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 Material Information
Title: Global gene expression of the inner cell mass and trophectoderm of the bovine blastocyst
Series Title: BMC Developmental Biology 12:33
Physical Description: Journal Article
Creator: Ozawa, Manabu
Sakatani, Miki
Yao, JiQiang
Shanker, Savita
Yu, Fahong
Yamshita, Rui
Wakabayashi, Shunichi
Nakai, Kenta
Dobbs, Kyle B.
Sudano, Mateus Jose
Farmerie, William G.
Hansen, Peter J.
Publisher: Biomed Central
 Notes
Abstract: Background: The first distinct differentiation event in mammals occurs at the blastocyst stage when totipotent blastomeres differentiate into either pluripotent inner cell mass (ICM) or multipotent trophectoderm (TE). Here we determined, for the first time, global gene expression patterns in the ICM and TE isolated from bovine blastocysts. The ICM and TE were isolated from blastocysts harvested at day 8 after insemination by magnetic activated cell sorting, and cDNA sequenced using the SOLiD 4.0 system. Results: A total of 870 genes were differentially expressed between ICM and TE. Several genes characteristic of ICM (for example, NANOG, SOX2, and STAT3) and TE (ELF5, GATA3, and KRT18) in mouse and human showed similar patterns in bovine. Other genes, however, showed differences in expression between ICM and TE that deviates from the expected based on mouse and human. Conclusion: Analysis of gene expression indicated that differentiation of blastomeres of the morula-stage embryo into the ICM and TE of the blastocyst is accompanied by differences between the two cell lineages in expression of genes controlling metabolic processes, endocytosis, hatching from the zona pellucida, paracrine and endocrine signaling with the mother, and genes supporting the changes in cellular architecture, stemness, and hematopoiesis necessary for development of the trophoblast.
Acquisition: Collected for University of Florida's Institutional Repository by the UFIR Self-Submittal tool. Submitted by Peter Hansen.
Publication Status: Published
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Source Institution: University of Florida Institutional Repository
Holding Location: University of Florida
Rights Management: All rights reserved by the submitter.
System ID: IR00001361:00001

Full Text

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Formula 1: Position-specific weight matrix (PSWM) scores where l : length of the matrix; nif: the observed number of base ‘ f ’ (A, C, G or T) at the i th position in the training; Ni: sum of the observed num ber of all bases at i -th position. Formula 2: Ratio of the score to the maximum score where min( score ): minimum value of each PSWM, max( score ): maximum value of each PSWM. Formula 3: Hypergeometric distribution where N : total number of promoters, p : expected frequency of t he promoters associated with a given PSWM (number of associated promoters/ N ), n : total number of promoters belonging to a dataset of concern and i : number of the promot ers associated with a given PSWM in a dataset.



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metabolism Linoleic acid Nicotinate andnicotinamidemetabolism N-glycan biosynthesis High-mannose type metabolism Sphingolipid biosynthesis O-Mannosyl glycan Benzoate degradationvia CoA ligation Tyrosine metabolism biosynthesis Peptidoglycan Fatty acid elongationin mitochondria degradation ofKetone bodies Synthesis and proline metabolism Arginine and Photosynthesis Fatty acidbiosynthesis biosynthesis Puromycin metabolism D-Alanine Lipopolysaccharidebiosynthesis ganglioseries Glycosphingolipidbiosynthesis Galactosemetabolism Ethylbenzenedegradation metabolism Propanoate Biphenyl degradation Pantothenate and CoAbiosynthesis Citrate cycle(TCA cycle) biosynthesis Novobiocin Flavonoidbiosynthesis metabolism Starch and sucrose biosynthesis Benzoxazinone nonribosomalpeptides siderophore group Biosynthesis of Carbazoledegradation metabolism Phenylalanine Naphthalene andanthracenedegradation P450 Substrates [B] Cytochrome phosphorylation Oxidative biosynthesis Folate Fructose andmannose metabolism biosynthesis Primary bile acid Inositol phosphatemetabolism biosynthesis Glucosinolate [B] Lipids tryptophan biosynthesis Phenylalanine tyrosine and biosynthesis Monoterpenoid Photosynthesis -antenna proteins metabolism Tryptophan D-glutamatemetabolism D-Glutamine and [B] Proteoglycans gamma-Hexachlorocyclohexanedegradation degradation Fluorene biosynthesis Clavulanic acid Lysinedegradation Reductivecarboxylate cycle(CO2 fixation) biosynthesis cephalosporin Penicillin and resistance beta-Lactam xylene degradation Toluene and Glycine, serine andthreonine metabolism D-ornithinemetabolism D-Arginine and Glycosaminoglycanbiosynthesis -chondroitin sulfate degradation Styrene [B] PhytochemicalCompounds ansamycins Biosynthesis of biosynthesis N-Glycan Biosynthesis of type IIpolyketide backbone biosynthesis Insect hormone Nitrogenmetabolism metabolism Thiamine metabolism Retinol fixation Carbon Pentose and glucuronateinterconversions polyketide products Biosynthesis of type II metabolism Pyruvate unit biosynthesis Polyketide sugar (GPI)-anchor biosynthesis Glycosylphosphatidylinositol Other glycandegradation metabolism Glycerolipid Gluconeogenesis Glycolysis / Glycosaminoglycanheparan sulfate biosynthesis beta-Alaninemetabolism Purine metabolism isoleucine biosynthesis Valine, leucine and metabolism Cystein and methionine 3-Chloroacrylic aciddegradation biosynthesis Streptomycin biosynthesis O-Glycan Terpenoid backbonebiosynthesis biosynthesis Isoflavonoid [B] Cytochrome P450 nucleotide sugarmetabolism Amino sugar and metabolism Lipoic acid biosynthesis Steroid hormone Glycosphingolipidbiosynthesis -lact and neolacto series metabolism Butanoate Biotinmetabolism Phosphonate andphosphinate metabolism biosynthesis Diterpenoid Biosynthesis ofunsaturated fatty acids Ether lipidmetabolism Tetracyclinebiosynthesis Sulfurmetabolism biosynthesis Sesquiterpenoid biosynthesis -keratan sulfate Glycosaminoglycan metabolism Caffeine Ascorbate andaldarate metabolism metabolism Porphyrin andchlorophyll biosynthesis Isoquinoline alkaloid biosynthesis Indole alkaloid biosynthesis Acridone alkaloid Riboflavinmetabolism Phenylpropanoidbiosynthesis Benzoate degradationvia hydroxylation Selenoamino acidmetabolism degradation Atrazine metabolism Pentose phosphate C5-Brancheddibasic acidmetabolism degradation Fluorobenzoate Fatty acidmetabolism biosynthesis Carotenoid 1,1,1-Trichloro-2,2-bis(4-chlorophenyl)ethane (DDT) degradation Steroid biosynthesis Glutathionemetabolism degradation Caprolactam Proteins [B] Photosynthesis globoseries Glycosphingolipidbiosynthesis Secondary bile acidbiosynthesis 1,4-Dichlorobenzenedegradation 1,2-Dichloroethanedegradation metabolism alpha-Linolenic acid Pyrimidine metabolism Taurine andhypotaurinemetabolism other terpenoid-quinonebiosynthesis Ubiquinone and pyridine alkaloid biosynthesis Tropane, piperidine and biosynthesis Flavone andflavonol metabolism Methane glutamate metabolism Alanine aspartate and Anthocyaninbiosynthesis Biosynthesis of vancomycingroup antibiotics Brassinosteroidbiosynthesis biosynthesis Zeatin metabolism dicarboxylate Glyoxylate and metabolism Vitamin B6 metabolism Glycerophospholipid metabolism Arachidonic acid [B] Glycosyltransferases 2,4-Dichlorobenzoatedegradation 16-membered macrolides Biosynthesis of 12-, 14and Tetrachloroethene degradation by folate One carbon pool Butirosin andneomycin biosynthesis isoleucine degradation Valine, leucine and degradation Bisphenol A Glycosaminoglycandegradation Cyanoamino acidmetabolism pinene degradation Limonene and Metabolism ofTerpenoids and Polyketides NucleotideMetabolism CarbohydrateMetabolism Cofactors and Vitamins Metabolism of Xenobiotics Biodegradationand Metabolism Metabolism Energy Amino AcidMetabolism Metabolism ofOther Amino Acid and Metabolism Glycan Biosynthesis Other Secondary Metabolites Biosynthesis of Metabolism Lipid



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Additional File 4. Differences in expression between inner cell mass (ICM) and trophectoderm (TE) for genes considered as bein g characteristically expressed by ICM, embr yonic stem cells and TE in human or mouse. a Genes Considered Characteristic of ICM or embryonic stem cells ABL2 ACOT9 AMD1 ARHGER18 BXL2L14 CANX CCNB1 CDCA2 CENPF CLDN7 CYP2S1 DAB2 DDX3X DNMT3L DSP EI24 EIF1AX EIF2S1 EIF5B EPRS FGFR2 FGFR3 FLG FOXD3 FOXH1 GATA2 GATA3 GATAD1 GJC1 GLTSCR2 GM2A GPD1L H19 HMGB1 HN1 HSD17B11 IFITM2 IGF2R IL17RA IL6R b ITGA5 ITGA6 JAK2 JUNB KDM2B KLF2 KTN1 LAMC1 LAMP2 LEFTY1 LRPAP 1 MAP3K3 MORC1 MRPL15 MTCH2 MUC15 MYC NANOG NANOS1 NODAL OTX2 PAX8 PDPN PEG3 PEX3 PLK1 POU2F1 POU5F1 PPARD PTP4A1 PTTG1IP PUM1 PUM2 PYGB RAB12 PYGB RAB12 RAB5C RAB6A RELL1 RHPN2 RPL14 RPL19 RPL32 RPL7A RUNX1 SALL4 SBNO1 SCD SIN3B SLIT3 SMAD2 SMAD4 SMAD5 SNX3 SOX2 SPATA13 SPETA5L1 SPIC SRRM1 SSFA2 STAG2 STAT3 TACC1 TAL1 TAXBP1 TFRC c TGFBR1 TGFBR3 TMED2 TOP2A TPRKB TRNAU1 AP VAV3 XIST ZBTB34 ZC3HAV1 ZFP42 ZNF296 Genes Considered Characteristic of TE AQP11 ARNT2 ASCL2 ATP1B3 BMP4 CDH19 CDH22 CDH24 CDX1 CDX2 CELSR2 CGB CGB1 CGB2 CGN d CLDN10 CLDN2 CSNK1A1 CYP11A DAAM1 DSC2 ELF5 EOMES ESRRB FGFR2 GCM1 HAND1 HLA-G HSD3B1 IFNT1 ID2 IRX3 JAM2 KRT1 KRT18 LEP LHCGR MSX2 OCLN PAG2 PCDH1 PCDHB7 PSG3 SFN TBX1 TEAD2 TEAD4 TKDP1 TJP2 a Genes in blue were upregulated in ICM and genes in red were upregulated in TE. The adjusted P value was <0.05 unless indicated by superscripts b adjusted P=0.05 c adjusted P=0.06. d adjusted P=0.09.



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RESEARCHARTICLEOpenAccessGlobalgeneexpressionoftheinnercellmassand trophectodermofthebovineblastocystManabuOzawa1,4,MikiSakatani3,JiQiangYao2,SavitaShanker2,FahongYu2,RuiYamashita5, ShunichiWakabayashi5,KentaNakai5,KyleBDobbs1,MateusJosSudano6,WilliamGFarmerie2andPeterJHansen1*AbstractBackground: Thefirstdistinctdifferentiationeventinmammalsoccursattheblastocyststagewhentotipotent blastomeresdifferentiateintoeitherpluripotentinnercellmass(ICM)ormultipotenttrophectoderm(TE).Herewe determined,forthefirsttime,globalgeneexpressionpatternsintheICMandTEisolatedfrombovineblastocysts. TheICMandTEwereisolatedfromblastocystsharvestedatday8afterinseminationbymagneticactivatedcell sorting,andcDNAsequencedusingtheSOLiD4.0system. Results: Atotalof870genesweredifferentiallyexpressedbetweenICMandTE.SeveralgenescharacteristicofICM (forexample, NANOG SOX2 ,and STAT3 )andTE( ELF5 GATA3 ,and KRT18 )inmouseandhumanshowedsimilar patternsinbovine.Othergenes,however,showeddifferencesinexpressionbetweenICMandTEthatdeviates fromtheexpectedbasedonmouseandhuman. Conclusion: Analysisofgeneexpressionindicatedthatdifferentiationofblastomeresofthemorula-stageembryo intotheICMandTEoftheblastocystisaccompaniedbydifferencesbetweenthetwocelllineagesinexpressionof genescontrollingmetabolicprocesses,endocytosis,hatchingfromthezonapellucida,paracrineandendocrine signalingwiththemother,andgenessupportingthechangesincellulararchitecture,stemness,andhematopoiesis necessaryfordevelopmentofthetrophoblast. Keywords: Blastocyst,Trophectoderm,Innercellmass,DevelopmentBackgroundFollowingitsformationbysyngamyofthepronucleiof theoocyteandsperm,themammalianembryobeginslife asatotipotent,singlecellorganism.Subsequentcyclesof celldivisionandtheformationoftightjunctionsbetween blastomeresleadtoaconditionwherebyblastomereson theouterfaceoftheembryoexhibitdifferentpatternsof cellpolarity,geneexpressionandproteinaccumulation thanblastomeresontheinnerpartoftheembryo[1-4]. Non-polarizedblastomeresintheinnerpartoftheembryo aredestinedtoformthepluripotentinnercellmass(ICM) thatgivesrisetotheembryowhilepolarizedcellsinthe outerfaceoftheembryoarefatedtodifferentiateintothe trophectoderm(TE),whichdevelopsintoextraembryonic membranes.Cellfatemaybedeterminedasearlyasthe 4 – 8cellstageinthemouseanddependupondifferences betweenblastomeresinthekineticsoftheinteraction betweenthetranscriptionfactorPou5f1andDNAbinding sites[5].Nonetheless,blastomeresdonotundergolineage commitmentuntilaboutthe32-cellstage(inmice),based onlossofabilityofblastomerestoformeitherICMorTE [2]. LineagecommitmenttowardsICMorTEisunderthe controlofspecifictranscriptionfactors.Theexactrole ofatleastsometranscriptionfactorsvarieswithspecies [6].Inthebeststudiedspecies,themouse,theICMis regulatedby Sall4 Pou5f1 Sox2 and Nanog whileTE formationresultsfromacascadeofeventsinvolving Yap1 Tead4 Gata3 Cdx2 Eomes and Elf5 [7].Functionalpropertiesofthetwocelllineagesisalsodivergent.Inpart,thisreflectstheprocessesresponsiblefor establishmentandmaintenanceofcelllineage,suchas *Correspondence: Hansen@animal.ufl.edu1DepartmentofAnimalSciencesandD.H.BarronReproductiveandPerinatal BiologyResearchProgram,POBox110910,Gainesville,FL32611-0910,USA Fulllistofauthorinformationisavailableattheendofthearticle 2012Ozawaetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and reproductioninanymedium,providedtheoriginalworkisproperlycited.Ozawa etal.BMCDevelopmentalBiology 2012, 12 :33 http://www.biomedcentral.com/1471-213X/12/33

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differencesintranscriptionfactorusage,cellsignaling pathwaysandepigeneticmarks[7,8].Inaddition,the functionoftheICM,whichisfatedtoundergoaseries ofdifferentiationeventsinthegastrulationprocess,is differentfromtheTE,whichisdestinedtointeractwith theliningofthematernalreproductivetract. Inthepresentstudy,wedescribe,forthefirsttime, differencesinthetranscriptomeoftheICMandTEwith theobjectiveofunderstandingtheconsequencesofthe differentiationofthesetwocelltypesforcellularfunction.ThiswasachievedbyseparatingICMandTEusing anewly-developedimmunomagneticprocedure[9]followedbynext-generationsequencing.Resultsrevealthe implicationsofthespatialanddevelopmentaldifferentiationofthesefirsttwolineagesofthepreimplantation embryowithrespecttometabolism,interactionwiththe maternalsystemandchangesincellulararchitecture.In addition,aspectsofmolecularcontroloftheprocessof lineagecommitmentanddifferentiationareillustrative ofsimilaritiesanddifferenceswiththeprototypical mousemodel.MethodsReagentsAllreagentswerepurchasedfromSigma-Aldrich(St. Louis,MO,USA)orFisherScientific(Pittsburgh,PA, USA)unlessotherwisespecified.EmbryocultureandICM/TEisolationBovineembryoswereproducedfromslaughterhousederivedoocytesusingproceduresforinvitrooocyte maturation,fertilization,andembryocultureas describedpreviously[10].OvariesweredonatedbyCentralPacking,CenterHillFlorida.Thedayoffertilization wasdefinedasDay0.Afterfertilizationfor18 – 20h, embryoswereculturedinSOF-BE1medium[11]at 38.5Cinahumidifiedatmosphereof5%CO2and5% O2withthebalanceN2.Embryoswereculturedin groupsof30ina50 lculturedropundermineraloil. AtDay6,anadditional5 lculturemediumwasadded. AtDay8,blastocystswereharvestedandusedtopreparepreparationsofICMandTEusingmagneticactivatedcellsortingasreportedpreviously[9]. ThreeseparatepoolsofTEandICMforeachtreatmentwereobtained.Eachpoolwaspreparedusing88to 102blastocysts.Atotalof15fertilizationprocedures wereusedtopreparetheblastocysts;asetofthreebulls wasusedforfertilizationforeachprocedure.RNApreparation,libraryconstructionandsequencing usingSOLiD4systemTotalRNAwasisolatedfromeachpoolofembryonic cellsusingthePicoPureRNAIsolationKit(AppliedBiosystems,FosterCity,CA,USA)accordingtothe manufacturer ’ sinstructions.ThequalityofRNAwas assessedusingtheAgilent2100Bioanalyzer(Agilent Technologies,SantaClara,CA).AmplifiedcDNAwas preparedfromtotalRNAforRNA-Seqapplications usingtheOvationRNA-Seqkit(NuGenTechnology, SanCarlos,CA).BarcodedfragmentlibrarieswereconstructedusingtheSOLiDTMv4fragmentlibrarykit accordingtothemanufacturer ’ sprotocol(AppliedBiosystems).Briefly,doublestrandedcDNAwasshearedto 150 – 180bpfragmentsusingaCovarisTMS2Sonication system(Covaris,Woburn,MA).ThefragmentedDNA wassubsequentlyend-repairedandblunt-endligatedto P1andP2adaptors.Theadaptorligated,purifiedand size-selected200 – 270bpfragmentswerenick-translated andthenamplifiedusingprimersspecifictoP1andP2 adaptorsandPlatinumWPCRAmplificationMix(AppliedBiosystems).Thequalityofthelibrariesandfragmentdistributionwereverifiedbyrunning1 lofeach libraryonAgilentDNA1000chip(AgilentTechnologies).Amplifiedlibraries(5differentlibrariespooledfor eachslide)wereimmobilizedontoSOLiDP1DNA beads(AppliedBiosystems).Thebead-boundlibraries werethenclonallyamplifiedbyemulsionPCRaccording totheAppliedBiosystemsSOLiDTM4SystemsTemplatedBeadPreparationGuide.Afteramplification, emulsionsweredisruptedwith2-butanolandthebeads containingclonallyamplifiedtemplateDNAwereP2enrichedandextendedwithabeadlinkerbyterminal transferase.Thequantityofthebeadswasdetermined usingaNanoDropWND1000spectrophotometer (ThermoScientific,Wilmington,DE).Approximately 600-700Mbeadsweredepositedoneachslide(ranin totalthreeslides)andsequencedusing ‘ sequencingby ligation ’ chemistryandthe50x5bpprotocolonthe SOLiDTMv4sequencer(AppliedBiosystems)atthe InterdisciplinaryCenterforBiotechnologyResearch, UniversityofFlorida.Resultswereobtainedascolor spacefastafiles.AnalysisofreaddataRawsequencingreadswereinitiallyprocessedwithGenomeQuesttools[12].Ambiguousresiduesweretrimmed offfrombothsidesofthesequence.BaseswithPhred qualitybelow12fromthe3 ’ endofthesequencewere removed.Readsthatwereshorterthan40basesorthat containedmorethan10baseswithqualitybelow12 werealsodiscardedaswerereadsconsistingofrepetitive singlebasesthataccountsformorethan60%ofthe lengthatthe3 ’ end.About53~64%ofreadswere retainedaftercleanup,proving102 – 157millionclean readsforthethreereplicatesofeachtreatment. Formappingtothegenome,the Bostaurus genomic sequence bosTau4 (repeatmasked)wasdownloaded fromtheUCSCgenomebrowser(http://genome.ucsc.Ozawa etal.BMCDevelopmentalBiology 2012, 12 :33 Page2of13 http://www.biomedcentral.com/1471-213X/12/33

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edu/).SequencingreadsofeachsampleweremappedindependentlytothereferencesequencesusingTopHat 1.2.0[13].TopHatsplitreadstosegmentsandjoinssegmentalignments.Amaximumofonemismatchineach ofthe25bpsegmentswasallowed.Thisstepmapped 36.8%readstothegenome.Theunmappedreadswere collectedandmappedtothereferenceusingBowtie 0.12.7[14]allowingthreemismatches.Unmappedreads werefurthermappedtocDNAsequencesusingbfast 0.6.4[15]whileallowingforthreemismatchesforeach read.ThecDNAsequencesof B taurus weredownloadedfromtheNationalCenterofBiotechnologyInformation.Scaffoldandchromosomesequenceswere clearedandatotalof35,842sequenceswereobtained (http://www.ncbi.nlm.nih.gov/nuccore/?term=txid9913 [Organism:noexp]).Bfastaligned27.6%ofthetotalreads tothecDNAsequences.Therefore,atotalof64.4%or 595millionreadsweremappedsuccessfully.Ofthe mappedreads,89.8%areuniquelymappedtoeitherthe genomeorcDNAsequences.Dataweredepositedinthe DDBJSequenceReadArchiveathttp://www.ddbj.nig.ac. jp/index-e.html(SubmissionDRA000504). Digitalgeneexpressionwasdeterminedasfollows. Thenumberofmappedreadsforeachindividualgene wascountedusingtheHTSeqtool(http://www-huber. embl.de/users/anders/HTSeq/doc/overview.html)with intersection-nonemptymode.HTSeqtakestwoinput files-bamorsam-formatfilesofmappedreadsanda genemodelfile.TheEnsemblegeneannotationfilein GTFformatwasdownloadedfromtheUCSCgenome browser.TheDESeqpackage[16]inRwasusedfor digitalgeneexpressionanalysis.DESequsesthenegative binomialdistribution,withvarianceandmeanlinkedby localregression,tomodelthenulldistributionofthe countdata.Significantup-anddownregulatedgenes wereselectedusingtwocutoffs:anadjustedPvalueof 0.05andaminimumfold-changeof1.5.Classificationofdifferentiallyexpressedgenesintogene ontology(GO)classesDifferentiallyexpressedgeneswereannotatedbythe DatabaseforAnnotation,VisualizationandIntegrated Discovery(DAVID;(DAVIDBioinformaticsResources 6.7,http://david.abcc.ncifcrf.gov/)[17].Mostgeneswere annotatedusingthebovinegenomeasareferenceand additionalgeneswereannotatedbycomparisontothe humangenome.TheDAVIDdatabasewasqueriedto identifyGOclassesenrichedforupregulatedanddownregulatedgenes.Functionsofdifferentiallyexpressed geneswerefurtherannotatedusingKyotoEncyclopedia ofGenesandGenomes(KEGG,http://www.genome.jp/ kegg/).OverviewofthedifferentiallyregulatedKEGG pathwaysweremappedonKEGGPathwayMapusing iPath2.0(http://pathways.embl.de/)[18]. Tofurtheranalyzepatternsofgenesdifferentially regulatedbetweenICMandTE,k-meanclusteringwas performed.Thereadscountdataofthe870significant genesfortheICM-controlversusTE-controlcomparisonwereclusteredusingk-meansstrategy[19].Toestimatethepremiumclusternumber,k-valuesfrom3to 100weretestedandthecorrespondingsumofsquares error(SSE)[20]wascalculatedforeachkvalue.SSEis definedasthesumofthesquareddistancebetweeneach memberofaclusteranditsclustercentroid.TheSSE valuesdroppedabruptlyuntilk=8(resultsnotshown). TobalancetheminimumnumberofSSEandtheminimumnumberofclusters,k=8wasselectedasthepremiumparameterforclusteringgenesandaheatmapwas generatedusing heatmap 2 ofRpackage.EnrichmentanalysisfortranscriptionfactorbindingsitesForeachdifferentiallyexpressedgene,thecandidatepromoterregionwasdefinedasthespanofnucleotides from200bpupstreamand50bpdownstreamfromthe transcriptionalstartsiteidentifiedinEnsembl.Todetect putativetranscriptionfactorbindingsites(TFBS)ineach promoter,wefollowedthemethodofWassermanand Sandelin[21].Position-specificweightmatriceswere obtainedfromtheJASPARdatabase[22].Thescorewas calculatedbyformula1inAdditionalFile1.Wealsocalculatedtheratioofthescoretothemaximumscoreby formula2(AdditionalFile1).Statisticalsignificanceof eachTFBSwasevaluatedbycalculatingthehypergeometricdistributionusingformula3(Additionalfile1). Weperformedthe ‘ match ’ programwith ‘ minSUM ’ and ‘ minFP ’ thresholdstodetectTFBS[23].StatisticalsignificanceofeachdetectedTFBSwasevaluatedbythe hypergeometricdistributionasdescribedabove.CalculationofGCcontentsanddetectionofCpGislandsThemethodbyGardiner-GardenandFrommer[24]was usedtoidentifyCpGislandsintheregionencompassing the100nucleotidesupstreamand100nucleotidesdownstreamfromthestartsite.Trans criptionalstartsitesfordifferentiallyexpressedgeneswereobtainedfromUMD3.1 [25].ForthedefinitionofCpGislands,TheGCcontent wascalculatedas([C]+[G])/200,where[N]denotesthe numberofnucleotides “ N ” withinthe200basewindow. TheCpGscorewascalculatedas[CG]/([C]*[G]*200).A genewasclassifiedasCpGpositivewhenitsGCcontentin theregionspanningthe100n ucleotidesupstreamandthe 100nucleotidesdownstreamfromthestartsiteexceeds0.5 andwhentheCpGscoreinthesameregionexceeds0.6. Otherwise,agenewasclassifiedasCpGnegative.Chisquareanalysiswasusedtodeterminewhetherthepercent ofgenesclassifiedasCpGpositivedifferedbetween1) genesoverexpressedinICMversusgenesoverexpressedinOzawa etal.BMCDevelopmentalBiology 2012, 12 :33 Page3of13 http://www.biomedcentral.com/1471-213X/12/33

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TEand2)genesoverexpressedinICMorTEversusthe referencepopulationof25118genesinthebovinegenome.Confirmationofdifferencesingeneexpressionbetween ICMandTEbyquantitativePCRAnexperimentwasperformedtoverifytheeffectofcell type(ICMvsTE)andCSF2onrelativemRNAabundance ofthe GATA3 ELF5 CDX2 NANOG and SOX2 .Embryos werepreparedasdescribedpreviouslyandblastocysts werecollectedatDay7.Poolsof25 – 34blastocystswere submittedtomagnetic-activatedcellsorting[9].Atotalof 6biologicalreplicatesofICMandTEwereprepared. mRNAextractionwasperformedusingtheAllPrep DNA/RNAminiKit(Qiagen,Inc.,Valencia,CA,USA)followedbyDNase(Qiagen)treatmentandreversetranscription(HighCapacitycDNAReverseTranscriptionKit, AppliedBiosystems,FosterCity,CA).Transcriptabundancefor GATA3 ELF5 CDX2 NANOG and SOX2 as wellashousekeepinggenes GAPDH SDHA and YWHAZ werequantifiedbyaBio-RadthermalcyclerCFX96-RealTimesystem(Bio-Rad,Hercules,CA,USA)usingSsoFast EvaGreenSupermixreagent(Bio-Rad,Hercules,CA, USA).PCRconditionswereasfollows:30secat95Cfollowedby40cycleseachof5secat95Cand1minat 60C.Datawereanalyzedusingthedelta-deltacycle threshold(Ct)method.Thereferencegenewasthe geometricmeanoftheCtvaluesof GAPDH SDHA and YWHAZ .Primersfor ELF5 werebasedon NM_001024569.1andweredesignedusingPrimerQuest fromidtDNA(http://www.idtdna.com)software,Efficiencywas95%andidentityofampliconswasverified bysequencingproducts.Theprimerswere5 ’ TGC CATTTCAACATCAGTGGCCTG3 ’ and5 ’ AAGGC CACCCTCAAAGACTATGCT3 ’ .Otherprimerpairs werepublishedpreviously: GATA3 [26], CDX2 and NANOG [9], SOX2 [27]and GAPDH SDHA and YWHAZ [28]. Datawereanalyzedbyleast-squaresanalysisofvariance usingtheGeneralLinearModel(GLM)procedureofthe StatisticalAnalysisSystem,version9.2(SASInstituteInc, Cary,NC,USA)Sourcesofvariationinthemodel includedcelltype(ICMandTE),replicateandtheinteraction;celltypewasconsideredfixedandreplicatewas consideredrandom.Logarithmictransformationwasappliedto CDX2 datatoimprovenormality.Alldataare reportedasuntransformedleast-squaresmeans.ResultsDifferentiallyexpressedgenesThelistsofdifferentiallyexpressedgenes,determined usinganadjustedPvalueof 0.05and 1.5-folddifferenceascut-offs,arepresentedinAdditionalfile2.There wereatotalof870genesthatweredifferentiallyexpressed betweenICMandTE,with411genesupregulatedinthe ICMand459downregulatedintheICM(i.e.,upregulated intheTE).Annotationofgenesdifferentiallyexpressedbetween ICMandTEDifferentiallyexpressedgeneswereannotatedusingthe GeneIDconversiontooloftheDAVIDBioinformatics Resources6.7(http://david.abcc.ncifcrf.gov/conversion. jsp);835ofthe870differentiallyexpressedgeneswere annotated(389genesupregulatedintheICMand424 genesupregulatedintheTE).ForthelistofgenesupregulatedinICM,10GOtermswerelistedintheBiologicalProcessgroup,4GOtermsintheCell Componentgroup,and5termsintheMolecularFunctiongroup(Table1).Termsrelatedtotranscriptionalactivitiesweredominantincludingregulationof transcription,DNA-dependent(25genes),regulationof transcriptionfromRNApolymeraseIIpromoter(11 genes),DNAbinding(29genes),transcriptionregulator activity(22genes)andtranscriptionfactoractivity(17 genes).TherewerealsoGOtermsrelatedtometabolic activityincludingregulationofRNAmetabolicprocess (25genes),positiveregulationofmacromoleculemetabolicprocess(12genes),negativeregulationofmacromoleculemetabolicprocess(10genes),andenzyme binding(10genes). ForgenesupregulatedinTE,12GOtermswerelisted intheBiologicalProcessgroup,12intheCellComponentgroup,and9intheMolecularFunctiongroup (Table2).GOtermsenrichedforTEweredistinctfrom thoseforICM.Alargenumberofgenesrepresentedby GOtermsrelatedwithmetabolismwereupregulatedin TEincludingproteolysis(27genes),oxidationreduction (23genes),lipidbiosyntheticprocessing(11genes),steroidmetabolicprocess(10genes),andpeptidaseactivity (actingonL-aminoacidpeptides)(22genes)aswellas genesinvolvedinbindingreactions[ionbinding(86 genes),cationbinding(83genes),metalionbinding(81 genes),calciumionbinding(34genes)andironion binding(12genes)].Therewasalsoenrichmentfor genesassociatedwithendo-orexocytosis,membrane transportandalterationsincellulararchitectureasindicatedbyGOtermsforvesicle-mediatedtransport(15 genes),actinfilament-basedprocess(14genes),actin cytoskeletonorganization(13genes),cytoskeleton organization(13genes),plasmamembrane(43genes), endoplasmicreticulum(32genes),cytoplasmicvesicle (14genes),vesicle(14genes),actincytoskeleton(13 genes),cellprojection(12genes),vacuole(11genes), endoplasmicreticulumpart(11genes),apicalpartofcell (10genes),andcytoskeletalarrangement(20genes). Functionsofdifferentiallyexpressedgeneswerefurther annotatedusingKEGG(http: //www.genome.jp/kegg/). GenesupregulatedinICMwereenrichedineighttermsOzawa etal.BMCDevelopmentalBiology 2012, 12 :33 Page4of13 http://www.biomedcentral.com/1471-213X/12/33

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(Table3A).Theseincludedp athwaysinvolvedinlineage commitment(e.g.,hematopoiet iccelllineage)anddifferentiation(axonguidance)aswellasthoseinvolvedinmaintenanceofstemnessandselfrenewal(e.g.,pathwayin cancerandJak-STATsignalingpathway).GenesupregulatedinTEwereenrichedin12terms(Table3B).Noneof thetermswereincommonwithKEGGtermsenrichedfor genesupregulatedforICM.Termswerepreferentiallyrelatedtotransmembranetransport(lysosome,aldosteroneregulatedsodiumresabsorption,andABCtransporters), lipidorsteroidmetabolism(PPARsignalingpathway, terpenoidbackbonebiosynthesis,sphingolipidmetabolism,steroidhormonebiosynthesis,fattyacidmetabolism)andothermetabolicprocesses(pantothenateand CoAbiosynthesis).Additionalfile3representsaKEGG metabolicpathwaymapinwhichpathwaysthatweredifferentiallyenrichedbetweenICMandTEwereidentified usingiPath2.0(http://pathways.embl.de/).Notethe increasedmetabolicactivityinTEascomparedtoICM.K-meanclusteringThe870genesthatweredifferentiallyexpressedbetween ICMandTEwereclusteredinto8clusters,with2,4,7, 9,23,48,149and628genesineachcluster(Additional file4).Thebiggestcluster(628genes)contained72.2% ofallthesignificantgenesandgeneswereincludedfrom almostalltheoverrepresentedpathways(Table3). Therefore,thek-meananalysisdidnotdisclosemuchinformationonfunctionalexpressionpatternsofdifferentiallyexpressedgenes.ComparisonofICM-TEdifferencesinthebovinewiththe mouseandhumanTheliteraturewasusedtoidentifyagroupofgenesthat havebeenidentifiedasbeingexpressedbyICM,TEor embryonicstemcellsinthemouse[29-32]orhuman [33-38](Additionalfile5).Amongthe119genesconsideredcharacteristicofICMorembryonicstemcells,8 weresignificantlyupregulatedinICM( KDM2B NANOG SOX2 SPIC STAT3 ZX3HAV1 ,and OTX2 ) andtwo( IL6R and TFRC )tended(P=0.06orless)tobe upregulatedinICM.Conversely,6genesconsideredas beingexpressedinICMorembryonicstemcellsinthe mouseorhumanwereupregulatedintheTE( DAB2 DSP GM2A SCD SSFA2 ,and VAV3 ).Of49genesconsideredcharacteristicofTE,12( AQP11 ATP1B3 CGN Table1GOtermsenrichedforgenesupregulatedintheICMascomparedtoTEaGOtermCountPercentPvalueFDRbBiologicalProcess Regulationoftranscription,DNA-dependent256.70.0443.9 RegulationofRNAmetabolicprocess256.70.0449.9 Neurologicalsystemprocess123.20.0116.9 Regulationofcellproliferation123.20.0224.9 Immuneresponse123.20.0335.4 Positiveregulationofmacromoleculemetabolicprocess123.20.0343.1 Cognition112.90.002.4 RegulationoftranscriptionfromRNApolymeraseIIpromoter112.90.0229.6 Responsetoorganicsubstance102.70.0116.8 Negativeregulationofmacromoleculemetabolicprocess102.70.0443.9 CellComponent Plasmamembrane349.10.0220.7 Extracellularregion308.00.004.2 Extracellularregionpart195.10.001.9 Extracellularspace123.20.0216.9 MolecularFunction DNAbinding297.80.0546.9 Transcriptionregulatoractivity225.90.0440.5 Calciumionbinding184.80.0332.1 Transcriptionfactoractivity174.60.0219.4 Enzymebinding102.70.017.7aOnlythoseGOtermswhichcontainedatleast10differentiallyexpressedgenesarelisted.bFalsediscoveryrate(x100).Ozawa etal.BMCDevelopmentalBiology 2012, 12 :33 Page5of13 http://www.biomedcentral.com/1471-213X/12/33

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CYP11A DSC2 ELF5 GATA3 HSD3B1 KRT18 MSX2 SFXNorTJP2 )wereupregulatedinTE. CDH24 ,acadherinreportedtobeupregulatedintheTEofthehuman [33],wasexpressedinhigheramountsintheICM. Wealsoexaminedexpressionofruminant-specificgenes knowntobeupregulatedinTE.Thethreeexamined, IFNT1 [39], PAG2 [40],and TKDP1 [41],wereupregulatedinTE. WeevaluateddifferencesinexpressionbetweenICM andTEforgenesthathavebeenshowninthemouse[7] tobeimportantforsegregationofICMandTElineages andsubsequentTEdifferentiation(Table4).Expression oftwogenesimportantforICMcommitment, NANOG and SOX2 ,wassignificantlyhigherforICMthanTE whileexpressionoftwoothergenesimportantforICM Table2GOtermsenrichedforgenesupregulatedintheTEascomparedtoICMaGOtermCountPercentPvalueFDRbBiologicalProcess Proteolysis276.40.006.26 Oxidationreduction235.40.0110.40 Intracellularsignalingcascade204.70.0343.10 Iontransport204.70.0450.64 Vesicle-mediatedtransport153.50.005.68 Regulationofcellproliferation153.50.0111.09 Actinfilament-basedprocess143.30.000.00 Actincytoskeletonorganization133.10.000.00 Cytoskeletonorganization133.10.001.22 Lipidbiosyntheticprocess112.60.0112.45 Steroidmetabolicprocess102.40.000.31 Negativeregulationofcellproliferation102.40.002.24 CellComponent Plasmamembrane4310.10.0440.40 Endoplasmicreticulum327.60.000.00 Cellfraction163.80.002.50 Cytoplasmicvesicle143.30.0331.04 Vesicle143.30.0436.80 Actincytoskeleton133.10.000.04 Membranefraction133.10.0111.72 Insolublefraction133.10.0115.08 Cellprojection122.80.0441.18 Vacuole112.60.000.87 Endoplasmicreticulumpart112.60.004.14 Apicalpartofcell102.40.000.04 MolecularFunction Ionbinding8620.30.000.14 Cationbinding8319.60.000.49 Metalionbinding8119.10.000.94 Calciumionbinding348.00.000.00 Peptidaseactivity,actingonL-aminoacidpeptides225.20.001.41 Cytoskeletalproteinbinding204.70.000.00 Actinbinding143.30.000.04 Ironionbinding122.80.0331.90 Lipidbinding112.60.0338.82aOnlythoseGOtermswhichcontainedatleast10differentiallyexpressedgenesarelisted.bFalsediscoveryrate(x100).Ozawa etal.BMCDevelopmentalBiology 2012, 12 :33 Page6of13 http://www.biomedcentral.com/1471-213X/12/33

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commitment, POU5F1 and SALL4 ,didnotdiffersignificantlybetweenICMandTE.Numerically,expressionof theselattertwogeneswashigherforICM.Fourgenes wereexaminedthatareimportantforTEcommitment – CDX2 GATA3 TEAD4 ,and YAP1 .Expressionof GATA3 wassignificantlyhigherforTEbuttherewere nosignificantdifferencesinexpressionbetweenICM andTEfortheotherthreegenes.Onegeneimportant fordifferentiationofTElaterindevelopment, ELF5 ,was expressedinhigheramountsinTE(adjustedP=0.022) whereasanother, EOMES ,wasbarelydetectableandnot differentbetweenICMandTE.Characteristicsofpromoterregionsofgenesdifferentially expressedbetweenICMandTETheregionspanningnucleotidesequenceslocated200bp upstreamto50bpdownstream ofthetranscriptionstart sitewasexaminedforpresenceofputativeTFBSforeach Table3KEGGPathwaysenrichedforgenesupregulatedintheinnercellmassortrophectodermTermGenes UpregulatedinInnerCellMass(A) Antigenprocessingandpresentation CD74 CD8B HSPA1L HSPA6 PSME1 BoLA DRB3 Complementandcoagulationcascades A2M F2R C1R PLAUR C4BPA Chemokinesignalingpathway ITK CCL24 CXCL7 GNAI1 GNB5 GNG7 PLCB1 STAT1 STAT4 STAT3 Axonguidance EPHA4 CHP DPYSL2 GNAI1 ROBO1 SEMA4G SLIT2 Arrhythmogenicrightventricularcardiomyopathy(ARVC) CDH2 DES GJA1 ITGA2 TCF7L2 Pathwaysincancer CDKN2B FGF12 FGF16 ITGA2MMP9 PDGFRA STAT1 STAT4 STAT3 TCF7L2 FOS KIT WNT Jak-STATsignalingpathway IL12RB2 IL19 IL6ST, IL7 STA1 STAT4 STAT3 SPRY2 Hematopoieticcelllineage CD1A CD8B ITGA2 IL7 KIT UpregulatedinTrophectoderm(B) Lysosome ATP6V0A4 GM2A NPC CTSB CTSH CTSL2 CTNS GLAA GALC MANBA PLA2G15 SCARB2 ATP6V0C SLC11A2 Steroidbiosynthesis NSDHL CYP41A1 FDFT1 SC4MOL Aldosterone-regulatedsodiumreabsorption ATP1B3 NEDD4L PRKCG SGK1 SFN Vascularsmoothmusclecontraction ACTA2 ACTG2 CALD1 CALML5 ITPR2 MYLK MYL6 PRKCH PRKCG PPARsignalingpathway ACSL4 AXSL6 FABP5 ACSL3 SCD SCP2 Phosphatidylinositolsignalingsystem CALML3 ITPR2INPP4B, INPP5D PRKCG SYNJ1 PantothenateandCoAbiosynthesis BCAT1 ENPP1 ENPP3 Terpenoidbackbonebiosynthesis HMGCR ACAT2 IDI1 Sphingolipidmetabolism UGCG GLA GALC SGPP1 Steroidhormonebiosynthesis UGT1A1 UGT1A6 CYP11A1 CYP3A28 HSD3B1 Fattyacidmetabolism ACAT2 ACSL4 ACSL6 ACSL3 ABCtransporters ABCA3 ABCB1 ABCC2 ABCG5 Table4DifferencesinexpressionbetweenICMandTEforgenesinvolvedinsegregationofICMandTEinmiceaGenesymbolRoleinmouseMeancounts ICMMeancount TEFoldchange TE / ICMAdjustedPvalue CDX2 TEcommitment5.72.80.490.780 ELF5 TEdifferentiation5.328.95.410.022 GATA3 TEcommitment363.6976.72.690.018 EOMES TEdifferentiation1.40.20.160.934 NANOG ICMcommitment3014.8620.90.210.000 POU5F1 ICMcommitment2394.11873.50.780.605 SALL4 ICMcommitment5.33.80.710.893 SOX2 ICMcommitment816.2360.70.440.005 TEAD4 TEcommitment7.112.01.690.894 YAP1 TEcommitment47.943.00.901.000aSource:Chenetal.[ 7 ].Ozawa etal.BMCDevelopmentalBiology 2012, 12 :33 Page7of13 http://www.biomedcentral.com/1471-213X/12/33

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genethatwasdifferentiallyexpressedbetweenICMand TE.Bindingsitesforthreetranscriptionfactors(PLAG1, RELAandRREB1)weresignificantlyenrichedforgenes overexpressedintheICMwhilebindingsitesforninetranscriptionfactors(EGR1,GAB PA,KLF4,MYF5,SP1,MZF1, NHLH1,PAX5andZFX)weresignificantlyenrichedfor TE.For11of12transcriptionfactorsidentifiedasbeing usedtoregulategenesoverexpressedinICMorTE,there wasnodifferenceinexpressionlevelbetweenICMandTE. Theexceptionwasfor EGR1 ,whereexpressionwasupregulatedinICM(Additionalfile2),eventhoughtheTFBS wasenrichedforgenesoverexpressedinTE.DifferencesinpromoterCpGislandsbetweengenes overexpressedinICMorTEThepercentofgenesoverexpressedinICMthatwere classifiedasCpGpositive(46.6%)waslower(P<0.05) thanforgenesoverexpressedinTE(55.3%).Moreover, thepercentofgenesclassifiedasCpGpositiveforgenes overexpressedineithertissuewashigherthanthepercentthatwereclassifiedasCpGpositivefortheentire bovinegenome(39.4%).Thus,DNAmethylationmay playagreaterroleforregulationofgenesdifferentially regulatedintheICMandTEthanitdoesforthegenomeasawhole. OfthegenesthatweredifferentiallyregulatedforICM andTE,threeweregenesinvolvedinepigeneticmodification.Thesewere DNMT1 and KDM2B ,overexpressed inICM,and DNMT3A likesequence,overexpressedin TE(Additionalfile2).Confirmationofdifferencesingeneexpressionbetween ICMandTEbyquantitativePCRUsingisolatedICMandTEfromaseparatesetofblastocyststhanusedforSOLiDsequencing,qPCRwasperformedtoverifytreatmenteffectsongeneexpression for6genes( GATA3 ELF5 CDX2 NANOG and SOX2 ). ResultsfordifferencesbetweenICMandTEweregenerallyconsistentwithresultsfromdeepsequencing (Figure1).Inparticular,expressionwashigherforTE thanICMfor GATA3 (P=0.07)and ELF5 (P<0.05)and washigherforICMthanTEfor NANOG (P<0.05)and SOX2 (P<0.05).Onediscrepancywithdeepsequencing resultswasfor CDX2 .WhiletherewasnosignificantdifferencebetweenICMandTEinthedeepsequencing database(Table4),mRNAfor CDX2 washigherforTE thanICMasdeterminedbyqPCR(Figure1).DiscussionDifferentiationinthemammalianembryoisdependent uponspatialposition-cellsontheinsideoftheembryoremainpluripotentforaperioduntilinitiationofgastrulation whilecellsontheouterfaceoftheembryodifferentiateinto TEandultimatelyformmuchoftheextraembryonic membranes.Here,usingmagnetic-assistedcellsortingand high-throughputnextgenerat ionsequencing,weshowthe consequencesofspatialdifferencesbetweenICMandTE andsubsequentdivergenceinlineagecommitmentforexpressionofgenesregulatingpluripotencyandlineage commitment,cellularmetabolism,andinteractionswith thematernalsystem. CommitmenttowardstheICMlineageinthemouseis maintainedbyactionsofPou5f1(Oct4),Sall4,Sox2and Nanog;Cdx2intheTEinhibits Pou5f1 expressionand allowsdifferentiationofextraembryonicmembranes [3,4,7].Inthebovine,too, SOX2 and NANOG wereoverexpressedinICMbutexpressionof POU5F1 and SALL4 werenotsignificantlydifferentbetweenICMandTE.A highdegreeofexpressionof POU5F1 intheTEwas expectedbecausedifferencesintheregulatoryregionof the POU5F1 geneincattleascomparedtothemousegene make POU5F1 resistanttoregulationbyCDX2[6].Nonetheless, POU5F1 expressionisgreaterintheICMofcattle [6,42].Inthepresentstudy,expressionofboth POU5F1 and SALL4 werenumericallygreaterforICM;failureto findsignificantdifferencesbetweenICMandTEmayrepresentthesmallsamplesize.Itshouldalsobekeptin mindthatembryosproducedinvitrohavealteredpatterns ofgeneexpressionrelativetoembryosproducedinvivo [43].SuchalterationscouldchangesomeofthedifferentialgeneexpressionbetweenICMandTE,ashasbeen reportedforthemouseembryo[44]. AnalysisofgenesupregulatedinICMprovidessome cluesastothesignalingpathwaysrequiredforspecification,pluripotency,andotherfunctionsoftheICM.A totalof8genesintheKEGGJak-STATsignalingpathway wereupregulated.Inmice,LIF,whichsignalsthroughthe Jak-STATpathway,canpromotepluripotencyofcells Figure1 Differencesbetweeninnercellmass(ICM)and trophectoderm(TE)inexpressionof6selectgenesas determinedbyquantitativePCR. Blastocystswereharvestedat Day7andICMandTEseparatedbymagneticactivatedcellsorting. Datarepresentleast-squaresmeansSEMofresultsfromsix biologicalreplicates.OpenbarsrepresentICMandfilledbarsTE. *=P<0.05. Ozawa etal.BMCDevelopmentalBiology 2012, 12 :33 Page8of13 http://www.biomedcentral.com/1471-213X/12/33

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derivedfromtheICM[45].WhileLIFcannotcausebovineICMcellstodevelopintostemcells[46],other moleculesthatsignalthroughtheJak-STATpathwayare likelytobeinvolvedinregulationoftheICM.Several genesrelatedtocellularmigrationwereupregulatedin ICM,asindicatedbyenrichmentofthechemokinesignalingpathway(10genes)andaxonguidance(7genes) GOterms.Inthemouse,blastomeresoftheICMcan changeposition,atleastinparttoalignpositionwith subsequentformationofprimitiveendoderm[47-49]. Perhaps,movementisdirectedbyguidancemolecules suchaschemokines. OutercellsofthemouseblastocystarecommittedtowardstheTElineagethroughtheactionsof Yap1 Tead4 Gata3 ,and Cdx2 ([3,4,7].Wefoundnodifferencein CDX2 expressionbetweenICMandTEusingdeepsequencing eventhoughitiswellestablishedthatthegeneisexpressed toagreaterextentinTEofthebovine[6,9,42]and CDX2 expressionwashigherinTEthanICMintheqPCRexperiment. CDX2 expressionwasverylowinthedeepsequencingexperiment,especiallycomparedtothatof POU5F1 Onepossibilityisthatdifferencesin CDX2 expressionbetweenTEandICMatDay7(asdetectedbyqPCR)become reducedatDay8.Likeseenearlier[6],otherhomologuesof CDX2 werenotdetected( CDX1 )orwerenearlynondetectable( CDX4 )(Additionalfile2). AnothergeneinvolvedinTElineage, GATA3 ,was expressedinhigheramountsinTE.AsimilarbutnonsignificantdifferenceinexpressionbetweenICMandTE wasnotedearlier[42].Therewasnosignificantdifferencein TEAD4 or YAP1 expressionbetweenICMand TE.Similarfindingswereobservedinthebovinefor TEAD4 [42].Ageneinvolvedindevelopmentofextraembryonicectoderminmice, ELF5 [7],wasoverexpressedinTEwhereasanothergeneinvolvedin developmentofextraembryonicmembranes, EOMES wasbarelydetectable.Infact,thereappearstobeanabsenceorverylowexpressionof EOMES inTEbetween day7and15ofgestationincattle[6].Inaddition,by Day11ofgestation,trophoblastexpressionof ELF5 is inhibitedandbecomeslimitedtotheepiblast[50]. Itisnotablethatseveralgenesc haracteristicallyexpressed inICMofmouseorhuman, DAB2 DSP GM2A SCD SSFA2 ,and VAV3 ,[30,32,37]weresignificantlyoverexpressedintheTEofthebovinewhile CDH24 ,reportedto beupregulatedintheTEofthehuman[33],wasexpressed inhigheramountsintheICMofthebovine. Dsp and Dab2 areindispensibleforembryonicdevelopmentinmiceand homologousrecombinationcausespostimplantationembryonicfailure[51,52].Clear ly,asfirstshownbyBergetal. [6],divergentevolutioninthecontrolofearlyembryonic developmentmeansthatstudyacrossawidearrayofspeciesisrequiredtounderstanddevelopmentalprocesses fully. Byvirtueofitspositionintheembryo,polarizedmorph-ology[53]andtightjunctionsbetweenitsmembercells [1],theTEisfatedtobethecelllineagethroughwhich theblastocystinteractsdirectlywiththemotherinterms ofnutrientexchange,maternal-conceptuscommunication, andplacentation.Itappearsthatexecutingthesefunctions placesincreasedmetabolicdemandsontheTEascomparedtotheICMasindicatedbyupregulationofgenes involvedinmetabolism,particularlythoseinvolvedinlipid metabolism.Lipidaccumulationinculturedbovine embryosisgreaterforTEthanICM,althoughthedifferencedependsuponmedium[54,55]. ItisthroughtheTEthatnutrientsentertheembryo andfromtheTEthatsecretoryproductsoftheembryo mustentertheuterineenvironment.Consistentwitha rolefortheTEinuptakeanddeliverywasupregulation ofgenesinvolvedinendo-orexocytosisandmembrane transport.Lysosomal-likestructureshavebeenreported tobemoreabundantinTEthanICMincattle,atleast forcertainmedia[54,55],andthemouse[53]. Moleculesinvolvedinsignalingtothemotherthat wereupregulatedinTEinclude IFNT1 PAG2 and TKDP1 .Therolefor IFNT1 istoactonthematernal endometriumtoblockluteolyticreleaseofprostaglandin F2 [39,56].Whilethisactionisinitiatedlaterinpregnancy,betweenDay15and17ofgestation,secretionof IFNToccursasearlyastheblastocyststage[57]. TKDP1 isamemberoftheKunitzfamilyofserineproteinase inhibitorsandmayfunctiontolimittrophoblastinvasivenessinspecieslikethecowwithepitheliochorialplacentation[41].Littleisknownabouttheroleof PAG2 whichisthemostlyabundantlyexpressedofatleast22 transcribed PAG genes[40].Unlikesome PAG genes (theso-called “ modern ” clade),whoseexpressionislimitedtotrophoblastgiantcellsformedlaterindevelopment, PAG2 isexpressedwidelyinthecotyledonary trophoblastandispredictedtobeanactiveasparticproteinase[58]. IFNT1 PAG2 and TKDP1 areallgenesthatare phylogenetically-restrictedtoruminants.Anotherconceptusproductthatisproducedmorewidelyinmammalsisestrogen.Theroleforembryonicestrogenisnot knownformostspeciesbutblastocystestrogenhasbeen suggestedtobeinvolvedinhatchingfromthezonapellucidainhamsters[59]andinconceptusgrowthinthe pig[60].Thebovineblastocyst,too,producesestrogen [61]andtheupregulationofgenesinvolvedinterpenoid backbonebiosynthesisandsteroidhormonebiosynthesis suggestthattheprimarysourceofblastocystestrogens istheTE. Followingblastocystformation,theruminanttrophoblastundergoesaseriesofdevelopmentalstepsthatare dependentonchangesincellshapeandspatialposition, includinghatching(whichrequiresactin-basedOzawa etal.BMCDevelopmentalBiology 2012, 12 :33 Page9of13 http://www.biomedcentral.com/1471-213X/12/33

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trophectodermalprojections[59]),elongation(which leadstoanincreaseinsizeoftheconceptusfromabout 0.16mmatDay8toasmuchas100mmormoreat Day16[62])andeventualattachmenttothematernal endometrium(commencingaroundDay20inthecow [63].Theupregulationofgenesinthetrophoblastfor ontologiessuchasactinfilament-basedprocess,actin cytoskeletonorganization,cellprojectionandcytoskeletalarrangementreflectstheextensivechangesincell architecturerequiredfortheseprocesses.Inaddition, threecathepsingenes, CTSB CTSH and CTSL2 ,were upregulatedinTE;theseproteinaseshavebeenimplicatedinblastocysthatching[59,64]. DifferencesingeneexpressionbetweenICMandTE areprobablydueinlargeparttodifferencesintranscriptionfactorusageandtoepigeneticmodifications.BindingsitesforthetranscriptionfactorsPLAG1,RELAand RREB1wereenrichedforgenesoverexpressedinICM whilebindingsitesforninetranscriptionfactors(EGR1, GABPA,KLF4,MYF,SP1,MZF1,NHLH1,PAX5and ZFX)weresignificantlyenrichedforTE.RELAisasubunitforNF B,whichinturnhasbeenimplicatedindifferentiationoftrophoblastlineagesfromembryonic stemcells[65]andinfunctionoftrophoblastgiantcells [66].Severalofthetranscriptionfactorsassociatedwith genesupregulatedinTEareinvolvedinhematopoiesis, includingEGR1[67],GABPA[68],MZF1[69],andZFX [70].Oneofthesetranscriptionalfactors,GABPA,can enhance Pou5f1 expressioninmouseembryonicstem cells[71]andanother,KLF4,isakeyregulatorofmaintenanceandinductionofpluripotency[72].Theoverall pictureisonewherehematopoiesisandstemnessis underpositiveregulationintheTE.AnothertranscriptionfactorassociatedwithregulationofgenesupregulatedinTEwasSP1.Thisproteinexertsseveralactions toregulatetrophoblastdevelopmentandfunction,includingactivationofexpressionofothertranscription factorssuchas Tfap2c [73]and Id1 [74].Inthecow,SP1 becomeslimitedtobinucleatecellsofthetrophoblastby Day25[75]. DNAmethylationcouldbeimportantforregulationof geneexpressionintheblastocystbecausethepromoter regionsofoverhalfofthegenesthatwereupregulatedin ICMorTEwereclassifiedasCpGpositive.Indeed,the percentofgenesclassifiedasCpGpositiveforgenesoverexpressedinICMorTEwashigherthanthepercentthat wereclassifiedasCpGpositivefortheentirebovinegenome.SlightlyfewergenesthatwereoverexpressedinICM wereclassifiedasCpG-positivethanforgenesthatwere overexpressedinTE,whichmightsuggestmoreinhibition ofgeneexpressionbymethylationinTE.Itisnoteworthy, however,thatNiemannetal.[76]didnotfindacorrelationbetweendegreeofCpGislandmethylationand amountofembryonicexpressionforeightgenes examined.Recentevidencehasbeeninterpretedtosignify thatitisnotthemethylationstateofindividualCpGthat determinegeneexpressionbutratherthemethylationstatusoflargeregionsofDNAthatspanmultiplegenes[77]. Incattle,thereareconflictingdataastowhether DNAmethylationislessextensiveforICMorforTE inbothembryosproducedinvitroandbysomaticcell nucleartransfer[78-80],Anotherepigeneticmark, H3K27me3,issimilarforbothcelltypes[81].Ofthe genesthatweredifferentiallyregulatedforICMand TE,threeweregenesinvolvedinepigeneticmodification.TwowereoverexpressedinICM: DNMT1 ,involved inmaintenanceofDNAmethylationduringsucceeding celldivisions[77],and KDM2B ,alysine-specifichistone dimethylasewhichcatalyzesdemethylationofH3K4and H3K6[82,83].Incontrast,a DNMT3A likesequence, whichestablishesDNAmethylationduringdevelopment andalsoparticipatesinmethylationmaintenance[77], wasoverexpressedinTE.Thepresenceofincreasedtranscriptabundancefor DNMT3A couldbeinterpretedto meanthat denovo DNAmethylationoccurstoagreater degreeinTE,asisindicatedbystudieswithembryos producedinvitro[79]andbysomaticcellnuclearcloning[80].FurtherresearchisnecessarytodeterminedifferencesinDNAmethylationbetweenTEandICMat thegene-specificandgenome-widelevel. Ingeneral,analysisofaseparatesetofisolatedICM andTEbyqPCRconfirmedtheresultsobtainedfordifferencesbetweencelltypesbydeepsequencing.Theexceptionwasfor CDX2 ,wheretherewasnodifferencein expressionasdeterminedbySOLiDsequencingbut whereexpressionwasgreaterforTEthanICMasdeterminedbyqPCR.Thediscrepancycouldreflecteither dayofsamplingdifferences(asdiscussedearlier)or, giventheoften-repeatedobservationthat CDX2 is expressedtoagreaterextentinTEthanICM[6,9,42], anerrorinducedbythedeepsequencingprocedure. Inconclusion,differentiationofblastomeresofthe morula-stageembryointotheICMandTEoftheblastocystisaccompaniedbydifferencesbetweenthetwocell lineagesinexpressionofgenescontrollingmetabolic processes,endocytosis,hatchingfromthezonapellucida,paracrineandendocrinesignalingwiththemother, andgenessupportingthechangesincellulararchitecture,stemness,andhematopoiesisnecessaryfordevelopmentofthetrophoblast.Muchoftheprocessleadingtothisfirstdifferentiationeventseemstobeunderthe controlofgenessuchas NANOG and GATA3 thatplay centralroleinlineagecommitmentinthemouse.As foundbyothersalso[6,42],therearefundamentaldifferencesfromthemouse.Understandingthenatureofthe processofpreimplantationdevelopmentinmammals willnecessarilyrequireacomparativeapproachbased onstudyofavarietyofanimalmodels.Ozawa etal.BMCDevelopmentalBiology 2012, 12 :33 Page10of13 http://www.biomedcentral.com/1471-213X/12/33

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ConclusionsAnalysisofgeneexpressionindicatedthatdifferentiation ofblastomeresofthemorula-stageembryointotheICM andTEoftheblastocystisaccompaniedbydifferences betweenthetwocelllineagesinexpressionofgenescontrollingmetabolicprocesses,endocytosis,hatchingfrom thezonapellucida,paracrineandendocrinesignaling withthemother,andgenessupportingthechangesin cellulararchitecture,stemness,andhematopoiesisnecessaryfordevelopmentofthetrophoblast.AdditionalfilesAdditionalfile1: Formulasusedforenrichmentanalysisfor transcriptionfactorbindingsites. Additionalfile2: DifferencesingeneexpressionbetweenICMand TE. GenesinwhichtheadjustedPvaluewas<0.05arecolorcoded(blue areupregulatedinICMandredareupregulatedinTE). Additionalfile3: KEGGmetabolicpathwaymapinwhichpathways thatweredifferentiallyenrichedbetweenICM(blue)andTE(red) wereidentifiedusingiPath2.0. Additionalfile4: Heatmapconstructedbyk-meanclusteringofthe 870genesthatdifferinexpressionbetweenICMandTE. Thecolors inthemapdisplaytherelativestandingofthereadscountdata;blue indicatesacountvaluethatislowerthanthemeanvalueoftherow whileredindicateshigherthanthemean.Theshadesofthecolor indicatehowfarawaythedatafromthemeanvalueoftherow. ColumnsrepresentindividualsamplesofICM(IC)andTE(TC). Additionalfile5: Differencesinexpressionbetweeninnercellmass (ICM)andtrophectoderm(TE)forgenesconsideredasbeing characteristicallyexpressedbyICMandTEinhumanormouse. Abbreviations DAVID:Databaseforannotation,visualizationandintegrateddiscovery; GO:Geneontology;KEGG:Kyotoencyclopediaofgenesandgenomes; ICM:Innercellmass;SSE:Sumofsquareserror;TFBS:Transcriptionfactor bindingsites;TE:Trophectoderm. Competinginterests Theauthorshavedeclaredthatnocompetinginterestsexist. Authorcontributions Conceivedanddesignedtheexperiments:MO,PJHWGF.Performedthe experiments:MO,SS,KBD,MJS.Analyzedthedata:MO,MS,J-QY,FY,RY,SW, KN,PJH.Wroteinitialdraftsofthepaper:MOPJH.Allauthorsreadand approvedthefinalmanuscript. Acknowledgments ThisworkwassupportedbyAgricultureandFoodResearchInitiativeGrant Nos.2009-65203-05732and2011-67015-30688fromUnitedStatesDept.of AgricultureNationalInstituteofFoodandAgricultureandbyagrantfrom theResearchOpportunityFund,UniversityofFloridaResearchFoundation. TheauthorsthankWilliamRembert,forcollectingoocytes,theChernin familyandCentralPacking(CenterHill,Florida),fordonatingovariantissue andScottRandellofSoutheasternSemen(WellbornFlorida),fordonating semen. Authordetails1DepartmentofAnimalSciencesandD.H.BarronReproductiveandPerinatal BiologyResearchProgram,POBox110910,Gainesville,FL32611-0910,USA.2InterdisciplinaryCenterforBiotechnologyResearch,UniversityofFlorida, Gainesville,FL32611-0910,USA.3Kyushu-OkinawaAgriculturalResearch Center,NationalAgricultureandFoodResearchOrganization,Kumamoto, Japan.4LaboratoryofDevelopmentalGenetics,InstituteofMedicalScience, UniversityofTokyo,Tokyo,Japan.5HumanGenomeCenter,Instituteof MedicalScience,UniversityofTokyo,Tokyo,Japan.6Departamentode ReproduoAnimaleRadiologiaVeterinria,FaculdadedeMedicina VeterinriaeZootecnia,UNESP,Botucatu,SoPaulo,Brasil. Received:6June2012Accepted:30October2012 Published:6November2012 References1.EckertJJ,FlemingTP: Tightjunctionbiogenesisduringearly development. BiochimBiophysActa 2008, 1778: 717 – 728. 2.MarikawaY,AlarcnVB: Establishmentoftrophectodermandinnercell masslineagesinthemouseembryo. MolReprodDev 2009, 76: 1019 – 1032. 3.Zernicka-GoetzM,MorrisSA,BruceAW: Makingafirmdecision: multifacetedregulationofcellfateintheearlymouseembryo. Nature Rev 2009, 10: 467 – 477. 4.RossantJ,TamPPL: Blastocystlineageformation,earlyembryonic asymmetriesandaxispatterninginthemouse. Development 2009, 136: 701 – 713. 5.PlachtaN,BollenbachT,PeaseS,FraserSE,PantazisP: Oct4kineticspredict celllineagepatterningintheearlymammalianembryo. NatCellBiol 2011, 13: 117 – 123. 6.BergDK,SmithCS,PeartonDJ,WellsDN,BroadhurstR,DonnisonM, PfefferPL: Trophectodermlineagedeterminationincattle. DevCell 2011, 20: 244 – 255. 7.ChenL,WangD,WuZ,MaL,DaleyGQ: Molecularbasisofthefirstcell fatedeterminationinmouseembryogenesis. CellRes 2010, 20: 982 – 993. 8.GasperowiczM,NataleDR: Establishingthreeblastocystlineages – then what? BiolReprod 2011, 84: 621 – 630. 9.OzawaM,HansenPJ: Anovelmethodforpurificationofinnercellmass andtrophectodermcellsfromblastocystsusingmagneticactivatedcell sorting. FertilSteril 2011, 95: 799 – 802. 10.LoureiroB,BonillaL,BlockJ,FearJM,BonillaAQS,HansenPJ: Colonystimulatingfactor2(CSF-2)improvesdevelopmentandposttransfer survivalofbovineembryosproducedinvitro. Endocrinology 2009, 150: 5046 – 5054. 11.FieldsSD,HansenPJ,EalyAD: Fibroblastgrowthfactorrequirementfor invitrodevelopmentofbovineembryos. Theriogenology 2011, 75: 1466– 1475. 12.ZhangJ,ChiodiniR,BadrA,ZhangG: Theimpactofnext-generation sequencingongenomics. JGenetGenom 2011, 38: 95 – 109. 13.TrapnellC,PachterL,SalzbergSL: TopHat:Discoveringsplicejunctions withRNA-Seq. Bioinformatics 2009, 25: 1105 – 1111. 14.LangmeadB,TrapnellC,PopM,SalzbergSL: UltrafastandmemoryefficientalignmentofshortDNAsequencestothehumangenome. GenomeBiol 2009, 10: R25. 15.HomerN,MerrimanB,NelsonS: BFAST:Analignmenttoolforlargescale genomeresequencing. PLoSOne 2009, 4: e7767. 16.AndersS,HuberW: Differentialexpressionanalysisforsequencecount data. GenomeBiol 2010, 11: R106. 17.HuangDW,ShermanBT,LempickiRA: Bioinformaticsenrichmenttools: pathstowardthecomprehensivefunctionalanalysisoflargegenelists. NucleicAcidsRes 2009, 37: 1 – 13. 18.YamadaT,LetunicI,OkudaS,KanehisaM,BorkP: iPath2.0:interactive pathwayexplorer. NuclAcidsRes 2011, 39: W412 – W415. 19.LiangJ,ZhangL,XiangZ,HeN: Expressionprofileofcuticulargenesof silkworm Bombyxmori BMCGenom 2010, 11: 173. 20.DashR,MishraD,RathAK,AcharyaM: AhybridizedK-meansclustering approachforhighdimensionaldataset. IntJEngSciTech 2011, 2: 59 – 66. 21.WassermanWW,SandelinA: Appliedbioinformaticsfortheidentification ofregulatoryelements. NatRevGenet 2004, 5: 276 – 287. 22.Portales-CasamarE,ThongjueaS,KwonAT,ArenillasD,ZhaoX, etal : JASPAR2010:thegreatlyexpandedopen-accessdatabaseof transcriptionfactorbindingprofiles. NuclAcidsRes 2010, 38: D105 – D110. 23.WingenderE: TheTRANSFACprojectasanexampleofframework technologythatsupportstheanalysisofgenomicregulation. Brief Bioinform 2008,9: 326 – 332. 24.Gardiner-GardenM,FrommerM: CpGislandsinvertebrategenomes. JMolBiol 1987, 196: 261 – 282. 25.HouY,BickhartDM,HvindenML,LiC,SongJ,BoichardDA,FritzS,Eggen A,DeniseS,WiggansGR,SonstegardTS,VanTassellCP,LiuGE: Fine mappingofcopynumbervariationsontwocattlegenomeassemblies usinghighdensitySNParray. BMCGenomics 2012, 13: 376.Ozawa etal.BMCDevelopmentalBiology 2012, 12 :33 Page11of13 http://www.biomedcentral.com/1471-213X/12/33

PAGE 12

26.SmithCS,BergDK,BergM,PfefferPL: Nucleartransfer-specificdefectsare notapparentduringthesecondweekofembryogenesisincattle. Cell Reprogram 2010, 12: 699 – 707. 27.OzawaM,SakataniM,HankowskiKE,TeradaN,DobbsKB,HansenPJ: Importanceofcultureconditionsduringthemorula-to-blastocystperiod oncapacityofinnercell-masscellsofbovineblastocystsfor establishmentofself-renewingpluripotentcells. Theriogenology 2012, 78: 1243 – 1251. 28.GoossensK,VanPouckeM,VanSoomA,VandesompeleJ,VanZeverenA, PeelmanLJ: Selectionofreferencegenesforquantitativereal-timePCR inbovinepreimplantationembryos. BMCDevBiol 2005, 5: 27. 29.HamataniT,DaikokuT,WangH,MatsumotoH,CarterMG,KoMS,DeySK: Globalgeneexpressionanalysisidentifiesmolecularpathways distinguishingblastocystdormancyandactivation. ProcNatlAcadSciUSA 2004, 101: 10326 – 10331. 30.SaitouM,YabutaY,KurimotoK: Single-cellcDNAhigh-density oligonucleotidemicroarrayanalysis:detectionofindividualcelltypes andpropertiesincomplexbiologicalprocesses. ReprodBiomedOnline 2008, 16: 26 – 40. 31.TesarPJ,ChenowethJG,BrookFA,DaviesTJ,EvansEP, etal : Newcelllines frommouseepiblastsharedefiningfeatureswithhumanembryonic stemcells. Nature 2007, 448: 196 – 199. 32.ChouY-F,ChenH-H,EijpeM,YabuuchiA,ChenowethJG, etal : Thegrowth factorenvironmentdefinesdistinctpluripotentgroundstatesinnovel blastocyst-derivedstemcells. Cell 2008, 135: 449 – 461. 33.AdjayeJ,HuntrissJ,HerwigR,BenKahlaA,BrinkTC, etal : Primary differentiationinthehumanblastocyst:comparativemolecularportraitsof innercellmassandtrophectodermcells. StemCells 2005, 23: 1514 – 1525. 34.EdwardsRG,HansisC: Initialdifferentiationofblastomeresin4-cell humanembryosanditssignificanceforearlyembryogenesisand implantation. ReprodBiomedOnline 2005, 11: 206 – 218. 35.KimberSJ,SneddonS,BloorDJ,El-BaregAM,HawkheadJA, etal : Expressionofgenesinvolvedinearlycellfatedecisionsinhuman embryosandtheirregulationbygrowthfactors. Reproduction 2008, 135: 635 –647. 36.CauffmanG,DeRyckeM,SermonK,LiebaersI,VandeVeldeH: Markers thatdefinestemnessinESCareunabletoidentifythetotipotentcellsin humanpreimplantationembryos. HumReprod 2009, 24: 63 – 70. 37.ReijoPeraRA,DeJongeC,BossertN,YaoM,HwaYangJY,AsadiNB,Wong W,WongC,FirpoMT: Geneexpressionprofilesofhumaninnercellmass cellsandembryonicstemcells. Differentiation 2009, 78: 18 – 23. 38.GalanA,SimonC: Monitoringstemnessinlong-termhESCculturesby real-timePCR. MethodsMolBiol 2010, 584: 135 – 150. 39.RobertsRM,ChenY,EzashiT,WalkerAM: Interferonsandthematernalconceptusdialoginmammals. SeminCellDevBiol 2008, 19: 170 – 177. 40.TeluguBP,WalkerAM,GreenJA: Characterizationofthebovine pregnancy-associatedglycoproteingenefamily – analysisofgene sequences,regulatoryregionswithinthepromoterandexpressionof selectedgenes. BMCGenom 2009, 10: 185. 41.ChakrabartyA,2ndMacLeanJA,HughesAL,RobertsRM,GreenJA: Rapid evolutionofthetrophoblastkunitzdomainproteins(TKDPs)-a multigenefamilyinruminantungulates. JMolEvol 2006, 63: 274 – 282. 42.FujiiT,MoriyasuS,HirayamaH,HashizumeT,SawaiK: Aberrantexpression patternsofgenesinvolvedinsegregationofinnercellmassand trophectodermlineagesinbovineembryosderivedfromsomaticcell nucleartransfer. CellReprogram 2010, 12: 617 – 625. 43.DriverAM,PeagaricanoF,HuangW,AhmadKR,HackbartKS,WiltbankMC, KhatibH: RNA-Seqanalysisuncoverstranscriptomicvariationsbetween morphologicallysimilarinvivo-andinvitro-derivedbovineblastocysts. BMCGenomics 2012, 13: 118. 44.GiritharanG,DellePianeL,DonjacourA,EstebanFJ,HorcajadasJA,Maltepe E,RinaudoP: Invitrocultureofmouseembryosreducesdifferentialgene expressionbetweeninnercellmassandtrophectoderm. ReprodSci 2012, 19: 243 – 252. 45.SmithAG,NicholsJ,RobertsonM,PathjenPD: Differentiationinhibiting activity(DIA/LIF)andmousedevelopment. DevBiol 1992, 151: 339 – 351. 46.CaoS,WangF,ChenZ,LiuZ,MeiC, etal : Isolationandcultureofprimary bovineembryonicstemcellcoloniesbyanovelmethod.JExpZoolA EcolGenetPhysiol 2009, 311: 368 – 376. 47.MeilhacSM,AdamsRJ,MorrisSA,DanckaertA,LeGarrecJF,ZernickaGoetzM: Activecellmovementscoupledtopositionalinductionare involvedinlineagesegregationinthemouseblastocyst. DevBiol 2009, 331: 210 – 221. 48.MorrisSA,TeoRT,LiH,RobsonP,GloverDM,Zernicka-GoetzM: Originand formationofthefirsttwodistinctcelltypesoftheinnercellmassinthe mouseembryo. ProcNatlAcadSciUSA 2010, 107: 6364 – 6369. 49.YamanakaY,LannerF,RossantJ: FGFsignal-dependentsegregationof primitiveendodermandepiblastinthemouseblastocyst. Development 2010, 137: 715 – 724. 50.PeartonDJ,BroadhurstR,DonnisonM,PfefferPL: Elf5 regulationinthe trophectoderm. DevBiol 2011, 360: 343 – 350. 51.VasioukhinV,BowersE,BauerC,DegensteinL,FuchsE: Desmoplakinisessentialinepidermalsheetformation. NatCell Biol 2011, 3: 1076 – 1085. 52.MorrisSM,TallquistMD,RockCO,CooperJA: DualrolesfortheDab2 adaptorproteininembryonicdevelopmentandkidneytransport. EMBO J 2002, 21: 1555 – 1564. 53.FlemingTP,WarrenPD,ChisholmJC,JohnsonMH: Trophectodermal processesregulatetheexpressionoftotipotencywithintheinnercell massofthemouseexpandingblastocyst. JEmbryolExpMorphol 1984, 84: 63 – 90. 54.AbeH,OtoiT,TachikawaS,YamashitaS,SatohT,HoshiH: Finestructureof bovinemorulaeandblastocystsinvivoandinvitro. AnatEmbryol 1999, 199: 519 – 527. 55.AbeH,YamashitaS,ItohT,SatohT,HoshiH: Ultrastructureofbovine embryosdevelopedfrominvitro-maturedand-fertilizedoocytes: comparativemorphologicalevaluationofembryosculturedeitherin serum-freemediumorinserum-supplementedmedium. MolReprodDev 1999, 53: 325 – 335. 56.BazerFW,BurghardtRC,JohnsonGA,SpencerTE,WuG: Interferonsand progesteroneforestablishmentandmaintenanceofpregnancy: interactionsamongnovelcellsignalingpathways. ReprodBiol 2008, 8: 179 –211. 57.deMoraesAA,DavidsonJA,FlemingJG,BazerFW,EdwardsJL, etal : Lack ofeffectofgranulocyte-macrophagecolony-stimulatingfactoron secretionofinterferon,otherproteins,andprostaglandinE2bythe bovineandovineconceptus. DomestAnimEndocrinol 1997, 14: 193 – 197. 58.GreenJA,XieS,RobertsRM: Pepsin-relatedmoleculessecretedby trophoblast. RevReprod 1998, 3: 62 – 69. 59.SeshagiriPB,SenRoyS,SireeshaG,RaoRP: Cellularandmolecular regulationofmammalianblastocysthatching. JReprodImmunol 2009, 83: 79 – 84. 60.BlombergL,HashizumeK,ViebahnC: Blastocystelongation,trophoblastic differentiation,andembryonicpatternformation. Reproduction 2008, 135: 181 – 195. 61.GadsbyJE,HeapRB,BurtonRD: Oestrogenproductionbyblastocyst andearlyembryonictissueofvariousspecies. JReprodFertil 1980, 60: 409 – 417. 62.BetteridgeKJ,FlechonJ-E: Theanatomyandphysiologyofpreattachmentbovineembryos. Theriogenology 1988, 29: 155 – 187. 63.KingGJ,AtkinsonBA,RobertsonHA: Developmentofthe intercaruncularareasduringearlygestationandestablishmentofthe bovineplacenta. JReprodFertil 1981, 61: 469 – 474. 64.BalboulaAZ,YamanakaK,SakataniM,HegabAO,ZaabelSM,TakahashiM: IntracellularcathepsinBactivityisinverselycorrelatedwiththequality anddevelopmentalcompetenceofbovinepreimplantationembryos. MolReprodDev 2010, 77: 1031 – 1039. 65.MarchandM,HorcajadasJA,EstebanFJ,McElroySL,FisherSJ,GiudiceLC: Transcriptomicsignatureoftrophoblastdifferentiationinahuman embryonicstemcellmodel. BiolReprod 2011, 84: 1258 – 1271. 66.MuggiaA,TeesaluT,NeriA,BlasiF,TalaricoD: Trophoblastgiantcells expressNFB2duringearlymousedevelopment. DevGenet 1999, 25: 23 – 30. 67.WilsonA,LaurentiE,TrumppA: Balancingdormantandself-renewing hematopoieticstemcells. CurrOpinGenetDev 2009,19: 461 – 368. 68.YuS,CuiK,JothiR,ZhaoDM,JingX, etal : GABPcontrolsacritical transcriptionregulatorymodulethatisessentialformaintenanceand differentiationofhematopoieticstem/progenitorcells. Blood 2011, 117: 2166 – 2178. 69.HromasR,DavisB,RauscherFJ3rd,KlemszM,TenenD, etal : Hematopoietictranscriptionalregulationbythemyeloidzincfinger gene,MZF-1. CurrTopMicrobiolImmunol 1996, 211: 159 – 164.Ozawa etal.BMCDevelopmentalBiology 2012, 12 :33 Page12of13 http://www.biomedcentral.com/1471-213X/12/33

PAGE 13

70.Galan-CaridadJM,HarelS,ArenzanaTL,HouZE,DoetschFK, etal : Zfx controlstheself-renewalofembryonicandhematopoieticstemcells. Cell 2007, 129: 345 – 357. 71.KinoshitaK,UraH,AkagiT,UsudaM,KoideH,YokotaT: GABP regulates Oct-3/4expressioninmouseembryonicstemcells. BiochemBiophysRes Commun 2007, 353: 686 – 691. 72.PappB,PlathK: Reprogrammingtopluripotency:stepwiseresettingof theepigeneticlandscape. CellRes 2011, 21: 486 – 501. 73.LiM,KellemsRE: Sp1andSp3Areimportantregulatorsof AP 2 gene transcription. BiolReprod 2003, 69: 1220 – 1230. 74.TakedaT,SakataM,IsobeA,YamamotoT,NishimotoF, etal : Involvement ofSp-1intheregulationoftheId-1geneduringtrophoblastcell differentiation. Placenta 2007, 28: 192 – 198. 75.DegrelleSA,MurthiP,Evain-BrionD,FournierT,HueI: Expressionand localizationofDLX3,PPARGandSP1inbovinetrophoblastduring binucleatedcelldifferentiation. Placenta 2011, 32: 917 – 920. 76.NiemannH,CarnwathJW,HerrmannD,WieczorekG,LemmeE, etal: DNA methylationpatternsreflectepigeneticreprogramminginbovine embryos. CellReprogram 2010, 12: 33 – 42. 77.JurkowskaRZ,JurkowskiTP,JeltschA: Structureandfunctionof mammalianDNAmethyltransferases. ChemBioChem 2011, 12: 206 – 222. 78.DeanW,SantosF,StojkovicM,ZakhartchenkoV,WalterJ, etal : Conservationofmethylationreprogramminginmammalian development:aberrantreprogramminginclonedembryos. ProcNatl AcadSciUSA 2011, 98: 13734 – 13738. 79.HouJ,LiuL,LeiT,CuiX,AnX,ChenY: GenomicDNAmethylation patternsinbovinepreimplantationembryosderivedfrominvitro fertilization. SciChinaCLifeSci2007, 50: 56 – 61. 80.KangYK,ParkJS,KooDB,ChoiYH,KimSU, etal : Limiteddemethylation leavesmosaic-typemethylationstatesinclonedbovinepre-implantation embryos. EMBOJ 2002, 21: 1092 – 1100. 81.RossPJ,RaginaNP,RodriguezRM,IagerAE,SiripattarapravatK, etal : PolycombgeneexpressionandhistoneH3lysine27trimethylation changesduringbovinepreimplantationdevelopment. Reproduction 2008, 136: 777 – 785. 82.TsukadaY,FangJ,Erdjument-BromageH,WarrenME,BorchersCH, etal : HistonedemethylationbyafamilyofJmjCdomain-containingproteins. Nature 2006, 439: 811 – 816. 83.FrescasD,GuardavaccaroD,BassermannF,Koyama-NasuR,PaganoM: JHDM1B/FBXL10isanucleolarproteinthatrepressestranscriptionof ribosomalRNAgenes. Nature 2007, 450: 309 – 313.doi:10.1186/1471-213X-12-33 Citethisarticleas: Ozawa etal. : Globalgeneexpressionoftheinnercell massandtrophectodermofthebovineblastocyst. BMCDevelopmental Biology 2012 12 :33. Submit your next manuscript to BioMed Central and take full advantage of: € Convenient online submission € Thorough peer review € No space constraints or color “gure charges € Immediate publication on acceptance € Inclusion in PubMed, CAS, Scopus and Google Scholar € Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Ozawa etal.BMCDevelopmentalBiology 2012, 12 :33 Page13of13 http://www.biomedcentral.com/1471-213X/12/33


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dochead Research article
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title
p Global gene expression of the inner cell mass and trophectoderm of the bovine blastocyst
aug
au id A1 snm Ozawafnm Manabuinsr iid I1 I4 email semil@ims.u-tokyo.ac.jp
A2 SakataniMikiI3 sakatani@ufl.edu
A3 YaoJiQiangI2 jiqiangyao@ufl.edu
A4 ShankerSavitasshanker@ufl.edu
A5 YuFahongfyu@ufl.edu
A6 YamashitaRuiI5 ryamasi@hgc.jp
A7 WakabayashiShunichis-wakaba@hgc.jp
A8 NakaiKentaknakai@hgc.jp
A9 Dobbsmi BKylekbd3z4@ufl.edu
A10 Sudanomnm JoséMateusI6 mjsudano@gmail.com
A11 FarmerieGWilliamwgf2@ufl.edu
A12 ca yes HansenJPeterHansen@animal.ufl.edu
insg
ins Department of Animal Sciences and D.H. Barron Reproductive and Perinatal Biology Research Program, PO Box 110910, Gainesville, FL, 32611-0910, USA
Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesville, FL, 32611-0910, USA
Kyushu-Okinawa Agricultural Research Center, National Agriculture and Food Research Organization, Kumamoto, Japan
Laboratory of Developmental Genetics, Institute of Medical Science, University of Tokyo, Tokyo, Japan
Human Genome Center, Institute of Medical Science, University of Tokyo, Tokyo, Japan
Departamento de Reprodução Animal e Radiologia Veterinária, Faculdade de Medicina Veterinária e Zootecnia, UNESP, Botucatu, São Paulo, Brasil
source BMC Developmental Biology
section Early developmentissn 1471-213X
pubdate 2012
volume 12
issue 1
fpage 33
url http://www.biomedcentral.com/1471-213X/12/33
xrefbib pubidlist pubid idtype doi 10.1186/1471-213X-12-33pmpid 23126590
history rec date day 6month 6year 2012acc 30102012pub 6112012
cpyrt 2012collab Ozawa et al.; licensee BioMed Central Ltd.note This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
kwdg
kwd Blastocyst
Trophectoderm
Inner cell mass
Development
abs
sec
st
Abstract
Background
The first distinct differentiation event in mammals occurs at the blastocyst stage when totipotent blastomeres differentiate into either pluripotent inner cell mass (ICM) or multipotent trophectoderm (TE). Here we determined, for the first time, global gene expression patterns in the ICM and TE isolated from bovine blastocysts. The ICM and TE were isolated from blastocysts harvested at day 8 after insemination by magnetic activated cell sorting, and cDNA sequenced using the SOLiD 4.0 system.
Results
A total of 870 genes were differentially expressed between ICM and TE. Several genes characteristic of ICM (for example, it NANOG, SOX2, and STAT3) and TE (ELF5, GATA3, and KRT18) in mouse and human showed similar patterns in bovine. Other genes, however, showed differences in expression between ICM and TE that deviates from the expected based on mouse and human.
Conclusion
Analysis of gene expression indicated that differentiation of blastomeres of the morula-stage embryo into the ICM and TE of the blastocyst is accompanied by differences between the two cell lineages in expression of genes controlling metabolic processes, endocytosis, hatching from the zona pellucida, paracrine and endocrine signaling with the mother, and genes supporting the changes in cellular architecture, stemness, and hematopoiesis necessary for development of the trophoblast.
bdy
Background
Following its formation by syngamy of the pronuclei of the oocyte and sperm, the mammalian embryo begins life as a totipotent, single cell organism. Subsequent cycles of cell division and the formation of tight junctions between blastomeres lead to a condition whereby blastomeres on the outer face of the embryo exhibit different patterns of cell polarity, gene expression and protein accumulation than blastomeres on the inner part of the embryo abbrgrp
abbr bid B1 1
B2 2
B3 3
B4 4
. Non-polarized blastomeres in the inner part of the embryo are destined to form the pluripotent inner cell mass (ICM) that gives rise to the embryo while polarized cells in the outer face of the embryo are fated to differentiate into the trophectoderm (TE), which develops into extraembryonic membranes. Cell fate may be determined as early as the 4–8 cell stage in the mouse and depend upon differences between blastomeres in the kinetics of the interaction between the transcription factor Pou5f1 and DNA binding sites
B5 5
. Nonetheless, blastomeres do not undergo lineage commitment until about the 32-cell stage (in mice), based on loss of ability of blastomeres to form either ICM or TE
2
.Lineage commitment towards ICM or TE is under the control of specific transcription factors. The exact role of at least some transcription factors varies with species
B6 6
. In the best studied species, the mouse, the ICM is regulated by Sall4, Pou5f1, Sox2 and Nanog while TE formation results from a cascade of events involving Yap1, Tead4, Gata3, Cdx2, Eomes and Elf5
B7 7
. Functional properties of the two cell lineages is also divergent. In part, this reflects the processes responsible for establishment and maintenance of cell lineage, such as differences in transcription factor usage, cell signaling pathways and epigenetic marks
7
B8 8
. In addition, the function of the ICM, which is fated to undergo a series of differentiation events in the gastrulation process, is different from the TE, which is destined to interact with the lining of the maternal reproductive tract.In the present study, we describe, for the first time, differences in the transcriptome of the ICM and TE with the objective of understanding the consequences of the differentiation of these two cell types for cellular function. This was achieved by separating ICM and TE using a newly-developed immunomagnetic procedure
B9 9
followed by next-generation sequencing. Results reveal the implications of the spatial and developmental differentiation of these first two lineages of the preimplantation embryo with respect to metabolism, interaction with the maternal system and changes in cellular architecture. In addition, aspects of molecular control of the process of lineage commitment and differentiation are illustrative of similarities and differences with the prototypical mouse model.
Methods
Reagents
All reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) or Fisher Scientific (Pittsburgh, PA, USA) unless otherwise specified.
Embryo culture and ICM/TE isolation
Bovine embryos were produced from slaughterhouse-derived oocytes using procedures for in vitro oocyte maturation, fertilization, and embryo culture as described previously
B10 10
. Ovaries were donated by Central Packing, Center Hill Florida. The day of fertilization was defined as Day 0. After fertilization for 18–20 h, embryos were cultured in SOF-BE1 medium
B11 11
at 38.5°C in a humidified atmosphere of 5% COsub 2 and 5% O2 with the balance N2. Embryos were cultured in groups of 30 in a 50 μl culture drop under mineral oil. At Day 6, an additional 5 μl culture medium was added. At Day 8, blastocysts were harvested and used to prepare preparations of ICM and TE using magnetic activated cell sorting as reported previously
9
.Three separate pools of TE and ICM for each treatment were obtained. Each pool was prepared using 88 to 102 blastocysts. A total of 15 fertilization procedures were used to prepare the blastocysts; a set of three bulls was used for fertilization for each procedure.
RNA preparation, library construction and sequencing using SOLiD 4 system
Total RNA was isolated from each pool of embryonic cells using the PicoPure RNA Isolation Kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. The quality of RNA was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). Amplified cDNA was prepared from total RNA for RNA-Seq applications using the Ovation RNA-Seq kit (NuGen Technology, San Carlos, CA). Barcoded fragment libraries were constructed using the SOLiDsup TMv4 fragment library kit according to the manufacturer’s protocol (Applied Biosystems). Briefly, double stranded cDNA was sheared to 150–180 bp fragments using a CovarisTMS2 Sonication system (Covaris, Woburn, MA). The fragmented DNA was subsequently end-repaired and blunt-end ligated to P1 and P2 adaptors. The adaptor ligated, purified and size-selected 200–270 bp fragments were nick-translated and then amplified using primers specific to P1 and P2 adaptors and Platinum® PCR Amplification Mix (Applied Biosystems). The quality of the libraries and fragment distribution were verified by running 1 μl of each library on Agilent DNA 1000 chip (Agilent Technologies). Amplified libraries (5 different libraries pooled for each slide) were immobilized onto SOLiD P1 DNA beads (Applied Biosystems). The bead-bound libraries were then clonally amplified by emulsion PCR according to the Applied Biosystems SOLiDTM 4 Systems Templated Bead Preparation Guide. After amplification, emulsions were disrupted with 2-butanol and the beads containing clonally amplified template DNA were P2-enriched and extended with a bead linker by terminal transferase. The quantity of the beads was determined using a NanoDrop® ND1000 spectrophotometer (Thermo Scientific, Wilmington, DE). Approximately 600-700M beads were deposited on each slide (ran in total three slides) and sequenced using ‘sequencing by ligation’ chemistry and the 50x5 bp protocol on the SOLiDTM v4 sequencer (Applied Biosystems) at the Interdisciplinary Center for Biotechnology Research, University of Florida. Results were obtained as color space fasta files.
Analysis of read data
Raw sequencing reads were initially processed with GenomeQuest tools
B12 12
. Ambiguous residues were trimmed off from both sides of the sequence. Bases with Phred quality below 12 from the 3’ end of the sequence were removed. Reads that were shorter than 40 bases or that contained more than 10 bases with quality below 12 were also discarded as were reads consisting of repetitive single bases that accounts for more than 60% of the length at the 3’ end. About 53 ~ 64% of reads were retained after clean up, proving 102–157 million clean reads for the three replicates of each treatment.For mapping to the genome, the Bos taurus genomic sequence bosTau4 (repeat masked) was downloaded from the UCSC genome browser (http://genome.ucsc.edu/). Sequencing reads of each sample were mapped independently to the reference sequences using TopHat 1.2.0
B13 13
. TopHat split reads to segments and joins segment alignments. A maximum of one mismatch in each of the 25 bp segments was allowed. This step mapped 36.8% reads to the genome. The unmapped reads were collected and mapped to the reference using Bowtie 0.12.7
B14 14
allowing three mismatches. Unmapped reads were further mapped to cDNA sequences using bfast 0.6.4
B15 15
while allowing for three mismatches for each read. The cDNA sequences of B. taurus were downloaded from the National Center of Biotechnology Information. Scaffold and chromosome sequences were cleared and a total of 35,842 sequences were obtained (http://www.ncbi.nlm.nih.gov/nuccore/?term=txid9913[Organism:noexp). Bfast aligned 27.6% of the total reads to the cDNA sequences. Therefore, a total of 64.4% or 595 million reads were mapped successfully. Of the mapped reads, 89.8% are uniquely mapped to either the genome or cDNA sequences. Data were deposited in the DDBJ Sequence Read Archive at http://www.ddbj.nig.ac.jp/index-e.html (Submission DRA000504).Digital gene expression was determined as follows. The number of mapped reads for each individual gene was counted using the HTSeq tool (http://www-huber.embl.de/users/anders/HTSeq/doc/overview.html) with intersection-nonempty mode. HTSeq takes two input files bam or sam-format files of mapped reads and a gene model file. The Ensemble gene annotation file in GTF format was downloaded from the UCSC genome browser. The DESeq package
B16 16
in R was used for digital gene expression analysis. DESeq uses the negative binomial distribution, with variance and mean linked by local regression, to model the null distribution of the count data. Significant up- and downregulated genes were selected using two cutoffs: an adjusted P value of 0.05 and a minimum fold-change of 1.5.
Classification of differentially expressed genes into gene ontology (GO) classes
Differentially expressed genes were annotated by the Database for Annotation, Visualization and Integrated Discovery (DAVID; (DAVID Bioinformatics Resources 6.7, http://david.abcc.ncifcrf.gov/)
B17 17
. Most genes were annotated using the bovine genome as a reference and additional genes were annotated by comparison to the human genome. The DAVID database was queried to identify GO classes enriched for upregulated and downregulated genes. Functions of differentially expressed genes were further annotated using Kyoto Encyclopedia of Genes and Genomes (KEGG, http://www.genome.jp/kegg/). Overview of the differentially regulated KEGG pathways were mapped on KEGG Pathway Map using iPath2.0 (http://pathways.embl.de/)
B18 18
.To further analyze patterns of genes differentially regulated between ICM and TE, k-mean clustering was performed. The reads count data of the 870 significant genes for the ICM-control versus TE-control comparison were clustered using k-means strategy
B19 19
. To estimate the premium cluster number, k-values from 3 to 100 were tested and the corresponding sum of squares error (SSE)
B20 20
was calculated for each k value. SSE is defined as the sum of the squared distance between each member of a cluster and its cluster centroid. The SSE values dropped abruptly until k = 8 (results not shown). To balance the minimum number of SSE and the minimum number of clusters, k = 8 was selected as the premium parameter for clustering genes and a heatmap was generated using heatmap.2 of R package.
Enrichment analysis for transcription factor binding sites
For each differentially expressed gene, the candidate promoter region was defined as the span of nucleotides from 200 bp upstream and 50 bp downstream from the transcriptional start site identified in Ensembl. To detect putative transcription factor binding sites (TFBS) in each promoter, we followed the method of Wasserman and Sandelin
B21 21
. Position-specific weight matrices were obtained from the JASPAR database
B22 22
. The score was calculated by formula 1 in Additional File supplr sid S1 1. We also calculated the ratio of the score to the maximum score by formula 2 (Additional File 1). Statistical significance of each TFBS was evaluated by calculating the hypergeometric distribution using formula 3 (Additional file 1). We performed the ‘match’ program with ‘minSUM’ and ‘minFP’ thresholds to detect TFBS
B23 23
. Statistical significance of each detected TFBS was evaluated by the hypergeometric distribution as described above.
suppl
Additional file 1
text
b Formulas used for enrichment analysis for transcription factor binding sites.
file name 1471-213X-12-33-S1.pdf
Click here for file
Calculation of GC contents and detection of CpG islands
The method by Gardiner-Garden and Frommer
B24 24
was used to identify CpG islands in the region encompassing the 100 nucleotides upstream and 100 nucleotides downstream from the start site. Transcriptional start sites for differentially expressed genes were obtained from UMD3.1
B25 25
. For the definition of CpG islands, The GC content was calculated as ([C]+[G])/200, where [N] denotes the number of nucleotides “N” within the 200 base window. The CpG score was calculated as [CG]/([C]*[G]*200). A gene was classified as CpG positive when its GC content in the region spanning the 100 nucleotides upstream and the 100 nucleotides downstream from the start site exceeds 0.5 and when the CpG score in the same region exceeds 0.6. Otherwise, a gene was classified as CpG negative. Chi-square analysis was used to determine whether the percent of genes classified as CpG positive differed between 1) genes overexpressed in ICM versus genes overexpressed in TE and 2) genes overexpressed in ICM or TE versus the reference population of 25118 genes in the bovine genome.
Confirmation of differences in gene expression between ICM and TE by quantitative PCR
An experiment was performed to verify the effect of cell type (ICM vs TE) and CSF2 on relative mRNA abundance of the GATA3, ELF5, CDX2, NANOG and SOX2. Embryos were prepared as described previously and blastocysts were collected at Day 7. Pools of 25–34 blastocysts were submitted to magnetic-activated cell sorting
9
. A total of 6 biological replicates of ICM and TE were prepared. mRNA extraction was performed using the All Prep DNA/RNA mini Kit (Qiagen, Inc., Valencia, CA, USA) followed by DNase (Qiagen) treatment and reverse transcription (High Capacity cDNA Reverse Transcription Kit, Applied Biosystems, Foster City, CA). Transcript abundance for GATA3, ELF5, CDX2, NANOG and SOX2 as well as housekeeping genes GAPDH, SDHA and YWHAZ were quantified by a Bio-Rad thermal cycler CFX96-Real-Time system (Bio-Rad, Hercules, CA, USA) using SsoFast EvaGreen Supermix reagent (Bio-Rad, Hercules, CA, USA). PCR conditions were as follows: 30 sec at 95°C followed by 40 cycles each of 5 sec at 95°C and 1 min at 60°C. Data were analyzed using the delta-delta cycle threshold (Ct) method. The reference gene was the geometric mean of the Ct values of GAPDH, SDHA and YWHAZ. Primers for ELF5 were based on NM_001024569.1 and were designed using PrimerQuest from idtDNA (http://www.idtdna.com) software, Efficiency was 95% and identity of amplicons was verified by sequencing products. The primers were 5’ TGCCATTTCAACATCAGTGGCCTG 3’ and 5’ AAGGCCACCCTCAAAGACTATGCT 3’. Other primer pairs were published previously: GATA3
B26 26
, CDX2 and NANOG
9
, SOX2
B27 27
and GAPDH, SDHA and YWHAZ
B28 28
.Data were analyzed by least-squares analysis of variance using the General Linear Model (GLM) procedure of the Statistical Analysis System, version 9.2 (SAS Institute Inc, Cary, NC, USA) Sources of variation in the model included cell type (ICM and TE), replicate and the interaction; cell type was considered fixed and replicate was considered random. Logarithmic transformation was applied to CDX2 data to improve normality. All data are reported as untransformed least-squares means.
Results
Differentially expressed genes
The lists of differentially expressed genes, determined using an adjusted P value of ≤0.05 and ≥ 1.5-fold difference as cut-offs, are presented in Additional file S2 2. There were a total of 870 genes that were differentially expressed between ICM and TE, with 411 genes upregulated in the ICM and 459 downregulated in the ICM (i.e., upregulated in the TE).
Additional file 2
Differences in gene expression between ICM and TE. Genes in which the adjusted P value was <0.05 are color coded (blue are upregulated in ICM and red are upregulated in TE).
1471-213X-12-33-S2.xlsx
Click here for file
Annotation of genes differentially expressed between ICM and TE
Differentially expressed genes were annotated using the Gene ID conversion tool of the DAVID Bioinformatics Resources 6.7 (http://david.abcc.ncifcrf.gov/conversion.jsp); 835 of the 870 differentially expressed genes were annotated (389 genes upregulated in the ICM and 424 genes upregulated in the TE). For the list of genes upregulated in ICM, 10 GO terms were listed in the Biological Process group, 4 GO terms in the Cell Component group, and 5 terms in the Molecular Function group (Table tblr tid T1 1). Terms related to transcriptional activities were dominant including regulation of transcription, DNA-dependent (25 genes), regulation of transcription from RNA polymerase II promoter (11 genes), DNA binding (29 genes), transcription regulator activity (22 genes) and transcription factor activity (17 genes). There were also GO terms related to metabolic activity including regulation of RNA metabolic process (25 genes), positive regulation of macromolecule metabolic process (12 genes), negative regulation of macromolecule metabolic process (10 genes), and enzyme binding (10 genes).
table
Table 1
caption
GO terms enriched for genes upregulated in the ICM as compared to TE
a
tgroup align left cols 5
colspec colname c1 colnum 1 colwidth 1*
c2 2
c3 3
c4 4
c5
thead valign top
row rowsep
entry
GO term
Count
Percent
P value
FDR
b
tfoot
a Only those GO terms which contained at least 10 differentially expressed genes are listed.
b False discovery rate (x 100).
tbody
Biological Process
 Regulation of transcription, DNA-dependent
25
6.7
0.04
43.9
 Regulation of RNA metabolic process
25
6.7
0.04
49.9
 Neurological system process
12
3.2
0.01
16.9
 Regulation of cell proliferation
12
3.2
0.02
24.9
 Immune response
12
3.2
0.03
35.4
 Positive regulation of macromolecule metabolic process
12
3.2
0.03
43.1
 Cognition
11
2.9
0.00
2.4
 Regulation of transcription from RNA polymerase II promoter
11
2.9
0.02
29.6
 Response to organic substance
10
2.7
0.01
16.8
 Negative regulation of macromolecule metabolic process
10
2.7
0.04
43.9
Cell Component
 Plasma membrane
34
9.1
0.02
20.7
 Extracellular region
30
8.0
0.00
4.2
 Extracellular region part
19
5.1
0.00
1.9
 Extracellular space
12
3.2
0.02
16.9
Molecular Function
 DNA binding
29
7.8
0.05
46.9
 Transcription regulator activity
22
5.9
0.04
40.5
 Calcium ion binding
18
4.8
0.03
32.1
 Transcription factor activity
17
4.6
0.02
19.4
 Enzyme binding
10
2.7
0.01
7.7
For genes upregulated in TE, 12 GO terms were listed in the Biological Process group, 12 in the Cell Component group, and 9 in the Molecular Function group (Table T2 2). GO terms enriched for TE were distinct from those for ICM. A large number of genes represented by GO terms related with metabolism were upregulated in TE including proteolysis (27 genes), oxidation reduction (23 genes), lipid biosynthetic processing (11 genes), steroid metabolic process (10 genes), and peptidase activity (acting on L-amino acid peptides) (22 genes) as well as genes involved in binding reactions [ion binding (86 genes), cation binding (83 genes), metal ion binding (81 genes), calcium ion binding (34 genes) and iron ion binding (12 genes)]. There was also enrichment for genes associated with endo- or exocytosis, membrane transport and alterations in cellular architecture as indicated by GO terms for vesicle-mediated transport (15 genes), actin filament-based process (14 genes), actin cytoskeleton organization (13 genes), cytoskeleton organization (13 genes), plasma membrane (43 genes), endoplasmic reticulum (32 genes), cytoplasmic vesicle (14 genes), vesicle (14 genes), actin cytoskeleton (13 genes), cell projection (12 genes), vacuole (11 genes), endoplasmic reticulum part (11 genes), apical part of cell (10 genes), and cytoskeletal arrangement (20 genes).
Table 2
GO terms enriched for genes upregulated in the TE as compared to ICM
a
GO term
Count
Percent
P value
FDR
b
a Only those GO terms which contained at least 10 differentially expressed genes are listed.
b False discovery rate (x 100).
Biological Process
 Proteolysis
27
6.4
0.00
6.26
 Oxidation reduction
23
5.4
0.01
10.40
 Intracellular signaling cascade
20
4.7
0.03
43.10
 Ion transport
20
4.7
0.04
50.64
 Vesicle-mediated transport
15
3.5
0.00
5.68
 Regulation of cell proliferation
15
3.5
0.01
11.09
 Actin filament-based process
14
3.3
0.00
0.00
 Actin cytoskeleton organization
13
3.1
0.00
0.00
 Cytoskeleton organization
13
3.1
0.00
1.22
 Lipid biosynthetic process
11
2.6
0.01
12.45
 Steroid metabolic process
10
2.4
0.00
0.31
 Negative regulation of cell proliferation
10
2.4
0.00
2.24
Cell Component
 Plasma membrane
43
10.1
0.04
40.40
 Endoplasmic reticulum
32
7.6
0.00
0.00
 Cell fraction
16
3.8
0.00
2.50
 Cytoplasmic vesicle
14
3.3
0.03
31.04
 Vesicle
14
3.3
0.04
36.80
 Actin cytoskeleton
13
3.1
0.00
0.04
 Membrane fraction
13
3.1
0.01
11.72
 Insoluble fraction
13
3.1
0.01
15.08
 Cell projection
12
2.8
0.04
41.18
 Vacuole
11
2.6
0.00
0.87
 Endoplasmic reticulum part
11
2.6
0.00
4.14
 Apical part of cell
10
2.4
0.00
0.04
Molecular Function
 Ion binding
86
20.3
0.00
0.14
 Cation binding
83
19.6
0.00
0.49
 Metal ion binding
81
19.1
0.00
0.94
 Calcium ion binding
34
8.0
0.00
0.00
 Peptidase activity, acting on L-amino acid peptides
22
5.2
0.00
1.41
 Cytoskeletal protein binding
20
4.7
0.00
0.00
 Actin binding
14
3.3
0.00
0.04
 Iron ion binding
12
2.8
0.03
31.90
 Lipid binding
11
2.6
0.03
38.82
Functions of differentially expressed genes were further annotated using KEGG (http://www.genome.jp/kegg/). Genes upregulated in ICM were enriched in eight terms (Table T3 3A). These included pathways involved in lineage commitment (e.g., hematopoietic cell lineage) and differentiation (axon guidance) as well as those involved in maintenance of stemness and self renewal (e.g., pathway in cancer and Jak-STAT signaling pathway). Genes upregulated in TE were enriched in 12 terms (Table 3B). None of the terms were in common with KEGG terms enriched for genes upregulated for ICM. Terms were preferentially related to transmembrane transport (lysosome, aldosterone-regulated sodium resabsorption, and ABC transporters), lipid or steroid metabolism (PPAR signaling pathway, terpenoid backbone biosynthesis, sphingolipid metabolism, steroid hormone biosynthesis, fatty acid metabolism) and other metabolic processes (pantothenate and CoA biosynthesis). Additional file S3 3 represents a KEGG metabolic pathway map in which pathways that were differentially enriched between ICM and TE were identified using iPath2.0 (http://pathways.embl.de/). Note the increased metabolic activity in TE as compared to ICM.
Additional file 3
KEGG metabolic pathway map in which pathways that were differentially enriched between ICM (blue) and TE (red) were identified using iPath2.0.
1471-213X-12-33-S3.pdf
Click here for file
Table 3
KEGG Pathways enriched for genes upregulated in the inner cell mass or trophectoderm
Term
Genes
center nameend namest
Upregulated in Inner Cell Mass (A)
Antigen processing and presentation
CD74, CD8B, HSPA1L, HSPA6, PSME1, BoLA-DRB3
Complement and coagulation cascades
A2M, F2R, C1R, PLAUR, C4BPA,
Chemokine signaling pathway
ITK, CCL24, CXCL7, GNAI1, GNB5, GNG7, PLCB1, STAT1, STAT4, STAT3
Axon guidance
EPHA4, CHP, DPYSL2, GNAI1, ROBO1, SEMA4G, SLIT2
Arrhythmogenic right ventricular cardiomyopathy (ARVC)
CDH2, DES, GJA1, ITGA2, TCF7L2
Pathways in cancer
CDKN2B, FGF12, FGF16, ITGA2 MMP9, PDGFRA, STAT1, STAT4, STAT3, TCF7L2, FOS, KIT,WNT
Jak-STAT signaling pathway
IL12RB2, IL19, IL6ST, IL7, STA1, STAT4, STAT3, SPRY2
Hematopoietic cell lineage
CD1A, CD8B, ITGA2, IL7, KIT
Upregulated in Trophectoderm (B)
Lysosome
ATP6V0A4, GM2A, NPC, CTSB, CTSH, CTSL2, CTNS, GLAA, GALC, MANBA, PLA2G15, SCARB2, ATP6V0C, SLC11A2
Steroid biosynthesis
NSDHL, CYP41A1, FDFT1, SC4MOL
Aldosterone-regulated sodium reabsorption
ATP1B3, NEDD4L, PRKCG, SGK1, SFN
Vascular smooth muscle contraction
ACTA2, ACTG2, CALD1, CALML5, ITPR2, MYLK, MYL6, PRKCH, PRKCG
PPAR signaling pathway
ACSL4, AXSL6, FABP5, ACSL3, SCD, SCP2
Phosphatidylinositol signaling system
CALML3, ITPR2INPP4B, INPP5D, PRKCG, SYNJ1
Pantothenate and CoA biosynthesis
BCAT1, ENPP1, ENPP3
Terpenoid backbone biosynthesis
HMGCR, ACAT2, IDI1
Sphingolipid metabolism
UGCG, GLA, GALC, SGPP1
Steroid hormone biosynthesis
UGT1A1, UGT1A6, CYP11A1, CYP3A28, HSD3B1
Fatty acid metabolism
ACAT2, ACSL4, ACSL6, ACSL3
ABC transporters
ABCA3,ABCB1, ABCC2, ABCG5
K-mean clustering
The 870 genes that were differentially expressed between ICM and TE were clustered into 8 clusters, with 2, 4, 7, 9, 23,48, 149 and 628 genes in each cluster (Additional file S4 4). The biggest cluster (628 genes) contained 72.2% of all the significant genes and genes were included from almost all the overrepresented pathways (Table 3). Therefore, the k-mean analysis did not disclose much information on functional expression patterns of differentially expressed genes.
Additional file 4
Heatmap constructed by k-mean clustering of the 870 genes that differ in expression between ICM and TE. The colors in the map display the relative standing of the reads count data; blue indicates a count value that is lower than the mean value of the row while red indicates higher than the mean. The shades of the color indicate how far away the data from the mean value of the row. Columns represent individual samples of ICM (IC) and TE (TC).
1471-213X-12-33-S4.png
Click here for file
Comparison of ICM-TE differences in the bovine with the mouse and human
The literature was used to identify a group of genes that have been identified as being expressed by ICM, TE or embryonic stem cells in the mouse
B29 29
B30 30
B31 31
B32 32
or human
B33 33
B34 34
B35 35
B36 36
B37 37
B38 38
(Additional file S5 5). Among the 119 genes considered characteristic of ICM or embryonic stem cells, 8 were significantly upregulated in ICM (KDM2B, NANOG, SOX2, SPIC, STAT3, ZX3HAV1, and OTX2) and two (IL6R and TFRC) tended (P=0.06 or less) to be upregulated in ICM. Conversely, 6 genes considered as being expressed in ICM or embryonic stem cells in the mouse or human were upregulated in the TE (DAB2, DSP, GM2A, SCD, SSFA2, and VAV3). Of 49 genes considered characteristic of TE, 12 (AQP11, ATP1B3, CGN, CYP11A, DSC2, ELF5, GATA3, HSD3B1, KRT18, MSX2, SFXN or TJP2) were upregulated in TE. CDH24, a cadherin reported to be upregulated in the TE of the human
33
, was expressed in higher amounts in the ICM.
Additional file 5
Differences in expression between inner cell mass (ICM) and trophectoderm (TE) for genes considered as being characteristically expressed by ICM and TE in human or mouse.
1471-213X-12-33-S5.pdf
Click here for file
We also examined expression of ruminant-specific genes known to be upregulated in TE. The three examined, IFNT1
B39 39
, PAG2
B40 40
, and TKDP1
B41 41
, were upregulated in TE.We evaluated differences in expression between ICM and TE for genes that have been shown in the mouse
7
to be important for segregation of ICM and TE lineages and subsequent TE differentiation (Table T4 4). Expression of two genes important for ICM commitment, NANOG and SOX2, was significantly higher for ICM than TE while expression of two other genes important for ICM commitment, POU5F1 and SALL4, did not differ significantly between ICM and TE. Numerically, expression of these latter two genes was higher for ICM. Four genes were examined that are important for TE commitment – CDX2, GATA3, TEAD4, and YAP1. Expression of GATA3 was significantly higher for TE but there were no significant differences in expression between ICM and TE for the other three genes. One gene important for differentiation of TE later in development, ELF5, was expressed in higher amounts in TE (adjusted P=0.022) whereas another, EOMES, was barely detectable and not different between ICM and TE.
Table 4
Differences in expression between ICM and TE for genes involved in segregation of ICM and TE in mice
a
6
c6
Gene symbol
Role in mouse
Mean counts, ICM
Mean count, TE
Fold change, TE/ICM
Adjusted P value
a Source: Chen et al.
7
.
CDX2
TE commitment
5.7
2.8
0.49
0.780
ELF5
TE differentiation
5.3
28.9
5.41
0.022
GATA3
TE commitment
363.6
976.7
2.69
0.018
EOMES
TE differentiation
1.4
0.2
0.16
0.934
NANOG
ICM commitment
3014.8
620.9
0.21
0.000
POU5F1
ICM commitment
2394.1
1873.5
0.78
0.605
SALL4
ICM commitment
5.3
3.8
0.71
0.893
SOX2
ICM commitment
816.2
360.7
0.44
0.005
TEAD4
TE commitment
7.1
12.0
1.69
0.894
YAP1
TE commitment
47.9
43.0
0.90
1.000
Characteristics of promoter regions of genes differentially expressed between ICM and TE
The region spanning nucleotide sequences located 200 bp upstream to 50 bp downstream of the transcription start site was examined for presence of putative TFBS for each gene that was differentially expressed between ICM and TE. Binding sites for three transcription factors (PLAG1, RELA and RREB1) were significantly enriched for genes overexpressed in the ICM while binding sites for nine transcription factors (EGR1, GABPA, KLF4, MYF5, SP1, MZF1, NHLH1, PAX5 and ZFX) were significantly enriched for TE. For 11 of 12 transcription factors identified as being used to regulate genes overexpressed in ICM or TE, there was no difference in expression level between ICM and TE. The exception was for EGR1, where expression was upregulated in ICM (Additional file 2), even though the TFBS was enriched for genes overexpressed in TE.
Differences in promoter CpG islands between genes overexpressed in ICM or TE
The percent of genes overexpressed in ICM that were classified as CpG positive (46.6%) was lower (P<0.05) than for genes overexpressed in TE (55.3%). Moreover, the percent of genes classified as CpG positive for genes overexpressed in either tissue was higher than the percent that were classified as CpG positive for the entire bovine genome (39.4%). Thus, DNA methylation may play a greater role for regulation of genes differentially regulated in the ICM and TE than it does for the genome as a whole.Of the genes that were differentially regulated for ICM and TE, three were genes involved in epigenetic modification. These were DNMT1 and KDM2B, overexpressed in ICM, and DNMT3A like sequence, overexpressed in TE (Additional file 2).
Confirmation of differences in gene expression between ICM and TE by quantitative PCR
Using isolated ICM and TE from a separate set of blastocysts than used for SOLiD sequencing, qPCR was performed to verify treatment effects on gene expression for 6 genes (GATA3, ELF5, CDX2, NANOG and SOX2). Results for differences between ICM and TE were generally consistent with results from deep sequencing (Figure figr fid F1 1). In particular, expression was higher for TE than ICM for GATA3 (P=0.07) and ELF5 (P<0.05) and was higher for ICM than TE for NANOG (P<0.05) and SOX2 (P<0.05). One discrepancy with deep sequencing results was for CDX2. While there was no significant difference between ICM and TE in the deep sequencing data base (Table 4), mRNA for CDX2 was higher for TE than ICM as determined by qPCR (Figure 1).
fig Figure 1Differences between inner cell mass (ICM) and trophectoderm (TE) in expression of 6 select genes as determined by quantitative PCR
Differences between inner cell mass (ICM) and trophectoderm (TE) in expression of 6 select genes as determined by quantitative PCR. Blastocysts were harvested at Day 7 and ICM and TE separated by magnetic activated cell sorting. Data represent least-squares means ± SEM of results from six biological replicates. Open bars represent ICM and filled bars TE. *=P<0.05.
graphic 1471-213X-12-33-1
Discussion
Differentiation in the mammalian embryo is dependent upon spatial position cells on the inside of the embryo remain pluripotent for a period until initiation of gastrulation while cells on the outer face of the embryo differentiate into TE and ultimately form much of the extraembryonic membranes. Here, using magnetic-assisted cell sorting and high-throughput next generation sequencing, we show the consequences of spatial differences between ICM and TE and subsequent divergence in lineage commitment for expression of genes regulating pluripotency and lineage commitment, cellular metabolism, and interactions with the maternal system.Commitment towards the ICM lineage in the mouse is maintained by actions of Pou5f1 (Oct4), Sall4, Sox2 and Nanog; Cdx2 in the TE inhibits Pou5f1 expression and allows differentiation of extraembryonic membranes
3
4
7
. In the bovine, too, SOX2 and NANOG were overexpressed in ICM but expression of POU5F1 and SALL4 were not significantly different between ICM and TE. A high degree of expression of POU5F1 in the TE was expected because differences in the regulatory region of the POU5F1 gene in cattle as compared to the mouse gene make POU5F1 resistant to regulation by CDX2
6
. Nonetheless, POU5F1 expression is greater in the ICM of cattle
6
B42 42
. In the present study, expression of both POU5F1 and SALL4 were numerically greater for ICM; failure to find significant differences between ICM and TE may represent the small sample size. It should also be kept in mind that embryos produced in vitro have altered patterns of gene expression relative to embryos produced in vivo
B43 43
. Such alterations could change some of the differential gene expression between ICM and TE, as has been reported for the mouse embryo
B44 44
.Analysis of genes upregulated in ICM provides some clues as to the signaling pathways required for specification, pluripotency, and other functions of the ICM. A total of 8 genes in the KEGG Jak-STAT signaling pathway were upregulated. In mice, LIF, which signals through the Jak-STAT pathway, can promote pluripotency of cells derived from the ICM
B45 45
. While LIF cannot cause bovine ICM cells to develop into stem cells
B46 46
, other molecules that signal through the Jak-STAT pathway are likely to be involved in regulation of the ICM. Several genes related to cellular migration were upregulated in ICM, as indicated by enrichment of the chemokine signaling pathway (10 genes) and axon guidance (7 genes) GO terms. In the mouse, blastomeres of the ICM can change position, at least in part to align position with subsequent formation of primitive endoderm
B47 47
B48 48
B49 49
. Perhaps, movement is directed by guidance molecules such as chemokines.Outer cells of the mouse blastocyst are committed towards the TE lineage through the actions of Yap1, Tead4, Gata3, and Cdx2 (
3
4
7
. We found no difference in CDX2 expression between ICM and TE using deep sequencing even though it is well established that the gene is expressed to a greater extent in TE of the bovine
6
9
42
and CDX2 expression was higher in TE than ICM in the qPCR experiment. CDX2 expression was very low in the deep sequencing experiment, especially compared to that of POU5F1. One possibility is that differences in CDX2 expression between TE and ICM at Day 7 (as detected by qPCR) become reduced at Day 8. Like seen earlier
6
, other homologues of CDX2 were not detected (CDX1) or were nearly non-detectable (CDX4) (Additional file 2).Another gene involved in TE lineage, GATA3, was expressed in higher amounts in TE. A similar but non-significant difference in expression between ICM and TE was noted earlier
42
. There was no significant difference in TEAD4 or YAP1 expression between ICM and TE. Similar findings were observed in the bovine for TEAD4
42
. A gene involved in development of extraembryonic ectoderm in mice, ELF5
7
, was overexpressed in TE whereas another gene involved in development of extraembryonic membranes, EOMES, was barely detectable. In fact, there appears to be an absence or very low expression of EOMES in TE between day 7 and 15 of gestation in cattle
6
. In addition, by Day 11 of gestation, trophoblast expression of ELF5 is inhibited and becomes limited to the epiblast
B50 50
.It is notable that several genes characteristically expressed in ICM of mouse or human, DAB2, DSP, GM2A, SCD, SSFA2, and VAV3,
30
32
37
were significantly overexpressed in the TE of the bovine while CDH24, reported to be upregulated in the TE of the human
33
, was expressed in higher amounts in the ICM of the bovine. Dsp and Dab2 are indispensible for embryonic development in mice and homologous recombination causes postimplantation embryonic failure
B51 51
B52 52
. Clearly, as first shown by Berg et al.
6
, divergent evolution in the control of early embryonic development means that study across a wide array of species is required to understand developmental processes fully.By virtue of its position in the embryo, polarized morphology
B53 53
and tight junctions between its member cells
1
, the TE is fated to be the cell lineage through which the blastocyst interacts directly with the mother in terms of nutrient exchange, maternal-conceptus communication, and placentation. It appears that executing these functions places increased metabolic demands on the TE as compared to the ICM as indicated by upregulation of genes involved in metabolism, particularly those involved in lipid metabolism. Lipid accumulation in cultured bovine embryos is greater for TE than ICM, although the difference depends upon medium
B54 54
B55 55
.It is through the TE that nutrients enter the embryo and from the TE that secretory products of the embryo must enter the uterine environment. Consistent with a role for the TE in uptake and delivery was upregulation of genes involved in endo- or exocytosis and membrane transport. Lysosomal-like structures have been reported to be more abundant in TE than ICM in cattle, at least for certain media
54
55
, and the mouse
53
.Molecules involved in signaling to the mother that were upregulated in TE include IFNT1, PAG2 and TKDP1. The role for IFNT1 is to act on the maternal endometrium to block luteolytic release of prostaglandin F2α
39
B56 56
. While this action is initiated later in pregnancy, between Day 15 and 17 of gestation, secretion of IFNT occurs as early as the blastocyst stage
B57 57
. TKDP1 is a member of the Kunitz family of serine proteinase inhibitors and may function to limit trophoblast invasiveness in species like the cow with epitheliochorial placentation
41
. Little is known about the role of PAG2, which is the mostly abundantly expressed of at least 22 transcribed PAG genes
40
. Unlike some PAG genes (the so-called “modern” clade), whose expression is limited to trophoblast giant cells formed later in development, PAG2 is expressed widely in the cotyledonary trophoblast and is predicted to be an active aspartic proteinase
B58 58
.
IFNT1, PAG2 and TKDP1 are all genes that are phylogenetically-restricted to ruminants. Another conceptus product that is produced more widely in mammals is estrogen. The role for embryonic estrogen is not known for most species but blastocyst estrogen has been suggested to be involved in hatching from the zona pellucida in hamsters
B59 59
and in conceptus growth in the pig
B60 60
. The bovine blastocyst, too, produces estrogen
B61 61
and the upregulation of genes involved in terpenoid backbone biosynthesis and steroid hormone biosynthesis suggest that the primary source of blastocyst estrogens is the TE.Following blastocyst formation, the ruminant trophoblast undergoes a series of developmental steps that are dependent on changes in cell shape and spatial position, including hatching (which requires actin-based trophectodermal projections
59
), elongation (which leads to an increase in size of the conceptus from about 0.16 mm at Day 8 to as much as 100 mm or more at Day 16
B62 62
) and eventual attachment to the maternal endometrium (commencing around Day 20 in the cow
B63 63
. The upregulation of genes in the trophoblast for ontologies such as actin filament-based process, actin cytoskeleton organization, cell projection and cytoskeletal arrangement reflects the extensive changes in cell architecture required for these processes. In addition, three cathepsin genes, CTSB, CTSH and CTSL2, were upregulated in TE; these proteinases have been implicated in blastocyst hatching
59
B64 64
.Differences in gene expression between ICM and TE are probably due in large part to differences in transcription factor usage and to epigenetic modifications. Binding sites for the transcription factors PLAG1, RELA and RREB1 were enriched for genes overexpressed in ICM while binding sites for nine transcription factors (EGR1, GABPA, KLF4, MYF, SP1, MZF1, NHLH1, PAX5 and ZFX) were significantly enriched for TE. RELA is a subunit for NFκB, which in turn has been implicated in differentiation of trophoblast lineages from embryonic stem cells
B65 65
and in function of trophoblast giant cells
B66 66
. Several of the transcription factors associated with genes upregulated in TE are involved in hematopoiesis, including EGR1
B67 67
, GABPA
B68 68
, MZF1
B69 69
, and ZFX
B70 70
. One of these transcriptional factors, GABPA, can enhance Pou5f1 expression in mouse embryonic stem cells
B71 71
and another, KLF4, is a key regulator of maintenance and induction of pluripotency
B72 72
. The overall picture is one where hematopoiesis and stemness is under positive regulation in the TE. Another transcription factor associated with regulation of genes upregulated in TE was SP1. This protein exerts several actions to regulate trophoblast development and function, including activation of expression of other transcription factors such as Tfap2c
B73 73
and Id1
B74 74
. In the cow, SP1 becomes limited to binucleate cells of the trophoblast by Day 25
B75 75
.DNA methylation could be important for regulation of gene expression in the blastocyst because the promoter regions of over half of the genes that were upregulated in ICM or TE were classified as CpG positive. Indeed, the percent of genes classified as CpG positive for genes overexpressed in ICM or TE was higher than the percent that were classified as CpG positive for the entire bovine genome. Slightly fewer genes that were overexpressed in ICM were classified as CpG-positive than for genes that were overexpressed in TE, which might suggest more inhibition of gene expression by methylation in TE. It is noteworthy, however, that Niemann et al.
B76 76
did not find a correlation between degree of CpG island methylation and amount of embryonic expression for eight genes examined. Recent evidence has been interpreted to signify that it is not the methylation state of individual CpG that determine gene expression but rather the methylation status of large regions of DNA that span multiple genes
B77 77
.In cattle, there are conflicting data as to whether DNA methylation is less extensive for ICM or for TE in both embryos produced in vitro and by somatic cell nuclear transfer
B78 78
B79 79
B80 80
, Another epigenetic mark, H3K27me3, is similar for both cell types
B81 81
. Of the genes that were differentially regulated for ICM and TE, three were genes involved in epigenetic modification. Two were overexpressed in ICM: DNMT1, involved in maintenance of DNA methylation during succeeding cell divisions
77
, and KDM2B, a lysine-specific histone dimethylase which catalyzes demethylation of H3K4 and H3K6
B82 82
B83 83
. In contrast, a DNMT3A like sequence, which establishes DNA methylation during development and also participates in methylation maintenance
77
, was overexpressed in TE. The presence of increased transcript abundance for DNMT3A could be interpreted to mean that de novo DNA methylation occurs to a greater degree in TE, as is indicated by studies with embryos produced in vitro
79
and by somatic cell nuclear cloning
80
. Further research is necessary to determine differences in DNA methylation between TE and ICM at the gene-specific and genome-wide level.In general, analysis of a separate set of isolated ICM and TE by qPCR confirmed the results obtained for differences between cell types by deep sequencing. The exception was for CDX2, where there was no difference in expression as determined by SOLiD sequencing but where expression was greater for TE than ICM as determined by qPCR. The discrepancy could reflect either day of sampling differences (as discussed earlier) or, given the often-repeated observation that CDX2 is expressed to a greater extent in TE than ICM
6
9
42
, an error induced by the deep sequencing procedure.In conclusion, differentiation of blastomeres of the morula-stage embryo into the ICM and TE of the blastocyst is accompanied by differences between the two cell lineages in expression of genes controlling metabolic processes, endocytosis, hatching from the zona pellucida, paracrine and endocrine signaling with the mother, and genes supporting the changes in cellular architecture, stemness, and hematopoiesis necessary for development of the trophoblast. Much of the process leading to this first differentiation event seems to be under the control of genes such as NANOG and GATA3 that play central role in lineage commitment in the mouse. As found by others also
6
42
, there are fundamental differences from the mouse. Understanding the nature of the process of preimplantation development in mammals will necessarily require a comparative approach based on study of a variety of animal models.
Conclusions
Analysis of gene expression indicated that differentiation of blastomeres of the morula-stage embryo into the ICM and TE of the blastocyst is accompanied by differences between the two cell lineages in expression of genes controlling metabolic processes, endocytosis, hatching from the zona pellucida, paracrine and endocrine signaling with the mother, and genes supporting the changes in cellular architecture, stemness, and hematopoiesis necessary for development of the trophoblast.
Abbreviations
DAVID: Database for annotation, visualization and integrated discovery; GO: Gene ontology; KEGG: Kyoto encyclopedia of genes and genomes; ICM: Inner cell mass; SSE: Sum of squares error; TFBS: Transcription factor binding sites; TE: Trophectoderm.
Competing interests
The authors have declared that no competing interests exist.
Author contributions
Conceived and designed the experiments: MO, PJH WGF. Performed the experiments: MO, SS, KBD, MJS. Analyzed the data: MO, MS, J-QY, FY, RY, SW, KN, PJH. Wrote initial drafts of the paper: MO PJH. All authors read and approved the final manuscript.
bm
ack
Acknowledgments
This work was supported by Agriculture and Food Research Initiative Grant Nos. 2009-65203-05732 and 2011-67015-30688 from United States Dept. of Agriculture National Institute of Food and Agriculture and by a grant from the Research Opportunity Fund, University of Florida Research Foundation. The authors thank William Rembert, for collecting oocytes, the Chernin family and Central Packing (Center Hill, Florida), for donating ovarian tissue and Scott Randell of Southeastern Semen (Wellborn Florida), for donating semen.
refgrp Tight junction biogenesis during early developmentEckertJJFlemingTPBiochim Biophys Acta20081778717lpage 72810.1016/j.bbamem.2007.09.031link fulltext 18339299Establishment of trophectoderm and inner cell mass lineages in the mouse embryoMarikawaYAlarcónVBMol Reprod Dev2009761019103210.1002/mrd.21057pmcid 287491719479991Making a firm decision: multifaceted regulation of cell fate in the early mouse embryoZernicka-GoetzMMorrisSABruceAWNature Rev200910467477Blastocyst lineage formation, early embryonic asymmetries and axis patterning in the mouseRossantJTamPPLDevelopment200913670171310.1242/dev.01717819201946Oct4 kinetics predict cell lineage patterning in the early mammalian embryoPlachtaNBollenbachTPeaseSFraserSEPantazisPNat Cell Biol20111311712310.1038/ncb215421258368Trophectoderm lineage determination in cattleBergDKSmithCSPeartonDJWellsDNBroadhurstRDonnisonMPfefferPLDev Cell20112024425510.1016/j.devcel.2011.01.00321316591Molecular basis of the first cell fate determination in mouse embryogenesisChenLWangDWuZMaLDaleyGQCell Res20102098299310.1038/cr.2010.10620628366Establishing three blastocyst lineages–then what?GasperowiczMNataleDRBiol Reprod20118462163010.1095/biolreprod.110.08520921123814A novel method for purification of inner cell mass and trophectoderm cells from blastocysts using magnetic activated cell sortingOzawaMHansenPJFertil Steril20119579980210.1016/j.fertnstert.2010.10.00621055741Colony-stimulating factor 2 (CSF-2) improves development and posttransfer survival of bovine embryos produced in vitroLoureiroBBonillaLBlockJFearJMBonillaAQSHansenPJEndocrinology20091505046505410.1210/en.2009-0481277597719797121Fibroblast growth factor requirement for in vitro development of bovine embryosFieldsSDHansenPJEalyADTheriogenology2011751466147510.1016/j.theriogenology.2010.12.00721295834The impact of next-generation sequencing on genomicsZhangJChiodiniRBadrAZhangGJ Genet Genom2011389510910.1016/j.jgg.2011.02.003TopHat: Discovering splice junctions with RNA-SeqTrapnellCPachterLSalzbergSLBioinformatics2009251105111110.1093/bioinformatics/btp120267262819289445Ultrafast and memory-efficient alignment of short DNA sequences to the human genomeLangmeadBTrapnellCPopMSalzbergSLGenome Biol200910R2510.1186/gb-2009-10-3-r25269099619261174BFAST: An alignment tool for large scale genome resequencingHomerNMerrimanBNelsonSPLoS One20094e776710.1371/journal.pone.0007767277063919907642Differential expression analysis for sequence count dataAndersSHuberWGenome Biol201011R10610.1186/gb-2010-11-10-r106321866220979621Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene listsHuangDWShermanBTLempickiRANucleic Acids Res20093711310.1093/nar/gkn923261562919033363iPath2.0: interactive pathway explorerYamadaTLetunicIOkudaSKanehisaMBorkPNucl Acids Res201139W412W41510.1093/nar/gkr313312574921546551Expression profile of cuticular genes of silkworm Bombyx moriLiangJZhangLXiangZHeNBMC Genom20101117310.1186/1471-2164-11-173A hybridized K-means clustering approach for high dimensional datasetDashRMishraDRathAKAcharyaMInt J Eng Sci Tech201125966Applied bioinformatics for the identification of regulatory elementsWassermanWWSandelinANat Rev Genet2004527628710.1038/nrg131515131651JASPAR 2010: the greatly expanded open-access database of transcription factor binding profilesPortales-CasamarEThongjueaSKwonATArenillasDZhaoXetal Nucl Acids Res201038D105D11010.1093/nar/gkp950280890619906716The TRANSFAC project as an example of framework technology that supports the analysis of genomic regulationWingenderEBrief Bioinform2008932633210.1093/bib/bbn01618436575CpG islands in vertebrate genomesGardiner-GardenMFrommerMJ Mol Biol198719626128210.1016/0022-2836(87)90689-93656447Fine mapping of copy number variations on two cattle genome assemblies using high density SNP arrayHouYBickhartDMHvindenMLLiCSongJBoichardDAFritzSEggenADeniseSWiggansGRSonstegardTSVan TassellCPLiuGEBMC Genomics20121337610.1186/1471-2164-13-37622866901Nuclear transfer-specific defects are not apparent during the second week of embryogenesis in cattleSmithCSBergDKBergMPfefferPLCell Reprogram20101269970710.1089/cell.2010.004020973678Importance of culture conditions during the morula-to-blastocyst period on capacity of inner cell-mass cells of bovine blastocysts for establishment of self-renewing pluripotent cellsOzawaMSakataniMHankowskiKETeradaNDobbsKBHansenPJTheriogenology2012781243125110.1016/j.theriogenology.2012.05.02022898023Selection of reference genes for quantitative real-time PCR in bovine preimplantation embryosGoossensKVan PouckeMVan SoomAVandesompeleJVan ZeverenAPeelmanLJBMC Dev Biol200552710.1186/1471-213X-5-27131535916324220Global gene expression analysis identifies molecular pathways distinguishing blastocyst dormancy and activationHamataniTDaikokuTWangHMatsumotoHCarterMGKoMSDeySKProc Natl Acad Sci USA2004101103261033110.1073/pnas.040259710147857115232000Single-cell cDNA high-density oligonucleotide microarray analysis: detection of individual cell types and properties in complex biological processesSaitouMYabutaYKurimotoKReprod Biomed Online200816264010.1016/S1472-6483(10)60554-818252045New cell lines from mouse epiblast share defining features with human embryonic stem cellsTesarPJChenowethJGBrookFADaviesTJEvansEPNature200744819619910.1038/nature0597217597760The growth factor environment defines distinct pluripotent ground states in novel blastocyst-derived stem cellsChouY-FChenH-HEijpeMYabuuchiAChenowethJGCell200813544946110.1016/j.cell.2008.08.035276727018984157Primary differentiation in the human blastocyst: comparative molecular portraits of inner cell mass and trophectoderm cellsAdjayeJHuntrissJHerwigRBenKahlaABrinkTCStem Cells2005231514152510.1634/stemcells.2005-011316081659Initial differentiation of blastomeres in 4-cell human embryos and its significance for early embryogenesis and implantationEdwardsRGHansisCReprod Biomed Online20051120621810.1016/S1472-6483(10)60960-116168219Expression of genes involved in early cell fate decisions in human embryos and their regulation by growth factorsKimberSJSneddonSBloorDJEl-BaregAMHawkheadJAReproduction200813563564710.1530/REP-07-035918411410Markers that define stemness in ESC are unable to identify the totipotent cells in human preimplantation embryosCauffmanGDe RyckeMSermonKLiebaersIVan de VeldeHHum Reprod200924637018824471Gene expression profiles of human inner cell mass cells and embryonic stem cellsReijo PeraRADeJongeCBossertNYaoMHwa YangJYAsadiNBWongWWongCFirpoMTDifferentiation200978182310.1016/j.diff.2009.03.00419398262Monitoring stemness in long-term hESC cultures by real-time PCRGalanASimonCMethods Mol Biol201058413515019907976Interferons and the maternal-conceptus dialog in mammalsRobertsRMChenYEzashiTWalkerAMSemin Cell Dev Biol20081917017710.1016/j.semcdb.2007.10.007227804418032074Characterization of the bovine pregnancy-associated glycoprotein gene family–analysis of gene sequences, regulatory regions within the promoter and expression of selected genesTeluguBPWalkerAMGreenJABMC Genom20091018510.1186/1471-2164-10-185Rapid evolution of the trophoblast kunitz domain proteins (TKDPs)-a multigene family in ruminant ungulatesChakrabartyA2nd MacLeanJAHughesALRobertsRMGreenJAJ Mol Evol20066327428210.1007/s00239-005-0264-316830095Aberrant expression patterns of genes involved in segregation of inner cell mass and trophectoderm lineages in bovine embryos derived from somatic cell nuclear transferFujiiTMoriyasuSHirayamaHHashizumeTSawaiKCell Reprogram20101261762510.1089/cell.2010.001720726774RNA-Seq analysis uncovers transcriptomic variations between morphologically similar in vivo- and in vitro-derived bovine blastocystsDriverAMPeñagaricanoFHuangWAhmadKRHackbartKSWiltbankMCKhatibHBMC Genomics20121311810.1186/1471-2164-13-118336872322452724In vitro culture of mouse embryos reduces differential gene expression between inner cell mass and trophectodermGiritharanGDelle PianeLDonjacourAEstebanFJHorcajadasJAMaltepeERinaudoPReprod Sci20121924325210.1177/193371911142852222383776Differentiation inhibiting activity (DIA/LIF) and mouse developmentSmithAGNicholsJRobertsonMPathjenPDDev Biol199215133935110.1016/0012-1606(92)90174-F1601171Isolation and culture of primary bovine embryonic stem cell colonies by a novel methodCaoSWangFChenZLiuZMeiCJ Exp Zool A Ecol Genet Physiol200931136837619340839Active cell movements coupled to positional induction are involved in lineage segregation in the mouse blastocystMeilhacSMAdamsRJMorrisSADanckaertALe GarrecJFZernicka-GoetzMDev Biol200933121022110.1016/j.ydbio.2009.04.036335312319422818Origin and formation of the first two distinct cell types of the inner cell mass in the mouse embryoMorrisSATeoRTLiHRobsonPGloverDMZernicka-GoetzMProc Natl Acad Sci USA20101076364636910.1073/pnas.0915063107285201320308546FGF signal-dependent segregation of primitive endoderm and epiblast in the mouse blastocystYamanakaYLannerFRossantJDevelopment201013771572410.1242/dev.04347120147376Elf5 regulation in the trophectodermPeartonDJBroadhurstRDonnisonMPfefferPLDev Biol201136034335010.1016/j.ydbio.2011.10.00722020251Desmoplakin is essential in epidermal sheet formationVasioukhinVBowersEBauerCDegensteinLFuchsENat Cell Biol2011310761085Dual roles for the Dab2 adaptor protein in embryonic development and kidney transportMorrisSMTallquistMDRockCOCooperJAEMBO J2002211555156410.1093/emboj/21.7.155512595511927540Trophectodermal processes regulate the expression of totipotency within the inner cell mass of the mouse expanding blastocystFlemingTPWarrenPDChisholmJCJohnsonMHJ Embryol Exp Morphol19848463906533258Fine structure of bovine morulae and blastocysts in vivo and in vitroAbeHOtoiTTachikawaSYamashitaSSatohTHoshiHAnat Embryol199919951952710.1007/s00429005024910350132Ultrastructure of bovine embryos developed from in vitro-matured and -fertilized oocytes: comparative morphological evaluation of embryos cultured either in serum-free medium or in serum-supplemented mediumAbeHYamashitaSItohTSatohTHoshiHMol Reprod Dev19995332533510.1002/(SICI)1098-2795(199907)53:3<325::AID-MRD8>3.0.CO;2-T10369393Interferons and progesterone for establishment and maintenance of pregnancy: interactions among novel cell signaling pathwaysBazerFWBurghardtRCJohnsonGASpencerTEWuGReprod Biol2008817921119092983Lack of effect of granulocyte-macrophage colony-stimulating factor on secretion of interferon-τ, other proteins, and prostaglandin E2 by the bovine and ovine conceptusde MoraesAADavidsonJAFlemingJGBazerFWEdwardsJLDomest Anim Endocrinol19971419319710.1016/S0739-7240(97)00002-79171977Pepsin-related molecules secreted by trophoblastGreenJAXieSRobertsRMRev Reprod19983626910.1530/ror.0.00300629509990Cellular and molecular regulation of mammalian blastocyst hatchingSeshagiriPBSen RoySSireeshaGRaoRPJ Reprod Immunol200983798410.1016/j.jri.2009.06.26419879652Blastocyst elongation, trophoblastic differentiation, and embryonic pattern formationBlombergLHashizumeKViebahnCReproduction200813518119510.1530/REP-07-035518239048Oestrogen production by blastocyst and early embryonic tissue of various speciesGadsbyJEHeapRBBurtonRDJ Reprod Fertil19806040941710.1530/jrf.0.06004096448924The anatomy and physiology of pre-attachment bovine embryosBetteridgeKJFlechonJ-ETheriogenology19882915518710.1016/0093-691X(88)90038-6Development of the intercaruncular areas during early gestation and establishment of the bovine placentaKingGJAtkinsonBARobertsonHAJ Reprod Fertil19816146947410.1530/jrf.0.06104697205793Intracellular cathepsin B activity is inversely correlated with the quality and developmental competence of bovine preimplantation embryosBalboulaAZYamanakaKSakataniMHegabAOZaabelSMTakahashiMMol Reprod Dev2010771031103910.1002/mrd.2125021104746Transcriptomic signature of trophoblast differentiation in a human embryonic stem cell modelMarchandMHorcajadasJAEstebanFJMcElroySLFisherSJGiudiceLCBiol Reprod2011841258127110.1095/biolreprod.110.08641321368299Trophoblast giant cells express NF-κB2 during early mouse developmentMuggiaATeesaluTNeriABlasiFTalaricoDDev Genet199925233010.1002/(SICI)1520-6408(1999)25:1<23::AID-DVG3>3.0.CO;2-K10402669Balancing dormant and self-renewing hematopoietic stem cellsWilsonALaurentiETrumppACurr Opin Genet Dev20091946136810.1016/j.gde.2009.08.00519811902GABP controls a critical transcription regulatory module that is essential for maintenance and differentiation of hematopoietic stem/progenitor cellsYuSCuiKJothiRZhaoDMJingXBlood20111172166217810.1182/blood-2010-09-306563306232621139080Hematopoietic transcriptional regulation by the myeloid zinc finger gene, MZF-1HromasRDavisBRauscherFJsuf 3rdKlemszMTenenDCurr Top Microbiol Immunol199621115916410.1007/978-3-642-85232-9_168585946Zfx controls the self-renewal of embryonic and hematopoietic stem cellsGalan-CaridadJMHarelSArenzanaTLHouZEDoetschFKCell200712934535710.1016/j.cell.2007.03.014189908917448993GABPα regulates Oct-3/4 expression in mouse embryonic stem cellsKinoshitaKUraHAkagiTUsudaMKoideHYokotaTBiochem Biophys Res Commun200735368669110.1016/j.bbrc.2006.12.07117194449Reprogramming to pluripotency: stepwise resetting of the epigenetic landscapePappBPlathKCell Res20112148650110.1038/cr.2011.28319341821321600Sp1 and Sp3 Are important regulators of AP-2γ gene transcriptionLiMKellemsREBiol Reprod2003691220123010.1095/biolreprod.103.01554512801994Involvement of Sp-1 in the regulation of the Id-1 gene during trophoblast cell differentiationTakedaTSakataMIsobeAYamamotoTNishimotoFPlacenta20072819219810.1016/j.placenta.2006.03.00216638616Expression and localization of DLX3, PPARG and SP1 in bovine trophoblast during binucleated cell differentiationDegrelleSAMurthiPEvain-BrionDFournierTHueIPlacenta20113291792010.1016/j.placenta.2011.08.01421937107DNA methylation patterns reflect epigenetic reprogramming in bovine embryosNiemannHCarnwathJWHerrmannDWieczorekGLemmeECell Reprogram201012334210.1089/cell.2009.006320132011Structure and function of mammalian DNA methyltransferasesJurkowskaRZJurkowskiTPJeltschAChemBioChem20111220622210.1002/cbic.20100019521243710Conservation of methylation reprogramming in mammalian development: aberrant reprogramming in cloned embryosDeanWSantosFStojkovicMZakhartchenkoVWalterJProc Natl Acad Sci USA2011981373413738Genomic DNA methylation patterns in bovine preimplantation embryos derived from in vitro fertilizationHouJLiuLLeiTCuiXAnXChenYSci China C Life Sci200750566110.1007/s11427-007-0003-717393083Limited demethylation leaves mosaic-type methylation states in cloned bovine pre-implantation embryosKangYKParkJSKooDBChoiYHKimSUEMBO J2002211092110010.1093/emboj/21.5.109212588311867537Polycomb gene expression and histone H3 lysine 27 trimethylation changes during bovine preimplantation developmentRossPJRaginaNPRodriguezRMIagerAESiripattarapravatKReproduction200813677778510.1530/REP-08-004518784248Histone demethylation by a family of JmjC domain-containing proteinsTsukadaYFangJErdjument-BromageHWarrenMEBorchersCHNature200643981181610.1038/nature0443316362057JHDM1B/FBXL10 is a nucleolar protein that represses transcription of ribosomal RNA genesFrescasDGuardavaccaroDBassermannFKoyama-NasuRPaganoMNature200745030931310.1038/nature0625517994099




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epdcx:valueString Global gene expression of the inner cell mass and trophectoderm of the bovine blastocyst
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Abstract
Background
The first distinct differentiation event in mammals occurs at the blastocyst stage when totipotent blastomeres differentiate into either pluripotent inner cell mass (ICM) or multipotent trophectoderm (TE). Here we determined, for the first time, global gene expression patterns in the ICM and TE isolated from bovine blastocysts. The ICM and TE were isolated from blastocysts harvested at day 8 after insemination by magnetic activated cell sorting, and cDNA sequenced using the SOLiD 4.0 system.
Results
A total of 870 genes were differentially expressed between ICM and TE. Several genes characteristic of ICM (for example, NANOG, SOX2, and STAT3) and TE (ELF5, GATA3, and KRT18) in mouse and human showed similar patterns in bovine. Other genes, however, showed differences in expression between ICM and TE that deviates from the expected based on mouse and human.
Conclusion
Analysis of gene expression indicated that differentiation of blastomeres of the morula-stage embryo into the ICM and TE of the blastocyst is accompanied by differences between the two cell lineages in expression of genes controlling metabolic processes, endocytosis, hatching from the zona pellucida, paracrine and endocrine signaling with the mother, and genes supporting the changes in cellular architecture, stemness, and hematopoiesis necessary for development of the trophoblast.
http:purl.orgdcelements1.1creator
Ozawa, Manabu
Sakatani, Miki
Yao, JiQiang
Shanker, Savita
Yu, Fahong
Yamashita, Rui
Wakabayashi, Shunichi
Nakai, Kenta
Dobbs, Kyle B
Sudano, Mateus J
Farmerie, William G
Hansen, Peter J
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BioMed Central Ltd
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Manabu Ozawa et al.; licensee BioMed Central Ltd.
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BMC Developmental Biology. 2012 Nov 06;12(1):33
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RESEARCHARTICLEOpenAccessGlobalgeneexpressionoftheinnercellmassand trophectodermofthebovineblastocystManabuOzawa1,4,MikiSakatani3,JiQiangYao2,SavitaShanker2,FahongYu2,RuiYamashita5, ShunichiWakabayashi5,KentaNakai5,KyleBDobbs1,MateusJosSudano6,WilliamGFarmerie2andPeterJHansen1*AbstractBackground: Thefirstdistinctdifferentiationeventinmammalsoccursattheblastocyststagewhentotipotent blastomeresdifferentiateintoeitherpluripotentinnercellmass(ICM)ormultipotenttrophectoderm(TE).Herewe determined,forthefirsttime,globalgeneexpressionpatternsintheICMandTEisolatedfrombovineblastocysts. TheICMandTEwereisolatedfromblastocystsharvestedatday8afterinseminationbymagneticactivatedcell sorting,andcDNAsequencedusingtheSOLiD4.0system. Results: Atotalof870genesweredifferentiallyexpressedbetweenICMandTE.SeveralgenescharacteristicofICM (forexample, NANOG SOX2 ,and STAT3 )andTE( ELF5 GATA3 ,and KRT18 )inmouseandhumanshowedsimilar patternsinbovine.Othergenes,however,showeddifferencesinexpressionbetweenICMandTEthatdeviates fromtheexpectedbasedonmouseandhuman. Conclusion: Analysisofgeneexpressionindicatedthatdifferentiationofblastomeresofthemorula-stageembryo intotheICMandTEoftheblastocystisaccompaniedbydifferencesbetweenthetwocelllineagesinexpressionof genescontrollingmetabolicprocesses,endocytosis,hatchingfromthezonapellucida,paracrineandendocrine signalingwiththemother,andgenessupportingthechangesincellulararchitecture,stemness,andhematopoiesis necessaryfordevelopmentofthetrophoblast. Keywords: Blastocyst,Trophectoderm,Innercellmass,DevelopmentBackgroundFollowingitsformationbysyngamyofthepronucleiof theoocyteandsperm,themammalianembryobeginslife asatotipotent,singlecellorganism.Subsequentcyclesof celldivisionandtheformationoftightjunctionsbetween blastomeresleadtoaconditionwherebyblastomereson theouterfaceoftheembryoexhibitdifferentpatternsof cellpolarity,geneexpressionandproteinaccumulation thanblastomeresontheinnerpartoftheembryo[1-4]. Non-polarizedblastomeresintheinnerpartoftheembryo aredestinedtoformthepluripotentinnercellmass(ICM) thatgivesrisetotheembryowhilepolarizedcellsinthe outerfaceoftheembryoarefatedtodifferentiateintothe trophectoderm(TE),whichdevelopsintoextraembryonic membranes.Cellfatemaybedeterminedasearlyasthe 4 – 8cellstageinthemouseanddependupondifferences betweenblastomeresinthekineticsoftheinteraction betweenthetranscriptionfactorPou5f1andDNAbinding sites[5].Nonetheless,blastomeresdonotundergolineage commitmentuntilaboutthe32-cellstage(inmice),based onlossofabilityofblastomerestoformeitherICMorTE [2]. LineagecommitmenttowardsICMorTEisunderthe controlofspecifictranscriptionfactors.Theexactrole ofatleastsometranscriptionfactorsvarieswithspecies [6].Inthebeststudiedspecies,themouse,theICMis regulatedby Sall4 Pou5f1 Sox2 and Nanog whileTE formationresultsfromacascadeofeventsinvolving Yap1 Tead4 Gata3 Cdx2 Eomes and Elf5 [7].Functionalpropertiesofthetwocelllineagesisalsodivergent.Inpart,thisreflectstheprocessesresponsiblefor establishmentandmaintenanceofcelllineage,suchas *Correspondence: Hansen@animal.ufl.edu1DepartmentofAnimalSciencesandD.H.BarronReproductiveandPerinatal BiologyResearchProgram,POBox110910,Gainesville,FL32611-0910,USA Fulllistofauthorinformationisavailableattheendofthearticle 2012Ozawaetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and reproductioninanymedium,providedtheoriginalworkisproperlycited.Ozawa etal.BMCDevelopmentalBiology 2012, 12 :33 http://www.biomedcentral.com/1471-213X/12/33

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differencesintranscriptionfactorusage,cellsignaling pathwaysandepigeneticmarks[7,8].Inaddition,the functionoftheICM,whichisfatedtoundergoaseries ofdifferentiationeventsinthegastrulationprocess,is differentfromtheTE,whichisdestinedtointeractwith theliningofthematernalreproductivetract. Inthepresentstudy,wedescribe,forthefirsttime, differencesinthetranscriptomeoftheICMandTEwith theobjectiveofunderstandingtheconsequencesofthe differentiationofthesetwocelltypesforcellularfunction.ThiswasachievedbyseparatingICMandTEusing anewly-developedimmunomagneticprocedure[9]followedbynext-generationsequencing.Resultsrevealthe implicationsofthespatialanddevelopmentaldifferentiationofthesefirsttwolineagesofthepreimplantation embryowithrespecttometabolism,interactionwiththe maternalsystemandchangesincellulararchitecture.In addition,aspectsofmolecularcontroloftheprocessof lineagecommitmentanddifferentiationareillustrative ofsimilaritiesanddifferenceswiththeprototypical mousemodel.MethodsReagentsAllreagentswerepurchasedfromSigma-Aldrich(St. Louis,MO,USA)orFisherScientific(Pittsburgh,PA, USA)unlessotherwisespecified.EmbryocultureandICM/TEisolationBovineembryoswereproducedfromslaughterhousederivedoocytesusingproceduresforinvitrooocyte maturation,fertilization,andembryocultureas describedpreviously[10].OvariesweredonatedbyCentralPacking,CenterHillFlorida.Thedayoffertilization wasdefinedasDay0.Afterfertilizationfor18 – 20h, embryoswereculturedinSOF-BE1medium[11]at 38.5Cinahumidifiedatmosphereof5%CO2and5% O2withthebalanceN2.Embryoswereculturedin groupsof30ina50 lculturedropundermineraloil. AtDay6,anadditional5 lculturemediumwasadded. AtDay8,blastocystswereharvestedandusedtopreparepreparationsofICMandTEusingmagneticactivatedcellsortingasreportedpreviously[9]. ThreeseparatepoolsofTEandICMforeachtreatmentwereobtained.Eachpoolwaspreparedusing88to 102blastocysts.Atotalof15fertilizationprocedures wereusedtopreparetheblastocysts;asetofthreebulls wasusedforfertilizationforeachprocedure.RNApreparation,libraryconstructionandsequencing usingSOLiD4systemTotalRNAwasisolatedfromeachpoolofembryonic cellsusingthePicoPureRNAIsolationKit(AppliedBiosystems,FosterCity,CA,USA)accordingtothe manufacturer ’ sinstructions.ThequalityofRNAwas assessedusingtheAgilent2100Bioanalyzer(Agilent Technologies,SantaClara,CA).AmplifiedcDNAwas preparedfromtotalRNAforRNA-Seqapplications usingtheOvationRNA-Seqkit(NuGenTechnology, SanCarlos,CA).BarcodedfragmentlibrarieswereconstructedusingtheSOLiDTMv4fragmentlibrarykit accordingtothemanufacturer ’ sprotocol(AppliedBiosystems).Briefly,doublestrandedcDNAwasshearedto 150 – 180bpfragmentsusingaCovarisTMS2Sonication system(Covaris,Woburn,MA).ThefragmentedDNA wassubsequentlyend-repairedandblunt-endligatedto P1andP2adaptors.Theadaptorligated,purifiedand size-selected200 – 270bpfragmentswerenick-translated andthenamplifiedusingprimersspecifictoP1andP2 adaptorsandPlatinumWPCRAmplificationMix(AppliedBiosystems).Thequalityofthelibrariesandfragmentdistributionwereverifiedbyrunning1 lofeach libraryonAgilentDNA1000chip(AgilentTechnologies).Amplifiedlibraries(5differentlibrariespooledfor eachslide)wereimmobilizedontoSOLiDP1DNA beads(AppliedBiosystems).Thebead-boundlibraries werethenclonallyamplifiedbyemulsionPCRaccording totheAppliedBiosystemsSOLiDTM4SystemsTemplatedBeadPreparationGuide.Afteramplification, emulsionsweredisruptedwith2-butanolandthebeads containingclonallyamplifiedtemplateDNAwereP2enrichedandextendedwithabeadlinkerbyterminal transferase.Thequantityofthebeadswasdetermined usingaNanoDropWND1000spectrophotometer (ThermoScientific,Wilmington,DE).Approximately 600-700Mbeadsweredepositedoneachslide(ranin totalthreeslides)andsequencedusing ‘ sequencingby ligation ’ chemistryandthe50x5bpprotocolonthe SOLiDTMv4sequencer(AppliedBiosystems)atthe InterdisciplinaryCenterforBiotechnologyResearch, UniversityofFlorida.Resultswereobtainedascolor spacefastafiles.AnalysisofreaddataRawsequencingreadswereinitiallyprocessedwithGenomeQuesttools[12].Ambiguousresiduesweretrimmed offfrombothsidesofthesequence.BaseswithPhred qualitybelow12fromthe3 ’ endofthesequencewere removed.Readsthatwereshorterthan40basesorthat containedmorethan10baseswithqualitybelow12 werealsodiscardedaswerereadsconsistingofrepetitive singlebasesthataccountsformorethan60%ofthe lengthatthe3 ’ end.About53~64%ofreadswere retainedaftercleanup,proving102 – 157millionclean readsforthethreereplicatesofeachtreatment. Formappingtothegenome,the Bostaurus genomic sequence bosTau4 (repeatmasked)wasdownloaded fromtheUCSCgenomebrowser(http://genome.ucsc.Ozawa etal.BMCDevelopmentalBiology 2012, 12 :33 Page2of13 http://www.biomedcentral.com/1471-213X/12/33

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edu/).SequencingreadsofeachsampleweremappedindependentlytothereferencesequencesusingTopHat 1.2.0[13].TopHatsplitreadstosegmentsandjoinssegmentalignments.Amaximumofonemismatchineach ofthe25bpsegmentswasallowed.Thisstepmapped 36.8%readstothegenome.Theunmappedreadswere collectedandmappedtothereferenceusingBowtie 0.12.7[14]allowingthreemismatches.Unmappedreads werefurthermappedtocDNAsequencesusingbfast 0.6.4[15]whileallowingforthreemismatchesforeach read.ThecDNAsequencesof B taurus weredownloadedfromtheNationalCenterofBiotechnologyInformation.Scaffoldandchromosomesequenceswere clearedandatotalof35,842sequenceswereobtained (http://www.ncbi.nlm.nih.gov/nuccore/?term=txid9913 [Organism:noexp]).Bfastaligned27.6%ofthetotalreads tothecDNAsequences.Therefore,atotalof64.4%or 595millionreadsweremappedsuccessfully.Ofthe mappedreads,89.8%areuniquelymappedtoeitherthe genomeorcDNAsequences.Dataweredepositedinthe DDBJSequenceReadArchiveathttp://www.ddbj.nig.ac. jp/index-e.html(SubmissionDRA000504). Digitalgeneexpressionwasdeterminedasfollows. Thenumberofmappedreadsforeachindividualgene wascountedusingtheHTSeqtool(http://www-huber. embl.de/users/anders/HTSeq/doc/overview.html)with intersection-nonemptymode.HTSeqtakestwoinput files-bamorsam-formatfilesofmappedreadsanda genemodelfile.TheEnsemblegeneannotationfilein GTFformatwasdownloadedfromtheUCSCgenome browser.TheDESeqpackage[16]inRwasusedfor digitalgeneexpressionanalysis.DESequsesthenegative binomialdistribution,withvarianceandmeanlinkedby localregression,tomodelthenulldistributionofthe countdata.Significantup-anddownregulatedgenes wereselectedusingtwocutoffs:anadjustedPvalueof 0.05andaminimumfold-changeof1.5.Classificationofdifferentiallyexpressedgenesintogene ontology(GO)classesDifferentiallyexpressedgeneswereannotatedbythe DatabaseforAnnotation,VisualizationandIntegrated Discovery(DAVID;(DAVIDBioinformaticsResources 6.7,http://david.abcc.ncifcrf.gov/)[17].Mostgeneswere annotatedusingthebovinegenomeasareferenceand additionalgeneswereannotatedbycomparisontothe humangenome.TheDAVIDdatabasewasqueriedto identifyGOclassesenrichedforupregulatedanddownregulatedgenes.Functionsofdifferentiallyexpressed geneswerefurtherannotatedusingKyotoEncyclopedia ofGenesandGenomes(KEGG,http://www.genome.jp/ kegg/).OverviewofthedifferentiallyregulatedKEGG pathwaysweremappedonKEGGPathwayMapusing iPath2.0(http://pathways.embl.de/)[18]. Tofurtheranalyzepatternsofgenesdifferentially regulatedbetweenICMandTE,k-meanclusteringwas performed.Thereadscountdataofthe870significant genesfortheICM-controlversusTE-controlcomparisonwereclusteredusingk-meansstrategy[19].Toestimatethepremiumclusternumber,k-valuesfrom3to 100weretestedandthecorrespondingsumofsquares error(SSE)[20]wascalculatedforeachkvalue.SSEis definedasthesumofthesquareddistancebetweeneach memberofaclusteranditsclustercentroid.TheSSE valuesdroppedabruptlyuntilk=8(resultsnotshown). TobalancetheminimumnumberofSSEandtheminimumnumberofclusters,k=8wasselectedasthepremiumparameterforclusteringgenesandaheatmapwas generatedusing heatmap 2 ofRpackage.EnrichmentanalysisfortranscriptionfactorbindingsitesForeachdifferentiallyexpressedgene,thecandidatepromoterregionwasdefinedasthespanofnucleotides from200bpupstreamand50bpdownstreamfromthe transcriptionalstartsiteidentifiedinEnsembl.Todetect putativetranscriptionfactorbindingsites(TFBS)ineach promoter,wefollowedthemethodofWassermanand Sandelin[21].Position-specificweightmatriceswere obtainedfromtheJASPARdatabase[22].Thescorewas calculatedbyformula1inAdditionalFile1.Wealsocalculatedtheratioofthescoretothemaximumscoreby formula2(AdditionalFile1).Statisticalsignificanceof eachTFBSwasevaluatedbycalculatingthehypergeometricdistributionusingformula3(Additionalfile1). Weperformedthe ‘ match ’ programwith ‘ minSUM ’ and ‘ minFP ’ thresholdstodetectTFBS[23].StatisticalsignificanceofeachdetectedTFBSwasevaluatedbythe hypergeometricdistributionasdescribedabove.CalculationofGCcontentsanddetectionofCpGislandsThemethodbyGardiner-GardenandFrommer[24]was usedtoidentifyCpGislandsintheregionencompassing the100nucleotidesupstreamand100nucleotidesdownstreamfromthestartsite.Trans criptionalstartsitesfordifferentiallyexpressedgeneswereobtainedfromUMD3.1 [25].ForthedefinitionofCpGislands,TheGCcontent wascalculatedas([C]+[G])/200,where[N]denotesthe numberofnucleotides “ N ” withinthe200basewindow. TheCpGscorewascalculatedas[CG]/([C]*[G]*200).A genewasclassifiedasCpGpositivewhenitsGCcontentin theregionspanningthe100n ucleotidesupstreamandthe 100nucleotidesdownstreamfromthestartsiteexceeds0.5 andwhentheCpGscoreinthesameregionexceeds0.6. Otherwise,agenewasclassifiedasCpGnegative.Chisquareanalysiswasusedtodeterminewhetherthepercent ofgenesclassifiedasCpGpositivedifferedbetween1) genesoverexpressedinICMversusgenesoverexpressedinOzawa etal.BMCDevelopmentalBiology 2012, 12 :33 Page3of13 http://www.biomedcentral.com/1471-213X/12/33

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TEand2)genesoverexpressedinICMorTEversusthe referencepopulationof25118genesinthebovinegenome.Confirmationofdifferencesingeneexpressionbetween ICMandTEbyquantitativePCRAnexperimentwasperformedtoverifytheeffectofcell type(ICMvsTE)andCSF2onrelativemRNAabundance ofthe GATA3 ELF5 CDX2 NANOG and SOX2 .Embryos werepreparedasdescribedpreviouslyandblastocysts werecollectedatDay7.Poolsof25 – 34blastocystswere submittedtomagnetic-activatedcellsorting[9].Atotalof 6biologicalreplicatesofICMandTEwereprepared. mRNAextractionwasperformedusingtheAllPrep DNA/RNAminiKit(Qiagen,Inc.,Valencia,CA,USA)followedbyDNase(Qiagen)treatmentandreversetranscription(HighCapacitycDNAReverseTranscriptionKit, AppliedBiosystems,FosterCity,CA).Transcriptabundancefor GATA3 ELF5 CDX2 NANOG and SOX2 as wellashousekeepinggenes GAPDH SDHA and YWHAZ werequantifiedbyaBio-RadthermalcyclerCFX96-RealTimesystem(Bio-Rad,Hercules,CA,USA)usingSsoFast EvaGreenSupermixreagent(Bio-Rad,Hercules,CA, USA).PCRconditionswereasfollows:30secat95Cfollowedby40cycleseachof5secat95Cand1minat 60C.Datawereanalyzedusingthedelta-deltacycle threshold(Ct)method.Thereferencegenewasthe geometricmeanoftheCtvaluesof GAPDH SDHA and YWHAZ .Primersfor ELF5 werebasedon NM_001024569.1andweredesignedusingPrimerQuest fromidtDNA(http://www.idtdna.com)software,Efficiencywas95%andidentityofampliconswasverified bysequencingproducts.Theprimerswere5 ’ TGC CATTTCAACATCAGTGGCCTG3 ’ and5 ’ AAGGC CACCCTCAAAGACTATGCT3 ’ .Otherprimerpairs werepublishedpreviously: GATA3 [26], CDX2 and NANOG [9], SOX2 [27]and GAPDH SDHA and YWHAZ [28]. Datawereanalyzedbyleast-squaresanalysisofvariance usingtheGeneralLinearModel(GLM)procedureofthe StatisticalAnalysisSystem,version9.2(SASInstituteInc, Cary,NC,USA)Sourcesofvariationinthemodel includedcelltype(ICMandTE),replicateandtheinteraction;celltypewasconsideredfixedandreplicatewas consideredrandom.Logarithmictransformationwasappliedto CDX2 datatoimprovenormality.Alldataare reportedasuntransformedleast-squaresmeans.ResultsDifferentiallyexpressedgenesThelistsofdifferentiallyexpressedgenes,determined usinganadjustedPvalueof 0.05and 1.5-folddifferenceascut-offs,arepresentedinAdditionalfile2.There wereatotalof870genesthatweredifferentiallyexpressed betweenICMandTE,with411genesupregulatedinthe ICMand459downregulatedintheICM(i.e.,upregulated intheTE).Annotationofgenesdifferentiallyexpressedbetween ICMandTEDifferentiallyexpressedgeneswereannotatedusingthe GeneIDconversiontooloftheDAVIDBioinformatics Resources6.7(http://david.abcc.ncifcrf.gov/conversion. jsp);835ofthe870differentiallyexpressedgeneswere annotated(389genesupregulatedintheICMand424 genesupregulatedintheTE).ForthelistofgenesupregulatedinICM,10GOtermswerelistedintheBiologicalProcessgroup,4GOtermsintheCell Componentgroup,and5termsintheMolecularFunctiongroup(Table1).Termsrelatedtotranscriptionalactivitiesweredominantincludingregulationof transcription,DNA-dependent(25genes),regulationof transcriptionfromRNApolymeraseIIpromoter(11 genes),DNAbinding(29genes),transcriptionregulator activity(22genes)andtranscriptionfactoractivity(17 genes).TherewerealsoGOtermsrelatedtometabolic activityincludingregulationofRNAmetabolicprocess (25genes),positiveregulationofmacromoleculemetabolicprocess(12genes),negativeregulationofmacromoleculemetabolicprocess(10genes),andenzyme binding(10genes). ForgenesupregulatedinTE,12GOtermswerelisted intheBiologicalProcessgroup,12intheCellComponentgroup,and9intheMolecularFunctiongroup (Table2).GOtermsenrichedforTEweredistinctfrom thoseforICM.Alargenumberofgenesrepresentedby GOtermsrelatedwithmetabolismwereupregulatedin TEincludingproteolysis(27genes),oxidationreduction (23genes),lipidbiosyntheticprocessing(11genes),steroidmetabolicprocess(10genes),andpeptidaseactivity (actingonL-aminoacidpeptides)(22genes)aswellas genesinvolvedinbindingreactions[ionbinding(86 genes),cationbinding(83genes),metalionbinding(81 genes),calciumionbinding(34genes)andironion binding(12genes)].Therewasalsoenrichmentfor genesassociatedwithendo-orexocytosis,membrane transportandalterationsincellulararchitectureasindicatedbyGOtermsforvesicle-mediatedtransport(15 genes),actinfilament-basedprocess(14genes),actin cytoskeletonorganization(13genes),cytoskeleton organization(13genes),plasmamembrane(43genes), endoplasmicreticulum(32genes),cytoplasmicvesicle (14genes),vesicle(14genes),actincytoskeleton(13 genes),cellprojection(12genes),vacuole(11genes), endoplasmicreticulumpart(11genes),apicalpartofcell (10genes),andcytoskeletalarrangement(20genes). Functionsofdifferentiallyexpressedgeneswerefurther annotatedusingKEGG(http: //www.genome.jp/kegg/). GenesupregulatedinICMwereenrichedineighttermsOzawa etal.BMCDevelopmentalBiology 2012, 12 :33 Page4of13 http://www.biomedcentral.com/1471-213X/12/33

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(Table3A).Theseincludedp athwaysinvolvedinlineage commitment(e.g.,hematopoiet iccelllineage)anddifferentiation(axonguidance)aswellasthoseinvolvedinmaintenanceofstemnessandselfrenewal(e.g.,pathwayin cancerandJak-STATsignalingpathway).GenesupregulatedinTEwereenrichedin12terms(Table3B).Noneof thetermswereincommonwithKEGGtermsenrichedfor genesupregulatedforICM.Termswerepreferentiallyrelatedtotransmembranetransport(lysosome,aldosteroneregulatedsodiumresabsorption,andABCtransporters), lipidorsteroidmetabolism(PPARsignalingpathway, terpenoidbackbonebiosynthesis,sphingolipidmetabolism,steroidhormonebiosynthesis,fattyacidmetabolism)andothermetabolicprocesses(pantothenateand CoAbiosynthesis).Additionalfile3representsaKEGG metabolicpathwaymapinwhichpathwaysthatweredifferentiallyenrichedbetweenICMandTEwereidentified usingiPath2.0(http://pathways.embl.de/).Notethe increasedmetabolicactivityinTEascomparedtoICM.K-meanclusteringThe870genesthatweredifferentiallyexpressedbetween ICMandTEwereclusteredinto8clusters,with2,4,7, 9,23,48,149and628genesineachcluster(Additional file4).Thebiggestcluster(628genes)contained72.2% ofallthesignificantgenesandgeneswereincludedfrom almostalltheoverrepresentedpathways(Table3). Therefore,thek-meananalysisdidnotdisclosemuchinformationonfunctionalexpressionpatternsofdifferentiallyexpressedgenes.ComparisonofICM-TEdifferencesinthebovinewiththe mouseandhumanTheliteraturewasusedtoidentifyagroupofgenesthat havebeenidentifiedasbeingexpressedbyICM,TEor embryonicstemcellsinthemouse[29-32]orhuman [33-38](Additionalfile5).Amongthe119genesconsideredcharacteristicofICMorembryonicstemcells,8 weresignificantlyupregulatedinICM( KDM2B NANOG SOX2 SPIC STAT3 ZX3HAV1 ,and OTX2 ) andtwo( IL6R and TFRC )tended(P=0.06orless)tobe upregulatedinICM.Conversely,6genesconsideredas beingexpressedinICMorembryonicstemcellsinthe mouseorhumanwereupregulatedintheTE( DAB2 DSP GM2A SCD SSFA2 ,and VAV3 ).Of49genesconsideredcharacteristicofTE,12( AQP11 ATP1B3 CGN Table1GOtermsenrichedforgenesupregulatedintheICMascomparedtoTEaGOtermCountPercentPvalueFDRbBiologicalProcess Regulationoftranscription,DNA-dependent256.70.0443.9 RegulationofRNAmetabolicprocess256.70.0449.9 Neurologicalsystemprocess123.20.0116.9 Regulationofcellproliferation123.20.0224.9 Immuneresponse123.20.0335.4 Positiveregulationofmacromoleculemetabolicprocess123.20.0343.1 Cognition112.90.002.4 RegulationoftranscriptionfromRNApolymeraseIIpromoter112.90.0229.6 Responsetoorganicsubstance102.70.0116.8 Negativeregulationofmacromoleculemetabolicprocess102.70.0443.9 CellComponent Plasmamembrane349.10.0220.7 Extracellularregion308.00.004.2 Extracellularregionpart195.10.001.9 Extracellularspace123.20.0216.9 MolecularFunction DNAbinding297.80.0546.9 Transcriptionregulatoractivity225.90.0440.5 Calciumionbinding184.80.0332.1 Transcriptionfactoractivity174.60.0219.4 Enzymebinding102.70.017.7aOnlythoseGOtermswhichcontainedatleast10differentiallyexpressedgenesarelisted.bFalsediscoveryrate(x100).Ozawa etal.BMCDevelopmentalBiology 2012, 12 :33 Page5of13 http://www.biomedcentral.com/1471-213X/12/33

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CYP11A DSC2 ELF5 GATA3 HSD3B1 KRT18 MSX2 SFXNorTJP2 )wereupregulatedinTE. CDH24 ,acadherinreportedtobeupregulatedintheTEofthehuman [33],wasexpressedinhigheramountsintheICM. Wealsoexaminedexpressionofruminant-specificgenes knowntobeupregulatedinTE.Thethreeexamined, IFNT1 [39], PAG2 [40],and TKDP1 [41],wereupregulatedinTE. WeevaluateddifferencesinexpressionbetweenICM andTEforgenesthathavebeenshowninthemouse[7] tobeimportantforsegregationofICMandTElineages andsubsequentTEdifferentiation(Table4).Expression oftwogenesimportantforICMcommitment, NANOG and SOX2 ,wassignificantlyhigherforICMthanTE whileexpressionoftwoothergenesimportantforICM Table2GOtermsenrichedforgenesupregulatedintheTEascomparedtoICMaGOtermCountPercentPvalueFDRbBiologicalProcess Proteolysis276.40.006.26 Oxidationreduction235.40.0110.40 Intracellularsignalingcascade204.70.0343.10 Iontransport204.70.0450.64 Vesicle-mediatedtransport153.50.005.68 Regulationofcellproliferation153.50.0111.09 Actinfilament-basedprocess143.30.000.00 Actincytoskeletonorganization133.10.000.00 Cytoskeletonorganization133.10.001.22 Lipidbiosyntheticprocess112.60.0112.45 Steroidmetabolicprocess102.40.000.31 Negativeregulationofcellproliferation102.40.002.24 CellComponent Plasmamembrane4310.10.0440.40 Endoplasmicreticulum327.60.000.00 Cellfraction163.80.002.50 Cytoplasmicvesicle143.30.0331.04 Vesicle143.30.0436.80 Actincytoskeleton133.10.000.04 Membranefraction133.10.0111.72 Insolublefraction133.10.0115.08 Cellprojection122.80.0441.18 Vacuole112.60.000.87 Endoplasmicreticulumpart112.60.004.14 Apicalpartofcell102.40.000.04 MolecularFunction Ionbinding8620.30.000.14 Cationbinding8319.60.000.49 Metalionbinding8119.10.000.94 Calciumionbinding348.00.000.00 Peptidaseactivity,actingonL-aminoacidpeptides225.20.001.41 Cytoskeletalproteinbinding204.70.000.00 Actinbinding143.30.000.04 Ironionbinding122.80.0331.90 Lipidbinding112.60.0338.82aOnlythoseGOtermswhichcontainedatleast10differentiallyexpressedgenesarelisted.bFalsediscoveryrate(x100).Ozawa etal.BMCDevelopmentalBiology 2012, 12 :33 Page6of13 http://www.biomedcentral.com/1471-213X/12/33

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commitment, POU5F1 and SALL4 ,didnotdiffersignificantlybetweenICMandTE.Numerically,expressionof theselattertwogeneswashigherforICM.Fourgenes wereexaminedthatareimportantforTEcommitment – CDX2 GATA3 TEAD4 ,and YAP1 .Expressionof GATA3 wassignificantlyhigherforTEbuttherewere nosignificantdifferencesinexpressionbetweenICM andTEfortheotherthreegenes.Onegeneimportant fordifferentiationofTElaterindevelopment, ELF5 ,was expressedinhigheramountsinTE(adjustedP=0.022) whereasanother, EOMES ,wasbarelydetectableandnot differentbetweenICMandTE.Characteristicsofpromoterregionsofgenesdifferentially expressedbetweenICMandTETheregionspanningnucleotidesequenceslocated200bp upstreamto50bpdownstream ofthetranscriptionstart sitewasexaminedforpresenceofputativeTFBSforeach Table3KEGGPathwaysenrichedforgenesupregulatedintheinnercellmassortrophectodermTermGenes UpregulatedinInnerCellMass(A) Antigenprocessingandpresentation CD74 CD8B HSPA1L HSPA6 PSME1 BoLA DRB3 Complementandcoagulationcascades A2M F2R C1R PLAUR C4BPA Chemokinesignalingpathway ITK CCL24 CXCL7 GNAI1 GNB5 GNG7 PLCB1 STAT1 STAT4 STAT3 Axonguidance EPHA4 CHP DPYSL2 GNAI1 ROBO1 SEMA4G SLIT2 Arrhythmogenicrightventricularcardiomyopathy(ARVC) CDH2 DES GJA1 ITGA2 TCF7L2 Pathwaysincancer CDKN2B FGF12 FGF16 ITGA2MMP9 PDGFRA STAT1 STAT4 STAT3 TCF7L2 FOS KIT WNT Jak-STATsignalingpathway IL12RB2 IL19 IL6ST, IL7 STA1 STAT4 STAT3 SPRY2 Hematopoieticcelllineage CD1A CD8B ITGA2 IL7 KIT UpregulatedinTrophectoderm(B) Lysosome ATP6V0A4 GM2A NPC CTSB CTSH CTSL2 CTNS GLAA GALC MANBA PLA2G15 SCARB2 ATP6V0C SLC11A2 Steroidbiosynthesis NSDHL CYP41A1 FDFT1 SC4MOL Aldosterone-regulatedsodiumreabsorption ATP1B3 NEDD4L PRKCG SGK1 SFN Vascularsmoothmusclecontraction ACTA2 ACTG2 CALD1 CALML5 ITPR2 MYLK MYL6 PRKCH PRKCG PPARsignalingpathway ACSL4 AXSL6 FABP5 ACSL3 SCD SCP2 Phosphatidylinositolsignalingsystem CALML3 ITPR2INPP4B, INPP5D PRKCG SYNJ1 PantothenateandCoAbiosynthesis BCAT1 ENPP1 ENPP3 Terpenoidbackbonebiosynthesis HMGCR ACAT2 IDI1 Sphingolipidmetabolism UGCG GLA GALC SGPP1 Steroidhormonebiosynthesis UGT1A1 UGT1A6 CYP11A1 CYP3A28 HSD3B1 Fattyacidmetabolism ACAT2 ACSL4 ACSL6 ACSL3 ABCtransporters ABCA3 ABCB1 ABCC2 ABCG5 Table4DifferencesinexpressionbetweenICMandTEforgenesinvolvedinsegregationofICMandTEinmiceaGenesymbolRoleinmouseMeancounts ICMMeancount TEFoldchange TE / ICMAdjustedPvalue CDX2 TEcommitment5.72.80.490.780 ELF5 TEdifferentiation5.328.95.410.022 GATA3 TEcommitment363.6976.72.690.018 EOMES TEdifferentiation1.40.20.160.934 NANOG ICMcommitment3014.8620.90.210.000 POU5F1 ICMcommitment2394.11873.50.780.605 SALL4 ICMcommitment5.33.80.710.893 SOX2 ICMcommitment816.2360.70.440.005 TEAD4 TEcommitment7.112.01.690.894 YAP1 TEcommitment47.943.00.901.000aSource:Chenetal.[ 7 ].Ozawa etal.BMCDevelopmentalBiology 2012, 12 :33 Page7of13 http://www.biomedcentral.com/1471-213X/12/33

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genethatwasdifferentiallyexpressedbetweenICMand TE.Bindingsitesforthreetranscriptionfactors(PLAG1, RELAandRREB1)weresignificantlyenrichedforgenes overexpressedintheICMwhilebindingsitesforninetranscriptionfactors(EGR1,GAB PA,KLF4,MYF5,SP1,MZF1, NHLH1,PAX5andZFX)weresignificantlyenrichedfor TE.For11of12transcriptionfactorsidentifiedasbeing usedtoregulategenesoverexpressedinICMorTE,there wasnodifferenceinexpressionlevelbetweenICMandTE. Theexceptionwasfor EGR1 ,whereexpressionwasupregulatedinICM(Additionalfile2),eventhoughtheTFBS wasenrichedforgenesoverexpressedinTE.DifferencesinpromoterCpGislandsbetweengenes overexpressedinICMorTEThepercentofgenesoverexpressedinICMthatwere classifiedasCpGpositive(46.6%)waslower(P<0.05) thanforgenesoverexpressedinTE(55.3%).Moreover, thepercentofgenesclassifiedasCpGpositiveforgenes overexpressedineithertissuewashigherthanthepercentthatwereclassifiedasCpGpositivefortheentire bovinegenome(39.4%).Thus,DNAmethylationmay playagreaterroleforregulationofgenesdifferentially regulatedintheICMandTEthanitdoesforthegenomeasawhole. OfthegenesthatweredifferentiallyregulatedforICM andTE,threeweregenesinvolvedinepigeneticmodification.Thesewere DNMT1 and KDM2B ,overexpressed inICM,and DNMT3A likesequence,overexpressedin TE(Additionalfile2).Confirmationofdifferencesingeneexpressionbetween ICMandTEbyquantitativePCRUsingisolatedICMandTEfromaseparatesetofblastocyststhanusedforSOLiDsequencing,qPCRwasperformedtoverifytreatmenteffectsongeneexpression for6genes( GATA3 ELF5 CDX2 NANOG and SOX2 ). ResultsfordifferencesbetweenICMandTEweregenerallyconsistentwithresultsfromdeepsequencing (Figure1).Inparticular,expressionwashigherforTE thanICMfor GATA3 (P=0.07)and ELF5 (P<0.05)and washigherforICMthanTEfor NANOG (P<0.05)and SOX2 (P<0.05).Onediscrepancywithdeepsequencing resultswasfor CDX2 .WhiletherewasnosignificantdifferencebetweenICMandTEinthedeepsequencing database(Table4),mRNAfor CDX2 washigherforTE thanICMasdeterminedbyqPCR(Figure1).DiscussionDifferentiationinthemammalianembryoisdependent uponspatialposition-cellsontheinsideoftheembryoremainpluripotentforaperioduntilinitiationofgastrulation whilecellsontheouterfaceoftheembryodifferentiateinto TEandultimatelyformmuchoftheextraembryonic membranes.Here,usingmagnetic-assistedcellsortingand high-throughputnextgenerat ionsequencing,weshowthe consequencesofspatialdifferencesbetweenICMandTE andsubsequentdivergenceinlineagecommitmentforexpressionofgenesregulatingpluripotencyandlineage commitment,cellularmetabolism,andinteractionswith thematernalsystem. CommitmenttowardstheICMlineageinthemouseis maintainedbyactionsofPou5f1(Oct4),Sall4,Sox2and Nanog;Cdx2intheTEinhibits Pou5f1 expressionand allowsdifferentiationofextraembryonicmembranes [3,4,7].Inthebovine,too, SOX2 and NANOG wereoverexpressedinICMbutexpressionof POU5F1 and SALL4 werenotsignificantlydifferentbetweenICMandTE.A highdegreeofexpressionof POU5F1 intheTEwas expectedbecausedifferencesintheregulatoryregionof the POU5F1 geneincattleascomparedtothemousegene make POU5F1 resistanttoregulationbyCDX2[6].Nonetheless, POU5F1 expressionisgreaterintheICMofcattle [6,42].Inthepresentstudy,expressionofboth POU5F1 and SALL4 werenumericallygreaterforICM;failureto findsignificantdifferencesbetweenICMandTEmayrepresentthesmallsamplesize.Itshouldalsobekeptin mindthatembryosproducedinvitrohavealteredpatterns ofgeneexpressionrelativetoembryosproducedinvivo [43].SuchalterationscouldchangesomeofthedifferentialgeneexpressionbetweenICMandTE,ashasbeen reportedforthemouseembryo[44]. AnalysisofgenesupregulatedinICMprovidessome cluesastothesignalingpathwaysrequiredforspecification,pluripotency,andotherfunctionsoftheICM.A totalof8genesintheKEGGJak-STATsignalingpathway wereupregulated.Inmice,LIF,whichsignalsthroughthe Jak-STATpathway,canpromotepluripotencyofcells Figure1 Differencesbetweeninnercellmass(ICM)and trophectoderm(TE)inexpressionof6selectgenesas determinedbyquantitativePCR. Blastocystswereharvestedat Day7andICMandTEseparatedbymagneticactivatedcellsorting. Datarepresentleast-squaresmeansSEMofresultsfromsix biologicalreplicates.OpenbarsrepresentICMandfilledbarsTE. *=P<0.05. Ozawa etal.BMCDevelopmentalBiology 2012, 12 :33 Page8of13 http://www.biomedcentral.com/1471-213X/12/33

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derivedfromtheICM[45].WhileLIFcannotcausebovineICMcellstodevelopintostemcells[46],other moleculesthatsignalthroughtheJak-STATpathwayare likelytobeinvolvedinregulationoftheICM.Several genesrelatedtocellularmigrationwereupregulatedin ICM,asindicatedbyenrichmentofthechemokinesignalingpathway(10genes)andaxonguidance(7genes) GOterms.Inthemouse,blastomeresoftheICMcan changeposition,atleastinparttoalignpositionwith subsequentformationofprimitiveendoderm[47-49]. Perhaps,movementisdirectedbyguidancemolecules suchaschemokines. OutercellsofthemouseblastocystarecommittedtowardstheTElineagethroughtheactionsof Yap1 Tead4 Gata3 ,and Cdx2 ([3,4,7].Wefoundnodifferencein CDX2 expressionbetweenICMandTEusingdeepsequencing eventhoughitiswellestablishedthatthegeneisexpressed toagreaterextentinTEofthebovine[6,9,42]and CDX2 expressionwashigherinTEthanICMintheqPCRexperiment. CDX2 expressionwasverylowinthedeepsequencingexperiment,especiallycomparedtothatof POU5F1 Onepossibilityisthatdifferencesin CDX2 expressionbetweenTEandICMatDay7(asdetectedbyqPCR)become reducedatDay8.Likeseenearlier[6],otherhomologuesof CDX2 werenotdetected( CDX1 )orwerenearlynondetectable( CDX4 )(Additionalfile2). AnothergeneinvolvedinTElineage, GATA3 ,was expressedinhigheramountsinTE.AsimilarbutnonsignificantdifferenceinexpressionbetweenICMandTE wasnotedearlier[42].Therewasnosignificantdifferencein TEAD4 or YAP1 expressionbetweenICMand TE.Similarfindingswereobservedinthebovinefor TEAD4 [42].Ageneinvolvedindevelopmentofextraembryonicectoderminmice, ELF5 [7],wasoverexpressedinTEwhereasanothergeneinvolvedin developmentofextraembryonicmembranes, EOMES wasbarelydetectable.Infact,thereappearstobeanabsenceorverylowexpressionof EOMES inTEbetween day7and15ofgestationincattle[6].Inaddition,by Day11ofgestation,trophoblastexpressionof ELF5 is inhibitedandbecomeslimitedtotheepiblast[50]. Itisnotablethatseveralgenesc haracteristicallyexpressed inICMofmouseorhuman, DAB2 DSP GM2A SCD SSFA2 ,and VAV3 ,[30,32,37]weresignificantlyoverexpressedintheTEofthebovinewhile CDH24 ,reportedto beupregulatedintheTEofthehuman[33],wasexpressed inhigheramountsintheICMofthebovine. Dsp and Dab2 areindispensibleforembryonicdevelopmentinmiceand homologousrecombinationcausespostimplantationembryonicfailure[51,52].Clear ly,asfirstshownbyBergetal. [6],divergentevolutioninthecontrolofearlyembryonic developmentmeansthatstudyacrossawidearrayofspeciesisrequiredtounderstanddevelopmentalprocesses fully. Byvirtueofitspositionintheembryo,polarizedmorph-ology[53]andtightjunctionsbetweenitsmembercells [1],theTEisfatedtobethecelllineagethroughwhich theblastocystinteractsdirectlywiththemotherinterms ofnutrientexchange,maternal-conceptuscommunication, andplacentation.Itappearsthatexecutingthesefunctions placesincreasedmetabolicdemandsontheTEascomparedtotheICMasindicatedbyupregulationofgenes involvedinmetabolism,particularlythoseinvolvedinlipid metabolism.Lipidaccumulationinculturedbovine embryosisgreaterforTEthanICM,althoughthedifferencedependsuponmedium[54,55]. ItisthroughtheTEthatnutrientsentertheembryo andfromtheTEthatsecretoryproductsoftheembryo mustentertheuterineenvironment.Consistentwitha rolefortheTEinuptakeanddeliverywasupregulation ofgenesinvolvedinendo-orexocytosisandmembrane transport.Lysosomal-likestructureshavebeenreported tobemoreabundantinTEthanICMincattle,atleast forcertainmedia[54,55],andthemouse[53]. Moleculesinvolvedinsignalingtothemotherthat wereupregulatedinTEinclude IFNT1 PAG2 and TKDP1 .Therolefor IFNT1 istoactonthematernal endometriumtoblockluteolyticreleaseofprostaglandin F2 [39,56].Whilethisactionisinitiatedlaterinpregnancy,betweenDay15and17ofgestation,secretionof IFNToccursasearlyastheblastocyststage[57]. TKDP1 isamemberoftheKunitzfamilyofserineproteinase inhibitorsandmayfunctiontolimittrophoblastinvasivenessinspecieslikethecowwithepitheliochorialplacentation[41].Littleisknownabouttheroleof PAG2 whichisthemostlyabundantlyexpressedofatleast22 transcribed PAG genes[40].Unlikesome PAG genes (theso-called “ modern ” clade),whoseexpressionislimitedtotrophoblastgiantcellsformedlaterindevelopment, PAG2 isexpressedwidelyinthecotyledonary trophoblastandispredictedtobeanactiveasparticproteinase[58]. IFNT1 PAG2 and TKDP1 areallgenesthatare phylogenetically-restrictedtoruminants.Anotherconceptusproductthatisproducedmorewidelyinmammalsisestrogen.Theroleforembryonicestrogenisnot knownformostspeciesbutblastocystestrogenhasbeen suggestedtobeinvolvedinhatchingfromthezonapellucidainhamsters[59]andinconceptusgrowthinthe pig[60].Thebovineblastocyst,too,producesestrogen [61]andtheupregulationofgenesinvolvedinterpenoid backbonebiosynthesisandsteroidhormonebiosynthesis suggestthattheprimarysourceofblastocystestrogens istheTE. Followingblastocystformation,theruminanttrophoblastundergoesaseriesofdevelopmentalstepsthatare dependentonchangesincellshapeandspatialposition, includinghatching(whichrequiresactin-basedOzawa etal.BMCDevelopmentalBiology 2012, 12 :33 Page9of13 http://www.biomedcentral.com/1471-213X/12/33

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trophectodermalprojections[59]),elongation(which leadstoanincreaseinsizeoftheconceptusfromabout 0.16mmatDay8toasmuchas100mmormoreat Day16[62])andeventualattachmenttothematernal endometrium(commencingaroundDay20inthecow [63].Theupregulationofgenesinthetrophoblastfor ontologiessuchasactinfilament-basedprocess,actin cytoskeletonorganization,cellprojectionandcytoskeletalarrangementreflectstheextensivechangesincell architecturerequiredfortheseprocesses.Inaddition, threecathepsingenes, CTSB CTSH and CTSL2 ,were upregulatedinTE;theseproteinaseshavebeenimplicatedinblastocysthatching[59,64]. DifferencesingeneexpressionbetweenICMandTE areprobablydueinlargeparttodifferencesintranscriptionfactorusageandtoepigeneticmodifications.BindingsitesforthetranscriptionfactorsPLAG1,RELAand RREB1wereenrichedforgenesoverexpressedinICM whilebindingsitesforninetranscriptionfactors(EGR1, GABPA,KLF4,MYF,SP1,MZF1,NHLH1,PAX5and ZFX)weresignificantlyenrichedforTE.RELAisasubunitforNF B,whichinturnhasbeenimplicatedindifferentiationoftrophoblastlineagesfromembryonic stemcells[65]andinfunctionoftrophoblastgiantcells [66].Severalofthetranscriptionfactorsassociatedwith genesupregulatedinTEareinvolvedinhematopoiesis, includingEGR1[67],GABPA[68],MZF1[69],andZFX [70].Oneofthesetranscriptionalfactors,GABPA,can enhance Pou5f1 expressioninmouseembryonicstem cells[71]andanother,KLF4,isakeyregulatorofmaintenanceandinductionofpluripotency[72].Theoverall pictureisonewherehematopoiesisandstemnessis underpositiveregulationintheTE.AnothertranscriptionfactorassociatedwithregulationofgenesupregulatedinTEwasSP1.Thisproteinexertsseveralactions toregulatetrophoblastdevelopmentandfunction,includingactivationofexpressionofothertranscription factorssuchas Tfap2c [73]and Id1 [74].Inthecow,SP1 becomeslimitedtobinucleatecellsofthetrophoblastby Day25[75]. DNAmethylationcouldbeimportantforregulationof geneexpressionintheblastocystbecausethepromoter regionsofoverhalfofthegenesthatwereupregulatedin ICMorTEwereclassifiedasCpGpositive.Indeed,the percentofgenesclassifiedasCpGpositiveforgenesoverexpressedinICMorTEwashigherthanthepercentthat wereclassifiedasCpGpositivefortheentirebovinegenome.SlightlyfewergenesthatwereoverexpressedinICM wereclassifiedasCpG-positivethanforgenesthatwere overexpressedinTE,whichmightsuggestmoreinhibition ofgeneexpressionbymethylationinTE.Itisnoteworthy, however,thatNiemannetal.[76]didnotfindacorrelationbetweendegreeofCpGislandmethylationand amountofembryonicexpressionforeightgenes examined.Recentevidencehasbeeninterpretedtosignify thatitisnotthemethylationstateofindividualCpGthat determinegeneexpressionbutratherthemethylationstatusoflargeregionsofDNAthatspanmultiplegenes[77]. Incattle,thereareconflictingdataastowhether DNAmethylationislessextensiveforICMorforTE inbothembryosproducedinvitroandbysomaticcell nucleartransfer[78-80],Anotherepigeneticmark, H3K27me3,issimilarforbothcelltypes[81].Ofthe genesthatweredifferentiallyregulatedforICMand TE,threeweregenesinvolvedinepigeneticmodification.TwowereoverexpressedinICM: DNMT1 ,involved inmaintenanceofDNAmethylationduringsucceeding celldivisions[77],and KDM2B ,alysine-specifichistone dimethylasewhichcatalyzesdemethylationofH3K4and H3K6[82,83].Incontrast,a DNMT3A likesequence, whichestablishesDNAmethylationduringdevelopment andalsoparticipatesinmethylationmaintenance[77], wasoverexpressedinTE.Thepresenceofincreasedtranscriptabundancefor DNMT3A couldbeinterpretedto meanthat denovo DNAmethylationoccurstoagreater degreeinTE,asisindicatedbystudieswithembryos producedinvitro[79]andbysomaticcellnuclearcloning[80].FurtherresearchisnecessarytodeterminedifferencesinDNAmethylationbetweenTEandICMat thegene-specificandgenome-widelevel. Ingeneral,analysisofaseparatesetofisolatedICM andTEbyqPCRconfirmedtheresultsobtainedfordifferencesbetweencelltypesbydeepsequencing.Theexceptionwasfor CDX2 ,wheretherewasnodifferencein expressionasdeterminedbySOLiDsequencingbut whereexpressionwasgreaterforTEthanICMasdeterminedbyqPCR.Thediscrepancycouldreflecteither dayofsamplingdifferences(asdiscussedearlier)or, giventheoften-repeatedobservationthat CDX2 is expressedtoagreaterextentinTEthanICM[6,9,42], anerrorinducedbythedeepsequencingprocedure. Inconclusion,differentiationofblastomeresofthe morula-stageembryointotheICMandTEoftheblastocystisaccompaniedbydifferencesbetweenthetwocell lineagesinexpressionofgenescontrollingmetabolic processes,endocytosis,hatchingfromthezonapellucida,paracrineandendocrinesignalingwiththemother, andgenessupportingthechangesincellulararchitecture,stemness,andhematopoiesisnecessaryfordevelopmentofthetrophoblast.Muchoftheprocessleadingtothisfirstdifferentiationeventseemstobeunderthe controlofgenessuchas NANOG and GATA3 thatplay centralroleinlineagecommitmentinthemouse.As foundbyothersalso[6,42],therearefundamentaldifferencesfromthemouse.Understandingthenatureofthe processofpreimplantationdevelopmentinmammals willnecessarilyrequireacomparativeapproachbased onstudyofavarietyofanimalmodels.Ozawa etal.BMCDevelopmentalBiology 2012, 12 :33 Page10of13 http://www.biomedcentral.com/1471-213X/12/33

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ConclusionsAnalysisofgeneexpressionindicatedthatdifferentiation ofblastomeresofthemorula-stageembryointotheICM andTEoftheblastocystisaccompaniedbydifferences betweenthetwocelllineagesinexpressionofgenescontrollingmetabolicprocesses,endocytosis,hatchingfrom thezonapellucida,paracrineandendocrinesignaling withthemother,andgenessupportingthechangesin cellulararchitecture,stemness,andhematopoiesisnecessaryfordevelopmentofthetrophoblast.AdditionalfilesAdditionalfile1: Formulasusedforenrichmentanalysisfor transcriptionfactorbindingsites. Additionalfile2: DifferencesingeneexpressionbetweenICMand TE. GenesinwhichtheadjustedPvaluewas<0.05arecolorcoded(blue areupregulatedinICMandredareupregulatedinTE). Additionalfile3: KEGGmetabolicpathwaymapinwhichpathways thatweredifferentiallyenrichedbetweenICM(blue)andTE(red) wereidentifiedusingiPath2.0. Additionalfile4: Heatmapconstructedbyk-meanclusteringofthe 870genesthatdifferinexpressionbetweenICMandTE. Thecolors inthemapdisplaytherelativestandingofthereadscountdata;blue indicatesacountvaluethatislowerthanthemeanvalueoftherow whileredindicateshigherthanthemean.Theshadesofthecolor indicatehowfarawaythedatafromthemeanvalueoftherow. ColumnsrepresentindividualsamplesofICM(IC)andTE(TC). Additionalfile5: Differencesinexpressionbetweeninnercellmass (ICM)andtrophectoderm(TE)forgenesconsideredasbeing characteristicallyexpressedbyICMandTEinhumanormouse. Abbreviations DAVID:Databaseforannotation,visualizationandintegrateddiscovery; GO:Geneontology;KEGG:Kyotoencyclopediaofgenesandgenomes; ICM:Innercellmass;SSE:Sumofsquareserror;TFBS:Transcriptionfactor bindingsites;TE:Trophectoderm. Competinginterests Theauthorshavedeclaredthatnocompetinginterestsexist. Authorcontributions Conceivedanddesignedtheexperiments:MO,PJHWGF.Performedthe experiments:MO,SS,KBD,MJS.Analyzedthedata:MO,MS,J-QY,FY,RY,SW, KN,PJH.Wroteinitialdraftsofthepaper:MOPJH.Allauthorsreadand approvedthefinalmanuscript. Acknowledgments ThisworkwassupportedbyAgricultureandFoodResearchInitiativeGrant Nos.2009-65203-05732and2011-67015-30688fromUnitedStatesDept.of AgricultureNationalInstituteofFoodandAgricultureandbyagrantfrom theResearchOpportunityFund,UniversityofFloridaResearchFoundation. TheauthorsthankWilliamRembert,forcollectingoocytes,theChernin familyandCentralPacking(CenterHill,Florida),fordonatingovariantissue andScottRandellofSoutheasternSemen(WellbornFlorida),fordonating semen. Authordetails1DepartmentofAnimalSciencesandD.H.BarronReproductiveandPerinatal BiologyResearchProgram,POBox110910,Gainesville,FL32611-0910,USA.2InterdisciplinaryCenterforBiotechnologyResearch,UniversityofFlorida, Gainesville,FL32611-0910,USA.3Kyushu-OkinawaAgriculturalResearch Center,NationalAgricultureandFoodResearchOrganization,Kumamoto, Japan.4LaboratoryofDevelopmentalGenetics,InstituteofMedicalScience, UniversityofTokyo,Tokyo,Japan.5HumanGenomeCenter,Instituteof MedicalScience,UniversityofTokyo,Tokyo,Japan.6Departamentode ReproduoAnimaleRadiologiaVeterinria,FaculdadedeMedicina VeterinriaeZootecnia,UNESP,Botucatu,SoPaulo,Brasil. Received:6June2012Accepted:30October2012 Published:6November2012 References1.EckertJJ,FlemingTP: Tightjunctionbiogenesisduringearly development. BiochimBiophysActa 2008, 1778: 717 – 728. 2.MarikawaY,AlarcnVB: Establishmentoftrophectodermandinnercell masslineagesinthemouseembryo. MolReprodDev 2009, 76: 1019 – 1032. 3.Zernicka-GoetzM,MorrisSA,BruceAW: Makingafirmdecision: multifacetedregulationofcellfateintheearlymouseembryo. Nature Rev 2009, 10: 467 – 477. 4.RossantJ,TamPPL: Blastocystlineageformation,earlyembryonic asymmetriesandaxispatterninginthemouse. Development 2009, 136: 701 – 713. 5.PlachtaN,BollenbachT,PeaseS,FraserSE,PantazisP: Oct4kineticspredict celllineagepatterningintheearlymammalianembryo. NatCellBiol 2011, 13: 117 – 123. 6.BergDK,SmithCS,PeartonDJ,WellsDN,BroadhurstR,DonnisonM, PfefferPL: Trophectodermlineagedeterminationincattle. DevCell 2011, 20: 244 – 255. 7.ChenL,WangD,WuZ,MaL,DaleyGQ: Molecularbasisofthefirstcell fatedeterminationinmouseembryogenesis. CellRes 2010, 20: 982 – 993. 8.GasperowiczM,NataleDR: Establishingthreeblastocystlineages – then what? BiolReprod 2011, 84: 621 – 630. 9.OzawaM,HansenPJ: Anovelmethodforpurificationofinnercellmass andtrophectodermcellsfromblastocystsusingmagneticactivatedcell sorting. FertilSteril 2011, 95: 799 – 802. 10.LoureiroB,BonillaL,BlockJ,FearJM,BonillaAQS,HansenPJ: Colonystimulatingfactor2(CSF-2)improvesdevelopmentandposttransfer survivalofbovineembryosproducedinvitro. Endocrinology 2009, 150: 5046 – 5054. 11.FieldsSD,HansenPJ,EalyAD: Fibroblastgrowthfactorrequirementfor invitrodevelopmentofbovineembryos. Theriogenology 2011, 75: 1466– 1475. 12.ZhangJ,ChiodiniR,BadrA,ZhangG: Theimpactofnext-generation sequencingongenomics. JGenetGenom 2011, 38: 95 – 109. 13.TrapnellC,PachterL,SalzbergSL: TopHat:Discoveringsplicejunctions withRNA-Seq. Bioinformatics 2009, 25: 1105 – 1111. 14.LangmeadB,TrapnellC,PopM,SalzbergSL: UltrafastandmemoryefficientalignmentofshortDNAsequencestothehumangenome. GenomeBiol 2009, 10: R25. 15.HomerN,MerrimanB,NelsonS: BFAST:Analignmenttoolforlargescale genomeresequencing. PLoSOne 2009, 4: e7767. 16.AndersS,HuberW: Differentialexpressionanalysisforsequencecount data. GenomeBiol 2010, 11: R106. 17.HuangDW,ShermanBT,LempickiRA: Bioinformaticsenrichmenttools: pathstowardthecomprehensivefunctionalanalysisoflargegenelists. NucleicAcidsRes 2009, 37: 1 – 13. 18.YamadaT,LetunicI,OkudaS,KanehisaM,BorkP: iPath2.0:interactive pathwayexplorer. NuclAcidsRes 2011, 39: W412 – W415. 19.LiangJ,ZhangL,XiangZ,HeN: Expressionprofileofcuticulargenesof silkworm Bombyxmori BMCGenom 2010, 11: 173. 20.DashR,MishraD,RathAK,AcharyaM: AhybridizedK-meansclustering approachforhighdimensionaldataset. IntJEngSciTech 2011, 2: 59 – 66. 21.WassermanWW,SandelinA: Appliedbioinformaticsfortheidentification ofregulatoryelements. NatRevGenet 2004, 5: 276 – 287. 22.Portales-CasamarE,ThongjueaS,KwonAT,ArenillasD,ZhaoX, etal : JASPAR2010:thegreatlyexpandedopen-accessdatabaseof transcriptionfactorbindingprofiles. NuclAcidsRes 2010, 38: D105 – D110. 23.WingenderE: TheTRANSFACprojectasanexampleofframework technologythatsupportstheanalysisofgenomicregulation. Brief Bioinform 2008,9: 326 – 332. 24.Gardiner-GardenM,FrommerM: CpGislandsinvertebrategenomes. JMolBiol 1987, 196: 261 – 282. 25.HouY,BickhartDM,HvindenML,LiC,SongJ,BoichardDA,FritzS,Eggen A,DeniseS,WiggansGR,SonstegardTS,VanTassellCP,LiuGE: Fine mappingofcopynumbervariationsontwocattlegenomeassemblies usinghighdensitySNParray. BMCGenomics 2012, 13: 376.Ozawa etal.BMCDevelopmentalBiology 2012, 12 :33 Page11of13 http://www.biomedcentral.com/1471-213X/12/33

PAGE 12

26.SmithCS,BergDK,BergM,PfefferPL: Nucleartransfer-specificdefectsare notapparentduringthesecondweekofembryogenesisincattle. Cell Reprogram 2010, 12: 699 – 707. 27.OzawaM,SakataniM,HankowskiKE,TeradaN,DobbsKB,HansenPJ: Importanceofcultureconditionsduringthemorula-to-blastocystperiod oncapacityofinnercell-masscellsofbovineblastocystsfor establishmentofself-renewingpluripotentcells. Theriogenology 2012, 78: 1243 – 1251. 28.GoossensK,VanPouckeM,VanSoomA,VandesompeleJ,VanZeverenA, PeelmanLJ: Selectionofreferencegenesforquantitativereal-timePCR inbovinepreimplantationembryos. BMCDevBiol 2005, 5: 27. 29.HamataniT,DaikokuT,WangH,MatsumotoH,CarterMG,KoMS,DeySK: Globalgeneexpressionanalysisidentifiesmolecularpathways distinguishingblastocystdormancyandactivation. ProcNatlAcadSciUSA 2004, 101: 10326 – 10331. 30.SaitouM,YabutaY,KurimotoK: Single-cellcDNAhigh-density oligonucleotidemicroarrayanalysis:detectionofindividualcelltypes andpropertiesincomplexbiologicalprocesses. ReprodBiomedOnline 2008, 16: 26 – 40. 31.TesarPJ,ChenowethJG,BrookFA,DaviesTJ,EvansEP, etal : Newcelllines frommouseepiblastsharedefiningfeatureswithhumanembryonic stemcells. Nature 2007, 448: 196 – 199. 32.ChouY-F,ChenH-H,EijpeM,YabuuchiA,ChenowethJG, etal : Thegrowth factorenvironmentdefinesdistinctpluripotentgroundstatesinnovel blastocyst-derivedstemcells. Cell 2008, 135: 449 – 461. 33.AdjayeJ,HuntrissJ,HerwigR,BenKahlaA,BrinkTC, etal : Primary differentiationinthehumanblastocyst:comparativemolecularportraitsof innercellmassandtrophectodermcells. StemCells 2005, 23: 1514 – 1525. 34.EdwardsRG,HansisC: Initialdifferentiationofblastomeresin4-cell humanembryosanditssignificanceforearlyembryogenesisand implantation. ReprodBiomedOnline 2005, 11: 206 – 218. 35.KimberSJ,SneddonS,BloorDJ,El-BaregAM,HawkheadJA, etal : Expressionofgenesinvolvedinearlycellfatedecisionsinhuman embryosandtheirregulationbygrowthfactors. Reproduction 2008, 135: 635 –647. 36.CauffmanG,DeRyckeM,SermonK,LiebaersI,VandeVeldeH: Markers thatdefinestemnessinESCareunabletoidentifythetotipotentcellsin humanpreimplantationembryos. HumReprod 2009, 24: 63 – 70. 37.ReijoPeraRA,DeJongeC,BossertN,YaoM,HwaYangJY,AsadiNB,Wong W,WongC,FirpoMT: Geneexpressionprofilesofhumaninnercellmass cellsandembryonicstemcells. Differentiation 2009, 78: 18 – 23. 38.GalanA,SimonC: Monitoringstemnessinlong-termhESCculturesby real-timePCR. MethodsMolBiol 2010, 584: 135 – 150. 39.RobertsRM,ChenY,EzashiT,WalkerAM: Interferonsandthematernalconceptusdialoginmammals. SeminCellDevBiol 2008, 19: 170 – 177. 40.TeluguBP,WalkerAM,GreenJA: Characterizationofthebovine pregnancy-associatedglycoproteingenefamily – analysisofgene sequences,regulatoryregionswithinthepromoterandexpressionof selectedgenes. BMCGenom 2009, 10: 185. 41.ChakrabartyA,2ndMacLeanJA,HughesAL,RobertsRM,GreenJA: Rapid evolutionofthetrophoblastkunitzdomainproteins(TKDPs)-a multigenefamilyinruminantungulates. JMolEvol 2006, 63: 274 – 282. 42.FujiiT,MoriyasuS,HirayamaH,HashizumeT,SawaiK: Aberrantexpression patternsofgenesinvolvedinsegregationofinnercellmassand trophectodermlineagesinbovineembryosderivedfromsomaticcell nucleartransfer. CellReprogram 2010, 12: 617 – 625. 43.DriverAM,PeagaricanoF,HuangW,AhmadKR,HackbartKS,WiltbankMC, KhatibH: RNA-Seqanalysisuncoverstranscriptomicvariationsbetween morphologicallysimilarinvivo-andinvitro-derivedbovineblastocysts. BMCGenomics 2012, 13: 118. 44.GiritharanG,DellePianeL,DonjacourA,EstebanFJ,HorcajadasJA,Maltepe E,RinaudoP: Invitrocultureofmouseembryosreducesdifferentialgene expressionbetweeninnercellmassandtrophectoderm. ReprodSci 2012, 19: 243 – 252. 45.SmithAG,NicholsJ,RobertsonM,PathjenPD: Differentiationinhibiting activity(DIA/LIF)andmousedevelopment. DevBiol 1992, 151: 339 – 351. 46.CaoS,WangF,ChenZ,LiuZ,MeiC, etal : Isolationandcultureofprimary bovineembryonicstemcellcoloniesbyanovelmethod.JExpZoolA EcolGenetPhysiol 2009, 311: 368 – 376. 47.MeilhacSM,AdamsRJ,MorrisSA,DanckaertA,LeGarrecJF,ZernickaGoetzM: Activecellmovementscoupledtopositionalinductionare involvedinlineagesegregationinthemouseblastocyst. DevBiol 2009, 331: 210 – 221. 48.MorrisSA,TeoRT,LiH,RobsonP,GloverDM,Zernicka-GoetzM: Originand formationofthefirsttwodistinctcelltypesoftheinnercellmassinthe mouseembryo. ProcNatlAcadSciUSA 2010, 107: 6364 – 6369. 49.YamanakaY,LannerF,RossantJ: FGFsignal-dependentsegregationof primitiveendodermandepiblastinthemouseblastocyst. Development 2010, 137: 715 – 724. 50.PeartonDJ,BroadhurstR,DonnisonM,PfefferPL: Elf5 regulationinthe trophectoderm. DevBiol 2011, 360: 343 – 350. 51.VasioukhinV,BowersE,BauerC,DegensteinL,FuchsE: Desmoplakinisessentialinepidermalsheetformation. NatCell Biol 2011, 3: 1076 – 1085. 52.MorrisSM,TallquistMD,RockCO,CooperJA: DualrolesfortheDab2 adaptorproteininembryonicdevelopmentandkidneytransport. EMBO J 2002, 21: 1555 – 1564. 53.FlemingTP,WarrenPD,ChisholmJC,JohnsonMH: Trophectodermal processesregulatetheexpressionoftotipotencywithintheinnercell massofthemouseexpandingblastocyst. JEmbryolExpMorphol 1984, 84: 63 – 90. 54.AbeH,OtoiT,TachikawaS,YamashitaS,SatohT,HoshiH: Finestructureof bovinemorulaeandblastocystsinvivoandinvitro. AnatEmbryol 1999, 199: 519 – 527. 55.AbeH,YamashitaS,ItohT,SatohT,HoshiH: Ultrastructureofbovine embryosdevelopedfrominvitro-maturedand-fertilizedoocytes: comparativemorphologicalevaluationofembryosculturedeitherin serum-freemediumorinserum-supplementedmedium. MolReprodDev 1999, 53: 325 – 335. 56.BazerFW,BurghardtRC,JohnsonGA,SpencerTE,WuG: Interferonsand progesteroneforestablishmentandmaintenanceofpregnancy: interactionsamongnovelcellsignalingpathways. ReprodBiol 2008, 8: 179 –211. 57.deMoraesAA,DavidsonJA,FlemingJG,BazerFW,EdwardsJL, etal : Lack ofeffectofgranulocyte-macrophagecolony-stimulatingfactoron secretionofinterferon,otherproteins,andprostaglandinE2bythe bovineandovineconceptus. DomestAnimEndocrinol 1997, 14: 193 – 197. 58.GreenJA,XieS,RobertsRM: Pepsin-relatedmoleculessecretedby trophoblast. RevReprod 1998, 3: 62 – 69. 59.SeshagiriPB,SenRoyS,SireeshaG,RaoRP: Cellularandmolecular regulationofmammalianblastocysthatching. JReprodImmunol 2009, 83: 79 – 84. 60.BlombergL,HashizumeK,ViebahnC: Blastocystelongation,trophoblastic differentiation,andembryonicpatternformation. Reproduction 2008, 135: 181 – 195. 61.GadsbyJE,HeapRB,BurtonRD: Oestrogenproductionbyblastocyst andearlyembryonictissueofvariousspecies. JReprodFertil 1980, 60: 409 – 417. 62.BetteridgeKJ,FlechonJ-E: Theanatomyandphysiologyofpreattachmentbovineembryos. Theriogenology 1988, 29: 155 – 187. 63.KingGJ,AtkinsonBA,RobertsonHA: Developmentofthe intercaruncularareasduringearlygestationandestablishmentofthe bovineplacenta. JReprodFertil 1981, 61: 469 – 474. 64.BalboulaAZ,YamanakaK,SakataniM,HegabAO,ZaabelSM,TakahashiM: IntracellularcathepsinBactivityisinverselycorrelatedwiththequality anddevelopmentalcompetenceofbovinepreimplantationembryos. MolReprodDev 2010, 77: 1031 – 1039. 65.MarchandM,HorcajadasJA,EstebanFJ,McElroySL,FisherSJ,GiudiceLC: Transcriptomicsignatureoftrophoblastdifferentiationinahuman embryonicstemcellmodel. BiolReprod 2011, 84: 1258 – 1271. 66.MuggiaA,TeesaluT,NeriA,BlasiF,TalaricoD: Trophoblastgiantcells expressNFB2duringearlymousedevelopment. DevGenet 1999, 25: 23 – 30. 67.WilsonA,LaurentiE,TrumppA: Balancingdormantandself-renewing hematopoieticstemcells. CurrOpinGenetDev 2009,19: 461 – 368. 68.YuS,CuiK,JothiR,ZhaoDM,JingX, etal : GABPcontrolsacritical transcriptionregulatorymodulethatisessentialformaintenanceand differentiationofhematopoieticstem/progenitorcells. Blood 2011, 117: 2166 – 2178. 69.HromasR,DavisB,RauscherFJ3rd,KlemszM,TenenD, etal : Hematopoietictranscriptionalregulationbythemyeloidzincfinger gene,MZF-1. CurrTopMicrobiolImmunol 1996, 211: 159 – 164.Ozawa etal.BMCDevelopmentalBiology 2012, 12 :33 Page12of13 http://www.biomedcentral.com/1471-213X/12/33

PAGE 13

70.Galan-CaridadJM,HarelS,ArenzanaTL,HouZE,DoetschFK, etal : Zfx controlstheself-renewalofembryonicandhematopoieticstemcells. Cell 2007, 129: 345 – 357. 71.KinoshitaK,UraH,AkagiT,UsudaM,KoideH,YokotaT: GABP regulates Oct-3/4expressioninmouseembryonicstemcells. BiochemBiophysRes Commun 2007, 353: 686 – 691. 72.PappB,PlathK: Reprogrammingtopluripotency:stepwiseresettingof theepigeneticlandscape. CellRes 2011, 21: 486 – 501. 73.LiM,KellemsRE: Sp1andSp3Areimportantregulatorsof AP 2 gene transcription. BiolReprod 2003, 69: 1220 – 1230. 74.TakedaT,SakataM,IsobeA,YamamotoT,NishimotoF, etal : Involvement ofSp-1intheregulationoftheId-1geneduringtrophoblastcell differentiation. Placenta 2007, 28: 192 – 198. 75.DegrelleSA,MurthiP,Evain-BrionD,FournierT,HueI: Expressionand localizationofDLX3,PPARGandSP1inbovinetrophoblastduring binucleatedcelldifferentiation. Placenta 2011, 32: 917 – 920. 76.NiemannH,CarnwathJW,HerrmannD,WieczorekG,LemmeE, etal: DNA methylationpatternsreflectepigeneticreprogramminginbovine embryos. CellReprogram 2010, 12: 33 – 42. 77.JurkowskaRZ,JurkowskiTP,JeltschA: Structureandfunctionof mammalianDNAmethyltransferases. ChemBioChem 2011, 12: 206 – 222. 78.DeanW,SantosF,StojkovicM,ZakhartchenkoV,WalterJ, etal : Conservationofmethylationreprogramminginmammalian development:aberrantreprogramminginclonedembryos. ProcNatl AcadSciUSA 2011, 98: 13734 – 13738. 79.HouJ,LiuL,LeiT,CuiX,AnX,ChenY: GenomicDNAmethylation patternsinbovinepreimplantationembryosderivedfrominvitro fertilization. SciChinaCLifeSci2007, 50: 56 – 61. 80.KangYK,ParkJS,KooDB,ChoiYH,KimSU, etal : Limiteddemethylation leavesmosaic-typemethylationstatesinclonedbovinepre-implantation embryos. EMBOJ 2002, 21: 1092 – 1100. 81.RossPJ,RaginaNP,RodriguezRM,IagerAE,SiripattarapravatK, etal : PolycombgeneexpressionandhistoneH3lysine27trimethylation changesduringbovinepreimplantationdevelopment. Reproduction 2008, 136: 777 – 785. 82.TsukadaY,FangJ,Erdjument-BromageH,WarrenME,BorchersCH, etal : HistonedemethylationbyafamilyofJmjCdomain-containingproteins. Nature 2006, 439: 811 – 816. 83.FrescasD,GuardavaccaroD,BassermannF,Koyama-NasuR,PaganoM: JHDM1B/FBXL10isanucleolarproteinthatrepressestranscriptionof ribosomalRNAgenes. Nature 2007, 450: 309 – 313.doi:10.1186/1471-213X-12-33 Citethisarticleas: Ozawa etal. : Globalgeneexpressionoftheinnercell massandtrophectodermofthebovineblastocyst. BMCDevelopmental Biology 2012 12 :33. Submit your next manuscript to BioMed Central and take full advantage of: € Convenient online submission € Thorough peer review € No space constraints or color “gure charges € Immediate publication on acceptance € Inclusion in PubMed, CAS, Scopus and Google Scholar € Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Ozawa etal.BMCDevelopmentalBiology 2012, 12 :33 Page13of13 http://www.biomedcentral.com/1471-213X/12/33