A cleavage clock regulates features of lineage specific differentiation in the development of a basal branching metazoan...

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Title:
A cleavage clock regulates features of lineage specific differentiation in the development of a basal branching metazoan, the ctenophore Mnemiopsis leidyi
Series Title:
EvoDevo
Physical Description:
Mixed Material
Creator:
Antje HL Fischer
Kevin Pang
Jonathan Q Henry
Mark Q Martindale
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EvoDevo
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Subjects / Keywords:
Ctenophore
Comb jelly
Photocyte
Comb cell
Cytochalasin
Cell cycle arrest

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Abstract:
Background: An important question in experimental embryology is to understand how the developmental potential responsible for the generation of distinct cell types is spatially segregated over developmental time. Classical embryological work showed that ctenophores, a group of gelatinous marine invertebrates that arose early in animal evolution, display a highly stereotyped pattern of early development and a precocious specification of blastomere fates. Here we investigate the role of autonomous cell specification and the developmental timing of two distinct ctenophore cell types (motile compound comb-plate-like cilia and light-emitting photocytes) in embryos of the lobate ctenophore, Mnemiopsis leidyi. Results: In Mnemiopsis, 9 h after fertilization, comb plate cilia differentiate into derivatives of the E lineage, while the bioluminescent capability begins in derivatives of the M lineage. Arresting cleavage with cytochalasin B at the 1-, 2- or 4-cell stage does not result in blastomere death; however, no visible differentiation of the comb-plate-like cilia or bioluminescence was observed. Cleavage arrest at the 8- or 16-cell stage, in contrast, results in the expression of both differentiation products. Fate-mapping experiments indicate that only the lineages of cells that normally express these markers in an autonomous fashion during normal development express these traits in cleavage-arrested 8- and 16-cell stage embryos. Lineages that form comb plates in a non-autonomous fashion (derivatives of the M lineage) do not. Timed actinomycin D and puromycin treatments show that transcription and translation are required for comb formation and suggest that the segregated material might be necessary for activation of the appropriate genes. Interestingly, even in the absence of cytokinesis, differentiation markers appear to be activated at the correct times. Treatments with a DNA synthesis inhibitor, aphidicolin, show that the number of nuclear divisions, and perhaps the DNA to cytoplasmic ratio, are critical for the appearance of lineage-specific differentiation. Conclusion: Our work corroborates previous studies demonstrating that the cleavage program is causally involved in the spatial segregation and/or activation of factors that give rise to distinct cell types in ctenophore development. These factors are segregated independently to the appropriate lineage at the 8- and the 16-cell stages and have features of a clock, such that comb-plate-like cilia and light-emitting photoproteins appear at roughly the same developmental time in cleavage-arrested embryos as they do in untreated embryos. Nuclear division, which possibly affects DNA-cytoplasmic ratios, appears to be important in the timing of differentiation markers. Evidence suggests that the 60-cell stage, just prior to gastrulation, is the time of zygotic gene activation. Such cleavage-clock-regulated phenomena appear to be widespread amongst the Metazoa and these cellular and molecular developmental mechanisms probably evolved early in metazoan evolution.

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University of Florida
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University of Florida
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Acleavageclockregulatesfeaturesoflineagespecificdifferentiationinthedevelopmentofa basalbranchingmetazoan,thectenophore MnemiopsisleidyiFischer etal. Fischer etal.EvoDevo 2014, 5:4 http://www.evodevojournal.com/content/5/1/4

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RESEARCH OpenAccessAcleavageclockregulatesfeaturesoflineagespecificdifferentiationinthedevelopmentofa basalbranchingmetazoan,thectenophore MnemiopsisleidyiAntjeHLFischer1,2 ,KevinPang3,4 ,JonathanQHenry5andMarkQMartindale6*AbstractBackground: Animportantquestioninexperimentalembryologyistounderstandhowthedevelopmental potentialresponsibleforthegenerationofdistinctcelltypesisspatiallysegregatedoverdevelopmentaltime. Classicalembryologicalworkshowedthatctenophores,agroupofgelatinousmarineinvertebratesthataroseearly inanimalevolution,displayahighlystereotypedpatternofearlydevelopmentandaprecociousspecificationof blastomerefates.Hereweinvestigatetheroleofautonomouscellspecificationandthedevelopmentaltiming oftwodistinctctenophorecelltypes(motilecompoundcomb-plate-likeciliaandlight-emittingphotocytes)in embryosofthelobatectenophore, Mnemiopsisleidyi Results: In Mnemiopsis ,9hafterfertilization,combplateciliadifferentiateintoderivativesoftheElineage,while thebioluminescentcapabilitybeginsinderivativesoftheMlineage.ArrestingcleavagewithcytochalasinBatthe 1-,2-or4-cellstagedoesnotresultinblastomeredeath;however,novisibledifferentiationofthecomb-plate-like ciliaorbioluminescencewasobserved.Cleavagearrestatthe8-or16-cellstage,incontrast,resultsintheexpressionof bothdifferentiationproducts.Fate-mappingexperimentsindicatethatonlythelineagesofcellsthatnormallyexpress thesemarkersinanautonomousfashionduringnormaldevelopmentexpressthesetraitsincleavage-arrested8-and 16-cellstageembryos.Lineagesthatformcombplatesinanon-autonomousfashion(derivativesoftheMlineage)do not.TimedactinomycinDandpuromycintreatmentsshowthattranscriptionandtranslationarerequiredforcomb formationandsuggestthatthesegregatedmaterialmightbenecessaryforactivationoftheappropriategenes. Interestingly,evenintheabsenceofcytokinesis,differentiationmarkersappeartobeactivatedatthecorrecttimes. TreatmentswithaDNAsynthesisinhibitor,aphidicolin,showthatthenumberofnucleardivisions,andperhapsthe DNAtocytoplasmicratio,arecriticalfortheappearanceoflineage-specificdifferentiation.(Continuedonnextpage) *Correspondence:mqmartin@whitney.ufl.eduEqualcontributors6WhitneyLabforMarineBioscience,Univ.Florida,9505OceanshoreBlvd,St, Augustine,FL32080,USA Fulllistofauthorinformationisavailableattheendofthearticle 2014Fischeretal.;licenseeBioMedCentralLtd.ThisisanopenaccessarticledistributedunderthetermsoftheCreative CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and reproductioninanymedium,providedtheoriginalworkisproperlycited.Fischer etal.EvoDevo 2014, 5 :4 http://www.evodevojournal.com/content/5/1/4

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(Continuedfrompreviouspage)Conclusion: Ourworkcorroboratespreviousstudiesdemonstratingthatthecleavageprogramiscausallyinvolvedin thespatialsegregationand/oractivationoffactorsthatgiverisetodistinctcelltypesinctenophoredevelopment. Thesefactorsaresegregatedindependentlytotheappropriatelineageatthe8-andthe16-cellstagesandhave featuresofaclock,suchthatcomb-plate-likeciliaandlight-emittingphotoproteinsappearatroughlythesame developmentaltimeincleavage-arrestedembryosastheydoinuntreatedembryos.Nucleardivision,whichpossibly affectsDNA-cytoplasmicratios,appearstobeimportantinthetimingofdifferentiationmarkers.Evidencesuggeststhat the60-cellstage,justpriortogastrulation,isthetimeofzygoticgeneactivation.Suchcleavage-clock-regulated phenomenaappeartobewidespreadamongsttheMetazoaandthesecellularandmoleculardevelopmental mechanismsprobablyevolvedearlyinmetazoanevolution. Keywords: Ctenophore,Combjelly,Photocyte,Combcell,CytochalasinB,Puromycin,Actinomycin,Cellcyclearrest, CelllineageBackgroundCtenophoresareamonophyleticgroupofseeminglysimplemarineanimalswithdistinctfeatures,suchasunique combrows,bodysymmetryandstereotypiccleavageprogram[1-11].ThenameCtenophoremeans comb-bearing andreferstoeightrowsofcomborcteneplates,eachpossessingthousandsofmotileciliaarrangedinlineararrays toformsmallpaddles[12].Eachcombplateinactenerow beatsinahighlysynchronizedmannerandtheyareused tomovetheanimalthroughthewatercolumn[12].The oral-aboralaxisisthemajorbodyaxisofctenophoresand itischaracterizedbythemouthatonepoleandthe statocyst-containingapicalorganattheopposite(aboral) pole[3,4,12](Figure1A,B).Theviewfromtheaboralpole showsthatctenophorescomprisefourhighlysimilarquadrants,whichareseparatedbytwoorthogonalplanes the tentacularplaneandtheesophagealorsagittalplane (Figure1A)[4,13-15].Theseplanesaredefinedbyaxesof rotationalsymmetryaroundtheoral-aboralaxisbecause diagonallyopposedquadrantsaremoreidenticaltoone anotherthanadjacentquadrants[3-610,13,15]. Manyctenophores,includingthelobatectenophore Mnemiopsisleidyi ,generatedistinctlight-producingphotocytesduringdevelopment,whichmigratetolocationssubjacenttoeachoftheeightcombrows.Genomesequencing of Mnemiopsis hasrevealedtwogenomicclusterscontainingtendistinctcopiesofphotoproteinscloselyresembling theluciferase-typephotoproteinsfoundatthebaseofthe Metazoa. Insitu hybridizationstudieshaveshownthat atleastsubsetsofthesemRNAsareexpressedinphotocytespriortowhentheseembryosarebioluminescent[16]. Thus,thepresenceorabsenceofbioluminescenceisa strongindicatorofthedevelopmentalfateofadifferentiatedphotocyte. Ctenophoredevelopmentishig hlystereotypicandunique withintheanimalkingdom[1,4,5].Inthelobatectenophore Mnemiopsisleidyi ,embryosaregenerallyfertilizedat thetimeofspawningandcleavagesareunipolarand holoblastic(Figure1,and2Additionalfile1)[4-6,10,17]. Subsequentroundsofdivisionoccurevery15to20minat roomtemperatureandthejuvenilecydippidstagehatches fromtheeggmembranewithin24hafterthefirstcleavage.Thesecondcleavage,whichisperpendiculartothe firstcleavage,givesrisetofoursimilar-sizedEMblastomeres[1,4,10,18].Thethirdcleavageisobliqueandresults intheformationoffourEcells(endcells)andfourMcells (middlecells)[1,4,10,14,18](Figure2andAdditionalfile1). EachMblastomereundergoestworoundsofasymmetric division,givingrisetotwosmallmmicromeres(m1and m2)towardstheaboralpoleandoneMmacromere towardstheoralpole[1,4,14].Eblastomeresundergothree asymmetriccelldivisionseach,producingthreesmalle micromeres(e1,e2ande3)attheaboralpoleandanE macromereattheoralpole[1,4,14].Themicromeresproliferatefurtherandbegintoenvelopthemacromeresduring gastrulationviaepibolyatapproximately3to4hourspost fertilization(hpf)[4,6](Figure2andAdditionalfile1). Laterduringdevelopment,themacromeres,whichare locatedattheoralpole,generateanothersetoforalmicromeres[4].Gastrulationiscompletearound5to6hpf. Ciliatedcombcellsappearby9hpfatthesametime bioluminescenceisdetected.Cydippidstagejuvenileshatch after18to24hpf[1,4,6,18]. Thestereotypedcleavageprograminctenophoresallows eachblastomeretobeidentifiedanditsfatefollowedby theinjectionofintracellularlineagetracers[1,4,7](summarizedinFigure1C).Forexample,themesoderm, includingmuscle,mesenchymalcellsandphotocytes,is generatedbythemicromeresbornfromendodermal precursorsatthefutureoralpole[4](summarizedin Figure1C).Earlylabelingexperimentsidentifiedthatthe e1micromeresgiverisetothecombplatecilia[18,19]; however,laterfate-mappingexperimentswereableto detectthem1micromere scontributionstotheformation ofcombplates(summarizedinFigure1C).Interestingly, whene1micromeresaredeleted,nocombplateciliaform,Fischer etal.EvoDevo 2014, 5 :4 Page2of19 http://www.evodevojournal.com/content/5/1/4

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Figure1 (Seelegendonnextpage.) Fischer etal.EvoDevo 2014, 5 :4 Page3of19 http://www.evodevojournal.com/content/5/1/4

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indicatingthate1micromeresareautonomouslyspecified togiverisetocombplateswhilem1micromeresrequire inductivesignals[18,20]. Here,weinvestigatetheroleofthecleavageprogramin thesegregationandexpressionofthedevelopmentalpotentialoftwodistinctcelltypes(motilecomb-plate-like ciliaandlight-emittingphotocytes)duringthedevelopmentofthectenophore Mnemiopsis .Usinganinhibitorof filamentousactinpolymerization(cytochalasinB)toblock cytokinesis(butnotnucleardivision),weshowthatthe formationofdifferentiationmarkersassociatedwitheach ofthesecelltypesappearsonlyaftertheyhavesegregated intotheirowndistinctlineagesandtheyarenevercoexpressedinthesamecells.Furthermore,usinginhibitors ofproteinsynthesis,transcriptionandDNAsynthesis,we provideevidencefortheexistenceofacleavageclockthat isbasedonthenumberofroundsofDNAsynthesis(and possiblycontrolledbythenuclear-to-cytoplasmicratio), whichdeterminesthetemporalappearanceofdifferentiationproductsincleavage-arrestedembryos.MethodsMnemiopsis collectionandembryosMnemiopsisleidyi adultswerecollectedfromtheNational OceanicandAtmosphericAdministrationjettyandEel PondinWoodsHole,MA,duringJuneandJulyandfrom 13660DeeringBayDr,CoralGables,FL33158,during December.Adultswerebroughtintothelabandinduced tospawnasdescribedpreviously[21].Eggswerewashed multipletimeswith0.2 mfilteredseawatertoremoveany jellyordebris.Onlybatchesinwhichahighpercentage ofembryosdevelopednormallywereusedinthestudy. Fertilizationintheseanimalsoccursatspawning,sofordevelopmentaltimingpurposesthiswasdesignatedas0hpf.PharmaceuticalinhibitorsAftertheembryoswerecollected,theyweretransferred bypipetto24-wellculturedishesfordrugtreatments. Thefollowingdrugswereused:cytochalasinB(Sigma,St. Louis,MO,USA,C6762),actinomycinD(Sigma,St.Louis, MO,USA,A1410),puromycin(Sigma,St.Louis,MO, USA,P7255)andaphidicolin(Sigma,St.Louis,MO,USA, A0781).Stocksolutionsofcytochalasin(1mg/ml),actinomycin(1mg/ml)andaphidicolin(1mg/ml)weremadeup indimethylsulfoxide,withaliquotsstoredat 20C.A puromycinstocksolution(12.5mg/ml)wasmadeupin distilledwaterandstoredat 20C.Freshlythawedaliquotswereusedineachexperiment.Workingsolutions weremadebydilutingstocksolutionswithfilteredseawater.Approximately1mlofsolutionwasaddedtoeach wellcontainingembryos.HoechststainingandimmunohistochemistryTheembryoswerefixedforantibodystainingin4% paraformaldehydeand0.02%glutaraldehyde,aspreviouslydescribedbyPangandMartindale[21].Following fixation,theembryoswereremovedfromtheirmembranesbygentlepipetting,washedwithPBSplus0.2% Triton(PBT),blockedin5%goatserumfor1handthen incubatedinanti-tyrosinetubulin(Sigma,T9028)overnightat4C.Theembryoswerewashedsixtimesfor 30mininPBTandincubatedwiththesecondaryantibody,goatanti-mouseconjugatedtoAlexa-594(Invitrogen,MolecularProbes,Carlsbad,CA,USA),Alexa-488 phalloidin(Invitrogen,MolecularProbes,Carlsbad,CA, USA)andHoechst33342(Invitrogen,MolecularProbes, Carlsbad,CA,USA)overnightat4C.Afterwardsthe embryoswerewashedtwicefor5mininPBSand mountedforimaging.LineagetracingAftertheembryoswerecollected,thevitellinemembranesweremechanicallyremovedusingsharpenedforceps.Thede-membranatedembryoswereallowedto developtothecorrectcleavagestagepriortoinjection withDiI,(CatalogNo.D-282;MolecularProbes,Inc., (Seefigureonpreviouspage.) Figure1 Ctenophoredevelopmentandcelllineage. Ctenophorebodyplan,with (A) lateralview,aboralpoleupand (B) aboralview.The combplatesareshowninyellowtoindicatethecontributionfrome1micromeres(yellow)andm1micromeres(pink).Thephotocytes,area derivativeofthe2Mcellsandassociatedwiththeendodermalcanalsunderlyingthecombrows[4,16]. (C) Simplifiedcelllineagefatemap showingthee1,m1,1E/and1M/micromeresublineages.Foreachofthedifferentearlycleavagestagestherearediagramsshowingtheembryo fromtheaboralpole.Modifiedafter[1,4,14]. Additionalfile1 Normalembryonicdevelopmentof Mnemiopsisleidyi. DICmicroscopytime-lapsemovieofdeveloping Mnemiopsis embryosbeginningwithazygoteuntil12hpf.The aboralsideisup.TheanimalshownisthesameasinFigure2A-P. Fischer etal.EvoDevo 2014, 5 :4 Page4of19 http://www.evodevojournal.com/content/5/1/4

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Eugene,OR)alipophilicmembranestainthatdiffuses laterallytostaintheentirecell,asdescribedin[4].FollowingDiIinjection,theembryoswerecarefullytransferredintoindividualwellsofaTerasakiplate(Nunc, Roskilde,Denmark)sothateachembryoremained separate.Eachwelloftheplatecontained10 loffilteredseawater(controls)orcytochalasinBsolution (1 g/ml).TheTerasakiplatewasthenstoredinahumidifiedchambertominimizeevaporation.ImagingTheembryoswerescoredforthepresenceorabsence ofcombcellsusingaStereo Discovery(Zeiss,Inc)or AxioScope(Zeiss,Inc)undertransmittedlight.Livecomb platecellswereimagedusingtime-lapsemicroscopywith timeintervalsoffivetotenimagespersecond.Normal Mnemiopsis developmentwasrecordedatthreeimagesper minuteusingDIC(Differentialinterferencecontrast). Visualizationofbioluminescencewasperformedonan Figure2 Embryonicdevelopmentof Mnemiopsisleidyi. DICimagesofdeveloping Mnemiopsis embryosatvariousstages,beginningwitha zygotein (A) until9hpfin (P) (A) Zygote. (B) 2-cellstage. (C) 4-cellstage. (D) 8-cellstage. (E) 16-cellstage. (F,G,H) 32-to60-cellstages. (I,J,K,L) Gastrulation. (M,N,O,P) Post-gastrulation.Theaboralsideisup.hpf,hourspostfertilization.Additionalfile1showstheembryonic developmentofthesamespecimeninatime-lapsemovie. Fischer etal.EvoDevo 2014, 5 :4 Page5of19 http://www.evodevojournal.com/content/5/1/4

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AxioScope,usingtheGFPfilt er(38HEGreenFluorescent Prot.filter,excitationBP470/40nm,emissionBP525/ 50nm)tostimulatephotoproteinemissionandwith0.5to 1secexposuretimeswithanAxioCamMrtocapturefaint signals.Priortovisualization,anembryowaskeptundisturbedindarknessfor10to20mintopreventactivation anddepletionofthephotoprotein. Confocalimagingafterimmunohistochemicalstaining wasperformedusingaZeiss710confocalmicroscope. ImageswereprocessedusingZensoftware(Zeiss,Inc) andVolocity(Improvision,Inc)tocreate3Dimagereconstructionsofconfocalsections.ResultsCytochalasinBinhibitsthepolymerizationofactinand thuspreventscytokinesis.Duringembryonicdevelopment, individualcleavagesandspecificdevelopmentalstagescan bearrestedbycytochalasinB,andthusthedevelopmental potentialofcellsthatwerepresentatthetimeofarrest canbeanalyzed.WeusedcytochalasinBonzygotes,the 2-,4-,8-and16-cellstagesandatlatertimepointsupto the60-cellstage(at3,3.5,4,5,6,7,8and9hpf).Anoverviewof Mnemiopsis developmentisshowninFigure2and Additionalfile1. CytochalasinBtreatmentof Mnemiopsis embryosledto theimmediatearrestofcytokinesis.Iftheembryowas undergoingcelldivisionatthetimeoftreatment,the cleavagefurrowimmediatelybegantoregressandthe blastomeresroundedup(Figure3,Additionalfile2). ThroughoutthecytochalasinBtreatment,theblastomeres didnotdieandcellnucleicontinuedtodivideas karyokinesisdoesnotrequireactinpolymerization[22]. Forthefirst4to5hoftreatment,thenucleidividedat thenormalrateandremainedintheperipheryofthecell (Figure4A),butafterapproximately5hpf,individual nucleifusedtogetherintooneorseverallargenuclei (Figure4B,C,D).Cleavage-arrestedblastomeresadhered tightlytoeachotheruntil7to8hpf,afterwhichindividual cellstendedtolosecontactwithoneanother.Although thenormalconfigurationoftheblastomereswaslost,they Additionalfile2 Regressionofthecleavagefurrowina Mnemiopsis embryoaftercytochalasinBtreatment. Cytochalasin Bwasaddedduringthefirstcleavageofa Mnemiopsis zygote.DIC microscopytime-lapseshowingtheingressionofthecleavage furrow.OncecytochalasinBhasbeenadded,cleavagestopsandthe cleavagefurrowregresses.Theanimalshownisthesameasin Figure3A-D. Figure3 Regressionofthecleavagefurrow(markedwitharrows)aftercytochalasinBtreatment. Cytochalasinwasaddedduringthefirst cleavageofa Mnemiopsis zygote.DICimagesshowingtheingressingcleavagefurrow(arrow) (A,B) .OncecytochalasinBhasbeenadded, cleavagestops (C) andfinallythecleavagefurrowregresses (D) .Additionalfile2showstheregressionofthecleavagefurrowinatime-lapse movieinthesamespecimen. Fischer etal.EvoDevo 2014, 5 :4 Page6of19 http://www.evodevojournal.com/content/5/1/4

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werestillcontainedwithinthevitellinemembrane,allowingfortheidentificationofalldescendantsfromanindividuallyarrestedembryo. Whilecytochalasin-treatedzygotesand2-and4-cell stagesdidnotshowanyvisiblesignofcelldifferentiation evenafter24hpf(Figure4),surprisingly,70%(31/44)of arrestedembryostreatedatthe16-cellstage(Figure5F) and83%(19/23)ofembryostreatedatthe32-cellstage hadctene-likeciliaaround9hpf.Whentreatedatthe 60-cellstageandonwards,nearly100%ofallarrested embryosformedctene-likecilia.Andwhentreatedwith cytochalasinBatthe8-cellstage,26%(9/35)ofthe arrestedembryoshadtwoormorecellsthatformedmotilecomb-plate-likecompoundcilia(Figure5,Additional file3,Additionalfile4,Additionalfile5).Theseciliawere approximatelythesamesizeandformedatthesame timeasforuntreatedcontrolembryos(Figure5G, Additionalfile6).Acloserlookatthectenecellsin treatedembryosrevealedthatindividualcomb-platelikeciliaappearedmorphologicallynormalwithrowsof ciliabeatingbackandforth(Figure5A,B,C,H,H',I,I', Additionalfile3,Additionalfile4,Additionalfile5). Ctenophoresmakeotherkindsofciliatedstructures(for example,domecilia,sensorypegs,ciliatedgroovesand balancingcilia)butmostofthesearenotmotile,andnone ofthemcomprisecompoundciliasuchasthosemadeby thecleavage-arrestedcells.Manyindividualcleavagearrestedcellsformedmultiplemotilecomb-plate-likecilia; thesecombswerenotarrangedindiscreterowsasisthe caseduringnormaldevelopment[23]andtheirbeating wasnotcoordinatedandappearedrandomwithrespectto oneanotherandtoneighboringcells.Theciliaofthe comb-plate-likegeneratingcellswereseenbeatingforup to18hpfandthesecellsquicklyseparatedfromtheother cellsintheembryos.Ctenecellsincleavage-arrestedembryosarederivedfrom e1micromeresDuringnormal Mnemiopsis development,ctenecellsare formedbye1micromeresandm1micromeres[4].However, whilee1micromerescandifferentiateintocomb-bearing cellsautonomously,m1micromeresrequireaninductive signalfromtheElineage[1].Todeterminewhichcell lineageswereformingthecomb cellsincleavage-arrested embryos,weutilizedDiIlabeling.LabelingofallfourE blastomeresatthe8-cellstageandarrestingtheembryoat the8-or16-cellstageledtoembryosinwhichallcells producingcomb-plate-likeciliawerelabeled(Table1, Figure6A,B,C,F,F',F'').Thesamewastrueafterlabeling allfourEmacromeresatthe8-cellstage,allowingthemto undergoonemorecelldivisi on(toformDiI-labeled1E macromeresande1micromeres)beforearrestingthemat the16-cellstage(Table1,Figure6F,F',F'').Theseexperimentsshowthatallcellsthatgaverisetocomb-plate-like ciliaincleavage-arrestedembryoswerederivedfromtheE lineage. Figure4 Nucleardivisionsincleavage-arrestedembryoscontinue. NucleicontinuetodivideaftercytochalasinBtreatmentresultingin multiplenucleiineachcell,whichlaterfuseintooneorseverallargenucleipercell.DNAstainingwithHoechst(blue)mergedwithDICimages. (A) EmbryoinhibitedwithcytochalasinBatthe2-cellstage.Multiplenucleiarevisiblealongtheperipheryofthecellsafter2hoftreatment. (B) AzygotearrestedwithcytochalasinBwithoneoverlylargenucleus. (C) EmbryoinhibitedwithcytochalasinBatthe2-cellstage,withone largenucleusintheleftcellandseverallargenucleiintherightcell. (D) EmbryoinhibitedwithcytochalasinBatthe4-cellstage.Eachcellhas onelargenucleus.Sincealltheembryoswereinhibitedpriortothe8-cellstage,noneofthemhavedifferentiatedcombcells. Fischer etal.EvoDevo 2014, 5 :4 Page7of19 http://www.evodevojournal.com/content/5/1/4

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ToruleoutthepossibilitythatDiIlabelingcouldinducethecellfateofthecomb-plate-likecilia,welabeled allfourMblastomeresatthe8-cellstageorallfour1M macromeresandm1micromeresatthe16-cellstageand arrestedtheembryoswithcytochalasinB.Inonlyone case(whichwaslikelytohavebeengeneratedfroma misinjectedcell),werethecellsproducingthecombplate-likecilialabeled(Figure6D,G). Todeterminewhethere1cellsspecificallygaveriseto thecombplates,welabeledallfoure1micromeresat the16-cellstageandimmediatelyafterwardstreatedthe embryoswithcytochalasinB.Alloftheresultingembryosformedcellsproducingcomb-plate-likeciliathat werelabeledwithDiI(Figure6E,Table1,Additional file7).Wealsolabeledallfour1Emacromeresafterthe divisionoftheEblastomeresintoe1and1Eatthe16cellstageandcleavagearrestedtheembryoswithcytochalasinB.Noneofthecellsproducingcomb-plate-like ciliathatformedwerelabeled(Table1).Thesedata demonstratethatonlyElineageblastomeresatthe8-cell stageande1micromeresatthe16-cellstagegiveriseto comb-bearingcells.1Emacromeres,butnotMlineage Figure5 Combcellsincleavage-arrestedembryos. Thearrowspointtotheciliaofthedifferentiatedcombcells. (A,B,C) DICimagesof cleavage-arrestedembryos. (A',B',C') Close-upviewsoftherectanglesin (A,B,C) (D,E,F) DNAstainingwithHoechst(blue)mergedwithDIC imagesofembryosinhibitedwithcytochalasinBatthe8-cellstage (D,E) or16-cellstage (F) (G) Controlembryoat10to12hpf.DICimages (H,I) andz-projectionsofconfocalimages (H',I') ofembryosstainedwithanti-tyrosinatedtubulin(red)showingthecilia,Alexa-488phalloidin (green)showingcellbordersandHoechst33342(blue)showingthenuclei.hpf,hourspostfertilization.Additionalfile3,Additionalfile4, Additionalfile5aremoviesshowingthemotilityoftheciliainthespecimensshownin A B and C respectively.Additionalfile6showsthe movementoftheciliainthecombrowsofthespecimenshownin G Fischer etal.EvoDevo 2014, 5 :4 Page8of19 http://www.evodevojournal.com/content/5/1/4

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descendants,appeartobeabletocontributetotheformationofcomb-plate-likecilia.PhotocytesarespecifiedbytheMlineageincleavagearrestedembryosTodeterminewhethertheMlineagealsogivesriseto productsspecifictodifferentiatedcellsincleavage-arrested Mnemiopsis embryos,welookedforthepresenceof bioluminescenceorlightproduction.In Mnemiopsis ,bioluminescenceisgeneratedbytheexpressionofluciferaselikephotoproteinsinaspecificcelltype,thephotocytes [11,16].Fate-mappingexperimentshaveshownthatduring development,the2Mmacromeresgiverisetophotocytes[4,16,24,25].Photo cytesarenormallylocatedin theendodermalcanalsunderneaththecombrows [11,26](Figure7A,A',A'',B).Tovisualizethephotoprotein,embryoswereacclimatizedtothedarkand imagedunderaGFPfiltersetonaZeisscompound microscope(Additionalfile8).Thebluefluorescentlight excitesthephotoproteininthesecellscausingthemto emitlightataround485nmto496nm[16,27].Inliving cydippids,theluminescenceappearedasgreenspecksand fadedawaywithin1to3sec,whichcanbeseenbythe nakedeyeandwasrecordedbyaCCDcamera. InembryosthatweretreatedwithcytochalasinBatthe 8-or16-cellstage,weobservedgreenbioluminescencein thecleavage-arrestedblastomeres.Thisbeganatthesame timeasincontrolembryosataround9hpf(Additional file9).BecauseDiIlabelingofMblastomeresmasked theweakflorescenceofthephotoproteinsininjected embryos,welabeledtheEblastomeresatthe8-cellstage withDiI.ObservationofDiI-injectedElineagecleavagearrestedembryosatthe8-or16-cellstagerevealedthat thephotoproteinwaslocatedintheunlabeledM(8-cell) and1Mmacromeres(precursorstothe2Mlineage)atthe 16-cellstage(Figure7C,D,E,F). Additionalfile3 Cleavage-arrestedembryowithbeatingcilia at2combcells. DICmicroscopymovieshowingthebeatingcilia oncomb-cellsinacleavage-arrestedembryo.Theembryoshownis thesameoneasinfigure5Aand5A'. Additionalfile4 Close-upononeacombcellwithbeatingcilia inacleavage-arrestedembryo. DICmicroscopymovieshowing thebeatingciliaoncomb-cellsinacleavage-arrestedembryo.The embryoshownisthesameoneasinfigure5Band5B'. Additionalfile6 Controlembryoataround10-12hpf. DIC microscopymovieshowingthebeatingciliaofthecombcells, whicharewellvisibleonbothsidesoftheembryo.Theembryo shownisthesameoneasinfigure5G. Additionalfile5 Cleavage-arrestedembryowithbeatingcilia atacombcells. DICmicroscopymovieshowingthebeatingcilia oncomb-cellsinacleavage-arrestedembryo.Theembryoshownis thesameoneasinfigure5Cand5C'. Fischer etal.EvoDevo 2014, 5 :4 Page9of19 http://www.evodevojournal.com/content/5/1/4

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Transcriptionandtranslationarerequiredforthe formationofcomb-plate-likeciliaincleavage-arrested embryosThedifferentiationofcomb-plate-likeciliainElineage cellsandphotoproteinsinMlineagedescendantsin cleavage-arrestedembryossuggeststhatdevelopmental potentialisfaithfullysegregatedtothecorrectspatiallineagesbytheearlycleavageprogram.However,itisunclear whatkeyfactorsmaybesegregatedintothelineages.To gaininsightintothemolecularnatureofthedeterminants fortheformationofcomb-plate-likecilia,weusedinhibitorsofmRNAandproteinsynthesistoseeiftheseclasses ofmoleculescouldberesponsiblefortheexpressionof thesestructures. Wetreatedembryosatvariousstages,beginningwith zygotesuptothetimethatcomb-plate-likeciliaare normallygeneratedinintactembryos(9hpf),with 10 g/mlofactinomycinD,aninhibitoroftranscription [28,29].Embryosthatweretreatedupto6hpf,didnot formcombplates(Table2),suggestingthatthelocalized determinantisnotsolelyanmRNA(possiblymaternal innature)thatisrequiredforcomb-plateformation. Theseresultssuggestthatadditionaltranscriptionisnecessaryforthecombplatestoformindescendantsof theElineage. Itisinterestingtonotethatembryosthatweretreated atthe1-,2-,4-,8-and16-cellstagescontinuedtodivide normallyandarrestedonlypriortogastrulationatabout the60-cellstage.EmbryosthatweretreatedwithactinomycinDatthe32-or60-cellstagedividedonlyafew moretimesandthenbecamearrested(Table2).This suggeststhatifactinomycinDisinhibitingthebulkof mRNAsynthesis,thenzygotictranscriptionappearsto beginataboutthe32-or60-cellstageandtherearesufficientstoresofmaternalmRNAstoallowtheembryo toreachthesestagesofdevelopment. Toblockproteinsynthesis,wetreatedembryosatvariousstageswithpuromycin(25to125 g/ml)[30].Comb formationwasonlyobservedinembryosthatweretreated after6hpf(at25 g/mlpuromycin),suggestingthatthe Table1Overviewofthecytochalasintreatmentexperimentson Mnemiopsisleidyi embryosDevelopmental stage TreatmentObservationConclusion 8-cellstageCytokinesisarrestedwithcytochalasinB50%havecombsAllcombsthatwereformedwerelabeled 16-cellstage75%havecombs 32-cellstage83%havecombs 8-cellstageCytokinesisarrestedwithcytochalasinB,labelingof allfourEblastomeres 62%(8/13)have combsthatarelabeled 38%(5/13)didnot formcombs 16-cellstageCytokinesisarrestedwithcytochalasinBat16-cell stage,labelingofallfourEblastomeresatthe8-cell stage 95/128(74%)formed combsthatarelabeled 99%ofembryosthatformedcombswerelabeled, Ecelllineageformscombs 32/128(25%)didnot formcombs 1/128(0.8%)formed combsthatarenot labeled 16-cellstageCytokinesisarrestedwithcytochalasinBatthe16-cell stage,labelingofalle1micromeresatthe16-cell stage 27/36(75%)formed combsthatarelabeled 100%ofembryosthatformedcombswere labeled,e1cellsformcombs 9/36(25%)didnot formcombs 16-cellstageCytokinesisarrestedwithcytochalasinBatthe16-cell stage,labelingofall1Emacromeresatthe16-cell stage NolabeledcombsOnlye1cellsat16-cellstageformcombsandE1 cellsdonotformcombs 8-cellstageCytokinesisarrestedwithcytochalasinBatthe8-cell stage,labelingofallMblastomeres 19/27(70%)formed combsthatarenot labeled 95%oftheembryosformedcombsthatwerenot labeledandcombsdonotarisefromMlineage cells 7/27(26%)didnot formcombs 1/27(4%)formed combsthatarelabeled 8-cellstageand 16-cellstage CytokinesisarrestedwithcytochalasinBatthe8-cell stageor16-cellstage,allEblastomereswerelabeled Unlabeledcellshave photoprotein Mcellscanmakephotoproteinevenwhenthey arecell-cyclearrested Fischer etal.EvoDevo 2014, 5 :4 Page10of19 http://www.evodevojournal.com/content/5/1/4

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keydeterminantisnotsolelythataproteinisrequiredfor combformation,butthatproteinsmustbetranslatedbetween5and6hpf(Table2).Thedifferentiationofcombsisregulatedbythenumber ofnucleardivisionsCellsproducingctene-likeciliaandphotocytesareformed atthesametimeincontrolembryosandinembryos treatedwithcytochalasinBatthe8-cellstageorlater, suggestingthattheembryohasamoleculartimingmechanismthatoperatesintheabsenceofcytokinesis. Becausenucleardivisionsappeartoproceednormallyin embryostreatedwithcytochalasinB(Figure4),thenumberofnucleardivisionsmaybeanimportantcomponent ofthetimingmechanism.Totestwhetherthetimingof theformationofthecomb-plate-likeciliaisdependenton thenumberofnucleardivisions,wetreatedembryoswith theDNAsynthesisinhibitoraphidicolin(2 g/ml)[31]at variouscleavagestagesupthrough5hpf(Table2).When treatedduringearlycleavage,uptothe32-cellstage,we didnotobservetheformationofanycomb-plate-likecilia. Whentreatedatthe60-cellstage,4hpf,and5hpf,wedid observecombformation,althoughitwasdelayedbyapproximately2to3h(12hpfasopposedto9hpf).Embryosthatweretreatedwithaphidicolinat5hpffor2h andthenremovedfromthedrug,formedcomb-plate-like ciliawithadelaycorrespondingapproximatelytothe lengthofthedrugtreatment.Theseresultsareconsistent Figure6 Lineagetracingexperimentsincleavage-arrestedembryosshowthate1micromeresgiverisetothecomb-cells. DICimages ofcleavage-arrestedembryoswithDiI-labelingshowninred.Whitearrowspointtolabeledcombplatesandblackarrowspointtounlabeled combplates.ThediagramsinthelowerleftcornersshowthestagewhentheembryowasinhibitedwithcytochalasinBandtheappropriate DiI-labeledcells. (A,B,C) AllfourEmacromereswerelabeledatthe8-cellstageandtheembryoswerearrestedatthe16-cellstage.Onlylabeled cellshavecombs. (D) AllfourMmacromereswerelabeledatthe8-cellstageandtheembryoswerearrestedatthe16-cellstage.Onlyunlabeled cellsformcombs. (E) Allfoure1micromereswerelabeledandtheembryoswerearrestedatthe16-cellstage.Allcombcellsarelabeled. (F,F'') Partofanembryothatwasarrestedatthe16-cellstage.AllEmacromeresarelabeled. (F) isaDICimage. (F') istheredchanneland (F'') shows bothimagesmerged.Thecombcellisclearlylabeled. (G,G',G'') Theembryowasarrestedatthe16-cellstage.AllfourMmacromeresare labeled. (G) istheDICimageand (G') theredchannel. (G'') showsbothimagesmerged.Allcombcellsareunlabeled. Fischer etal.EvoDevo 2014, 5 :4 Page11of19 http://www.evodevojournal.com/content/5/1/4

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withthefactthattheaphidicolintreatmentisreversible, thatDNAsynthesisisabletoresumefollowingtherescue (forexample,thedrugisnotlethal)andthatthecorrect totallineage-specificDNAcontentisimportantforthe timingoftheformationofcomb-plate-likecilia.Wedid notseeanycombformationinembryosthatweretreated withbothcytochalasinBandaphidicolin,evenafter6hpf. ThedoubletreatmentwithcytochalasinBandaphidicolin isnotreversibleandembryosthatweretreatedfor2,3or 4hpfdidnotrecoveranddidnotformcombcells.These resultsshowedthatthatthecombinedeffectsofblocking DNAsynthesisandcytokinesisaremoredetrimentalthan eitherdrugaloneandindicatethatacriticalnumberof nucleardivisionsisessential.DiscussionWeusedcytochalasinBtoinhibitcytokinesisinearlyctenophoreembryostoexaminehowdevelopmentalpotentialis partitionedintotwodistinctce lllineages:thosegivingrise tocombplatesandthoseformingthephotocytes.When cytokinesisisarrestedinearly Mnemiopsisleidyi embryos, treatedembryosdonotundergoapoptosis,butrathernucleardivisionscontinueandgiverisetomultinucleated cells.CytochalasinBwasusedtostudythesegregationof developmentalpotentialin Mnemiopsis byFreeman[18]. Freeman[18]inhibitedcertaindivisionsincludingthefirst, secondorthirdcleavageorthesecondandthirdcleavage byshorttreatmentswithcytochalasinB.Whenonlythefirst cleavagewasinhibitedbyabriefexposure,mostembryos developedlikethecontrolembryosbutweredelayedbyone cleavagecycle[18].However,whenthesecond,thirdorsecondandthirdcleavageswerereversiblyblocked,theinhibitedcleavageswereskippedandtheembryoscontinuedthe cleavageprogramwithareducednumberofcells[18]. Surprisingly,aswedemonstratehere,ifcytokinesisis arrestedatthe8-cellstageorlaterandispermittedto remainarrestedforthefollowinghoursofdevelopment, comb-plate-likeciliaandthephotoprotein-mediated bioluminescencecharacteristicofphotocytesformatthe correcttimeindevelopmentcomparedwithcontrolembryos.Lineage-tracingexperimentsrevealthatcomb-platelikecellsarederivedfromEcellsatthe8-cellstageand fromthee1micromeresatthe16-cellstate.Thesearethe Figure7 Photocytesincontrolembryosandcleavage-arrestedembryos.(A,A',A'') Innormalembryos,photocytes(whitearrows)are visibleat9to10hpfintheendodermbeneaththecombrows.Inthisliveanimal,viewedfromtheaboralpole, (A) showsnucleiinblue (Hoechst33342staining)overlaidwithphotoproteinfluorescence(green). (A') PhotoproteinfluorescenceasviewedundertheGFPchannel. (A'') DICimageofthesameembryoshownin (A) and (A') (B) Photoproteinfluorescenceofacontrolanimal. (C,D,E) Emacromereswere labeledwithDiIatthe8-cellstageandembryoswerecleavagearrestedatthe16-cellstage.Photoproteinisvisibleasgreendots(arrowheads), incellsthatarenotlabeled. (F) Photoproteininanembryothatwascleavagearrestedatthe16-cellstage.hpf,hourspostfertilization.Additional files8and9showvideosofthelight-emittingphotocytesofthespecimensshowninfigures B and F Additionalfile7 Cleavage-arrestedembryowithbeatingcilia andlabelede1-celllineage. DICmicroscopymovieincombination withfluorescentlightshowinganembryoinwhichthee1-cellswere DiIlabeledatthe16-cellstage.Theembryowasarrestedwith cytochalasinBatthe32-cellstage.Pleasenotethatonlylabeledcells bearcilia.TheembryoshownisthesameoneasinFigure6E. Fischer etal.EvoDevo 2014, 5 :4 Page12of19 http://www.evodevojournal.com/content/5/1/4

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samecelllineagesthatautonomouslygeneratecomb-platelikeciliawhenisolatedfromtherestoftheembryo[1].Previousintracellularfate-mappingexperimentsshowedthat m1micromeredescendantsalsohavethecapacitytomake comb-plate-likecilia[4]butMlineagedescendantswere notfoundtogiverisetocomb-plate-likeciliaincleavagearrestedembryos.Likewise,thebioluminescencecharacteristicofM-lineage-derivedphotocyteswasonlyseeninMlineage-derivedcellsincleavage-arrestedembryos.Earlier experimentsshowedthatthem1micromeresrequireinductivesignalsfirstfromthee11ore12micromeresand laterfromendomesodermalcellsderivedfromEandM Table2Overviewoftheaphidicolin,actinomycinDandpuromycintreatmentexperimentson Mnemiopsisleidyi embryosDevelopmentalstageTreatment Observation Conclusion 2-cellstage ArrestofDNAsynthesisfor 2hwithaphidicolin, 2 g/ml Nocombsformed(0/30) DNAsynthesisisessentialfor combformation. 16-cellstage 32-cellstage 60-cellstage 27/30(90%)formedcombs,comb formationwasdelayedby2h 4hpf 29/30(97%)formedcombs,comb formationwasdelayedby2h 5hpf 29/30(97%)formedcombs,comb formationwasdelayedby2h 60-cellstageand4hpfArrestofDNAsynthesisand cytokinesiswithaphidicolin, 2g/ml,pluscytochalasinB, 1to2 g/ml Immediatelyarrested,singlenuclei observed,neithercombsnor photoproteinwereformedinany embryo(0/30) DNAsynthesisisessentialforcomb formationevenincleavage-arrested embryos. 1-to2-cellstageBlockingoftranscriptionwith actinomycinD,10 g/ml 0/30gastrulated,developmentarrests at32-to60-cellstage Zygotictranscriptionbeginsaroundthe32to60-cellstageandthespecificationofthe combsrequiresmorethanonlymaternalRNAs. 4-to8-cellstage 0/30gastrulated,developmentarrests at32-to60-cellstage 16-to32-cellstage 10/30(33%)gastrulate,20/30(67%) arrestat32-to60-cellstage 60-cellstage 28/30(93%)gastrulatebutnopharynx isformed,2/30(7%)donotgastrulate 5hpf(post-gastrulation) Nopharynxisformed 2-cellstage Arrestoftranslation withpuromycin, 125 g/ml,25 g/ml 0/30formedcombs Combproteinistranslatedpriorto6hpf, combformationrequiresproteinthatis translatedintheembryoandnotmaternally loaded. 16-cellstage 0/30formedcombs 60-cellstage 0/30formedcombs 6hpf(post-gastrulation) 32/30(97%)formedcombs Additionalfile9 Bioluminescenceinphotocytesofacleavage arrestedembryos. Lightmicroscopymovieshowinglightemission fromthephotocytesthatwasstimulatedbyabriefilluminationwith fluorescentlight.Thosecellsthatcontainthebioluminescentprotein arephotocytes.TheembryoshownisthesameoneasinFigure7C. Additionalfile8 Bioluminescenceinphotocytesofacontrol embryos. Lightmicroscopymovieshowinglightemissionfromthe photocytesthatwasstimulatedbyabriefilluminationwith fluorescentlight.Thephotocytesarewellvisibleoneithersideof theembryo.TheanimalshownisthesameoneasinFigure7B. Fischer etal.EvoDevo 2014, 5 :4 Page13of19 http://www.evodevojournal.com/content/5/1/4

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macromeresbeforetheyformcombs[14,20].Ourresults supportthefindingthatm1micromeresrequireadditional inductivesignalsfromendomesodermalcellsforcombformation,sincenocombswereformedbym1micromeresin cleavage-arrestedembryos.Thissupportsthefindingthat thepresenceofe1micromeresisnotsufficienttoinduce combformationinm1micromeres[14,20]. Interestingly,neithercomb-plate-likecilianorbioluminescencewasdetectedincleavage-arrestedzygotes or2-or4-cellstageembryos,suggestingthatthesegregationofdistinctEandMlineagesiscrucialfortheexpressionofthelineage-specificdifferentiationproducts. Theseresultssuggestthatfactorsthatarerequiredfor comb-plateandphotocytedeterminationarealready presentandlocalizedatthe8-and16-cellstagesand mayhavemutuallyexclusiveactivity.ThesegregationofdevelopmentaldeterminantsOneofthefundamentalprocessesofdevelopmentisthe localizationoffactorsthatarerequiredtoestablishcell polarity,breaksymmetryanddrivetheasymmetric specificationofcellfates.Experimentalembryologists haveprovidedevidenceforthesegregationoffactors thatdeterminespecificcellfatesinadiversevariety ofdifferentorganisms(examplesaresummarizedin [32-34]).Insomeembryos,segregatedfactorsareasymmetricallylocalizedmaternallytothecellcortexsuchas thedevelopmentalpotentialrequiredforgastrulationin echinodermembryos[35-39]andfortheestablishmentof dorso-ventralpolarityinmanyembryosthatundergospiralcleavage[40-43],orthematernalgradientsininsect eggs[44-46].Othercytoplasmicfactorsaresegregatedactivelyatthetimeoffertilizationasinascidians[47-49], amphibians[50,51]andsoilnematodes[52,53]. Inctenophoreembryos,allevidencesuggeststhatthe asymmetricallocalizationofdevelopmentalpotentialis activelysegregatedbythecleavageprocessitself.Eachof theearlycelldivisionsleadstoadefiniteasymmetric cellfate.Thesiteoffirstcleavagegivesrisetothe oral-aboralaxis[10].Interestingly,asimilarcorrelation betweenthesiteofthefirstcleavageandthesiteofgastrulation,andthustheformationoftheoral-aboralaxis, hasbeenobservedinthehydrozoan Clytiahemisphaerica (previouslycalled Phialidiumgregarium )[54].Freeman [54]showedthatthesiteofthefirstcleavagecanbealteredfromthesideofthepolarbodyformationexperimentallyinctenophoresaswellasinhydrozoans[10,54]. Incontrast,theeggsofseveralseaurchinspeciesare alreadypolarizedalongtheanimal-vegetalaxisatthetime offertilization(summarizedin[55]).Starfisheggsand evenascidianeggsshowasimilarpolarization[56,57]. Basedoncurrentlyavailabledata,itcouldbeassumed thatanirrevocablepolarizationoftheunfertilizedeggwas anevolutionarynoveltyofbilaterianswhereasinbasally branchingtaxa,suchasctenophoresandcnidarians,the eggispolarizedbythepositionofthefemalepronucleus, whichdeterminesthesiteofthefirstcleavage,after fertilization[17].However,abroadertaxonsamplingisdesiredtofurthersupportthishypothesis. Inctenophores,theseconddivisiongivesrisetotheanal axis[4,15,19].ThethirddivisionseparatestheEandMlineages[4,18],andthefourthdivisionsegregatesfatesbetweenmicromerelineages(forexample,combrows)and macromerelineages(forexample,photocytecells)[4,18]. Consequently,whencytokinesisisinhibited,thefactorsremainassociatedwiththeproperlineagesthatwouldhave generatedthedescendantsduringnormaldevelopment. Interestingly,photoproteinformationandcomb-plateformationneverappearedtooccurinthesamecell.Itispossiblethatthedevelopmentaldeterminants,whichare requiredforcomb-plateandphotoproteindifferentiation, inhibiteachotherandonlyafterthesefactorshavebeen spatiallysegregatedcantheybeactivated. Althoughwehavelittleinformationonthecellbiological natureofthesegregationprocessinctenophores,centrosomeshavebeenshowntobeacausalfactorinthesegregationoffactorsthatspecifycellfatesinothersystems.In Caenorhabditiselegans ,thesperm-derivedcentrosome,a complexofseveralproteinsthatactsasthemicrotubuleorganizingcenter,breaksthe symmetryoftheoocyteand setsoffaseriesofeventsthatreliesonmaternallydeposited proteinsandeventuallyleadstotheestablishmentofthe anterior-posterior,dorsal-ventralandleft-rightbodyaxes [58,59].AsurprisinglylargenumberofmRNAsarelocalizedtospecificcelllineagesinthesnail Ilyanassa [60]. ThesemRNAsareassociatedwiththecentrosomes,andas thecellsdividethemRNAsaresubsequentlydistributedin anasymmetricmannertospecificdaughtercells[61].Experimentalevidenceshowsthatthesefactorsplayanactive roleintheuniquedevelopmentofthesecells[60,61].It wouldbeinterestingtoinvest igatewhetheracentrosomedependentmechanismisalsoinvolvedinthesegregationof developmentalfactorsin Mnemiopsis ,wheredistinctdevelopmentalfatesaredecidedateachoftheearlydivisions.Whatisthenatureofsegregateddevelopmental determinants?Ctenophoredevelopmentisrapid,withcleavagesoccurringevery15to20min.Theasymmetricallocalizationof proteinsormRNAsintodistinctlineagesduringeachdivisioncouldbeasimplewaytodistributecomponents quicklysoastodistinguishdifferentcellfates.Freeman [18]showedthatthedevelopmentaldeterminantsthatare requiredforcombformationbegintobelocalizedtothe aboralregionalreadyduringthe2-cellstage.Although crudeandnotgenespecific,ourexperimentsthatinhibit transcriptionandtranslationsuggestthatthedistributed determinantsarenotsimplyrepresentedbythefullsetofFischer etal.EvoDevo 2014, 5 :4 Page14of19 http://www.evodevojournal.com/content/5/1/4

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comb-plateproteinorcomb-platemRNA.Insteadsubsequenttranscriptionandtranslationarerequiredforcomb formation.Thenatureofthecomponentsthatareasymmetricallylocalizedduringtheearlycleavagestagesin Mnemiopsis remainsunknown. Inothersystems,RNAs,proteins,proteincomplexesor amixofthesearethedevelopmentaldeterminantsthat aredistributedunevenlyduringdevelopmentandwhich breakthesymmetries.Awell-knownexampleisthe localizationoftheproteinDishevelled akeyplayerof theWnt/ -cateninpathway toonesideofan unfertilizedegg.Thisisoneofthefirstsymmetry-breaking eventsinmanybilaterians[17,62,63].Otherwell-known examplesofproteinswithasymmetriclocalizationsare Miranda,ProsperoandStaufenduring Drosophila neuroblastdivision[64]andtheParproteinsduring C.elegans development[65]. Besidesthelocalizationofproteinsorproteincomplexes,RNAsareoftenasymmetricallystoredandthusestablishcellpolarityanddeterminethedifferentdaughter cellfates.OneofthemostfamousexamplesofRNA localizationisthelocalizationofthe Oskar mRNA,which isoneofthekeycomponentsusedtoestablishpolarityin the Drosophila oocyteduring Drosophila oogenesis[66-69]. OtherexamplesofRNAlocalizationare ASH1 inbudding yeast, bicoid in Drosophila embryos, Vg1 in Xenopus oocytes and CamKIIa indistaldendritesinmammalianneurons (summarizedin[70]). Futureexperimentswillbeneededtorevealthenature ofthedevelopmentaldeterminantsthataresegregated duringearlyctenophoredevelopment.Temporalregulationofcomb-plateformation:counting celldivisionsTheregulationofgrowthanddifferentiationarekeyto developmentalprocesses.Moreover,thetimingofdevelopmentaleventsisaparticularlyimportantaspectofdevelopment.Weshowedthatthedifferentiationofcomb cellsin Mnemiopsis embryos,whicharecleavagearrested atthe8-cellstageorlater,subsequentlyoccursatthesame timeasinuntreatedcontrolembryos.Furthermore,our resultsshowthatcomb-plateformationisblockedifDNA replicationisinhibitedbyaphidicolininadditiontotheinhibitionofcytokinesisbycytochalasinB.Thisindicates thatthenumberofnucleardivisioncyclesortheamount ofDNA(thenuclear-to-cytoplasmicratio)appeartobe importantfactorsforcomb-celldifferentiationandnot simplytheamountoftimethathaselapsedsincethethird cleavage.Aspreviouslyshown,afterthesecondand/or thirdcleavageisblocked, Mnemiopsis embryosskipthe respectivecleavagesandcontinueinatimelymanner withtheirsubsequentcleavageprogram,suggestingthata timingsystemdeterminestheorientationofeachdivision [18].Oncethisprocessisactivated,thecleavageplaneis determinedbythistimingmechanismandcleavagesdo notfollowasetorderwithrespecttoeachpriorcleavage division[18].Sincetheinhibitionofthefirstcleavageonly causesadelayoftheprogram,whichotherwiseoccurs normally,Freeman[18]concludedthatthetimingmechanismisinitiatedwiththecompletionofthefirstcleavage. Embryosthatundergoexactnumbersofcelldivisions areknownfromabroadrangeofdevelopmentalsystems; however,littleisunderstoodabouttheunderlyingmechanisms[71-75].Astunningexampleisthehighlystereotypiccelldivisionpatternoftheventralgermband inmalacostracancrustaceans.In Cheraxdestructor ,for example,allectoteloblastsundergoexactly15roundsof asymmetriccelldivisionandgivebirthtoectodermalblast cells,whichundergotwodistinctdivisionseach[74]. FundamentalexperimentsbyWhittakershowedthatascidianembryosdevelopedmuscle-specificacetylcholinesteraseandbrainpigmentcelltyrosinaseinspecific blastomeresevenincleavage-arrestedembryosandconcludedthatspecificpositionalinformationisdifferentially segregatedduringearlydevelopment[76].Sincethen severalstudieshaveemployedcytochalasinBtoinvestigate thecellfatespecificationandcelllineageofascidian embryos(forexample,[77-80]).SatohandIkegami[75] performedaseriesofexperimentscombiningcytochalasin Bandaphidicolininascidianembryos,showingthatfuture musclecellsmustundergoeightcell-divisioncyclesbefore theystartexpressingthemusclecelllineagemarkeracetylcholinesterase.Nevertheles s,thequestionofhowcells count thenumberofcellcyclesremainsunanswered. Anumberofcountingmechanismshavebeensuggested forthisphenomenon.Basedontheobservationthat genomicDNAishighlymethylatedinzygotesandgradually demethylatedduringdevelopment,whichtendstoderepresstranscriptionalacti vityinmammals[81],Kataoka etal .[73]suggestedthatchangesinDNAmethylationcould beinvolvedinkeepingcount ofthenumbercellcyclesin ascidians.However,theirownresultsdonotsupportthis hypothesisastheywereunabletodetectanychangesin DNAmethylation[73].Anotherpossibleexplanationisthat aspecificratioofDNAtocytoplasmisrequired.Thisratio iscrucialduringearly Xenopus development,wherezygotic transcriptionisinitiatedafter11to12roundsofcelldivision [82].TherequiredratioofDNA-to-cytoplasmicvolumecan alsobeacquiredwhencytokinesisisblockedwithcytochalasinBbutDNAreplicationisallowedtocontinuefurther [82].Incontrast,in C.elegans ,thetimingofgutmarker geneexpressiononlydependsonanearlyperiodofDNA synthesisuntilthe8-cellstagewhenthegutisclonally established[83].Subsequentro undsofcelldivisions,which usuallyoccurafterthe8-cellstage,canbeinhibitedwithout preventingtheexpressionofthemarkergeneatalater stage,thusthisisindependentoftheDNA-to-cytoplasmic ratioorthenumberofDNAsynthesisrounds[83].Fischer etal.EvoDevo 2014, 5 :4 Page15of19 http://www.evodevojournal.com/content/5/1/4

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Futurestudiesarerequiredtounravelthemechanism thatregulatesthetimingofcelldifferentiationinctenophores,whetherthisistheratioofDNAtocytoplasm, DNAmethylation,proteindegradation,telomericalterations,acombinationofdifferentfactorsoranentirely differentmechanismthatiscurrentlyunknown.TheonsetofzygotictranscriptionEarlydevelopmentaleventsareregulatedbygeneproducts,whichmaybeprovidedmaternally[84].Theactivationofthezygoticgenomemarksthematernal-to-zygotic transition(MZT),whichwasfirstdescribedfor Xenopus [82]andhassincebeenstudiedinmanymodelorganisms [84].Itisestimatedthataround40%(inmice)to75%(in seaurchins)ofallproteincodinggenesarerepresentedas maternalmRNAsandaredegradedthroughoutearlydevelopment[85,86]. Otherthanbilaterians,theonsetofzygotictranscriptionhasonlybeendescribedincnidarians.In Clytia hemisphaerica ,zygotictranscriptionstartsatthelate blastulatoearlygastrulastage[63,87].Theseaanemone Nematostellavectensis exhibitsanupregulationofgene expressionaround10hpf[88],atatimeindevelopment whencelldivisionisasynchronous[89]. WeusedactinomycinDtoinhibittranscriptionand weshowedthattreatmentpriortothe60-cellstagedid notvisiblyaffectdevelopmentuntiltheanimalreached the60-cellstage,suggestingthatmaternallyloadedtranscriptsaresufficienttosupporttheearlycleavageprogramuptothisstagebutnotsubsequentdevelopment ordifferentiation.Theseresultssuggestthattheonsetof zygotictranscriptionstartsataboutthe60-cellstage, justpriortogastrulation.Therapidearlydevelopment ofctenophoresalsosuggeststhattheremaybelittletime forthetranscriptionofnewmessagesuntilthecellcycle begantoslowdown.Futureexperimentsusinglabeled nucleotideswillhelptodeterminethepreciseonsetof zygotictranscriptionin Mnemiopsis. Thetransitionfrommaternaltozygotictranscription ismostlikelyanancientfeatureinanimalevolutionand Figure8 Summaryofmechanismscontrollingthedevelopmentofcombplatesandphotocytes.(A) Overviewofthenormalembryonic developmentof Mnemiopsis andthedifferenttreatmentswithcytochalasinB,aphidicolin,cytochalasinBplusaphidicolin,actinomycinDand puromycin.Theblackarrowindicateswhencombsformduringnormaldevelopment.Theyellowpartofeachbarindicateswhenatreatment startsthatpermitscombformation,andthegreenpartindicateswhenatreatmentstartsthatpermitsphotocyteformation,inthosecaseswhere ithasbeentested.Thewhitepartofeachbarindicatesthedevelopmentalperiod,whentreatmentwiththeinhibitorblockscomborphotocyte formation. (B) Overviewofthesegregationofdevelopmentaldeterminantsthroughoutearlycleavages.Allembryosareshownfromtheaboral pole.Atthe4-cellstage,thedevelopmentaldeterminantsarenotyetsegregated.Atthe8-cellstage,theMblastomeresinheritfactorsthat specifythephotocytelineage(greencloudslabeledwith photo )andtheEblastomeresinheritfactorsthatspecifythecomb-celllineage(orange cloudslabeledwith comb ).Atthe16-cellstage,factorsthatspecifythecomb-celllineagearesegregatedtothee1micromeres.Them1micromeres canformcombsbuttheyrequireaninductivesignalfromtheecells(pinkclouds)inadditiontosignalsprovidedbytheendomesoderm[20]. (C) SchematicoverviewofchangesDNAtocytoplasmicratioduringnormaldevelopmentandembryostreatedwithcytochalasinB,whichisa potentialmechanismforcountingthenumberofcelldivisions. Fischer etal.EvoDevo 2014, 5 :4 Page16of19 http://www.evodevojournal.com/content/5/1/4

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mightbetightlylinkedwiththeevolutionofmulticellularityandsexualreproduction.Ifctenophoresarethe sistergroupofallmetazoans,assuggestedbyrecent studies[90,91],onecouldconcludethattheinclusionof theMZTispartofthemetazoangroundpattern.However,sofardataabouttheMZTinspongesarenotavailableandthephylogeneticpositionofctenophoresisstill verymuchunderdebate[92].Therearecurrentlyseveral contradictoryhypotheses,including:(a)ctenophoresand cnidariansaresistergroups[93-95]and(b)ctenophores arethesistergrouptoallbilaterians[96].Recentdevelopmentalstudiessupporttheideathatctenophoreslack severalcharactersthataresharedbetweencnidariansand bilaterians[13,97-101]andthustheysupporttheideathat (c)ctenophoresarethesistergrouptoallremaining eumetazoans[102]oreven,asmentionedabove,that(d) ctenophoresarethesistergrouptoallmetazoans[90,91]. Aphylogeneticcomparisonofmaternallyloadedproteinsandthemolecularregulationofthematernal-tozygotictransitioninbilaterianandnon-bilateriantaxa mightprovidefurtherinsightintotheevolutionofsexualreproductionandlifehistory.ConclusionsAsinotheranimalswithamosaicdevelopment,embryos ofthecombjelly Mnemiopsisleidyi showdifferentiationof selectedcellfatesevenwhencytokinesisisarrestedusing cytochalasinBduringthecourseofearlyembryogenesis [48,77,78,103,104].AnoverviewisgiveninFigure8.We demonstratedthatembryosarrestedpriortothe8-cell stagedonotshowvisiblesignsofcellfatedifferentiation fromeithertheEorMlineages.However,ifcytokinesisis blockedatthe8-cellstageorlater,comb-plate-likecilia, whicharederivedautonomouslyfromtheElineage,and thebioluminescencecharacteristicofphotocytesformat thecorrecttimecomparedwithcontrolembryos.TreatmentwiththeDNAsynthesisinhibitoraphidicolinreveals thatthenumberofnucleardivisionsisessentialforthe propertimingofthedifferentiationofthecomb-plate-like cilia.Lineagetracingexperimentsshowthatcellsproducingcomb-plate-likeciliaareformedautonomouslyfrom theElineage,particularlythee1micromeres,andnotany oftheMorm1lineages.Likewise,bioluminescenceis onlyobservedintheMlineageandspecificallyinM macromeres.Inaddition,weshowedthatdevelopmental determinantsthatarerequiredtogeneratemotilecombplate-likeciliaandphotoproteinexpressionarealready presentandlocalizedatthe8-cellstageandfurtherlocalize totheappropriatecellsinthesubsequentcelldivisions. Furtherstudiesarerequiredtounravelthenatureofthedevelopmentaldeterminantsandwhichmechanismsareused tosegregatethem.Thetimedinhibitionoftranscription andtranslationwithactinomycinDandpuromycin,respectively,showsthatbothtranscriptionandtranslationare requiredforcombcellstoform,suggestingthatnotallthe necessaryfactorsarematernallydeposited.Whiletheseinhibitorstudiesarenotdefinitivegene-specificapproaches, theydoprovideinsightintothepotentialnatureofmaterialsthataredifferentiallysegregatedintodifferentembryoniclineages.Finally,wepresentedevidencethatsuggests thatzygotictranscriptionin Mnemiopsis beginsaround the60-cellstage,justbeforetheonsetofgastrulation.Abbreviations hpf: hourspostfertilization;MZT:Maternal-to-zygotictransition; PBS:Phosphate-bufferedsaline;PBT:PBSplus0.2%Triton; DIC:Differentialinterferencecontrast;AO:Apicalorgan. Competinginterests Theauthorsdeclarethattheyhavenocompetinginterests. Authors contributions AHLF,KP,JQHandMQMconceivedthestudyandanalyzedthedata.AHLF andKPconductedandanalyzedthedrugtreatmentsandproducedthe figures.KPanalyzedtheimmunohistochemistry,producedtheconfocal micrographsandrecordedthetime-lapseimages.JQHandMQMperformed themicroinjection.KP,JQHandMQMimagedtheinjectedembryos.AHLF wrotethefirstdraftofthemanuscript.Allotherauthorshelpedtocomplete andeditthemanuscript.Allauthorsreadandapprovedthefinalmanuscript. Acknowledgements WeareexceptionallythankfultoProfWilliamBrowne(UniversityofMiami, FA)forallowingustoperformsomeoftheexperimentsinhislaboratory andprovidinganimals.WealsowanttothankthecommunityoftheMarine BiologicalLaboratoryforfacilitatingthesestudies,inparticularthe2009 Embryologyclass,thefacultyandthecoursedirectors,LeeNiswanderand NipamPatel,wherethisprojectwasinitiated. ThisworkissupportedbyaBoehringerIngelheimTravelgrantandanMBL FinancialAidAwardtoAHLFtoattendtheEmbryologycourse2009.Itis furthersupportedbyNationalScienceFoundationgrantsawardedtoJQH (No.1121268)andKP(No.1158629).MQMwassupportedbytheNational ScienceFoundationandNASA. Authordetails1DevelopmentalBiologyUnit,EuropeanMolecularBiologyLaboratory Heidelberg,MeyerhofStrasse1,Heidelberg69117,Germany.2current address:MolecularandCellBiologyDepartment,HarvardUniversity,16 DivinityAvenue,Cambridge,MA02138,USA.3KewaloMarineLaboratory, PacificBiosciencesResearchCenter,UniversityofHawaiiatManoa,Honolulu, HI,USA.4currentaddress:SarsInternationalCentreforMarineMolecular Biology,Thormhlensgt.55,BergenN-5008,Norway.5DepartmentofCell andStructuralBiology,UniversityofIllinois,601S.GoodwinAve,Urbana,IL 61801,USA.6WhitneyLabforMarineBioscience,Univ.Florida,9505 OceanshoreBlvd,St,Augustine,FL32080,USA. 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