An Oct4-Sall4-Nanog network controls developmental progression in the pre-implantation mouse embryo.

2.4 MB·English

by  Tan, Meng How

MolecularSystemsBiology9;Articlenumber632;doi:10.1038/msb.2012.65
Citation:MolecularSystemsBiology9:632
&2013EMBOandMacmillanPublishersLimited Allrightsreserved1744-4292/13
www.molecularsystemsbiology.com
An Oct4-Sall4-Nanog network controls developmental
progression in the pre-implantation mouse embryo
MengHowTan1,4,7,KinFaiAu2,7,DeniseELeong1,KiraFoygel1,5,WingHWong2,3,*andMyleneWMYao1,6,*
1 DepartmentofObstetricsandGynecology,StanfordUniversitySchoolofMedicine,Stanford,CA,USA,2 DepartmentofStatistics,SchoolofHumanitiesand
Sciences,StanfordUniversity,Stanford,CA,USAand3 DepartmentofHealthResearchandPolicy,StanfordUniversitySchoolofMedicine,Stanford,CA,USA
4Presentaddress:DepartmentofGenetics,StanfordUniversitySchoolofMedicine,Stanford,CA94305,USA
5Presentaddress:DepartmentofRadiology,MolecularImagingProgram,StanfordUniversitySchoolofMedicine,Stanford,CA94305,USA
6Presentaddress:UnivfyInc.,LosAltos,CA94022,USA
7Theseauthorscontributedequallytothiswork
* Correspondingauthors.WHWong,DepartmentofStatistics,SchoolofHumanitiesandSciences,StanfordUniversity,390SerraMall,Stanford,CA94305,USA.
Tel.:[phone];Fax:[phone];E-mail:[email protected],DepartmentofObstetricsandGynecology,StanfordUniversitySchoolof
Medicine,Stanford,CA94305,USA.Tel.:[phone];Fax:[phone];E-mail:[email protected]
Received28.7.11;accepted30.11.12
Landmark events occur in a coordinated manner during pre-implantation development of the
mammalian embryo, yet the regulatory network that orchestrates these events remains largely
unknown.Here, wepresent the first systematic investigation of the network in pre-implantation
mouseembryosusingmorpholino-mediated gene knockdowns ofkeyembryonicstemcell(ESC)
factors followed by detailed transcriptome analysis of pooled embryos, single embryos, and
individualblastomeres.WedelineatedtheregulonsofOct4,Sall4,andNanogandidentifiedasetof
metabolism- and transport-related genes that were controlled by these transcription factors in
embryosbutnotinESCs.Strikingly,theknockdownembryosarrestedatarangeofdevelopmental
stages.WeprovidedevidencethattheDNAmethyltransferaseDnmt3bhasaroleindeterminingthe
extenttowhichaknockdownembryocandevelop.Wefurthershowedthatthefeed-forwardloop
comprisingDnmt3b,thepluripotencyfactors,andthemiR-290-295clusterexemplifiesanetwork
motifthatbuffersembryosagainstgeneexpressionnoise.OurfindingsindicatethatOct4,Sall4,and
Nanogformarobustandintegratednetworktogovernmammalianpre-implantationdevelopment.
MolecularSystemsBiology9:632;publishedonline8January2013;doi:10.1038/msb.2012.65
SubjectCategories:simulationanddataanalysis;development
Keywords: pluripotencyfactors;pre-implantationdevelopment;transcriptionalnetworks
Introduction indefinitely in culture. The transcription factor Oct4, also
known as Pou5f1, has been shown to have a key role in the
Manycriticalcellulareventsoccurwithinafewdaysduringthe
maintenance ofan undifferentiatedstate in bothhuman and
pre-implantationdevelopmentofamammalianembryo.First,
mouseESCs.Chromatinimmunoprecipitation(chIP)assaysin
the paternal and maternal genomes are reprogrammed to a
ESCsrevealthatOct4bindstomorethanathousandlociinthe
totipotent state. Second, the embryonic genome is activated
genome, suggesting that it may regulate many genes as a
afteraperiodofrelianceonmaternalfactors.Third,polarityis
guardian of pluripotency (Boyer et al, 2005; Loh et al, 2006;
established when the inner and outer cells of a multicell
Chenetal,2008a).Inaddition,otherkeytranscriptionfactors
embryo are biased toward different cell fates. Finally, two
that control the self-renewing and pluripotent properties of
importantcellfatedecisionsoccurbeforeimplantation.Inthe
ESCsincludeSall4andNanog.Previousstudiesindicatethat
first cell fate decision, the outside cells develop into extra-
Oct4,Sall4,andNanogshareaclosefunctional relationship.
embryonic trophectoderm, while cells that are inside the
embryoretainpluripotencyandformtheinnercellmass(ICM). Co-immunoprecipitationexperimentsshowthattheyinteract
Inthesecondcellfatedecision,someoftheICMcellsfurther with one another in vivo (Wu et al, 2006; Liang et al, 2008;
differentiateintotheprimitiveendoderm(Zernicka-Goetzetal, Yangetal,2008).Furthermore,thethreetranscriptionfactors
2009). Given their complexities, all of these early cellular co-occupy the promoters of many target genes (Boyer et al,
eventshavetobecarefullyorchestratedbya generegulatory 2005; Wu et al, 2006; Yang et al, 2008), indicating that they
networktoensuretheproperdevelopmentoftheembryo. formanintegratednetworkinESCs.
Thegeneregulatorynetworkofembryonicstemcells(ESCs) Compared with ESCs, much less is known about the gene
hasbeenextensivelyinvestigatedinrecentyears.ESCs,which regulatorynetworkinearlymammalianembryoslargelydue
are derived from the ICM of mammalian embryos, are to technical challenges. In particular, limited number of
pluripotentlikecellsintheICMbuttheycanalsoself-renew embryos or cells has so far precluded extensive studies of
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any regulatory network operating during pre-implantation multiple one-cell embryos (Supplementary Figure S1). Hence,
development. For example, chIP experiments to map tran- wechosetofocusonthethreetranscriptionfactorsinthisstudy.
scriptionfactorbindingsitesinpre-implantationembryosare TodetermineifOct4,Sall4,orNanog playedanimportant
currentlyunfeasible.Fortunately,advancesingeneexpression roleduringpre-implantationdevelopmentofthemammalian
profilingarenowallowingstudiesintosmallnumberofcellsor embryo, we depleted each of the proteins by injecting
even single cells. Several laboratories used microarrays to translation-blocking morpholinos (MOs) (Supplementary
profile the transcriptome during pre-implantation develop- Figure S2), which targeted both maternal and embryonic
ment of mouse embryos (Hamatani et al, 2004; Wang et al, transcripts, into one-cell fertilized zygotes and then cultured
2004;Zengetal,2004;Xieetal,2010).Expressionanalysisof theinjectedembryostogetherwithcontrolembryosinserum
48 genes in individual cells from the one-cell zygote to the supplementedHumanTubalFluid(SAGEIn-VitroFertilization
blastocyst identified markers of outer and inner cells (Guo Inc.). For each MO injection, we confirmed that the protein
etal,2010).Inaddition,next-generationsequencingtechnol- wasabsentbyimmunocytochemistry(Figure1A).After4days
ogy was used to investigate the pre-implantation transcrip- ofinvitroculture,97.7%ofuninjectedembryosand85.1%of
tome in single mouse blastomeres (Tang et al, 2011). While control MO-injected embryos developed at least until the
thesepreviousstudieswereimportantforunderstandingearly morula stage, but surprisingly, o25% of embryos that were
mammaliandevelopment,theyreliedprimarilyontranscrip- depletedofanyofthethreepluripotencyfactorscompacted.In
tome profiling of wild-type embryos and did not perform addition, 40–60% of embryos injected with a MO targeting
Oct4,Sall4,orNanogarrestedbythe4-cellstageafter4daysof
perturbation experiments to dissect interactions between the
differentgenes. in vitro culture, compared with o10% for the uninjected or
Here,weshowthatthekeypluripotencyfactorsinESCsalso control MO-injected embryos (Po0.05, Student’s t-test). To
verifythatourfindingswerenotduetooff-targeteffects,we
form critical nodes in the regulatory network governing pre-
performed rescue experiments for each pluripotency factor
implantation development of the mammalian embryo. We
(Figure1BandC).Thepercentageofembryosthatarrestedby
used gene-specific knockdowns followed by genome-wide
the4-cellstagedecreasedby25.3%whenweco-injectedOct4
microarray analysis as well as extensive single-embryo and
MOwithOct4mRNA,whilethepercentagedecreaseforSall4
single-cellexpressionprofilingto deconstruct theOct4-Sall4-
Nanog transcriptional network in pre-implantation embryos. orNanogwas18.3or24.7%respectively(Po0.05,Student’s
t-test).Theseandotheradditionalrescuedata(Supplementary
Our study yielded novel and unexpected insights into the
ResultsandSupplementaryFiguresS3andS4)indicatedthat
regulatory mechanisms that control the earliest stages of
theMOswerespecificfortheirintendedtargetsandthatour
mammaliandevelopment.
observedphenotypeswereadirectconsequenceofknocking
down the pluripotency factors. Taken together, our experi-
Results ments indicate that Oct4, Sall4, and Nanog are essential for
pre-implantation development. Interestingly, these results
Oct4, Sall4, and Nanog are essential for pre-
contradicted published knockout mouse models, where
implantation development of the mouse embryo
embryosthatwerehomozygousnullforOct4,Sall4,orNanog
Since a network of transcription factors has been shown to appearedtodevelopinvivoatleastuntiltheblastocyststage,
regulate pluripotency in ESCs (Zhou et al, 2007; Chen et al, althoughtheywerestillembryoniclethal(Nicholsetal,1998;
2008a), we hypothesize that some of these factors may also Mitsuietal,2003;Sakaki-Yumotoetal,2006).Alikelyreason
haveanintegralroleincontrollingdevelopmentalprogression for the apparent discrepancy is that maternal transcripts are
of the pre-implantation embryo. We previously found that noteliminatedinknockoutmousemodels(seeSupplementary
Oct4 may play an important role at the maternal-zygotic informationformorediscussion).
transition(Foygeletal,2008).Here,wesoughttoundertakea
morecomprehensivestudyofthefunctionsofkeyESCfactors
in pre-implantation embryos. We first measured the abun-
Global transcriptome analysis identifies genes
dance of several pluripotency factors in individual one-cell
dependent on Oct4, Sall4, and Nanog
zygotesusing amicrofluidicreal-time PCRsystem known as
Biomark (Fluidigm). While the Kru¨ppel-like factor Klf5 was Tounderstandthemolecularmechanismsthatmightcausethe
absent,wedetectedthetranscriptsofOct4,Sall4,andNanogin embryostoarrestduringknockdownofapluripotencyfactor,
Figure1 MO-mediatedknockdownofOct4,Sall4,orNanoginpre-implantationmouseembryoswaseffectiveandspecificandresultedindevelopmentalarrest.
(A)Confocalmicroscopyimagesofembryosstainedwitha-Oct4(leftpanel),a-Sall4(middlepanel),ora-Nanog(rightpanel)antibodies.Theembryoswerestainedfor
theprotein-of-interest2daysaftermicroinjection.Oct4andNanogproteinswerecompletelyundetectablebyimmunostainingin100%oftheembryosinjectedwiththe
correspondingMO,whiletheuninjectedembryosandthecontrolMO-injectedembryoswerestronglystained.Inaddition,whileasmallamountofSall4proteinwas
detectedinembryosinjectedwithaSall4MO,theproteinlevelintheseembryoswassignificantlylessthanthatinallthecontrolembryos.(Scalebar¼45mm).
(B)Representativeimagesofpre-implantationembryosundervariousexperimentalconditions.Foreverysetofrescueexperiments,therewerefourconditions:(i)no
injection,(ii)injectionwithacontrolMO,(iii)injectionwiththeexperimentalMOalone,and(iv)co-injectionwiththeexperimentalMOandthecorrespondingmRNAthat
wasinvitrotranscribed.(C)Percentagesofembryosthatarrestedbythe4-cellstageduring4daysofinvitroculture.Leftpanel(greenbars):comparedwithembryos
injectedwiththeOct4MOonly,weobservedthatco-injecting0.6mMOct4MOwith0.20–39ng/mlOct4mRNAresultedinasmallerpercentageofembryosarrestingby
the4-cellstage(9independentexperiments)(Po0.05,Student’st-test).Middlepanel(bluebars):comparedwithembryosinjectedwiththeSall4MOalone,wefound
thatco-injecting0.6mMSall4MOwith7–146ng/mlSall4mRNAloweredtherateofdevelopmentalarrestbythe4-cellstage(8independentexperiments)(Po0.05,
Student’st-test).Rightpanel(redbars):comparedwithembryosinjectedwiththeNanogMOalone,wediscoveredthatco-injecting0.6mMNanogMOwith5ng/ml
NanogmRNAalsoresultedinpartialrescueofthephenotype(7independentexperiments)(Po0.05,Student’st-test).
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we investigated transcriptome changes in pooled embryos theuninjectedembryosareatthemulticellstage.Comparison
using Affymetrix microarrays at B20 and 44h after MO oftheearliertimepointwiththelatertimepointrevealedthat
injection (Supplementary Files S1–S5). At 20h, uninjected depletionofapluripotencyfactorreducedtheextentofzygotic
controlembryosareatthe2-cellstage,whileat44h,almostall genome activation (see Supplementary information and
A
DNA Oct4 Overlay DNA Sall4 Overlay DNA Nanog Overlay
d d d
e e e
ct ct ct
e e e
nj nj nj
ni ni ni
U U U
O O O
M M M
ol ol ol
ntr ntr ntr
o o o
C C C
Oct4 MO Sall4 MO Nanog MO
B
Uninjected Control MO Oct4 MO Oct4 MO+Oct4 mRNA
Uninjected Control MO Sall4 MO Sall4 MO+Sall4 mRNA
Uninjected Control MO Nanog MO Nanog MO+Nanog mRNA
C
100 100 100
90 90 90
e
g 80 80 80
a
st 70 70 70
cell 60 60 60
y 4- 50 50 50
st b 40 40 40
e 30 30 30
Arr 20 20 20
%
10 10 10
0 0 0
d O O +A d O O +A d O O +A
Uninjecte Control M Oct4 M Oct4 MOOct4 mRN Uninjecte Control M Sall4 M Sall4 MOSall4 mRN Uninjecte Control M Nanog M Nanog MOanog mRN
N
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Supplementary Figure S5). We also found that MO-mediated that were downregulated during Sall4 knockdown were also
knockdownofOct4hadthemostprominenteffect,compared downregulated during Oct4 knockdown and 133 out of 245
withknockdownsofSall4andNanog.Atthe20-htimepoint, genes that were downregulated during Nanog knockdown
a total of 634 genes were differentially expressed in Oct4 werealsodownregulatedduringOct4knockdown,indicating
MO-injected embryos, while o10 genes were differentially that the three transcription factors shared a close functional
expressedinSall4MO-andNanogMO-injectedembryos(false relationshipduringpre-implantationembryonicdevelopment
discovery rate (FDR)o0.1) (Figure 2A) (see Materials and (Po10(cid:2)100,chi-squaretest).
methods fordetails). However, at 44hpost injection, a clear We asked which of the differentially expressed genes
effect on global expression levels was observed for all three are directly controlled by Oct4, Sall4, or Nanog. Since
transcriptionfactors(Figure2B).Notably,505outof799genes chIP experiments could not be readily performed on
A B
Sall4 Nanog Sall4 Nanog Sall4 Nanog Sall4 Nanog
2 0 0 1 0 4 286 8 104 200 20 244
0 0 54 53
0 0 0 1 451 79 230 130
456 177 1476 1125
Oct4 Oct4 Oct4 Oct4
Downregulated Upregulated Downregulated Upregulated
C D
2 2
Vesicle-mediated transport 148 (76)
Endocytosis 78 (38) 1 1
Lysosomal transport 13 (2)
Exocytosis 55 (25) 0 0
5 10 15
Phosphate metabolism 34 (15)
RNA localization 16 (5) EDownregulated (embryos and ESCs):
Localization 20 (7) 2 2
Lipid metabolism 116 (80)
Coenzyme metabolism 18 (7) 1 1
Carbohydrate metabolism 90 (66)
0 0
Nitrogen compound metabolism 12 (5) 5 10
Mitochondrion organization 7 (2)
Neurotransmitter secretion 35 (21) Upregulated (embryos):
2 2
Oxygen species metabolism 11 (4)
Peroxisomal transport 6 (2) 1 1
0 2 4 6 8 10 12 14
0 0
–log10 (P-value) 5 10
Ion transport 62 (35) Upregulated (ESCs):
Cation transport 52 (29) 2 2
Carbohydrate metabolism 74 (47) 1 1
Meiosis 27 (13)
Vesicle-mediated transport 81 (54) 0 0
Phosphate metabolism 20 (11) 5 10
0 2 4 6 8 10 12 14 Core motif:
–log (P-value)
10
Figure2 Microarrayanalysisofpooledembryosidentifiedtheprotein-codingregulonsofOct4,Sall4,andNanog.(A)Venndiagramshowingthenumberofgenesthat
weredifferentiallyexpressed20hafterMOinjectiontoknockdownoneofthethreepluripotencyfactors.Atthisearliertimepoint,onlyknockdownofOct4exerteda
strongeffectonglobalgeneexpressionlevels.(B)Venndiagramshowingthenumberofgenesthatwasdifferentiallyexpressed44hafterMOinjection.Atthislater
timepoint,knockdownofanyofthethreetranscriptionfactorscausedhundredsofgenestobesignificantlydownregulatedorupregulated(FDRo0.1).(C)GOanalysis
ofgenesthatweredifferentiallyexpressedduringknockdownofOct4,Sall4,orNanoginpre-implantationembryosbutshowednoevidenceofbeingcontrolledbythe
pluripotencyfactorsinESCs.Thesegenesmaybenoveldirecttargetsofthethreetranscriptionfactorsintheembryosortheymayalsobeindirectlyregulated.Upper
panel:GOanalysisoftheembryo-specificgenesthatweredownregulated.Lowerpanel:GOanalysisoftheembryo-specificgenesthatwereupregulated.Allthe
biologicalprocessesthatareuniquetotheembryo(Po0.01)areplotted.Theactualnumbersofgenesforeachprocess(withtheexpectednumbersgiveninbrackets)
areshownattheendofthebars.Manyofthefunctionalcategoriesarerelatedtoeithertransport(inred)ormetabolism(inblue).(D)A15-bpmotifwithinOct4-bound
regulatoryregionsofgenesthatwerealsodifferentiallyexpressedduringknockdownofOct4inpre-implantationembryosorESCs.(E)A7-bpcoremotifwasfoundtobe
enrichedinNanog-boundregulatoryregionsofgenesthatwerealsodifferentiallyexpressedduringknockdownofNanoginpre-implantationembryosorESCs.Thecore
motifwasextendedatthe50endforgenesthatweredownregulatedduringNanogknockdownanditwasextendedatthe30endforgenesthatwereupregulatedduring
Nanogknockdown.
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pre-implantation embryos, we compared our microarray majorrolesinpre-implantationdevelopmentalprocesses.We
resultswithpubliclyavailablechIPdatasetsinESCsinstead thereforequantifiedmiRNAexpressionfromthe1-celltothe
(Boyeretal,2005;Lohetal,2006;Limetal,2008;Yangetal, blastocyst stage in uninjected mouse embryos and detected
2008; Chen et al, 2008a; Ouyang et al, 2009). 20.4% of the 297 distinct miRNAs in at least one of these stages
downregulated genes and 18.8% of the upregulated genes (SupplementaryFileS10).K-meansclusteringfurtherrevealed
have promoters or enhancers that are bound by the corre- that many of the miRNAs were temporally regulated over
sponding transcription factor (Supplementary Figure S6). development(Figure3A).
GeneOntology(GO)analysisusingthePantherclassification WehypothesizethatsomeofthehighlyexpressedmiRNAs
system(Thomaset al,2003)furtherrevealed thatthe down- are integral components of the Oct4-Sall4-Nanog gene
regulated genes are involved in the establishment or main- regulatory network in the pre-implantation embryo. To
tenance of chromatin architecture, while the upregulated determine which miRNAs are regulated by Oct4, Sall4, or
genes are involved in stress response and differentiation or Nanog,weknockeddowneachtranscriptionfactorseparately
developmental processes. Manyof the directtargetsarealso andmeasuredmiRNAexpressionlevels46haftermicroinjec-
involvedinthecellcycleorarecomponentsofkeysignaling tionwhentheknockdownphenotypeswerereadilyobserva-
pathways(SupplementaryFigureS7;SupplementaryFileS6). ble. More miRNAs were downregulated than upregulated
SinceESCsdonotexistinanaturaldevelopmentalcontext, (Figure 3B; Supplementary File S11), indicating that Oct4,
thepluripotencyfactorsmaycontroladditionalgenesinpre- Sall4, and Nanog served primarily as positive regulators of
implantation embryos but not in ESCs. To determine the miRNAexpressioninpre-implantationembryos.Inaddition,
embryo-specific genes, we subtracted away from our micro- mostofthedownregulatedmiRNAsweresignificant(Po0.05)
arrayresultsgenesthatwerepresentintheESCchIPdatasets inatleasttwodifferentknockdownconditions,suggestingthat
as well as genes that responded to Oct4, Sall4, or Nanog the expression of many miRNAs was under combinatorial
depletion in ESCs (Supplementary Figure S6). GO analysis control by the transcription factors(Figure 3B). In total, 120
revealed that many of these embryo-specific genes were distinct miRNAs were differentially expressed. 40 of them
associated with metabolism- and transport-related functions (33.3%) have promoters or enhancers that are occupied by
(Figure2C;SupplementaryFileS7),whichreflectedtheunique Oct4,Sall4,orNanoginESCs(Limetal,2008;Marsonetal,
embryonicconditionsthatareabsentfromESCs. 2008). The remaining 80 miRNA genes may be regulated
Toidentifycis-actingelementsthatmediatethedirecttargeting indirectly by the three transcription factors or they may
of Oct4, Sall4, or Nanog to the genome in pre-implantation represent novel direct targets of Oct4, Sall4, or Nanog in the
embryos,wesearchedtheboundlociforknownmotifsandalso pre-implantationembryo.
performed de novo motif discovery using CisGenome (Ji et al, Next,wesoughttounderstandhowthepluripotencyfactors
2008). The Oct4-Sox2 consensus sequence was present in the and their protein-coding and non-coding regulons are inter-
promoterorenhancerregionsof83downregulatedgenes(outof connected. 61.1% of the protein-coding genes (2646 out of
284 genes) and 35 upregulated genes (out of 153 genes) 4329 genes) thatweredifferentiallyexpressedduringknock-
(Figure 2D; Supplementary File S8). In addition, we obtained downofOct4,Sall4,orNanogwerepredictedbythemiRanda
withhighconfidenceacoremotif,TGACCTT,thatwasenrichedin algorithm (www.microrna.org) (Betel et al, 2008) to be
the Nanog-bound promoter or enhancer regions of 25 down- targetedbyatleastonemiRNAthatwasalsoregulateddirectly
regulatedgenes(outof40genes)and31upregulatedgenes(outof orindirectlybythethreetranscriptionfactors(Supplementary
46 genes) (Supplementary File S9). Notably, the motif for FileS12).Similarresultswereobtainedwhenwerestrictedour
downregulatedgenesisextendedatthe50endbytwobasepairs, target predictions to protein-coding genes and miRNAs that
whilethemotifforupregulatedgenesisextendedatthe30 end werepresentintheESCchIPdatasets(62.7%;622outof992
by one to three basepairs (Figure 2E). A de novo search for genes) (Supplementary File S13). In comparison, when we
cis-regulatory elements using a different program, SCOPE, also randomlyselected10000distinctsetsofprotein-codinggenes
returnedthe same coremotif (Carlsonet al, 2007; Chakravarty andmiRNAs,wefoundthatonly52.8±0.7%oftheprotein-
etal,2007;SupplementaryFigureS8).Takentogether,ourresults codinggeneswerepredictedtobemiRNAtargets.Hence,our
suggestthatdifferentcofactorsmaymediateNanog’sfunctionasa analysis indicates that Oct4, Sall4, and Nanog and their
transcriptionalactivatororrepressor.Furthermore,thecoremotif protein-coding and non-coding regulons are more intercon-
is similar to previously identified motifs for the retinoic acid nectedthanrandom(Po10(cid:2)6,Z-test).Toincreaseconfidence
receptorRaraaswellastheestrogen-relatedreceptorsEsrraand inthemiRNA-targetpredictions,wefurtherdeterminedwhich
Esrrb(SupplementaryFigureS9),whichsuggeststhatNanogmay ofthemiRNAsoutputtedbymiRandawerealsopredictedbyat
co-regulatemanygenestogetherwithRaraandEsrra/Esrrb. least one other algorithm (see Materials and methods and
Supplementary Files S14 and S15). An example of a subnet-
workisillustratedinFigure3C.
MicroRNAs form an integral component of the
Oct4-Sall4-Nanog regulatory network in
pre-implantation embryos Knockdown of Oct4, Sall4, or Nanog causes gene
expression changes even in embryos that appear
MicroRNAs (miRNAs) are an important class of post-tran-
to develop normally
scriptional regulators that have diverse and critical roles
duringanimaldevelopmentandregeneration(YangandWu, Asourmicroarraydataweregeneratedfrompoolsofembryos
2007;StefaniandSlack,2008).Itislikelythattheyalsohave and the ensemble averages may have masked interesting
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A B
1-cell2-cell4-cellMulticellBlastocyst
I Sall4 Nanog Sall4 Nanog
II 9 3 15 14 3 5
22 0
III 10 8 2 2
IV
32 3
Oct4 Oct4
V
Downregulated Upregulated
Repression Induction
–1 0 1
C
Klf5 Klf2 miR-20a
Pou2f3 miR-92a
Pou5f1
miR-191
Dnmt3a
miR-709
Dnmt3b
miR-720
Jarid2 miR-805
Smarcd1 Oct4 miR-291a-3p
Sall4
Fgfr2 Nanog miR-292-3p
Fzd4
miR-293
Fzd7
miR-294
Spry4
miR-295
Dazl
Sf3b2
Lhx2
Sf1
Meis2
Neurog2 Atg2a Atg7
Figure3 Oct4,Sall4,andNanogcontroltheexpressionofnumerousdevelopmentallyregulatedmiRNAsinthepre-implantationembryo.(A)K-meansclustered
expressionprofilesfor235miRNAsthatwereclearlyexpressedinuninjected(normal)pre-implantationembryos.Genesareinrows,withtemporalprogressionfromleft
toright.Redindicateshighexpressionlevels,whilegreenindicateslowexpressionlevels.ThemajorityofthesemiRNAsshowageneralincreaseinexpressionlevels
(clusterIandclusterV)fromthe1-celltotheblastocyststage.(B)VenndiagramshowingthenumberofmiRNAsthatweredifferentiallyexpressedduringknockdownof
Oct4,Sall4,orNanog(Po0.05).MiRNAsinthemiR-290-295clusterweredifferentiallyexpressedunderallthreeknockdownconditions.(C)Anexampleofan
integratednetworkcontrolledbyOct4,Sall4,andNanoginpre-implantationembryos.Theprotein-codinggenesaredepictedinvariouscolorstosignifydifferent
functionalcategories.Darkbrown:pluripotencytranscriptionfactors;lightblue:epigeneticregulators;lightbrown:signalingmolecules;pink:differentiationfactors;
purple:embryo-specificfactors.ThelistedmiRNAsarewithinthetop20mostabundantmiRNAsintheembryos.X-Ymeans‘XregulatesY.’Forthearrowsemanating
fromthecenter,asolidarrowindicatesconfirmeddirectregulation,whileadottedarrowindicateseitherindirectregulationorpotentialdirectregulation.
embryo-to-embryo variability or important expression (Fluidigm). We focused our single embryo analysis on 48
dynamics at the single embryo level, we sought to monitor protein-coding genes, 43 of which were found from our
gene expression levels in individual embryos using BioMark microarray experiments to be differentially expressed during
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Oct4,Sall4,orNanogknockdown.The43geneswerechosen Hence, the expression of these seven genes (highlighted in
because they perform essential functions in ESCs, are key green in Figure 4C and Table I) is low in 2-cell and 3-cell
epigenetic regulators or signaling molecules, have important knockdownembryos,highin4-cellknockdownembryos,and
roles during differentiation events, or may have embryo- evenhigherinmulticellknockdownembryos.
specific functions. The five remaining genes had unchanged Since promoter DNA methylation contributes to ESC gene
expressionlevelsandservedasnegativecontrols. regulation and methylation-deficient ESCs are restricted in
In our first set of analyses, we compared between the theirdevelopmental potential (Fouseet al, 2008),we decided
various experimental conditions, namely (1) no injection, to investigate the DNA methyltransferase Dnmt3b in greater
(2)injectionwithacontrolMO,and(3)injectionwithaMO depth.KnockdownofDnmt3busingatranslation-blockingMO
targeting Oct4, Sall4, or Nanog. For each condition, we revealedamoreseverephenotypethanthatforanyofthethree
collected between 80 and 120 embryos at 44h after MO pluripotency factors. Specifically, 66.3±21.6% of embryos
injection. When all the embryos were considered in the injectedwiththeDnmt3bMOarrestedbythe2-cellstage,even
analysis, we found that our BioMark and microarray results though the percentages for Oct4, Sall4,and Nanog were only
agreed reasonably well with each other (Supplementary between5.5and19.0%(Po0.001,Student’st-test)(Figure5A).
Information, Supplementary Figure S10, and Supplementary Rescue experiments with in vitro transcribed Dnmt3b mRNA
File S16). However, since depletion of a pluripotency factor confirmed the specificity of the Dnmt3b MO (Figure 5B–D).
caused embryos to arrest at different developmental stages These results suggest that Dnmt3b is essential for pre-
(Supplementary Figure S11), some of the transcript changes implantation development and that it may have a role in
observed between experimental conditions could be due to determining the extenttowhich an embryo depleted ofOct4,
expression changes between the various stages. Hence, we Sall4,orNanogcandevelop.Inaddition,althoughDnmt3b(cid:2)/(cid:2)
next compared 4-cell and multicell control embryos with embryos had been reported to develop normally before
knockdownembryosthatwerealsoatthe4-cellandmulticell E9.5 (Okano et al, 1999b; Ueda et al, 2006), it is likely that
stages (red boxes in Figure 4A). Intriguingly, we found that maternalDnmt3b,whichwedetectedin1-cellmousezygotes
manygeneswerestillsignificantlydownregulatedorupregu- (Supplementary Figure S1), may be driving the earliest
lated (Figure 4B). In particular, at least four genes, namely developmentalstagesintheknockoutmousemodel.
Jarid2,Aqp3,Atg2a,andSf3b2,weredifferentiallyexpressed
inallthreeknockdownconditions. Hence, although someof
the knockdown embryos appeared to be morphologically
A coherent feed-forward loop controls the
normal,numerousmolecularchangeshadalreadyoccurredin
expression of Dnmt3b
them.
In ESCs, chIP experiments showed that the pluripotency
factors can control the transcription of Dnmt3b directly.
Interestingly,thefactorscanalsoregulateDnmt3bexpression
Expression of Dnmt3b distinguishes early-
indirectly via the ESC-specific miRNAs in the miR-290-295
arrested embryos from late-arrested embryos
cluster. Specifically, Oct4 and Nanog directly control the
during Oct4, Sall4, or Nanog knockdown
expressionofthesemiRNAs,whichshowsageneralupward
Strikingly, we observed that, while uninjected and control- trend from the 1-cell to the blastocyst stage (Supplementary
injected embryos were able to develop in a stereotypical Figure S12). The miRNAs then target the 30UTR of the
manner, embryos that lacked any of the three pluripotency retinoblastoma-like 2 (Rbl2) mRNA, whose protein product
factorsarrestedoverarangeofdevelopmentalstagesinstead functions as a transcriptional repressor of Dnmt3b (Benetti
(Supplementary Figure S11). At 44h after MO injection, the et al, 2008; Sinkkonen et al, 2008). In the pre-implantation
knockdownembryosweredistributedmainlyoverthe2-cell, embryo,wefoundfromourmicroarrayanalysisthatalthough
3-cell, 4-cell, and more advanced multicell stages. Hence, in Rbl2didnotpassourcutoffcriterionforsignificantgenes,its
our second set of analyses, we asked if our BioMark expression is still upregulated 1.6- to 2.9-fold during knock-
experiments could identify genes that would differentiate downof Oct4,Sall4, orNanog.Our microarrayanalysis also
early-arrestedembryos(2-celland3-cellembryos)fromlate- revealedthatDnmt3bexpressionwasdownregulatedby3-to
arrested embryos (4-cell and multicell embryos) during 19-foldwhenOct4,Sall4,orNanogwasdepleted(FDRo0.1;
knockdownofapluripotencyfactor(blueboxesinFigure4A). Supplementary Files S3 and S5). Hence, a coherent feed-
Interestingly,theexpressionofahandfulofgenescorrelates forward loop (FFL) consisting of the pluripotency factors,
with the extent to which a knockdown embryo develops. the miR-290-295 cluster, Rbl2, and Dnmt3b regulates
Figure 4C shows a heatmap of the top 10 genes that are DNA methylation in the pre-specified embryo and in ESCs
differentially expressed between early-arrested embryos and (Figure8B).
late-arrested embryos. Most of them encode DNA-binding Toobtainaquantitativeunderstandingofthefeed-forward
proteins,suchasthetranscriptionfactorTbx3,whichshares architecture, we constructed a model of non-linear ordinary
manycommontargetswithOct4andNanog(Hanetal,2010). differential equations (ODEs) based on Michaelis-Menten
Wefurthercompared4-cellknockdownembryoswithmulti- kinetics with cooperativity (Figure 6A) and performed the
cellknockdownembryos(TableI)andfoundthat7ofthe10 simulation using a Runge-Kutta algorithm. We chose the
genes that were differentially expressed between early- parameters so that the model would reproduce known
arrested embryos and late-arrested embryos were also more experimental observations. In uninjected (wild-type)
highlyexpressedinmulticellembryosthanin4-cellembryos. embryos, the pluripotency factors are present at high levels
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and activate the expression of the miR-290-295 cluster. The approximately bigger than 5, the hypersensitive response is
miRNAsthendownregulatetheproductionofRbl2.Whenthe lost and the FFL essentially behaves like the simple two
level of Rbl2 is low, Oct4, Sall4, and Nanog can then fully componentsystem.However,whentheratiobecomessmaller,
activate the expression of Dnmt3b (Figure 6B, left panel). the switch-like behavior becomes more pronounced
However, the converse is true during knockdown of the (Figure 6D; Supplementary Figure S14). In contrast, the
pluripotencyfactors(Figure6B,rightpanel).Furthermore,in steepest slope of the response curve is unaffected by the
Dicer-null ESCs, both the transcript and protein levels of parameter b (see Supplementary information and
c
Dnmt3bweresignificantlylowerthanthatinwild-typeESCs SupplementaryFigureS15).Hence,ouranalysisshowedthat
(Benettietal,2008;Sinkkonenetal,2008).Inagreementwith inordertoachieveahypersensitiveresponse,theregulationof
theseobservations,whenwesettheexpressionofthemiRNAs Dnmt3b transcription cannot be dominated by the pluripo-
to zero in our model, we found that the peak Dnmt3b tencyfactors,sothatRbl2canexertitseffect.
expression level dropped by 1.7-fold for our chosen set of A prediction from our model is that at high levels of
parameters(SupplementaryFileS17). pluripotency factors, for example in pre-specified embryos
WenextinvestigatedtheadvantagesthattheFFLconferson and in ESCs, the concentration of Dnmt3b becomes more
the pluripotency factors’ regulation of DNA methylation. susceptibletointrinsicgeneexpressionnoisewhentheFFLis
Superficially, the pathway through the miRNAs and Rbl2 broken.To test the prediction, wedetermined the cell-to-cell
appears to be unnecessary, as the pluripotency factors can variabilityofDnmt3btranscriptlevelforwild-typeESCsand
simply activate the expression of Dnmt3b. To identify for Dicer(cid:2)/(cid:2) ESCs (Calabrese et al, 2007). Specifically,
differencesbetweenthisstraightforwarddesignandtheFFL, we performed quantitative real-time PCR (RT-qPCR) experi-
wealsosimulatedthetwo-componentmodelconsistingofthe ments on fourdifferent sets of cells: (1) Dicer(cid:2)/(cid:2) ESCs,(2)
pluripotency factors and Dnmt3b only (Supplementary File Dicer(cid:2)/(cid:2) ESCstransfectedwithacontrolmiRNAmimicthat
S18). When we varied the concentration of the pluripotency hasminimal sequence identitywithmiRNAsin humansand
factors from low (0) to high (1) and examined the output mice(Dharmacon),(3)Dicer(cid:2)/(cid:2)ESCstransfectedwithamiR-
(normalized Dnmt3b expression level) of both models, we 295 mimic (Dharmacon), and (4) wild-type ESCs. To verify
foundthatthesimpletwo-componentmodelproducedamore thatthetransfectionofmiR-295wassuccessful,wemeasured
gradedoutput,whiletheFFLgaveamoreswitch-likeresponse thelevelsofitstargetgenesRbl2,p21,andCasp2(Benettietal,
(Figure 6C). Hence, the miRNA-Rbl2 pathway served to 2008; Sinkkonen et al, 2008; Wang et al, 2008; Zheng et al,
sharpen the system output so that it has a more switch-like 2011) and confirmed that they were all downregulated
behavior. (SupplementaryFigureS16).Wealsotransfectedeachofthe
Sincetheperformanceofanon-lineardynamicalsystemis four cell populations with a plasmid encoding green fluor-
dependent on the precise values of the parameters, we escentprotein(GFP),sothatwecouldsortforsinglecellsby
wondered how our results may change with different flowcytometry, and then determined the transcript levels of
parameter choices. We first found that the feed-forward Dnmt3b, Oct4, Sall4, and Nanog in individual ESCs using
network motif is robust with respect to the synthesis rate of Biomark(Fluidigm).Strikingly,Dnmt3bexpressionexhibited
the Rbl2 transcriptional repressor (see Supplementary the widest spread of Ct values in Dicer-null ESCs. The
informationandSupplementaryFigureS13).Next,westudied interquartilerangeforDicer-nullESCsandforcellstransfected
the behavior of the dynamical system when we varied the with the control miRNA was 8.94 and 3.33 respectively,
synthesisrateofDnmt3b.TheexpressionofDnmt3bisunder whiletherangeforDicer-nullESCstransfectedwithmiR-295
combinatorialregulationbythepluripotencyfactorsandRbl2. and for wild-type ESCs was 2.87 and 2.79 respectively
InourmathematicaldescriptionoftheFFL,wehavemodeled (Figure 6E, upperpanel). The standarddeviation of Dnmt3b
the influences of each transcription factor as both mutually expressionlevelwasalsolargerinDicer-nullESCsandcontrol-
independent(secondandthirdtermsontheright-handsideof transfected cells than in miR-295-transfected cells and wild-
thethirdODE)andcooperative(fourthtermofthethirdODE). type ESCs (Supplementary Figure S17A). Furthermore, these
However, the relative contributions of each regulator to the resultsarenotduetotheexpressionofthepluripotencyfactors
transcription of Dnmt3b are unknown. Thus, we varied the exhibitingdifferentdegreesofvariabilitybetweenthefoursets
ratioofthetwoparameters,b andb ,andobservedhowthe of cells (Figure 6E, lower panel, and Supplementary Figure
a b
concentrationofDnmt3brespondstochangesinthelevelof S17B). When corrected for the standard deviation of the
the pluripotency factors. We found that when b /b is pluripotencyfactors,theexpressionofDnmt3bwasstillmore
a b
Figure4 Geneexpressionanalysisofindividualembryosrevealshiddenmolecularchangesandtranscriptdifferencesbetweenearly-andlate-arrestedembryos.
(A)Aschematicdiagramshowingthedevelopmentalprogressionofuninjectedandinjectedembryos.Embryoswerecollectedat44hpost-injectionforexpression
analysis.Twomainanalyseswereperformed.First,wecomparedtheexpressionofcontrolembryosthatwereatthe4-cellandmulticellstageswiththeexpressionof
knockdownembryosthatwerealsoatthe4-cellandmulticellstages(redboxes).Second,wecomparedtheexpressionofknockdownembryosthatwereatthe2-celland
3-cellstages(early-arrested)withtheexpressionofknockdownembryosthatwereatthe4-cellandmulticellstages(late-arrested)(blueboxes).(B)Thefirstcomparison.
Althoughalltheembryosinthisanalysisweremorphologicallysimilar(4-cellandmulticellstages),manygeneswerestilldifferentiallyexpressedintheknockdown
embryos.TheindividualCtvaluesforthetop10rankinggenesofeachknockdownconditionareplottedasheatmaps.P-valuesaregiveninbrackets.Toppanel:Oct4
knockdown;middlepanel:Sall4knockdown;bottompanel:Nanogknockdown.Mostofthegenesshownaredownregulateduponknockdownofapluripotencyfactor.
Thegenesthatareupregulatedaremarkedbyanasterisk(*).Inaddition,genesthatarehighlightedinpinkarecommonamongallthethreeknockdownconditionsand
theyparticipateincriticalfunctionssuchasepigeneticgenesilencing(Pengetal,2009;Lietal,2010;Pasinietal,2010),autophagy(Tsukamotoetal,2008),and
alternativesplicing(Tangetal,2010).(C)Thesecondcomparison.TheindividualCtvaluesforthetop10rankinggenesareplottedasaheatmap,withtheP-values
giveninbrackets.Theexpressionlevelsofthegeneshighlightedingreenarealsosignificantlydifferentbetweenthe4-cellstageandthemulticellstage.
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variable in Dicer-null cells and control-transfected cells that the FFL serves to buffer Dnmt3b expression against
than in miR-295-transfected cells and wild-type ESCs intrinsic variabilityin the concentrations of the pluripotency
(Supplementary Figure S17C). Hence, our results indicate factors.
A
44 h later
Uninjected
Injected with 44 h later
control MO
Injected with 44 h later
Oct4/Sall4/Nanog
MO
B Uninjected Control MO Oct4 MO
Hat1 (P = 4.46×10–56)
Jarid2 (P = 1.35×10–55)
Aqp3 (P = 1.81×10–54)
Atg2a (P = 7.82×10–54)
Klf2 (P = 8.72×10–50)
Sf3b2 (P = 3.84×10–48)
Vps72 (P = 5.80×10–48)
Sf1 (P = 1.74×10–46)
Dnmt3b (P = 4.51×10–46)
Trim28 (P = 3.35×10–42)
Uninjected Control MO Sall4 MO
Atg2a (P = 6.47×10–46)
Sf1 (P = 2.20×10–44)
Jarid2 (P = 1.47×10–43)
Sf3b2 (P = 1.52×10–43)
Trim28 (P = 1.46×10–42)
Dnmt3a (P = 2.07×10–41)
Dppa5a (P = 3.28×10–41)
Aqp3 (P = 5.61×10–41)
Pou2f3 (P = 1.73×10–37)
*Spry4 (P = 3.93×10–35)
Uninjected Control MO Nanog MO
Sf3b2 (P = 8.23×10–25)
Smarcd1 (P = 1.10×10–20)
Dnmt3b (P = 2.15×10–20)
Jarid2 (P = 4.30×10–20)
Atg2a (P = 3.27×10–19)
Aqp3 (P = 3.41x10–18)
Hmgb1 (P = 3.84×10–18)
Klf2 (P = 1.58×10–17)
*Fzd4 (P = 1.70×10–17)
Tcfap2c (P = 1×10–17)
≤–3–2 –1 0 1 2 ≥3
Ct relative
to mean
C 2-cell and 3-cell 4-cell Multicell
Hmgb1 (P = 1.16×10–33)
Sf3b2 (P = 1.86×10–22)
Dnmt3b (P = 1.29×10–20)
Klf2 (P = 2.76×10–17)
Klf5 (P = 1.10×10–16)
Trim24 (P = 1.07×10–13)
Pou5f1 (P = 1.50×10–13)
Wwtr1 (P = 2.84×10–13)
Atg7 (P = 2.21×10–12)
Tbx3 (P = 1.28×10–11)
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Single-cell analysis reveals an increase in gene development,whileotherembryosmightexpressalowerlevel
expression noise during knockdown of Oct4 or of Dnmt3b and arrest earlier in development. In uninjected
Sall4 embryos, the pluripotency factors are present at high levels
andfluctuationsintheirconcentrationsduetointrinsicnoise
Our simulations suggest an intriguing explanation for why
do not affect the level of Dnmt3b significantly (right side of
during knockdown of a pluripotency factor, some embryos
bluegraphinFigure6C).However,whenapluripotencyfactor
mightexpressahigherlevelofDnmt3bandprogressfurtherin
has been knocked down to a low level, the gradient of the
responsecurveincreasesdramaticallyandthesystemexistsin
theswitchregimeinstead(leftsideofbluegraphinFigure6C).
Table I The top 10 genes that were differentially expressed between 4-cell In this case, even small fluctuations in the concentrations of
knockdownembryosandmulticellknockdownembryos
the pluripotency factors can affect the level of Dnmt3b
Gene P-value Foldchange appreciably.
To test the hypothesis that the expression of Dnmt3b and
Fgfr2 1.80(cid:3)10(cid:2)12 2.44
possiblymanyothergenesbecomenoisierduringknockdown
Hmgb1 2.13(cid:3)10(cid:2)12 2.20
Pou5f1 8.16(cid:3)10(cid:2)12 2.46 of a pluripotency factor, we performed single-cell real-time
Dnmt3b 1.47(cid:3)10(cid:2)11 1.91 PCRexperiments using BioMark (Fluidigm). In these experi-
Tbx3 1.52(cid:3)10(cid:2)11 2.45 ments, we focused on Oct4 and Sall4, since knockdown of
Sf3b2 6.92(cid:3)10(cid:2)8 1.48
Atg7 4.53(cid:3)10(cid:2)7 2.02 Nanog gave a milder phenotype that is consistent with the
Racgap1 6.17(cid:3)10(cid:2)7 1.49 expression of Nanog being significantly lower than that of
Klf5 8.43(cid:3)10(cid:2)7 1.64 Oct4 or Sall4 in the earliest developmental stages. For each
Dnmt3a 2.31(cid:3)10(cid:2)6 1.46
experimental condition (no injection, injection with control
MO,injectionwithOct4MO,andinjectionwithSall4MO),we
Thegeneshighlightedingreenwerealsoexpressedatsignificantlylowerlevels
in2-celland3-cellembryoswhencomparedwithlaterstageembryos. collectedembryosthatweremorphologicallyatthe4-cellstage
A C D
100 100 110
90 90 100
80 80 90
age 70 age 70 ent 80
Arrest by 2-cell st 34560000 Arrest by 2-cell st 34560000 Morula developm 45670000
% % % 30
20 20 20
10 10 10
0 0 0
O O O O d O O +A d O O +A
Oct4 M Sall4 M Nanog M Dnmt3b M Uninjecte Control M Dnmt3b M nmt3b MOmt3b mRN Uninjecte Control M Dnmt3b M nmt3b MOmt3b mRN
Dn Dn
D D
B
Uninjected Control MO Dnmt3b MO Dnmt3b MO+Dnmt3b mRNA
Figure5 TheDNAmethyltransferaseDnmt3bisessentialforpre-implantationdevelopment.(A)Percentagesofembryosthatarrestedbythe2-cellstageforfour
differentknockdownconditions.Unexpectedly,weobservedthatthemajorityofembryosinjectedwithaMOtargetingDnmt3barrestedaroundthematernal-zygotic
transition(Po0.001,Student’st-test,whencomparingDnmt3bwithanyofthepluripotencyfactors).(B)Representativemicroscopyimagesofpre-implantationmouse
embryosundervariousexperimentalconditions.(C)Quantificationofobservedphenotypesafter4daysofinvitroculture.Comparedwithembryosthatwereinjected
with0.6mMDnmt3bMOonly,co-injectionof0.6mMDnmt3bMOwith1–10ng/mlDnmt3bmRNAreducedthefractionofembryosthatarrestedbythe2-cellstage
(7independentexperiments)(Po0.05,Student’st-test).(D)Percentagesofembryosthatdevelopeduntilatleastthemorulastageafter4daysofculture.Compared
withembryosthatwereinjectedwithDnmt3bMOonly,co-injectionofDnmt3bMOwithinvitrotranscribedDnmt3bmRNAincreasedthepercentageofembryosthat
reachedthemorulastage(Po0.05,Student’st-test).
10 MolecularSystemsBiology2013 &2013EMBOandMacmillanPublishersLimited