Non-canonical Notch signaling represents an ancestral mechanism to regulate neural differentiation

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Title:
Non-canonical Notch signaling represents an ancestral mechanism to regulate neural differentiation
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English
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Layden, Michael J.
Martindale, Mark Q.
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Bio-Med Central (Evo Devo)
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Abstract:
Background: Cellular differentiation is a critical process during development of multicellular animals that must be tightly controlled in order to avoid precocious differentiation or failed generation of differentiated cell types. Research in flies, vertebrates, and nematodes has led to the identification of a conserved role for Notch signaling as a mechanism to regulate cellular differentiation regardless of tissue/cell type. Notch signaling can occur through a canonical pathway that results in the activation of hes gene expression by a complex consisting of the Notch intracellular domain, SuH, and the Mastermind co-activator. Alternatively, Notch signaling can occur via a non-canonical mechanism that does not require SuH or activation of hes gene expression. Regardless of which mechanism is being used, high Notch activity generally inhibits further differentiation, while low Notch activity promotes differentiation. Flies, vertebrates, and nematodes are all bilaterians, and it is therefore unclear if Notch regulation of differentiation is a bilaterian innovation, or if it represents a more ancient mechanism in animals. Results: To reconstruct the ancestral function of Notch signaling we investigate Notch function in a non-bilaterian animal, the sea anemone Nematostella vectensis (Cnidaria). Morpholino or pharmacological knockdown of Nvnotch causes increased expression of the neural differentiation gene NvashA. Conversely, overactivation of Notch activity resulting from overexpression of the Nvnotch intracellular domain or by overexpression of the Notch ligand Nvdelta suppresses NvashA. We also knocked down or overactivated components of the canonical Notch signaling pathway. We disrupted NvsuH with morpholino or by overexpressing a dominant negative NvsuH construct. We saw no change in expression levels for Nvhes genes or NvashA. Overexpression of Nvhes genes did not alter NvashA expression levels. Lastly, we tested additional markers associated with neuronal differentiation and observed that non-canonical Notch signaling broadly suppresses neural differentiation in Nematostella. Conclusions: We conclude that one ancestral role for Notch in metazoans was to regulate neural differentiation. Remarkably, we found no evidence for a functional canonical Notch pathway during Nematostella embryogenesis, suggesting that the non-canonical hes-independent Notch signaling mechanism may represent an ancestral Notch signaling pathway. Keywords: Notch, Nvnotch, Nematostella vectensis, Cellular differentiation, Evolution
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Layden and Martindale EvoDevo 2014, 5:30 http://www.evodevojournal.com/content/5/1/30; Pages 1-14
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doi:10.1186/2041-9139-5-30 Cite this article as: Layden and Martindale: Non-canonical Notch signaling represents an ancestral mechanism to regulate neural differentiation. EvoDevo 2014 5:30.

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© 2014 Layden and Martindale; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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RESEARCHOpenAccessNon-canonicalNotchsignalingrepresentsan ancestralmechanismtoregulateneural differentiationMichaelJLayden1andMarkQMartindale2*AbstractBackground: Cellulardifferentiationisacritical processduringdevelopmentofmulticellularanimalsthatmustbetightly controlledinordertoavoidprecociousdifferentiationorfailedgenerationofdifferentiatedcelltypes.Researchinflies, vertebrates,andnematodeshasledtotheidentificationofaconservedroleforNotchsignalingasamechanismto regulatecellulardifferentiationregardl essoftissue/celltype.Notchsignalingcanoccurthroughacanonicalpathwaythat resultsintheactivationof hes geneexpressionbyacomplexconsistingoftheNotchintracellulardomain,SuH,andthe Mastermindco-activator.Alternatively,Notchsignalingcanoccurviaanon-canonicalmechanismthatdoesnotrequire SuHoractivationof hes geneexpression.Regardlessofwhichmechanis misbeingused,highNotchactivitygenerally inhibitsfurtherdifferentiation,whilelowNotchactivityprom otesdifferentiation.Flies,vertebrates,andnematodesareall bilaterians,anditisthereforeuncleari fNotchregulationofdifferentiationisabilaterianinnovation,orifitrepresentsa moreancientmechanisminanimals. Results: ToreconstructtheancestralfunctionofNotchsignalingw einvestigateNotchfunctioni nanon-bilateriananimal, theseaanemone Nematostellavectensis (Cnidaria) Morpholinoorpharmacologicalknockdownof Nvnotch causes increasedexpressionoftheneuraldifferentiationgene NvashA .Conversely,overactivationof Notchactivityresultingfrom overexpressionofthe Nvnotch intracellulardomainorbyoverexpressionoftheNotchligand Nvdelta suppresses NvashA. WealsoknockeddownoroveractivatedcomponentsofthecanonicalNotchsignalingpathway.Wedisrupted NvsuH withmorpholinoorbyoverexpressingadominantnegative NvsuH construct.Wesawnochangeinexpressionlevelsfor Nvhes genesor NvashA. Overexpressionof Nvhes genesdidnotalter NvashA expressionlevels.Lastly,wetestedadditional markersassociatedwithneuronaldifferentiationandobservedthatnon-canonicalNotchsignalingbroadlysuppresses neuraldifferentiationin Nematostella Conclusions: WeconcludethatoneancestralroleforNotchinme tazoanswastoregulatene uraldifferentiation Remarkably,wefoundnoevidenceforafunctionalcanonicalNotchpathwayduring Nematostella embryogenesis, suggestingthatthenon-canonical hes -independentNotchsignalingmechanismmayrepresentanancestralNotch signalingpathway. Keywords: Notch, Nvnotch Nematostellavectensis ,Cellulardifferentiation,Evolution *Correspondence: mqmartin@whitney.ufl.edu2WhitneyLaboratoryforMarineBioscience,UniversityofFlorida,St Augustine,FL32080,USA Fulllistofauthorinformationisavailableattheendofthearticle 2014LaydenandMartindale;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsofthe CreativeCommonsAttributionLicense(http://creativecommons.org/licenses/by/4.0),whichpermitsunrestricteduse, distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycredited.TheCreativeCommonsPublic DomainDedicationwaiver(http://creativecommons.org/publicdomain/zero/1.0/)appliestothedatamadeavailableinthis article,unlessotherwisestated.LaydenandMartindale EvoDevo 2014, 5 :30 http://www.evodevojournal.com/content/5/1/30

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BackgroundMetazoandevelopmentrequi resamechanismtocontrol thebalancebetweenpoolsofcellsthatareabletodifferentiateintodistinctspecializedcelltypesandcellsthatremain undifferentiatedtocontributetogrowthordifferentiateata latertime.Identifyingthemechanismsthatregulatethis balanceprovidesinsightsintotheevolutionofanimaldevelopmentalprogramsandcluesastotheputativemolecularchangesthatunderscoredtheemergenceofmetazoans fromsingle-celledan cestors.FunctionalstudieshaveidentifiedtheNotchsignalingpat hway(describedbelow)asa conservedregulatorofcellula rdifferentiation,butthisis onlyknownfrombilateriananimals.Thereareatleastfour metazoanlineagesthatdivergedpriortotheemergenceof bilaterians.Theyarethectenophores,poriferans,placozoans,andcnidarians,withcnidariansbeingthemostclosely relatedtobilaterians[1-4]. Notchsignalingisimplicatedasaregulatorofcellular differentiationinmultiplebilateriantissuetypesincluding neural,blood,epidermal,endothelial,muscle,andbone [5-10].AwellknownandstudiedexampleofNotchregulationofdifferentiationisinbilaterianneurogenesis.Duringtheformationofthe Drosophila ventralnervecord, cellswithhighNotchactivitysuppresstheformationofa neuroblastprogenitorcellinfavorofmaintainingundifferentiatedneuralectodermfate[11,12].Similarly,invertebrateneurogenesis,highNotchactivityinneuralstem cellsactstomaintainaneuralstemcellfateidentity,while lownotchactivityindaughtercellspromotesneuronal differentiation[8,13].Inbothvertebrateandinvertebrate neurogenesis,Notchinhibitsneurogenesisbyrepressing theexpressionofproneuralgenetranscriptionfactors [11,13-15].Proneuralgenetranscriptionfactorsarebasic helix-loop-helixtranscriptionfactorsthatbelongtoeither the achaete-scute or atonal genefamilies[15]. TherearetwomechanismsbywhichNotchcanregulate differentiation.Theyarethecanonical[16]andnoncanonicalpathway[16,17].Thecoreminimalcomponentssharedbybothpathwaysarethe notch receptor, delta ligand,andthe -secretaseandADAMprotease cleavagecomplexes[9,16].Additionalcorecomponents requiredspecificallyforcanonicalNotchsignalingare hes suH smrt, and mastermind [9,18,19].Bothcanonicalandnon-canonicalpathwaysaretypicallyinitiated bythebindingofDeltatotheNotchreceptor,whichinducescleavageandreleaseoftheNotchintracellulardomainbyAdamproteaseand -secretasecleavageevents. Inthecanonicalpathway,theNotchintracellulardomain interactswithSuHanddisplacestheSMRTco-repressor normallyboundtoSuHandrecruitstheMastermind transcriptionalco-activator.Thiscomplextheninduces expressionofthe hes genes,whichregulateexpressionof Notchtargets,suchastheproneuralgenes[9,16,18].NoncanonicalNotchsignalingbypassesinteractionswithSuH andactivationof hes geneexpression,toregulatetarget geneexpressionviaalternativemechanisms[16,20]. GenomicanalysesofcoreconservedNotchcomponentssuggestthatthecoreNotchpathwayevolvedspecificallyinthemetazoanlineage. notch,delta, and hes homologsdonotexistoutsideofthemetazoans[19,21], andallfivemajoranimalcladespossessaNotchhomolog.Thectenophoresaretheonlynon-bilaterianslackingadefinitiveDeltaligand,althoughtheypossessmany Delta-likeproteinsthatcouldpotentiallyactivateNotch ligands[1,19],and,recently,Delta-likegeneshavebeen identifiedtoactivateNotchinbilaterians[22].Ofthe remainingcoreconservedgenes,themembersofthe -secretasecomplexandADAMproteasesallpredate themetazoandivergence[19].KeyregulatorycomponentsofthecanonicalNotchpathwaywerenotpresent untiltheemergenceofthecnidarian-bilateriancommon ancestor.The suH geneevolvedpriortotheearliest metazoans,butthe mastermind co-activatorevolvedin thecnidarian-bilaterianancestor[19],andtheSMRTcorepressorisnotpresentoutsideofthebilaterianlineage [1,3,4,19,23,24].Thus,althoughNotchsignalingevolved earlyinthemetazoanlineage,itisunclearifthecanonicalornon-canonicalpathwayrepresentstheancestral Notchsignalingmechanism. Onewaytodeterminetheevolutionofaparticular signalingpathwayistodeterminehowitfunctionsin phylogeneticallyinformativeextantanimalsthatallow reconstructionoftheancestralrole(s)ofthepathwayat deepevolutionarynodeswithintheanimalphylogeny. However,gene-specificfunctionalstudiesaddressing Notchsignalinginnon-bilaterianmetazoansiscurrentlylacking.Characterizationoftheexpressionpatternsof Notchsignalingcomponentsandpharmacologicaldisruptionof -secretaseimplicateNotchasaregulatorof differentiationinthenon-bilaterians[25-28].First,inthe poriferan Amphimedonqueenslandica Amqdelta homologsareexpressedindifferentiatingcelltypesthroughout development[25].Inthecnidarians,treatmentwithDAPT, whichinhibits -secretasecleavageoftheNotchintracellulardomain[26,28,29],increasesexpressionofdifferentiated cellmarkers(particularlyneuronalmarkers)[26].Oneof thesemarkersis NvashA ,whichisan achaete-scute gene familyhomologknowntoregulateembryonicneurogenesis in Nematostella [30].Marlowandcoworkers[26]alsoinvestigatedtheroleof NvsuH ondevelopmentofthecnidocytes,whicharethestingingcellsin Nematostella ,usinga spliceblockingmorpholino(MO)againstthe NvsuH gene andadominantnegativeconstruct.Theyfoundthatmaturecnidocyteswerelackingin Nematostella planulawhen NvsuH functionwasreducedandthatthisphenotypewas similartothereductionincnidocytesresultingfromtreatinganimalswithDAPT[26].However,inthisstudythe authorsdidnotcompareotherphenotypesresultingfromLaydenandMartindale EvoDevo 2014, 5 :30 Page2of14 http://www.evodevojournal.com/content/5/1/30

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DAPTtreatmentwithadisruptionof NvsuH .DAPTtreatmenthasbeenfoundtoinhibitmaturation,butnotspecification,ofcnidocytesinpolypsofthehydrozoancnidarian Hydra [28].Takentogether,thepreviousstudiesinnonbilaterianssuggestthatNotchsignalingplayedarolein regulatingtheprocessofneuronalcelldifferentiationin thecnidarian-bilaterianancestor,butthelackofdetailed gene-specificstudiesdoesnotclarifyifthecanonicalor non-canonicalNotchsignalingpathwayrepresentsthe ancestralmechanismofNotchsignaling. Herewetakeadvantageoftheabilitytoconductfunctionalgeneticexperimentsinthecnidarianseaanemone Nematostellavectensis tocharacterizetheroleofNotch signalingduringembryonicdevelopment.Weshowthat Notchactivityin Nematostella suppressesexpressionof NvashA -dependentneuraldifferentiationmarkers[30], andthatthesuppressionof NvashA -dependentneural markersoccursviaspecificinhibitionof NvashA expressionby Nvnotch .WealsoshowthatNotchactivitybroadly inhibitsexpressionofneuronaldifferentiationmarkersfor otherneuralcelltypesinthe Nematostella embryo.AlthoughsomecomponentsofcanonicalNotchsignaling arepresentinthe Nematostella genome,ourexperiments indicatethatinhibitionofdifferentiatedcellmarkersoccursviathenon-canonical( Nvhes -independent)mechanismduringembryonicdevelopment.MethodsGenesusedinthisstudyThegenesusedinthisstudywerepreviouslypublished [26,30,31].Embryomanipulationsand insitu analysisAllembryosweregrowntoeitherearlygastrulastages, byraisinganimalsfor24hourspost-fertilization(hpf)at 17C,ortolategastrulastages,byraisinganimalsfor24 hpfat25C.Allfixation, insitu probesynthesis,and in situ hybridizationswerecarriedoutaspreviouslydescribed[30,32,33].ImageswereobtainedonaZeissImager M2inconjunctionwiththeAxiocamHRcandZenPro software(CarlZeissLLC,Thornwood,NY,USA).For gastrulastageanalysis,10 MDAPTtreatmentwasbegun 3hpfaspreviouslydescribed[26].Forlarvalstageanalysis ofDAPT-treatedanimals,animalswereallowedtogrowto desiredstage(either24or48hpfat25C)andthen, animalswereincubatedin10 MDAPTfor24hours.mRNAinjectionsThe Nvnicd fusionconstructwasgeneratedbyPCR amplifyingtheintracellulardomainof Nvnotch using (Forward5 CACCATGGTTGTTGTGCTCGCAGGCG GTAAG3 andReverse5 GTCTGATAATAACTCCA CTATGTC3 )PCRprimers.ThePCRproductwasthen cloned Nvnicd inframeand5 tothe venus coding sequenceusingtheGatewaycloningvectorsystem (Invitrogen,Carlsbad,CA,USA).Fulllength Nvdelta wasamplifiedusingthe(Forward5 CACCATGCAGC TACTACCACTCCAGCCATCAC3 andReverse5 ATATTTCCACTTCCACTTCTTGCCAG3 )primers andclonedinframe5 tothe venus codingsequence. Nvhes2 and Nvhes3 constructswereclonedusing(Forward5 CACCATGGAAAAAATGCGGAGGGCGAG 3 andReverse5 TCAAATTGTCCTCCCCATTCAC 3 )and(Forward5 CACCGCCGTTGACTGCATCG ATAGC3 andReverse5 TCACCATGGGCGCCAC AGTG)PCRprimers,respectively.Theywereboth clonedinframetothe3 endofthe venus coding sequence.Injectionconcentrationofthe Nvnicd:venus was300ng/ l.Injectionconcentrationof Nvdelta:venus was900ng/ l.Injectionconcentrationsof venus:hes2 and venus:hes3 were300ng/ land150ng/ l,respectively.Weinjectedthepreviouslypublished NvashA: venus and NvsuHDN:venus mRNAasdescribedpreviously(Laydenandcolleagues[30]andMarlowandcolleagues[26]).mRNAwaspreparedandinjectedasprevious described[30,34].Animalsweresortedpriortoanalysisto identifyembryosexpressingtheVenusreporterproteinand toeliminatethenon-expressinganimals.MorpholinoinjectionsFluoresceinlabeled NvashA translation-blockingMOwas injectedaspublished[30]. NvSuH splice-blockingMO wasinjected,andspliceblockingwasobservedaspreviouslydescribed[26].An Nvnotch splice-blockingMO(5 GTCCTTTGATTTCGTACCTCATGGA3 )(GeneTools Inc.,Philomath,OR,USA)that resultsinatruncationofthe Nvnotch intracellulardomainand Nvdelta splice-blocking MO(5 GCGACCTGACAAGAACAGTGAAGTC3 ) (GeneToolsInc.)thatremovestheexoncontainingthe MNNLdomainweredesignedandinjectedat1mMand 600nM,respectively.Splice-blockingefficiencywasestimatedusingPCRandDNAelectrophoresistoobserve shiftsinthesizeofthewild-typeormorphantmRNA.A controlMO(5 AGAGGAAGAATAACATACCCTGTCC 3 )wasalsoinjectedataconcentrationof1mMandgene expressionwascomparedtouninjectedcontrolanimals. Animalsweresortedafterinjectiontoeliminatetheuninjectedanimalsasindicatedbythelackoffluorescence.QuantificationofcellnumberTocountthenumberof NvashA -expressingcellswe mountedanimalswiththeaboralendup,visualized usingthe10objectiveontheZeissImagerM2(Carl ZeissLLC).Wenormalizedthefocalplanebyfocusing onthemostsuperficialleveloftheaboralectodermand thencountedthetotalnumberofvisiblecells.LaydenandMartindale EvoDevo 2014, 5 :30 Page3of14 http://www.evodevojournal.com/content/5/1/30

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QuantitativePCRanalysisRNAisolationandquantitative(q)PCRanalyseswere conductedaspreviouslydescribed[30]. Nvactin Nvef1B, and Nvatpsynthase house-keepinggeneswereusedto normalizefoldchangeinqPCRexperiments.AllqPCR primersusedhavebeenpreviouslydescribed[26,30,35]. EachqPCRanalysiswasrepeatedintriplicatepoolsof embryosinjectedinindependentsessions.Basedonpreviousstudies,weconsiderafoldchangegreaterthan1.5 meaningful.Weoftenfailtodetectchangesinexpression viaalternativeapproachesforfold-changeslessthan1.5.ResultsNvnotch and Nvdelta spatiotemporalexpressionis consistentwiththatofaregulatorofcellular differentiationPreviousstudiesshowedthat Nvnotch and Nvdelta are expressedintissuesknowntobeundergoingdifferentiationinlategastrulathroughjuvenilepolypdevelopmental stages[26].However,multiplestudiessuggestdifferentiationin Nematostella isfirstobservedintheearlygastrula whenexpressionofneuralgenes NvashA [30]and Nvelav [31,36]aredetected.Wetestedforboth Nvnotch and Nvdelta expressionbymRNA insitu hybridizationinearly gastrulaanimals(Figure1).Initially, Nvnotch and Nvdelta expressionisdistributedina  saltandpepper Ž pattern (Figure1A,C),meaningthatthecellsthatareexpressing Nvdelta and Nvnotch aredistributedthroughouttheectodermandappearlikepeppergranulesmixedintoapileof salt.The Nvnotch  saltandpepper Ž patternisslightlyvariableinthatitappearspatchyasifthereareclustersof Nvnotch expressingcellsdistributedinthe  saltandpepper Ž pattern(Figure1C,yellowarrow).Theexpressionof bothgenesexpandsovertime,andbothgenesareubiquitouslyexpressedbythelategastrulastage(Figure1B,D). Interestingly,withintheubiquitous Nvdelta expression, thereisa  saltandpepper Ž distributionofcellsthatappear enrichedfor Nvdelta (Figure2B,whitearrows).Basedon thespatiotemporalexpressionpatternspreviouslyreported [26]andextendedhere, Nvnotch and Nvdelta expressionis consistentwiththeearliestonsetofcellulardifferentiation.Nvnotch inhibitsexpressionoftheneurogenic transcriptionfactor NvashATodetermineifNotchsignalingin Nematostella functions toregulatecellulardifferentiation,wechosetocharacterize theeffectsofNvNotchactivityontheexpressionofthe previouslyidentifiedneuraldifferentiationgene NvashA Figure1 Nvnotch and Nvdelta embryonicexpression. Expression of Nvdelta (A,B) and Nvnotch (C,D) isshownatearlygastrula (A,C) and lategastrula (B,D) stages. Nvdelta isexpressedina “ saltandpepper ” expressionpatternatearlygastrula (A) ,andubiquitouslyexpressedat lategastrula (B) ,thoughtherearecellsenrichedfor Nvdelta inthelate gastrula( B ,arrows).Clustersofcellsdistributedina “ saltandpepper ” patternexpress Nvnotch intheearlygastrulastages (C) .Bylategastrula, Nvnotch appearstohavelow-levelubiquitousexpression (D) .Images arelateralviewstakenfromasuperficialfocalplane;oralistotheleft. Figure2 Activationof Nvnotch suppresses NvashA expression. Imagesofanimalsstainedfor NvashA by insitu hybridizationareshown (A-F) .Allimagesarelateralviewswithoraltotheleft.Therelativefocal planeisindicatedtotheleftofe achrowofimages.Animalswith controlwild-typeNotchactivity (A,D), withNotchactivityreducedby injectionofa Nvnotch morpholino(MO) (B,E) ,andwithNotchactivity overactivatedbyoverexpression(OE)ofthe Nvnotch intracellular domain(Nvnicd) (C,F) areshown. (G) Quantitative(q)PCRanalysisofthe relativeexpressionof NvashA iscomparedinanimalswithreduced Notchactivity(DAPT, Nvnotch MO, Nvdelta MO)andincreasedNotch activity( Nvnicd OE),andtoanimalsinjectedwithacontrolMO.Thered rectangleindicatesarelativefoldchangeof 1.5to1.5,whichwe considertocorrespondwithno changeinexpressionlevel. (H) Quantificationoftheaveragenumberof NvashA -positivecellscounted intheaboraldomain(seeMethods).N 20animalscountedfor eachtreatment. LaydenandMartindale EvoDevo 2014, 5 :30 Page4of14 http://www.evodevojournal.com/content/5/1/30

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Previousreportsshowedthatcontinuoustreatmentwith DAPTfor72hoursresultedinanupregulationof NvashA [26] butthisstudydidnotcharacterizeearlierDAPTphenotypes.Weassayedgastrulatreatedwith10 MDAPT for NvashA expressionbymRNA insitu hybridizationand qPCR(Additionalfile1;Figure2) .NvashA expressionlevels increasedbyapproximatelytwo-foldinDAPTtreatedanimals(Additionalfile1;Figure2G).BecauseDAPTdoesnot directlyinhibitNotchsignaling,wewereconcernedthat theDAPT NvashA phenotypemaybecausedbyadisruptionofapathwayotherthanNotch.ToconfirmthatNotch signalingspecificallyinhibits NvashA expression,wegeneratedsplice-blockingMOsdirectedagainstthe Nvdelta ligandandthe Nvnotch receptor(Additionalfile2A).The splice-blocking Nvnotch MOresultsin Nvnotch mRNAs containingstopcodonsthatresultinaprematuretruncationoftheNotchintracellulardomain(datanotshown). Injectionofthe Nvnotch splice-blockingMOresultedina cellthatappearedtoexpressrelativelyhigherlevelsof NvashA comparedtocontrol(compareFigure2AandD toBandE),atwo-foldincreasein NvashA expression measuredbyqPCR(Figure2G),anda60%increaseinthe numberof NvashA positivecells(Figure2H).Thesimilarincreasein NvashA expressionobservedinDAPTtreatedand Nvnotch MO-injectedanimalssuggestthat the NvashA phenotypeobservedinDAPT-treatedanimalsisspecificallyduetoinhibitionofNvNotch.A splice-blockingMOgeneratedagainst NvDelta generatesamiss-splicedtranscriptthatencodesan Nvdelta transcriptonlymissingtheMNNLdomainpresentin theextracellularregionoftheprotein(Additionalfile2A; datanotshown).Injectionofthe Nvdelta splice-blocking MOresultsinanapproximate1.6-foldincreasein NvashA expression(Figure2G).ThisdemonstratesthatNvNotch andNvDeltaarebothrequiredtorepress NvashA inthe embryonicectoderm. TofurtherconfirmthatNotchactivityfunctionsto repress NvashA ,weusedtwoapproachestooveractivate Notchactivity.First,wemimickedconstitutivelyactive NotchbyinjectinganmRNAencodingthe Nvnotch intracellulardomainfusedinframetothe venus codingsequence( Nvnicd:venus )[37].WeobservedNvNicd:Venus nuclearlocalization(Additionalfile2D),andanearly completerepressionof NvashA expressionasdetectedby mRNA insitu hybridization(CompareFigure2AandD toCandF),andanapproximatesix-foldreductionin NvashA levelsasdetectedbyqPCR(Figure2G).Second, weoveractivatedNotchactivitybyinjectingmRNA encodingforthefulllength Nvdeltagenefusedtothe venus reporter( Nvdelta:venus ).Overexpressionof Nvdelta showedlowerlevelsof NvashA expressionbymRNA in situ hybridization(Figure3).Weobservedweak NvashA expressionin57%ofthe Nvdelta:venus injectedanimals (Figure3A,B)andanapproximatethree-foldreductionin NvashA expressionasmeasuredbyqPCR(Figure3C,light greybar).Todetermineifthesuppressionof NvashA by Nvdelta requiredNvNotch,wetreated Nvdelta:venus injectedanimalswithDAPT.Treating Nvdelta:venus injectedanimalswithDAPTresultedinatwo-fold upregulationof NvashA (Figure3C,darkgreybar).This isconsistentwiththepreviouslyobservedphenotypes followingDAPTtreatmentand NvNotch MOinjection (Figure2),andsuggeststhatNvNotchactstoinhibit NvashA expressionwhenactivatedbyinteractionswith NvDelta .Notchactivitysuppressesneurogenesisthrough repressionof NvashA expressionTodetermineifchangesin NvashA levelsdownstreamof Notchactivitycorrespondtochangesin NvashA -dependent neurogenesis weassayedforchangesinexpressionof thepreviouslyidentified NvashA neuraltargetgenes Figure3 Nvdelta activates Nvnotch activitytosuppress NvashA .(A-B) ShownareAboralviewsof NvashA expressionincontrol (A) and Nvdelta overexpressing(OE) (B) animals.Phenotypicclasseswerescoreda snoexpression,weak,wild-type(WT)levels,andstrongexpression.Thekeyisshownin theimageandbarsatthebaseofeachimagerepresent thepercentageofanimalsineachphenotypicclass. (C) Relativefoldchangeof NvashA and previouslyidentified Nvasha neuralgenetargetsandinanimalsoverexpressing Nvdelta (lightgreybars)andanimalsthatareoverexpressing Nvdelta and treatedwithDAPT(darkgreybars).Redrectangledenotesrelativefoldchange 1.5to1.5,whichcorrespondstonochangeinrelativeexpression. LaydenandMartindale EvoDevo 2014, 5 :30 Page5of14 http://www.evodevojournal.com/content/5/1/30

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[30].OveractivationofNotchactivitybyeitherinjectionofthefulllength Nvdelta:venus orinjectionofthe Nvnicd:venus constructresultedinadramaticdownregulationof NvashA neuraltargetgenes(Figure4A,D,G, darkbluebars;Additionalfile3,lightgreybars).Coinjectionof NvashA:venus mRNAwiththe Nvnicd:venus mRNAwassufficienttosuppressthereductionofneural geneexpressionphenotyperesultingfromoveractivationof Notchactivity(Figure4C,F,G,lightbluebars).Manyofthe NvashA:venusNvnicd:venus co-injectedembryosassayed by insitu hybridizationshowedneuralgeneexpression phenotypesconsistentwiththeincreasednumberof neuronsobservedwhen NvashA isexpressedalone (Figure4C,F)[30].TreatmentwithDAPTincreased thelevelsofneuralgeneexpression(Figure4A,dark orangebars).Co-injectionofthe NvashA translationblockingMO[30]suppressestheDAPTinducedupregulationofneuralgeneexpression(Figure4A,light orangebars).ThesedatasuggestthatNotchactivity suppresses NvashA -dependentneurogenesisprimarily Figure4 Nvnotch suppressesneurogenesisbyregulating NvashA expression.(A) Relativeexpressionlevelsof NvashA targetgenesin animalswithoverexpressing(OE) Nvnicd (darkbluebars),overexpressing Nvnicd and NvashA (lightbluebars),animalstreatedwithDAPT(dark orangebars),andDAPTtreatedanimalsinjectedwiththe NvashA morpholino(MO)(lightorangebars).Redrectanglerepresentsrelativefold change – 1.5to1.5,whichcorrespondstonochangeinrelativeexpression.Eachtreatmentwasrepeatedatleastthreetimes. (B-G) Aboralviews ofmRNA insitu imagesfromtwo NvashA neuraltargetgenesareshown.Animalswithoveractive Nvnotch (B,E) ,control (C,F) ,andbothoveractive Nvnotch andoveractive NvashA (D,G) areshown.Animalsin(B-G)werequantifiedintophenotypicclassesbasedonhavingno,weak,wild-type (WT)-like,orstrongexpressionlevels.Thekeyisshownintheimageandbarsatthebaseofeachimagerepresentthepercentageofanimalsineach phenotypicclass. LaydenandMartindale EvoDevo 2014, 5 :30 Page6of14 http://www.evodevojournal.com/content/5/1/30

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throughthespecificinhibitionof NvashA expression ratherthanbroadlytargetingdownstreamgenesexpressed indifferentiatingneurons.Post-embryonictreatmentwithDAPTincreases NvashA expressioninthelarvalectodermandendodermWewantedtotestwhetherNotchactivityregulates NvashA atlaterdevelopmentalstagesindependentlyof theearlierrolesdescribedabove.Inordertodisrupt NotchsignalingatlaterstageswithoutdisruptingNotch signalingatearlystagesweoptedtouseDAPTtreatments. AlthoughDAPTtreatmentmaynotspecificallydisrupt Notchsignaling,theincreasein NvashA followingtreatmentwithDAPTorinjectionofthe Nvnotch MOinthe embryoareidentical(Figure1),whichsuggeststheDAPT NvashA phenotypeisduetoadisruptionofNotchsignaling.WeperformedtwoDAPTtreatments(Figure5).The firsttreatmentbeganatthelategastrulastageandcontinuedfor24hoursintotheearlyplanulalarvalstages (Figure5A-C).Wedetected NvashA expressioninthe formingpharynx(Figure5A,arrow),ina  saltandpepper Ž patternintheectoderm(Figure5A,inset),andsomeweak stainingina  saltandpepper Ž patternwithintheendoderm incontrolplanulae(Figure5A,arrowhead).Treatment withDAPTresultedinanincreaseinpharyngealstaining (Figure5B,arrow)andanincreaseinthenumberofectodermalcellsexpressing NvashA (Figure5B,inset).Itwas difficulttobecertainthatendodermal NvashA wasincreasedbecauseofthestrongectodermalexpression,butit appearsasifthereisanexpansionof NvashA expressionin theendodermaswell.Wewerealsoabletoclassifyanimals intogroupsofanimalshavin gno,weak,normalwild-type, orstrong NvashA expressionforbothcontrolandDAPTtreatedanimals.Incontrolanimals,approximately70%of theanimalshadwild-typelevelsof NvashA expression,and onlyapproximately10%oftheanimalshadstrongexpressionof NvashA. InDAPT-treatedanimals90%ofthe animalsdisplayedthestrongexpressionphenotype.We alsoobservedathree-foldincreasein NvashA expressioninDAPT-treatedanimalsbyqPCR(Figure5C).We alsotreatedanimalswithDAPTfrom48to72hpf,which ensuredanimalswereallwithinthelarvalstagesofdevelopmentduringthetreatment(Figure5D-F). NvashA expressionincontrol72hpfplanulaewasdetectedinthe pharynxandformingmesenterystructures(Figure5D, arrow)andina  saltandpepper Ž endodermalpattern.We Figure5 DAPTtreatmentincreases NvashA expressionintheplanulalarva.(A-C) Forty-eighthourspostfertilization(hpf)animalseithertreated withcontrolDMSO (A) orwithDAPT (B-C). (A) NvashA expressionincontrolanimalsisdetectedinthedevelopingpharynx(arrow),intheendoderm (arrowhead),andintheectoderm(inset).(B)TreatmentwithDAPTincreases NvashA expressionineachtissue.(C)Quantitative(q)PCRanalysisrevealsa three-foldincreaseintherelativelevelsof NvashA inDAPT-treatedanimals. (D-F) Seventy-twohpfanimalseithertreatedwithcontrolDMSO(D)orwith DAPT(E-F).(D) NvashA expressionincontrolanimalsisdetectedinthedevelopingpharynx(arrow)andintheendoderm.(E)TreatmentwithDAPT increases NvashA expressionineachtissue.(F)qPCRanalysisrevealsathree-foldincreaseintherelativelevelsof NvashA inDAPT-treatedanimals.The keyin(C)and(F)showsthatanimalsweregrowninnormal1/3Xseawater(blacklinebetweentimeintervals)orinthepresenceofDAPT(redline betweentimeintervals).Animalsin(A,B,D,E)werequantifiedintophenotypicclassesbasedonhavingno,weak,wild-type-like,orstrongexpressi on levels.Thekeyisshownintheimageandbarsatthebaseofeachimagerepresentthepercentageofanimalsineachphenotypicclass.Redboxin (CandF)indicatestheregionbetween0and1.5-foldchange,whichweconsidertoindicatenochangeinexpression.Allanimalsareshownina lateralviewwiththeoralsidetotheleft. LaydenandMartindale EvoDevo 2014, 5 :30 Page7of14 http://www.evodevojournal.com/content/5/1/30

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didnotdetectanyectodermal NvashA expressionin72 hpfanimals.AnimalstreatedwithDAPTfrom48to72 hpfshowedastrongincreasein NvashA intheforming pharynxandmesenteries(Figure5E,arrow),andtheendodermhasanincreasein NvashA expressionlevels.As before,wecouldeasilygroupphenotypicclassesfor NvashA expression:incontrolanimals,80%oftheanimals showedwild-typeexpressionlevelsandonlyapproximately7%showedthestrong NvashA expressionphenotype(Figure5D).However,intheDAPT-treatedanimals 86%oftheanimalsdisplayedthestrong NvashA expressionphenotype(Figure5E).DAPT-treatedanimalsalso hadanapproximatethree-foldincreasein NvashA expressionlevelsbyqPCR(Figure5F).Thesedatademonstrate thatDAPTtreatmentpromotesanincreasein NvashA at laterstages,andthatsimilarmechanismsregulateboth embryonicandlarvaldifferentiation.Moreover,theseresultsarguethatthedynamicexpressionpatternsobserved for Nvnotch and Nvdelta (ectoderminearlyembryoand movingintotheendoderminlarvalstages[26])supportsthehypothesisthat Nvnotch regulatescellular differentiationinmultipletissuesthroughoutdevelopment in Nematostella.Nvnotch broadlyinhibitsexpressionofgenesassociated withneuronaldifferentiationLastly,wewonderedifNotchactivitymightinfluence expressionlevelsofotherdifferentiationgenesunrelated to NvashA .Weusedpreviouslydescribeddifferentiation genes, Nvgcm,Nvsoxb2,Nvsox2,Nvmef2.iv, and Nvminicol4 [31,38-40],aswellastworecentlyidentifiedgenes, Nvcoup1 and Nvath-like1 (Figure6) that,like NvashA ,areall expressedina  saltandpepper Ž pattern.Itshouldbe notedthatallofthesegenesareassociatedwithneuronal differentiation,thoughonly Nvmef2.iv and Nvminicol4 havebeendefinitivelylinkedtoneuraldevelopment.They regulateformationofthecn idocyteneuralcelltype [39,40].Asweobservedfor NvashA, inhibitingNotch activitybytreatingwithDAPT(Figure6,bluebars)or injectingthe Nvnotch MO(Additionalfile4,greenbars) increasedexpressionlevelsfornearlyallthe  saltandpepper Ž genesassayed.Theonlygenesassayedthatshowed nosignificantincreaseinexpressionlevelsfollowingtreatmentwithDAPTwere Nvmef2.iv and Nvminicol4, though Nvminicol4 wasupregulatedfollowing Nvnotch MOinjection(Additionalfile4).Wealsoincluded Nvsox1 Nvsox3, Nvsoxe1 ,and Nvets1a becausetheyareexpressedindistinctbroaddomainsratherthanina  saltandpepper Ž pattern,whichsuggeststhattheyareinvolvedinpatterning regionaldomainsratherthandifferentiation.Expression levelsofthe  broadlyexpressed Ž genesdidnotchange followingDAPTtreatmentorinjectionofthe Nvnotch MO.OveractivationofNotchsignalingbyinjecting Nvnicd:venus suppressedexpressionofallofthe  salt andpepper Ž genes(Figure6,darkorangebars),including Nvmef2.iv and Nvminicol4 .Again,thebroadlyexpressed geneswereunaffectedby Nvnicd injection. Toconfirmthesegenesareindependentof NvashA weattemptedtorescuethelossof  saltandpepper Ž geneexpressionresultingfromoveractivationofNotch signalingbyco-injectingthe Nvnicd:venus andthe NvashA:venus constructs(Figure6,lightorangebars).Only Nvgcm wasrescuedbyexpressionof NvashA. Thissuggeststhat, withtheexceptionof Nvgcm, the  saltandpepper Ž genes arenottargetsof NvashA. Therefore,wesuggestNotch activitybroadlyregulatesexpressionofgenesassociated withneuraldifferentiationinthe Nematostella embryo.Thenon-canonicalNotchsignalingpathwayinhibits NvashA expressionSuppressionof NvashA byactivatedNotchsignaling canoccurthroughthecanonical( suH and hes genedependent),thenon-canonical( suH and hes geneindependent),orthroughbothpathways.Wetestedthe putativecontributionsofthecanonicalandnon-canonical pathwaysin Nematostella .First,wetestedif Nvnotch regulatedtheexpressionof Nvhes genes.Four Nvhes genes, Nvhes1,2,3,Nvhl1 ,areexpressedin Nematostella embryosandcouldpotentiallyberegulating NvashA [26]. However,only Nvhes2 and Nvhes3 expressionisdetected bymRNA insitu hybridizationintheearlygastrulawhen theearliestonsetofdifferentiationof NvashA positivecells isoccurring[26].Wecomparedchangesinexpressionfor eachofthesegenesusingqPCRfollowingtreatmentwith DAPT(Figure7,bluebars),injectionofthe Nvnotch MO (Figure7,orangebars),andfollowinginjectionofthe Figure6 Nvnotch regulates “ saltandpepper ” differentiation genes. Relativefoldchangeof “ saltandpepper ” genesinanimals followingtreatmentwithDAPT(bluebars),injectionwith Nvnicd (darkorangebars),injectionwith Nvnicd and NvashA (lightorange bars),or NvashA alone(greenbars).Redrectangledenotesrelative foldchange 1.5to1.5,whichindicatesnochangeinrelative expression. “ saltandpepper ” differentiationgenesaresuppressedby Nvnotch activitywhilegeneswithbroadexpressiondomainsare unaffectedbyanyofourtreatments.OE,overexpressing. LaydenandMartindale EvoDevo 2014, 5 :30 Page8of14 http://www.evodevojournal.com/content/5/1/30

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Nvnicd:venus mRNA(Figure7,purplebars).Treatment withDAPTresultedinanapproximatetwo-foldreduction in Nvhes1 and Nvhl1 levels.The Nvhes2 and Nvhes3 genes bothshowedagreaterthaneight-foldreductioninexpressionfollowingDAPTtreatment(Figure7A,bluebars). However, Nvnotch MOinjectedanimalsshowednochange in Nvhes1 or Nvhl1 expression,andarelativelyminor decreasein Nvhes2 and Nvhes3 levels(Figure7A,orange bars).Wefailedtodetectanyreductionof Nvhes2 or Nvhes3 in Nvnotch morphantsbymRNA insitu hybridization(Figure7B-E).Becausethe NvashA phenotyperesultingfrominjectionofthe Nvnotch MOis assevereastreatmentwithDAPT(Figure1),andthere islittlewild-type Nvnotch transcriptinthe Nvnotch morphantanimals(Additionalfile2A),webelievethe NotchMOtobehighlyefficient.However,wewere concernedthatlowlevelsof Nvnotch activitymaybe sufficienttopromote Nvhes geneexpressionintheembryo.ToaddressthisweoveractivatedNotchsignalingby injectingthe Nvnicd:venus and Nvdelta:venus constructs, whichshouldincrease Nvhes expressionifthecanonical pathwaywasintact.Weobservednosignificantchangefor Nvhes1-3 andonlyaminorincreasein Nvhl1 expression followinginjectionof Nvnicd:venus (Figure7E,purplebars) Similarly,injectionofthe Nvdelta:venus mRNAfailedto induceexpressionofanyofthe Nvhes genes.Thus,ourdata suggestthat,althoughDAPTtreatmentreducestheexpressionlevelsof Nvhes1-3 or Nvhl1 in Nematostella embryos, theobserveddownregulationis Nvnotch -independent. Eventhough Nvnotch doesnotregulate Nvhes geneswe stillwantedtotestif NvsuH regulated Nvhes genes,andif Nvhes genesweresufficienttosuppress NvashA expression.Nvhes2 and Nvhes3 aretheonlytwo Nvhes genesthathaveexpressionthatinitiatesintheearlyembryowhenthefirstcellulardifferentiationisobservedin Nematostella .Weoverexpressed Nvhes2 and Nvhes3 by injecting venus:Nvhes2 and venus:Nvhes3 mRNAs. Nvhes2 or Nvhes3 overexpressiondidnotresultinanychanges inthelevelsof NvashA asdetectedbymRNA insitu hybridization(Figure8A-F)orqPCR(Figure8G).We testedif NvsuH regulated Nvhes genesor NvashA by injectingbothan NvsuH MOandadominantnegative NvsuH [26].Neitherofthesemanipulationsresultedindetectablechangesof Nvhes or NvashA expressionbyqPCR (Figure8H).ThesedatasuggestthatthecanonicalNotch pathwaydoesnotregulate NvashA -dependentneuraldevelopmentintheearly Nematostella embryo. TodetermineifcanonicalNotchsignalingcouldregulate the NvashA -independent  saltandpepper Ž expressedgenes associatedwithcellulardifferentiation,wetestedwhether overexpressing Nvhes2 or Nvhes3 viainjectionofthe venus:Nvhes2 or venus:Nvhes3 mRNAcouldsuppressexpressionofthe  saltandpepper Ž genes.Wesawnochange intheexpressionlevelsbyqPCRforanyofthe  saltand pepper Ž genesassayedhere(Additionalfile4,lightand darkbluebars).Thus,itappearsthatnon-canonicalNotch signalingbroadlysuppressesexpressionofgenesthat promoteneuraldifferentiationin Nematostella embryos.DiscussionModelofNotchsignalingin NematostellaOurdatashowthatNvNotchisactivatedbyNvDeltato regulatecellulardifferentiationin Nematostella ,butbased onourobservationshereitislikelythatNotchactivityin Figure7 Nvnotch doesnotregulate Nvhes expressioninthe Nematostella embryo.(A) Averagerelativefoldchangeof Nvhes gene expressioninanimalsinjectedwith Nvnotch morpholino(MO;orangebars),treatedwithDAPT(bluebars),injectedwith Nvnicd:venus (darkpurple bars),injectedwith Nvdelta:venus (lightpurplebars),oracontrolMO(greybars.Redrectanglecoverstheregionwheretherelativefoldchange ratioisequalto 1.5to1.5andcorrespondstonochangeinrelativeexpressionlevel. (B-E) Lateralviewsoflatestagegastrulaexpressing Nvhes2 (B-C) or Nvhes3 (D-E) .Oralistotheleft.Deepfocalplaneisshownandsuperficialfocalplaneisshownininset.Weobservednodiscernable differencein Nvhes2 or Nvhes3 expressionby insitu analysisbetweenwild-typeand Nvnotch MOinjectedanimals.WescoredN>80embryosfor eachtreatment.OE,overexpressing. LaydenandMartindale EvoDevo 2014, 5 :30 Page9of14 http://www.evodevojournal.com/content/5/1/30

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Nematostella regulatesthecompetenceofcellstorespond toavarietyofinstructivedifferentiationcues.Elevated levelsofNotchactivitysuppressdifferentiatedcell markers,whiledecreasedlevelsofNotchactivityincrease expressionofdifferentiatedcellmarkers(Figures1and8; Additionalfile1) However,inhibitionofNotchsignaling isnotsufficienttoinduceatotaltransformationofcells intodifferentiatedcells.ThissuggeststhatNotcheither actsatdefinedtimepointsinthedifferentiationprocessor thatNotch-independentinstructivecuesacttoinduce particulardifferentiatedcelltypes.Ourmodelpredictsthat therelativelevelofNotchactivityandtheamountof inductivesignalcoordinatetodetermineifdifferentiationwilloccur.Consistentwiththisprediction,extending treatmentwithDAPTto3daysresultsinanimalsthat haveamorepronouncedexpansionofdifferentiatedcell markersthanourearlyshortertreatment[26].OneinterpretationisthatextendedinhibitionofNotchactivity providesmoreopportunityforundifferentiatedcellsto encounterandrespondtoexternalinductivecues.Additionally,quantificationofthenumberofcellsexpressing anyone  saltandpepper Ž geneisnotoftenreproducible fromanimaltoanimal(Figure1H;unpublishedobservations)[30],suggestingthatthemechanismgoverningthe numberofcellsofadistinctcelltypeissomewhat stochastic.Takentogethertheseobservationsarguethat, inanygivenanimalatagiventime,therearevariable numbersofcellscompetenttorespondtodistinctdifferentiationcues.Ourdatasupportsthehypothesisthatthe competenceisinpartregulatedby Nvnotch activity. Notchappearstofunctionbroad lytoinhibitneuraldifferentiation.Wetestedanumberofgenesthathavebeenpreviouslyreportedtobeassociatedwithdifferentiationduring Nematostella development(Figure6;Additionalfile3).We foundthatinhibiting Nvnotch byinjectingthe Nvnotch MO orbytreatingwithDAPTresultedinupregulationofthe differentiatedmarkers.Conversely,overactivationofNotch byoverexpressingthe Nvnicd:venus mRNAsuppressed expressionofthedifferentiationmarkers.Themarkersthat weused( NvashA,Nvsox2,Nvgcm,Nvsoxb2,Nvath-like, Nvmef2.iv,Nvminicol4,Nvcoup1-like )areallpredictedtobe associatedwithneurogenesis and/orcnidocytedevelopment in Nematostella ,although(withtheexceptionof NvashA Nvmef2.iv, and Nvminicol4 )noneofthemareconfirmed regulatorsofneuraldevelopment.Thus,wecannotconcludeatthispointifNotchbroadlyregulatesexpressionof alldifferentiatedcelltypesorspecificallyregulatesneural developmentin Nematostella .Evenifthedifferentiation geneswechosearespecificton euraldevelopmentweargue thattheyareindependentof NvashA -dependentneuraldevelopment.Weshowthat,otherthan Nvgcm ,noneofthe differentiationgenesassayedherecanberescuedwhen NvashA isoverexpressedinanimalswithincreasedNotch activity(Figure5).Also,wehavenotobservedanycoexpressionof Nvsoxb2 or Nvsox2 with NvashA neuraltargets,andboth NvsoxB2 and Nvsox2 areexpressedinwhat appearstobemanymorecellsthan NvashA [38](unpublishedobservation) Thus,weareconfidentthatNotch activitybroadlyinhibitsexpres sionofgenesassociatedwith neuraldifferentiation,butcannotdeterminewhatothercell typesmightberegulatedbyNotchactivity. WealsoproposethatNotchregulationofdifferentiation isareiterativeprocessduringNematostella development. Differentiationbeginsduringtheearlygastrulastageof Nematostella development,butcontinuesthroughout embryonicandlarvalstages.Theexpressionpatternsof NvashA andotherknowndevelopmentalgenesareknown tobedynamicthroughoutthesestages[30,31,36,38]. ExpressionofNotchsignalingcomponentsappearstobe enrichedintissueslikelytobeundergoingcellulardifferentiationduringdevelopment.Forexample,theembryonicexpressionof Nvnotch and Nvdelta initiateinthe ectoderm,andaremaintainedthereuntillateplanula stages(Figure2)[26].Inearlyplanulastagestheendodermbeginstoshowexpressionofdifferentiatedcell markers[31,36].Endodermalexpressionof Nvnotch and Nvdelta arecoincidentwithendodermaldifferentiation. Nvnotch and Nvdelta areexpressedintheformingand growingtentaclebuds[26](unpublishedobservation),and Figure8 Nvhes2 and Nvhes3 overexpressiondoesnotrepress NvashA expression.(A-F) Lateralviewsofembryosexpressing NvashA ;oralistotheleft.Thereisnodiscernabledifferencein NvashA expressionincontrol(A-B), Nvhes2 overexpressing(OE) (C-D) ,or Nvhes3 (E-F).N>65scoredforeachexperiment. (G) Relativefoldchangeof NvashA inembryostreatedoverexpressing Nvhes2 or Nvhes3 (H) Averagerelativefoldchangeof NvashA neuralgenes,and Nvhes genesinanimalsinjectedwiththe NvsuH morpholino(MO;darkgreybars)oradominantnegative NvsuH (DN;lightgreybars).Redrectangledenotesrelativefoldchange 1.5 to1.5,whichindicatesnochangeinrelativeexpression.Eachinjection wasrepeatedthreetimes. LaydenandMartindale EvoDevo 2014, 5 :30 Page10of14 http://www.evodevojournal.com/content/5/1/30

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expressioninjuvenileandadultpolypsismaintainedin theendodermalportionoftheeightmesenteries[26], whereconstantdifferentiationofnematosomesisknown tooccur[41].WealsofoundthattreatingwithDAPTfor distincttimewindowsthroughoutlarvaldevelopmentresultedintheincreased NvashA expression(Figure5).This suggeststhatthesameorasimilarmechanismcontrols NvashA expressionatlatertimepointsandindifferenttissues(endodermversusectoderm)thanduringembryonic development.Wewouldliketoextendthistemporalanalysistogene-specificknockdownsof Nvnotch .However, wefocusedthisinitialstudyontheearlyembryonic rolesof Nvnotch becauseconditionalknockdownof Nvnotch functionspecificallyatlatertimepointsisstill difficultin Nematostella .Asthetechnologyofconditional allelestodisruptgenefunctionspecificallyatdistinctlife stagesin Nematostella advances,andasidentificationof genesthatserveasmarkersforcellsdifferentiatedwithin distincttemporalwindowsarefound,ourmodelcanbe testedfurther.WepredictthatdisruptingNotchactivity indistincttemporalwindowsshoulddisruptonlythecell typesthatarenormallybornwithinthattimeframe.Notchsignalingpathwaymayhaveemergedtoregulate metazoancellulardifferentiationTheemergenceofmulticellularanimalswithspecialized celltypeshadtorequireamechanismtoregulatewhether cellsdifferentiateorremainpluripotent.Notchhasbeen showntohaveahighlyconservedroleasaregulatorof differentiationinnearlyallbilateriantissues.However,prior tothisstudyitwasunclearhowNotchfunctionedinnonbilateriananimals,andthustherewaslittleinferenceabout ancestralNotchfunction.Weshowthatnon-canonical Notchsignalinginthecnidarianseaanemone, Nematostellavectensis ,broadlyinhibitscellulardifferentiation duringdevelopment.Thisprovidesaclearexampleof NotchregulatingdifferentiationoutsideofBilateria.Given howhighlyconservedtheroleforNotchasaregulatorof differentiationappears,andthefactthatcoreNotchcomponentsevolvedspecificallyinmetazoans,itislikelythat Notchregulatesdifferentiationinallmetazoans.Tofully supportthishypothesisweneedtoreconstructthefunctionofNotchsignalinginthecommonancestorofall metazoansbycharacterizingtheroleofNotchinanimals representingtheearliestdivergedmetazoanlineage.The sisterlineagetotherestofanimalsisstillbeingdebated, butthecurrentconsensusisthatitiseitherCtenophora orPorifera.Disruptionofgenefunctionineitherofthese groupshasprovendifficult,butwecaninferputativefunctionbasedonexpressionpatterns.Expressionof notch and delta homologsintheporiferan A.queenslandica initiatesexpressioninaspatiotemporalpatternconsistentwithregulatorsofcellulardifferentation[25].The amqdelta homologsappeartobeexpressedin differentiatinganddifferentiatedcelltypesconsistent withtheideathattheyactivateNotchtosuppressdifferentiationinthesurroundingcells,whilehavinglowNotch activitythemselves[25].TheexpressionpatternsofNotch signalinghomologsinctenophoresarenotknown,and definitivehomologsfor delta havenotbeenfound.Thus, wecannotpredictputativefunctionsforNotchsignaling inthatlineage.EvolutionofcanonicalNotchsignalingOurresultssuggestthatcanonicalNotchsignalingisnot presentinthecnidarianlin eageandthatthecanonical pathwayevolvedinthestemofthebilaterianlineage.In Nematostella ,gene-specificknockdownof Nvnotch,NvsuH, oroveractivationof Nvnicd didnotsignificantlyaffectexpressionlevelsof Nvhes genes,whichareanimportant targetofthecanonicalNotchsignalingpathway.OveractivationofNotchsignalingbyoverexpressingeither Nvnicd or Nvdelta wassufficienttosuppressexpression ofdifferentiatedcellmarkers,butbothfailedtoupregulateanyofthe Nvhes genesmonitored(Figure5).Furthermore,overexpressionof Nvhes2 or Nvhes3 failedto suppress NvashA orothergenesassociatedwithcellular differentiation(Figure5;Additionalfile4).Inaddition,the expressionof Nvhes homologsthroughout Nematostella developmentareinconsistentwiththenotionthatthey aretargetsof Nvnotch signaling.Most Nvhes genesshow minimaloverlapwith Nvnotch expressionoutsideofthe embryonicectoderm[26].Threeexceptionstothisare Nvhes3andNvhl1, whichoverlapwith Nvnotch expressionintheoralectodermandaboralectodermduring planulastages[26],and Nvhes1 ,whichoverlapswiththe Nvnotch expressionintheplanulaendoderm.However, Nvhes1 expressionappearsubiquit ousintheplanulastages, whereas Nvnotch expressionbecomeslimitedtotheendoderm,suggestingthatthe Nvhes1 expressionisregulatedby factorsotherthan Nvnotch .Thereportedexpressionof NvsuH isalsoinconsistentwiththeideathatcanonical Notchsignalingregulatesdifferentiation. NvsuH isnot expressedinthedifferentiatingectodermattheonsetof cellulardifferentiationintheearlygastrulawhenexpression of NvashA andthe  saltandpepper Ž genesisinitiated[26]. However, NvsuH isexpressedubiquitouslylaterintheplanulalarvalstages. Acloserexaminationofthephylogeneticdistributionof canonicalNotchsignalingco mponentsinthethreepublishedcnidariangenomesalsosupportsthelackofanintact canonicalNotchpathwayin cnidarians[4,23,24] Previous analysissuggestedthatthecnidarian-bilateriancommon ancestorwasthefirstanimalwithacomplimentofgenes thatparticipateincanonical Notchsignaling[19].However, thecnidarianhomologsofthetranscriptionalco-activator mastermind thatisrecruitedtoactivate hes expressionareonlyweaklyconservedatbestwithbilaterianhomologsLaydenandMartindale EvoDevo 2014, 5 :30 Page11of14 http://www.evodevojournal.com/content/5/1/30

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[1,19].Moreover,SuHalsointeractswiththeSMRTcorepressortosuppressexpressionof hes homologswhen Notchsignalingisnotactive. smrt homologshavenotbeen identifiedinanyofthecurrentlypublishedcnidariangenomes[4,19,23,24]. Itshouldbenotedthatmostofthe Nvhes genesareseverelydownregulatedfollowingDAPTtreatment(Figure5) [26].However,ourdataarguethattheDAPT-induced Nvhes phenotypesoccurindependentlyof Nvnotch .The currentdraftofthe Nematostella genomedescribesonlya single Nvnotch gene.However,thereareadditionalsingle passtransmembraneproteinsthat,like Nvnotch ,haveEFG repeatsintheirextracellulardomain(unpublishedobservation)[24].Theintracellulardomainsoftheseproteins lackthetypicalintracellulardomainslinkingNotchsignalingto hes generegulation[19,26],butbecausethe -secretasecomplexisbelievedtocleavemostsingle passtransmembranesignalingproteins,itisreasonable tohypothesizethatDAPTisaffectingoneormoreof these  Notch-like Ž proteins,andthattheymayregulate hes expression.Giventhatactivationof hes expression isahallmarkofcanonicalNotchsignaling,wespeculate thatsomeaspectof hes biologyunderliestheemergenceofthecanonicalpathway.Oneexplanationcould bebasedonthefactthat hes genesfunctionasoscillatorsthatpromotecellproliferation[13,42].Interestingly, weobserve Nvhes2 expressioninproliferatingcells (unpublishedobservation).BecausehighNotchactivity oftensuppressesdifferentiation,perhapsincorporating regulationofproliferationdownstreamofNotchactivity providedamechanismtobothsuppressdifferentiation andpromoteproliferation.Thisisconsistentwiththe observationthatcanonicalNotchactivityinthedevelopmentofmanybilateriantissuesisoftenassociatedwith maintainingtissue-specificstemcells[8]. ToverifythatcanonicalNotchsignalingisnotintactin thecnidarian-bilaterianancestorgenespecificfunctional studiesneedtobeconductedinothercnidarianspecies. Additionalanalysesneedtobedonein Nematostella once toolsemergetoinvestigaterolesforNotchsignalingspecificallyduringpost-embryonicdevelopment.Currently, attemptingtointerpretlate-stagephenotypesinmorphant animalsiscomplicatedbecauseitisunclearhowearly disruptionof Nvnotch influenceslaterdevelopment. Temporal-specifictreatmentswithDAPTwouldnot beinformativebecauseweshowedthattheresponses of Nvhes genestoDAPTintheembryoare Nvnotch independentphenotypes.ConclusionsBasedonourfunctionalanalysisinthecnidarian Nematostellavectensis andpreviouspharmacologicalexperimentsinothercnidarianspecies,weproposethatthe Notchsignalingpathwayregulatedcellulardifferentiation inthecnidarian-bilaterianancestor.Thisarguesthatthe roleofNotchasaregulatorofcellulardifferentiation evolvedpriortothelastcommonancestorofbilaterian animals.FunctionalstudiesarerequiredinothernonbilaterianlineagestoreconstructtheroleofNotchsignalingatmorebasalnodesinthemetazoanphylogeny. BecauseallcomponentsofcanonicalNotchsignaling likelydidnotevolveuntilthecnidarian-bilateriancommon ancestor,afullcomplementofcanonicalsignalingcomponentsonlyexistsinthebilaterians,andbecausecanonical Notchsignalingisnotrequiredfor Nvnotch toregulateembryonicneuraldifferentiationin Nematostella ,wespeculate thatnon-canonicalNotchsignalingistheancestralnotch mechanismandthatthecanonicalpathwaylikelyevolved specificallyinthebilaterianlineage.AdditionalfilesAdditionalfile1: DAPTtreatmentupregulates NvashA. (A-D)Shown arelateralviewsofembryosexpressing NvashA .Oralistotheleft. DAPT-treatedanimalshavehigherlevelsof NvashA expression.Phenotypic classeswescoredasbeingwild-type,strong,weak,orno NvashA expression. Keyisshowninimageandbarsatthebaseofeachimagerepresentthe percentageofanimalsineachphenotypicclass. Additionalfile2: Controlexperiments. (A)Spliceblockingefficiency foreachspliceMOusedinthisstudyisshown.(B-D)InjectionofmRNAs encodingforthe Nvnicd:venus (B), venus:Nvhes2 (C),and venus:Nvhes3 (D) resultedintranslatedproteinandcanbedetectedinthenucleiofthe developingembryo. Additionalfile3: Relativefoldchangeof NvashA neuronaltargets in Nvdelta OEanimals. Relativefoldchangeof NvashA neuraltarget genesinanimalsoverexpressingthe Nvdelta:venus mRNA(lightgreybars) oroverexpressingthe Nvdelta:venus mRNAandtreatedwithDAPT(dark greybars).Redboxindicatesregionwherefoldchangeratioisbetween 1.5 and1.5indicatingnochangeinexpression. Additionalfile4: Relativefoldchangeof “ saltandpepper ” genes in Nvnotch morphantand Nvhes overexpressinganimals. Relative foldchangeof “ saltandpepper ” andbroaddomainexpressedcontrols areshownforanimalsinjectedwiththe Nvnotch MO(greenbars), venus: Nvhes2 (lightbluebars),or venus:Nvhes3 (darkbluebars).Eachinjection wasrepeatedatleastthreetimes.Redboxindicatesregionwherefold changeratioisbetween 1.5and1.5indicatingnochangeinexpression. Abbreviations hpf: Hourspost-fertilization;MO:morpholino;(q)PCR:(quantitative) polymerasechainreaction. Competinginterests Theauthorsdeclarethattheyhavenocompetinginterests. Authors'contributions MJLconceivedandcarriedoutthegenerationofconstructs,collectionof data,anddataanalysis.MJLandMQMcarriedoutanimalinjections. MJLandMQMwrotethemanuscript.Bothauthorsreadandapprovedthe finalmanuscript. Acknowledgements WewishtoacknowledgeDrLeslieBabonis,DrTimothyDubuc,andDr JosephRyanfortheircriticalreadingofthismanuscript.Thisworkwas fundedbygrantnumberR21RR034343fromtheNationalInstitutesofHealth.LaydenandMartindale EvoDevo 2014, 5 :30 Page12of14 http://www.evodevojournal.com/content/5/1/30

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