regenerative neurogenesis is induced from glia by ia-2 driven ......2019/08/01 · involved in...

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RegenerativeneurogenesisisinducedfromgliabyIa-2drivenneuron-gliacommunication

NealeHarrison1,2*,ElizabethConnolly1*,AliciaGascónGubieda1,3,ZidanYang1,4,BenjaminAltenhein5,

MariaLosada-Perez6,MartaMoreira1andAliciaHidalgo1#

*Theseauthorscontributedequally

1,NeuroDevelopmentGroup,SchoolofBiosciences,UniversityofBirmingham,UK.2,Current

address:TomlinsonLab,SchoolofBiosciences,UniversityofBirmingham,UK.3,Currentaddress:

InstituteforCellandMolecularBiosciences,NewcastleUniversity,NewcastleuponTyneNE24HH,

UK.4,Currentaddress:MaxPlanckFloridaInstituteforNeuroscience,1MaxPlanckWay,Jupiter,

Florida33458,USA;5,InstituteofZoology,UniversityofCologne,Germany;6,InstitutoCajal,

ConsejoSuperiordeInvestigacionesCientificas(CSIC),Madrid,Spain.

.

#Authorforcorrespondence:

ProfessorAliciaHidalgo ph0044(0)1214145416 [email protected]

Wordcount:6,711inmaintext

Figures:8mainfigures,plus4supplementaryfigures.

Keywords:Drosophila,CNS,neuron,glia,neuralstemcell,regeneration,kon-tiki,NG2,insulin,ia-2,

prospero,Notch,Deadpan,Repo,glialregenerativeresponse,neurogenesis,damage,injury,spinal

cord,brain,ventralnervecord,larva.

.CC-BY 4.0 International licensenot certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (which wasthis version posted August 1, 2019. . https://doi.org/10.1101/721498doi: bioRxiv preprint

https://doi.org/10.1101/721498

http://creativecommons.org/licenses/by/4.0/

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ABSTRACT

Akeygoaltopromotecentralnervoussystemregenerationistodiscovermechanismsofinjury-

induceddenovoneurogenesis.Glialcellsmightinduceneurogenesisuponinjury,butthisisdebated,

andunderlyingmechanismsareunknown.Acriticalmissinglinkistheidentificationofneuronal

factorsthatcouldinteractwithglialNG2tofacilitateregeneration.Here,weusedDrosophila

geneticstosearchforneuronalpartnersoftheNG2homologueKon-tiki(Kon),andidentifiedIa-2,

involvedininsulinsecretion.Ia-2isexclusivelyneuronal,andalterationsinIa-2functiondestabilized

cellfate.Injuryincreasedia-2expressionandinducedneuralstemcells.Usingglialmarkers,genetic

epistasisanalysisandlineagetracing,wedemonstratethatIa-2functionstogetherwithKonand

Dilp6toinducedenovoneuralstemcellsfromglia.Altogether,Ia-2andKoninitiateaneuron-glia

interactionloopthatcoordinatesdenovoproductionofbothneuronsandgliaforcentralnervous

systemregeneration.

INTRODUCTION

Humanscannotregeneratethecentralnervoussystem(CNS)afterinjury,butsomeanimalsdoand

regenerationmostofteninvolvesdenovoneurogenesis(TanakaandFerretti,2009).Thereafter,

newlyformedneuronsintegrateintofunctionalneuralcircuits.Thisenablestherecoveryoffunction

andbehavior,whichishowCNSregenerationismeasured(TanakaandFerretti,2009).Thebrainsof

humansandmostvertebratescontinuetoproducenewneuronsinresponsetotheenvironment

throughoutlife,theyalsointegrateintofunctionalcircuits,andthisconstitutesoneofthekey

manifestationsofstructuralbrainplasticity(Gage,2019;TanakaandFerretti,2009).Thus,eventhe

adulthumanbrainhascellsthatcanrespondtoenvironmentalchallenge.Ifwecouldunderstandthe

molecularmechanismsunderlyingnaturalregenerativeneurogenesis,wewouldbeabletofurther

enhancedenovoneurogenesistopromoteregenerationinthehumanCNS,afterdamageordisease.

Thefactthatregenerativeneurogenesisisfoundinmanydiverseanimalsmayreflectanancestral,

evolutionarilyconservedgeneticmechanism,whichmanifestsitselftovariousdegreesinfully


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regeneratingandnon-regeneratinganimals(TanakaandFerretti,2009).Onthisbasis,itmaybe

possibletodiscovermolecularmechanismsofinjury-inducedneurogenesisinthefruit-flyDrosophila,

whichisthemostpowerfulgeneticmodelorganism.

Regenerativeneurogenesiscouldoccurthrougheitheractivationofquiescentneuralstem

cells,de-differentiationofneuronsorglia,ordirectconversionofgliatoneurons.Acrossmany

regeneratinganimals,newneuronsoriginatemostlyfromglialcells(FalkandGotz,2017;Tanakaand

Ferretti,2009).Thus,unravellingthemolecularmechanismsthatswitchglialcellsintobecoming

neuralstemcellsorneuronsisofparamountimportance.InthemammalianCNS,glialcellscanoften

behavelikeneuralstemcells,eveninnormaldevelopment(FalkandGotz,2017).Forinstance,radial

glianormallyproduceneuronsduringdevelopment,andintheadultbrain,newneuronsare

produceddailyinthehippocampusfromtheseglialcells(FalkandGotz,2017).Thereisevidencethat

inthemammalianCNS,astrocytesandNG2-glia(alsoknownasoligodendrocyteprogenitorcells,

OPCs),canproduceneurons,mostparticularlyuponinjury(DimouandGotz,2014;FalkandGotz,

2017;Valny,etal.,2017;ViganoandDimou,2016).NG2-gliaaremostrelevant,astheyaretheonly

progenitorcelltypeintheadultbrain,constitute5-10%ofcellsinthetotalhumanCNSandremain

proliferativethroughoutlife(DimouandGotz,2014).InthenormalintactCNS,NG2-gliaare

progenitorsofastrocytes,OPCsandoligodendrocytes,anduponinjurytheycanalsoproduce

neurons(DimouandGotz,2014;Torper,etal.,2015;Valny,etal.,2017).Theycanalsobedirectly

reprogrammedintoneuronsthatintegrateintofunctionalcircuits(Pereira,etal.,2017;Torper,etal.,

2015).ThefunctionsanddiversityofNG2-gliaarenotyetfullyunderstood,buttheyareparticularly

closetoneurons:theyreceiveandrespondtoactionpotentialsintegratingthemintocalcium

signaling,theysurveyandmodulatethestateofneuralcircuitsbyregulatingchannels,secreting

chondroitinsulfateproteoglycanperineuralnets,andinducingtheirownproliferationtogenerate

moreNG2glia,astrocytesthatsustainneuronalphysiology,andoligodendrocytesthatenwrapaxons

(DimouandGotz,2014;SakryandTrotter,2016;Sun,etal.,2016).Towhatextentthesefunctions

dependontheNG2geneandprotein,isnotknown.


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IntheCNS,NG2isexpressedbyNG2-gliaandpericytes,butnotbyoligodendrocytes,

neurons,orastrocytes.NG2isalargetransmembraneproteinthatcanbecleaveduponneuronal

stimulationbyα−andγ−secretases,toreleasesecretedandintra-cellularforms(Sakry,etal.,

2014;SakryandTrotter,2016).Theintracellulardomain(ICD)-NG2ICD-ismostlycytoplasmic,andit

activatesproteintranslationandinducescellcycleprogression(Nayak,etal.,2018).NG2ICDlacksa

DNAbindingdomainandthereforedoesnotdirectlyfunctionasatranscriptionfactor,butithasa

nuclearWW4domain,nuclearlocalizationsignals,itwasfoundinthenucleusandcanregulategene

expression(Nayak,etal.,2018;SakryandTrotter,2016;Sakry,etal.,2015).NG2isabundantin

proliferatingNG2-gliaandglioma(Nayak,etal.,2018;SakryandTrotter,2016;Sakry,etal.,2015).

NG2isrequiredforOPCproliferationandmigrationindevelopmentandinresponsetoinjury,andit

participatesinglialscarformation(Biname,etal.,2013;Kucharova,etal.,2011;Kucharovaand

Stallcup,2010).Potentially,NG2mayendowOPCswithplastic,homeostaticandrepairpropertiesin

interactionwithneurons(DimouandGotz,2014;SakryandTrotter,2016).However,whetherNG2

itselfmaybeinvolvedindenovoneurogenesisremainsunresolved.Acriticalmissinglinkisthe

identificationofneuronalpartnersthatmightinteractwithNG2toinduceregenerative

neurogenesis.

Thefruit-flyDrosophilaisparticularlypowerfulforidentifyingnovelmolecularmechanisms.

TheDrosophilaNG2homologueiscalledkon-tiki(kon)orperdido(Perez-Moreno,etal.,

2017;Schnorrer,etal.,2007).Konpromotesglialproliferationandcellfatedeterminationin

developmentanduponinjury(Losada-Perez,etal.,2016).KonworksinconcertwithNotchand

Prospero(Pros)todrivetheglialregenerativeresponsetoCNSinjury(Kato,etal.,2011;Losada-

Perez,etal.,2016).Notchsignalingactivateskonexpression,andtogethertheypromoteglial

proliferation;Konisalsoessentialtodetermineneuropileglialcellfate,whichultimatelydependson

Pros;andProsinhibitsglialproliferation,inhibitskonexpressionandmaintainsglialcell

differentiation(GriffithsandHidalgo,2004;Kato,etal.,2011;Losada-Perez,etal.,2016).Thus,Notch

andKonpromoteneuropileglialproliferation,andProstheirdifferentiation.Therelationship


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betweenthesegenesisalsoconservedinthemouse,wherethehomologueofpros,prox1,iscritical

foroligodendrocytedifferentiation(Kato,etal.,2015).Together,Notch,KonandProsforma

homeostaticgenenetworkthatsustainsneuropileglialintegritythroughoutthelife-courseand

drivesglialregenerationuponinjury(HidalgoandLogan,2017;Kato,etal.,2018).

Remarkably,wenoticedthatinjurytotheDrosophilalarvalCNSresultedinspontaneous,yet

incomplete,repairalsooftheaxonalneuropile(Kato,etal.,2011).Thisstronglysuggestedthatinjury

mightalsoinduceimprovementsinneurons.Thesecouldcorrespondtoaxonalregrowth,or

generationofnewneurons.Here,weaskedwhetherKonmayinteractwithneuronalpartnersthat

couldcontributetoregenerativeneurogenesisafterinjury.

RESULTS

Ia-2isafunctionalpartnerofKonexpressedinneurons

Geneticmanipulationofgliainducedaxonalneuropilerepair,andup-regulationofkoningliawas

sufficienttoinduceCNSrepair(Kato,etal.,2011;Losada-Perez,etal.,2016),implyingthatKonmight

interactwithneuronalfactorsduringregeneration.TosearchforneuronalpartnersofKon,we

carriedoutgeneticscreensthataimedtoidentifygenesexpressedinneuronswithnon-autonomous

effectsonglia.WetestedwhetherRNAiknock-downofcandidategenesinneuronsorgliarescued

theextendedventralnervecordphenotypeofover-expressedfull-lengthkon(Supplementary

FiguresS1andS2).WetestedfactorspredictedorknowntointeractwithKonand/orNG2(Perez-

Moreno,etal.,2017;Schnorrer,etal.,2007),andfactorsinvolvedinNotchsignaling,tovalidatethe

approach;phosphatases,asarelationshipofKon/NG2hadpreviouslybeenreportedforPrl1and

Ptp99A(Song,etal.,2012);andothertransmembraneproteinsexpressedinneurons.Rescueby

knock-downofknowninteractors,suchasintegrins(Perez-Moreno,etal.,2017),factorsinvolvedin

Notchsignaling(e.g.Mtm,Akap200),secretases(i.e.kuz,kuz-l)thatcleavebothNotchandNG2/Kon

(SakryandTrotter,2016)andprl-1(Song,etal.,2012),validatedtheapproach(Supplementary

Figure1A-F).Amongstthenovelhits,mostprominentweregenesencodingtransmembraneprotein


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phosphatasesandinsulin-relatedfactors,includingphosphataseLAR,Aktandphosphatase-deadia-2

(SupplementaryFigure2A-D).Ptp2Anegativelyregulatesinsulinreceptorsignaling,maintaining

neuralstemcellquiescence(Gil-Ranedo,etal.,2019).LARisinvolvedinneuronalaxonguidance,and

isresponsibleforde-phosphorylating,andthusinactivating,insulinreceptorsignaling(Mooney,et

al.,1997;Wills,etal.,1999).Aktisakeyeffectorofinsulinreceptorsignallingdownstream(VanDer

Heide,etal.,2006).Ia-2isahighlyevolutionarilyconservedphosphatase-deadtransmembrane

proteinphosphataserequiredindensecorevesiclesforthesecretionofinsulin,insulin-related

factor-1(IGF-1)andneurotransmitters.Italsohassynapticfunctionsandinfluencesbehaviourand

learning(Cai,etal.,2009;Cai,etal.,2011;Cai,etal.,2001;Carmona,etal.,2014;Harashima,etal.,

2005;Henquin,etal.,2008;Hu,etal.,2005;Nishimura,etal.,2010).

Konisrequiredinglia,itinfluencesgeneexpression,andlossofkonfunctionprevents

expressionofglialdifferentiationmarkers(Losada-Perez,etal.,2016).Thus,tofurthertestthe

functionalrelationshiptokon,weusedquantitativereal-timereversetranscriptionPCR(qRT-PCR)on

dissectedlarvalCNS,toaskwhetherkonlossorgainoffunctionaffectedtheexpressionofgenes

identifiedfromthegeneticscreens.Consistenly,konknock-downinneurons(withkonc452,

elavGAL4>UAS-konRNAi)hadnoeffect,whereasinglia(withkonc452,repoGAL4>UAS-konRNAi)it

resultedina3-foldincreaseinia-2mRNAlevels(SupplementaryFigure3A).Conversely,over-

expressionoffull-lengthkonineitherneuronsorgliadown-regulatedia-2mRNAlevelsby25%

(SupplementaryFigure3B).Wevalidatedtheseresultsbyincreasingtherepeatsofthemost

promisingsubsetofgenes(SupplementaryFigure3C,D),andthisconfirmedthestrongesteffectof

konlossandgainoffunctiononia-2mRNAlevels(Figure1A).ThisdatashowedthatKonpreventsia-

2expression.Next,weaskedwhetherknock-downorover-expressionofia-2inneurons(with

elavGAL4)hadanyeffectonkonmRNAlevels,butnonedid(Figure1B).However,over-expressionof

ia-2inglia(withrepoGAL4>ia-2[GS11438])decreasedkonmRNAlevels(Figure1B).Thesedata

indicatethatKonandIa-2restricteachother’sexpressiontogliaorneurons,respectively.Sinceboth


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KonandIa-2aretransmembraneproteins,thiseffectispresumablyindirect.Together,thesedata

identifiedIa-2asafactorthatinteractsgeneticallywithKon.

KonfunctionsinconcertwithNotchandProsduringglialregeneration(Kato,etal.,

2011;Losada-Perez,etal.,2016).Thus,toaskhowia-2mightrelatetothisregenerativegene

network,wetestedwhetherlossorgainoffunctionofprosorNotchmightaffecttheexpression

levelsofia-2indissectedlarvalCNSs.Notchtsmutantscausedanalmosttwo-foldincreaseinia-2

expression,whereasNotchICDover-expressioninglia(repoGAL4>NotchICD)causedamild

downregulationofia-2(Figure1C).So,Notchpreventsia-2expressioninglia.Thisresemblesthe

effectofkononia-2,consistentlywiththefactthatkondependsonNotch(Losada-Perez,etal.,

2016).Ia-2mRNAlevelsalsoincreasedinprosmutantlarvae,butmostlywhenproswasover-

expressedinglia(Figure1D).Thelossoffunctionphenotypeismostlikelyindirect,asinglialcells

ProsandNotchdependoneachother(Kato,etal.,2011),solossofproscausesthedown-regulation

ofNotch,whichwouldincreaseia-2expression.Instead,thestrongereffectofprosgainoffunction

onia-2,andthefactthatProsisatranscriptionfactor,indicatethatProsmaydirectlyregulateia-2

expression.Importantly,prosisexpressed,aswellasinglia,inallganglionmothercellsandsome

neurons,raisingthepossibilitythatProsmayactivateia-2expressionduringacell-fatetransition.

Altogether,thesedatashowthatia-2expressionisrepressedbykonandNotchinglia,andactivated

bypros.Thesedatameanthatia-2isfunctionallyrelatedtothekon,Notch,prosgenenetworkthat

drivestheregenerativeresponsetoCNSinjury.

Theabovedatasuggestedthatia-2expressionisnormallyrepressedinglia.Totestwhat

cellsnormallyexpressia-2,weknocked-downia-2withRNAiineitherneuronsorgliaandmeasured

ia-2mRNAlevelswithqRT-PCRindissectedlarvalCNSs.ia-2-RNAiknock-downinglia(with

repoGAL4)didnotaffectmRNAlevelscomparedtowild-type,howeverknock-downinneurons(with

elavGAL4)down-regulatedia-2transcriptstoabout20%ofwild-typelevels,showingthatia-2is

expressedinneurons(Figure1E).Tovisualizeia-2expressioninvivo,weusedatransgenicprotein

fusionofIa-2toyellowfluorescentprotein(YFP).Ia-2YFP+cellsdidnothavetheglialmarkeranti-


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Repo,noranti-Deadpan(Dpn),whichisthegeneralneuroblastmarkerandalsolabelstransit

amplifyingganglionmothercellsintypeIIneuroblastlineages(BooneandDoe,2008),butallIa-

2YFP+cellswereElav+(Figure1G,H,J).Thisdemonstratesthatia-2isexpressedexclusivelyin

neurons.

Altogether,thesedatashowthatIa-2andKonarerestrictedtoneuronsandglia,respectively

(Figure1F),andthatIa-2isafunctionalneuronalpartnerofKon.

Ia-2canregulateneurogenesis

Next,wecarriedoutafunctionalanalysisofia-2intheCNS.Askonknock-downincreasedia-2mRNA

levels,wesoughttoverifythisusingIa-2-YFP.Wefoundthatkonlossoffunctioninglia(konc452/+;

repoGAL4>kon-RNAi)increasedthenumberofIa-2-YFP+cellsalongthemidline(Figure2A,B).The

ectopiccellsdidnothavetheglialmarkerRepo(Figure2C).Midlinecellswereunaffectedbykon

over-expressionineitherneuronsorglia(Figure2A,B,elavGAL4>konandrepoGAL4>kon).Thus,in

theabsenceofkon,ectopicIa-2-YFP+neuronswerefoundatthemidline.Theseresultscouldalso

partlyexplaintheincreasedia-2mRNAlevelsseenwithkonlossoffunction.

Toaskwhatfunctionia-2mighthaveinneurons,wealteredia-2expressionandvisualized

theeffectusingstandardneuronalmarkers.ia-2knock-downinneurons(elavGAL4>ia-2RNAi)hadno

obviousdetectableeffectonFasIIorBP102(SupplementaryFigure4A,B),anditdidnotchangeEve+

neuronnumbereither(Figure2D,E).AsProsactivatesia-2(Figure1D),weaskedwhetheria-2might

inturnaffectPros.Over-expressionofia-2ineitherneuronsorgliahadnoeffectonPros+cells

(Figure2F,G).Bycontrast,ia-2knock-downinneurons(elavGAL4>ia-2RNAi)increasedPros+cell

number,andthesecellslookedsmall(Figure2F,G).Prosisnormallyfoundinneuropileglia,some

neuronsandallganglionmothercells,suggestingthatectopicPros+cellsmightbeganglionmother

cellsorneurons,whicharegenerallysmallerthanglia.

TotestwhetherectopicPros+cellsoriginatedfromneuralstemcells,weaskedwhether

alteringia-2functionmightaffecttheexpressionofdeadpan(dpn),ageneralneuroblastmarker.


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Bothia-2gainoffunction(elav>ia-2)andlossoffunction(Df(2L)ED7733/+;elav>ia-2RNAi)inneurons

increasedthenumberofabdominalVNCDpn+cells(Figure2H,I,J).TheincreaseinDpn+cellnumber

alsocorrelatedwithtumorousovergrowthsintheVNC(Figure2H),characteristicofgenotypes

causingectopicneuroblastproliferation.TheectopicDpn+cellsincludedcellsalongthemidline,and

cellssurroundingtheneuropile,inpositionsnormallyoccupiedbyglialcells(Figure2I).Theectopic

Dpn+cellsweredistinctfromnormallarvalneuralstemcells,whichareventro-lateralandfurther

fromtheneuropile.Furthermore,theywerevisualisedat120hafteregglaying(AEL),afterthe

normaldisappearanceofnormalabdominalneuralstemcells.Thus,alterationsinthenormallevels

ofneuronalIa-2inducedneuroblastmarkerexpressionectopically.

ThesedatashowedthatinterferencewithnormalneuronalIa-2levelsdestabilizescellfate,

andinducesganglionmothercellandneuralstemcellmarkers.Thiseffectappearedtobenon-

autonomous,asneuronsthemselveswereunaffected.AsIa-2andKonarefunctionallyrelatedand

confinedtoeitherneuronsorglia,respectively,thissuggestedthatcommunicationbetweenneurons

andgliamightmodulatecellfatestability.

Injuryup-regulatesia-2expressionandinducesregenerativeneurogenesis

Datahadshownthatalteringnormalia-2levelsinducedexpressionoftheneuralstemcellmarker

Dpn.CNSinjuryinducedtheup-regulationofkonexpression(Losada-Perez,etal.,2016).Thus,we

askedwhetherinjurymightaffectia-2expressionand,consequently,induceneurogenesis.Crush

injurywascarriedoutat74-76hafteregglaying(AEL)inearlythird-instarlarvalVNCslabelledwith

theendoplasmicreticulumGFPmarkerG9(Figure3A).qRT-PCRininjuredVNCsrevealedavirtually

2-foldincreaseinia-2mRNAlevelsat5-7hpost-injury,whichrecoveredhomeostaticallyby24hpost-

injury(Figure3C).Thus,CNSinjurycausedanincreaseinia-2expression.

Sinceincreasedia-2levelsinducedectopicDpn+cells(Figure2I,J),andia-2wasup-regulated

ininjury,weaskedwhetherinjuryinducedneurogenesis.WefocusedintheabdominalVNConly,

whichhas3neuroblastsperhemi-segmentintheearlythirdinstarlarva,thatoccupyventro-lateral


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positions.CrushinjuryintheabdominalVNCat74-76hAELresultedinectopicDpn+cellsby5-7h

later(Figure3A,B,D,inn=6/17VNCs).Theseweremorenumerousthanthenormaldevelopmental

abdominallarvalneuroblasts,andincludedcellslocatedindorsalpositionsnotnormallytakenby

them(Figure3B,D;see(Froldi,etal.,2015;Sousa-Nunes,etal.,2011)).ThenumerousDpn+cells

couldcorrespondtoinjury-induceddivisionsofneuroblastsnormallyfoundduringlarval

development.Totestwhetherinjurymightinduceectopicneuralstemcellsdistinctfrom

developmentalneuroblasts,wenextcarriedoutcrushinjuryatthreelatertimepoints(Figure3E,F):

(1)at96hAELandanalysedtheVNCs6post-injury(PI,102hAEL),whenincontrolVNCs,abdominal

hemi-segmentshave0or1Dpn+cellremaning.(2)At105handanalysed24hPI(129hAEL),whenin

controlstherearenoventro-lateralneuroblasts,onlyDpn+cellsalongthemidline;and(3)at117h

AELandanalysedtheVNCs12hpost-injury(129hAEL),takingalsoadvantageofthedelayed

pupariationofinjuredlarvae.At129hAELtherearenoremainingabdominalventro-lateralneural

stemcellsinintactcontrols,onlysomeDpn+cellsalongthemidline(Figure3I).Injuryinducedat

thesethreetimepoints,causedectopicDpn+cellscomparedtocontrols(Figure3G-J,EctopicDpn+

cellsinabdominalVNC:Injury96hAEL,analysis6hPI(102hAEL)inn=4/9VNCs;injuryat105,

analysisat24hPIinn=2/11VNCs;Injury117hAEL,analysis12hPI(129hAEL)inn=9/32).Mostbut

notallectopicDpn+cellslackedIa-2YFP.Importantly,mostectopicDpn+cellssurroundedthe

neuropile,andsomeweredorsal,inpositionsneveroccupiedbydevelopmentalneuralstemcells

(Figure3H,J).Thesedatashowthatinjuryinducesectopicneuralstemcells.Sinceia-2levels

increaseduponinjury,andia-2gainoffunctioninducedneuralstemcells,thissuggestedthatia-2

wasresponsiblefortheincreaseinDpn+cellscausedbyinjury.

Thesedataraisedthequestionofhowmightia-2induceneurogenesis.

Dilp6dependsonneuronalIa-2andglialKon

Ia-2ishighlyconservedanditfunctionsindensecorevesiclestoreleaseinsulinand

neurotransmitters(Cai,etal.,2011;Harashima,etal.,2005;Kim,etal.,2008;Nishimura,etal.,2010).


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ThereareeightDrosophilainsulin-like-peptides(Dilps)andIa-2affectsonlyDilp-6(Kim,etal.,2008).

dilp-6isexpressedincortexandbloodbrainbarrierCNSglia,andactivatesneuroblastproliferation

followingaperiodofquiescenceinnormallarvaldevelopment(ChellandBrand,2010;Sousa-Nunes,

etal.,2011).Thus,weaskedwhethertheincreaseinDpn+cellsinia-2lossandgainfunction

observedaboveinvolveddilp-6.Wevisualizeddilp-6expressingcellsinwanderinglarvaeusingdilp6-

GAL4(ChellandBrand,2010;Sousa-Nunes,etal.,2011)todriveexpressionofthenuclearreporter

HistoneYFP.Mostdilp-6>YFP+cellswerealsoRepo+,buttheydidnotsurroundtheneuropileand

lackedtheneuropileglialmarkerPros(Figure4A,B).Thus,mostdilp-6expressingcellsinthe

abdominallarvalVNCwerecortexandsurfaceglia,aspreviouslyreported(ChellandBrand,

2010;Sousa-Nunes,etal.,2011).Somedilp6>his-YFP+cellswereRepo-negativeandElav+,andthus

wereneurons(Figure4A,B).Thus,dilp-6isexpressedinafewneuronsperVNCsegment,andmostly

innon-neuropileglia.

ToaskwhatcellsmightreceiveDilp6,wevisualizedtheexpressionofitsreceptor,theinsulin

receptor(InR),usinganavailableGAL4line,InRNP2552,todrivehistone-YFP,andtestedco-localisation

withglialandneuronalmarkers.InRNP2552>his-YFP+cellsweremostlyRepo+neuropilegliaandafew

wereElav+neurons(Figure4C,D).WecannotruleoutthatInRmayalsobeexpressedinothercell

types,andthisprofilecouldalsobedynamic.

UsingqRT-PCR,wefoundthatia-2RNAiknock-downinneuronsdidnotsignificantlyalterthe

levelsofdpnorelavmRNA,butdecreasedthelevelsofdilp-6expression(Figure4E).Theectopic

abdominalDpn+cellsobservedwithia-2knock-down(Figure2H-J)weresmallerandhadlowerDpn+

levelsthannormalneuralstemcellswhichwerestillabundantinthethorax,soanyeffectindpn

mRNAlevelsinthisexperimentwouldhavebeenmaskedbythisbackgroundexpression,becoming

undetectable.Over-expressionofia-2inneuronsincreasedthelevelsofdpn,elavanddilp-6mRNA

(Figure4F).ThesedatawereconsistentwiththeinvivodatashowinganincreaseinDpn+cells.

Importantly,thesedatashowedthatia-2functioninneuronspositivelyregulatesdilp-6expression,

presumablyindirectly.konknock-downingliareduceddilp6mRNAlevelsevenmore(Figure4G),


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meaningthatkonisprominentlyrequiredfordilp-6expressioninglia.However,over-expressionof

full-lengthkonalonewasnotsufficienttoincreasetheexpressionofneitherdilp-6nordpn(Figure

4H),perhapsbecausethefull-lengthformdoesnotgetactivated.Thesedatashowedthatdilp6

expressiondependspartlyonia-2fromneurons,andmostlyonkonfromglia.

Altogether,inthethirdinstarlarvaDilp6isproducedbysomeneurons,butmostlybynon-

neuropileglia,anditisreceivedatleastbyInRinneuropileglia.Thissuggestedthataninitial‘Dilp6

signal’originatingfromneuronswasthenamplifiedbycortexgliaandreceivedbyneuropileglia.

SincebothIa-2andKonaretransmembraneproteins,thisraisedthequestionofhowthis

amplificationloopmaywork.

Apositiveneuron-gliacommunicationloopboostsDilp-6productionfromglia

SignalamplificationcouldoccurifDilp-6werefirstsecretedfromneuronsbyIa-2,tothenactivate

InRinglia,andInRsignallinginturndrovetheKon-dependentup-regulationofdilp-6expressionin

glia.ThiswouldrequirethatKonfunctionsinnucleitoregulategeneexpression,butthisisnot

known.BothinmammalsandDrosophila,NG2andKonareresponsibleforglialproliferation

(KucharovaandStallcup,2010;Losada-Perez,etal.,2016).Inmammals,anintracellulardomainof

NG2(NG2ICD)isgenerateduponcleavageofthefull-lengthform(Nayak,etal.,2018;Sakry,etal.,

2014).CytoplasmicNG2ICDinteractswithcomponentsofthePI3K-Akt-mTORpathway,toactivate

proteintranslationandcellcycleprogression(Nayak,etal.,2018;Sakry,etal.,2015).NG2ICDalso

positivelyregulatestheexpressionofmultiplegenes,includingdownstreamtargetsofmTOR,thus

exertingpositivefeedback(Nayak,etal.,2018;Sakry,etal.,2015),butwhetherthisrequiresnuclear

localizationofNG2ICDisunknown.InDrosophila,Konhadbeenreportedtolackanuclearlocalization

signal(Schnorrer,etal.,2007).Altogether,whetherNG2orKonregulateglialproliferationandgene

expressionthroughcytoplasmicornucleareventsremainedunsolved.Thus,toaskwhetherthe

intracellulardomainofKon(KonICD)mightfunctioninthenucleus,wegeneratedanHA-taggedform

ofKonICD.Glialover-expressionofKonICD-HA(repoGAL4>UAS-KonICD-HA)revealedHAcolocalisationwith


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theglialnucleartranscriptionfactorRepoinneuropilegliacells,showingthatKonICDwasinnuclei

(Figure5A).Next,totestwhetherKonICDissufficienttoinduceglialproliferation,weover-expressed

itingliaandautomaticallycountedglialcellslabeledwiththenuclearmarkerhistone-YFP,using

DeadEasysoftware.Over-expressionoffull-lengthkoninducesproliferationandincreasesglialcell

number(Losada-Perez,etal.,2016).Over-expressionofkonICDingliaincreasedglialcellnumbertoo

(UAShisYFP;repoGAL4>UASkonICD,Figure5B,C).Thus,KonICDcaninduceglialproliferation.Thesedata

showedthatKoncanfunctioninthenucleustoregulatedilp-6expression,andstronglysuggested

thatfull-lengthKonisnormallycleaved,releasingKonICDtopromoteglialproliferationandregulate

geneexpression.

Next,totestwhetherDilp6activatesInRinglia,weasked:(1)whetherover-expressionof

dilp-6couldmimictheincreaseinglialcellnumbercausedbyKonICD,and(2)whetherthiscouldbe

rescuedbyover-expressionofadominantnegativeformoftheinsulinreceptor(InRDN)inglia.We

foundthatover-expressionofdilp-6inglialcellsincreasedglialcellnumbercomparablytoKonICD

(Figure5B,C),andthiswasrescuedwithconcomitantover-expressionofInRDNinglia(Figure5B,C).

ThesedatameantthatDilp-6activatesInRsignalinginglia,andinducesglialproliferation.Dilp6and

InRsignalingalsoreactivatequiescentdevelopmentalneuralstemcells(ChellandBrand,

2010;Sousa-Nunes,etal.,2011),butKonfunctionsinglia(Losada-Perez,etal.,2016).Tofurther

verifywhetherKonfunctionisrestrictedtoglia,weaskedwhetherKonmightalsoberequiredin

neuralstemcells.RNAikonknock-downinneuralstemcellswithinscutable-GAL4(ins-GAL4>UAS-

kon-RNAi)didnotaffectthenumberordistributionoflarvalDpn+cells(Figure5D,E),meaningthat

Konisnotrequiredinnormalneuralstemcells.SinceglialproliferationdependsonKon(Losada-

Perez,etal.,2016),thefactthatdilp-6couldreproducetheincreaseincellnumbercausedbykonICD,

andthisdependedonInRinglia,stronglysuggestedthatInRsignallingcanactivateKoncleavage

downstream,specificallyinglia.

Toconclude,altogetherthesedatasuggestedthatIa-2triggersthereleaseofDilp-6from

neurons,whichthenisreceivedbyglialcells,whereInRsignalingactivatesKon,whichinturninduces


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glialproliferationandexpressionofdilp-6.Dilp6secretedfromgliainturnpositivelyfeeds-backon

glia,furtheramplifyingDilp-6production.Thus,anon-autonomouspositivefeedbackloopbetween

neuronalIa-2andglialKonpromotesglialproliferation,triggersdilp-6expressionandsustainsDilp-6

productioninglia(Figure5F).ThisraisedthequestionofwhetherneuronalIa-2inconcertwiththe

Kon-Dilp-6gliallooponlyproducedmoreglia,orwhethertheycouldalsoinduceneurogenesis.

Ia-2andDilp6caninduceneuralstemcellsfromglia

ToaskwhetherKon,Ia-2orDilp6wereresponsibleforinjury-inducednon-developmental

neurogenesis,weover-expressedtheminglia(withrepoGAL4).Over-expressionoffull-lengthkon

didnotinduceectopicDpn+cells(Figure6A-D).Bycontrast,over-expressionofia-2couldinduce

ectopicDpn+cellsprominentlyalongthemidlineandinlowerlevelsalsoinlaterallocations

surroundingtheneuropile,whichcouldbeglia(Figure6A-D).Over-expressionofdilp-6hadaneven

strongereffect,andthereweremanyDpn+cellssurroundingtheneuropile(Figure6A-D).Dpnlevels

inectopiccellsweregenerallylowerthaninnormalneuralstemcells.Thesedatashowedthatboth

Ia-2andDilp-6caninducedpnexpression.However,Kon-full-lengthalonecan’t(althoughthiscould

beduetopartialcleavage),meaningthatinsulinsignalingisrequiredtoinduceneuralstemcells.

SinceIa-2drivesDilp-6productionandsecretion,thissuggestedthatultimatelyDilp-6induced

neurogenesis.

TofurthertestwhetherIa-2up-regulateddpnectopicallyviaDilp-6,wecarriedoutepistasis

analysis.Over-expressionofia-2togetherwithdilp-6knock-downinglia(repoGAL4>UAS-ia-2,UAS-

dilp-6-RNAi),rescuedthenumberofDpn+cells(Figure6A-D),demonstratingthatIa-2induces

neurogenesisviaDilp-6.Furthermore,over-expressionofia-2togetherwithkonRNAiinglia

(repoGAL4>UAS-ia-2,UAS-konRNAi)alsorescuedtheDpn+phenotype(FigureA-D),consistentlywith

thefactthatdilp-6expressiondependsonKoninglia(seeFigure4G)andthatKonandDilp6engage

inapositivefeedbackloop(seeFigure5).Finally,theectopicDpn+phenotypewasalsorescuedby

over-expressionofdilp-6togetherwithInRDNinglia(Figure6A-D,repoGAL4>UAS-dilp-6,UAS-InRDN),


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meaningthatneurogenesisdependsonInRsignalinginglia.Together,thesedatashowedthatIa-2

inducesneurogenesisviaDilp-6andInRsiganllinginglia,andthatectopicDpnoriginatedfromglial

cells(Figure5E).

TheectopicDpn+cellsweredistributedalongthemidlineandsurroundingtheneuropile,in

locationsthatseemedtocorrespondtoglialcells.Thus,toverifythis,wetestedwhetherDpn

colocalisedwiththeglialmarkerRepo.Manyofthelateral,ectopicDpn+cellsobservedwithdilp-6

over-expressionwerealsoRepo+(Figure7A),meaningtheyoriginatedfromglialcells.Dpnlevels

werelowerthaninnormalneuralstemcells.Bycontrast,theectopicmidlineDpn+cellswerenot

Repo+(Figure7A,magentacells).ThismeantthattherearetwodistinctpopulationsofectopicDpn+

cells:lateralcellsthatareneuropileglia,andmidlinecellswhichcouldoriginatefromRepo-negative

midlineglia.

Tofurthertestwhethertheectopicneuralstemcellsoriginatedfromglia,weusedthecell-

lineagemarkerG-TRACE.ThisGAL4-dependenttoolresultsinthepermanentlabelingofUAS-

expressingcellsandtheirprogenycells.Thus,repoGAL4>G-TRACEmarkscellsthatwereonceglia,

eveniftherepopromoterweretobesilencedduetoachangetoaneuroblastcellstate.Cellsthat

wereoriginallygliabutmaynolongerbesowouldbelabelledingreen(GFP+),andrecentlyspecified

glialcellswouldbelabelledinred(RFP+).G-TRACEexpressioninallneuronswithelavGAL4oringlia

withrepoGAL4togetherwithdilp-6causedlarvallethalityandthuscouldnotbeanalysed.By

contrast,over-expressionofbothG-TRACEandia-2inglia(repoGAL4>G-TRACE,UAS-ia-2)revealed

G-TRACE+Dpn+cellsaroundtheneuropile(Figure7C,D).MostofthesecellshadGFP,butalsoRFPat

somewhatlowerlevels(Figure7C,D).ThesedatademonstratethatectopicDpn+wereonceglial

cells.SinceRFPwasalsopresent,thedataalsoshowedthatglialcellfatehadnotbeensuppressed,

andinsteadsuggestedthatglialcellsmaybeintheprocessofde-differentiation.

Altogether,thesedatashowedthatIa-2,Dilp-6,InRsignalingwithKoncaninducedenovo

formationofneuralstemcellsinneuropileassociatedglialcells.


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DISCUSSION

AcriticalmissinglinktounderstandhowtoinduceCNSregenerationinnon-regeneratinganimals

suchashumans,hadbeentoidentifyfactorsthatmightinteractwithNG2toinduceregenerative

neurogenesis.NG2iskeybecauseNG2-gliaaretheonlypopulationofprogenitorcellsthatare

presentthroughoutlifeintheadulthumanbrain(DimouandGotz,2014;Torper,etal.,2015;Valny,

etal.,2017).Thus,theyaretheidealcellstorespondtoinjuryandbemanipulatedtopromote

regeneration.Still,whetherNG2-gliacangiverisetoneuronsishighlydebated,andifso,the

underlyingmechanismwasunknown(DimouandGotz,2014;FalkandGotz,2017;Valny,etal.,

2017;ViganoandDimou,2016).Here,usingDrosophilainvivofunctionalgeneticanalysiswehave

identifiedneuronalIa-2andinsulinsignalingasthekeyinteractorsoftheNG2homologuekon,that

caninduceneurogenesisfromNG2-likeglialcells.

WeprovideevidencethatIa-2isaneuronalpartnerofKonresponsibleforinducing

neurogenesisfromNG2-likegliaafterCNSinjury(Figure8A).Weshowthatintheun-injuredCNS,

KonandIa-2mutuallyrepresseachother’sexpression,confiningeachothertogliaandneurons,

respectively.Normallevelsofia-2arerequiredtopreventthenon-autonomousinductionofneural

stemcellsfromneighbouringglialcells.Areductioninia-2levelsnon-autonomouslyincreaseskon

expression,whichup-regulatesdilp-6fromglia;anincreaseinia-2levelsup-regulatesDilp-6

secretionandproductionfromneurons.Eitherway,theconsequenceisraisedDilp-6,whichswitches

onapositiveamplificationloopfromgliathatresultsinfurtherDilp-6production.Thereafter,Dilp6

caninducedpnexpressioninglia.Injurycausesanup-regulationinia-2expression,aswellaskon

(Losada-Perez,etal.,2016)(Figure8B).Ia-2drivessecretionofDilp-6fromneurons,andan

amplificationpositivefeedbackloopinvolvingKon,Dilp6andInRdrivesthefurtherKon-dependent

productionofDilp-6fromcortexglia.Dilp-6increasesKon-dependentglialproliferation,meaning

thatInRsignalingmayfacilitateKoncleavage.Dilp-6alsotriggersInRsignalingandtheexpressionof

theneuralstemcellmarkerdpninneuropile–butnotcortex-glia(Figure8B,C).DpnisabHLH

transcriptionalrepressorofneuronaldifferentiation,andcommonneuralstemmarkerinDrosophila


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(Sousa-Nunes,etal.,2010).EctopicDpn+cellsarefounduponinjuryandgeneticmanipulationofIa-2

andDilp-6.WeshowthattheseectopicDpn+cellsoriginatefromglia.Altogether,thisrelayfrom

neuronstocortexandthentoneuropilegliaenablesconcertedglio-andneuro-genesis,matching

interactingcellpopulationsforregeneration(Figure8B).

Invertebratesandalsoininvertebrates,neuralstemcellsfoundafterdevelopmentinthe

adultCNSanduponinjury,aregenerallydistinctfromdevelopmentalones,andcanoriginatefrom

hemocytes,butmostoften,fromglialcells(FalkandGotz,2017;SimoesandRhiner,2017;Tanakaand

Ferretti,2009).OurfindingsthatDilp6andInRcaninducedpnexpressionarereminiscentoftheir

functionsintheinductionofneuralstemcellsfromquiescentprogenitorsindevelopment(Chelland

Brand,2010;Gil-Ranedo,etal.,2019;Sousa-Nunes,etal.,2011).However,theDpn+cellsinduced

uponinjuryandafterdevelopment,aredistinctfromthedevelopmentalneuralstemcellsnormally

inducedbyDilp-6.Firstly,ininjuriescarriedoutinthirdinstarlarvae,theinducedneuralstemcells

weremorenumerousthannormalneuralstemcells.Secondly,ininjuriescarriedoutlatein

wanderinglarvae,Dpn+cellswerefoundafternormaldevelopmentalneuralstemcellshavebeen

eliminatedthroughapoptosis(Bello,etal.,2003).Thirdly,inbothcasesDpn+cellswerefoundin

ectopiclocationsnotnormallyoccupiedbydevelopmentalneuralstemcells(Bello,etal.,2003).For

instance,ectopicDpn+cellsoftenoccupieddorsalpositionsovertheneuropile.EctopicDpn+cells

werelocatedalongthemidlineandsurroundingtheneuropile,inpositionsthatcorrespondto,

possiblymidline,andoftenneuropileglia.Indeed,ourdatademonstratedthatatleastthenon-

midlineectopicDpn+originatefromglialcells:1,allectopicDpn+cellsfromgeneticmanipulations

didnothaveIa-2-YFP,whichisexpressedinallneurons.Thefactthatuponinjury,aminorityof

ectopicDpn+cellswerealsoIa-2YFP+couldmeanthatuponinjuryia-2isover-expressedalsoinglia,

orthatasneuronsacquireDpntheydown-regulateia-2.2,EctopicDpn+cellssurroundedthe

neuropile,occupyingpositionsofneuropileglia;3,Repo-negativeDpn+cellsoccupiedpositions

alongthemidline,characteristicofmidlineglia;4,allDpn+cellssurroundingtheneuropilealsohad


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Repo;5,lineagetracingwithG-TRACEdemonstratedthatectopicDpn+cellsoriginatedfromglial

cells.

Inprinciple,regenerativeneurogenesiscouldoccurviadirectconversionofgliaintoneurons,

glialde-differentiation,orneuronalde-differentiation.Neuronalde-differentiationoccursbothin

mammalsandinDrosophila(Froldi,etal.,2015).However,inmammals,neurogenesisafter

developmentandinresponsetoinjurymostoftenoriginatesfromglialcells(DimouandGotz,

2014;FalkandGotz,2017;TanakaandFerretti,2009).Intheadultmammalianbrain,radialgliainthe

hippocampusrespondtoenvironmentalchallengebydividingasymmetricallytoproduceneural

progenitors,thatproduceneurons(Shtaya,etal.,2018).AstrocytesandNG2gliacangenerate

neuronsinresponsetostrokeorexcitoxicinjury,andgeneticmanipulations(DimouandGallo,

2015;Heinrich,etal.,2014;PeronandBerninger,2015).Geneticmanipulationcanleadtothedirect

conversionofNG2gliaintoneuronsthatintegrateintofunctionalneuralcircuits(Pereira,etal.,

2017;Torper,etal.,2015).Importantly,injurycreatesadistinctcellularenvironmentthatprompts

glialcellstogeneratedifferentcelltypesthanintheun-injuredCNS.Forinstance,elevatedSox-2is

sufficienttodirectlyreprogrammeNG2gliaintoneurons,butonlyuponinjury(Heinrich,etal.,

2014).AndwhereasduringnormaldevelopmentNG2glialcellsmayonlyproduceoligodendrocyte

lineagecells,uponinjurytheycanproducealsoastrocytesandneurons(DimouandGallo,

2015;Huang,etal.,2018).Whattheinjurycuesare,isunknown.

InourcontextintheDrosophilalarva,Dpnmaynotbesufficienttocarryneurogenesis

through.Firstly,lossofia-2functionresultedinectopicDpn+andPros+cellsthatcouldbeganglion

mothercellsaswellasneuralstemcells,butgainofia-2functionresultedonlyinDpn+cellsbutnot

Pros+.ThissuggestedthatIa-2andDpnarenotsufficientforneuroblaststoprogresstoganglion

mothercells.Secondly,alterationsinia-2levelsinducedectopicDpn+cells,butnotectopicEve+

neurons.Andfinally,ectopicDpn+cellsstillhadalsoRepo.Thesedatasuggestthattogenerate

neurons,glianotonlyrequiretoexpressdpn,butalsotoreceiveotheryetunknownsignalsfor

neuronaldifferentiation(Figure8B).AlimitationofinjuryinthelarvalVNCistime.Injuryisbest


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19

carriedoutinthelatelarvatoavoidinterferencewithdevelopmentalneuroblasts.However,justa

fewhoursafterinjury,larvaepupate.Cellsmaynothaveenoughtimetogothroughneuronal

lineagesfromneuralstemcelltodifferentiatedneuron.Pupariationandmetamorphosisbringina

differentcellularcontextthatcouldinterferewithregeneration.Withourdata,wecanconcludethat

aneuron-gliacommunicationloopinvolvingIa-2,Dilp-6,KonandInRisresponsiblefortheinduction

oftheneuralstemcellmarkerDpninglia.

OurworkhasrevealedakeylinkbetweenNotch,Pros,Konandinsulinsignalingtodrive

regenerativeneurogenesisfromglia.Ia-2hasuniversalfunctionsindensecorevesiclestorelease

insulinandneurotransmitters(Cai,etal.,2011;Cai,etal.,2001;Harashima,etal.,2005;Kim,etal.,

2008;Nishimura,etal.,2010).Dilp-6reactivatesquiescentdevelopmentalneuralstemcellsin

Drosophila(ChellandBrand,2010;Sousa-Nunes,etal.,2011),andinmammals,insulin-likegrowth

factor1(IGF-1)inducestheproductionofastrocytes,ologendendrocytesandneuronsfrom

progenitorcellsintheadultbrain,alsoinresponsetoexercise(Mir,etal.,2017;Nieto-Estevez,etal.,

2016).ThetranscriptionfactorSox-2thatcanswitchastrocytestobecomingneuralstemcellsand

produceneurons,isadownstreameffectorofInR/AKTsignaling(Mir,etal.,2017).Together,these

findingsmeanthatinsulinsignalingisinvolvedinswitchingglialcellsintobecomingneuralstemcells.

Intriguingly,dpnwasinducedonlyinneuropileassociatedglialcells,butnotincortexglia.Neuropile

gliaproliferateuponinjury,toregenerateglialcells(Kato,etal.,2011;Losada-Perez,etal.,2016).

Thus,someRepo+gliacellsmayproduceonlygliaafterinjury,andthosethatbecomeDpn+cells

couldgiverisetobothneuronsandglia.Neuropileglia,identifiedbythealrmGAL4driverandknown

asDrosophilaastrocytes,uniquelyexpressNotch,ProsandKon,aswellasInR,andweshowedthat

ia-2isfunctionallylinkedtothesegenes.Inmammals,thecombinationofNotch1,Prox1andNG2is

uniquetoNG2-glia,alsoafterdevelopment,andisabsentfromastrocytes(Cahoy,etal.,2008).This

wouldsuggestthatIa-2andDilp-6canonlyinducedpninNG2-likegliabearingthiscombinationof

factors.Infact,dpnisregulatedbybothNotchandPros:Notchactivatesdpnexpressionpromoting

stemness,andProsinhibitsit,promotingtransitiontoganglionmothercellandneuron(Babaoglan,


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etal.,2013;BiandKuang,2015;San-JuanandBaonza,2011;Vaessin,etal.,1991).Thus,onlyglialcells

withNotchandProsmaybepoisedtomodulatestemnessandneuronaldifferentiation.

Notch,KonandProsregulateglialproliferationinresponsetoinjury(Kato,etal.,

2011;Losada-Perez,etal.,2016),andIa-2isfunctionallyrelatedtothem(Figure1).Inthe

mammalianCNS,NotchdeterminesNG2-gliaproliferationandmaintainstheprogenitorstate,

whereasitsdownregulationisrequiredtoinducebothglialandneuronaldifferentiationfollowing

proliferation(Ables,etal.,2010;FalkandGotz,2017;Piccin,etal.,2013;Yamamoto,etal.,2001).

Notchalsoactivateskonexpression(Losada-Perez,etal.,2016).NG2interactswithAKTandother

downstreamcomponentsoftheInRsignallingpathwaytopromoteproteinsynthesisandcellcycle

progression,andtoregulatetheexpressionofitsdownstreameffectorsinapositivefeedbackloop

(Nayak,etal.,2018).Insulinsignalingalsoactivatesdpnexpression,byrepressingFoxO,which

repressesdpn(Siegrist,etal.,2010).Consistentwiththesedata,wehaveshownthatinglia,the

abilityofDilp-6andInRtoinduceglialproliferationdependsonKon,andthatKondrivespositive

feedbackoninsulinsignalingbyregulatingdilp-6expression.Importantly,Kondoesnotappearto

functionindevelopmentalneuralstemcells.AsNotch,Prosandinsulinsignalingareknownto

positivelyregulatedpnexpression(Siegrist,etal.,2010;Babaoglan,etal.,2013;BiandKuang,

2015;San-JuanandBaonza,2011;Vaessin,etal.,1991),andinjuryinducesaNotch-dependentup-

regulationofKon(Losada-Perez,etal.,2016),ourdatasuggestthattheNotch-Kon-InRsynergycan

triggertheactivationofdpnexpression.Inducedneuralstemcellsthereafterwouldhavethe

potentialtogenerateonlygliafromdaughtercellsthathaveKonandPros,onwhichRepoandglial

cellfatedepend,orneurons,fromdaughtercellsthatlackKon,andexpressProsonwhichIa-2

depends(Figure8B).Thus,uponinjury,Ia-2,insulinsignalling,Notch,ProsandKonfunctioning

togetherenabletheregenerativeproductionofbothmoreglialcells,andofneuralstemcellsfrom

glia(Figure8B,C).

Toconclude,wehaveshownthatIa-2triggerstwodistinctresponsesinglia:inglialcells

withKon,Ia-2andinsulinsignalingboostKon-dependentglialproliferationandamplificationofDilp-


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6.InNG2-likeglialcellsthatalsoexpressNotchandPros,theircombinationwithKonandinsulin

signalinginresponsetoIa-2alsounlockstheirneurogenicpotential,inducingneuralstemcellfate.

Asaresult,thesegenescoulddrivetheproductionofbothglialcellsandneuronsafterinjury,

enablingthematchingofinteractingcellpopulations,whichisessentialforregeneration.

MATERIALSANDMETHODS

Flystocksandgenetics.FlystocksusedarelistedinTable1below.Stockscarryingcombinationsof

over-expressionandRNAi,orRNAiandmutants,etc.,weregeneratedbyconventionalgenetics.Nts

mutantswereraisedat18°Ctoenablenormalembryogenesis,andswitchedto25°Cfromlarval

hatchingtothethirdinstarlarvalwanderingstagetocauseNlossoffunction.Forallexperiments,

larvaebearingbalancerchromosomeswereidentifiedbyeitherusingthefluorescentbalancersCyO

Dfd-YFPandTM6BDfd-YFPorusingthebalancerSM6a-TM6BTb—,whichbalancesboththesecond

andthirdchromosomes,anddiscarded.Forthegeneticscreens,larvaewithfluoresencentVNCs(i.e.

repoGAL4>UAS-FlyBoworelavGAL4>UAS-FlyBow)wereselected.

What Genotype FromControl yw Hidalgolab

konc452/CyO,Dfd-YFP Losada-Perezetal2016ia-2deficiency:w1118;P{w+mW.Scer\FRT.hs3=3'.RS5+3.3'}ED7733/SM6a KyotoDGRC150199prosS044116/TM6B Katoetal,2011prosvoila1/TM6B Katoetal,2011

Mutants

Notchts1/FM7(sn+)actGFP Katoetal,2011Reporter IA2-YFP:w1118;PBac{566.P.SVS-1}ia-2CPTI100013 KyotoDGRC115077

Neurons:w;;elavGal4 HidalgolabGlia:w;;repoGal4/TM6B HidalgolabwDilp-6Gal4/FM7 KyotoDGRC103877w*;P{GawB}InRNP2552/TM6B KyotoDGRC104236

GAL4drivers

w*;P{GawB}insc[Mz1407] BSC8751UAS-ia-2:w;;UASia-2-@attp2/SM6aTM6B ThisworkUAS-ia-2:y1w67c23;P{GSV6}GS11438/SM1 KyotoDGRC203335w;UASpros-k Katoetal,2011w;UAS-HA-KonFL4-1/TM3Twi-GFP Losada-Perezetal2016w;UAShistone-YFP Katoetal,2011w;UASkon-ICD::HA Thisworky1w1118;;UAS-dilp-6 GiftofAlexGouldy1w1118;P{UAS-InR.K1409A}2 BSC8252

UASlines

G-TRACE:w[*];P{w[+mC]=UAS-RedStinger}4,P{w[+mC]=UAS-FLP.D}JD1,P{w[+mC]=Ubi-p63E(FRT.STOP)Stinger}9F6/CyO

BSC28280


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UAS-ia-2-RNAi:y1sc1v1;;P{TRIPHM50V536}attP2 BSC33672UAS-konRNAi:P{KK102101}VIE-260B VDRCUAS-Dilp6-RNAi:P{KK111727}VIE-260B VDRCUAS-ptp4ERNAi:yscv;;P{TripHMSO1838}attp2 BSC38369UAS-ptp10DRNAi:yscv;;P{TripHMSO1917}attp2 BSC39001UAS-ptp61FRNAi:yscv;;P{TripHMCO43446}attp2 BSCUAS-ptp69ARNAi:yv;;P{TripJFO3399}attp2 BSC29462UAS-ptp99ARNA:yv;;p{Trip1858}attp2 BSC25840UAS-HpoRNAi:yv;;p{TripHMSO0006}attp2 BSC33614UAS-Prl-1RNAi:yscvp;;{TripPMAS01826}attp2/TM3,Sb BSC38358UAS-LarRNAi:w/yv;;p{TripHMSO2156}attp2 BSC40938UAS-PP2A-B'RNAi:yw;;p{TripHMO5256}attp2 BSCUAS-GRIPRNAi:yw;;P{tRIPjfo2969}attp2 BSC28334UAS-Akap200RNAi:yv;;p{TripHMSO18}attp2 BSC28532UAS-arfrp1RNAi:y[1]v[1];P{y[+t7.7]v[+t1.8]=TRiP.JF02651}attP2 BSC27501UAS-cadNRNAi:yv[+t1.8]=TRiP.JF02653}attP2 BSC27503UAS-cadN2RNAi:yv[+t1.8]=TRiP.JF02658}attP2 BSC27508UAS-cerkRNAi:yv[+t1.8]=TRiP.GL00273}attP2 BSC35361UAS-CG42327RNAi:yv[+t1.8]=TRiP.HMS00227}attP2 BSC33356UAS-dsRNAi:yv[+t1.8]=TRiP.HMS00759}attP2 BSC32964UAS-eyaRNAi:yv[+t1.8]=TRiP.JF03160}attP2 BSC28733UAS-eysRNAi:yv[+t1.8]=TRiP.JF01069}attP2 BSC31513UAS-fak56DRNAi:yv[+t1.8]=TRiP.JF02484}attP2 BSC29323UAS-ftRNAi:yv[+t1.8]=TRiP.JF03245}attP2 BSC29566UAS-fu12RNAi:yv[+t1.8]=TRiP.HMC04917}attP40 BSC57728UAS-fzRNAi:yv[+t1.8]=TRiP.HMS01308}attP2 BSC34321UAS-ifRNAi:yv[+t1.8]=TRiP.JF02695}attP2 BSC27544UAS-inaDRNAi:yv[+t1.8]=TRiP.HMC03170}attP2 BSC52313UAS-kulRNAi:yv[+t1.8]=TRiP.HMC03803}attP40 BSC55653UAS-kuzRNAi:w;P{KK103555}VIE-260B VDRCUAS-metroRNAi:yv[+t1.8]=TRiP.HMC04629}attP40 BSC57242UAS-mewRNAi:yv[+t1.8]=TRiP.JF02694}attP2 BSC27543UAS-mtmRNAi:yv[+t1.8]=TRiP.JF01114}attP2 BSC31552UAS-mysRNAi:yv[+t1.8]=TRiP.JF02819}attP2 BSC27735UAS-sdcRNAi:yv[+t1.8]=TRiP.HMC03265}attP2 BSC51723UAS-wnt5RNAi:yv[+t1.8]=TRiP.HMS01119}attP2 BSC34644UAS-wdeRNAi:yv[+t1.8]=TRiP.HMS00205}attP2 BSC33339UAS-vangRNAi:yv[+t1.8]=TRiP.HMS01343}attP2 BSC34354UAS-stanRNAi:yv[+t1.8]=TRiP.JF02047}attP2 BSC26022UAS-ssu72RNAi:yv[+t1.8]=TRiP.HMS04461}attP40 BSC57018UAS-shgRNAi:yv;;TRiP.JF02769}attP2/TM3,Sb[1] BSC27689UAS-psnRNAi:yv;;TRiP.JF02760}attP2/TM3,Sb[1] BSC27681UAS-prosbeta7RNAi:yv[+t1.8];;TRiP.HMS00122}attP2 BSC34812UAS-prl-1RNAi:yvTRiP.HMS01826}attP2/TM3,Sb[1] BSC38358UAS-larRNAi:yv[+t1.8]=TRiP.HMS02186}attP40 BSC40938UAS-lanB2RNAi:yv[+t1.8]=TRiP.HMC04076}attP40 BSC55388

UAS-RNAilines

UAS-lanARNAi:yv[+t1.8]=TRiP.JF02908}attP2 BSC28071

CrushinjuryinthelarvalVNC.CrushinjuryinthelarvalCNSwascarriedoutaspreviouslyreported

(Losada-Perez,etal.,2016),andonlylesionsintheabdominalVNCwereanalysed.Larvalcollections

werestagedbyputtingtheGofliesinanegglayingchamberfor2h,thencollectingtheF1larve74h


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later.Crushinjurywascarriedout:(1)at74-76hafteregglaying(AEL);afterinjury,VNCswereleftto

carryondevelopingat25°C,andweredissected5-7hlater;(2)at96h,andafterinjurytheywere

incubatedat25°Canddissectedandfixed6hlater;(3)at117hAEL,andafterinjury,wereincubated

at25°C,anddissected12hourslater.DissectedandfixedVNCwhenthenprocessedforantibody

stainingsfollowingstandardprocedures.

Molecularcloning

TheUAS-konICD-VenusconstructsweregeneratedfromESTLD31354viaPCRamplificationwith

KappaHiFiPCRkit(Peqlab)andsubsequentcloningusingtheGatewaycloningsystem(Invitrogen)

accordingtomanufacturersinstructions.PrimersusedwerekonICDfwdcomprisingtheCACC-

sequenceatthe5’-end(CCACAGGAAACTGAGAAAGCACAAGGC)fordirectcloningofthePCRproduct

(482bp)intotheentryvectorpENTR/D-Topo,andkonICDrev(AAACCTTACACCCAATACTGATTCC)

includingtheendogenousstop-codon,underlined.DestinationvectorwaspTVWfortaggingtheICD

ontheN-terminuswithVenus,includinga5xUAScassetteandP-elementendsfortransformation.

ThesedestinationvectorsweredevelopedbytheMurphy-LabatCarnegieInstitutionofScience,

Baltimore,MD,USA,andcanbeobtainedfromDrosophilaGenomicsResourceCenteratIndiana

University,USA.TransformantflystrainsweregeneratedbyBestGeneInc,ChinoHills,CA,USA

followingastandardtranposase-mediatedgermlinetransformationprotocol.

AUAS-ia-2constructwasgeneratedusingGatewaycloning(Invitrogen,asabove).Ia-2cDNA

wasgeneratedbyreverse-transcriptionPCRfrompurifiedmRNAfromOregonRflies,andclonedinto

pDONR.Subsequently,astandardPCRamplificationwasperformedusingPhusionHigh-Fidelity

(FisherScientific),primersIa-2F(5’-ATGGCACGCAATGTACAACAACGGC)andia-2-stopR(5’-

CTTCTTCGCCTGCTTCGCCGATTTG),andtheresultingPCRproduct(3918bp)wasclonedintopGEM®-T

EasyVector(Promega).Subsequently,aPhusionHighFidelityPCRamplificationwascarriedoutusing

GatewayprimersIa-2attBF1(5’-

ggggacaagtttgtacaaaaaagcaggcttcATGGCACGCAATGTACAACAACGGC)andIa-2attBR1(5’–


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ggggaccactttgtacaagaaagctgggtcCTTCTTCGCCTGCTTCGCCGATTTG),andplasmidpGEM-ia-2as

template.UsingGatewaycloning,thePCRproduct(3979bp)wasclonedintofirstpDONR221and

subsequentlyintothepUAS-gw-attBdestinationvector,forφC31transgenesis.Theconstructwas

injectedbyBestGene,togeneratetransgenicfliesbearingUAS-ia-2attheattP2landingsite.

QuantitativerealtimereversetranscriptionPCR(qRT-PCR).

qRT-PCRwaspreformedaccordingtostandardmethodsandaspreviouslydescribed(Losada-Perez,

etal.,2016),withthefollowingalteration.Foreachsample,10thirdinstarlarvaewereusedper

genotypeperreplicate.Atleastthreeindependentbiologicalreplicateswereperformedforall

experimentsotherthaninSupplementaryFigureS3AandBwheretworeplicateswerecarriedouton

allcandidatesandthoseofinterestwheretakenforwardtocarryouttwofurtherreplicates.Foralist

oftheprimersusedinthisstudypleaseseeTable2below:

Table2PrimersPRIMER SEQUENCE5’TO3’RpL32qPCRF AAGCGGCGACGCACTCTGTT

RpL32qPCRR AAGCGGCGACGCACTCTGTTGAPDH2FqPCR GCCCAGCATACAGGCCCAAGGAPDH2RqPCR GTGAAGCTGATCTCTTGGTACGACKonqPCR(Ex10-11)F3 CCGCGCCCTAATCTTTAACTTTTACKonqPCR(Ex10-11)F3 CCCAAGCGATTTCTTTACCAptp99AqPCRF2 TCGCTATCCCAACATCACGGptp99AqPCRR2 TGAACGCATGTCCCTTCTGGFzqPCRF1 AGTCGCACTTATTCCACCTGGFzqPCRR1 CTGGCCCACGAAACAAACGHippoqPCRF2 ACCCATAGCCACAGAGTATTCTHippoqPCRR2 TGCTGTTCATCCTGCTGTTGia-2qPCRF1 ACGGTCACCCAGTTTCACTTia-2qPCRR1 CGGTAGGACTTGTTCACTTTCCInaDqPCRF1 TCATTGAGTTGAAGGTGGAAAAGAInaDqPCRR1 CTGCCACTTGTCCCTCCGlarqPCRF1 TTGCTGAGTACAACATGCCGlarqPCRR1 TGAAGTCGATGAAGCCCTCGPrl-1qPCRF1 TGACGAGTGGTTTGAGGTCTTAAPrl-1qPCRR1 GCCCAATTCAATCAGTGCCAPtp10DqPCRF1 TGCAACAGATCAACACGTCTGPtp10DqPCRR1 TATACTGCTGCTCCGTCTGCPtp61FqPCRF1 GTCCAAGGTGCTCTGCGAGPtp61FqPCRR1 ATGAGGGGTTCTTCAGCGTC


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Ptp69DqPCRF1 TGTAGTGTGGGCGAAAACGAPtp69DqPCRR1 CGCATCGGAAGTGGTGTTAGYorkieqPCRF1 ATCAGCCCCATTCAGTTGAACYorkieqPCRR1 CCTCCCACTGCGTAGATTTTGTADpnqPCRF1 ACGCATGTCCAATCCCAATGDpnqPCRR2 GCGACGTTTCTCCATAATCGGTElavqPCRF1 CTACTTGCCGCAAACAATGACElavqPCRR1 CTTCACCGACTCAATCTCGCdilp6qPCRF1 CGATGTATTTCCCAACAGTTTCGdilp6qPCRR1 AAATCGGTTACGTTCTGCAAGTC

Immunostainingswerecarriedoutfollowingstandardprocedures.Thefollowingprimaryantibodies

wereused:mouseanti-Repo(1:100,DSHB);guineapiganti-Repo(1:1000,BenAltenhein);ratanti-

Elav(1:250,DSHB);mouseanti-FasIIID4(1:500,DSHB);mouseanti-Prospero(1:250,DSHB);guinea

piganti-Dpn(1:1000,giftofJ.Skeath);mouseanti-Eve3C10(1:20,DSHB);rabbitanti-GFPat1:250

(MolecularProbes).SecondaryantibodieswereAlexaconjugated:Donkeyanti-rabbit488(1:250,

MolecularProbes),goatanti-mouse488(1:250,MolecularProbes),goatanti-mouse647(1:250,

MolecularProbes),goatanti-guineapig488(1:250,MolecularProbes),goatanti-guineapig633

(1:250,MolecularProbes),goatanti-rat647(1:250,MolecularProbes).

Microscopyandimaging.ImagedatawereacquiredusingZeissLSM710andLeicaSP8laserscanning

confocalmicroscopes,usinga25xlens,1.25zoom,resolution512x512or1024x1024,step0.96µm

and3xaveragingforallsamplesexceptforcellcountingwithDeadEasythathavenoaveraging.

ImageswereanalysedusingImageJ.Imagesofhorizontalsectionsareprojectionsfromthe

stacksofconfocalimagesthatspanthethicknessoftheentireVNC,usingImageJ.Transverseviews

weregeneratedusingtheResliceoption.ImageswereprocessedusingAdobeCreativeSuite6

PhotoshopandcompiledwithAdobeIllustrator.

Automaticcellcounting

Glialcellslabelledeitherwithanti-RepoorwithrepoGAL4>UAShistone-YFPwerecounted

automaticallyin3DacrossthethicknessoftheVNCusingDeadEasyLarvalGliasoftware,as


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26

previouslydescribed.Prospero+andDpn+cellswerecountedmanuallyin3D(i.e.notinprojections),

asthesignalwasnoisyforDeadEasy.

Statisticalanalysis

StatisticalanalysiswascarriedoutusingGraphpadPrism.Alldatainthisworkarecontinuous.Tests

todeterminewhetherdataweredistributednormallyandvarianceswereequalwereinitiallycarried

out,andthereafterifso,parametricOneWayANOVAtestswherecarriedwhencomparingmore

thantwosampletypesgroup.Multiplecomparisoncorrectionswerecarriedoutwithpost-hoc

Dunnetttestscomparisonstosetcontrols,orBonferronicomparisonsofallsamplesagainstall.Box

plotswereusedtorepresentthedistributionofdata.

ACKNOWLEDGEMENTS

WethankourlabsandC.Rezavalfordiscussionsandcommentsonthemanuscript;S.Corneliussen,

T.SchunkeandS.Dietzandfortechnicalhelp;Y.Jan,J.Skeath,A.GouldandF.Schnorrerfor

reagents;BloomingtonDrosophilaStockCentreforfruit-fliesandDevelopmentalStudiesHybridoma

Bank,Iowaforantibodies.

FUNDING

ThisworkwasfundedbyBBSRCProjectGrantsBB/L008343/1andBB/R00871X/1toA.H.,andBBSRC

MIBTPPhDstudentshiptoE.C.

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San-Juan,BPandBaonza,A.(2011).ThebHLHfactordeadpanisadirecttargetofNotchsignalingandregulatesneuroblastself-renewalinDrosophila.DevBiol352,70-8210.1016/j.ydbio.2011.01.019Schnorrer,F,Kalchhauser,IandDickson,BJ.(2007).ThetransmembraneproteinKon-tikicouplestoDgriptomediatemyotubetargetinginDrosophila.DevCell12,751-76610.1016/j.devcel.2007.02.017Shtaya,A,Sadek,AR,Zaben,M,Seifert,G,Pringle,A,Steinhauser,CandGray,WP.(2018).AMPAreceptorsandseizuresmediatehippocampalradialglia-likestemcellproliferation.Glia66,2397-241310.1002/glia.23479Siegrist,SE,Haque,NS,Chen,CH,Hay,BAandHariharan,IK.(2010).InactivationofbothFoxoandreaperpromoteslong-termadultneurogenesisinDrosophila.CurrBiol20,643-64810.1016/j.cub.2010.01.060Simoes,ARandRhiner,C.(2017).ACold-BloodedViewonAdultNeurogenesis.FrontNeurosci11,32710.3389/fnins.2017.00327Song,Y,Ori-Mckenney,KM,Zheng,Y,Han,C,Jan,LYandJan,YN.(2012).RegenerationofDrosophilasensoryneuronaxonsanddendritesisregulatedbytheAktpathwayinvolvingPtenandmicroRNAbantam.GenesDev26,1612-162510.1101/gad.193243.112Sousa-Nunes,R,Cheng,LYandGould,AP.(2010).RegulatingneuralproliferationintheDrosophilaCNS.CurrOpinNeurobiol20,50-5710.1016/j.conb.2009.12.005Sousa-Nunes,R,Yee,LLandGould,AP.(2011).FatcellsreactivatequiescentneuroblastsviaTORandglialinsulinrelaysinDrosophila.Nature471,508-51210.1038/nature09867Sun,W,Matthews,EA,Nicolas,V,Schoch,SandDietrich,D.(2016).NG2glialcellsintegratesynapticinputinglobalanddendriticcalciumsignals.Elife5,10.7554/eLife.16262Tanaka,EMandFerretti,P.(2009).Consideringtheevolutionofregenerationinthecentralnervoussystem.NatRevNeurosci10,713-72310.1038/nrn2707Torper,O,Ottosson,DR,Pereira,M,Lau,S,Cardoso,T,Grealish,SandParmar,M.(2015).InVivoReprogrammingofStriatalNG2GliaintoFunctionalNeuronsthatIntegrateintoLocalHostCircuitry.CellRep12,474-48110.1016/j.celrep.2015.06.040Vaessin,H,Grell,E,Wolff,E,Bier,E,Jan,LYandJan,YN.(1991).prosperoisexpressedinneuronalprecursorsandencodesanuclearproteinthatisinvolvedinthecontrolofaxonaloutgrowthinDrosophila.Cell67,941-953Valny,M,Honsa,P,Kriska,JandAnderova,M.(2017).MultipotencyandtherapeuticpotentialofNG2cells.BiochemPharmacol141,42-5510.1016/j.bcp.2017.05.008VanDerHeide,LP,Ramakers,GMandSmidt,MP.(2006).Insulinsignalinginthecentralnervoussystem:learningtosurvive.ProgNeurobiol79,205-22110.1016/j.pneurobio.2006.06.003Vigano,FandDimou,L.(2016).TheheterogeneousnatureofNG2-glia.BrainRes1638,129-13710.1016/j.brainres.2015.09.012


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Wills,Z,Bateman,J,Korey,CA,Comer,AandVanVactor,D.(1999).ThetyrosinekinaseAblanditssubstrateenabledcollaboratewiththereceptorphosphataseDlartocontrolmotoraxonguidance.Neuron22,301-312Yamamoto,S,Nagao,M,Sugimori,M,Kosako,H,Nakatomi,H,Yamamoto,N,Takebayashi,H,Nabeshima,Y,Kitamura,T,Weinmaster,G,Nakamura,KandNakafuku,M.(2001).TranscriptionfactorexpressionandNotch-dependentregulationofneuralprogenitorsintheadultratspinalcord.JNeurosci21,9814-982321/24/9814[pii]

FIGURELEGENDS

FIGURE1 ia-2interactsgeneticallywithkon,Notchandpros.

(A)Quantitativereal-timePCR(qRT-PCR)showingthatlossofkonfunctioningliacausedover2-fold

increaseinia-2mRNAlevels.konLOF:heterozygousmutanttogetherwithkonRNAiinglia

(genotype:konc452/UASkonRNAi;repoGAL4/+).OneWayANOVAp<0.0001(RNAi).Over-expressionof

konmarginallydecreasedia-2mRNAlevels.OneWayANOVAp=0.045(GOF).Post-hocDunnett’stest

multiplecomparisonstocontrol.N=4replicates.(B)qRT-PCRshowingthatover-expressionofia-2in

gliadownregulatedkonmRNAlevels.Thus,ia-2represseskonexpression.Left:UnpairedStudentt-

testwithWelchcorrectionp=0.457.Right:OneWayANOVAp<0.045,post-hocDunnett’stest

multiplecomparisonstocontrol.N=4-6replicates.(C)Ia-2isfunctionallyrelatedtoNotch:qRT-PCR

showingthatia-2mRNAlevelsincreasedinNtsmutantlarvaeattherestrictivetemperatureof25°C.

UnpairedStudentt-testwithWelchcorrectionLeft:p=0.4123;Right:p=0.2182.N=3replicates.(D)ia-

2isfunctionallyrelatedtopros:qRT-PCRshowingthatover-expressionofprosingliaincreasedia-2

mRNAlevelsby2fold.UnpairedStudentt-testwithWelchcorrection.Left:p=0.1368;Right:

p=0.0428.N=3replicates.(E)qRT-PCRshowingthatia-2RNAiknock-downinneuronsloweredia-2

mRNAlevelsto20%,whereasingliaithasnoeffect,meaningthatia-2isexpressedinneurons.One

WayANOVAp=0.0004,post-hocmultiplecomparisonstocontrolDunnett’stest.N=3replicates.

(G,H,I)FusionproteinIa-2-YFPrevealedexpressionexclusivelyinneurons,asallia-2-YFP+cellswere

alsoElav+,butRepo—andDpn—.N=4-16larvalVNCs.(F)Illustrationtoshowthatkonandia-2

functionsarerestrictedtogliaandneurons,respectively,andtheyrepresseachother.(G,horizontal

views;H,transverseview;I,highermagnificationview).Withmorethantwosampletypes,asterisks


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32

indicatemultiplecomparisonpost-hocteststocontrols:*p<0.05,**p<0.01,***p<0.001,

****p<0.0001.

FIGURE2 ia-2influencesneuralcellfatestability.(A,B)Lossofkonfunctioninglia

(konc452/UASkonRNAi;repoGAL4/+)increasedthenumberofIa-2-YFP+cellsalongthemidline.One

WayANOVAp<0.0001,post-hocTukey’stest.N=5-8VNCs.(C)TheectopicIa-2-YFP+cellsinkonloss

offunctionwerenotRepo+glia.N=7VNCs.(D,E)Neitherlossnorgainofia-2functionaffected

numberofEve+neurons.OneWayANOVAp=0.2277.N=7-12VNCs.(F,G)Lossofia-2functions

(elavGAL4>UASia-2RNAi)increasedPros+cellnumber.OneWayANOVAp=0.0002,post-hoc

Dunnett’stest.N=8-10VNCs.(H)Lossofia-2function(Df(2L)ED7733/+;elavGAL4>UASia-2RNAi)

causedVNCovergrowth.(I,J)Bothlossandgainofia-2functionincreasedDpn+cellnumber.One

WayANOVAp=0.0021,post-hocDunnett.N=7-12.Asterisksindicatemultiplecomparisonpost-hoc

teststoafixedcontrol:*p<0.05,**p<0.01,***p<0.001,****p<0.0001.

FIGURE3 Injuryinducedia-2expressionandneurogenesis.(A-D)Crushinjuryinthelarval

abdominalVNCcarriedoutat74-76hafteregglaying(AEL)caused:(B,D)formationofmultiple

ectopicDpn+neuralstemcells(arrowheads)by5-7hourspost-injury(N=6/17VNCs).Dpn+cellswere

ia-2-YFP—;and(C)anincreaseinthelevelsofia-2mRNAat5-7hpost-injury,whichrecovered

homeostaticallyby24h,detectedbyqRT-PCR.N=3replicates.(E,G,H)Crushinjuryinthelarval

abdominalVNCat96hAELcausedectopicDpn+cellsby6hourspost-injury(arrowheads).Most

Dpn+cellswereia-2-YFP—,butsomewereia-2-YFP+.N=4/9VNCs.(H)TherewereectopicDpn+cells

dorsally,ontheedgeoftheneuropile.(I,J)CrushinjuryinthelarvalabdominalVNCat117hAEL

causedectopicDpn+cellsby12hourspost-injury(arrowheads).Dpn+cellswerefoundinectopic

dorsalpositionsdevoidofdevelopmentalneuralstemcells(arrowheadsinJ).N=9/32VNCs.(B,G,I)

Horizontalviews,(H,J)sagittalviewsand(D,J)transverseviews.


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FIGURE4 Ia-2andKonregulatedilp-6expressioninneuronsandglia,respectively.(A,B)Dilp-

6GAL4>UAShisYFPcellsaremostlyRepo+glia(arrows),andPros—,meaningtheyarenotastrocyte-

likegliasurroundingtheneuropile,asseenintransverseviewsin(B);frompositiontheyappearto

becortexandsurfaceglia.SomecellsareRepo—(arrowheads,leftcolumn)andElav+(arrows,right

column)meaningtheyareneurons.(C,D)inrexpressionvisualizedwithreporterInRNP2552GAL4>

UAShistoneYFPisexpressedinsomeRepo+neuropilegliaandinsomeinElav+neurons.(E-H)qRT-

PCRsshowingthat:(E,F)ia-2knock-downinneurons(elavGAL4>UASia-2RNAi)downregulatesdilp-6

mRNAlevels,andover-expressionofia-2(withUAS-ia-2GS11438)up-regulatesdpn,dilp-6andelav

mRNA.(E)UnpairedStudent-ttestscomparingeffectoneachgeneseparately,differencesnot

significant;(F)OneWayANOVApergenegroup,notsignificantlydifferent.N=4replicatesforboth.

(G,H)konknock-downinglia(konc452/UASkonRNAi;repoGAL4/+)downregulatesdilp-6mRNAlevels,

whereasover-expressiononkondoesnothavearemarkableeffect.N=3replicatesforboth.(G)One

WayANOVApergenegroup,onlydifferencesindilp-6mRNAsignificantp=0.0362.OneWayANOVA

pergenegroup,notsignificantlydifferent.*p<0.05.

FIGURE5 Ia-2,KonandDilp-6arelinkedthoughaneuron-gliacommunicationloop.(A)Over-

expressedHA-taggedKonICDinglia(repoGAL4>UASkonICd::HA)visualizedinstage16embryoswith

anti-HAantibodies,localizestoglialnuclei(arrowheads).(B,C)Over-expressionofeitherthe

intracellulardomainofeitherkon(konICD)ordilp-6issufficienttoincreaseglialcellnumber

(visualizedwithrepoGAL4>UAShistone-YFP).Over-expressionofadominantnegativeformofthe

insulinreceptorrescuestheincreaseincellnumbercausedbydilp-6(repo>hisYFP,dilp-6,InRDN),

meaningthatautocrineInRsignalingregulatesglialproliferation.OneWayANOVAp<0.0001,post-

hocTukey’stestmultiplecomparisonsbetweenallsamples.N=15-28VNCs.(D,E)kon-RNAiknock-

downinneuralstemcellswithinscGAL4doesnotaffectDpn+expressionorcellnumber.Unpaired

Studentt-test,p=0.3111.N=10VNCs(F)Illustrationsummarisingthatapositivefeedbackautocrine


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loopinvolvingDilp-6,InRandKonpromotesbothglialproliferationandDilp-6production.Asterisks

refertomultiplecomparisonpost-hoctest,allsamplesvs.all:**p<0.01,****p<0.0001.

FIGURE6 Ia-2andDilp-6induceectopicneuralstemcellsthatdonotexpressia-2andresult

fromInRsignalinginglia.(A-D)Over-expressionofia-2anddilp-6,butnotkon-full-length,increased

Dpn+cellnumber.BothIa-2andDilp-6inducedDpn+atthemidlineandinlateralpositions:here

showingia-2inductionmostprominentlyalongthemidline(arrowheads),anddilp-6inlateral

positionsaroundneuropile(arrowheads;arrowsindicatemidlineDpn+cellsinC).(B)EctopicDpn+

cellsdidnotexpressia-2-YFP(arrowheads).(D)Geneticepistasisanalysisshowingthat:theincrease

inDpn+cellnumbercausedbyia-2over-expressionwasrescuedbydilp-6RNAiandkon-RNAiknock-

downinglia,meaningthatia-2increasesDpn+cellsviaglialKonandDilp-6;andpreventinginsulin

signalingwithInRDNingliarescuestheincreaseinDpn+cellnumbercausedbydilp-6over-

expression,meaningthatectopicDpn+originatefromInRsignalinginglia.OneWayANOVA

p<0.0001,post-hocTukey’stestmultiplecomparisonsallsamplesvs.all.N=8-13VNCs.(E)Illustration

ofneuropilegliaconversiontoDpn+cells.(A)Horizontalviews;(B)highermagnification;(C)

transverseviews.Asterisksrefertomultiplecomparisonpost-hoctests:*p<0.05,***p<0.0001,

****p<0.0001.

FIGURE7 Ectopicneuralstemcellsoriginatefromglia.(A,B)Over-expressionofdilp-6induced

DpnexpressioninRepo+neuropileglialcells(arrowheads).N=10VNCs.(C,D)G-TRACEexpressionin

gliawithrepoGAL4revealedGFP+cellsthatwereglialcellsfromcelllineage’sorigin,andRFP+newly

generatedglialcells.DpncolocalisedinneuropilegliawithbothGFPandRFP,meaningthatDpn+

cellsoriginatedfromglia,andatthatpointintimethesecellsstillretainedglialfeatures.N=8VNCs.

(A,C)horizontal,and(B,D)transverseviews.


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FIGURE8 Injury-inducedneurogenesisistriggeredbyaneuron-gliacommunicationloop

involvinginsulingsignallingandKon.(A,C)IntheabdoinallarvalVNC,neuronsexpressia-2,andglia

expresskonatverylowlevels.NeuropileNG2-likegliahaveactiveNotchandPros.Inthenormal,

uninjuredabdominalVNC,InRisexpressedbyglialcellsandneurons;ia-2expressionismaintainedin

neurons,konanddilp-6areswitchedoff,andtherearenoneuralstemcells(NBs).(B)Injurytothe

abdominalVNCprovokesadramaticsurgeinia-2andinkonmRNAlevels.Thisdrivestheinitial

secretionofDilp-6fromasmallgroupofneurons.SecretedDilp-6bindsInRinglia,andInRsignaling

mayfacilitatecleavageandactivationofKon.KonICDactivatesglialproliferationandexpressionof

dilp-6inglia.SecretedDilp6bindsInRincortex/surfaceglia,boostingapositivefeedbackloopthat

amplifiesDilp-6productionfromnon-neuropileglia.InNotch+Pros+NG-2-likegliasurroundingthe

neuropile,Dilp-6andInRsignalingcausetheKon-dependentup-regulationofDpn+.Dpn+neural

stemcellscouldpotentiallythereafterresultintheproductionofnewneuronsandglia.The

productionofDpn+cellsingliadependsonKon,butKon-fulllengthaloneisnotabletoinducedpn

expressionornewDpn+cells.Thus,neurogenesisrequiresinsulinsignalingandKondowsntream.

Together,Ia-2,Dilp-6,InRandKoncaninduceneurogensesisandgliogenesis,matchingcell

populationsduringregeneration.

SupplementaryFigure1 Modifiergeneticscreenstoidentifygenesinteractingwithkon

(A,B)Over-expressionofkoningliawithrepoGAL4(repo>UASFlyBow,UASkon-full-length)causeda

phenotypeofverylongventralnervecord(A),andinneuronswithelavGAL4too,althoughtoa

lesserextent(B).ThesephenotypeswerequantifiedbyusingthereporterUASFlyBow,andtheVNC

measuredusingImageJtools.RNAiknock-downofcandidategenescanrescuethesephenotypes,

someexamplesareshownhere.(C-G)Thekongainoffunction(GOF)phenotyperesultingfromover-

expressingkon-full-lengthineitherneuronsorglia,canberescuedbyRNAiknock-downof:(C)

predictedinteractorsofKonorNG2,mostprominentlyinglia;KruskalWallisANOVAp<0.0001,post-

hocDunntestto>FlyBow,koncontrols.N=4-24VNCs.(D)α andγ-secretasesthatcleaveNG2and


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36

Notch,fromglia.;KruskalWallisANOVAp<0.0001,post-hocDunntestto>FlyBow,koncontrols.

N=4-24VNCs.(E)knownKonpartners,e.g.integrins,andothertransmembraneproteins,alsofrom

neurons;;KruskalWallisANOVAp<0.0001,post-hocDunntestto>FlyBow,koncontrols.N=3-24

VNCs.(F)cytoplasmicphosphatases,fromeithergliaorneurons;KruskalWallisANOVAp<0.0001,

post-hocDunntestto>FlyBow,koncontrols.N=7-24VNCs.VNClengthindicatedinyellowin(A,B).

*Asterisksindicatemultiplecomparisonpost-hocteststocontrols:*p<0.05,**p<0.01,***p<0.001,

****p<0.0001.

SupplementaryFigure2 Modifiercandidategeneticscreensidentifygenesencoding

transmembranephosphatasesandinsulinsignalingfactorsasinteractingwithkon.(A,B)Over-

expressionofkoningliacausesaverylongVNC(A),andinneuronstoo,buttoalesserextent(B).

RNAiknock-downofcandidategenescanrescuethesegainoffunctionphenotypes,someexamples

aregiven.(C,D)QuantificationofnormalizedVNClengthshowsrescueprominentlybymost

transmembranephosphatases,theNotch-relatedAkap200,andgenesfunctionallyrelatedtothe

insulinsignalingpathway,andincludingAkt,larandia-2.Normalisedmeasurementsaregivenasa

ratiooftheVNCovertotallarvallength.Kruskal-WallisANOVAp<0.0001,post-hocDunn’stest

comparisontocontrolsrepo>konorelav>kon.(C)N=2-28;(D)N=2-31.*Asterisksindicatemultiple

comparisonpost-hocteststocontrols:*p<0.05,**p<0.01,***p<0.001,****p<0.0001.

SupplementaryFigure3 Lossandgainofkonfunctionprominentlyaffectedia-2expression.

(A,B)Exploratoryquantitativereal-timePCR(qRT-PCR),N=2replicateseach:(A)showingthechange

inmRNAlevelsforcandidategenesuponkonRNAitargetedtoeitherneurons(withelavGAL4)orglia

(withrepoGAL4).ia-2mRNAlevelsincreasedatleast3foldwhenkonwasknocked-downinglia;(B)

showingtheeffectofkongainoffunction.konover-expressionineitherneuronsorgliadecreased

ia-2mRNAlevels.Thefirsttwocolumnshavebeenleftcutoutastheyarecontrolswiththeincrease

inkonmRNAwithkonover-expression,whichareveryhighcomparedtotherest.(C,D)Further


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37

replicateswerecarriedoutforaselectedgroupofgenes,andtheyvalidatethatkonprominently

regulatesia-2expression.N=4replicateseach.Thefirstcolumnsrepresenttheveryhighincreasein

konmRNAwithkonover-expression,andtheyhavebeencutastheygowellbeyondthisscale

comparedtotherest.

SupplementaryFigure4 Alterationsinia-2levelscausenoobviousneuronalphenotypes.

(A)Neuronsandtheiraxonalfasciclesarevisualisedwithanti-FasII.N=7-11VNCs.(B)Neuronsand

theirdendritesarevisualizedwithanti-BP102.N=9-10VNCs.Noabnormalphenotypeswere

observed.


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Figure 1 ia-2 interacts genetically with kon, Notch and pros

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elav>Konrepo>Konkon ; elav>kon-RNAic452

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Figure 2 Ia-2 influences neural cell fate stability

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0.0

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Figure 3 Injury induces ia-2 expression and neurogenesis

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mer

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Figure 4 Ia-2 and Kon regulate dilp-6 expression in neurons and glia, respectively

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Figure 5 Ia-2, Kon and Dilp-6 are linked though a neuron-glia communication loop.

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cell division Dilp-6

Dilp-6

Dilp-6

IR

C

E

GLIA

Repo

Repo

GLIARepo

A merge anti-Repo anti-HA

repo>konICD

>dilp-6,InRDN


https://doi.org/10.1101/721498


0

20

40

60

80

ia-2-YFP, repo>KonControl: ia-2-YFP

ia-2-YFP, repo>ia-2

No

of D

pn+

NB

s

ia-2-YFP, repo>ia-2, Kon-RNAi

ia-2-YFP, repo>dilp-6

***

ia-2-YFP, repo>ia-2, dilp-6RNAi

ia-2-YFP, repo>dilp-6, InRDN

***** ********

mer

geia

-2-Y

FPD

pn

ia-2-YFP Dpn

A

ia-2-YFP, repo>Kon ia-2-YFP, repo>ia-2 ia-2-YFP, repo>dilp-6ia-2-YFP

mer

geia

-2-Y

FPD

pn

mergeB

C

ia-2-YFP Dpn

3x 1μ

m o

ptic

al s

ectio

ns

ia-2-YFP Dpn

Dia-2-YFP, repo>Kon ia-2-YFP, repo>ia-2 ia-2-YFP, repo>dilp-6ia-2-YFP

Figure 6 Ia-2 and Dilp-6 induce ectopic neural stem cells that do not express ia-2 and result from InR signaling in glia.

neuropile

cortex glia

neuropile glia

Repo- midline cells

Dpn+ neuropile glia

Dpn+ Repo- midline cellsia-2, Dilp-6


https://doi.org/10.1101/721498


Repo Dpnm

erge

Rep

oD

pn

repo>dilp-6

Repo Dpn

ventrallateraldorsal

DpnRFP GFP

repo>ia-2, G-TRACE

A B

D

mer

geR

epo

Dpn

transverse

ventrallateraldorsal transverse

Dpn

GFP

RFP

mer

ge

Dpn

GFP

RFP

mer

ge

CDpnRFPGFP

Figure 7 Ectopic neural stem cells originate from glia


https://doi.org/10.1101/721498


NO INJURY INJURY INDUCES REGENERATIVE NEUROGENESIS

ia-2

NotchPros

system OFF

ia-2

NotchProsKonICD

Kon

Dilp-6 Dilp-6

dilp-6cortex glia neuropile glia

NG2-like

neuron

KonICD

Kon Ia-2

proliferationglia

GLIA

Kon, Repo GLIA

RepoRepo

NEURON

KonInR

ProsPros

ia-2

Repo Repo

NEURAL STEM CELL: GLIA, NEURONS

system OFF Injury

Dilp-6

Dilp-6neuron

cortex glia

neuropile glia, NG2-like

Figure 8 Injury-induced neurogenesis is triggered by a neuron-glia communication loop involving insuling signalling and Kon.

A B

C

Notch, Pros, Kon, DpnKon

Dpn

cortex glia NG2-like glia

Pros

insulin signalling

KonInR

neuron

cortex glia NG2-like glia


https://doi.org/10.1101/721498


A repo>FlyBow

>+ control >kon >kon, lar RNAi

>kon, wnt5 RNAi

>+ control >kon >kon, lar RNAi

>kon, mys RNAi

elav>FlyBowB

Supplementary Figure 1 Modifier genetic screens to identify genes interacting with kon

repo>

FlyBow

repo>

FlyBow

, Kon

>fak5

6D R

NAi

>grip

RNAi

>metr

o RNAi

>sdc

RNAi

>fz R

NAi

>van

g RNAi

>wnt5

RNAi

0.0

0.5

1.0

1.5

2.0

2.5

VNC

leng

ht (m

m)

******

*****

elav>

FlyBow

elav>

FlyBow

, Kon

>fak5

6D R

NAi

>grip

RNAi

>metr

o RNAi

>sdc

RNAi

>fz R

NAi

>van

g RNAi

>wnt5

RNAi0.0

0.5

1.0

1.5

2.0

2.5

VNC

leng

ht (m

m)

***

C

repo>

FlyBow

repo>

FlyBow, K

on

>cad

N RNAi

>cad

N2 RNAi

>ds R

NAi

>ft R

NAi

>shg

RNAi

>stan

RNAi

>if R

NAi

>mew

RNAi

>mys

RNAi

>eys

RNAi

>lanB

2 RNAi

>wde

RNAi

0.0

0.5

1.0

1.5

2.0

2.5

VNC

leng

ht (m

m)

********

*****

elav>

FlyBow

elav>

FlyBow

, kon

>cad

N RNAi

>cad

N2 RNAi

>ds R

NAi

>ft R

NAi

>shg

RNAi

>stan

RNAi

>if R

NAi

>mew

RNAi

>mys

RNAi

>eys

RNAi

>lanB

2 RNAi

>wde

RNAi0.0

0.5

1.0

1.5

2.0

2.5

VNC

leng

ht (m

m)

*** *

repo>

FlyBow

repo>

FlyBow

, Kon

>psn

RNAi

>kuz R

NAi

>kul R

NAi0.0

0.5

1.0

1.5

2.0

2.5

VNC

leng

ht (m

m)

**** ******

elav>

FlyBow

elav>

FlyBow

, Kon

>psn

RNAi

>kuz

RNAi

>kul

RNAi0.0

0.5

1.0

1.5

2.0

2.5

VNC

leng

ht (m

m)

-secretase-secretase

***

D

Eall are repo>FlyBow, kon

all are elav>FlyBow, kon

transmembrane proteins and known partners

secretasesall are repo>FlyBow, kon

predicted interactors of Kon/NG2

all are repo>FlyBow, kon

all are elav>FlyBow, kon all are elav>FlyBow, kon

fibronectinlaminin

integrinscadherins

repo>

FlyBow

repo>

FlyBow

, Kon

>eya

RNAi

>mtm

RNAi

>prl1

RNAi

>Ssu

72 R

NAi0.0

0.5

1.0

1.5

2.0

2.5

cytoplasmic phosphatases

VNC

leng

ht (m

m)

*

****** *

elav>

FlyBow

elav>

FlyBow

, Kon

>eya

RNAi

>mtm

RNAi

>prl1

RNAi

>Ssu

72 R

NAi0.0

0.5

1.0

1.5

2.0

2.5

VNC

leng

ht (m

m)

****

all are elav>FlyBow, kon

all are repo>FlyBow, kon

F


https://doi.org/10.1101/721498


C D

>Akt RNAi

repo>FlyBow

>lar RNAi

>Akap200 RNAi

>ia-2RNAi

>Akap200 RNAi

elav>kon

repo>koncontrol

>grip RNAi

>Akt RNAi

>ptp4E RNAi

A Ball are repo>FlyBow, kon all are elav>FlyBow, kon

Supplementary Figure 2 Modifier candidate genetic screens identify genes encoding transmembrane phosphatases and insulin signaling factors as interacting with kon.

VNC length

larval length

VNC

VN

C le

ngth

/tota

l lar

val l

engt

h

transmembrane phosphatases and insulin-relatedall are repo>FlyBow, kon

transmembrane phosphatases and insulin-relatedall are elav>FlyBow, kon

rescue from glia rescue from neurons

**

elav>

kon

elav>

kon; A

kap20

0 RNAi

elav>

kon,g

rip R

NAi

elav>

kon,Lar

RNAi

elav>

kon,p

tp99A

RNAi

elav>

kon,ia

-2 RNAi

elav>

kon,p

tp69D

RNAi

elav>

kon;p

tp10D

RNAi

Elav>k

on;pt

p4E R

NAi

elav>

kon,p

tp61F

RNAi

elav>

kon,A

kt RNAi

elav>

kon,

hpo R

NAi

Elav>k

on;in

aD R

NAi 0.0

0.2

0.4

0.6

repo>

FlyBow

repo>

kon

repo>k

on;Aka

p200R

NAi

repo>

kon,g

rip R

NAi

repo>k

on,Lar RNAi

repo>

kon,p

tp99A

RNAi

repo>k

on,ia-2

RNAi

repo>

kon,p

tp69D

RNAi

repo>

kon;p

tp10D

RNAi

repo>

kon;p

tp4E R

NAi

repo>

kon,

ptp61

F

Repo>k

on, Akt

RNAi

repo>

kon,

hpo R

NAi

repo>

kon;i

naD R

NAi 0.0

0.2

0.4

0.6

0.8

****

****

****

****

**** *** **** ******** ****

**

*

***

VN

C le

ngth

/tota

l lar

val l

engt

h


https://doi.org/10.1101/721498


qRT-PCR: effect of kon over-expressionqRT-PCR: effect of kon RNAi knock-down

A B

kon

hpo

ia-2

ptp99

A

ptp69

Fprl

1 yki

inaD

kon

hpo ia-

2

ptp99

A

ptp69

F

ptp10

Dprl

-1

ptp61

F lar fzyo

rkie

inaD

0

1

2

3

4

control kon mRNA: test mRNA:

kon repo>kon-RNAic452kon elav>kon-RNAic452

qRT-PCR: effect of kon RNAi knock-down

mRNA

folo

d ch

ange

0.0

0.5

1.0

1.5

elav>Kon

repo>Kon

elav>Kon

repo>Kon

control kon mRNA: test mRNA:

kon

hpo ia-

2

ptp99

A

ptp69

F

ptp10

Dprl

-1

ptp61

F lar fzyo

rkie

inaDmRNA

kon repo>kon-RNAic452kon elav>kon-RNAic452

folo

d ch

ange

qRT-PCR: effect of kon over-expression

0

1

2

3

4

mRNA

folo

d ch

ange

0.0

0.5

1.0

1.5

folo

d ch

ange

kon

hpo

ia-2

ptp99

A

ptp69

Fprl

1 yki

inaDmRNA

C D

Supplementary Figure 3 Loss and grain of kon function prominently affects ia-2 expression


https://doi.org/10.1101/721498


wt elav>ia-2 RNAi elav>ia-2

FasII

wt elav>ia-2 RNAi elav>ia-2

BP102A B

Supplementary Figure 4 Alterations in ia-2 levels cause no obvious neuronal phenotypes


https://doi.org/10.1101/721498


regenerative neurogenesis is induced from glia by ia-2 driven ......2019/08/01 · involved in...

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