Thomas W. Holstein University, Austria. Vienna, *Author forcorrespondence (e-mail:[email protected]) und Limnologie,UniversitätInnsbruck,Technikerstrasse 25,6020Innsbruck,Austria. Niehrs, 2003). able toantagonizeWntsignalling(Krupniketal.,1999;Mao and 2000). Dkk3isuniquewithintheDickkopf family in thatitisnot depending onthecellularcontext (MaoandNiehrs,2003;Wu etal., addition, Dkk2canactasanLrp6agonist,wellantagonist, Krupnik etal.,1999;MaoandNiehrs,2003;Wu etal.,2000).In and Dkk4areabletoinhibitWntsignalling(Glinkaetal.,1998; et al.,2000). 2000; Kazanskayaetal.,Mukhopadhyay2001;Shinya Glinka etal.,1998;Grotewold andRuther, 2002b;Hashimotoetal., and limbpatterninginthevertebrate embryo(Davidson etal.,2002; of canonicalWntsignallingbyDkk1isessentialforheadformation Mao etal.,2002;2001;Semenov etal.,2001). Inhibition receptor KrementoinhibitWntsignalling(Davidson etal.,2002; a majorsecretedWntantagonist,bindstoLrp5/Lrp6anditsco- activates thecanonicalWnt- Frizzled toformafunctionalliganddual-receptorcomplex that currently assumedthatWntligandsbindtobothLrp5/Lrp6and Frizzled andthelow-density lipoproteinreceptorLrp5/Lrp6.Itis types ofreceptormolecules:theseven-pass trans-membraneprotein development, Wntligandsinitiatesignallingbyinteractingwithtwo cellular animals(Kusserow etal.,2005).Invertebrate embryonic evolution was probablylinked totheoriginandevolution ofmulti- Nusse, 2004;2003),andtheirappearanceinearlymetazoan cell signallingmolecules(LoganandNusse,2004;Nelson Secreted Wntglycoproteinsconstituteoneofthemajorfamilies of INTRODUCTION KEY WORDS:Dickkopf, signal peptidescreen,ofanovel control ofcellfate,proliferation,andpolarityduringdevelopment.Here,wereporttheisolation,fromaregeneration-s The Corina Guder An ancientWnt-Dickkopfantagonismin Development 133,901-911doi:10.1242/dev.02265 Accepted 28December 2005 1 Freiburg, Breisacher Strasse117,79106Freiburg, Germany. Technology, Germany. 280, 69120Heidelberg,Germany. 230, 69120Heidelberg,Germany. 1030 Vienna, Austria. 1030 Vienna, (CIBIV), Max F.Bioinformatics Vienna PerutzLaboratories(MFPL), DrBohrGasse9, cnidarians andbilaterians.Insteady-state that theWntantagonisticsubfamilyDkk1/Dkk2/Dkk4andnon-modulatingDkk3separatedpriortodivergenceof although oncetheheadhasregeneratedtheyareexcludedfromit.ActivationofWnt/ early injuryandregenerationresponsivegene, body columnofHydra the downregulation ofhydkk1/2/4 Molecular EvolutionandGenomics,HeidelbergUniversity, ImNeuenheimerFeld Of thefourDickkopf (Dkk)proteinfamily members, Dkk1, Dkk2 Xenopus dickkopf gene Dkk1.Basedonthecorrespondingexpressionpatternsof ( dkk) genefamilyencodessecretedantagonistsofWntsignallingproteins,whichhaveimportantfunctionsinthe 1,2 6 3 University ofVeterinary Austria. Medicine,Vienna, Deutsches Krebsforschungszentrum, ImNeuenheimerFeld , SoniaPinho 8 Vienna University,Vienna Austria. Vienna, dkk, Wntsignalling,antagonism,Regeneration,Axisformation,Neurogenesis, Cnidaria, is aneurogenicenvironmentsuppressingWntsignallingandfacilitatingneurogenesis. 1,2, 4 2 ␤ * Klinik fürTumorbiologie anderUniversität Molecular CellBiology, DarmstadtUniversityof -Catenin pathway. Dickkopf 1(Dkk1), transcripts. Whenoverexpressedin dickkopf 3 , Tanju G.Nacak Hydra, gene fromthefreshwatercnidarian 5 Center forIntegrative 9 Institut fürZoologie xrsinisinverselyrelatedtothatof hydkk1/2/4-expression hydkk1/2/4-expressing glandcellsareessentialforheadregenerationin 2,4 7 , HeikoA.Schmidt Medical Xenopus, HyDkk1/2/4hassimilarWnt-antagonizingactivityto Hydra Hydra culture andexperiments bilaterian setting upaneurogenicenvironment inthe bodycolumnofthepre- phylogenetically very old,andprobablyhadamajorfunctionin analyses supporttheview thattheWnt-Dickkopf antagonismis by geneduplication.Moreover, ourexpression andfunctional homologue, fromwhichthevertebrate phylogenetic analysisindicatesthatthis specific inayeastsignalpeptidesecretionscreen.Structural and recently identifiedin the insectandnematodegenomes.ADkk3-relatedproteinwas homologues ininsects,noDickkopf proteincouldbeidentifiedin receptor proteinsFrizzledandLrp5/Lrp6fromvertebrates have evolution Wnt-Dickkopf antagonism was established.Althoughthe function onCRD2(BrottandSokol, 2002;Lietal.,2002). to theLrp5/Lrp6receptor. CRD1is thoughttohave amodulating signalling bycompetingwiththeWnt-Frizzledcomplex forbinding CRD2 isnecessaryandsufficient torepresscanonicalWnt diagnostic forgroupingofDkkproteins.Ithasbeenshown that domains (CRD1andCRD2),whichareseparatedbyaspacerregion, Fujisawa, 1978),aswell MATERIALS ANDMETHODS The novel freshwater polyp radiation ofvertebrates. relatively lateinmetazoanevolution, i.e.concomitantwiththe to thisproposal,theDickkopf-Wnt antagonismwas established the vertebrate Dkk1/2/4subfamily (Feddersetal.,2004).According furthermore proposedthatsubsequentgenomeduplicationcreated suggested thatDkk3representstheancestralDickkopf type.Itwas (Rentzsch etal.,2005). was inducedinpolyps of a week,andused24hoursafterfeeding forexperiments. Gametogenesis 1997), wereculturedat18°C(Takano andSugiyama,1983),fedfive times Hitherto, itiscompletelyunknown atwhatpointinmetazoan Vertebrate Dickkopf moleculesconsistoftwo cysteine-rich We describehereanew Dickkopf-related moleculefromthe strains hydkk1/2/4 Hydra. 5,6,7,8 Hydra Hydra. Comparativesequenceanalysisdemonstrates Hydra magnipapillata , BertHobmayer Hydra andthestarletseaanemone Dickkopf proteinwas isolatedasregeneration ␤ -Catenin signallingleadstothecomplete and neuronalgenes,wesuggestthatthe Hydra Hydra Hydra vulgaris Hydra vulgaris (Fedders etal.,2004),anditwas yn3.hydkk1/2/4 hywnt3a. RESEARCH ARTICLE 9 105 andsf-1(Sugiyama , ChristofNiehrs Dkk1, Basel andAEP(Martinetal., strain AEPasdescribed dkk Dkk2 Hydra, gene isa and Nematostella Xenopus Dkk4 is an dkk1/2/4 Hydra, 3 pecific and arose 901 .

DEVELOPMENT (T)17ATAATTTAACTCG-3 After washing for5minutes withPBS/PBS covered object slideandallowed todryfor 45minutesatroomtemperature. macerated cells(David, 1973)dropped(100 Labelling Kit(Roche). 1158) andfromfull-length ng ng TOPFLASH vector, 8ng all experiments theHydra vulgarisclonewas used. substitutions withintheORF, resultinginseven amino acidexchanges. For hydkk1/2/4 T vector (Promega). Asequencecomparisonofthethreestrainsrevealed that primers (5Ј magnipapillata Baseland additionally amplifiedfromboth,Hydra vulgarisstrain TTTTGCAAGACTCGGC-3 TCTTTAACATAGCTATTACATTGC-3 (Invitrogen), and specificprimers(5 magnipapillata complete polyAtail.Thefull-lengthsequencewas amplifiedfrom fromthehydraESTcollection(http:// mpc.uci.edu/hampson/public_html/blastlif9). Thissequencecontainsa (CA303262) taa05h01 al., 1999).Blastsearchrevealed amatchwithclone Jacobset the signalpeptidesecretionapproach(Fig.1)(Jacobsetal.,1997; transcripts fromthei-celldepleted A Cloning ofhydkk1/2/4 System III(Promega). magnipapillata vulgaris We isolatedpolyA+RNA fromtotalRNA (Suzukietal.,2001)of Isolation ofmRNA Molecular techniques for 24or48hours. mediumto5 hydra Alsterpaullone (Calbiochem)was dissolved inDMSOanddilutedwith Alsterpaullone treatment All animalsweretransferredintofreshmediumaftertreatment. one deepcutwas setintothemiddleofbodycolumn(30%width). off withoutleaving anopenwound withexposed endoderm.For wounding, described (Newman, 1974).Thiscausedtheheadtobegraduallypinched a knotwiththinhairaroundthesubhypostomalregion ofthepolypas scalpel. Insomeexperiments, headswereremoved withoutinjurybytying Polyps werebisectedat20%,50%or80%bodylengthbyusingasmall Regeneration andwoundingexperiments Temperature-sensitive interstitialcellsofHydra magnipapillata Elimination ofinterstitialcelllineage 902 riboprobes wereproducedfroma 534 bpfragmentof followed by Fast Redsubstrate.Senseandantisense substrate NBT/BCIP was performedasdescribed(Hansenetal.,2000;Philipp2004),with et al.,1997)ataprobeconcentrationof0.05ng/ Whole-mount ISHwas performedasdescribed(Grensetal.,1996;Martinez In situhybridization(ISH) normalized againstrenilla. checked bymonitoringGFPfluorescence.Fireflyluciferaseactivity was vector to100ngDNA. After24hours,thetransformationefficiency was well plates,intriplicate,with1ngeachof described (Wu etal.,2000).Transformation ofcells was carriedoutin96- TOPFLASH assayinHEK293T cellswas performedaspreviously Cell cultureexperiments interstitial cellsandderivatives was examined inmacerates(David, 1973). starved polypswerekept at18°Cforuptosixweeks(seeResults).Lossof were eliminatedbyculturingpolypsat28-30°Cfor5days;afterwards hydkk1/2/4 Macerate ISHwas performedbypreparingafixed cellsuspension of xdkk1 RESEARCH ARTICLE strain Basel,Hydra magnipapillata and 3.125ng -GAAAACATACATCTTTTCTGATTTATCAATC-3 from DAcoewas isolatedinascreenfororganizer-specific cDNA clone strain 105cDNA by5Ј tansf-1byusingoligo-dTprimedcDNA andspecific strain strain 105,byusingthePolyATtract mRNA Isolation Hydra magnipapillata ␮ M. Dailyfedpolypswereincubatedinalsterpaullone hkrm1. SamplesweresupplementedwithpCS2 Ј ghumanLRP6,20ng mwnt1, 3ng ). PCRampliconswereclonedintothepGEM- Ј hydkk1/2/4 ). Completehydkk1/2/4 Hydra magnipapillata RACE-PCR using theGeneRacerKit Ј and 5Ј using theDIGorFITCRNA GFP sf-1 exhibited 10nucleotide Tween strain sf-1(nf-1),andHydra Ј ␮ -CCGCAGAGTGCACCT- ␮ , l for36hours.DoubleISH l) ontoapoly-L-Lysine- -GCAGTCTGCATCCT- renilla , preparationswere pre- strain sf-1byusing hywnt3a and cDNAs were hfrizzled1 hydkk1/2/4, 5 strain sf-1 Ј (bp 624- and 5Ј Hydra Hydra Hydra , 10 + - case of all blastomeresoffour-cell-stage Xenopuslaevis embryos(100pg),orin purified withP6SpinColumns(Bio-Rad).Injectionsweredoneradially into mesoderm-free caps, pg belcheri tsingtaunese and 9370);Acropora millepora (EST CN559480);Nematostellavectensis the VAG modelofevolution. PUZZLE 5.2(Schmidtetal.,2001)(10,000intermediatetrees),bothusing 2004) (300repetitions)andquartetpuzzlingasimplementedinTREE- likelihood treeswerecomputedusing IQPNNI (Vinh andvon Haeseler, (http://igs-server.cnrs-mrs.fr/Tcoffee) andadjustedmanually. Maximum Protein sequencealignmentwas performed usingtheTCoffee alignmenttool Phylogenetic analyses The completehydkk1/2/4 In vitrotranscriptionofhydkk1/2/4and mount ISH. diluted, overnight 4°C).Detectionwas carriedoutasdescribedforwhole- room temperature)andanti-digoxigeninmonoclonalantibody(4000-fold and 40°C),thensequentiallyincubatedwithblockingreagent(3hoursat and washed (2 hybridized (2hours,60°C),(2460°C,0.1ng/␮ luciferase p01234 aloneorinconjunctionwith stage, andsubequatoriallywith the Wnt-reporterconstructsiamois- Xenopus laevis embryoswereinjectedinallblastomeres atthefour-cell Luciferase assays at thefour-cell stage:xwnt8 Xenopus laevis embryoswereinjectedintheanimalpoleofallblastomeres Animal capassay ORF andthe produced withtheMessageMachineSP6Kit(Ambion)from explants werecarriedoutasdescribed(Gawantka etal.,1995).mRNA was In vitrofertilization,embryoculture,staging,microinjectionandculture of Xenopus combination withMetaMorphorAdobePhotoshopsoftware. Micrographs wereprocessedusingthemanufacturer’s software in Spot-II, aCanonPowerShot G5oraNikon DSL-1camerawereused. interference contrast.For microphotography, eitheraDiagnosticInstruments Axiovert 100,oraNikon Eclipse-80imicrocope,bothequippedwith Specimens wereanalyzedusingaZeissStemiSV11binocular, aZeiss Microscopy 10 fortheinductionof Animal capswereexplanted atstage8-9andanalyzedbyRT-PCR atstage BAA85488, BAA33475); Dictyosteliumdiscioideum NM_145592); humanDkk1,Dkk2,Dkk3,Dkk4(BAA34651, BAA85465, Dkk1, Dkk2,Dkk3,Dkk4(NM_015789,NM_020265,NM_015814, (BAA82135); fragment usingtheoligonucleotides5Ј (myc-tag) expression vectors (Ruppetal.,1994).The AAL40731). (Sanger Institute);humanandmouseColipase(Col)(AAP35458, hydkk1/2/4 temperature was 55°Cforthecomplete 30 secondsatannealingtemperatureand1minute72°C;the amplicons were:2minutesat94°C,then30cycles of30secondsat94°C, vulgaris amplified fromHydra 5 GATC Company (Konstanz). on anABIPRISM310GeneticAnalyzer(AppliedBiosystems)orbythe TGTATTCAG-3 including theKOZAK sequence,and5 oligonucleotides5 the Ј -GTATTTAAATCGATACAAAGATCCAC-3 The following sequenceswere used:HyDkk1/2/4-A;HyDkk1/2/4-B 12.5pgxwnt8)was injectedaswell. xdkk1, hywnt3a experiments ORF and52°Cforthehywnt3a hywnt3a ϫ Xenopus radially intoventral blastomeres(1ng).ControlmRNA (10 SSC, 2ϫ Ј , andclonedintopCS2+vector. PCRconditionsforall histone-4 ORF inthe (EST AY608670); Zebrafish( Ј -CATCGATTTGCCGCCACCATGGGCACG-3 Dkk1, Dkk2(AAC02427, XLA300197);mouse 5 minutes,60°C;2 ORF was amplifiedandclonedasa siamois (100 pg), expression was monitoredfornormalization. NotI linearizedpCS2+vector; mRNA was cDNA asaClaI/ expression. (EST GS01bF09.b1);Branchiostoma -CTTTTCGGATCCATCAAT-3Ј xdkk1 hydkk1/2/4 Ј -TTTCTAGACTATTTACAGG- ORF. Sequencingwas performed Dkk1/2/4, Dkk3(contigs7341 ϫ hywnt3a mRNA (200 pg), SSC+0.1 %CHAPSat50°C Ј xwnt8 xbra into pCS2+andpCS2+MT Development 133(5) sequence, 46°Cforthe XbaI fragmentusing expression validates (150 pg),xdkk1 Danio rerio hywnt3a hydkk1/2/4 WGS_BC5V2_0 BamHI/ hydkk1/2/4 ORF was l probe) Dkk1 ) (6 ng). (300 ClaI and Ј ,

DEVELOPMENT raffinose plates. with anintact expression intheyeaststrain YTK12.Clonesexpressing afusionprotein motif oftheDkkfamily sharesahighstructuralsimilaritywith Dickkopf subfamily membersDkk1,Dkk2andDkk4.TheCRD2 highest score(E-value 0.005)similaritytotheCRD2ofvertebrate supplementary material).BLASTsearchesatNBCIrevealed with signal peptideof19aminoacids(seeFig.S1Ainthe a proteinof73aminoacids,containingcysteine-rich motifanda (88 bpand31bp,respectively). Theopenreadingframeencodes sequence obtaininga395bptranscriptwithshort5 encoding acysteine-rich protein.Using5 in thesignalpeptidesecretionscreencontaineda328bpfragment specific cDNA library(Fig.1A).Themostabundant clone(10%) head removal (0.5to24hours),andpreparedaregeneration- heads andisolatedregenerating stumpsatvarious time-pointsafter nematocytes. Afterinducingheadregeneration, wecollectedthe magnipapillata, whichhadlosttheirinterstitialstemcellsand heat-shocked animalsofthemutantstrainsf-1from eliminate thehighlyabundant transcriptsofthiscellline.We used nematocytes andinterstitialcellsdevelop normally, wetriedto Campbell, 1978)have shown that Klein etal.,1996).BecauseMarcumandCampbell(Marcum we performedasignalpeptidesecretionscreen(Jacobsetal.,1999; In ordertoidentifygrowth factors andtheirantagonistsinHydra, Hydra Identification ofaDickkopf-related moleculefrom RESULTS four-cell stagewithwnt8 Xenopus laevis Secondary axesassay presented asRelative LuciferaseUnits(RLU). 10.5 inPassive Lysis Buffer (Promega) (25 pg) and Wnt-Dickkopf antagonisminHydra cloned adjacenttoasignalpeptide-deficient yeast cDNA synthesis.( shocked polypsatvarioustimesafter headremoval formRNAand cells. Heads,aswellregenerating tips,were isolatedfrom theheat- exposed toheatshockfor3days, causingtheeliminationofinterstitial the temperature-sensitive strain organizer inayeastsignalpeptidesecretion screen. Isolationofsecreted moleculesfrom the Fig. 1. A B rmtrEoIXhoI EcoRI Promotor ADH size fractionatedcDNA hydkk1/2/4 in ayeastsignalpeptidesecretion screen etsokcut heat shock 3 d Hydra signalpeptidewere selectedbygrowth on embryos wereinjectedintotwo oppositeblastomeresatthe Kex2 cleavagesite B ) Cloningstrategy. Size-fractionated cDNAwas (750 pgand3ng).Embryoswerecollectedatstage10- Invertase (12.5 pg),xdkk1 Hydra magnipapillata emntrpSUC2T7M13ORI Terminator ADH regeneration (10 pg)andhydkk1/2/4 Hydra 0.5-24 hr 17,100regenerating tips Ј RACE wecompletedthe 24,000 headsand ␮ l/embryo). Resultsare lacking nerve cells, cDNA from invertase genefor Hydra head sf-1 were Ј ( and 3Ј A cut ) Polypsof Screening in Yeast (1 ng). Hydra UTRs isolated inthesecretionscreenandsecond sequences, theothercontaining cnidarian Dkksubtrees:onecontainingthemore subfamilies, but againnotwiththecolipases(Fig.2A). number ofaminoacidswithboththeDkk1/2/4andDkk3 found inthevertebrate Dkk3subfamily. CnidarianDkkssharea Dkk1, Dkk2,andDkk4shareanumberofmotifsthatcannotbe and lysines,arepartiallyconserved amongthespecies. Vertebrate are completelyconserved, andsomeotheraminoacids, like glycines to vertebrate colipases.IncnidarianDkks,alltencysteine residues reveal ahighersimilarityofcnidarianDkkstovertebrate Dkksthan TCoffee (Fig.2A),but alsoClustalWandMuscle(datanotshown), Fig. S1A,Binthesupplementarymaterial).Alignmentsusing proteins, eachcomprisingtwo completecysteine-rich domains(see in insectandnematodegenomes. cellular slimemold urochordate fromtwo othercnidarians, Nematostella vectensisand ESTs invertebrates. We found members oftheDkkfamily usingESTdatafromseveral other in cnidarians.For abetteroverview, weperformedasearchfor we hypothesizedthatitmightbeanew memberof theDkkfamily higher similaritytoCRD2ofthevertebrate Dkk1/2/4subfamilies, novel classified asaDkk3orthologue(Feddersetal.,2004).Becausethe which sharesstructuralfeatureswiththevertebrate Dkk3andwas Another Dkk-relatedmoleculewas recentlyidentifiedin precursor molecule reveals features ofaputativeDkk1/2/4 Phylogenetic analysisoftheDickkopf-related value 0.62). expressing glandcells,weinvestigated the hydkk1/2/4 nematocytes andsex cells.To verify thei-cellnatureof stem celllineage(i-cells),which alsogives risetonerve cells, present inglandcells.Glandcells arederivatives oftheinterstitial (Hobmayer etal.,2000;Technau andBode,1999). of peduncle region. Thisexpression patternisinversely relatedtothat specimens, wefoundagradedexpression, diminishingtowards the (see alsoHobmayeretal.,1990a;1990b).Inmany with asharpboundarybelow thetentacleformationzone(Fig.3A,B) whereas theentirebodyexhibited astrongendodermalexpression Hydra’s headwas completelyfreeofhydkk1/2/4-expressing cells, analyzed itsexpression patternbyinsituhybridization.Strikingly, In ordertounravel thefunctionofputative of theinterstitialstemcelllineage hydkk1/2/4 cnidarian DkksasNvDkk1/2/4andHyDkk1/2/4(seeDiscussion). subfamilies was never contradicted.For clarity, werefer tothenovel However, abasalpositionbetweentheDkk3andDkk1/2/4 inner branchesduetothehighdivergence oftheDkkfamily. PUZZLE norbootstrapanalyseswereabletoresolve any ofthe vertebrate Dkksubfamilies 1,2and4.Unfortunately, neitherTREE- subtree ispositionedclosertothehighlysupportedclusterof of thenovel Hydra colipase fold(Aravind andKoonin, 1998).However, thesimilarity colipases (coenzymesoflipases)andhasbeenassignedtothe The IGPNNIML-tree(Fig.2C)oftheCRD2shows two distinct The hydkk1/2/4 hywnt3a Hydra Nematostella , Dkk-related moleculeidentifiedinthescreenshows a Branchiostoma belcheri tsingtaunese is notexpressed inendodermal epithelialcells,but is hytcf, is expressed inendodermal derivatives Dictyostelium. Dkk proteintocolipaseswas muchlower (E- brachyury contigs encodefortwo different Dkk-like Acropora millepora and otherhead-specificgenes No Dkk-like proteinswerefound RESEARCH ARTICLE Hydra Dkk-related protein hydkk1/2/4 nvdkk. Thelatter n fromthe and , , fromthe hydkk1/2/4- -expression dkk3-like gene, we Hydra, 903

DEVELOPMENT red. (C 904 in budding polyps.Surprisingly, there werenochangesinthe pathway inHydra,weanalyzed theexpression patternof As HyDkk1/2/4isaputative antagonisttotheWntsignalling hydkk1/2/4 of i-cellsintonursecells. their i-cellorigin,asoogenesisis accompaniedbythedifferentiation G), but notspermatogenesis(Fig.3H).Thisisinaccordance with hydkk1/2/4 full ofvesicles (Fig.3D).We alsofoundadownregulation of by morethan90%toabout50.Theseresidualhydkk1/2/4 shock, thenumberof slowly thannematocytes andnerve cells.Thirtydaysafterheat proliferating capacity(SchmidtandDavid, 1986),they arelostmore after aheatshock(Fig.3C).Becauseglandcellshave alimited 1978). Asexpected, neurons, but alsoglandcellsdisappear(Sugiyama and Fujisawa, and, over time,allnon-dividing derivatives, i.e.nematocytes and sf-1. Thismutantlosesinterstitialstemcellsafterheatshockat28°C pattern inthetemperature-sensitive Hydra magnipapillata methods). (B Fig. 2.Sequenceanalysisofhydkk1/2/4. Dkk1/2/4-A_hydra Dkk3_nematostella Dkk1/2/4_nematostella Dkk3_mouse Dkk3_human Dkk4_mouse Dkk4_human Dkk2_mouse Dkk2_xenopus Dkk2_human Dkk1_xenopus Dkk1_mouse Dkk1_human Dkk3_dictyostelium Dkk3_acropora Dkk3_amphioxus Dkk1/2/4-B_hydra Dkk3_hydra Dkk1_zebrafish Col_mouse Col_human A B AX2 GX2 GX2 QNIM reo k R2dmiswt REPZL support values(>50). ) IQPNNIMLtree ofDkkCRD2domainswithTREE-PUZZLE C1 X4DC2X2G-C3C4 C1 X4EC2X2GL C1 X4DC2X2GL RESEARCH ARTICLE Mouse Dkk4 Nematostella Dkk1/2/4 expression inglandcellsatsitesofoogenesis(Fig.3E- ) Domainstructure ofmouseDkk4,NvDkk1/2/4andHyDkk1/2/4;shadedboxesindicateconservedresidues withinCRD2;cysteinesin expression duringbudding C3C4 C3C4 hydkk1/2/4-expressing glandcellsdiminished hydkk1/2/4-expressing glandcellswerelost VX5KQ AX5WI AX5KI SP AES LQF FSK LAP AKS GTY GTF GAR GTI GTI GES GES GDP GDL GDP GDV GSV GSV GEN GEI GEL C5 KX2LXEGX2C6 C5 KX2LXENX2C6X14C7 C5 C C C C C C C C C C C C C C C C C C C C C LN MN NIIGP WN NDHHQ KTDA K D D S DNQR DNQR L L L L L L L L L KDA K RHE KDK R R R R R R R R R NX2VXEGX2 SMQ SAQ SS TS TS TF SS ST SS ST SS SS SS D E D E D E D D D D D D D D D D D C C C C C C C C C C C C C C C C C C C C C CRD1 K-S---R K-S---N -PT HP-K--E -PA E-N K-N D-E--NS AGE S-A QP- QP- GP- GP- I-D I-E I-E AP- A-A A-S A-E HyDkk 1/2/4 G--Q G--Y G--- G--- GKAA G--L G--L G--L G--L G--L G--F G--F G--F G--L G--L G--L G--L C6 X7 X16 ( CRD1 CC CC CC CC CC CC CC CC CC CC CC CC CC CC CC CC CC CC CC CC CC A C7 XC8GXGLX C7 XC8EXGLX ) TCoffee alignmentofHyDkk1/2/4withtheCRD2availableDkkmolecules(seeMaterialsand T-----SVL- AGVS--HKIRGA------RYST--VVL- --V--N------V--N------V --V A----PT AFQ AFQ A-- A-- A-- A-- A-- A-- A-- A-- A-- --Q------Q---- XC8GXGFX RELSLSTRNSV---- REPAINPHISI---- R---GLL R---GLL R--- R--- R--- R--- R--- R--- R--- R--- R--- SP HGE-WI----- H-- H-- H-- H-- H-- H-- H-- H-- H-- H--DTILGIAR H--SSALGLAR FGY----- FQL----- FLLTKQ-- FLLTKQ-- FPV----- FPV----- FWTK-I-- FWTK-I-- FWTK-I-- FWTK-I-- FWTK-I-- FWSK-I-- FWSK-I-- FWSK-I-- FWSK-I-- CRD2 CRD CRD2 C9 RX16C10 C9 VX12C10 C9 hydkk1/2/4 RX20C10 cells were strain C C C C C C C C C C C C C C C C C C C C C Q VRQPQLK ELRPNLDWP NSY NSY K K KK T T K K K K K K K K K THKAM TS P P PP P P P P P P P P P P P >175 221 LGVL M M LPV LPV V V V V V V V V V MAS VK VK LN LA LR LR LL LH LH LH LD LK LK LK 92 Dkk1/2/4-B hydra E E EY ENM EN E E E E Q Q Q E E E E ENSE ENSE Dkk1/2/4-A hydra GGP GA GA G G G G G G G G G G G ER ES E E E Q Q E E E Q Q Q Q expression. apicalregion of allregenerates was freeof the With theemergence oftentacles,30hours afterheadremoval, completely downregulated inthepresumptive head (Fig.4B). for wound closure(1 hour). Afterwards, hydkk1/2/4 (Fig. 4B,C),whichissignificantly longerthanthetimerequired sustained upto6hoursafterhead removal in50%ofallanimals cutting within30minutes(Fig.4).Highlevels ofexpression were hydkk1/2/4 is highlydynamic.Inanimalsbisectedat80%bodylength, During regeneration thetranscriptionalregulation of hydkk1/2/4 apoptosis. transcriptional downregulation, theactive retreatofglandcellsor remains unclearwhetherthisdecreaseinhydkk1/2/4 formation, reflectingtheexpression patternindetachedpolyps.It tentacles emerge, was hydkk1/2/4 in mid-bud stages(Fig.4A).Onlyinlatestages,just beforethe expression level ofhydkk1/2/4 L L V I L L V V V V V V V V V C C C C C C C C C C C C C C C C C C C C C NTKTDS------NPFLRP------GT DPTFAS------S-- GFR------DR GFR------DK G-PINLFHQVY G-PINFFRNVYVGAQ------V TVPAG-GL-AY HDPTSQLLDLI HDPASRLLDLI SRRGHKD---- SRRGHKD---- TKQRKKG---- TKLRKKG---- TKQRKKG---- TKHRRKG---- TKHKRKG---- TKHRRKG---- TKHKRKG---- SPKTLYG------I SVKTLYG------I Dkk3 nematostella Dkk3 hydra Dkk1/2/4 nematostella C expression was markedly upregulated atthesiteof TDGR------V S------VNHN TW-E-LEPEGALD TW-E-LEPDGALD T-AQA--PE-I T-AQA--PE-I S--HGL--E-I S--HGL--E-I S--HGL--E-I S--HGL--E-I S--HGL--E-I S--HGL--E-I T--HGL--E-I xrsinduringregeneration expression 93 Dkk3 mouse Dkk4 mouse Dkk3 human F FT-- F--A F F F F F F F F F YY YY Dkk4 human EHI E-- EPV Q Q Q Q Q Q Q Q Q Q KA R- R- R- R- R- R- R- R- R- R- R- R- K- Dkk2 human Dkk2 mouse 80 C C C C C C C C C C C C C C C C C C C C C G- -P E- G- G- GP GP -P -P -P D- D- D- D- D- H- Y- Y- D- -P -P in theentirebud, neitherinearly, nor 96 98 C C C C C C C C C C C C C C C C C C C C C EK RV ND EP EP KQ KQA GE AS AS GP GP AK AK AK GA GE GE GE ER ER 91 downregulated atthesiteofhead G G G G G G GFV G G G G G G G G G G G G G Dkk3 acropora 52 85 77 LK MT LT LE LE LQ LI LL LL LT LL LS LS LS LS LA LS LS LT LT 97 C C C C C C C C C C C C C C C C C C C C C 73 VDSV------VTKNEQVGE------GR QQDPATAQ------FT VKVRGTLTG------M VKVRGTLTG------M KNVGT--KG------KQVGL--FG------RKVRS-QHRKKRPMRM QPH------SHS-- QPH------SHS-- RSQVT---SNRQHSR- RSQLT---SNRQHAR- KVWKD--ATYSSKAR- KVWKD--ATYSSKSR- KVWKD--ATYSSKAR- RLQKGEFTTVPKTSR- RIQKD-HHQASNSSR- RIQKD-HHQASNSSR- RTQRGDGGKAS-RS-- -EGDRSIIGAITNTNY----GI EGDKT-IVGSITNTNF----GI Dkk1 human Dkk1 zebrafish 69 Dkk2 xenopus Dkk1 mouse Dkk1 xenopus Dkk3 amphioxus 100 Development 133(5) Dkk3 dictyostelium colipase mouse signal isdueto colipase human IRK--- VRR--- LH--YA IH--EI LRKERR L--VYM L--VYV LR-V-- LR-V-- LH-V-- LR-V-- LH-V-- LH--T- LH--T- LH--T- LH--T- hydkk1/2/4 hydkk1/2/4 C C C C C C C C C C C C C C C C C C C C C KAS VYV QLE VDH VDN L M VST - - Q Q Q Q Q Q Q Q Q LDS HDA was KE KE KP KP RI KI KI KI KI RH RH RH RH

DEVELOPMENT repair. dramatic increaseinhydkk1/2/4 head regenerates (datanotshown). Thus,regeneration inducesa but thenumberofhydkk1/2/4-expressing cellswas notashighin hydkk1/2/4 compared withcontroltissuefromthebodycolumn(Fig.4D,E). gland cellswas alsomarkedly elevated intheregenerating tipwhen the siteofregeneration. Theexpression level insmallandlarge doubled from0.33±0.12inuncutcontrolanimalsto0.62±0.06at % oftheentirebodylength)1hourafterheadremoval. Thisratio control animalsandinisolatedregenerating tips(correspond to10 ratio ofhydkk1/2/4-expressing cellstoepithelialinuncut gland cellsincreasedatthesiteofregeneration. We determinedthe macerated cellsrevealed thatthenumberof of theentirebodylength(Fig.4B,0.5and4hours).ISHon roughly 5-10epithelialcelldiameters,whichcorrespondsto10% Wnt-Dickkopf antagonisminHydra The region ofstimulated expression was alsoupregulated infootregeneration, hydkk1/2/4 expression atthesiteoftissue hydkk1/2/4-expressing expression measured of the siteofinjury(Fig.5A).Even afterwound closure,increasedlevels a strongstimulationof body columnandleaving theheadintact.Injuredanimalsexhibited possibilities. (1)Animalswereinjuredbymakingadeepcutintothe from theheadorbyinjurystimulusitself.We testedboth could beeithercausedbytheremoval ofinhibitingsignalsemanating The rapidincreaseofhydkk1/2/4 hydkk1/2/4 causes astimulationof In conclusion,ourresultssuggestthatitistheinjurysignalitself Newman’s observations, tiedregenerates didnotregenerate normally. (Fig.5B).Inagreementwith expression occurredinsuchpolyps (Newman, 1974).Nosignificantupregulation of tying aknotwiththinhairaroundthepolyp’s subhypostomalregion (2)Headsofpolypswereremoved withoutwounding by the wound. of aninhibitorysignalfromthehead. hydkk1/2/4 transcripts werestillrecognizableincellsatthesiteof is involvedintheinjuryresponse spermatogenesis. undergoing gametogenesis.(E-G)Oogenesis;(H) individual glandcellsare showninD.( epithelial animalsofstrainsf-1afterheatshock; Fig. 3. hydkk1/2/4 animal. (B hybridization. whole-mount insitu hydkk1/2/4 hydkk1/2/4 hydkk1/2/4- DoubleISH:hywnt3a ) (red). ( message duringheadregeneration regenerating tissue. body columntissue;reg, cells from macerated cells.bc,cellsofnormal ( silenced upregulation, lightbarsrepresent animals withhydkk1/2/4 sample): darkbarsrepresent determined (n apical 10%ofbodylengthwas regenerating tips.Expression inthe hydkk1/2/4- (see text).(C regenerating tipsforquantification cutting positiontoisolate times indicated;arrows indicatethe and allowedtoregenerate forthe decapitated at80%bodylength regeneration. Polypswere developing budstages;( regeneration. during buddingand Fig. 4. D transcripts foratleast6hours expression, andnottheremoval , RESEARCH ARTICLE C E , ) hydkk1/2/4 xrsinanalysisby expression D ) Residualglandcellsin hydkk1/2/4 expression hydkk1/2/4 expression dynamicsin ) Quantificationof =14 to24polypsper ( A (blue) and expression in ) Earlytolate expression. E-H) Animals ( hydkk1/2/4 A B ) head ) Whole 905

DEVELOPMENT activates thecanonicalWntsignalling pathway (Brounetal.,2005). Alsterpaullone specificallyinhibitsGsk3inHydra alsterpaullone (Knockaertetal.,2002;Leost2000). overexpression situationbytreatingpolypswiththekinaseinhibitor gene expression inanadditionalexperiment. We createdahywnt3a We testedtheputative antagonismbetweenWntand alsterpaullone-treated polyps Inhibition ofhydkk1/2/4expression in (seeDiscussion). expression Hydra hydkk1/2/4 spontaneously activated. Theseareunexpected data,indicatingthat hydkk1/2/4-depleted backgroundhywnt3a were formedalongthebodycolumn.Thissuggeststhatina ( and 1hour, respectively. dynamics ininjured (A)and ligated(B)animalsafter6hours expression We foundasignificantnumberofectopic shown). In47%ofallanalyzedhydkk1/2/4-depletedsf-1polyps control animalsthatwerestarved forthesametime (datanot domains insuchanimals(Fig.6H-J),but never innon-heat-shocked number ofregenerated tentaclesdroppedfrom6.1at regenerative capacityoftheseanimalswas severely reduced:the hydkk1/2/4 regeneration (Fig.6E-G).Mostregenerating pieceshadno animals at4hoursafterheadremoval (Fig.6B-D)andat9daysof regenerates lacked pieces regenerated ahead(Fig.6G).Althoughmost ofthese lacked (Fig.6C,D).Following regeneration, almostall‘regenerates’ cells their hydkk1/2/4-expressing cellsandcontinuedtoregenerate fairly (Fig. 6A).Non-heat-shocked controlpolypslostonlyabout20%of strongly correlatedtothedepletionof regeneration capacityofheat-shocked, starved sf-1animalswas gland cellsafterlong-termstarvation (Fig.6).We foundthatthe analyzed themutantstrainsf-1,whichloses To assessfurthertherelevance ofHyDkk1/2/4forregeneration, we capacity ofepithelialHydra hydkk1/2/4 906 Fig. 5. successful regenerates had regeneration. normally (att an average of270cellsatt cells. Thenumberofhydkk1/2/4-expressing cellswas reducedfrom n =55), several different-sized patchesofhywnt3a-expressing cells Figure 6shows representative examples of35-day-starved We alsotestedwnt hydkk1/2/4- hydkk1/2/4 RESEARCH ARTICLE and have afunctionintheregulation of + gland cellsarerequiredfornormalregeneration in cells 35 3.9±1.3tentaclesinsf-1and3.9±1.1105animals). , is involvedintheloss-of-regeneration (Fig. 6B),althoughafew hadupto50 + hydkk1/2/4 xrsindynamics. expression cells andfailed toregenerate properly. Very few expression inhydkk1/2/4- + 0 hydkk1/2/4 cells (Fig.6G),wepresumethatsuch to fourcellsatt hydkk1/2/4- ( A + , B hydkk1/2/4- cells atthestartof ) hydkk1/2/4- hywnt3a expression canbe 46 depleted animals. expressing gland (Fig. 6A).The hywnt3a t 0 and thereby hydkk1/2/4 hydkk1/2/4 to 0.4at expression expressing gene t 46 + (B-G) and upto9daysfordetermination ofregeneration behaviour. length andregenerated at least 4hourspriortoinsituhybridization 69, 178and140.Barsindicates.d. Animalswere cutat50%body as theaveragenumberoftentacles perregenerate (right); 22 withincreasing time.Efficiency ofheadregeneration wasmeasured glandcellsinbodycolumnpieces(left); positive polyps.( epithelial sf-1 Fig. 6. in regenerating epithelialsf-1polyps(5-9days regeneration). and 9days(E-G)afterheadremoval. ( H-J) Ectopic Double ISHindicatesthatthedownregulation of completely absentwhenectopictentacleswereformed(Fig.7B,C). hours aftertheonsetofalsterpaullonetreatment,andwere 7A,B). days (Fig.7A,B)andectopichead-like structuresafter8days(Fig. supplementary material),followed byectopictentacles after3-4 hywnt3a Alsterpaullone-treated polyps(24hours)formednumerousspot-like negatively regulated bycanonicalWntsignalling. between distinctheads(Fig.7C).Thus, hydkk1/2/4 column (Fig.S2inthesupplementarymaterial).Atlaterstages, from thecentreofhywnt3a hydkk1/2/4 hydkk1/2/4 andhywnt3a hydkk1/2/4 -expression domainsafter3days(seeFig.S2inthe expression was restoredinthetentacle-freetissue xrsininindividualregenerates, 4hours( expression transcription levels successively decreased24-48 A ) Quantificationofresidual -expression domainsinthebody expression inregenerating hydkk1/2/4 n Development 133(5) =6, 3,5,48,37and hywnt3a hydkk1/2/4 hydkk1/2/4 expression is n =20, 101, expression B-D) - starts

DEVELOPMENT though itwas lesseffective than formation couldbeblocked bytheco-injectionofhydkk1/2/4,even secondary axisformationinXenopus hydkk1/2/4, weco-injected not abolished.To furtherdemonstratetheWnt-inhibitoryactivity of enlarged, whereasposteriortrunkregions wereseverely reducedif phenotype (Fig.8A):anteriorstructures,like thecementgland,were in severe morphologicaldefectscomparabletotheDickkopf hydkk1/2/4 enhanced inhibitionoflatecanonicalWntsignalling.Injection and reducedposteriorstructures(Dickkopf phenotype)dueto xdkk1 As demonstratedbyGlinkaetal.(Glinkaal.,1998),endogenous possible, wehave chosenaheterologousapproachin upregulation was abolishedbyco-injectionwithxdkk1 gene, asreadout(Fig.8C).Embryoswereinjectedwith adirectWnttarget animal capassaywas performedusingsiamois, HyDkk1/2/4 ondownstream targets ofcanonicalWntsignalling,an vertebrate Dkk1andDkk4.To determinetheinhibitingeffect of clearly show thatHyDkk1/2/4isafunctional homologueof function. Becausehydkk1/2/4 Overexpression isaninstructive experiment toelucidateprotein HyDkk1/2/4 isfunctionallyactiveinXenopus Wnt-Dickkopf antagonisminHydra with anantisense experiments, determinedasthepercentage of squares) andtentacleformation (blackcircles) from three independent micrographs. (B animals. Fig. 7. indicate s.d.;solidlineindicatesthe lengthofAPtreatment. ( polyps andthenumberoftentacles from atotalof120polyps.Bars transferred toHydramediumfortimeindicated.( transcripts. Non-injected controlembryosshowed nodetectable mesodermal contaminationandinternalnormalization,respectively. Detection of then carriedoutonanimalcapstomeasuretheexpression of in presenceorabsenceof overexpression intheembryoleadstoenlarged headstructures hydkk1/2/4 Polyps were incubatedfor 24 hoursin5 mRNA ineachblastomereoffour-cell embryosresulted siamois ) Quantificationofhydkk1/2/4 brachyury hydkk1/2/4 xrsininalsterpaullone(AP)-treated expression was upregulated upon xdkk1 and probe (for detailsseeResults). and xdkk1 histone-4 overexpression inHydra hydkk1/2/4 embryos. Thissecondaryaxis (Fig. 8B).Theseexperiments with xwnt8 served ascontrolsfor expression (white hydkk1/2/4- hc induces xwnt8, which A mRNA. RT-PCR was ␮ ) Dark-field M APandthen injection, andthis Xenopus laevis. or xwnt8 expressing hydkk1/2/4. C ) ISH was not siamois. siamois mRNA construct (stage10 to10.5). Inhibition of induction byxdkk1 Dickkopf phenotype.( nue activationof a xdkk1 blocksxwnt8-induced h4, histone-4.(D was assayedbyRT-PCR. WE,wholeembryos;xbra, embryos. Fig. 8.Heterologous expression of protein, whichhasaninhibitoryfunctioninWntsignalling. an invertebrate, theidentificationofanovel Dickkopf-related 2000; BrottandSokol, 2002).Here,wedescribe,forthefirsttimein Frizzled complex; Dkk3doesnotinhibitWntsignalling(Wu etal., Dkk2 andDkk4areinhibitoryligandsoftheLrp5/Lrp6-Wnt- signalling invertebrates. Amongthefoursubfamilies onlyDkk1, Dickkopf proteinshave beenidentifiedasmajorinhibitorsofWnt evolution The originoftheDkkproteins inmetazoan DISCUSSION inhibiting Wntsignalling. indicate thatHyDkk1/2/4isaDickkopf homologuecapableof dose-dependent, Wnt-inhibitoryactivity. Thesefindingsstrongly (Korinek etal.,1997)(Fig.8D).Inthisassay, HyDkk1/2/4showed a This resultwas furtherverified usingtheTOPFLASH Wntreporter ( A siamois ) Overexpression of xdkk1 ) Co-injectionof or byhydkk1/2/4 induction byco-injectionofxdkk1 B ) Inhibitionofxwnt8-mediatedsecondaryaxis hydkk1/2/4 co-injection. ( hydkk1/2/4 RESEARCH ARTICLE or hydkk1/2/4 siamois-luciferase reporter (750 pgand3ng)or C in Xenopuslaevis ; Xenopus brachyury ) Animalcapassay. induces the or hydkk1/2/4 907

DEVELOPMENT expansion invitroandduringtheprocess oftissuerepair. Itprobably (MSCs) fromthebonemarrow secreteandrequireDkk1forcell process ofmammaliantissue repairthatmarrow stromalcells underestimated sofar. This roleofglandcellsinpatterning processeshascertainlybeen Hydra therefore presumethatanessentialtriggerforheadregeneration in second cutatthesiteofinjury(SugiyamaandFujisawa, 1977).We to theinjurystimulus:regenerates develop normallyaftersettinga lost and couldnotregenerate normally. Furthermore,animalsthathave minimal oreven withoutinjury, exhibit nohydkk1/2/4 removed bymeansoftheligaturetechnique(Newman, 1974),with indispensable forregeneration, becauseanimalswhoseheadswere any siteinthebodycolumn.Thisearlyupregulation seemstobe injury stimulus,asitalsooccurredbysimplycuttingtheanimals at mammalian Dkk1andDkk4.Thereisasecondhydkk1/2/4 vertebrate Dkk1,Dkk2andDkk4;thesimilarityishighestto 908 head removal. hydkk1/2/4 of during earlyregeneration. We foundarapidanddramaticincrease The mostobvious roleHyDkk1/2/4playsin gene hydkk1/2/4 ancestor ofcnidariansandbilaterians. dkk1/2/4 features commontothe regions. Thisfindingisconsistentwiththehypothesis thatthe DKK3 dkk4 2000; Luke etal.,2003).Hence,weconcludethat were duplicatedearlyinvertebrate evolution (PollardandHolland, other duplicatedgenefamilies. Geneswithinthisparalogyregion Leveugle etal.,2004;PollardandHolland,2000),numerous (Birnbaum etal.,2000;Coulier2000a;2000b; which alsocontainsFGFreceptorsandNKhomeoboxgenes sites liewithinthewell-characterized4/5/8/10paralogygroup, ( DKK2 (http://wolfe.gen.tcd.ie/dup/human5.28/) shows thathuman al., 2003;Lundin,1993;McLysaght etal.,2002) paralogon databaseHuman5.28ofWolfe andMcLysaght (Luke et analysis ofparalogyregions inthehumangenomeusing is supportedbythechromosomallocationofvertebrate Dkks.An analysis becauseofthehighdivergence ofthesequences,thisnotion Although thiscouldnotbeclarifiedwithcertaintybyphylogenetic and related tothenematocyte-specific hydkk3 hydkk1/2/4. presume thattheCRD1ofthisgeneislostin there isadkk1/2/4 hydrozoan-specific geneduplication).In than 79%nucleotideidentity(probablyaproductofrecent diversification ofcnidarian features ofchordate 2004). the DKK1 Interestingly, Prockop etal.(Prockop etal.,2003) found inthe The regeneration deficientmutantstrainreg-16 isalsosensitive Structurally, thenovel proteincorrespondstotheCRD2of Based ontheirconserved CRD2,allcnidarian hydkk1/2/4 Hydra hydkk1/2/4 dkk/1/2/4 most likely originatedbygeneduplication.Bycontrast,human maps to11p15.3,whichisnotpartofthesamesetparalogy is theearlyreleaseofDickkopf proteinsatthesiteofcutting. and RESEARCH ARTICLE maps to10q11, subgroup werelikely tohave beenpresentinthecommon EST database(CN559480; DKK4 There isalsoadkk3 message atthesiteofinjurywithinfirsthourafter -expressing cellsalsolosetheirregeneration capacity. is anearlyregeneration-responsive gene families duringearlymetazoanevolution. are locatedwithinrelatedchromosomalregions gene thatcontainstheN-terminalCRD1.We dkk3 DKK2 dkk upregulation was clearlyrelatedtothe Hydra and genes suggestsadeepsplitinto to 4q25andDKK4 dkk1/2/4 , gene inNematostella,whichis Nematostella Hydra in Nematostella vectensis genes. Thesignificant Hydra EST-Kiel) withmore Hydra dkk to 8p11).These and vertebrate dkk1 (Fedders etal., is itsfunction upregulation genes share , dkk2 gene in DKK1, Hydra dkk3 and ␤ Ruther, 2002a;Grotewold andRuther, 2002b).Anothercandidateis that isupregulated duringembryonicwounding (Grotewold and Jun), astress-responsive transcriptionfactor andactivator ofdkk1 transcripts. OnemoleculartriggercouldbeJun(alsoknown asc- signal actuallycausestheextremely fast upregulation of signals andtheinitiationoftissuerepair. Itisasyetunclearwhich HyDkk1/2/4 inHydra cutting (Holsteinetal.,1991;Holstein2003),weproposethat changes inthepatternofcellcycle andproliferationatthesiteof 2002b). Becauseregeneration in tissue (Grotewold andRuther, 2002a;Grotewold andRuther, apoptosis duringvertebrate limbdevelopment, andinUV-irradiated Dkk1 al., 2005;Gregory etal.,2003;Horwitz,2004;Prockop etal.,2003). interacts withWnt5a in theregulation of 2000). 2004). Bothgeneshave beenidentifiedin dependent manner(Gonzalez-Sanchoetal.,2005;Niida it mightsuppressWnt/ to bestablysilencedbyWnt/ Colon tumoursexhibit activated Wnt/ transcriptional downregulation of found during transient. Theshiftinthetranscriptionalregulation of early activation ofhydkk1/2/4 silencing of catenin pathway bytreatmentwithalsterpaullone,acomplete regeneration, i.e.inlatebud stagesandafteractivation oftheWnt/ generating thedistinctcompartmentsof Sancho etal.,2005).Thus,in and todkk3 dkk1 It was proposedthathypermethylationofthe downregulation of colipases, which facilitate theinteraction of pancreaticlipaseswith cnidarians suggestapossible scenario. TheCRD2issimilarto al., 1999;MaoandNiehrs,2003). been shown toinhibit ormodulateWntsignallingatall(Krupniket CRD1 andCRD2withoutaproteolytic cleavage site,andhasnot (Fig. 2C).Bycomparison,Dkk3 hasalinker sequencebetween represent anancientfeatureofthebasalDkk4withinvertebrates from thefull-lengthprotein(Krupniketal.,1999),whichmight was alsoshown thattheCRD2ofDkk4isproteolyticallycleaved is consistentwithsimilarfindingsfortheCRD2ofvertebrates. It rich domainofDkks,canexert aninhibitoryeffect onWntsignalling that HyDkk1/2/4,correspondingtothecarboxy-terminalcysteine- the canonicalWntpathway HyDkk1/2/4 blockstheinductionofdownstream target geneof can blockXWnt8-inducedsecondaryaxisformation(Fig.8B). (3) anteriorizing capacityin HyDkk1/2/4 andtheendogenousXDkk1have thesame similar Wnt-inhibitorypropertiestoendogenousXDkk1. (1) Xenopus We testedthefunctionofHyDkk1/2/4asaputative Wntinhibitor in antagonist ofWntsignalling HyDkk1/2/4 isanevolutionaryconserved the sharpboundaryunderneathhead. -Catenin, whichcanalsoactivate dkk1 Our datashow thatthereexists anadditionallevel ofcomplexity The evolutionary originofDickkopf proteinsremainsunclear, but silencing, similartothesilencingofotherWntinhibitorygenes has alsobeenfoundtobestronglyupregulated atthesitesof embryos. Inthisheterologoussystem,HyDkk1/2/4has (Suzuki etal.,2004;CaldwellGonzalez- hydkk1/2/4 Hydra dkk1 hydkk1/2/4 in thegrowth regulation ofMSCs(Gregory et has asimilarfunctionintheresponsetostress regeneration sharessimilaritieswiththe Ctnnsgaln ntebodycolumn, ␤-Catenin signallinginthe expression (Gonzalez-Sanchoetal.,2005). expression. Thisclearlyindicatesthatthe Xenopus embryogenesis.(2)HyDkk1/2/4 siamois ␤ Hydra, xrsinduringregeneration isonly expression -Catenin signallinginthehead,while Hydra expression. We foundduringlate dkk1 in animalcapassays.Thefact hydkk1/2/4 is accompaniedbydramatic Hydra’s body, asimpliedby ␤-Catenin signallingand in humancolontumours. Hydra expression inadose- dkk1 Development 133(5) expression appears (Hobmayer etal., promotor leadsto hydkk1/2/4 hydkk1/2/4 ␤ -

DEVELOPMENT be morecomplicated.Duringbudding, region. Itshouldbeemphasized,however, thatthisinteractionmight hywnt3a clusters allover thebodycolumn.Thispatchyupregulation of hydkk1/2/4-expressing cells, is alsoconsistentwiththefact that,inpolypsthatweredepletedof help tosuppresstheexpression ofWntgenesinthebody column.It with theideathatHyDkk1/2/4isinvolved inamechanismthatmight column, but notinthehypostomalregion (Fig. 3).Thisisconsistent whereas C.G., B.H.andT.W.H., unpublished)(Hobmayeretal.,2000), (Hobmayer etal.,2000). regularly ariseinatissuethatstronglyexpresses hypostomal region aroundthemouthof hywnt3a hywnt3a/hy Nevertheless, theexpression patternsof similar toDkk2inXenopus cells 1.5-to2-fold(seeFig.S3inthesupplementarymaterial), human LRP6-andmouseWnt1-inducedWntsignallingin293T In accordancewiththisidea,wefoundthatHyDkk1/2/4enhances the plasmamembrane,Dkk/Wntantagonismmayhave evolved. membranes. Fromsuchafacilitated interactionofWntandDkkat Dkk mayhave initiallyserved totetherWntligandstarget sequences (Nusse,2003;Willert etal.,2003).Thus,lipidbindingof hydrophobic thanispredictedfromtheirprimaryaminoacid Wnts arepalmitoylated proteinsandarethereforemuchmore associated withthecellsurface (Nusse,2003;Smolichetal.,1993). (Aravind andKoonin, 1998).Wntproteinsareindeedtightly proteins tointeractwithlipidsinorderregulate Wntfunction similarity, itwas proposedthattheCRD2ofDkkcouldhelpother lipid micelles(Krupniketal.,1999).Basedonthisstructural Wnt-Dickkopf antagonisminHydra Fig. 9. of neuronal andpro-neuronal genesin mature nematocytes;nmb, nematoblast;nmp,nematocyteprecursors; nv, neuronal cells. ( endoderm ispostulatedtofacilitate stemcellgrowth andtheformationofneuronal precursor cell precursors; cellsintheectoderm.nvp,nerve nm, The evidence foraninhibitoryfunctionin hydkk1/2/4- was never observed innormalpolypsexcept inthebudding hydkk1/2/4 n te n genesfrom and otherWnt ␤ -catenin/hytcf ectoderm A nmp nvp stem nmb nm nv cell expression pattern andneuronal pattern differentiation in expression is uniformlyexpressed intheentirebody hywnt3a endoderm dkk1/2/4 are mutuallyexclusive andsuggestive: embryos (BrottandSokol, 2002). gl-cells hywnt3a Hydra. Modified,withpermission, from Meinhardt (Meinhardt, 2002; Meinhardt, 2004). and Hydra is expressed insmallcell hywnt3a- hydkk1/2/4 B are expressed inthe Hydra Hydra hydkk1/2/4 expression spots (F. Rentzsch, are alsoco- is lessclear. hydkk1/2/4 neurogenic column body and Xenopus and thefact thathydkk1/2/4 done. However, thecharacteristicexpression patternof speculate, asnoexperiments addressingthisquestionhave been an additionalroleinsteady-stateanimals.Atthispoint,wecanonly process inHydra, itremainstobeclarifiedwhetherthemoleculehas Although Evolutionary considerations signalling inHydra. unclear astowhatextent HyDkk1/2/4actuallyantagonizesWnt molecule hastheabilitytoantagonizeWntsignalling,itremains assays inXenopus expressed duringearlyregeneration. Thus,althoughourfunctional hypothesis issupportedbytheexpression patternsofcnidarian Wnt evolution isthefacilitation of neuronal differentiation. This a primaryfunctionofBmpandWntantagonisminmetazoan C.G., B.H.andT.W.H., unpublished),supportingthehypothesisthat antagonist Chordinisalsoexpressed inthesametissue(F. Rentzsch, in theectodermof differentiation byinhibitingthe␤ HyDkk1/2/4 fromglandcellsintheendodermpromotesneuronal neurogenic region ofthe We thereforefavour thedefinitionofbodycolumnasbeing 1996; Lindgensetal.,2004;Smith1999;Technau etal.,1996). from multipotentinterstitialstemcellstakes place(Grensetal., 9 shows schematicallythatinthisregion theneuronaldifferentiation In Kazanskaya etal.,2000;Mukhopadhyay2001;Niehrs,2004). anterior anddorsalregion ofthevertebrate brain(Glinkaetal.,1998; (Glinka etal.,1997),andisnecessarytoactivate neuralgenesinthe vertebrates. Invertebrates, Dkk1caninducesecondaryheads state Hydra. Hydra, Hydra ( A suggests thathydkk1/2/4hasasimilarfunctioninsteady- ) Therelease ofHyDkk1/2/4 from glandcellsinthe hydkk1/2/4 hydkk1/2/4 B polyps asduringheadandneuronalinductionin ) Thehydkk1/2/4- ndraBilateria Cnidaria (tentacles) bmp 5-8b dkk1/2/4 wnt 3a provide clearevidence thattheHyDkk1/2/4 aboral dkk3 gsc bra oral ash chd otx zic nk2 is onlyexpressed inthebodycolumn.Figure is evidently requiredfortheregeneration Hydra Hydra. We presumethatthereleaseof can inducetheDkk1phenotypein expression domaincorrelates tothat body column(Fig.9A).TheBMP trunk -Catenin/Wnt signallingpathway RESEARCH ARTICLE posterior dkk1, -2,-3 anterior cluster wnt hox nk2 otx hydkk1/2/4 909

DEVELOPMENT Brott, B.K.andSokol,S.Y. D.,Coulier,Birnbaum, F., Pebusque,M. J.andPontarotti, P. http://dev.biologists.org/cgi/content/full/133/4/901/DC1 Supplementary materialforthisarticleisavailableat Supplementary material was fundedbytheDFG. Meinhardt forcriticallyreading anearlyversionofthe manuscript.Thiswork (Darmstadt) andKirstenWehner (Darmstadt)fortechnicalassistance;andHans for sharingunpublisheddataandhelpfuldiscussions;AnneLehmkuhl chromosome analysisformammalianDkks;UliTechnau (Darmstadt,Bergen) their essentialhelpwiththephylogeneticanalysisandforproviding the this manuscript.We alsothankRebeccaFurlongandPeterHolland(Oxford) for We thankCharlesN.David(Munich)forcriticallyreading thefinalversionof related expression patternsof Dkks, thechromosomallocationofvertebrate Dkks,theinversely lack ofphylogeneticinformationbecausethehighdivergence of regeneration inHydra The vitalroleoftheexpression ofhydkk1/2/4 Conclusion posterior andanteriorend. the bilateriantrunkevolved later, andintercalatedbetweenthe brain, whereproneuronalandneuronalgenesareexpressed, whereas idea, theHydra of bilaterians(Meinhardt,2002;Meinhardt,2004).Accordingtothat theoretical considerationsonmidlineformationduringtheevolution genes (Hobmayeretal.,2000;Kusserow etal.,2005)andby 910 Aravind, L.andKoonin,E.V. References Broun, M.,Gee,L.,Reinhardt, B.andBode,H.R. David, C.N. Coulier, F., D.(2000b).MetaHoxgene R.andBirnbaum, Popovici,C.,Villet, Coulier, F., Burtey, S.,Chaffanet, M.,Birg,F. D. andBirnbaum, Caldwell, G.M.,Jones,C.,Gensberg,K.,Jan,S.,Hardy, R.G.,Byrd, P., Davidson, G.,Mao,B.,delBarco, Barrantes,I.andNiehrs,C. Gonzalez-Sancho, J.M.,Aguilera,O., Garcia, J.M.,Pendas-Franco, N.,Pena, Glinka, A.,Wu, W., Delius, H.,Monaghan,A.P., Blumenstock,C.andNiehrs, Gawantka, V., Delius,H.,Hirschfeld,K.,Blumenstock,C.andNiehrs, Fedders, H.,Augustin,R.andBosch,T. C. Glinka, A.,Wu, W., Onichtchouk,D.,Blumenstock,C.andNiehrs, C. together withotherdevelopmental genes. Caenorhabditis ancestor ofcnidariansandbilaterians.Itthusappearsthat Dkk/Wnt antagonismwas alreadypresentinthelastcommon Xenopus the functionalantagonismofHyDkk1/2/4andcanonicalWntsin “Paleogenomics”: lookinginthepasttofuture. domain oftheWntantagonists–Dickkopfs. domains ofDickkopfproteins. clusters. J.Exp.Zool.288,345-351. 444. Ancestrally-duplicated paraHOXgeneclustersinhumans. Wnt antagonistsFRP1incolorectal tumorigenesis. Chughtai, S.,Wallis, Y., Matthews,G.M.andMorton,D. 2907-2916. head organizerinhydrainvolvesthecanonicalWntpathway. Arch. Dev. Biol.171,259-268. and isdownregulated inhumancoloncancer. Wnt antagonistDICKKOPF-1geneisa downstream targetofbeta-catenin/TCF C., Cal,S.,Garcia deHerreros, A.,Bonilla, F. and Munoz,A. functions inheadinduction. C. Xenopus. Nature389,517-519. Head inductionbysimultaneousrepression ofBmpandWntsignallingin Xvent-1. EMBOJ. AntagonizingtheSpemannorganizer:role ofthehomeoboxgene (1995). Genes Evol.214,72-80. expressed indifferentiating nematocytesinthebasalmetazoanHydra. Development 129,5587-5596. proteins interactwithDickkopf1 toregulate anteroposterior CNSpatterning. (1998). Dickkopf-1isamemberofnew familyofsecreted proteins and RESEARCH ARTICLE and (1973). Aquantitativemethodformacerationof Hydra 15, 6268-6279. body columnisthecounterparttovertebrate and insectshave losttheDkk-Wnt-antagonism, are allconsistentwiththehypothesisthat was unexpected. Furthermore,despitethe (2002). RegulationofWnt/LRPsignalingbydistinct Nature 391,357-362. (1998). Acolipasefoldinthecarboxy-terminal Mol. Cell.Biol. hydkk1/2/4 (2004). ADickkopf-3-related geneis 22, 6100-6110. Oncogene 24,1098-1103. and Curr. Biol. Cancer Res.64,883-888. 20) Formationofthe (2005). J. Exp.Zool. hywnt3a in glandcellsforhead Int. J.Oncol.17,439- 8 Hydra tissue. , R477-R478. Development (2000). (2002). Kremen in (2005). The 288, 21-22. (2000a). (2004). The and Hydra, Dev. (1997). Roux’s 132, Gregory, C.A.,Singh,H.,Perry, A.S.andProckop, D.J. Knockaert, M.,Wieking,K.,Schmitt,S.,Leost,Grant,K.Mottram,J. Holstein, T. W., Hobmayer, E.andTechnau, U. Horwitz, E.M. Holstein, T. W., Hobmayer, E.andDavid,C.N. Hobmayer, E.,Holstein,T. W. andDavid,C.N. Grotewold, L.andRuther, U. Gregory, C.A.,Perry, A.S.,Reyes,E.,Conley, A.,Gunn,W. G.andProckop, Leost, M.,Schultz,C.,Link,A.,Wu, Y. J.,Mandelkow, Z.,Biernat, E.M., Kusserow, A.,Pang,K.,Sturm,C.,Hrouda, M.,Lentfer, J.,Schmidt,H.A., Krupnik, V. E.,Sharp,J.D.,Jiang,C.,Robison,K.,Chickering,T. W., Korinek, V., Barker, N.,Morin,P. J.,vanWichen,D.,deWeger, R.,Kinzler, K. Klein, B.,LeMoullac,G.,Sellos,D.andVan Wormhoudt, A. Kazanskaya, O.,Glinka,A.andNiehrs,C. Jacobs, K.A.,Collins-Racie,L.Colbert,M.,Duckett,Evans,C., Jacobs, K.A.,Collins-Racie,L.Colbert,M.,Duckett,Golden-Fleet, Hobmayer, E.,Holstein,T. W. andDavid,C.N. Hobmayer, B.,Rentzsch,F., Kuhn,K.,Happel,C.M.,vonLaue,C.,Snyder, Hashimoto, H.,Itoh,M.,Yamanaka, Y., Yamashita, S.,Shimizu,T., Solnica- Grotewold, L.andRuther, U. Grens, A.,Gee,L.,Fisher, D.A.andBode,H.R. Leveugle, M., Prat, K., Popovici, C., Birnbaum, D.andCoulier,Leveugle, M.,Prat,K.,Popovici,C.,Birnbaum, F. Hansen, G.N.,Williamson,M.andGrimmelikhuijzen,C.J.P. Li, L.,Mao,J.,Sun,Liu,W. andWu, D. Decapoda): useinassessinggeneexpr cloning andsequencingoftrypsincDNAsfrom Penaeusvannamei(Crustacea, Biochem. CellBiol. cycling inhydra. and abatterycell. morphogenesis inhydraII:Formationofacomplexbetweensensorynervecell adult stemcellsfrom bonemarrow. signalling inhibitordickkopf-1isrequired forreentry intothecellcycleofhuman Chem. 277,25493-25501. Identification followingaffinity purificationonimmobilizedinhibitor. C., Kunick,C.andMeijer, L. evolutionarily conservedmodelsystemforregeneration? Biotechnol. 22,386-388. 895. morphogenesis inhydraI:Therole ofheadactivator. development: therole ofDickkopf-1. programmed celldeathandchondrogenesis duringvertebratelimb Biol. 180,473-488. homeobox gene,hasarolethebasalendofaxisinhydra. inpatterning and recovery ofadultstemcellsfrom bonemarrow. D. J. dependent kinase5/p25. Paullones are potentinhibitorsofglycogensynthasekinase-3beta and cyclin- Bibb, J.A.,Snyder, G.L., Greengard, P., Zaharevitz, D.W. etal. Nature 433,156-160. (2005). UnexpectedcomplexityoftheWntgenefamilyinaseaanemone. Technau, U.,vonHaeseler, A.,Hobmayer, B.,Martindale,M.Q.etal. 238, 301-313. Functional andstructuraldiversityofthehumanDickkopfgenefamily. Amaravadi, L.,Brown, D. E., Guyot,D.,Mays,G.,Leiby, K.etal. 275, 1784-1787. activation byabeta-catenin-Tcf complexinAPC–/–coloncarcinoma. W., Vogelstein, B.andClevers,H. 127, 4981-4992. dickkopf1 inprechordal platespecificationandneuralpatterning. peptides. MethodsEnzymol. (1999). AgeneticselectionforisolatingcDNAclonesthatencodesignal Golden-Fleet, M.,Kelleher, K.,Kriz,R.,LaVallie, E.R.,Merberg,D.etal. Gene (1997). AgeneticselectionforisolatingcDNAsencodingsecreted proteins. Kelleher, K.,Kriz,R.,LaVallie, E.R.,Merberg,D.,Spaulding,V., etal. axis formationinthediploblasticmetazoanHydra. P., Rothbacher, U.andHolstein,T. W. 152. forebrain specificationandaxialmesendodermformation. Krezel, L.,Hibi,M.andHirano,T. Res. colocalization. CellTissue probes forfivedifferent Hydraneuropeptide preprohormones: evidencefor color double-labelinginsituhybridizationofwhole-mountHydrausingRNA EMBO J. regulated byBmpsignalingandc-Junmodulatesprogrammed celldeath. 2323. hypothesis ofsuccessivegeneexpansions. Phylogenetic analysisofCionaintestinalis genesuperfamiliessupportsthe independently ofdishevelled. of Dickkopf-2activatescanonicalWntsignaling pathwayviaLRP-6 (2005). Dkk1-derivedsyntheticpeptidesandlithiumchlorideforthecontrol 198, 289-296. 21, 966-975. (2004). Dkk1-mediatedexpansionofadultstemcells. Dev. Biol. Development 109,897-904. 28, 551-563. Eur. J.Biochem.267,5983-5994. 148, 602-611. 303, 468-479. (2002a). Bmp,FgfandWntsignallingin (2002b). TheWntantagonistDickkopf-1is J. Biol.Chem277,5977-5981. (2002). IntracellularTargets ofPaullones. 301, 245-253. J. Biol.Chem. (2000). ZebrafishDkk1functionsin (1997). Constitutivetranscriptional Int. J.Dev. Biol.46,943-947. ession duringthemoultcycle. (2000). WNTsignallingmoleculesactin (2002). Secondcysteine-richdomain J. Mol.Evol.58,168-181. (2000). Therole ofXenopus (2003). Cnidarians:an (1991). Pattern ofepithelialcell (1991). Pattern (1990b). Tentacle (1990a). Tentacle (1996). CnNK-2,anNK-2 278, 28067-28078. Nature 407,186-189. J. Biol.Chem. Development 109,887- Development 133(5) Dev. Dyn. (2003). TheWnt Dev. Biol. (1996). Molecular (2000). Two- Development 226, 257-267. 280, 2309- (2004). Trends (2000). 217, 138- J. Biol. Int. J. Science (1999). Gene Dev.

DEVELOPMENT Niehrs, C. Newman, S.A. Nelson, W. J.andNusse,R. Mukhopadhyay, M.,Shtrom, S.,Rodriguez-Esteban,C.,Chen,L.,Tsukui, T., Martinez, D.E.,Dirksen,M.L.,Bode,P. M.,Jamrich,Steele,R.E.and Niida, A.,Hir Mao, B.andNiehrs,C. Lundin, L.G. Luke, G.N.,Castro, L.F., McLay, K.,Bird, C.,Coulson,A.andHolland,P. W. Logan, C.Y. andNusse,R. Lindgens, D.,Holstein,T. W. andTechnau, U. Wnt-Dickkopf antagonisminHydra Meinhardt, H. Meinhardt, H. McLysaght, A.,Hokamp, K.andWolfe, K.H. Martin, V. J.,Littlefield,C.L.,Archer, W. E.andBode,H.R. Pollard, S.L.andHolland,P. W. Philipp, I.,Holstein,T. W. andHobmayer, B. Nusse, R. Marcum, B.A.andCampbell,R.D. Mao, B.,Wu, W., Davidson,G.,Marhold,J.,Li,M.,Mechler, B.M.,Delius,H., Mao, B.,Wu, W., Li,Y., Hoppe,D.,Stannek,P., Glinka,A.andNiehrs,C. organizer. Morphol. 31,541-555. possible relation totherole ofthecutendinregeneration. cadherins pathways. morphogenesis inthemouse. (2001). Dickkopf1isrequired forembryonicheadinduction andlimb Gomer, L.,Dorward, D.W., Glinka,A.,Grinberg,Huang,S.P. etal. paralogous chromosomal regions inmanandthehousemouse. and disease.Annu.Rev. CellDev. Biol. Nat. Rev. Neurosci. bilateral bodyplan:anoldbecameayoungbrain. duplication duringearlychordate evolution. during axisformationandheadspecificationinhydra. Bode, H.R. Embryogenesis inhydra. signaling, isatargetofthebeta-catenin/TCFpathway. Sugano, S.andAkiyama,T. 1-19. Proc. Natl.Acad.Sci.USA (2003). DispersalofNKhomeoboxgeneclustersinamphioxusandhumans. nematocytes. Development131,191-201. of thezic/odd-paired gene,isinvolvedintheearlyspecificationofsensory and interstitialcells. Wnt/LRP6 signaling. in humangenomeancestry. differentiation. GeneExpr. Patterns of thec-JunNH2-terminalkinasegenefamily, isexpressed duringnematocyte signaling mechanismsatthecellsurface. 8526. receptors thatregulate Wnt/beta-cateninsignalling. Hoppe, D.,Stannek,P., Walter, C.etal. 411, 321-325. (2001). LDL-receptor-related protein 6isareceptor forDickkopfproteins. (2003). WntsandHedgehogs:lipid-modifiedproteins andsimilaritiesin (2004). RegionallyspecificinductionbytheSpemann-Mangold Nat. Rev. Genet. oko, T., Kasai,M.,Furukawa,Y., Nakamura,Y., Suzuki,Y., (1993). Evolutionofthevertebrategenomeasreflected in (1997). Budhead,aforkhead/HNF-3homologue,isexpressed (2004). Different strategiesformidlineformationinbilaterians. (2002). Theradial-symmetrichydraandtheevolutionof (1974). Theinteractionoftheorganizingregions inhydraandits 5 J. CellSci.29,17-33. Gene 302,179-183. , 502-510. Science (2003). Kremen2 modulatesDickkopf2activityduring Biol. Bull.192,345-363. 100, 5292-5295. (2004). TheWntsignalingpathwayindevelopment 5 (2004). ConvergenceofWnt,beta-catenin,and Curr. Biol.10,1059-1062. , 425-434. 303, 1483-1487. Dev. Cell1 (2004). DKK1,anegativeregulator ofWnt (2000). Evidencefor14homeoboxgeneclusters 5 (1978). DevelopmentofHydralackingnerve , 397-402. 20, 781-810. , 423-434. Development 130,5297-5305. (2002). Kremen proteins are Dickkopf Nat. Genet.31,200-204. (2005). HvJNK,aHydramember (2002). Extensivegenomic (2004). Hyzic,theHydrahomolog Nature 417,664-667. Dev. Biol. Oncogene BioEssays 24,185-191. J. Embryol. Exp. J. Embryol. (1997). 192, 523-536. Genomics 16, 23, 8520- Nature Semenov, M.V., Tamai, K.,Brott, B.K.,Kuhl,M.,Sokol,S.andHe,X. Schmidt, T. andDavid,C.N. Schmidt, H.A.,Strimmer, K.,Vingron, M.andvonHaeseler, A. Rupp, R.A.,Snider, L.andWeintraub, H. Suzuki, H.,Watkins, D.N.,Jair, K.W., Schuebel, K.E.,Markowitz,S.D., Sugiyama, T. andFujisawa,T. Shinya, M.,Eschbach,C.,Clark,Lehrach,H.andFurutani-Seiki,M. Rentzsch, F., Hobmayer, B.andHolstein,T. W. Prockop, D.J.,Gregory, C.A.andSpees,J.L. V Technau, U.andBode,H.R. Takano, J.andSugiyama,T. Suzuki, Y., Makino,A.andMae,T. Sugiyama, T. andFujisawa,T. Smolich, B.D.,McMahon,J.A.,A.P. andPapkoff, J. Smith, K.M.,Gee,L.,Blitz,I.L.andBode,H.R. Willert, K.,Brown, J.D.,Danenberg,E.,Duncan,A.W., Weissman, I.L., Wu, W., Glinka,A.,Delius,H.andNiehrs,C. Technau, U.andHolstein,T. W. inh, leS.andVon Haeseler, A. computing. BioInformatics PUZZLE: maximumlikelihoodphylogeneticanalysisusingquartetsandparallel deficient strain. mechanisms inHydra.II.Isolationandcharacterizationofaninterstitialcell- Head inducerDickkopf-1isaligandforWntcoreceptor LRP6. development. J.CellSci.85,197-215. the nuclearlocalizationofXmyoD. 12. kinase 3hasaproapoptotic functioninHydragametogenesis. signalling incolorectal cancer. al. Dong Chen,W., Pretlow, T. P., Yang, B.,Akiyama,Y., Van Engeland,M.et Exp.Morphol.42,65-77. Embryol. mechanisms inHydra.III.Characterisationofaregeneration deficientstrain. during headformationinHydra. the prechordal theanteriorneuralplate. plateandpatterns (2000). ZebrafishDkk1,inducedbythepre-MBT Wntsignaling,issecreted from 951-961. Natl. Acad.Sci.USA100,11917-11923. thepowerofadultstemcellstorepairgene therapy:harnessing tissues. and stoppingintime.Mol.Biol.Evol. Hydra. Dev. Biol. continuous precursor migrationintheformationofpedunclenervenet Exp.Morphol.78,141-168. slow-budding strain(L4).J.Embryol. mechanisms inhydra.VIII.Head-activationandhead-inhibitionpotentialsofa 1575-1579. RNA from riceleavesatdifferent agesusingbenzylchloride. 4 family proteins are secreted andassociatedwiththecellsurface. 404. the Otxgenefamily, hasarole incellmovementhydra. modified andcanactasstemcellgrowth factors. Reya, T., Yates, J.R.,3rd andNusse,R. Biol. 10,1611-1614. between dickkopf1anddickkopf2regulates Wnt/beta-cateninsignalling. , 1267-1275. (2004). EpigeneticinactivationofSFRPgenesallowsconstitutiveWNT J. CellSci.29,35-52. 177, 599-615. 18, 502-504. (1983). Geneticanalysisofdevelopmental (1986). GlandcellsinHydra:cellcyclekineticsand (1999). HyBra1,aBrachyuryhomologue,acts (1978). Geneticanalysisofdevelopmental (1977). Geneticanalysisofdevelopmental Nat. Genet.36,417-422. (1996). Phenotypicmaturationofneurons and (2004). IQPNNI:movingfastthrough tree space Development 126 Genes Dev. (2001). Anefficient methodforextractionof 21, 1565-1571. RESEARCH ARTICLE (2003). Wntproteins are lipid- (1994). Xenopusembryosregulate (2000). Mutualantagonism (2003). Onestrategyforcelland 8 20) Glycogensynthase (2005). , 1311-1323. (1999). CnOtx,amemberof Nature 423,448-452. , 999-1010. Dev. Biol. Mech. Dev. J. Exp.Bot.52, Curr. Biol.11, Dev. Biol. Mol. Biol.Cell (2002). TREE- (1993). Wnt 212, 392- 98, 3-17. 278, 1- Proc. (2001). Curr. 911 J.

DEVELOPMENT