Apolipoprotein E Associated Astrocytic Glia of theCentral ...
Regenerative neurogenesis is induced from glia by Ia-2 driven ......2019/08/01 · involved in...
Transcript of 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.
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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
.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
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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
.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
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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,
.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
20
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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21
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
.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
22
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
.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
23
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’–
.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
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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
.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
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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
.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
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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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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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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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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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.
.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
Figure 1 ia-2 interacts genetically with kon, Notch and pros
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Kon Ia-2
***
.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
Figure 2 Ia-2 influences neural cell fate stability
0
50
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200
No.
abd
omin
al
Eve
+ ce
lls
D
control: yw/+ elav>ia-2-RNAi elav>ia-2
control elav>ia-2RNAi
elav>ia-2
No.
abd
omin
al
Dpn
+ ce
lls
H
**
0
10
20
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40
50
*
control Df ia-2,elav>ia-2RNAi
elav>ia-2
J
Dpn
control Df ia-2, elav>ia-2-RNAi
elav>ia-2
F
yw control elav>ia-2RNAi repo>ia-2elav>ia-2
Pros
yw
elav>
ia2RNAi
repo>
ia2
elav>
ia20
100
200
300 **
A
control elav>konrepo>konkon, repo>kon-RNAic452
kon, repo>kon-RNAic452
mer
geBIa-2YFP
Ia-2YFP Repo
ia2-YFP0
50
100
150
200
ia2-YFP, kon LOFia2-YFP, repo>konia2-YFP, elav>kon
C
****
No.
mid
line
YFP
+ ce
lls
E
G
Eve
No.
abd
omin
al
Pro
s+ c
ells
IDpn
Df ia-2, elav>ia-2-RNAi
.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
0.0
0.5
1.0
1.5
2.0
2.5
5-7h 24h
control: non-injured G9
injured, G9
qRT-PCR: ia-2 mRNA
folo
d ch
ange
vs.
con
trol
Figure 3 Injury induces ia-2 expression and neurogenesis
C
Merge Dpn
non-
inju
red
inju
red
ia2-YFP
non-
inju
red
inju
red
Merge Dpn ia2-YFPD
A
E F
I
LI LII LIII LIII
2 3day 1
74h AEL injuryegg laying
5-7h 24h after injury
LI LII LIII LIII
2 3day 1
96h AEL injury
6h after injury
B
LI LII LIII LIII
2 3day 1
injury at 117h AEL
fix at 12h after injury
Merge Dpn ia2-YFP Dpn ia2-YFP
injured at 96h AEL analysed 6h after injury (102h AEL)
inju
red
non-
inju
red
DpnMerge Dpn ia2-YFP
Dpn
inju
red
horizontal sections
v d
d
horizontal sections
G
non-
inju
red
H
J
non-
inju
red
inju
red
non-
inju
red
inju
red
ia2-YFPinjured at 117h AEL analysed 12h after injury (129h AEL)
Dpn
transverse
transverse
.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
mer
ge
ElavProsRepo
dilp
-6>h
isYF
P
dilp-6>hisYFPElavProsRepo
dilp-6>hisYFP
Figure 4 Ia-2 and Kon regulate dilp-6 expression in neurons and glia, respectively
0.0
0.5
1.0
1.5
0.0
0.5
1.0
1.5
2.0
2.5
mRNA: dpn dilp6 elav
Control: UAS-ia-2RNAi/+elav>ia-2RNAi
folo
d ch
ange
qRT-PCR in ia-2 LOF
folo
d ch
ange
qRT-PCR in ia-2 GOF
mRNA: dpn dilp6 elav
Control: UAS-ia-2 (GS11438) /+elav>ia-2 (GS11438)repo>ia-2 (GS11438)
E F
0.0
0.5
1.0
1.5
mRNA: dpn dilp6 elav
elav>Kon
repo>Kon
Control: UAS-kon FL /+
folo
d ch
ange
qRT-PCR in kon LOF qRT-PCR in kon GOF
0.0
0.5
1.0
folo
d ch
ange
mRNA: dpn dilp6 elav
G HControl: UAS-kon-2RNAi/+
kon ; elav>Kon-RNAic452
kon ; repo>Kon-RNAic452
A B
ElavRepoRepo ElavRepoC D InR >hisYFPNP2552InR >hisYFPNP2552
dorsalventral lateral transverse
*
.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
Figure 5 Ia-2, Kon and Dilp-6 are linked though a neuron-glia communication loop.
**
No.
his
-YFP
+ ce
lls
repo>hisYFP
B repo>hisYFP
0
200
400
600**
****
****
>konICD >dilp-6+>kon >dilp-6 >dilp-6, IR-DN+ ICD
0
20
40
60
No.
abd
omin
al D
pn+
NB
s
insc>+
insc>kon-RNAi
ns
insc>+ insc>kon-RNAi
Danti-Dpn
FKon
KonICD
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
.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
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
.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
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
.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
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
.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
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
.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
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
.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
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
.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
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
.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