiling Three complementary experimental approaches were chosen for expression profiling of (a b) TSCs and (c)mES cells with inducible expression of Eomes or Cdx2 (a) For the expression profiling of bona fide TSCs and early stages of differentiation119864119900119898119890119904

GFP TSCs were FACS-enriched for GFPhigh cells and forced towards differentiation by withdrawal of stemness maintaining conditionsfor 3d (b) For the identification of Eomes-regulated target genes TSCs harbouring Eomes conditional alleles (119864119900119898119890119904CA) in combination witha tamoxifen-inducible Cre-estrogen receptor allele (R26CreER) were used for conditional inactivation in vitro Cells were tamoxifen-treatedfor 24 h and (b1015840) Cre-mediated excision was monitored by genotyping PCR and (b10158401015840) presence of EOMES protein was analysed by Westernblot (c) To transcriptionally profile the initiation of Eomes- and Cdx2-induced target genes during ES cell to TSC conversion mES cellswith doxycycline-regulated expression were induced for 48 h (c1015840) The expression of V5-tagged EOMES or CDX2 protein was monitored byWestern blot MEF mouse embryonic fibroblast TSC trophoblast stem cell asterisks (lowast) indicate the different time points of gene expressionprofiling by gene arrays

Supplementary Table S4) All experimental interventionsresulted in gross changes of differential gene expression thatwere defined by changes in expression above 15-fold with a119901 value below 005 in three biological replicates and a meanlevel of gene expression above a set background threshold Toidentify genes that showed differential expression in multipleexperiments we analysed the intersections of genes thatwere among (1) the 300 most downregulated genes during

differentiation of FACS-enriched EomesGFP-high TSCs (2) the189 genes that were downregulated following induced Eomesdeletion (3) the 300 genes that showed the highest relativeexpression in TSCs compared to mES cells as assessed bythe ratio of median expression values in TSCs to the medianexpression values in mES cells (4) the 498 genes that wereupregulated in response to induced Cdx2 expression and (5)the 147 genes that were upregulated in response to induced


1733

474

1637

513

151

1907

192

1585

335221

0

500

1000

1500

2000

1 2 3 4 5

Diff

eren

tially

expr

esse

d

Upregulated (ge15x)

Downregulated (le15x)

Median TSC ESC ge 2

Median ESCTSC ge 2

gene

s

(a)

(b) (c) (d)

(1) Forced EomesGFP differentiation

(2) Induced Eomes LOF

(3) Median expression TSCESC

(4) Induced Cdx2 expression

(5) Induced Eomes expression

A 221

G 16

F 7

E 26D 37

C 251B 129

EomesElf5

Cdx2

1

2 3

ELy6aGdf6Bves

Gm1673Nat8lApln

Adamts7A330050F15Rik

Hs3st3b1Rgs5Ttc39cCdx2Popdc3Mmp9Pth1rSlc16a5

4833419F23RikSh3pxd2bMeis1GpnmbLy6g

Map3k8Lcp1Cd83Cpm

Ccdc85b

GDuox2Duoxa2Il5ra

Cyp26a1Inpp1Cldn26Htr5b

Tmem181c-psEomesTrim9Fgfr4Zfp382Sox21Elf5Nlrc3

Ppp2r2c

DSocs2Dusp6Nr0b1Prss46Zic2

Fermt1D430041D05Rik

Zic3Zic5

C77370Etv4Ehd3ProdhKcnab3Hdgfrp3Prps1Bok

B830017H08RikCth

Kcnk13MestPcsk6Scmh1

Fam124aFam81aSp4Hipk2Robo4Myef2Ntf3Ninl

Asphd2Slc4a11Depdc1aPrkar2aLrp12Sesn3

FTph1

Ubash3bSema7aEfnb2Fv1

Dram1Emp2

LDuox2Duoxa2Il5ra

Cyp26a1Gdf6Cldn26Htr5b

Adamts7A330050F15RikTmem181c-ps

Rgs5Trim9Ttc39cMmp9Slc16a5

4833419F23RikSh3pxd2bFgfr4Zfp382Sox21Meis1Map3k8Nlrc3Lcp1Cd83

Ppp2r2cCcdc85b

NLy6aInpp1Bves

Gm1673Nat8lApln

Hs3st3b1EomesCdx2Popdc3Pth1rGpnmbLy6gElf5Cpm

KZic2Zic5Prss50Fbp2Tbx18Stac2Dusp4

D130040H23RikAmphO3far1Nnt

BambiMpped2Ssbp2Sp4

Smad6Cxcl14Usp27xEpb41l3Dcaf12l1Gpx3Slc40a1Rbp4Smad7Zdhhc9Ppap2bSpry1Plekhg3Slitrk4Abr

MMorc4Pla2g7Slc44a3Emp2Eps8l2H19Dsc2Igf2

Capn6Afap1l1Msx2Nxf7CtdsplScn1bBhmt2Gpr126Igf2asPdzrn3PpargRassf8Gata2Satb2AcppFignl2Dhrs9Rab6bMgat4bCobll1Sync

3830417A13RikNrkGm9Bcl9l

1600029D21RikIrx3

H 228K 30

I 418

N 15

M 35

J 223

L 27

EomesCdx2Elf5

1

34

O 114R 29

U 2S 2

P 419

T 48

Q 248Cdx2Elf5

5

34

Eomes

SClic5Dram1

UEomesNxf7

RMef2bBhlhe41Cubn

Gm5091Srgn

Fam100bGm364Slc17a9TfecTaf9bBollKlb

Slc25a31Slc40a1Zdhhc9Fam129a

OttGm15107Snora62Gm5634HpgdApoeNostrinSlc15a2Flywch2

LOC100862646Cd55Nxnl2

Gm15127

TMorc4Cdx2Pla2g7Slc44a3Popdc3Emp2BvesEps8l2H19Dsc2Igf2

Capn6Ly6g

Afap1l1Msx2

Gm1673Ly6aCtdsplScn1bBhmt2Hs3st3b1Gpr126Igf2asAplnPdzrn3Nat8lPth1rPpargRassf8Cpm

GpnmbGata2Satb2AcppFignl2Inpp1Dhrs9Rab6bMgat4bCobll1Sync

3830417A13RikNrkElf5Gm9Bcl9l

1600029D21RikIrx3

Blue genes for validation

Red core TSC factors

Orange previously published TSC markers

Figure 4 Comparative gene expression analysis reveals candidate TSC marker genes (a) Genome-wide expression analysis generated fivedata sets of differentially expressed genes resulting from different experimental settings as shown in Figure 3 (1) The first group containsregulated genes after 3 days of differentiation of 119864119900119898119890119904GFP-high FACS-enriched TSCs when compared to undifferentiated 119864119900119898119890119904GFP-high TSCs(2) The second group represents differentially regulated genes 3 days following Eomes gene deletion in TSCs (LOF loss-of-function) (3)The third group comprises genes which show significantly increased or decreased median expression in TSCs versus mES cells (4 5) andthe fourth and fifth group are differentially expressed genes 2 days after induced expression of Cdx2 or Eomes respectively The bar chartindicates numbers of differentially expressed genes for each gene group (bndashd) Venn diagrams indicate groups of intersecting differentiallyexpressed genesThe genes of resulting groups are listed in the table below eachVenn diagram (b) Comparison between the 300most stronglydownregulated genes in differentiating 119864119900119898119890119904GFP FACS-enriched TSCs the 189 genes downregulated following Eomes deletion and the 300genes with highest expression ratio between TSCs and mES cells Of note Cdx2 Eomes and Elf5 expression levels decrease during TSCdifferentiation The loss of Eomes function does not significantly reduce Cdx2 expression after 3d In (c) the set of differentially regulatedgenes after Eomes gene deletion was exchanged with those 498 genes that were upregulated following Cdx2-induction in mES cells In (d)genes that were induced by Eomes or Cdx2 expression in mES cells were compared to 300 genes with the highest expression ratio in TSCsversus mES cells Note that Eomes is neither significantly upregulating Elf5 nor Cdx2 expression Differences in number of genes listed in (a)and in (bndashd) result from genes with multiple transcripts but identical gene symbols The selected cut-off criteria for genes to be included indatasets were a positive or negative fold change ge 15 in response to treatment and a 119901 value le 005 Genes selected for further analyses arehighlighted in blue core genes of the TSC positive feedback loop in red and genes that were previously identified as TSC markers in orange


Eomes expression in mES cells (Figure 4 and SupplementaryTable S4) The intersections of differentially expressed genesare represented in Venn diagrams and corresponding groupsof genes are listed in tables (Figures 4(b)ndash4(d) and Supple-mentary Table S4)

In a first comparative analysis we investigated the inter-section of those genes that were (1) downregulated in dif-ferentiating 119864119900119898119890119904GFP-high TSCs (2) downregulated in TSCsfollowing induced deletion of Eomes and (3) presented witha high relative expression in TSCs in comparison tomES cells(Figure 4(b)) 16 genes (group G) matched these criteria andthus were considered candidate Eomes target genes whichare lost during differentiation of TSCs Within this groupwere the known specific TSC markers Elf5 and Eomes itselfAdditionally this group contained novel genes that were notpreviously described in TSCs such as Duox2 and Duoxa2or Cldn26 Cdx2 was not included in the intersection sincethe deletion of Eomes caused only a mild reduction of Cdx2within the short time interval of the experiment

Next we compared genes that were (1) downregulatedduring differentiation of 119864119900119898119890119904GFP-high TSCs that were (4)positively regulated by Cdx2 in mES cells and that were (3)TSC-specific (Figure 4(c)) The resulting group of 15 genesincluded Eomes Elf5 and Cdx2 itself

To compare the early transcriptional changes inducedby Cdx2 or Eomes that initiate the conversion of mEScells to TSC-like cells we analysed the intersecting TSC-specific genes (3) that were upregulated following two days ofCdx2 (4) or Eomes (5) induction (Figure 4(d)) Surprisinglyexcept for Eomes none of the known TSC factors wassignificantly upregulated by both Cdx2 and Eomes after twodays of induced expression However this might reflect theprevious observations that Eomes is not as effective duringthe conversion of mES cells to TSCs in comparison to Cdx2WhileCdx2 expression initiates TSC-specific gene expressionwithin 48 hours including upregulation of Eomes and Elf5Eomes expression fails to effectively induce TSC-specificgenes within the 2-day interval despite positive regulationof published Eomes targets such as Fgf5 Mesp2 and Mixl1[50 51]

To validate the gene array data of differentially expressedgenes we performed quantitative RT-PCRs for establishedTSC marker genes and selected genes that were at inter-sections of differentially regulated genes in more than oneexperiment To validate specific expression in bona fideTSCs we compared expression levels in FACS-enriched119864119900119898119890119904

GFP-high TSCs with levels during forced differentiationby removal of stemness maintaining conditions (Figure 5)All tested known and novel TSC marker genes showedhighest expression in undifferentiated TSCs and significantlyreduced levels following induction of differentiation withchanges in expression over a minimum of two magnitudes(Figure 5)

35 NovelMarkerGenes Label TSCs duringDevelopment of theEarly Placenta To generally validate if the approach revealednovel markers for TSCs during embryonic developmentcandidate genes were further analysed by in situ hybridisation

0

02

04

06

08

1

d0d1

d2d3

ns

ns

ns

Relat

ive m

RNA

expr

essio

n (d

0=1)

Cdx2

Eomes

Elf5

Bok

Cldn

26

Cyp26a1

Duox2

Duoxa2

Nr0b1

Sox21

Forced EomesGFP differentiation

Figure 5 Genes identified by expression arrays and comparativeanalyses are downregulated during TSC differentiation when val-idated by qPCR mRNA expression levels for genes selected bydataset comparisons were quantified at 24 h intervals by qRT-PCRof undifferentiated FACS-enriched 119864119900119898119890119904GFP-high TSCs at day 0 (d0)and after forced differentiation for three days (d1ndashd3) Expressionlevels are depicted as means of biological triplicates relative to d0which was set to 1 All genes were significantly (119901 value le 005)downregulated at different time points unless indicated otherwise(ns not significant) Error bars indicate the standard error of themean 119901 values were calculated according to two-sided Studentrsquos 119905-test All marker genes show grossly reduced expression during thecourse of 3 days of differentiation

using histological sections of E75 embryos We focused ongenes with previously unknown functions in the murine TEsuch as BokMtd Duox2 Duoxa2 Nr0b1 and Sox21 To ourknowledge specific surface markers for TSCs that wouldallow for antibody-mediated FACS-sorting have not beenidentified so farThus we additionally analysed expression ofthe transmembrane protein-coding gene Cldn26 (Tmem114)For Sox21 it was shown that it is involved in the regulation ofintestinal and pluripotent stem cells and is induced by Sox2an important stem cell factor in bothmES cells and TSCs [5253] Accordingly Sox21 was also included into the followinganalysis Expression of Cyp26a1 in the trophectoderm waspreviously described and together with Eomes served as apositive control for expression in the chorion Of the seventested novel marker genes six showed expression in entirechorion (Bok Cldn26 and Sox21) or were more specificallyrestricted to the chorionic hinge (Nr0b1Duox2 andDuoxa2)(Figure 6)

In conclusion the in situ hybridisation analysis revealedthat the multimodal expression profiling of TSCs in cultureserves as a valuable resource for the identification of novelTSCmarker genes in vitro but also for TSCs of the developingplacenta

4 Discussion

In the present report we describe a resource for the tran-scriptional signature of TSCs We used genetically modifiedTSCs andmES cells to experimentally interfere with stemness


Nr0b1

E75

Sox21

E75

Duox2

E75

Duoxa2

E75

Cldn26

E75

Cyp26a1

E75

BokMtd

E75

AlB

Eomes

Ch

PS

E75

VE

Figure 6 Differentially expressed genes show regional specific expression in the chorion of E75 embryos In situ hybridisation analysis onsagittal sections of E75mouse embryos reveals specific expression of Eomes as positive control and novel TSCmarker genes BokMtdCldn26Cyp26a1 Duox2 Duoxa2 Nr0b1 and Sox21 in the entire chorion or limited expression in the chorionic hinge (indicated by arrowheads)AlB allantoic bud Ch chorion PS primitive streak VE visceral endoderm Scale bar 200 120583M

maintaining regulation of TSCs downstream of Eomes andCdx2 followed by the assessment of transcriptional changesThe combined analysis of expression data resulted in acomprehensive list of candidate TSC marker genes of whicha handful were tested and positively validated for theirspecific expression in cultured undifferentiated TSCs and inthe chorion of the gastrulating embryo Thus the presentedexpression data will serve as a valuable resource for furtherstudies of stemness maintaining regulatory circuitries ofTSCs

Using TSCs that harbour the 119864119900119898119890119904GFP reporter alleleallowed for the purification of GFPhigh TSCs that by mor-phology and in their transcriptional signature likely resem-ble bona fide TSCs without contaminating early differ-entiating TSCs that are normally found in TSC cultures

Corresponding expression data of GFPhigh TSCs thus resem-ble the actual signature of bona fide TSCs underscored bythe rapid gross and early changes in gene expression thatwe found during their differentiation Expression profilesof TSCs in stemness maintaining conditions and duringinduced differentiation either by removal of MCM and F4Hor by genetic interference with the TSC regulatory circuitrywere previously reported and had revealed partially overlap-ping gene lists [53ndash55] However the validation of candidatesfrom our comparative expression analysis identified severaladditional and novel TSC marker genes Among those is thetranscription factor Sox21 that is strongly expressed in thechorion of the E75 embryo and is downregulated duringdifferentiation of TSCs in culture Interestingly it was previ-ously demonstrated that Sox21 is induced by Sox2 and unlike


Sox2 [53] Sox21 negatively regulates transcription of Cdx2 inmES and colon cancer cells [52] This obvious difference inexpression in mES cells and TSCs makes Sox21 an interestingcandidate that like Sox2 [53] might act differently in thecircuitry of TSC and mES stemness factors Functions ofSox21 and other novel candidates in TSC biology and TEdevelopment will be addressed in ongoing studies

The importance ofCdx2 as lineage determining transcrip-tion factor was apparently demonstrated by the ability toconvert mES cells to TSCs when overexpressed in mES cells[3] even though more recent studies suggest that this lineageconversion might not be complete at the phenotypic tran-scriptional and epigenetic level [56] Eomes similarly has thecapacity to induceTSC fate howeverEomes seems less potentand possibly induces even less complete lineage conversion[3]This notion is underscored by our datasets which displaymore profound transcriptional responses following Cdx2 incomparison to Eomes expression when induced from theidentical doxycycline-inducible locus This difference mightat least partially arise from the reduced induction of Elf5in Eomes-expressing mES cells which is known as a centralcomponent of a positive feed-forward regulation of the coreTSC circuitry [25]

While both CDX2 and EOMES were previously used asmarkers for TSCs coimmunofluorescence staining of cul-tured TSCs revealed a remarkable heterogeneity of labellingOnly a subset of TSCs that can be morphologically dis-tinguished by a small cell and nucleus size consistentlyshowed colabelling of CDX2 and EOMES together withELF5 We suspect that these cells resemble bona fide TSCsIt will require further fate analysis to reveal if the presenceof individual key TSC factors primes future fate decisionssimilar to lineage specifying roles of key pluripotency factorsduring embryonic development and in differentiating mEScells [50 57] It is noteworthy that EOMES is also detectedin parietal trophoblast giant cells (pTGC) (Figure 1(e)) andthus it is tempting to speculate that differentiation towards thetrophoblast giant cell lineage might be promoted by EOMES

The immune-detection of EOMES CDX2 and ELF5 inthe chorion at late gastrulation stages revealed the presenceof TSCs until E90ndashE95 in the remaining chorion andthe absence of EOMES-positive cells at later stages Thesefindings are in line with studies byUy et al that demonstratedthe presence of TSCs that can be isolated and cultureduntil the 11-somite stage but not afterwards [31] Anotherreport that used an EomesGFP BAC-transgenic reporter[32] detected GFP positive cells in the margins of the E145placenta However the nature of these cells was not defined indetail We were unable to detect similar cells using EOMES-antibody staining Thus we conclude that TSCs are indeedonly maintained until E95 in the remaining portions ofthe chorion from which they can be isolated as previouslyreported [31]

In summary this study contributes to the characterisationof TSCs during early phases of placentogenesis and of TSCsin culture We demonstrate that TSCs exhibit a remarkabledegree of heterogeneity with respect to protein levels of keyTSC transcription factors TSCs are lost around E95 fromtheir stem cell niche in the chorion We used complementary

genetic tools of cultured TSCs and ES cells to determine thetranscriptional profile of TSCs in their fully undifferentiatedand early differentiating state Comparative analysis revealedseveral new TSC marker genes Presented data can be usedas a valuable resource for future studies of TSCs and thecorresponding transcriptional regulatory network

Conflict of Interests

The authors declare that there is no conflict of commercialinterests

Authorsrsquo Contribution

Sebastian J Arnold conceived the study and Georg Kualesand Sebastian J Arnold performed experiments and analysedthe data Matthias Weiss and Oliver Sedelmeier assisted inand supervised some experiments Dietmar Pfeifer per-formed microarray experiments and data analysis GeorgKuales assembled the figures Georg Kuales and Sebastian JArnold wrote and edited the paper

Acknowledgments

The authors thank Klaus Geiger for FACS of 119864119900119898119890119904GFPcells Marie Follo for supporting the qRT-PCR analysisMichael Kyba for A2lox ES cells Hynek Wichterle for theP2lox gateway cloning vector and Gerd Walz for thoughtfuldiscussion and generous support This work was supportedby an Exploration Grant from the Boehringer IngelheimFoundation to Sebastian J Arnold a project grant (A03)of the Collaborative Research Centre 850 (SFB 850) andthe Emmy Noether Programme (AR7321-1) of the GermanResearch Foundation (DFG) to Sebastian J Arnold

References

[1] S J Arnold and E J Robertson ldquoMaking a commitmentcell lineage allocation and axis patterning in the early mouseembryordquo Nature Reviews Molecular Cell Biology vol 10 no 2pp 91ndash103 2009

[2] M Zernicka-Goetz S A Morris and A W Bruce ldquoMaking afirm decision multifaceted regulation of cell fate in the earlymouse embryordquoNature Reviews Genetics vol 10 no 7 pp 467ndash477 2009

[3] H Niwa Y Toyooka D Shimosato et al ldquoInteraction betweenOct34 and Cdx2 determines trophectoderm differentiationrdquoCell vol 123 no 5 pp 917ndash929 2005

[4] A Ralston and J Rossant ldquoCdx2 acts downstream of cellpolarization to cell-autonomously promote trophectoderm fatein the early mouse embryordquoDevelopmental Biology vol 313 no2 pp 614ndash629 2008

[5] K Cockburn and J Rossant ldquoMaking the blastocyst lessonsfrom the mouserdquo The Journal of Clinical Investigation vol 120no 4 pp 995ndash1003 2010

[6] M Gasperowicz and D R C Natale ldquoEstablishing three blas-tocyst lineages-then whatrdquo Biology of Reproduction vol 84 no4 pp 621ndash630 2011

[7] R Yagi M J Kohn I Karavanova et al ldquoTranscription factorTEAD4 specifies the trophectoderm lineage at the beginning


of mammalian developmentrdquo Development vol 134 no 21 pp3827ndash3836 2007

[8] B Zhao X Wei W Li et al ldquoInactivation of YAP oncoproteinby the Hippo pathway is involved in cell contact inhibition andtissue growth controlrdquo Genes and Development vol 21 no 21pp 2747ndash2761 2007

[9] M Ota and H Sasaki ldquoMammalian Tead proteins regulate cellproliferation and contact inhibition as transcriptional media-tors ofHippo signalingrdquoDevelopment vol 135 no 24 pp 4059ndash4069 2008

[10] N Nishioka S Yamamoto H Kiyonari et al ldquoTead4 is requiredfor specification of trophectoderm in pre-implantation mouseembryosrdquo Mechanisms of Development vol 125 no 3-4 pp270ndash283 2008

[11] N Nishioka K-I Inoue K Adachi et al ldquoThe Hippo signalingpathway components Lats and Yap pattern Tead4 activityto distinguish mouse trophectoderm from inner cell massrdquoDevelopmental Cell vol 16 no 3 pp 398ndash410 2009

[12] H Sasaki ldquoMechanisms of trophectoderm fate specificationin preimplantation mouse developmentrdquo Development Growthand Differentiation vol 52 no 3 pp 263ndash273 2010

[13] P A Latos andM Hemberger ldquoReview the transcriptional andsignalling networks of mouse trophoblast stem cellsrdquo Placentavol 35 pp S81ndashS85 2014

[14] T Rayon S Menchero A Nieto et al ldquoNotch and hippo con-verge on Cdx2 to specify the trophectoderm lineage in themouse blastocystrdquo Developmental Cell vol 30 no 4 pp 410ndash422 2014

[15] Z Cao T S Carey A Ganguly C A Wilson S Paul andJ G Knott ldquoTranscription factor AP-2 induces early Cdx2expression and represses HIPPO signaling to specify the tro-phectoderm lineagerdquo Development vol 142 no 9 pp 1606ndash1615 2015

[16] A Ralston B J Cox N Nishioka et al ldquoGata3 regulatestrophoblast development downstream of Tead4 and in parallelto Cdx2rdquo Development vol 137 no 3 pp 395ndash403 2010

[17] S Tanaka T Kunath A-K Hadjantonakis A Nagy and JRossant ldquoPromotion to trophoblast stem cell proliferation byFGF4rdquo Science vol 282 no 5396 pp 2072ndash2075 1998

[18] M Guzman-Ayala N Ben-Haim S Beck and D B ConstamldquoNodal protein processing and fibroblast growth factor 4 syn-ergize to maintain a trophoblast stem cell microenvironmentrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 101 no 44 pp 15656ndash15660 2004

[19] D G Simmons and J C Cross ldquoDeterminants of trophoblastlineage and cell subtype specification in the mouse placentardquoDevelopmental Biology vol 284 no 1 pp 12ndash24 2005

[20] R M Roberts and S J Fisher ldquoTrophoblast stem cellsrdquo Biologyof Reproduction vol 84 no 3 pp 412ndash421 2011

[21] D J Pearton R Broadhurst M Donnison and P L PfefferldquoElf5 regulation in the trophectodermrdquo Developmental Biologyvol 360 no 2 pp 343ndash350 2011

[22] Q Winger J Huang H J Auman M Lewandoski and TWilliams ldquoAnalysis of transcription factor AP-2 expression andfunction during mouse preimplantation developmentrdquo Biologyof Reproduction vol 75 no 3 pp 324ndash333 2006

[23] P Kuckenberg S Buhl T Woynecki et al ldquoThe transcriptionfactor TCFAP2CAP-2120574 cooperates with CDX2 to maintaintrophectoderm formationrdquoMolecular and Cellular Biology vol30 no 13 pp 3310ndash3320 2010

[24] M Donnison A Beaton H W Davey R Broadhurst PLrsquoHuillier and P L Pfeffer ldquoLoss of the extraembryonic ecto-derm in Elf5 mutants leads to defects in embryonic patterningrdquoDevelopment vol 132 no 10 pp 2299ndash2308 2005

[25] R K Ng W Dean C Dawson et al ldquoEpigenetic restriction ofembryonic cell lineage fate by methylation of Elf5rdquo Nature CellBiology vol 10 no 11 pp 1280ndash1290 2008

[26] F Wen J A Tynan G Cecena et al ldquoEts2 is required fortrophoblast stem cell self-renewalrdquo Developmental Biology vol312 no 1 pp 284ndash299 2007

[27] P Kuckenberg C Kubaczka and H Schorle ldquoThe role oftranscription factor Tcfap2cTFAP2C in trophectoderm devel-opmentrdquoReproductive BioMedicine Online vol 25 no 1 pp 12ndash20 2012

[28] F Beck T Erler A Russell and R James ldquoExpression of Cdx-2in the mouse embryo and placenta possible role in patterningof the extra-embryonic membranesrdquo Developmental Dynamicsvol 204 no 3 pp 219ndash227 1995

[29] J Luo R Sladek J-A Bader A Matthyssen J Rossant andV Giguere ldquoPlacental abnormalities in mouse embryos lackingthe orphan nuclear receptor ERR-120573rdquoNature vol 388 no 6644pp 778ndash782 1997

[30] B G Ciruna and J Rossant ldquoExpression of the T-box geneEomesodermin during early mouse developmentrdquoMechanismsof Development vol 81 no 1-2 pp 199ndash203 1999

[31] G D Uy K M Downs and R L Gardner ldquoInhibition oftrophoblast stem cell potential in chorionic ectoderm coincideswith occlusion of the ectoplacental cavity in the mouserdquoDevelopment vol 129 no 16 pp 3913ndash3924 2002

[32] G S Kwon and A-K Hadjantonakis ldquoEomesGFPmdasha toolfor live imaging cells of the trophoblast primitive streak andtelencephalon in the mouse embryordquo Genesis vol 45 no 4 pp208ndash217 2007

[33] A Erlebacher K A Price and L H Glimcher ldquoMaintenanceof mouse trophoblast stem cell proliferation by TGF-120573activinrdquoDevelopmental Biology vol 275 no 1 pp 158ndash169 2004

[34] A P Russ S Wattler W H Colledge et al ldquoEomesoderminis required for mouse trophoblast development and mesodermformationrdquo Nature vol 404 no 6773 pp 95ndash99 2000

[35] K Chawiengsaksophak R James V E Hammond F Kontgenand F Beck ldquoHomeosis and intestinal tumours in Cdx2 mutantmicerdquo Nature vol 386 no 6620 pp 84ndash87 1997

[36] H J Auman T Nottoli O Lakiza Q Winger S Donaldsonand T Williams ldquoTranscription factor AP-2gamma is essentialin the extra-embryonic lineages for early postimplantationdevelopmentrdquoDevelopment vol 129 no 11 pp 2733ndash2747 2002

[37] A A Avilion S K Nicolis L H Pevny L Perez N Vivianand R Lovell-Badge ldquoMultipotent cell lineages in early mousedevelopment depend on SOX2 functionrdquo Genes and Develop-ment vol 17 no 1 pp 126ndash140 2003

[38] D Zhu S Holz E Metzger et al ldquoLysine-specific demethylase1 regulates differentiation onset and migration of trophoblaststem cellsrdquo Nature Communications vol 5 article 3174 2014

[39] M Hemberger W Dean andW Reik ldquoEpigenetic dynamics ofstem cells and cell lineage commitment digging waddingtonrsquoscanalrdquo Nature Reviews Molecular Cell Biology vol 10 no 8 pp526ndash537 2009

[40] S Roper and M Hemberger ldquoDefining pathways that enforcecell lineage specification in early development and stem cellsrdquoCell Cycle vol 8 no 10 pp 1515ndash1525 2009


[41] D Strumpf C-A Mao Y Yamanaka et al ldquoCdx2 is requiredfor correct cell fate specification and differentiation of trophec-toderm in the mouse blastocystrdquo Development vol 132 no 9pp 2093ndash2102 2005

[42] H Yamamoto M L Flannery S Kupriyanov et al ldquoDefectivetrophoblast function in mice with a targeted mutation of Ets2rdquoGenes and Development vol 12 no 9 pp 1315ndash1326 1998

[43] P Georgiades and J Rossant ldquoEts2 is necessary in trophoblastfor normal embryonic anteroposterior axis developmentrdquoDevelopment vol 133 no 6 pp 1059ndash1068 2006

[44] T Boroviak and J Nichols ldquoThe birth of embryonic pluripo-tencyrdquo Philosophical Transactions of the Royal Society B Biolog-ical Sciences vol 369 no 1657 2014

[45] S J Arnold J Sugnaseelan M Groszer S Srinivas and E JRobertson ldquoGeneration and analysis of a mouse line harboringGFP in the EomesTbr2 locusrdquo Genesis vol 47 no 11 pp 775ndash781 2009

[46] S M Gordon J Chaix L J Rupp et al ldquoThe transcription fac-tors T-bet and Eomes control key checkpoints of natural killercell maturationrdquo Immunity vol 36 no 1 pp 55ndash67 2012

[47] S J Arnold U K Hofmann E K Bikoff and E J Robert-son ldquoPivotal roles for eomesodermin during axis formationepithelium-to-mesenchyme transition and endoderm specifi-cation in the mouserdquo Development vol 135 no 3 pp 501ndash5112008

[48] E O Mazzoni S Mahony M Iacovino et al ldquoEmbryonicstem cell-based mapping of developmental transcriptional pro-gramsrdquo Nature Methods vol 8 no 12 pp 1056ndash1058 2011

[49] M Kyba R C R Perlingeiro and G Q Daley ldquoHoxB4 confersdefinitive lymphoid-myeloid engraftment potential on embry-onic stem cell and yolk sac hematopoietic progenitorsrdquoCell vol109 no 1 pp 29ndash37 2002

[50] A K K Teo S J Arnold M W B Trotter et al ldquoPluripotencyfactors regulate definitive endoderm specification througheomesoderminrdquo Genes amp Development vol 25 no 3 pp 238ndash250 2011

[51] I Costello I-M Pimeisl S Drager E K Bikoff E J Robertsonand S J Arnold ldquoThe T-box transcription factor Eomeso-dermin acts upstream of Mesp1 to specify cardiac mesodermduring mouse gastrulationrdquo Nature Cell Biology vol 13 no 9pp 1084ndash1092 2011

[52] A N Kuzmichev S-K Kim A C DrsquoAlessio et al ldquoSox2 actsthrough Sox21 to regulate transcription in pluripotent anddifferentiated cellsrdquo Current Biology vol 22 no 18 pp 1705ndash1710 2012

[53] KAdachi INikaidoHOhta et al ldquoContext-dependentwiringof Sox2 regulatory networks for self-renewal of embryonic andtrophoblast stem cellsrdquo Molecular Cell vol 52 no 3 pp 380ndash392 2013

[54] B L Kidder and S Palmer ldquoExamination of transcriptionalnetworks reveals an important role for TCFAP2C SMARCA4and EOMES in trophoblast stem cell maintenancerdquo GenomeResearch vol 20 no 4 pp 458ndash472 2010

[55] D J Pearton C S Smith E Redgate J van Leeuwen M Don-nison and P L Pfeffer ldquoElf5 counteracts precocious trophoblastdifferentiation by maintaining Sox2 and 3 and inhibiting Hand1expressionrdquoDevelopmental Biology vol 392 no 2 pp 344ndash3572014

[56] F Cambuli A Murray W Dean et al ldquoEpigenetic memory ofthe first cell fate decision prevents complete ES cell reprogram-ming into trophoblastrdquo Nature Communications vol 5 p 55382014

[57] M Thomson S J Liu L-N Zou Z Smith A Meissner andS Ramanathan ldquoPluripotency factors in embryonic stem cellsregulate differentiation into germ layersrdquoCell vol 145 no 6 pp875ndash889 2011

[58] M Vooijs J Jonkers and A Berns ldquoA highly efficient ligand-regulated Cre recombinase mouse line shows that LoxP recom-bination is position dependentrdquo EMBOReports vol 2 no 4 pp292ndash297 2001

[59] M Iacovino D Bosnakovski H Fey et al ldquoInducible cassetteexchange a rapid and efficient system enabling conditional geneexpression in embryonic stem and primary cellsrdquo Stem Cellsvol 29 no 10 pp 1580ndash1588 2011

[60] A Nagy M Gertsenstein K Vintersten and R BehringerManipulating the Mouse Embryo A Laboratory Manual ColdSpring Harbor Laboratory Press 3rd edition 2003


additional transcriptional regulators including Eomes [4]and Elf5 [21] initiate expression in the TE lineage or becomespecifically restricted to TE cells such as AP2120574 [22 23]Following implantation around E45 the polar TE givesrise to the extraembryonic ectoderm (ExE) which containsTSCs and the ectoplacental cone (EPC) which mediates theembryonic invasion into the decidual wall then connects theembryo to the maternal uterus TSCs retain their stem cellcharacteristics namely self-renewal capacity and multipo-tency only in the polar TE and the ExE while cells of themural TE lack supportive signalling from the ICM and thusdifferentiate to primary trophoblast giant cells (TGCs) [17]TSC maintenance is controlled by transcriptional circuitriesinvolving Elf5 [24 25] Ets2 [26] andAP2120574 [23 27] which actin positive feed forward loops tomaintainCdx2 andorEomesexpression After E75 the core set of TSC marker genesincluding Cdx2 Eomes Elf5 and Esrr120573 remain expressed inthe chorion in addition to other embryonic and extraembry-onic expression domains [24 28ndash30]

TSCs can be routinely isolated and cultured from E35blastocysts and E65 ExE explants under defined condi-tions [17] Assessing the potential to isolate cells with TSCcharacter beyond E65 Uy and colleagues have shown thatthe frequency to obtain TSC clones increases until the 4-somite pair stage (simE80 to E825) but then rapidly drops andno TSCs can be isolated following the 11-somite pair stagearound E85 to E875 [31] The T-box transcription factorEomes was previously used to mark TSCs through differentdevelopmental stages and expression of an EomesGFP BACtransgene was detected until E145 in the outer peripheryof the murine placenta [32] To date it was not clearlyshown until which embryonic stage cells with TSC characterare maintained in the TSC niche which is functionallydefined by high levels of Fgf and Tgf120573 signals [17 33] Inaddition toEomes [34] andCdx2 [35] also other transcriptionfactors share essential functions for TSC self-renewal andmultipotency including Elf5 [24] Esrr120573 [29] Ets2 [26] AP2120574[23 27 36] and Sox2 [37] A recent report demonstrated thatthe functional loss of the histone demethylase Lsd1 in TSCsresults in premature migration of TSCs from their nichedemonstrating additional requirements of proper epigeneticregulation for the propagation of TSCs [38 39]

In addition to their functional importance during TSCmaintenance Cdx2 [3 11] Eomes [3] Elf5 [25] Tead4 [11]AP2120574 [23] andGata3 [16] also evoke lineage conversion frommouse embryonic stem (mES) cells to the TE lineage whenoverexpressed (reviewed in [40]) While the transcriptionalprogramme involving Cdx2 Eomes Elf5 AP2120574 and Ets2is key for the maintenance of the trophoblast stemnessstate genetic deletions of each of these transcription fac-tors generate remarkable different embryonic phenotypesCdx2-deficiency results in implantation defects [41] Eomes-deficient embryos show early postimplantation [34] and thedeletions of Elf5 [24] Ets2 [26 42 43] or AP2120574 [36] lead topostgastrulation lethality This diversity in loss-of-functionphenotypes might indicate differential requirements of thesefactors for the stemness maintaining regulatory circuitryAlternatively different states of TSCs might exist with dif-ferential developmental potential and distinct requirements

of transcriptional regulation in analogy to different statesof pluripotency such as that found in naıve or primedpluripotent stem cells [39 44]

In the current study we have analysed the endoge-nous expression of key stemness-maintaining factors Cdx2Eomes and Elf5 in the chorion of gastrulation stage embryosto characterise TSCs by overlapping marker gene expressionWe traced Eomes expression through later gastrulation stagesand demonstrate that it is lost around E95 in the regionemanating from the chorionic hinge Surprisingly TSCmark-ers consistently show heterogenous partially nonoverlappingexpression in different areas of the late TSC niche poten-tially indicating different states of TSCs during placentaldevelopmentThe analysis of TSCs cultured under stemness-maintaining conditions additionally revealed heterogeneousTSC marker expression in vitro underscoring our findingsfrom embryonic analyses in the chorion To determine thetranscriptional state that describes undifferentiated bona fideTSCs and to monitor transcriptional changes during earlyTSC differentiation we used genetic tools to manipulateEomes and Cdx2 expression in TSC cultures followed bygenome wide transcriptional profiling First we generatedTSCs that harbour an 119864119900119898119890119904GFP reporter allele therebymarking bona fide TSCs that were enriched by fluorescenceactivated cell sorting (FACS) and forced towards differentia-tion by removal of stemness maintaining conditions Secondwe employed TSCs that allow for the inducible deletion ofEomes gene function and third we used inducible expressionof key TSC transcriptional regulators Cdx2 and Eomes inmouse ES (mES) cells for the identification of downstreamtarget genes Resulting differential expression profiles allowfor a detailed description of the TSC signature and thechanges during early differentiation which might be directlyor indirectly regulated by a combination of Fgf- and Tgf120573-signalling and the key regulatory factors of TSCs Cdx2 andEomesWe used resulting expression profiles to identify novelcandidate TSC marker genes at stemness state To validatethis approach a handful of differentially expressed geneswere selected and tested by qRT-PCRs of bona fide TSCsand during early differentiation Additionally expression ofnovel candidates was analysed in the TSC compartment ofpostimplantation embryos by in situhybridisation analysis Insummary this study characterises and describes the expres-sion signature of TSCs within the embryo and in culturedTSCs

2 Materials and Methods

21 Cell Culture Genetically modified TSCs were isolatedfromE35 blastocysts of animals carrying alleles for119864119900119898119890119904GFP

[45]119864119900119898119890119904CA [47] and R26CreER [58] according to standardprotocols [17] TSCs cultured in stemnessmaintaining condi-tions (SMC) were kept in 70 mouse embryonic fibroblast-(MEF-) conditioned TSC-medium (MCM) supplementedwith hrFGF4 and heparin (F4H) on MEFs [17] For dif-ferentiation conditions (DC) TSCs were cultured in TSC-medium without MCM and F4H on gelatinized plates with-out feeder cells [17] Cre-mediated excision of the conditional119864119900119898119890119904

CA allele was induced by administration of 1120583gmL














3 Results










Eomes Merge

E65

E75

E85

E95

E10

5

ChH

ChH

EPC

ExE

(a)

(b)

(d)

(e)

(f)

(c)

PS

VE

EPC

Ch

Al

Ch

EPC

ChH ChH

Pl

Pl

pTGC

NK

EPCav

Cdx2Eomes

Eomes

Eomes

Elf5

Eomes

EomesGFP

lowast

lowast

lowast

100120583m

100120583m

500120583m

500120583m

100120583m

500120583m

e998400

d998400

d998400998400

f998400

(a998400) (a998400998400)

(b998400)

(d998400) (d998400998400)

(e998400)

(f998400)




100120583m

(a)

(b)

(c)

(d)


100120583m

Cdx2 Elf5 DAPIMerge

100120583m


100120583m






(b)

(c)


FACS

MEF feeder layer



minus1d

Induced expression

ESCsMEF depletion





(b998400998400)(b998400)

(c998400)

NullCA+


Eomes

CA+

Eomes

CAC

A

R26

CreER

T

R26

CreER

TEo

mes

CA+

3d Tx

10075

10075


+Tx

0d 1d 2d 3d 4d +Tx

120572 Eomes

120572 Eomes

EomesCA+ R26CreERT

(a)


10075

+minus

p2lox-Cdx2-V5

50

37



Induced Eomes LOF







1733

474

1637

513

151

1907

192

1585

335221

0

500

1000

1500

2000

1 2 3 4 5

Diff

eren

tially

expr

esse

d

Upregulated (ge15x)


Median TSC ESC ge 2

Median ESCTSC ge 2

gene

s

(a)

(b) (c) (d)






A 221

G 16

F 7

E 26D 37

C 251B 129

EomesElf5

Cdx2

1

2 3

ELy6aGdf6Bves

Gm1673Nat8lApln




Map3k8Lcp1Cd83Cpm

Ccdc85b

GDuox2Duoxa2Il5ra



Ppp2r2c


Fermt1D430041D05Rik

Zic3Zic5


B830017H08RikCth




FTph1


Dram1Emp2

LDuox2Duoxa2Il5ra





Ppp2r2cCcdc85b

NLy6aInpp1Bves

Gm1673Nat8lApln




BambiMpped2Ssbp2Sp4





1600029D21RikIrx3

H 228K 30

I 418

N 15

M 35

J 223

L 27

EomesCdx2Elf5

1

34

O 114R 29

U 2S 2

P 419

T 48

Q 248Cdx2Elf5

5

34

Eomes

SClic5Dram1

UEomesNxf7

RMef2bBhlhe41Cubn

Gm5091Srgn





Gm15127


Capn6Ly6g

Afap1l1Msx2




1600029D21RikIrx3













0

02

04

06

08

1

d0d1

d2d3

ns

ns

ns

Relat

ive m

RNA

expr

essio

n (d

0=1)

Cdx2

Eomes

Elf5

Bok

Cldn

26

Cyp26a1

Duox2

Duoxa2

Nr0b1

Sox21





4 Discussion



Nr0b1

E75

Sox21

E75

Duox2

E75

Duoxa2

E75

Cldn26

E75

Cyp26a1

E75

BokMtd

E75

AlB

Eomes

Ch

PS

E75

VE
















Acknowledgments


References













































































3 Results










Eomes Merge

E65

E75

E85

E95

E10

5

ChH

ChH

EPC

ExE

(a)

(b)

(d)

(e)

(f)

(c)

PS

VE

EPC

Ch

Al

Ch

EPC

ChH ChH

Pl

Pl

pTGC

NK

EPCav

Cdx2Eomes

Eomes

Eomes

Elf5

Eomes

EomesGFP

lowast

lowast

lowast

100120583m

100120583m

500120583m

500120583m

100120583m

500120583m

e998400

d998400

d998400998400

f998400

(a998400) (a998400998400)

(b998400)

(d998400) (d998400998400)

(e998400)

(f998400)




100120583m

(a)

(b)

(c)

(d)


100120583m

Cdx2 Elf5 DAPIMerge

100120583m


100120583m






(b)

(c)


FACS

MEF feeder layer



minus1d

Induced expression

ESCsMEF depletion





(b998400998400)(b998400)

(c998400)

NullCA+


Eomes

CA+

Eomes

CAC

A

R26

CreER

T

R26

CreER

TEo

mes

CA+

3d Tx

10075

10075


+Tx

0d 1d 2d 3d 4d +Tx

120572 Eomes

120572 Eomes

EomesCA+ R26CreERT

(a)


10075

+minus

p2lox-Cdx2-V5

50

37



Induced Eomes LOF







1733

474

1637

513

151

1907

192

1585

335221

0

500

1000

1500

2000

1 2 3 4 5

Diff

eren

tially

expr

esse

d

Upregulated (ge15x)


Median TSC ESC ge 2

Median ESCTSC ge 2

gene

s

(a)

(b) (c) (d)






A 221

G 16

F 7

E 26D 37

C 251B 129

EomesElf5

Cdx2

1

2 3

ELy6aGdf6Bves

Gm1673Nat8lApln




Map3k8Lcp1Cd83Cpm

Ccdc85b

GDuox2Duoxa2Il5ra



Ppp2r2c


Fermt1D430041D05Rik

Zic3Zic5


B830017H08RikCth




FTph1


Dram1Emp2

LDuox2Duoxa2Il5ra





Ppp2r2cCcdc85b

NLy6aInpp1Bves

Gm1673Nat8lApln




BambiMpped2Ssbp2Sp4





1600029D21RikIrx3

H 228K 30

I 418

N 15

M 35

J 223

L 27

EomesCdx2Elf5

1

34

O 114R 29

U 2S 2

P 419

T 48

Q 248Cdx2Elf5

5

34

Eomes

SClic5Dram1

UEomesNxf7

RMef2bBhlhe41Cubn

Gm5091Srgn





Gm15127


Capn6Ly6g

Afap1l1Msx2




1600029D21RikIrx3













0

02

04

06

08

1

d0d1

d2d3

ns

ns

ns

Relat

ive m

RNA

expr

essio

n (d

0=1)

Cdx2

Eomes

Elf5

Bok

Cldn

26

Cyp26a1

Duox2

Duoxa2

Nr0b1

Sox21





4 Discussion



Nr0b1

E75

Sox21

E75

Duox2

E75

Duoxa2

E75

Cldn26

E75

Cyp26a1

E75

BokMtd

E75

AlB

Eomes

Ch

PS

E75

VE
















Acknowledgments


References








































































Eomes Merge

E65

E75

E85

E95

E10

5

ChH

ChH

EPC

ExE

(a)

(b)

(d)

(e)

(f)

(c)

PS

VE

EPC

Ch

Al

Ch

EPC

ChH ChH

Pl

Pl

pTGC

NK

EPCav

Cdx2Eomes

Eomes

Eomes

Elf5

Eomes

EomesGFP

lowast

lowast

lowast

100120583m

100120583m

500120583m

500120583m

100120583m

500120583m

e998400

d998400

d998400998400

f998400

(a998400) (a998400998400)

(b998400)

(d998400) (d998400998400)

(e998400)

(f998400)




100120583m

(a)

(b)

(c)

(d)


100120583m

Cdx2 Elf5 DAPIMerge

100120583m


100120583m






(b)

(c)


FACS

MEF feeder layer



minus1d

Induced expression

ESCsMEF depletion





(b998400998400)(b998400)

(c998400)

NullCA+


Eomes

CA+

Eomes

CAC

A

R26

CreER

T

R26

CreER

TEo

mes

CA+

3d Tx

10075

10075


+Tx

0d 1d 2d 3d 4d +Tx

120572 Eomes

120572 Eomes

EomesCA+ R26CreERT

(a)


10075

+minus

p2lox-Cdx2-V5

50

37



Induced Eomes LOF







1733

474

1637

513

151

1907

192

1585

335221

0

500

1000

1500

2000

1 2 3 4 5

Diff

eren

tially

expr

esse

d

Upregulated (ge15x)


Median TSC ESC ge 2

Median ESCTSC ge 2

gene

s

(a)

(b) (c) (d)






A 221

G 16

F 7

E 26D 37

C 251B 129

EomesElf5

Cdx2

1

2 3

ELy6aGdf6Bves

Gm1673Nat8lApln




Map3k8Lcp1Cd83Cpm

Ccdc85b

GDuox2Duoxa2Il5ra



Ppp2r2c


Fermt1D430041D05Rik

Zic3Zic5


B830017H08RikCth




FTph1


Dram1Emp2

LDuox2Duoxa2Il5ra





Ppp2r2cCcdc85b

NLy6aInpp1Bves

Gm1673Nat8lApln




BambiMpped2Ssbp2Sp4





1600029D21RikIrx3

H 228K 30

I 418

N 15

M 35

J 223

L 27

EomesCdx2Elf5

1

34

O 114R 29

U 2S 2

P 419

T 48

Q 248Cdx2Elf5

5

34

Eomes

SClic5Dram1

UEomesNxf7

RMef2bBhlhe41Cubn

Gm5091Srgn





Gm15127


Capn6Ly6g

Afap1l1Msx2




1600029D21RikIrx3













0

02

04

06

08

1

d0d1

d2d3

ns

ns

ns

Relat

ive m

RNA

expr

essio

n (d

0=1)

Cdx2

Eomes

Elf5

Bok

Cldn

26

Cyp26a1

Duox2

Duoxa2

Nr0b1

Sox21





4 Discussion



Nr0b1

E75

Sox21

E75

Duox2

E75

Duoxa2

E75

Cldn26

E75

Cyp26a1

E75

BokMtd

E75

AlB

Eomes

Ch

PS

E75

VE
















Acknowledgments


References


































































100120583m

(a)

(b)

(c)

(d)


100120583m

Cdx2 Elf5 DAPIMerge

100120583m


100120583m






(b)

(c)


FACS

MEF feeder layer



minus1d

Induced expression

ESCsMEF depletion





(b998400998400)(b998400)

(c998400)

NullCA+


Eomes

CA+

Eomes

CAC

A

R26

CreER

T

R26

CreER

TEo

mes

CA+

3d Tx

10075

10075


+Tx

0d 1d 2d 3d 4d +Tx

120572 Eomes

120572 Eomes

EomesCA+ R26CreERT

(a)


10075

+minus

p2lox-Cdx2-V5

50

37



Induced Eomes LOF







1733

474

1637

513

151

1907

192

1585

335221

0

500

1000

1500

2000

1 2 3 4 5

Diff

eren

tially

expr

esse

d

Upregulated (ge15x)


Median TSC ESC ge 2

Median ESCTSC ge 2

gene

s

(a)

(b) (c) (d)






A 221

G 16

F 7

E 26D 37

C 251B 129

EomesElf5

Cdx2

1

2 3

ELy6aGdf6Bves

Gm1673Nat8lApln




Map3k8Lcp1Cd83Cpm

Ccdc85b

GDuox2Duoxa2Il5ra



Ppp2r2c


Fermt1D430041D05Rik

Zic3Zic5


B830017H08RikCth




FTph1


Dram1Emp2

LDuox2Duoxa2Il5ra





Ppp2r2cCcdc85b

NLy6aInpp1Bves

Gm1673Nat8lApln




BambiMpped2Ssbp2Sp4





1600029D21RikIrx3

H 228K 30

I 418

N 15

M 35

J 223

L 27

EomesCdx2Elf5

1

34

O 114R 29

U 2S 2

P 419

T 48

Q 248Cdx2Elf5

5

34

Eomes

SClic5Dram1

UEomesNxf7

RMef2bBhlhe41Cubn

Gm5091Srgn





Gm15127


Capn6Ly6g

Afap1l1Msx2




1600029D21RikIrx3













0

02

04

06

08

1

d0d1

d2d3

ns

ns

ns

Relat

ive m

RNA

expr

essio

n (d

0=1)

Cdx2

Eomes

Elf5

Bok

Cldn

26

Cyp26a1

Duox2

Duoxa2

Nr0b1

Sox21





4 Discussion



Nr0b1

E75

Sox21

E75

Duox2

E75

Duoxa2

E75

Cldn26

E75

Cyp26a1

E75

BokMtd

E75

AlB

Eomes

Ch

PS

E75

VE
















Acknowledgments


References

































































(b)

(c)


FACS

MEF feeder layer



minus1d

Induced expression

ESCsMEF depletion





(b998400998400)(b998400)

(c998400)

NullCA+


Eomes

CA+

Eomes

CAC

A

R26

CreER

T

R26

CreER

TEo

mes

CA+

3d Tx

10075

10075


+Tx

0d 1d 2d 3d 4d +Tx

120572 Eomes

120572 Eomes

EomesCA+ R26CreERT

(a)


10075

+minus

p2lox-Cdx2-V5

50

37



Induced Eomes LOF







1733

474

1637

513

151

1907

192

1585

335221

0

500

1000

1500

2000

1 2 3 4 5

Diff

eren

tially

expr

esse

d

Upregulated (ge15x)


Median TSC ESC ge 2

Median ESCTSC ge 2

gene

s

(a)

(b) (c) (d)






A 221

G 16

F 7

E 26D 37

C 251B 129

EomesElf5

Cdx2

1

2 3

ELy6aGdf6Bves

Gm1673Nat8lApln




Map3k8Lcp1Cd83Cpm

Ccdc85b

GDuox2Duoxa2Il5ra



Ppp2r2c


Fermt1D430041D05Rik

Zic3Zic5


B830017H08RikCth




FTph1


Dram1Emp2

LDuox2Duoxa2Il5ra





Ppp2r2cCcdc85b

NLy6aInpp1Bves

Gm1673Nat8lApln




BambiMpped2Ssbp2Sp4





1600029D21RikIrx3

H 228K 30

I 418

N 15

M 35

J 223

L 27

EomesCdx2Elf5

1

34

O 114R 29

U 2S 2

P 419

T 48

Q 248Cdx2Elf5

5

34

Eomes

SClic5Dram1

UEomesNxf7

RMef2bBhlhe41Cubn

Gm5091Srgn





Gm15127


Capn6Ly6g

Afap1l1Msx2




1600029D21RikIrx3













0

02

04

06

08

1

d0d1

d2d3

ns

ns

ns

Relat

ive m

RNA

expr

essio

n (d

0=1)

Cdx2

Eomes

Elf5

Bok

Cldn

26

Cyp26a1

Duox2

Duoxa2

Nr0b1

Sox21





4 Discussion



Nr0b1

E75

Sox21

E75

Duox2

E75

Duoxa2

E75

Cldn26

E75

Cyp26a1

E75

BokMtd

E75

AlB

Eomes

Ch

PS

E75

VE
















Acknowledgments


References

































































1733

474

1637

513

151

1907

192

1585

335221

0

500

1000

1500

2000

1 2 3 4 5

Diff

eren

tially

expr

esse

d

Upregulated (ge15x)


Median TSC ESC ge 2

Median ESCTSC ge 2

gene

s

(a)

(b) (c) (d)






A 221

G 16

F 7

E 26D 37

C 251B 129

EomesElf5

Cdx2

1

2 3

ELy6aGdf6Bves

Gm1673Nat8lApln




Map3k8Lcp1Cd83Cpm

Ccdc85b

GDuox2Duoxa2Il5ra



Ppp2r2c


Fermt1D430041D05Rik

Zic3Zic5


B830017H08RikCth




FTph1


Dram1Emp2

LDuox2Duoxa2Il5ra





Ppp2r2cCcdc85b

NLy6aInpp1Bves

Gm1673Nat8lApln




BambiMpped2Ssbp2Sp4





1600029D21RikIrx3

H 228K 30

I 418

N 15

M 35

J 223

L 27

EomesCdx2Elf5

1

34

O 114R 29

U 2S 2

P 419

T 48

Q 248Cdx2Elf5

5

34

Eomes

SClic5Dram1

UEomesNxf7

RMef2bBhlhe41Cubn

Gm5091Srgn





Gm15127


Capn6Ly6g

Afap1l1Msx2




1600029D21RikIrx3













0

02

04

06

08

1

d0d1

d2d3

ns

ns

ns

Relat

ive m

RNA

expr

essio

n (d

0=1)

Cdx2

Eomes

Elf5

Bok

Cldn

26

Cyp26a1

Duox2

Duoxa2

Nr0b1

Sox21





4 Discussion



Nr0b1

E75

Sox21

E75

Duox2

E75

Duoxa2

E75

Cldn26

E75

Cyp26a1

E75

BokMtd

E75

AlB

Eomes

Ch

PS

E75

VE
















Acknowledgments


References








































































0

02

04

06

08

1

d0d1

d2d3

ns

ns

ns

Relat

ive m

RNA

expr

essio

n (d

0=1)

Cdx2

Eomes

Elf5

Bok

Cldn

26

Cyp26a1

Duox2

Duoxa2

Nr0b1

Sox21





4 Discussion



Nr0b1

E75

Sox21

E75

Duox2

E75

Duoxa2

E75

Cldn26

E75

Cyp26a1

E75

BokMtd

E75

AlB

Eomes

Ch

PS

E75

VE
















Acknowledgments


References

































































Nr0b1

E75

Sox21

E75

Duox2

E75

Duoxa2

E75

Cldn26

E75

Cyp26a1

E75

BokMtd

E75

AlB

Eomes

Ch

PS

E75

VE
















Acknowledgments


References











































































Acknowledgments


References
































































A Resource for the Transcriptional Signature of Bona Fide ......ResearchArticle A Resource for the...

Documents

Transcript of A Resource for the Transcriptional Signature of Bona Fide ......ResearchArticle A Resource for the...