67404 Illkirch Cedex,CUdeStrasbourg,France. et deBiologieMoléculaire etCellulaire, UMR7104,CNRS/INSERM/ULP, BP10142,F- KEY WORDS: embryos, theTv1-Tv5neuroblasts,whichnormallyexpress neuronal identity the tritocerebrumshowsthatinactivationof development. Expressionof this report,weanalysetheroleofcolumnarpatterninggene anteroposterior anddorsoventralaxes,suchasHoxgenesforcolumnarpatterning.In In Simon G.Sprecher of neuromere formationduringembryonicbraindevelopment The columnargene Development 133,4331-4339(2006)doi:10.1242/dev.02606 Accepted 31August 2006 ‡ † 1 defective 1999; von OhlenandDoe,2000).Thus, subdivision oftheneuroectodermintolongitudinaldomains(Bhat, whereas columnargeneactivity resultsinatripartitedorsoventral segment alongtheAPaxisintofourparalleltransverse rows, These studiesshow thatsegment polaritygeneactivity divides each (VNC) alongtheanteroposterior(AP)anddorsoventral (DV) axes. cascades thatpatternandspecifytheembryonicventral nerve cord In INTRODUCTION rn Hirth Frank London, DeCrespigny Park,LondonSE58AF, UK York University, NY10003-6688, USA *Present address: CenterforDevelopmentalGenetics,DepartmentofBiology, New Mainz, D-55099Germany. Programmed celldeath loss of partially restorestritocerebralcellsaswellaxontracts.Taken together, ourresultsindicatethat an almostcompleteabsenceofthetritocerebralneuromere.Correspondingly, geneticblockofapoptosisin brain phenotypecausedbymutationoftheHoxgene 2003). columnar genes(reviewed bySkeath, 1999;Skeath andThor, columns areinfluencedbyexpression ofthecorresponding the individual neuroblaststhatformindifferent dorsoventral ventral, intermediateandlateralneuroblasts.Thedifferent fates of neuroectodermal cellsinturngive riseto thethreecolumnsof cells (D’AlessioandFrasch,1996;Isshikietal.,1997).These defective intermediate neuroblasts (Jimenez etal.,1995;MellerickandNirenberg, 1995),while and properidentityspecificationoftritocerebralneurallineagesduringembryonicbraindevelopment specific homeobox intermediate neuroectodermalcells(Weiss etal.,1998)and Author forcorrespondence (e-mail:[email protected]) Present address: MRCCentre forNeurodegeneration Research, King’s College Biozentrum, UniversityofBasel,CH-4056Switzerland. Drosophila Drosophila Drosophila lab -expressing cellsbecauseofincreasedapoptoticactivity, resultinginagap-likebrainphenotypethatischaracterizedby ( vnd Drosophila , evolutionarilyconservedtranscriptionfactorsarerequiredforthespecificationofneurallineagesalong 1,†,‡ , moleculargeneticstudieshave identifiedgenetic ) isexpressed inventral neuroectodermalcells vnd , ( msh 1,2, is requiredfortheformationandspecificationoftritocerebralneurallineages.Thus,inearly , Braindevelopment,Neuromere, DVpatterning, ) isexpressed inlateralneuroectodermal *, RolfUrbach vnd is observedinspecificsubsetsofcellsallbrainneuromeres.Loss-of-functionanalysisfocussedon ( ind 3 Institute ofGenetics,University ) expression isrestrictedto eta evu system ventral nervous 3 , Gerhard M.Technau vnd vnd 2 Institut deGénétique results inregionalizedaxonalpatterningdefects,whicharecomparablewiththe is requiredfortritocerebral labial muscle lab ( lab , donotform.Laterinembryogenesis, ). However, incontrastto ventral nervoussystemdefective defective formationofasubset oftritocerebralneuroblast andthe vnd formation ofthetritocerebrumin thedeveloping brain.Thus,lossof during embryonicbraindevelopment of later embryonicbraindevelopment islacking. However, informationontheexpression andfunctionof (Urbach andTechnau, 2003a; UrbachandTechnau, 2003b). neuroectoderm aswellinsubsetsofidentifiedbrainneuroblasts neurogenesis shows that in thedeveloping brainof known abouttheexpression andfunctionof thecolumnargene Modica, 2002). neuroblasts (Chuetal.,1998;McDonaldMellerickand transformations intheidentityofintermediateandlateral number. Conversely, overexpression of in neurons show pathfindingdefectsandmidlinegliaarereduced function mutants,commissuresarefused,ventral unpaired medial of embryonicdevelopment. In within thedeveloping VNCfromcellularizationuntilthecompletion an and tritocerebralneuromeres, show that out afunctionalanalysisof protocerebrum, deuterocerebrum andtritocerebrum.We thencarry that itisconfinedtosubsets of neuralcellsinthedeveloping immunocytochemistry, wefirstmaptheexpression of developing VNCof in axonalpathfindingandanoverall reducednumberofcellsinthe correlate withlossormis-specificationofneuronalprogeny, errors neuroblasts areabsentormis-specified.Theseprecursorcelldefects 3 , FilippoM.Rijli Here, weanalysetheroleofcolumnarpatterninggene In contrasttothesituationinembryonicVNC,muchless is The functionalroleofthecolumnargenesisexemplified by ventral nervoussystemdefective Nkx2- function leadstoagap-like brainphenotypethatisduetothe type homeoboxgene,whichiscontinuouslyexpressed 2 vnd , HeinrichReichert lab Drosophila mutant embryos.Thus,in ciiyinspecifyingtritocerebral activity vnd vnd vnd vnd focussed ontheintercalarysegment ( vnd is expressed intheprocephalic vnd loss-of-function mutants,ventral , Hoxgene, is requiredforthegenesis RESEARCH ARTICLE ) inembryonicbrain . Arecentstudyonearlybrain mutants showanextensive Drosophila vnd 1 vnd mutant embryos and Drosophila labial vnd is essentialforthe vnd . , Neuroblast, vnd a leadto can mutant vnd vnd and show . Using loss-of- during 4331 vnd vnd vnd ,

DEVELOPMENT crossed backtowildtype. MATERIALS ANDMETHODS immunolabelling. Embryosweremounted inVectashield H-1000(Vector). washed for3 minutes withStop/Wash solution(fromtheApoTaq kit),andsubsequently incubation, supernatantwas removed andembryoswerewashed for2 strength TdTmixture(fromtheApoTaq kit)for1hourat37°C.After the ApoTaq kit)for2minutes.Embryoswereincubatedintheworking hb-lacZ Bienz, 1992).Homozygousnullmutantswereidentifiedbytheabsenceof ␤ analyse thedevelopment ofthetritocerebral Homozygous nullmutantswereidentifiedbytheabsenceof (Jimenez andCampos-Ortega, 1990)was used,balancedover FM7,ftz- The wildtypewas Oregon-R. For Drosophila neural lineagesinembryonicbraindevelopment of essential fordevelopment andidentityspecificationoftritocerebral These findingssuggestthatthedorsoventral patterninggene results inpartialrestorationoftritocerebralcellsandaxontracts. neuromere, whereasblockingapoptosisin and thelongitudinalconnectives thatnormallyrunthroughthis apoptotic activity, resultinginthelossoftritocerebralcommissure show thatthislossofneuraltissueisassociated withincreased subsequent lossofneuraltissueinthemutantdomain.Moreover, we 4332 512 ranged from0.2-1.5 For laserconfocalmicroscopy, aLeica TCSSPwas used.Opticalsections Laser confocalmicroscopy andgenerationof3Ddigital models washed inPBTfor2 et al.,1998)withthefollowing modifications.Afterfixation,embryoswere commercial TUNELkit(ApoTag, Oncor)aspreviously described(Richter (Dianova). Apoptoticactivity was assayedbyTUNELanalysisusinga alkaline phosphatase-conjugatedantibodiesgeneratedingoatallat1:500 preparations analyzedusingNomarskiopticswereeitherbiotinylated or Probes), allat1:150dilution.Secondaryantibodiesusedforflat-mount 488, Alexa-568 andAlexa-647 antibodiesgenerated ingoat(Molecular Secondary antibodiesusedforconfocalmicroscopicanalysiswereAlexa- 1:20 (DSHB),mouseanti-Engrailed[4D9,1:6(Patel etal., 1989);DSHB]. (DSHB), mouseanti-PROS 1:4(SpanaandDoe,1995),mouseanti-REPO mouse anti-Fasciclin II1:5(LinandGoodman,1994),ratanti-ELAV 1:30 anti- VND at1:200(McDonaldetal.,1998)(kindlyprovided byC.Q.Doe),rabbit unpublished), mouseanti-NRT at1:20(BP106antibody, DSHB),rabbitanti- rabbit anti-LABat1:100(F.H., unpublished),ratanti-LABat1:500(F.H., [FITC-conjugated; 1:100(JanandJan,1982);JacksonImmunoresearch], [1:300 (Bieretal.,1992);kindlyprovided byH.Vaessin], rabbitanti-HRP 1995; Urbachetal.,2003).Primaryantibodieswererabbitanti-Deadpan according topreviously published protocols(Patel, 1994;Therianosetal., Embryos weredechorionated,fixed, immunostained,flattenedandstaged Immunocytochemistry andTUNELassay line inordertoactivate UAS:: we usedline former (Tremml andBienz,1992)crossedinto vnd within neurallineagesof to earlyneurallineageformation.For theubiquitousblockofapoptosis al., 2004)whichintheembryonicCNSisactive fromneurectodermalstage As aGal4-driver line,weusedthe Perrimon, 1993)was usedinordertoperform overexpression experiments. al., 2001).TheUAS/Gal4 transcriptionalactivation system(Brandand expression patternsinthedeutocerebralanlage(Hirthetal.,1998;Hirth (Campos-Ortega andHartenstein,1997). 2003). EmbryoswerestagedaccordingtoCampos-Ortega andHartenstein gal ϫ ␤ mutant background,weusedtheline GAL 1:200-1:400(MilanAnalytika),mouseanti- 512 pixels, or1024 and reflectsendogenous lab . For comparisonwiththewild-typesituation, RESEARCH ARTICLE -expressing tritocerebralcellsinthe 7.31 lab-lacZ/7.31lab-lacZ;lab strains andgenetics ϫ 2 minutes, ϫ ␮ m recordedinlineaverage mode withpicturesizeof 5 minutes,thenwashed inEquilibrationBuffer (from vnd 7.31 lab-lacZ ϫ 6 1024 pixels. Capturedimagesfromoptical mutant embryos,weusedthe p35 ϫ lab sca:: 30 minutesinPBTbeforestarting vnd transcription (Mergliano andMinden, expression withadditionalectopic mutant analysis,thenullallele Gal4 (Klaesetal.,1994;Sprecher vnd shows cytoplasmic distribution of 6 . To identify vd1 lab 7.31 lab-lacZ/7.31lab-lacZ vnd /TM3, hb-lacZ labial expression territoryinthe -null mutantembryos ␤ mutant background, GAL 1:50(DSHB), 7.31 lab-lacZ Drosophila vnd sca:: expression in (Tremml and ftz-lacZ Gal4 driver vnd . lacZ vnd . To was ϫ is 2 6 . deuterocerebral; red, tritocerebral). developing brain( reconstructed modelsshow relative locationofdomainswithin become apparent thatare stillobservableatstage15.(B,D)3D (green, yellow).(A,C)Atstage late12,three immunolabelling withanti-HRPantibody (red) andanti-VNDantibody antibody (red) andanti-VND antibody(green/yellow). (C)Double- Embryodouble-immunolabelled withanti-Neurotactin (NRT) (A) shown forembryoniclatestage12( microscopic stacks,coveringcorresponding opticalsections (B,D)are sections, lateralviews(A,C)and3Dreconstructed modelsofconfocal development. Fig. 1.Spatiotemporalexpression of the tritocerebralanddeuterocerebral observed inthedeutocerebrumandtritocerebrum. Although neuromere (Fig.1A,B)andtwo smallerexpression domainsare late stage12,alarge expression domainisseenintheprotocerebral seen inspecificcellclusterswithinthesebrainneuromeres.Thus,at 2006)]. Duringlaterstagesofembryogenesis, during theearlyphaseofneurogenesis,Urbachetal.(Urbachal., brain neuromeres[foradetaileddescriptionof ventral domainsoftheprotocerebral,deutcerebralandtritocerebral is seenintheneurectodermanddelaminatingneuroblasts During aninitialphaseofembryonicneurogenesis, embryonic braindevelopment Neuromere-specific RESULTS Photoshop. Hartenstein, 2004).FigureswerearrangedandlabelledusingAdobe and transparency ofindividual materialsurfaces (fordetails, seePereanuand were generatedshowing themodelinappropriateangle,virtuallighting smoothening ofthesurface. For 2Drepresentationsinfigures,screenshots was furtherprocessedbyincreasingthenumberoftrianglespermaterialand synthesizes asurface bytriangulationaroundthedefinedmaterials,which which weretobeincludedinthemodel.Subsequently, theprogram (immunoreactivity ofagiven antibody)ineachlayerofagiven tiff stack, materials weredrawn manuallyaroundthelabelledstructures were importedintoAMIRA(MercuryComputerSystems).User-defined generation of3Ddigitalmodels,raw tiff stacks(stacksofopticalsections) series ofopticalsectionswereimportedandprocessedusingImageJ.For the sections werearrangedandprocessedusingIMARIS(Bitplane).Complete Laser confocalmicroscopy, reconstructions ofoptical vnd expression domains:blue, protocerebral; green, vnd expression during A , B ), andstage15( vnd vnd expression clustersarein vnd during embryonicbrain Development 133(21) vnd expression domains expression isalso vnd vnd C , D ). expression expression

DEVELOPMENT protocerebral b1 to bepresent butare severely misplaced(arrow). (E)Inwildtype,the expressing cellsinresidual tritocerebral/deuterocerebral region appear embryonic wild-typebrain.(D)In specific markerREPOreveals localizationofglialcellbodiesin seen inthetritocerebral/deuterocerebral region (arrow). (C)Theglia- neural cellbodies.(B)Bycontrast,in (yellow/green). (A)Inwildtype,neuron-specific marker ELAV reveals all immunolabelling withanti-HRPantibody(red) andananti-ENantibody a cellulargapindeuto- andtritocerebral region (arrow). spot are present (arrowheads), andneuron-specific HRPmarkerreveals mutant embryosonlyb1 head spot(shs)are visible(arrowheads). (F)Bycontrast,in tritocerebral b3 indicate generaltrito-deutocerebral region. ( (red) andglial-specificanti-REPOantibody(yellow/green). Arrows (yellow/green). ( immunolabelling withanti-HRPantibody(red) andanti-ELAV antibody that noneofthegliacellsembryonicbrainexpress Immunolabelling withglia-specificanti-REPOantibodyindicates with anti-PROS andneuron-specificanti-ELAV antibodies. ganglion mothercellsandneuronsasjudgedbyimmunolabelling embryonic neurogenesis,wefind visible inthesethreeneuromericdomains(Fig.1C,D).Throughout of embryogenesis,atembryonicstage15,expression of close proximitytoeachother, they donotoverlap. Towards theend Vnd controls brainneuromere formation reconstructions ofopticalsections,lateralviews.( embryos atembryonicstage15. Fig. 2.Mutantbrainphenotypeobservedin not shown). vnd en C -expressing cellsarealsoseenintheneuromeresof en , D -stripe andanteriormost -stripe (b1),deuterocerebral b2 ) Double-immunolabellingwithanti-HRPantibody en -stripe and vnd Laser confocalmicroscopy vnd mutant embryos,REPO- vnd en -null mutantsalargegapis expressing secondaryhead expression inneuroblasts, en E , F expressing secondary ) Double vnd A , en B -null mutant ) Double- -stripe (b2), vnd vnd -null vnd is still (data Mutational inactivation of function mutants Gap-like braindefectsoccurin further inthisreport. sense organs; these the suboesophagealganglionandVNC,aswellinperipheral were notstudiedfurther. (see alsoMellerickandModica,2001).Theselattertwo phenomena expressing tritocerebral neuroblastsareaffected in (Urbach andTechnau, 2003a),thesefindingssuggestthat overlapping expression inasubsetoftritocerebralneuroblasts phenotype observed fortheHoxgene described above is,inpart,reminiscentof the mutantbrain suboesophageal ganglionandtheVNCisaffected inthe seen in a lessmarked reductioninoverall sizeoftheprotocerebrumisalso to thecelllossdefectintritocerebral/deutocerebralbrainregion, parts ofthedeutocerebrumarelackingin observation thatmajorpartsoftheembryonictritocerebrumand the b3 and thesecondaryheadspotarevisible;neitherb2 mutant brain(embryonicstage13onwards), onlytheb1 stripe) delimitstheposteriortritocerebrum(Fig.2E).Inlate posterior deutocerebrum,andtheb3 head spot),theb2 also seenmoreanteriorlyintheprotocerebrumassecondary head spot)delimitstheposteriorprotocerebrum(several studied theexpression of tissue (Fig.2C,D). affected region, mostprobablyowing totheabsenceofneuronal are presentinthemutantbut fail tobecorrectlylocalizedinthe Glia-specific anti-REPOimmunoreactivity reveals thatglialcells arrows) andthetritocerebralcommissureiscompletelyabsent. suboesophageal ganglionaremissingorstronglyreduced(Fig.2, connectives thatnormallyrunfromtheprotocerebrumto axonal patterningdefectsintheembryonicbrain.Thelongitudinal suboesophageal ganglion(Fig.2B).Thiscelllossisassociatedwith remains andinterconnectstheprotocerebrum 46/74) athinstrandofELAV- andHRP-immunoreactive cells (not shown), whileinthemajorityof gap iscompletelydevoid ofElav- andHRP-immunoreactive cells phenotype reveals thatin36.5%ofthemutantembryos(27/74)this 2A,B). Evaluation ofthepenetrance the neuromeresofposteriorsuboesophagealganglion(Fig. large gapseparatingtheanteriordeutocerebralbrainregion from neuron-specific anti-HRPandanti-ELAV antibodiesidentifiesa brain.Immunolabellingwith phenotype inthelatestageembryonic tritocerebrum, andspecificallyon the embryos. To investigate this, wefocussedonthedeveloping 1998; Page, 2000;Hirthetal.,2001).As connectives andlackofthetritocerebralcommissure (Hirthetal., axogenesis defectsoccur, includingthedisruption oflongitudinal however, thesecellsfail todifferentiate intoneuronsandmarked mutants, tritocerebralcellsaregeneratedandpositionedcorrectly; Ott andTechnau, 1992;Hirthetal.,1995).Theb1 demarcate theposteriorboundaryofbrainneuromeres(Schmidt- embryonic brainislocatedinseveral smallclustersofcellsthat neuromere, andfirstdetermined whetherlossof formation of At thegrosshistologicallevel, the To delineatetheregion affected in en vnd -stripe canbeidentified(Fig.2F).Thissupportsthe lab mutant embryos.Moreover theorganization ofthe -expressing neuroblasts. en vnd -stripe (or -expressing cellswillnotbeconsidered engrailed vnd en results inapronouncedbrain RESEARCH ARTICLE vnd antennal stripe)delimitsthe ( lab vnd en vnd en mutants inmoredetail,we ), whichinthewild-type vnd expression domainofthis -stripe (or labial mutant brainphenotype mutant embryos(63%; lab vnd loss-of- vnd and mutant. Inaddition ( vnd lab en function affects vnd en ). In -null mutant -stripe (or en vnd vnd en intercalary -stripe nor also show en cells are lab mutant mutant -stripe 4333 -null lab vnd en -

DEVELOPMENT of neuroblasts encircled bybroken line,B).Fg,foregut; Lr, labrum; MdandMx,mandibular andmaxillarysegment,respectively detectable levels;similarly, inwildtype aneuroblast expressing DPNatsignificantlylower levelsisfoundinsamerelative p NumberofDPN-positiveneuroblasts (whiteasterisks)isdiminished,whencompared with B.Oneneuroblast (whitedot)doesno (D) invagination asmarkedbyred arrowheads inAandC). (Fg) isaffected (compare lateralextensionofforegut compared withwildtype(A), andinvaginationoftheforegut expansion ofLABdomainappears tobereduced when mutants (compare withD). (C)In dorsal neuroblasts thatare assumedtoberetained in deutocerebral neuroblasts. Broken lineencircles group of Td1-8, dorsaltritocerebral neuroblasts; Dv2,Dv4ventral (Urbach etal.,2003)]:Tv1-5,ventraltritocerebral neuroblasts; indicated [according tonomenclature ofUrbachetal. tritocerebral neuroblasts developingfrom LABdomainare stage 11.(B)Two deutocerebral andcompletesetof wild-type embryos,allbrainneuroblasts havedevelopedby in A,Cbyblackframes,atlevelofbrainneuroblasts. (A)In preparations. (B,D)Highermagnificationofregions indicated are presentandexpress (Urbach andTechnau, 2003a).Bystage11,alloftheseneuroblasts all ofthetritocerebralneuroblastsandtwo deutocerebralneuroblasts neuroectodermal domaingives riseto15neuroblasts,whichinclude During theearlyphaseofbrainneurogenesis, vnd Defective tritocerebral neuroblast formationin 4334 (A,B) and anti-LAB (brown), atembryonicstage11,inwildtype(WT) immunolabelling withneuroblast-specific anti-DPN(blue)and tritocerebral domainof Fig. 3.Defectiveneuroblast formationin responsible forthegap-like phenotypeobserved in defective neuroblastformation,otherphenomenamustbe mutant brains(compareFig.3Bwith3D).Hence,inadditionto neural progeny aregeneratedinthetritocerebrumofstage 11 tritocerebral neuroblasts,aswelllarge numberof deutocerebrum. Thesedataimplythat neuroblasts ofthetritocerebrumandadjacentpart expressing neuroblastsappearstoaffect preferentiallyventral Technau, 2003a;Urbachetal.,2006)],thisreductionin [e.g. expression ofmolecularmarkers indicative ofdorsalneuroblasts expression aresometimesobserved inthisregion). Basedonthe enlarged roundedcellsinsub-ectodermalpositionlackingDeadpan marker Deadpan(thismaybeaslightunderestimateasfew cells areobserved thatco-express diminished (Fig.3C,D).Generallyonlyfourtosixlarge rounded of domain appearstobereducedinsizeandaccordingly, thenumber between stages8and11(UrbachTechnau, 2003a). neuroblasts originate,dynamicallyco-expresses tritocerebral neuroblastsTv1-Tv5andthetwo deutocerebral ventral partoftheneuroectodermaldomain,fromwhich neuroblasts, Dv2andDv4(Fig.3A,B).Inthewildtype,most tritocerebral neuroblasts,Td1-Td8,andtwo deutocerebral tritocerebral neuroblasts,Tv1-Tv5,amoredorsalgroupof mutant brain.Thisisbecauseadorsalsubsetofthe massive celllossphenotypeobserved inthelateembryonic mechanism aloneisunlikely tobetheexclusive causeforthe vnd developing tritocerebrum. formation ofaventral subsetof In Although thereductionintritocerebralneuroblastnumberseen lab mutants canaccountforsomeofthetritocerebraldefects,this mutants vnd -expressing neuroblaststhatderive fromthisbrainregion is ladybird early RESEARCH ARTICLE vnd mutants thisventral-most partofthe -null mutants(C,D).(A-D)Ventral viewsofflat , empty spiracles vnd lab mutants. ; they includeaventral groupof vnd lab -null mutants,overall lab -expressing neuroblastsinthe ( and theneuroblast-specific A-D , lab vnd wingless ) Double- -expressing is requiredforthe vnd lab lab lab lab (Urbach and lab -null -expressing -expressing vnd -expressing -expressing and mutant lab vnd vnd vnd - by a7.31 the tritocerebralmutantdomainaregeneratedandcanbevisualized this, wetookadvantage ofthefact thatin mutant tritocerebrumbystudying (Fig. 4C,D). remaining cellsoftheinterconnectingstranddoshow ganglion. Despitetheextensive celllossseenin remaining partofthedeutocerebrumwithsuboesophageal strand ofcellsthatinterconnectstheprotocerebrumand vnd loss-of-function mutantbrains.Owingtoextensive celllossinthe neuromere (Fig.4A,B).Next, weanalysed prominent intheventral region (accordingtoneuraxis)ofthis differentiating tritocerebrumthroughoutembryogenesis andis through a either byactingdirectlyon brains, implyingthat labelled bythe7.31 investigate this,wefocussedon the tritocerebralcellsthatare differentiating cellsduringtritocerebralneuromereformation.To possibility isthat neuromere development duringlaterstagesofembryogenesis?One What isthenatureof tritocerebrum Increased apoptosisinthe stages ofembryonicbraindevelopment. expression of the partial overlap withtritocerebral (see alsoHirthetal.,1998), neuronal differentiation markers incellsofthe Hirth etal.,1998).Surprisingly, despitethelackofexpression of a partialoverlap of neuromere formation.Immunocytochemical analysisindicatesthat overlapping expression duringlaterstagesoftritocerebral To investigate this,wefirstdeterminedwhether vnd We next investigated whetherexpression of lab- mutant tritocerebrum,thisanalysiswas limitedtotheremaining and specific reporterconstruct(Fig.4F).Thisindicatesthat lab lab lab -independent requirement. - vnd lacZ in tritocerebral neuromere formation is notaffected bytheabsenceof lab-lacZ reporter construct(Tremml andBienz,1992; vnd vnd vnd vnd is requiredalsolaterinembryogenesis– might beinvolved in maintaining lab and reporter constructandcomparedwild- vnd requirement forpropertritocerebral expression intritocerebralcellsor lab is expressed normallyandshows lab lab vnd mutant cells,asvisualizedby loss-of-function mutants.For expression persistsinthe mutant lab lab osition (seeTd5incluster Development 133(21) -null mutants,cellsin expression inlate vnd . lab vnd vnd occurs inthe t express DPNat mutant domain lab mutant brains, lab and during late expression lab show vnd lab

DEVELOPMENT and express (blue) andanti-LAB(green); onlyafewcellsremain in thetritocerebrum ( 5F). Thus,anaverage of27cellsexpressing the tritocerebral domainissignificantlyreducedin Fig. 3B,D).Bycontrast,bylatestage12,the somewhat reducedinsizebut notdeletedin 12 indicatesthatthe ( expressing tritocerebral domain(arrow); AandBare from samesection. reporter geneexpression in comparison ofreportergeneexpression inwildtype(Fig.5B)with anti-VND (red); co-expression of brain triple-immunolabelledwithanti-HRP(blue),anti-LAB(green) and immunolabelled withanti-HRPantibody(blue).( and Fare from samesection. expression overlapswith tritocerebral domain.( anti-HRP (blue);noimmunoreactivity isdetectedin endogenous of thisreporterconstructinawild-typebackgroundreflects type with Vnd controls brainneuromere formation Arrows indicatetritocerebral region. ( stage 15embryos,reconstructions ofopticalsections,lateralviews. tritocerebral neuromere formation. Fig. 4. expression isseeninapart oftritocerebral domainmutantfor (red) andanti- embryonic braintriple-immunolabelled withanti-HRP(blue),anti-VND LacZ};;lab D C ) ) vnd vnd vnd mutant embryonicbrainimmunolabelledwithanti-HRP(blue). mutant embryonicbraindouble-immunolabelledanti-HRP vd1 vnd / lab and theanteriorHoxgene lab lab ␤ null mutantembryonicbrains.Atstage12,expression ; CandDare from samesection.( GAL, revealing 7.31 vd1 expression inthetritocerebrum(Fig.5A,B,D,E).A : nullmutantembryonicbrainimmunolabelled with F ) P{ry lab-lacZ lab + vnd 7.31 lab-LacZ};;lab -expressing tritocerebraldomainis vnd -null mutants(Fig.5C)atearlystage specific reporter geneexpression; E lab and A ) Wild-type embryonicbrain ) Wild-type - Laser confocalmicroscopy of LacZ lab labial is seeninpartof reporter (green). B vd1 vnd act independentlyin ) Wild-type embryonic ) Wild-type E ) / lab P{ry vnd mutants (seealso lab-lacZ vd1 lab + mutants (Fig. 7.31 lab- : nullmutant -expressing vnd lab lab reporter - and 1998).] Inearlystage12 developing brainatthisstage(Abramsetal.,1993;Nassif (Fig. 5G).[Alow level of apoptosis isseenthroughoutthe tritocerebral wild type,low level apoptotic activity isassociatedwiththe apoptotic activity inthedeveloping tritocerebrum.Inearlystage12 cells inthe tritocerebral vnd suggests thatinadditiontodefective neuroblastformation,lossof with anaverage of43cellsdetectableinwildtype(Fig.5K).This identifiable withinthe 5I,J). Thus,onaverage, 20TUNEL-positive apoptoticcellsare has occurredin 12, asignificantreductionofthenumberanti-LABpositive cells expressing cellsinthisregion (Fig.5H).Accordingly, bylatestage type andthiscorrelateswithadecreaseinthenumberof the tritocerebral number ofTUNEL-positive apoptoticcellsisseenassociatedwith gene aredetectableinthe arrow). Moreover, apronounced connectives thattransverse thetritocerebrum aredetectable(Fig.6C, longitudinal axon tractsdisplayfasciculation defectsandappear anteroposterior neuraxis.However, neural fibrescontributing to connectives aredetectableandformacontinuousbandalongthe loss-of-function mutants.Thus, FAS2-immunoreactive longitudinal apoptosis significantlyreduces the gap-like defectsobserved in compared withwild-typeand tracts, areseverely perturbedorlacking(Fig.7C,D;arrow). When positive cells,aswellFAS2-immunoreactive longitudinalaxon (Fig. 7B).Inthe 7A), whereasanti-ELAV labelsdifferentiating postmitoticneurons number ofearlydifferentiating neurons,aswellaxontracts(Fig. the wild-typeembryonicbrain,anti-FAS2 immunostaininglabelsa against Fasciclin II(FAS2) (LinandGoodman,1994)ELAV. In of apoptosis,wecarriedoutimmunocytochemistry usingantibodies restoration ofthe by theabsenceof development: theexpression of genetically independentmannerintritocerebralneuromere 2003), ina an inhibitorofcell-deatheffector caspases(Mergliano andMinden, (Sprecher etal.,2004)inordertoactivate UAS:: neural lineageformation,weusedthe ubiquitous blockofapoptosisfromneurectodermalstagetoearly misexpression experiments in activation system(BrandandPerrimon,1993)inordertoperform vnd determined whetherblockingapoptosiscanprevent celllossinthe this reductioniscausedbyincreasedapoptosisofneuraltissue. lab 5K). Thesefindingssuggestthatmarked reductionoftritocerebral domain whencomparedwith10apoptoticcellsinwildtype(Fig. 6D withFig.4B).Thesedatasuggestthat in thecelldeathprevented contrast tothe tissue inthe apoptosis leadstoaremarkablerestorationofHRP-immunoreactive type (Fig.6A)and Analysis ofprogrammedcelldeathreveals thatthislossofneural To characterizeinmore detailtheextent ofneuraltissue In ordertofurthersubstantiatetheseobservations, wenext -expressing cellsoccursasearlyembryonicstage12 andthat mutant brain.For this,weusedtheUAS/Gal4 transcriptional function affects properdevelopment ofneuralcellsinthe vnd lab lab lab vnd -null mutantbackground.Whencomparedwithwild -expressing domainasassayedbyTUNELstaining -expressing domainisassociatedwithincreased -expression domain. vnd lab vnd vnd mutant tritocerebrum(Fig.6C).Notably, andin vnd vnd vnd -expressing domainwhencomparedwithwild mutants whencomparedwithwildtype(Fig. mutant tritocerebrum,bothFAS2 andELAV- mutant brainphenotyperesultingfromblock as longapoptosisisprevented. mutant situation,descendinglongitudinal mutant brain(Fig.6B),ubiquitousblockof vnd vnd vnd vnd mutant tritocerebrumwhencompared mutant tritocerebral vnd mutants, asignificantincreaseinthe mutant tritocerebrum(compareFig. lab lab vnd mutant brain,ubiquitousblockof appears tobelargely unaffected -expression domainisobserved RESEARCH ARTICLE mutant embryos.For the sca:: lab p35 Gal4 driver line and lab- transcription, vnd expression act ina 4335 lab vnd -

DEVELOPMENT late stage12,numberof domain (arrow). (B,E)P{ry indicated asstars(**).Byearlystage12,thenumberofTUNEL-positiveapoptoticcellsin (red) showing (green)anti-NRT andanti-LAB double-immunolabelled with type Wild bottom row. (A,D) projected ontoeachfigure in outlined (whiteline,arrow) and domain inwild-typeembryosis extent of late stage12;averagemaximal case: wtTUNEL=10, positive 7.31 showing increased levelofapoptoticactivityin vnd embryogenesis (reviewed bySkeath andThor, 2003).Inthecaseof neuroblasts alongthedorsoventral axisduring neuroectoderm andsubsequent formationanddeterminationof msh top row. ( projected ontoeachfigure in outlined (whiteline,arrow) and domain inwild-typeembryosis extent of stage 12;averagemaximal (C,F) P{ry endogenous reporter constructmimics shows that7.31 (green) showinglowlevelofapoptoticactivityin (red) andTUNELstaining immunolabelled withanti-LAB double- (G,I) Wild-type lab domain, asassayedby7.31 reveals extentof ( sections, lateralviews. reconstructions ofoptical confocal microscopy, embryonic stage12. vnd Fig. 5.Increased apoptosisin 4336 specification of theventral neuroectodermal columnandspecific Previous analyses have demonstratedthatthegenes brain development Expression andfunction of DISCUSSION tritocerebral domain. apoptotic activity andthesubsequentlossofneuralcellsin brain defectobserved in 7F, arrow). Thesedataprovide furtherevidence that thegap-like detectable inthecelldeathprevented arrow). Moreover, alarge numberofELAV-positive cellsare only looselybundled whencomparedwithwildtype (Fig.7E, NRT (green)NRT andanti- immunolabelling usinganti- background. Double- lab NRT (green)NRT andanti- immunolabelling usinganti- null background. Double- A-C - -LacZ} inwild-type lacZ , detailedstudieshave shown that -null mutantembryosat , G are requiredforthecolumnar subdivision oftheVNC , H RESEARCH ARTICLE reporter construct. + ) Embryosatearly lab lab D-F 7.31 -expressing -expressing lab , lab I lab , J lab ) Embryosat - expression. lab lab lacZ -expression -lacZ} in - LacZ expression vnd -expressing cellsdetectableinwild-typeand ␤ ␤ GAL GAL Laser TUNEL=20; wtcells=43, + lab-lacZ 7.31 vnd vnd - mutants islargely duetoincreased expressing cellsin vnd vnd mutant tritocerebrum(Fig. in embryonic lab vnd lab vnd expression domainatearlystage12.( vnd domain. (H,J) cells=27. Standard deviationsare indicatedasbars( is requiredfor mutant tritocerebrum are significantlyreduced. vnd Drosophila , ind vnd vnd and mutant background. Values are meansof -null mutant;anti-LABimmunolabelling(red) andTUNELstaining(green) development: precursorcell development andneuralprogeny suggests that focused onthetritocerebralneuromeres, whereourmutantanalysis investigation ofthefunctionalrole of differentiating neural cellsineachbrainneuromere.For an course ofembryogenesis, and Technau, 2003b;Urbachetal.,2006)andinthesubsequent seen inthedeveloping neuromeresoftheembryonic brain(Urbach neuroectoderm andbrainneuroblastformation, embryonic braindevelopment. Starting atearlystagesof 1998; McDonaldetal.,MellerickandModica,2002). formation inthedeveloping VNC(Jimenez etal.,1995;Chu resulting inaxonalpathfindingdefectsanddefective commissure lost, andtheRP2neuronisfrequentlyabsentin progeny. For example, theaCC/pCCanddMP2/vMP2neuronsare neuroblasts correlateswiththelossormis-specificationofneural neuroblasts, andthatabsenceormisspecificationofventral In thisstudy, weshow thatthe K vnd ) QuantitationofTUNEL-positivecellsand vnd mutant tritocerebrum are significantlyincreased; by acts atleastduringtwo important stepsinits vnd P <0.000005 each)ofStudent’s expression isseeninspecific clusters n vnd =17 preparations countedineach vnd gene isalsorequiredduring in braindevelopment, we Development 133(21) vnd vnd mutant embryos, expression is ␤ -gal- t -test are

DEVELOPMENT anti-LAB (green). Whencompared withwild-type(A)and Embryonicbraindouble-immunolabelledwith anti-HRP(red) and (D) Embryonicbrainimmunolabelledwithanti-HRP (red). (A-C) like brainphenotype observed inlate their survival andeventually leadtocelldeath.In thissense,thegap- Vnd controls brainneuromere formation tritocerebral neuroblastsarelackingin maintenance. Thus,earlyinneurogenesis,aventral subsetof with Fig.4B). brain (B), ( in Fig. 6.Partialrestoration ofbrainstructures and Arrows indicatetritocerebral region. ( of stage15embryos,reconstructions ofopticalsections,lateralviews. like detectable (C).Restorationofneuraltissuealsoresults inwild-type- longitudinal connectivesthattransversethetritocerebrum are immunoreactive tissuein residual tritocerebral absence of ectopic activity of genes suchasEmsandMsh,togetherwiththe tritocerebrum (Urbachetal.,2006). Itisthereforeconceivable that as EmsandMshinresidualventral neuroblastsofthe characterized bytheectopicexpression ofdorsalmarker genessuch neural lineages.Indeed, tritocerebrum. Laterinembryogenesis, vnd be aconsequenceofidentitychangesimposedon increased apoptoticactivity inthe tritocerebral neurallineages.Accordingly, ourobservation of is requiredforproperformationandidentityspecification of McDonald etal.,1998;MellerickandModica,2002), of theembryonicVNC(Jimenezetal.,1995;Chu1998; tracts. embryos resultsinpartialrestorationoftritocerebralcellsandaxon apoptotic activity, andblockingapoptosisin tritocerebrum. Thisgap-like phenotypeisassociatedwithincreased loss ofneuraltissue,togetherwithaxonalpatterningdefectsinthe C , Together, thesedatasuggestthat,similartoitsroleindevelopment D vnd lab ) is requiredfortheformationofneuroblastsindeveloping sca mutants byblockingapoptosis. expression domainin ::Gal4/UAS:: p35- vnd mediated blockofapoptosispartiallyrestores HRP- activity ultimately imposeidentitychangeson p35 vnd vnd in mutant lineagesthatareincompatible with vnd vnd mutant tritocerebrum, anddescending vnd null mutantbackground. mutant tritocerebrum (D,compare loss-of-function embryosare vnd A ) Wild type;( ) Wild vnd vnd vnd mutant tritocerebrummight Laser confocalmicroscopy mutants, suggestingthat mutants displayasevere mutant embryos canbe B vnd ) lab vnd vnd -null mutant expression vnd vnd vnd mutant; deficient mutant activity mutant death prevented significant numberofELAV-expressing cellsare detectableinthecell detectable, althoughneuralfibres displayfasciculationdefects,anda function mutants:FAS2-immunoreactive longitudinalconnectives are apoptosis partiallyrestores thegap-likedefectsobserved in expressing cellsare lackinginthisdomain.(E,F)Ubiquitous blockof in thearea ofthetritocerebral region andthemajorityofELAV- the tritocerebrum. (C,D)Bycontrast,in apparent withinpostmitoticneurons ofallbrainneuromeres, including differentiating neurons aswellaxontracts, and ELAV expression is wild type,FAS2 immunostainingreveals anumberofearly immunolabelled withanti-FAS2 (red) andanti-ELAV (green). (A,B)In immunolabelled withanti-FAS2 (red). (B,D,F)Embryonicbraindouble- are notthesameasinA,C,E,respectively. (A,C,E)Embryonic brain sca Integrated actionof during embryonicbraindevelopment. and ofimproperidentityspecificationtritocerebralneurallineages regarded asaconsequencebothofdefectsinneuroblastformation manner but independentlyintheformation andspecificationofthe which isinvolved indorsoventral patterning, actinanintegrated anteroposterior patterningsystem, andthecolumnargene Our resultsindicatethatthehomeotic gene brain development tritocerebral region. ( reconstructions ofopticalsections,lateralviews.Arrows indicate vnd Fig. 7.Partialrestoration ofbraintractsincelldeathprevented ::Gal4/UAS:: mutant. Laser confocalmicroscopy ofstage15embryos, p35 vnd in A mutant tritocerebrum (F, arrow; compare withB). vnd , B ) Wild-type; ( ) Wild-type; -null mutantbackground. EmbryosinB,D,F vnd and C RESEARCH ARTICLE , vnd D ) lab vnd -null mutants,agapisseen lab in embryonic mutant; ( , whichispartofthe E , F vnd ) loss-of- 4337 vnd,

DEVELOPMENT expression asthecompleteabsenceof unaffected in posterior tritocerebrum(thisstudy),expression of Urbach andTechnau, 2004)andsubsequentlyinneuralcellsofthe expression intritocerebralneuroblasts(UrbachandTechnau, 2003b; and hindbrain. specification duringembryonicdevelopment ofthetelencephalon Nkx2 Technau, 2003b;Urbachand Technau, 2004). Drosophila vnd/Nkx2 development. Acomparisonoftheanteroposteriororder function of 2002). Moreover, thisevolutionary conservation inexpression and missing ortransformed(Cornellandvon Ohlen,2000;Ralluetal., ventral-most cellsinthespinalcordand neuroectodermandintheabsenceof ventral tube, intopologicallysimilarpositionsasis vertebrate homologuesof conserved, bothintermsofexpression andfunction.Thus,the 2000). Notably, the animals, includingmammals(Harvey, 1996;Cornellandvon Ohlen, Nkx2 tritocerebral neuromere.Although 4338 The evolution vnd/Nkx2 of theHoxgene lineages thatsubsequentlybecomefurtherspecifiedbytheactivity ensuring properformationanddevelopment oftritocerebralneural into patternformationalongtheanteroposteriorneuraxisby This indicatesthattheactivity ofthecolumnargene et al.,1998;Page, 2000;Hirthetal.,2001;Sprecher al.,2004). neuronal identitywithinthesameterritoryduringlaterstages(Hirth appears tobeindependentlyrequiredforthespecificationof developing tritocerebralneuromere,whereastheHoxgene that the developing tritocerebrumisdiscriminable.Ourresultssuggest normally runthroughthisneuromere),theirmodeofactionwithin commissure andperturbationofthelongitudinalconnectives that comparable axonalpatterningdefects(lossofthetritocerebral tritocerebral further supportedbythefact thatblockingapoptosisrestores express secondary defectbecauseoftheabsencecellsthatnormally (with theexception ofararethinstrandneuronalcells)reflects Thus, comparablewiththerole of adjacent moredorsolateralganglioniceminence(Susseletal.,1999). medial ganglioniceminence)becomestrans-fated tothat ofthe telencephalon; andtheventral-most aspectofthetelencephalon(the complete absenceofTrkA-expressing cellsinthedeveloping 1996); thenumberofcorticalinterneuronsishalved; thereisa patterning defects:theentirepituitaryismissing(Kimuraet al., particular, thesequentialgeneration ofvisceralmotoneurons and neural lineagespecification in thedeveloping hindbrain.In brain development inmice(Ralluetal.,2002). in patternformationandcellfate determinationduringembryonic development (thisstudy)(Urbachetal.,2006), In termsoffunction,geneticknockouts inmicehave shown that Thus, althoughthe In addition,recentstudieshave shown that vnd Drosophila ee appeartoplayacrucialroleinpatterning andneuronal genes class oftranscriptionfactors thathave beenfoundinvarious RESEARCH ARTICLE is requiredforthespecificationofneurallineageswithin lab gene expression intheearlyembryonicbrainsof vnd/Nkx2 . Thisindependentgeneticactivity of and mousereveals remarkablesimilarities (Urbachand lab genes inbraindevelopmentand lab columnar gene lab expression in Nkx2.1 mutant cells.Conversely, . vnd/Nkx2 genes appearstoapplysomeextent tobrain lab and mutant micedisplaynumerousbrain vnd vnd family ofgenesisexceptionally well vnd r expressed intheneuralplate,or are vnd mutant brainphenotypesresultin -null mutantembryos belongs tothehighlyconserved vnd vnd lab and expression in during vnd lab vnd Drosophila Nkx2.2 does notacton Nkx2.1 show overlapping in the Drosophila vnd/Nkx2 vnd vnd is involved in vnd is integrated vnd Drosophila . is involved and VNC are mutants appears genes, lab brain lab lab is Jimenez, F., Martin-Morris, L.E.,Velasco, L.,Chu,H.,Sierra,J.,Rosen, D.R. Jimenez, F. andCampos-Ortega,J.A. Bhat, K.M. F.M.R. andF.H.). Swiss NSF(toH.R.),theDFGR.U.andG.M.T.) and ELTEM-NEUREX (to Vaessin andK.Whiteforfliesantibodies.Thisworkwassupportedbythe Doe, P. Fichelson,R.Finkelstein,K.Matthews, D.M.Mellerick,J.B.Skeath,H. We thankA.H.Brand,theDevelopmentalStudiesHybridomaBank,C.Q. comparable withtheintegrated activity of Hox1 andPbxfactors (Samadetal.,2004).Thesedatasuggestthat Phox2b enhancerenhancestranscriptionalactivation ofPhox2bby of Nkx2.2atorinvicinityPbx/Hox-bindingsitesproximaltothe fate (Pattyn etal.,2003a;Samad2004).De-repressive activity switch intheselectionofmotorneuronorserotonergic neuronal of thehomeodomainproteinPhox2b,whichinturnactsasabinary of theseproteinsistocoordinatethespatialandtemporalactivation (Pattyn etal.,2003a;Pattyn etal.,2003b).Animportantfunction activities ofNkx2.2-andHox1/2-classhomeodomain proteins located intheventral hindbraincruciallydependontheintegrated serotonergic neuronsfromacommonpoolofneuralprogenitors Abrams, J.M.,White,K.,Fessler, L.I.andSteller, H. 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DEVELOPMENT