Conservation and Diversification of an Ancestral Chordate Gene Regulatory Network for ...

21 Pages·2011·4.46 MB·English

Conservation and Diversification of an Ancestral
Chordate Gene Regulatory Network for Dorsoventral
Patterning
Iryna Kozmikova, Jana Smolikova, Cestmir Vlcek, Zbynek Kozmik*
InstituteofMolecularGenetics,Prague,CzechRepublic
Abstract
Formationofadorsoventralaxisisakeyeventintheearlydevelopmentofmostanimalembryos.Itiswellestablishedthat
bone morphogenetic proteins (Bmps) and Wnts are key mediators of dorsoventral patterning in vertebrates. In the
cephalochordate amphioxus, genes encoding Bmps and transcription factors downstream of Bmp signaling such as Vent
areexpressedinpatternsreminiscentofthoseoftheirvertebrateorthologues.However,thekeyquestioniswhetherthe
conservationofexpressionpatternsofnetworkconstituentsimpliesconservationoffunctionalnetworkinteractions,andif
so, how an increased functional complexity can evolve. Using heterologous systems, namely by reporter gene assays in
mammalian cell lines and by transgenesis in medaka fish, we have compared the gene regulatory network implicated in
dorsoventral patterning of the basal chordate amphioxus and vertebrates. We found that Bmp but not canonical Wnt
signaling regulates promoters of genes encoding homeodomain proteins AmphiVent1 and AmphiVent2. Furthermore,
AmphiVent1andAmphiVent2promotersappeartobecorrectlyregulatedinthecontextofavertebrateembryo.Finally,we
show that AmphiVent1 is able to directly repress promoters of AmphiGoosecoid and AmphiChordin genes. Repression of
genes encoding dorsal-specific signaling molecule Chordin and transcription factor Goosecoid by Xenopus and zebrafish
Vent genes represents a key regulatory interaction during vertebrate axis formation. Our data indicate high evolutionary
conservationofacoreBmp-triggeredgeneregulatorynetworkfordorsoventralpatterninginchordatesandsuggestthat
co-option of the canonical Wnt signaling pathway for dorsoventral patterning in vertebrates represents one of the
innovations throughwhichan increased morphologicalcomplexity of vertebrateembryo isachieved.
Citation: Kozmikova I, Smolikova J, Vlcek C, Kozmik Z (2011) Conservation and Diversification of an Ancestral Chordate Gene Regulatory Network for
DorsoventralPatterning.PLoSONE6(2):e14650.doi:10.1371/journal.pone.0014650
Editor:PatrickCallaerts,KatholiekeUniversiteitLeuven,Belgium
ReceivedJuly23,2010;AcceptedJanuary4,2011;PublishedFebruary3,2011
Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsPublicDomaindeclarationwhichstipulatesthat,onceplacedinthepublic
domain,thisworkmaybefreelyreproduced,distributed,transmitted,modified,builtupon,orotherwiseusedbyanyoneforanylawfulpurpose.
Funding:ThisworkwassupportedbytheGrantAgencyofCzechAcademyofSciences(IAA500520604andIAA500520908toZ.K.).InstituteofMolecular
GeneticsissupportedbytheAcademyofSciencesoftheCzechRepublic(AV0Z50520514).Thefundershadnoroleinstudydesign,datacollectionandanalysis,
decisiontopublish,orpreparationofthemanuscript.
CompetingInterests:Theauthorshavedeclaredthatnocompetinginterestsexist.
*E-mail:[email protected]
Introduction patternshomologoustotheirvertebratecounterparts[2,3].Itwas
shown previously that teleost and amphibian Vent proteins can
Establishmentofadorsoventral(DV)axisisakeyeventinearly suppress theexpression ofdorsalgenes duringearly development
developmentofanybilateriananimalembryo.Thecrucialstepin [4,5,6].Xvent-2(alsoknownasXvent-2B,Xom,Xbr-1andVox)
DV axis formation is specification of the dorsal and ventral directly represses the Goosecoid promoter in Xenopus embryo [6].
mesoderm. In vertebrates, the establishment of the organizer Noting mutually exclusive expression of AmphiVent1 and Amphi-
involvesactivationofseveralgenes[1].Theirproteinproductsare Chordin[2],itcanbesuggestedthatAmphiVent1islikewiseableto
mostly transcription factors (such as Otx2, XFD1, Goosecoid) or antagonize expression of organizer-specific genes as do its
secretedproteins(suchasADMP,Nodal,Noggin,Chordin).The vertebrate homologues [7]. During the gastrula stage, AmphiVent1
organizer secreted proteins Chordin and Noggin are capable to isexpressedthroughoutthemesendoderm[3].Bylategastrula,it
inactivate BMP signaling molecules that play a key role in the isdown-regulatedventrallybutremainsexpresseddorsolaterallyin
induction and maintainance of ventral and lateral mesoderm. the paraxial mesoderm. Then, at the mid-neurula stage,
Conversely, the expression of Chordin is negatively regulated by AmphiVent1-expressing ventral mesoderm forms as outgrowth
Bmp2 and Bmp4 proteins through their targets, ventralizing from the somites [3,8]. The developmental expression of
homeobox genes Vent and Vox [1]. Recently it was demonstrated amphibian and teleost Vent genes during gastrula stages is most
that theexpressionpatternsof genesmediatingDVpatterningin conspicuous in ventral mesoderm and is down-regulated in the
early development are highly conserved between basal chordates regions of organizer, chordamesoderm and neural plate [5,7]. At
(cephalochordateamphioxus)andvertebrates[2].Orthologuesof theneurulastage,amphioxusaswellasvertebrateVentgenesare
the vertebrate organizer-specific genes such as Goosecoid, Chordin, expressed along the edges of the neural plate, in the tail bud/
Nodal are expressed in early chordate embryo [2]. Amphioxus proctodeal region, and in the foregut [3,9]. Even though there
ventral-specific genes encoding Bmp signaling molecules, and appearstobeatemporaldifferencebetweentheventralexpression
Hex, Evx and Vent transcription factors demonstrate expression of AmphiVent1 and vertebrate Vent genes during early develop-
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ment, their dorsal expression is similar as exemplified by are present in the zebrafish genome while four Vent genes are
downregulation at the dorsal lip of the blastopore and neural presentinXenopuslaevis.HighernumberofVentgenesinXenopus
plate[3].Itisinterestingtonotethatwithintheanimalkingdom laevismaybecausedbyarecentduplicationofitsgenomeasonly
Vent genes are present in chordates only. Moreover, although in twoVentgenesarefoundinanotherfrogXenopustropicalis(http://
humans the Vent-like homeobox gene has been described, no www.ensembl.org). Only a single Vent gene is found in the
Vent gene has been found in the mouse. Amphioxus genome genome of humans and chimpanzee. Interestingly, a functional
contains two Vent genes, which are situated on the same copyofaVentgenehasbeenlostfromthemousegenome;onlya
chromosome incloseproximity of eachother [10]. fragment of the Vent-type homeodomain in the mouse genome
Multipletranscriptionalinputsarelikelyrequiredforthecorrect canbeidentified(thisstudy;seediscussion).Phylogeneticanalysis
regulation of Vent genes. Among those Bmp-mediated activation suggests that independent lineage-specific duplication is responsi-
of vertebrate Vent genes is well documented. Bmp2 and Bmp4 blefortheincreasedcopynumberofVentgenes(Fig.1A).Recent
activateXvent-2promoterviaSmad1inXenopus[11,12]andinP19 lineage-specific duplication of AmphiVent1 and AmphiVent2 is
murine embryonal cells [13]. This activation is mediated consistent with high sequence similarity (83% nucleotide identity
synergistically by OAZ zinc finger transcription factor, which withinrespectiveORF’s;EMBOSSPairwiseAlignmentathttp://
caninteractwithMH2domainsofSmad1andSmad4proteinsin www.ebi.ac.uk/Tools/emboss/align/). Since AmphiVent2 has not
responsetotheBmpsignal[13].Recentinvestigationsrevealthat been previously characterized at all we performed an expression
inadditiontoBmp,thecanonicalWntsignalingpathwayplaysan analysisusingreal-timequantitativeRT-PCR.AsshowninFig.1B
important role in patterning of ventral mesoderm in Xenopus and AmphiVent2 displays similar but not identical temporal and
zebrafish.InthezebrafishembryoWnt8directlyactivatesVentand quantitative regulation of mRNA expression as compared to
Voxgenesthroughb-catenin[14].BothXenopusXvent-1andXvent-2 AmphiVent1.
genes contain conserved Lef/Tcf binding sites in the promoter. It is well established that a relatively short (approximately
Xwnt-8 protein can activate Xvent-1 promoter and the activation 300bp)promoter(59genomicnon-codingregion)ofXVent-2Bgene
depends on the functional Lef/Tcf binding site [15]. Likewise, is sufficient for Bmp-mediated regulation [12,13]. Given the
transgenic analysis of Xvent-2 promoter revealed that mutation of known position of vertebrate Vent genes in the gene regulatory
theLef/Tcfbindingsitedecreasesexpressionofthereportergene network governing DV patterning, we hypothesized that amphi-
[16]. oxusVentgenesmightberegulatedbyBmpsignaling.Totestthis
In this study we have investigated the role of AmphiVent1 possibility, we isolated approximately 1kb of 59genomic non-
homeodomainproteininthemoleculareventsresponsibleforDV coding regions of AmphiVent1 (21230/+20) and AmphiVent2
patterning in amphioxus. We have specifically focused on three (2912/+22)genesputativelycontainingpromotersandgenerated
main areas: the role of Bmp and canonical Wnt signaling in luciferasereportergeneconstructs.Wenexttestedtheiractivityin
AmphiVent1generegulation,functionalpropertiesofVentproteins, P19 cells in the absence and presence of exogenous Bmp
and identification of direct targets of AmphiVent1 transcription (heterologoushumanBMP2wasusedinthisstudyunlessindicated
factor. Using luciferase reporter assays in P19 murine embryonal otherwise). The promoter of Xenopus Xvent-2B gene known to be
cells we have demonstrated Bmp-mediated activation of the activatedbyBmpsignalingintheembryoandinP19cells[12,13]
AmphiVent159genomicnon-codingregions(putativepromoter)via was used as a control in all experiments. We observed BMP2-
Smad1/Smad4 proteins. Similar to vertebrate Xvent-2B gene induced stimulation of AmphiVent1-luc and AmphiVent2-luc
promoter,AmphiVent1promoterresponsivenesstoBmpsignalingis reportergeneactivityinP19 cellsthat wascomparable tothat of
co-stimulated by zinc finger transcription factor OAZ. Further- Xvent-2B-luc (Fig. 1B). In contrast, the 59genomic non-coding
more,reporterplasmidswhereexpressionofGFPiscontrolledby region(2300/+65)oftheorthologoushumanVENTX2genewas
Xenopus Xvent-2B and amphioxus AmphiVent1 promoters show a not inducible by BMP2. However, it is very likely that the
highly similar expression pattern in transgenic medaka embryos. 59genomic non-coding region of VENTX2 used in our study did
We found that AmphiVent1 protein acts as a transcriptional notcontainacompletepromoterandsoBmp-responsiveelements
repressorwiththerepressiondomainlocatedatitsN-terminusthat mighthavebeenmissing.ApplyingdifferentdosesofBMP2(from
appears to interact with groucho family co-repressor Grg4. As in 12ng/ml up to 400ng/ml) resulted in rather similar promoter
the case of its vertebrate orthologues, AmphiVent1 protein can inductions (Fig. S1A). Similar results were obtained by using
suppress the activity of amphioxus Chordin and Goosecoid gene humanBMP4orBMP7forthepathwaystimulation(Fig.S1B).In
promoters. Our data thus provide evidence for a remarkable the same experimental setting, AmphiVent1 promoter was not
conservationofBmp-triggeredgeneregulatorynetworkmediating stimulated by treatment with either human TGF-b or human
DV patterning in vertebrates and basal chordates. On the other activin (Fig. S1C) that are, together with Bmp, members of the
hand, our data suggest an increased complexity of DV pattern TGF-b super-family [19]. Combined, our data show that
regulationinvertebrates.ThecanonicalWntsignalingregulatory 59genomicnon-codingregionsofAmphiVent1andAmphiVent2genes
inputforventral-specificgeneexpressionappearstobelackingin contain functional regulatory regions that are stimulated by Bmp
cephalochordates(thisstudy)[17]andhaslikelybeenco-optedin signaling in P19 cells like their Xenopus counterparts and are
vertebrates. therefore referred toas promotersinthismanuscript.
Results Bmp responsiveness of AmphiVent1 promoter is
mediated by Smad transcription factors
59genomicnon-codingregionsofamphioxusVentgenes
We next decided to molecularly dissect AmphiVent1 promoter
are activated by Bmp signaling regulation. We have chosen AmphiVent1 since the corresponding
Two Vent-like genes, AmphiVent1 [3] and AmphiVent2 [10], can gene has previously been characterized and represents an
be identified in the genome of cephalochordate amphioxus important marker of ventral mesoderm in amphioxus [2,3]. We
(http://genome.jgi-psf.org/Brafl1/Brafl1.home.html). Both of tested if Bmp responsiveness of the AmphiVent1 gene promoter is
them encode the Q50 homeodomain protein with the Vent- mediated by the members of the Smad group of proteins. Co-
specificT47substitution[18](Fig.1A).Likewise,twoVentgenes transfection of common partner human Smad4 with receptor-
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Figure1.AmphioxusVentgenesareregulatedbyBMPsignaling.(A)PhylogeneticanalysisofVentgenesinthechordatelineage.Please,note
thatspecies-specificduplicationisresponsiblefortheincreasedcopynumberofVentgenes.AminoacidsequencealignmentofVenthomeodomainsis
shownwithcharacteristicaminoacidsQ50andT47markedbyarrowheads.Numbersshownindicatebootstrapsupportvalues.(B)QuantitativeRT-PCR
expressionanalysisofAmphiVent1andAmphiVent2duringB.floridaedevelopment.(C)59genomicnon-codingregionsofAmphioxusandXenopusVent
genesareregulatedbyBmpsignaling.P19cellsweretransfectedwithluciferasereporterscontainingAmphiVent1,AmphiVent2,Xvent-2BandVENTX2
59genomicnon-codingregionsintheabsence(openbars)andpresence(blackbars)ofexogenoushumanBMP2.**P,0.01.
doi:10.1371/journal.pone.0014650.g001
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activatedhumanSmad1intoP19cellsresultedinactivationofthe cells [13], it appears to be a limiting factor for Bmp-dependent
AmphiVent1 promoter (Fig. 2A). To explore whether intrinsic regulation. Finally, cotransfection of expression vectors encoding
Smad4DNA-bindingactivityisrequiredforpromoteractivation, caAlk2, hOAZ, Smad1 and Smad4 into P19 cells led to
we used a Smad4-deficient cell line, MDA-MB-468. Wild-type remarkable activation of the AmphiVent1 reporter gene (70- fold
Smad4, but not DNA-binding-deficient mutant Smad4-D4 [20], activation)(Fig.3B).Theseresultssuggestthattranscriptionfactor
was able to induce promoter activity when cotransfected with OAZmediatesBmpregulationoftheAmphiVent1geneinasimilar
Smad1 (Fig. 2B). We identified six putative Smad-binding wayasitdoesinthecaseoftheXvent-2gene.Toprovideaninsight
elements (SBE;cAGAC)inthepromoterofAmphiVent1(Fig.2C). into the molecular mechanism of OAZ-mediated regulation of
TofunctionallyanalyzeSBE’swithintheAmphiVent1promoter,59- AmphiVent1 promoter we used a dominant-negative hOAZ
truncated promoter fragments (2750/+20, 2350/+20 and construct.ItisknownthatZF’s6-13constitutetheDNA-binding
2150/+20) were cloned upstream of the luciferase reporter gene domainofhOAZ,whichishoweverlackingtheabilitytointeract
(Fig. 2C). These truncated reporter genes were transfected into withSMAD’sandtherebytoactivatetargetpromoters[13].Based
P19cellsandcellswerestimulatedbyBMP2.Wehaveobserveda on these properties hOAZzf6-13 was previously used as a
gradualdecreaseofBmpresponsivenessofreportergenesthatwas dominant-negative protein [13]. We cotransfected hOAZzf6-13
directlycorrelatedwiththeextentofpromotertruncation(Fig.2C). togetherwithcaAlk2intoP19cellsandweexaminedtheresponses
ThestimulatoryeffectofBMP2wascompletelyabolishedonlyin ofXvent-2andAmphiVent1reportergenes.Incongruencewithour
the case of AmphiVent(2150)-luc reporter gene construct, which previous results, caAlk2-induced activation of AmphiVent1 and
doesnotcontainanyputativeSBE’s(Fig.2C).Theseresultsshow Xvent-2 reporter genes was significantly suppressed by cotransfec-
that in vitro all putative SBE’s are relevant for Bmp-mediated tion of the hOAZzf6-13 construct (Fig. 3C). The most likely
inducibilityoftheAmphiVent1promoter.Wenextconfirmedthese explanation of the observed suppressive effect of hOAZzf6-13 is
data by a mutational analysis of the AmphiVent1 promoter. that the dominant-negative form of hOAZ competes with
Reportergeneconstructscontainingpointmutationsofindividual endogenous OAZ expressed in P19 cells for DNA-binding on
SBE’swithinAmphiVent1promoterweregenerated.Wefoundthat AmphiVent1 promoter. It was shown previously that the same
destroying any single SBE does not have a significant effect on mechanism, namely DNA-binding displacement of endogenous
Bmp responsiveness (data not shown). Only when all six SBE’s OAZ by hOAZzf6-13, was responsible for attenuation of Bmp-
weremutated,theBmpresponsivenessofAmphiVent1reportergene mediated activation of Xvent-2 [13]. Summarized, our data
was completely lost (Fig. 2C). We were further interested in indicate deep homology in the molecular mechanisms of Bmp-
whether the upstream promoter region of AmphiVent1 is able to mediated regulationof chordate ventgenes.
function as an autonomous Bmp response element (BRE). Such
BREactivitywaspreviouslyascribedtoaspecificregionofXenopus Amphioxus Vent genes are not regulated by Wnt/b-
Xvent-2Bpromoter[13].Tothisendtworeportergeneconstructs catenin signaling
were generated that contained either a cluster of three proximal
It was shown that Wnt/b-catenin signaling directly regulates
(2669/2218)orthreedistal(21214/2669)SBE’supstreamofa
Xvent-1BandXvent-2BgenesviabindingoftheLef/Tcf/b-catenin
minimal promoter (constructs designated pTAZ-BRE/P and
complextotheirpromoters[15,16].Toanalyzeaplausiblerolein
pTAZ-BRE/D, respectively). Constructs were tested for their
the regulation of AmphiVent1 and AmphiVent2 genes, we first
activity in P19 cells in the absence and presence of exogenous
searched for Lef/Tcf binding motifs within their promoters. We
humanBMP2.Althoughdeletionandmutationanalysesrevealed
found putative Lef/Tcf elements (59-CTTTGTT-39) in both
that all SBE’s are functional within the context of the natural
AmphiVent1 (position 2596/2590) and AmphiVent2 (position
AmphiVent1 promoter, only the cluster of proximal SBE’s can
2450/2446) promoters (Fig. 4A). Promoters of Xenopus Vent
function as an autonomous BRE when fused to a heterologous
genes, however, contain conserved consensus Lef/Tcf binding
promoter (Fig. 2D). Taken together, our data suggest that the
sequences in a more proximal position (265/259 in Xvent-1B
promoter of amphioxus AmphiVent1 gene is directly activated by
promoter and 276/270 in Xvent-2B promoter, respectively)
theSmad-mediated Bmpsignaling pathway.
(Fig.4A).Incontrast,the59genomicnon-codingregionofhuman
VENTX2 (2248/+65) does not contain any Lef/Tcf binding
Smadproteinsco-operatewithzincfingerproteinOAZin sequence (data not shown). To examine whether promoters of
amphiVent1 promoter activation AmphiVent1, AmphiVent2, Xvent-1B and Xvent-2B genes and 59geno-
OAZ is a 30-zinc finger (ZF) protein, which associates with mic non-coding region of VENTX2 gene are responsive to
Smad1inresponsetoBMP2,allowingselectiverecognitionofthe canonical Wnt signaling, we cotransfected their reporter genes
BRE in Xenopus Xvent-2 promoter [13]. ZF’s 6-13 of OAZ bind into293TcellstogetherwithN-terminallytruncatedb-catenin(b-
directlytotheBREofXvent-2promoterwhereasZF’s14-19atthe cateninDN). It is well established that b-cateninDN is a
C-terminus of OAZ interact with Smad1 and Smad4 (Fig. 3A). constitutively active form of b-catenin (proteolytically stabilized),
The human OAZ protein (hOAZ) is homologous to Xenopus and whichisabletointeractwithendogenousLEF/TCFtranscription
amphioxus OAZ transcription factors [13] (data not shown). To factors, thus mimicking activation of canonical (Wnt/b-catenin)
investigatewhetherOAZisinvolvedinBmp-dependentregulation signaling. Mouse Sp5 promoter is known to be responsive to
of AmphiVent1 promoter, we cotransfected human hOAZ with canonical Wnt signaling and was used as a positive control [22].
constitutively active human receptor caAlk2 into P19 cells. Only the activity of Xvent-1B and Xvent-2B promoters was
Expression of caAlk2 is known to trigger Bmp signaling, thus significantly stimulated by cotransfection of human b-cateninDN
mimickingadditionofaBmpligand[21].TransfectionofhOAZ (Fig. 4B). Conversely, cotransfection of b-cateninDN with
cDNA alone did not stimulate the AmphiVent1 reporter gene. As AmphiVent1-luc, AmphiVent2-luc and VENTX2-luc did not
expected, expression of the constitutively active caAlk2 alone leadtoanysignificantstimulationoftherespectivereportergenes.
activated the AmphiVent1 reporter gene about 4-fold (Fig. 3B). On the contrary, cotransfection of b-cateninDN with Amphi-
Cotransfection of the hOAZ expression vector together with Vent2-luc resulted in a modest but significant repression of the
caAlk2 resulted in potentiation of caAlk2-mediated response (12- reporter gene. Similar data were obtained when Wnt3A-
fold) (Fig. 3B), suggesting that, although OAZ is present in P19 conditioned medium was applied to 293T cells transfected with
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Figure2.BmpresponsivenessofAmphiVent1promoterismediatedbySmadtranscriptionfactors.(A)AmphiVent1reportergenewas
cotransfectedwithorwithoutplasmidscodingSmad1andSmad4proteinsintoP19cells.(B)AmphiVent1reportergenewascotransfectedwithor
withoutplasmidsexpressingSmad1pluswild-type(Smad4wt)orDNA-binding-deficientmutantSmad4(Smad4-D4)intoSmad4-deficientcellline,
MDA-MB-468.(C)MappingoffunctionalSmad-bindingelements(SBE)inAmphiVent1promoter.Luciferasereporterplasmidscontainingwild-type,
deletedormutatedAmphiVent1promoterfragmentsweretransfectedintoP19cellsandcellswerestimulatedbyBMP2.Fold-inductionbyBMP2is
indicated.PositionofindividualSBE’sisindicatedbyblackovals,andmutatedSBE’sbycrossedoval.(D)IdentificationofanautonomousBREin
AmphiVent1promoter.P19cellsweretransfectedwithreporterscontainingaminimalpromoterfusedtoeitherthreeproximalSBEelements(pTAZ-
BRE/P) or three distal SBE elements (pTAZ-BRE/D). Reporter genes were stimulated by exogenous human BMP2 (50ng/ml). *P,0.05, **P,0.01,
***P,0.001.
doi:10.1371/journal.pone.0014650.g002
theindividualventreportergenes(Fig.S2).NextwemutatedLef/ signaling,participationoftheWnt/b-cateninsignalingpathwayin
TcfbindingsitesinAmphiVent1,AmphiVent2,Xvent-1BandXvent-2B regulation ofVent genes isnot conservedamong chordates.
promoters and performed cotransfections with b-cateninDN into
293T cells. As shown in Fig. 4C, responsivness of mutated Xvent- Functional diversification of amphioxus Vent gene
1B and Xvent-2B promoters to b-cateninDN was abolished promoters: a case of possible regulation by dorsal-
indicating that single Lef/Tcf binding sites in these promoters
specific forkhead transcription factors
mediate canonical Wnt signaling. As expected, mutating putative
ByanalyzingtheproximalregionsofAmphiVent1andAmphiVent2
Lef/TcfbindingsitesinAmphiVent1andAmphiVent2promotersdid
promotersinsilicousingFamilyRelationssoftware(http://family.
nothaveanysignificanteffectinreportergeneassaysascompared
caltech.edu) we identified highly conserved sequence motifs that
to wild-type constructs. Cotransfection of b-cateninDN with
are80%similarwithinasliding20bpwindow(Fig.5Aanddata
AmphiVent2mut-luc (like AmphiVent2-luc, see above) resulted
not shown). A FoxD-binding element GTAAC was found within
inamodestrepressionofthereportergenesuggestinganindirect
this region in the AmphiVent2 gene whereas the promoter of
typeofregulation.Theseresultsconfirmpreviouslypublisheddata
AmphiVent1 contains a single nucleotide change (GcAAC) in the
showing direct regulation of the two Xenopus vent genes by
FoxDmotif(Fig.5A).InamphioxusAmphiFoxDisexpressedinthe
canonical Wnt signaling [15,16]. Our data suggested that
axial mesendoderm within the dorsal lip of blastopore at early
AmphiVent1 and AmphiVent2 genes are not directly regulated by
gastrula stage [28]. Taking into account the expression pattern
canonicalWntsignaling.However,ourconclusionswerebasedon
data and our in silico analysis we hypothesized that AmphiVent
rather limited 59genomic non-coding regions that might be
genesmightbetargetsofAmphiFoxD.Fromthispointofview,it
sufficient for Bmp-responsivness but not necessarily for respon-
isinterestingtonotethatanegativeregulationbetweenFoxDand
siveness to canonical Wnt signaling. To provide more definitive
ventgeneshasbeendescribedinXenopus[29].XFD-19,theXenopus
answer about the possible role of Wnt/b-catenin signaling in the
FoxDortologue,wasshowntobesuppressedbytheXvent-1gene
regulation of AmphiVent1 and AmphiVent2 expression we pharma-
andplaysaroleinDVpatterning.Infact,XFD-19isadorsallip-
cologically manipulated Wnt pathway in vivo in the developing
specific transcription factor, which is specifically activated in
amphioxusembryos.ToactivatethecanonicalWntsignaling,we
Xenopus organizer. To investigate whether AmphiFoxD protein
used 6-Bromoindirubin-39-oxime (BIO), a potent and less toxic
can bind to putative sites within the conserved region of
inhibitorofglycogensynthasekinase-3b(GSK-3b)ascomparedto
lithium (Li+)[23]. BIO was added to developing amphioxus AmphiVent1 and AmphiVent2 promoters, double-stranded oligonu-
cleotidesderivedfromthecorrespondingregionsofeachpromoter
embryos at blastula stage and embryos were allowed to develop
weretestedbyinvitroDNA-bindingassay(electrophoreticmobility
until mid-neurula stage at which point mRNA was isolated and
shift assay, EMSA). AmphiFoxD formed a specific complex with
gene expression interrogated by real-time quantitative RT-PCR.
theprobe,whichcorrespondedtotheAmphiVent2promoterregion
Amphioxus FoxQ2 and Axin genes were used as controls to test
(Fig. 5B, C). In contrast, AmphiFoxD did not bind to the probe
for effectiveness of Wnt pathway stimulation. It was previously
whichcorrespondedtotheAmphiVent1promoterregion,consistent
shown that AmphiFoxQ2 expression is downregulated upon
pharmacological manipulation of canonical Wnt signaling (Li+ withtheobservedmutationintheFoxDbindingmotifortoanon-
specific(unrelated)probe.Bindingsitespecificitywasconfirmedby
administration; [24]). Axin is a functional component of Wnt/b-
EMSA in the presence of increasing amounts of non-specific
catenin signaling that associates directly with b-catenin, GSK-3b
(unrelated) double-stranded oligonucleotideor AmphiFoxD bind-
and APC and is implicated in down- regulating Wnt signaling
ing site derived from AmphiVent2 promoter region. As shown in
[25].VertebrateAxin2isadirecttargetofWnt/b-cateninsignaling
Fig.5D,onlyAmphiFoxDbindingsitecaneffectivelycompetefor
whose expression is induced by activated Wnt signaling and acts
the formation of the complex. To provide further evidence for
therefore in a negative feedback loop [26,27]. Axin2 is currently
AmphiFoxD-mediated regulation of AmphiVent2, P19 cells were
the most reliable and frequently used natural readout of Wnt/b-
transfected with luciferase reporters containing AmphiVent1 or
cateninsignalinginvivo(http://www.stanford.edu/group/nusselab
AmphiVent2promotersinthepresenceorabsenceofanexpression
/cgi-bin/wnt/reporters). As shown in Fig. 4D, constitutive
plasmid encoding AmphiFoxD. AmphiFoxD can significantly
activation of Wnt/b-catenin signaling in the developing amphi-
repress AmphiVent2-luc but not AmphiVent1-luc promoter
oxusembryosresultedinmarkedincreaseofAmphiAxinexpression
construct (Fig. 5E). Our data suggest that AmphiVent2, but not
and downregulation of AmphiFoxQ2 expression. In contrast,
AmphiVent1,mightbesubjecttoFoxDregulation.Inaddition,the
however, expression of AmphiVent1 and AmphiVent2 has not been
data exemplify functional diversification of promoter sequences
significantly upregulated in the presence of activated Wnt/b-
after duplicationof ventgenes in theamphioxuslineage.
catenin signaling.
Available evidence thus suggests that although canonical Wnt
ActivationofAmphiVent1andAmphiVent2promotersin
signaling plays a prominent role in the early establishment of
ventral mesoderm in Xenopus and zebrafish, amphioxus does not early developing medaka embryos
usethispathwayforspecificationoftheventralfate.Insummary, We next asked whether the amphioxus AmphiVent1 (1.2kb),
our data argue that, in contrast to the situation with Bmp AmphiVent2 (0.9kb) and Xenopus Xvent-2B (0.3 kb) promoters are
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Figure3.ZincfingerproteinOAZmediatesinductionofAmphiVent1promoterbyBMPsignaling.(A)SchematicstructureofOAZand
dominant-negative construct ZF6-13. Individual zinc fingers are shown as black boxes. DNA-binding and Smad-interacting domains of OAZ are
indicated.(B)OAZpotentiatesBmp-mediatedinductionofAmphiVent1promoter.P19cellsweretransientlycotransfectedwithAmphiVent1reporter
(21230+20-luc)andindicatedexpressionplasmids.(C)Thedominant-negativeformofOAZattenuatesBmpinducibilityofAmphiVent1andXvent-2B
promoters.AmphiVent1 andXvent-2BreportergeneswerecotransfectedinP19 cellswithorwithoutdominant-negativeZF6-13constructinthe
absenceorpresenceofBmppathwaystimulationmediatedbyexpressionplasmidencodingcaAlk2.*P,0.05,**P,0.01,***P,0.001.
doi:10.1371/journal.pone.0014650.g003
activated in medaka embryos. Corresponding EGFP reporter gastrulation and its pattern resembled the activation of EGFP in
constructs p817-AmphiVent1, p817-AmphiVent2 and p817- the embryos injected with p817-Xvent-2B (Fig. 6 F-G9). At mid-
Xvent-2B were injected into medaka embryos at the single cell gastrula stage the strongest EGFP signal was observed laterally
stage and their transient expression was monitored during early from growing embryonic shield. Neither AmphiVent1 nor Xvent-2B
embryogenesis. At early gastrula stage AmphiVent1(Fig. 6A-A9), promoterwasactivatedintheareaoftheembryonicshield,where
Xvent-2B (Fig. 6B-B9), and AmphiVent2 (Fig. S3A-A9) promoters dorsal mesodermal marker Goosecoid is expressed in the medaka
were activated throughout the dorsal blastoderm of the embryo, embryo (Fig. 6H) [31]. We detected ventrolateral expression of
demarcatingtheregionofthemostdorsalembryonicshield,where EGFPdrivenbytheXvent-2Bpromoter(Fig.6G).Incontrast,the
dorsalmesodermalmarkerChordinisexpressed(Fig.6C)[30].The AmphiVent1 promoter was not activated ventrally in medaka
EGFP signal fromthep817-AmphiVent1 (Fig.6D-E9) and p817- embryo at mid-gastrula stage (Fig. 6E). It is interesting to note
AmphiVent2 (Fig. S3B-C9) constructs remained evident during that, in contrast to the Xvent-2B promoter, AmphiVent1 and
Figure 4. Canonical Wnt signaling activates XenopusXvent-1BandXvent-2Bbut notAmphiVent1andAmphiVent2promoters. (A)
Schematic diagram of the Xvent-1B, Xvent-2B, AmphiVent1 and AmphiVent2 promoter-luciferase constructs with putative Tcf/Lef binding sites
depicted by black rectangles. Nucleotide changes within Tcf/Lef binding site introduced into mutant luciferase reporter genes used in (C) are
indicated.(B,C)Wild-type(B)ormutant(C)luciferasereporterplasmidswerecotransfectedwithexpressionplasmidencodingastabilizedformofb-
catenin(b-cateninDN)into293Tcells.Please,notethatfoldinductionofindividualreportergeneswasnormalizedtoactivationofthepromoter-less
construct pGL3-basic. (D) Quantitative RT-PCR expression analysis of AmphiAxin, AmphiFoxQ2, AmphiVent1 and AmphiVent2 in control embryos
(DMSO)andinembryostreatedwithcanonicalWntsignalingactivator(BIO)[23].*P,0.05,**P,0.01,***P,0.001.
doi:10.1371/journal.pone.0014650.g004
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Figure5.PromoterofAmphiVent2containsabindingsitefordorsal-specificforkheadtranscriptionfactorAmphiFoxD.(A)Family
RelationssoftwarewasusedforidentificationofhighlyconservedsequencesincludingaputativeFoxDbindingsiteinAmphiVent1andAmphiVent2
promoters.(B) Sequencesofwild-typeand mutatedforkheadbinding sites. Previously characterized bindingsites for vertebrateFoxD and FoxC
familymemberswerealignedwithputativesitesderivedfromAmphiVent1andAmphiVent2promoters.(C)EMSAofAmphiFoxDinteractionwith
bindingsitesindicatedin(B).Please,notethatonlythebindingsitederivedfromAmphiVent2promoter(designatedAmphiFoxDBSV2)isrecognized
by AmphiFoxD transcription factor. Non-specific (unrelated) double-stranded oligonucleotide is not able to bind AmphiFoxD. (D) EMSA of
AmphiFoxD with binding site derived from AmphiVent2 promoter in the presence of increasing amounts (106, 206, 406, 806) of non-specific
(unrelated)double-strandedoligonucleotideorAmphiFoxDbindingsite.Please,notethatonlyAmphiFoxDbindingsitecaneffectivelycompetefor
the formation of the complex. (E) P19 cells were transfected with luciferase reporters containing AmphiVent1 or AmphiVent2 in the presence or
absence of an expression plasmid encoding AmphiFoxD. AmphiFoxD can significantly repress AmphiVent2 but not AmphiVent1 promoter.
***P,0.001.
doi:10.1371/journal.pone.0014650.g005
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AmphiVent2 promoters were activated dorsolaterally, but not AmphiVent1 transcription factor represses
ventrally, in the medaka embryo. These observations are in AmphiGoosecoid and AmphiChordin promoters
agreementwithknownexpressionpatterndataforvertebrateand
One of the most prominent functions of vertebrate Vent
cephalochordateVentgenes.TheexpressionofXenopusventgenes transcription factors is repression of organizer-specific genes such
and AmphiVent1 was demonstrated to be highly similar in as Goosecoid and chordin. To analyze whether AmphiVent1 acts as
dorsolateralbutnotventrolateralmesoderm[3].Whereassomites transcriptionalrepressorofAmphiGoosecoidandAmphiChordingenes,
and their derivatives originate from dorsolateral mesoderm, we first generated luciferase reporters containing AmphiGoosecoid
ventrolateral mesoderm gives rise to the heart and other (2580/+101) and AmphiChordin (21354/+118) promoter regions.
components of circulatory system, which is generally much Cotransfections of AmphiGoosecoid-luc and AmphiChordin-luc
simpler incephalochordates than invertebrates. plasmids with an expression vector carrying the AmphiVent1
Takentogether,ourdatasuggestthattheAmphiVent1promoter codingsequenceresultedindownregulationofreportergenes2.8-
is correctly regulated in the vertebrate embryo and that its fold and 6.8-fold, respectively (Fig. 8A). Paired-type homeodo-
spatiotemporalactivityisbylargesimilartotheactivityofXvent-2B mainsinteractwithacoreTAATmotifasmonomersorashomo/
promoter inthesame context. heterodimers with dimer sites containing inverted TAAT core
motifsseparatedbyseveralnucleotides[38,39,40,41].Asynthetic
All Vent proteins function as transcriptional repressors homeodomain reporter gene designated 3xHD(P3)-luc that
and interact with groucho co-repressors contains three palindromic homeodomain binding sites (TAAT-
XenopusXvent-2BandzebrafishVentgeneswereshowntoactas cagATTA)wasrepressed2-foldbyAmphiVent1(Fig.8A).Incase
of 36HD(P3)-luc the two TAAT core motifs are separated by
transcriptional repressors [4,6,11]. Besides, activating function of
threenucleotides(P3).InordertobetterdefinetheDNA-binding
Xvent-2 was described [15,32]. To examine the transcriptional
specificity of AmphiVent1 and spacing requirements, we per-
properties of chordate vent proteins, a Gal4 reporter assay was
formed EMSA with AmphiVent1 homeodomain and a series of
employed. Plasmids encoding Gal4 fusions with AmphiVent1,
bindingsites(Fig.8B).AlthoughAmphiVent1homeodomainwas
AmphiVent2, Xvent-1b, Xvent-2b and VENTX2 were cotrans-
abletointeractwithasingleTAATmotifintheP1/2bindingsite,
fectedwithGal4-dependentreportergene.AsshowninFig.7A,all
homeodomaindimerizationwasobservedonP2-P4bindingsites.
vent proteins strongly repressed expression of the reporter gene
In comparisonwitharelated paired-type homeodomainofPax6,
whentetheredtothepromoterviaGal4bindingsites.Wefurther
AmphiVent1 has a conspicuous preference for a three-nucleotide
focusedonidentifyingthefunctionaldomainswithinAmphiVent1
spacer (P3) (Fig. 8B). It was shown previously that the sequence
thatmediatetranscriptionalrepression.AmphiVent1andAmphi-
CTAATTGiscriticalforXvent-2Bbinding,andthatthebinding
Vent2showhighaminoacidsequencehomologywithintheentire
is enhanced by the presence of an additional ATTA motif six or
open reading frame (80% identity, 84% similarity) suggesting
sevennucleotides39ofthecoreTAAT[6].Inadditiontomultiple
similar molecular properties. From the two amphioxus vent
TAAT core motifs we found two CTAATTG motifs in
proteins we selected AmphiVent1 since it is encoded by a
AmphiChordin promoter at positions 2574/2580 and 2687/
previouslycharacterizedgenethatrepresentsanimportantmarker
2682 (Fig. 8A and data not shown). The CTAATTG motif is
ofventralmesoderminamphioxus[2,3].Tothisend,Gal4fusion
notpresentinAmphiGoosecoidpromoter,butinstead,weidentifieda
constructs encoding different domains of AmphiVent1 were
putative P3-like palindromic homeodomain-binding sequence
cotransfected together with the Gal4-dependent reporter plasmid
ATTAttgTAAT at a position 256/268. To investigate whether
into 293T cells. The Gal4 fusion proteins containing the N-
AmphiVent1 is able to repress AmphiGoosecoid promoter through
terminusorthehomeodomainrepressedtranscription21-foldand
this binding site, AmphiGoosecoid promoter-containing reporter
10-fold,respectively(Fig.7B).Incontrast,theGal4fusionprotein
plasmid with a mutated homeodomain motif (CTTCttgTCCT)
containingtheC-terminusofAmphiVent1activatedtranscription
wasgeneratedanddesignatedAmphiGoosecoid(mut)-luc(Fig.8C).
2.2-fold(Fig.7B).Theseresultssuggestthatoverall,AmphiVent1
BindingoftheAmphiVent1homeodomainproteininEMSAwas
acts as a transcriptional repressor and has strong repression
readily detectable to the wild-type AmphiGoosecoid promoter
domains at its N-terminus and within the homeodomain. In
sequence, but not to its mutated version (Fig. 8C, lower right
addition,thereisaweaktranscriptionalactivationdomainlocated
panel).Inaccordancewiththisdata,AmphiVent1wasnotableto
within the C-terminus of AmphiVent1 like in Xenopus Xvent-2B repress the AmphiGoosecoid(mut)-luc reporter gene (Fig. 8C,
protein [15]. Next, we tried to identify specific amino acid lowerleftpanel).Tofurthercorroborateourresultsweconverted
sequences within the N-terminus which are responsible for the AmphiVent1toanactivatorbyfusingittoastrongtransactivation
repressionfunctionofAmphiVent1.WecotransfectedGal4fusion domain derived from the VP16 transcription factor. As shown in
constructsencodingAmphiVent1aminoacids1-116,23-116,41- Fig.8C,AmphiVent1-VP16wasabletostronglyactivatethewild-
116, 67-116, 1-74 and 1-42, respectively. All fusion proteins typeAmphiGoosecoid-lucreportergene,butnottheAmphiGoo-
stronglyrepressedexpressionofthereportergene(Fig.7C).These secoid(mut)-luc in which the homeodomain binding site was
data suggest that the AmphiVent1 protein likely has multiple mutated. Combined, these data suggest that repression of the
independent repressor domainswithinitsN-terminus.Sin3Aand AmphiGoosecoid promoter by AmphiVent1 is mediated via P3-like
Groucho family members appear to be widely used cofactors binding site. Likewise, AmphiVent1-VP16 was able to strongly
mediating transcriptional repression of many DNA-binding activate the AmphiChordin-luc reporter gene, whereas fusion of
proteins, including those containing a homeodomain AmphiVent1 to the engrailed repression domain (AmphiVent1-
[33,34,35,36,37]. We therefore tested a possible interaction of EN)generatedatranscriptionfactorwithpropertiescomparableto
the AmphiVent1 N-terminal domain with these obligatory co- wild-type AmphiVent1 (Fig. 8D). It is well established that the
repressors. As shown in Fig. 7D, the AmphiVent1 N-terminal homeodomain can both bind DNA and mediate protein-protein
domain is able to interact with mouse Grg4 but not with human interactions [42]. To investigate whether the DNA-binding
Sin3A.InteractionofAmphiVent1withgroucho-likeco-repressors function of AmphiVent1 homeodomain is critical for the
mayberesponsible,atleastinpart,forfunctionofAmphiVent1as downregulation of both AmphiGoosecoid and AmphiChordin genes,
a potent transcriptional repressor. we generated two mutants, AmphiVent1(R53A) and Amphi-
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