Serotonergic neuron ribosomes regulate the neuroendocrine ......2021/06/10 · 1 Serotonergic...

Serotonergicneuronribosomesregulatetheneuroendocrinecontrolof1

Drosophiladevelopment.2

LisaP.Deliu,DeeshpaulJadir,AbhishekGhosh,SavrajS.Grewal4

ClarkHSmithBrainTumourCentre,ArnieCharbonneauCancerInstitute,AlbertaChildren’sHospital6

ResearchInstitute,andDepartmentofBiochemistryandMolecularBiologyCalgary,UniversityofCalgary,7

AlbertaT2N4N1,Canada.8

Correspondence:grewalss@ucalgary.ca10

ABSRACT12

Theregulationofribosomefunctionisaconservedmechanismofgrowthcontrol.Whilestudiesin14

singlecellsystemshavedefinedhowribosomescontributetocellgrowth,themechanismsthatlink15

ribosomefunctiontoorganismalgrowtharelessclear.HereweexplorethisissueusingDrosophila16

Minutes,aclassofheterozygousmutantsforribosomalproteins(Rps).Theseanimalsexhibita17

delayinlarvaldevelopmentcausedbydecreasedproductionofthesteroidhormoneecdysone,the18

mainregulatoroflarvalmaturation.Wefoundthatthisdevelopmentaldelayisnotcausedby19

decreasesineitherglobalribosomenumbersortranslationrates.Instead,weshowthattheyare20

dueinparttolossofRpfunctionspecificallyinasubsetofserotonin(5-HT)neuronsthatinnervate21

theprothoracicglandtocontrolecdysoneproduction.Wefoundthatthese5-HTneuronshave22

defectivesecretioninMinuteanimals,andthatoverexpressionofsynapticvesicleproteinsin5-23

HTergiccellscanpartiallyreversetheMinutedevelopmentaldelay.Theseresultsidentifyacell-24

specificroleforribosomalfunctionintheneuroendocrinecontrolofanimalgrowthand25

development.26

INTRODUCTION28

Theregulationofribosomeandproteinsynthesisareconservedmechanismsofgrowthcontrol.Several30

decadesofstudiesinunicellularsystemssuchasE.coli,yeastandculturedmammaliancellshavedefined31

boththesignalingpathwaysthatcouplegrowthcuestoribosomesynthesisandfunction,andthe32

mechanismsbywhichchangesinmRNAtranslationdrivecellgrowthandproliferation(DaiandZhu,2020;33

LempiainenandShore,2009;RudraandWarner,2004;Warner,1999).However,themechanismsthat34

operateinwholeanimalsduringdevelopmentalgrowtharelessclear.Inthesecontexts,bodygrowthisnot35

(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted June 10, 2021. ; https://doi.org/10.1101/2021.06.10.447971doi: bioRxiv preprint

determinedsolelybyprocessesthatgoverncell-autonomousgrowth,butalsobyinter-organ36

communicationtoensurecoordinatedgrowthanddevelopmentacrossalltissuesandorgans(Boulanetal.,37

2015;DroujinineandPerrimon,2016;Grewal,2012).Hence,tissuespecificchangesinribosomefunction38

havethepotentialtomediatenon-autonomouseffectsonwhole-bodyphysiologytocontrolorganismal39

development.40

Thecomplexlinksbetweenribosomefunctionandanimaldevelopmentareexemplifiedbytheorganismal42

biologyofribosomalproteins(Rps)(TerzianandBox,2013;XueandBarna,2012).Metazoanribosomes43

have70-80Rps,andmutantsforalmostallofthesearehomozygouslethalinanimals,emphasisingtheir44

essentialroleinribosomesynthesisandfunction.However,inmanycasesRpmutantsshowdominant45

phenotypesasheterozygotes.ThesephenotypesareoftenspecifictotheaffectedRpandcangiveriseto46

tissue-specificeffectsthatcannotbeexplainedsimplybyloweredoverallproteinsynthesisandgrowth47

rates.Forexample,inzebrafishcertainrp/+mutantscandevelopperipheralnervetumors(Amsterdamet48

al.,2004).Similarly,someDrosophilaRpmutantsdevelopselectivetissueovergrowthphenotypes(Torok49

etal.,1999;Watsonetal.,1992).SeveralRp/+mutantsinmicehavealsobeenshowntoeachexhibittissue50

specificdevelopmentaldefectsthatdifferbasedontheRpaffected.Forexample,rpl38/+miceshowspecific51

skeletalsegmentationdefects(Kondrashovetal.,2011),rps14/+miceshowdefectsinblooddevelopment52

(Barlowetal.,2010),andrpl27a/+miceshowdefectsincerebellardevelopment(Terzianetal.,2011).The53

dominanteffectsofrp/+mutationsalsoextendtohumans,whereseveralpathologies,collectivelytermed54

ribosomopathies,arecausedbyheterozygosityforRpmutations,andleadtotissue-specificeffectssuchas55

blooddisorders,congenitalgrowthdefects,andpredispositiontocancer(Farley-Barnesetal.,2019;56

Kampenetal.,2020;YelickandTrainor,2015).Themechanismsthatdeterminethesedominanteffectsof57

rp/+mutationsarenotfullyclearbutarethoughttoinvolveselectivealterationsinmRNAtranslation.58

Thesealterationsmayoccureitherasaresultofloweredribosomenumbersorduetoribosome59

heterogeneity,whereribosomeswithdifferentcomplementsofRpshavebeenproposedtohavedifferent60

translationalproperties(Dinman,2016;GenuthandBarna,2018a,b;Khajuriaetal.,2018;MillsandGreen,61

2017).ThesestudiesemphasisetheimportanceoffurtherworktounderstandhowRpfunctioncontributes62

toorganismalgrowthanddevelopment.63

Drosophilalarvaehaveprovidedanexcellentmodelsysteminwhichtodefinethecell-,tissue-andbody-65

levelmechanismsthatcontroldevelopmentalgrowth(Andersenetal.,2013;Boulanetal.,2015;Texadaet66

al.,2020).Larvaegrowalmost200-foldinmassover4-5daysbeforeundergoingmetamorphosistothe67

pupalstage.Thisdevelopmentaltransitioniscontrolledbyapulseofsecretionofthesteroidhormone,68

ecdysone,fromtheprothoracicgland(PG),whichthenactsontissuestostimulatepupationattheendof69

thelarvalperiod(Kannangaraetal.,2021;Panetal.,2021;Yamanakaetal.,2013).Thetimingofthispulse70

isundercontroloftwoseparatesubsetsofneuronsexpressingeithertheneuropeptide,PTTH,orthe71

neuromodulatorserotonin(5-HT),thateachinnervatethePGandstimulateecdysoneproduction72

(McBrayeretal.,2007;Shimada-NiwaandNiwa,2014;Shimelletal.,2018).Thisneuroendocrinenetwork73

integratessignalsfromtheenvironmentandothertissuestoensurepropertimingoftheecdysonepulse74

andthelarval-pupaltransition.Forexample,nutrientsignalscanactonboththe5-HTneuronsandthePG75

toensurepropercouplingofdevelopmentmaturationwithnutrients(Layalleetal.,2008;Shimada-Niwa76

andNiwa,2014).Epithelialdiscdamagealsoleadstoadelayinlarvaldevelopmenttoallowtimeforproper77

tissueregenerationbeforetransitiontothepupalstage.Onewaythatthisdelayismediatedisby78

suppressionofPTTHsignalingbydilp8,aninsulin/relaxin-likepeptidethatsignalsfromdamageddiscsto79

asubsetofLgr3receptorexpressingneuronsthatinhibitPTTHneuronalactivity(Colombanietal.,2015;80

Colombanietal.,2012;Garellietal.,2012;Garellietal.,2015;Jaszczaketal.,2016).Inaddition,the81

inflammatorycytokine,Upd3cansignaldirectlyfromdamageddiscstothePGtosuppressecdysoneand82

delaydevelopment(Romaoetal.,2021).83

AninterestingclassofmutantsthatexhibitalterationsinlarvaldevelopmentaretheMinutes(Lambertsson,85

1998;Marygoldetal.,2007).Thesearedominantmutantsthatareclassicallydescribedbytheir86

developmentaldelayandshortbristles.AlmostallMinutesarerp/+mutantsandtheyhaveperhapsbeen87

beststudiedincontextofcellcompetition,aprocessinwhichmosaicclonesofrp/+cellsinimaginaldisc88

epitheliaareoutcompetedandkilledbysurroundingwild-type(+/+)cells.Severalmechanismshavebeen89

describedtoaccountwhyrp/+cellsareoutcompetedincludingalteredproteostasis(Baumgartneretal.,90

2021;Recasens-Alvarezetal.,2021),competitionfordppgrowthfactor(Morenoetal.,2002),inductionof91

innateimmunesignaling(Germanietal.,2018;Meyeretal.,2014),andinductionofthetranscriptionfactor92

Xrp1(Baillonetal.,2018;Leeetal.,2018).Interestingly,someofthesedisc-intrinsic,cellcompetition93

effectshavealsobeenshowntopartiallyaccountfortheorganismaldelayindevelopmentseeninrp/+94

animals.Forexample,disc-specificRpknockdownstimulatesXrp1inductionofdilp8(Boulanetal.,2019),95

andbothlossofXrp1anddisc-specificknockdownofdilp8caneachpartiallyreversethedelayin96

developmentseeninrp/+animals(Akaietal.,2021;Jietal.,2019;Leeetal.,2018).Ithasalsobeenshown97

thatlossofRpfunctionspecificallyinthePGcanalsoexplaintheoveralldelayindevelopmentinrps6/+98

animals(Linetal.,2011).Theseresultssuggestthattheoveralldelayinorganismaldevelopmentseenin99

rp/+animalsmayresultfromtissuenon-autonomouseffectsofRps.HenceMinutesprovideanexcellent100

systemtoexplorehowtissue-selectivefunctionsofRpscontributetowhole-bodyphenotypes.101

Hereweprovidefurtherevidencefortissue-specificeffectsofRpinthecontroloflarvaldevelopment.We103

describehowlossofRpfunctionresultsindefectsinvesicle-mediatedsecretionspecificallyinthe5-HT104

neuronsthatinnervatethePGleadingtodevelopmentaldelayinMinuteanimals.105

RESULTS107

Rps13/+animalsdonotshowaglobaldecreaseinribosomelevelsorproteinsynthesis109

ForourstudyweusedfliesheterozygousforpreviouslycharacterizedalleleofribosomalproteinS13,111

P[lacW]M(2)32A(hereafterreferredtoasrpS13/+animals),whichhavedecreasedexpressionofrps13112

mRNA(FigureS1A)andhavebeenobservedtohavetheclassicMinutephenotypeofshorterandthinner113

bristlesandadelayinlarvaldevelopment(Saeboe-Larssenetal.,1998).Wequantifiedthedelayin114

developmentofrpS13/+andcontrols(w1118)bymeasuringthetimeittookforanimalstoreachthepupal115

stageafteregglaying.WefoundthatrpS13/+animalsweredelayedindevelopmentbyabout40hours,116

whichcorrespondstoadelayofapproximately20%comparedtocontrolanimals(Figure1A).Wealso117

measuredbodysizeasthelarvaedeveloped,andwesawthatrpS13/+larvaehadasmallersizecompared118

toage-matchedcontrolanimalsatdifferentstagesoflarvaldevelopment(FigureS1B).However,dueto119

theirprolongedlarvalperiod,therpS13/+animalsgrewforalongertime.Hence,whenwemeasuredboth120

finallarvalandpupalsize,wefoundthat,inbothcases,rpS13/+animalswereabout12%largerthan121

controls(Figure1B,C,FigureS1C).Wemeasuredmouthhookmovementsasmeasureoffeedingrateand122

sawasmall,butsignificant,increaseinrpS13/+larvaewhencomparedtocontrols(FigureS1D).This123

indicatesthatthegrowthanddevelopmentalphenotypesofrpS13/+animalsdonotresultsimplyfrom124

reducedfeeding.ThesedatasuggestthatrpS13/+animalsexhibitareducedgrowthanddevelopmental125

rate.126

Studiesindifferentmodelsystemshaveshownthatthephenotypesseeninrp/+animalsareoften128

associatedwithloweredribosomenumbersandreducedproteinsynthesis.Wethereforeinvestigated129

ribosomelevelsandproteinsynthesisinrpS13/+animals.Inordertomeasureribosomenumbers,we130

measuredmature18Sand28SrRNAinwanderingL3wholelarvallysates.Wesawnosignificantdifference131

inrRNAlevelsbetweenrpS13/+andcontrollarvae(Figure1D).TotalproteincontentinwanderingL3132

larvallysatesalsoshowednosignificantdifferenceinrpS13/+larvaecomparedtocontrollarvae(Figure133

1E).Finally,weinvestigatedwhetherrpS13/+animalsshowadecreaseinproteinsynthesisrate.Todothis134

weusedapuromycinlabellingassay(Deliuetal.,2017).Wefirstquantifiedthelevelsofpuromycin135

incorporationofrpS13/+andcontrolanimalsatthewanderinglarvalstageinordertodevelopmentally136

matchcontrolandrpS13/+animalsandfoundnosignificantdifferenceinproteinsynthesisrates(Figure137

1F,G).Werepeatedthisassayattwootherearliertimepointswithaged-matchedlarvae,andonceagain138

foundnodecreaseintranslationratesinrpS13/+larvaecomparedtocontrollarvae(FigureS2).This139

suggeststhattheMinutedelayeddevelopmentisnotduetoagloballossofribosomenumbersor140

translationalcapacity.141

rpS13/+animalsshowadefectinecdysonesignalling143

Thedurationofthelarvalperiodiscontrolledinlargepartbythesteroidhormone,ecdysone(Panetal.,145

2021).Inparticular,attheendoflarvaldevelopment,aneuro-endocrinecircuitstimulatesapulseof146

ecdysoneproductionandsecretionfromtheprothoracicgland(PG).Thiscirculatingecdysonethenactson147

larvaltissuestotriggerthelarvaltopupaltransition.Anydefectsinthisneuro-endocrinecircuitleadstoa148

delayinlarvaldevelopmenttothepupalstage.Giventheirdelayeddevelopment,weexaminedwhether149

rpS13/+animalsshowadefectinecdysonesignaling.Wedidthisbymeasuringthetranscriptlevelsof150

phantomandspookierbothofwhichencodeenzymesforPGecdysoneproduction.Aspreviouslydescribed,151

bothshowedmaximalexpressionpeaksat120hoursAELincontrolanimals,consistentwiththeecdysone152

pulsethattriggerspupation(Figure2A).However,inrpS13/+animalsthesepeaksweredelayedbyabout153

oneday(144hours)andcontinuedtoshowexpressionevenat168hoursforlarvaethatwerestill154

wandering(Figure2A),suggestingadelayinecdysonesignalling.Wealsofoundthatfeedinglarvae20155

hydroxyecdysone(20HE)wasabletopartiallyreversethedevelopmenttimingdelayseenintherpS13/+156

byaboutonethirdofthetotaldelay(Figure2B).EcdysonesynthesisinthePGcanbestimulatedbyseveral157

differentsignalingpathways,includingtheRas/ERKandTORkinasepathways(Cruzetal.,2020;Layalleet158

al.,2008;Rewitzetal.,2009).WhenweoverexpressedtheTORactivator,RhebinthePG,wefoundthat159

whileithadnoeffectondevelopmentaltimingincontrolanimals,itwassufficienttopartiallyreversethe160

developmenttimingdelayseenintherpS13/+animalsagainbyaboutonethirdofthetotaldelay(Figure161

2C).Together,thesedataindicatethatrpS13/+animalsexhibitadelayindevelopmentthatcanbe162

explainedinpartduetobluntedecdysonesignaling.163

5-HTneuronalrpS13isrequiredforproperdevelopmentaltiming165

OurearlierdataindicatedthatrpS13/+animalsdidnotshowanyglobalchangesinwhole-bodyribosome167

orproteinsynthesislevels.Hence,itispossiblethatthedelayeddevelopmentinrpS13/+animalsreflectsa168

moreselectiveroleforRpS13,perhapsinspecificcellsortissuesinvolvedincontrollingecdysone.To169

investigatethispossibility,ourapproachwastousetheGal4/UASsystemtore-expressaRpS13transgene170

(UAS-rpS13)inspecifictissuesinrpS13/+animalsandthenexaminewhetherthiscouldreversethedelay171

indevelopment.Wefocusedinparticularonexaminingcellsandtissuesimportantforstimulatingthe172

ecdysonepulse.Wefirstre-expressedUAS-rpS13thePGusingthePGdriver,P0206-Gal4.Wesawno173

significantchangeintimingincontrolanimalswiththedriveraloneorwiththeover-expressionofUAS-174

rpS13incontrolanimals.Moreover,wefoundthatPG-specificexpressionofUAS-rpS13inrpS13/+larvae175

wasunabletoreversethedelayindevelopmentseenintherpS13/+animals(Figure3A).Wethen176

examinedtheimaginaldiscs.GrowthaberrationscausedbyreducedRpexpressionleadtoreleaseofdilp8177

whichultimatelyhasanegativeeffectonecdysonesynthesis.Weusedtheesg-Gal4tswhichdirects178

temperature-inducibleexpressioninallimaginalcellsinthelarvae.WefoundthatexpressionofUAS-rpS13179

throughoutlarvaldevelopmentusingesg-Gal4tshadlittleeffectondevelopmentaltimingincontrolanimals,180

butinsteadhadanexacerbationofdevelopmentaldelayinrpS13/+animals(Figure3B).ThePG-induced181

expressionofecdysoneattheendofthelarvalperiodiscontrolledbyneuronalsignalstothePG.We182

thereforeexaminedtheeffectofre-expressingrpS13inneuronsusingapan-neuronaldriver,elav-Gal4.We183

foundthatwhileneuronalexpressionofUAS-rpS13inthecontrolanimalsdidnotaffecttiming,itwas184

sufficienttopartiallyrescuethedevelopmentaldelayinrpS13/+larvae.Indeed,thisrescuewassimilarto185

thatseenwitheither20HEfeedingorbystimulationofTORsignalinginthePG(Figure3C).Weconfirmed186

thisresultbyusingasecondpan-neuronaldriver,nSyb-Gal4,whichalsorescuedtimingbyroughlyone187

thirdwhilenotacceleratingtiminginthewildtypeanimals(Figure3D).SincerpS13/+animalsalsohavean188

increasedfinalbodysizephenotype,wemeasuredpupalvolumeinanimalswithneuronalUAS-rpS13189

expression.WefoundthatexpressionofUAS-rpS13intherpS13/+larvaewitheitherelav-Gal4ornsyb-Gal4190

ledtoasignificantreversaloftheincreasedbodysizeseeninrpS13/+animals(Figure3E,F).Thesedata191

pointtoaneuronalrequirementforRpS13inlarvaldevelopmentthataccountsfortheecdysonedefectin192

Minuteanimals.193

Weexaminedwhetherwecouldseeanyglobalchangesineitherthesizeorproteinsynthesislevelsof195

brainsfromrpS13/+animalscomparedtocontrols.However,whenwemeasuredwanderinglarvalbrain196

size(ventralnervecordwidth)ortranslationrates(usingpuromycinlabelling)andfoundnodecreasein197

theMinuteanimals(Figures3G,H).WethereforeexaminedwhethertherequirementforneuronalRpS13198

forproperdevelopmentaltimingmightreflectaroleinaspecificsubsetofneurons,inparticularthose199

knowntoinfluencePGfunction.OneimportantsubsetisapairofbilateralPTTH-expressingneuronsthat200

directlyinnervatethePG.TheserespondtodevelopmentalcuestosecretethepeptidePTTHwhichactson201

thePGtostimulatepeaklevelsofecdysonebiosynthesisattheendofthelarvalstage(McBrayeretal.,202

2007;Shimelletal.,2018).ThePTTHneuronsarealsothemselvesdirectlyregulatedbyanothersubsetof203

neurons(Lgr3-expressingneurons)thatarecontrolledbytissuedamage(Colombanietal.,2015;Garelliet204

al.,2015;Jaszczaketal.,2016).WethereforeexaminedtheeffectsofexpressionofUAS-rpS13inthese205

neuronsusingtheptth-Gal4andlgr3-Gal4drivers.WefoundthatwhenweexpressedUAS-rpS13inPTTH206

neuronswesawnoeffectondevelopmentaltimingincontrolanimalsandasmallrescueofthe207

developmentaldelayinrps13/+animals(Figure4A).ExpressionofUAS-rpS13usingthelgr3-Gal4driver208

hadnoeffectondevelopmentaltimingeitherincontrolorrps13/+animals(Figure4B).Theseresults209

suggestthatexpressionoftheRpS13inneuronsthatcontrolPTTHsignalingdoesnotfullyaccountforthe210

rescueofMinutedevelopmentaltimingthatweobservedwithpan-neuronalRpS13expression.211

ThePGisalsodirectlyinnervatedbyserotonergic(5-HT)neurons(Shimada-NiwaandNiwa,2014).These213

5-HTneuronsarerequiredforproperecdysoneproductionattheendofthelarvalstage,particularlyin214

responsetodietarynutrients.Whenweusedthe5-HTneuronaldrivertrh-Gal4toexpressUAS-rpS13we215

foundthatwecouldreversethedelayindevelopmentseeninrpS13/+animalsbyaboutone-third.This216

recapitulatestheextentoftherescueseenwithpan-neuronalUAS-rpS13expressionandissimilartothat217

seenwitheither20HEfeedingorbyTOR-dependentactivationinthePG(Figure4C).Thereare218

approximately100serotoninergicneuronsinthelarvalbrain,ofwhichthreepairsinnervatethe219

prothoracicglanddirectly(Shimada-NiwaandNiwa,2014).Usinganotherneuronaldriverwhichhasa220

morelimitedexpressionpatternwhichincludesthesethreepairsof5-HTneuronswere-expressedUAS-221

rpS13inrpS13/+andcontrolanimalsandfoundthattimingwasagainrescuedbyapproximatelyone-third222

intherpS13/+animalswhiledevelopmentwasunaffectedincontrolanimals(Figure4D).Thesedata223

suggestaspecificroleforRpS13in5-HTneuronsthatinnervatethePGintheregulationofdevelopmental224

timing.225

WespeculatedthatreducedRpS13levelsin5-HTneuronsmayleadtodecreasedproteinsynthesis,thus227

weexaminedtheeffectsofexpressingtwoknownstimulatorsoftranslation,dMycandrhebincontroland228

rpS13/+animals.WefoundthatbothdMycandrhebover-expressioninrpS13/+animalsrescuedtimingto229

thesameextentasrpS13re-expression,whilehavingminimaleffectsinthecontrolanimals(Figure5A-B).230

RecentstudieshaveidentifiedthetranscriptionfactorXrp1isaneffectorofMinutephenotypes,particularly231

cellcompetition(Jietal.,2019).However,whenweusedRNAitoknockdownXrp1specificallyin5-HT232

neuronswefoundthattherewasnoeffectondevelopmentaltimingintherpS13/+animals(Figure5C).233

ThedevelopmentaldelayinrpS13/+animalsispartiallyreversebyserotonergicexpressionof235

synapticvesicleproteins.236

Wenextfocusedonwhatspecificaspectsofserotonergicneuronalbiologymightbealteredinrps13/+238

larvaetoexplaintheirdelayeddevelopment.Ithasbeenpreviouslyreportedthatthe5-HTneuronsthat239

projecttothePGareregulatedbynutrientavailabilityandthatinlownutrientconditionstheseneuronal240

projectionsarereduced,leadingtodiminished5-HTsignalingtothePGand,asaresult,reducedecdysone241

releaseanddelayeddevelopment.WethereforeinvestigatedwhetherrpS13/+animalsalsoshoweda242

reductioninaxonalprojectionsintothePG.Westained5-HTneuronsinbothcontrolandrpS13/+animals243

andinbothcases,weobservedprojectionstothePG.Whenwecountedboutonnumbersfrom5-HT244

neuronsthatprojectedtothePGwefoundnodifferencebetweencontrolsandrpS13/+animalssuggesting245

noalterationsin5-HTneuronaloutgrowth(Figure6A-B).Itispossiblethatthe5-HTneuronsinrpS13/+246

larvaehavereducedactivity,leadingtodecreasedstimulationofecdysoneproductioninthePG.Toexplore247

thispossibility,weexaminedtheeffectsofgeneticactivationoftheseneuronsbyexpressingtheNaChBac248

sodiumchannel,whichleadstodepolarizationandactivationofneurons.However,wefoundthat249

expressionofUAS-NaChBacdidnotreversethedelayindevelopmentseeninrpS13/+animals(Figure6C).250

Wealsofoundthatexpressionoftryptophanhydroxylase(Trh),akeyenzymeinthesynthesisof5-HT,also251

didnotalterthedelayeddevelopmentinrpS13/+larvae(Figure6D).252

Akeyprocessinneuronsisthevesiclemediatedsecretionofneurotransmittersandneuropeptides.We254

thereforeexaminedwhethersecretionmightbealteredinthe5-HTneuronsofrpS13/+animals.Todothis255

weexpressedasecretedformofGFP(sGFP)inthe5-HTneuronsofbothcontrolandrpS13/+animals.In256

controllarvaewesawbrightGFPstaininginaxonsandintheterminiofneuronsthatinnervatethePG257

(Figure6E,F).However,thissGFPexpressionwasstronglysuppressedinrpS13/+animals(Figure6E,F)258

suggestingthattheyhaveasecretorydefectintheirserotonergicneurons(Figure6F).Incontrastwith259

theseeffectswithsGFP,wefoundthatexpressionofunmodifiedGFPwassimilarincontrolsandrpS13/+260

animals,indicatingthattheMinuteanimalsdonotshowageneraldisruptionoftransgeneexpression261

(FigureS3).262

SincewesawadefectinthesecretoryprocessofrpS13/+animalswewantedtofurtherinvestigatethisby264

lookingatproteinsrequiredinsynapticsecretion.Thesynthesis,axonaltransport,andsynapticreleaseof265

vesiclecontentsiscontrolledbyanumberofproteins,includingmembersoftheSNAPReceptor(SNARE)266

complex.Interestingly,previousstudieshaveshownthatthesesynapticvesicleproteinsneedtobe267

continuallysynthesizedforproperneuronalfunction(Truckenbrodtetal.,2018)andthattheirsynthesisis268

oftentranslationallyregulated(Batistaetal.,2017;DalyandZiff,1997).Wethereforeexaminedtheeffects269

ofexpressingsynapticvesicleproteinsinserotonergicneurons.Strikingly,wefoundthatexpressionofany270

ofoneofthreesynapticvesicleproteins-nsyb,syt1orSNAP29-wasabletorescuedevelopmentaldelay271

seeninrpS13/+animals,tothesamemagnitudeasthatseenwithexpressionofUAS-S13(Figure7A-C).We272

wantedtoseeifthisphenotypewasuniquetorpS13/+animalsbyexaminingtwootherMinutes,rpS24/+273

andrpS26/+.LikerpS13/+,thesetwoadditionalMinutesalsoshowadelayindevelopment,albeitslightly274

weaker.However,aswithrpS13/+animals,wesawthatthisdelayintimingwasalsorescuedbyabouta275

third,byoverexpressionofnsyb(Figure7D-E).276

DISCUSSION280

SincethediscoverythatMinutesaremutantsforRps,aprevailinghypothesis(the“balancehypothesis”)282

hasbeenthattheirdevelopmentalphenotypesresultfromanimbalanceofcytoplasmicribosomalprotein283

concentrationsleadingtoincompleteribosomalsubunitassemblyandreducedoveralltranslation284

(Marygoldetal.,2007).However,ourdataindicatethatdespiteallcellsinaMinuteanimalbeing285

heterozygousforanRpgene,thisdoesnotresultinawhole-bodydecreaseinribosomenumbersorprotein286

synthesis.Rather,thedevelopmentaldelayphenotypesinMinuteanimalslikelyresultfrommoreselective287

effectsofRploss.Hereweidentify5-HTneuronsasoneimportantcell-typeinwhichRpfunctionis288

requiredforproperlarvaldevelopmentaltiming.289

InterestinglywefoundthattheeffectofRpS13in5-HTneuronscouldaccountfor30-40%ofthetotal291

developmentaldelayinrps13/+animals.ThissuggeststhatRpS13containingribosomesmaybeplaying292

rolesinothertissuestocontroldevelopmentaltiming.Indeed,previousstudieshavealsoshowntissue-293

selectiveeffectsofRpsondevelopment.Forexample,re-expressionofrpS6inprothoracicglandscould294

partiallyrescuetheanimal’sdevelopmentaldelayinrpS6/+larvae(Linetal.,2011),althoughwedidnot295

findsuchaPGspecificroleforrpS13.Ithasalsobeenshownthatdisc-specificknockdownofdilp8in296

rpS3/+animalscanpartiallyreversetheirlarvaldevelopmentaldelay(Akaietal.,2021).Onepossibility,297

therefore,isthatRpfunctioninacombinationoftissuesisrequiredforproperendocrinecontrolof298

developmentaltiming.Furtherstudiesarerequiredtoseeifthesetissue-specificcontributionsaresimilar299

forallRpsormayshowsomeheterogeneitydependingontheRpbeingexamine.The5-HTneuronsthat300

innervatethePGhavebeenshowntobeparticularlyimportantincouplingnutritiontothecontrolof301

ecdysoneanddevelopmentaltiming(Shimada-NiwaandNiwa,2014).Giventhatthecontrolofribosome302

synthesisisaconservedfunctionofnutrient-signalingpathways,ourresultspinpointingakeyroleforRps303

inthefunctionoftheseneuronssuggestonewaythatnutrientsmaymodulatethe5-HTcontrolof304

ecdysone.305

OurworksuggeststhattherequirementforRpS13in5-HTneuronsmayreflectaroleinthecontrolof307

vesicle-mediatedsecretion.WesawthatexpressionofasecretedformofGFPwaslostontheaxonsand308

terminiof5-HTneurons,andwesawthatexpressingdifferentvesicleproteinsintheseneuronsinrps13/+309

animalswasabletorescuethedevelopmentaldelaytothesameextentasUAS-rpS13re-expression.How310

mightlossofS13affectsecretionin5-HTneurons?Oneplausiblemechanismisthroughreduced311

translation.Thisnotionissupportedbyourresultsshowingthatserotonergicexpressionoftwoconserved312

inducersodproteinsynthesis,MycandRheb,couldrescuethedelayindevelopmentseeninrp13/+313

animals.Currentmodelssuggestthatrp/+phenotypesarisefromeitherheterogeneousribosomesor314

selectivemRNAtranslationalcontrol(Dinman,2016;GenuthandBarna,2018a,b;Khajuriaetal.,2018;315

MillsandGreen,2017).WesawapartialrescueintimingwithUAS-nsyboverexpressioninthreedifferent316

minutes–rps13/+,rps24/+andrpS26/+-suggestingperhapsthatribosomeheterogeneitymaynotexplain317

the5-HTneuronalcontributiontotheMinutephenotypes.Alternatively,areductionofRpS13inthese318

neuronsmayresultinselectivechangesinmRNAtranslationofproteinsinvolvedincontrollingneuronal319

secretoryfunction.Inmammaliansystems,synapticvesicleproteinsarerequiredtobecontinually320

synthesizedforproperneuronalfunction(Truckenbrodtetal.,2018).Moreover,theirexpressionhasbeen321

showntobepost-transcriptionallyregulated(DalyandZiff,1997).Hence,thevesicleproteinsthemselves322

maybesubjecttoselectivetranslationalregulation.ItcouldbethatSNAREcomplexmRNAssharea323

commonfeatureintheir5’or3’UTRregionsthatleadstothembeingtranslationallyregulatedinsimilar324

ways.Indeed,UTR-mediatedregulationoftranslationisaprevalentmodeofcontrollinggeneexpressionin325

neurons(Blairetal.,2017).Thesubcellularcontroloftranslationisalsoimportantinneurons(Bieveretal.,326

2019;Cionietal.,2018;Holtetal.,2019).Here,localtranslationinregionssuchascellbodies,axonsand327

terminiallowsforselective,spatialcontroloverproteinsynthesis.Forexample,ithasbeenfoundthat328

SNAP25synapticvesicleproteinscanbelocallytranslatedinaxons(Batistaetal.,2017).Thus,itispossible329

thatthedefectsinMinuteanimalsmayoccurduetoalterationsinlocaltranslationin5-HTneurons.330

Ourfindingsmayalsoberelevanttohumanbiology.Ithasbeenobservedthatmanyneurologicaldisorders332

arisefromabnormalsynapticvesicleformationandfunction.These“synaptopathies”includedisorders333

suchasschizophrenia,AHDH,andautism,andareoftenassociatedwithaberrantmRNAtranslation(Bagni334

andZukin,2019;Chenetal.,2019).Interestinglysomeribosomopathiesinhumanshavealsobeenshown335

topresentwithvariousneurologicaldisorderssuchasmicrocephalyandmentalretardation.Inparticular,336

mutationsinrpS23andrpL10havebeenshowntobeassociatedwithautismspectrumdisorder(Klaucket337

al.,2006;Paolinietal.,2017).Perhapscertainribosomopathiesthatpresentmentaldisorderscouldinpart338

beduetodefectinvesiclefunctionand,asaresult,disruptedsynapticfunction.339

MATERIALSANDMETHODS341

Flystrainsandhusbandry343

Flieswereraisedonfoodwiththefollowingcomposition:150gagar,1600gcornmeal,770gtorulayeast,344

675gsucrose,2340gD-glucose,240mlacidmixture(propionicacid/phosphoricacidper34litersof345

water).Forallexperimentslarvaeweremaintainedat25°C,unlessotherwiseindicated.Unlessotherwise346

statedtheflystrainsusedwereobtainedfromtheBloomingtonStockCenter(BDSC):w1118,yw,347

yw*;P[w[+mC]=lacW]rpS13[1]/cyoGFP(2246),UAS-rpS13GFP/cyo(GiftfromS.Brogna),P0206-GAL4(Gift348

fromC.Mirth),esgts-GAL4,elav-GAL4(GiftfromF.Buldoc),nsyb-GAL4(51635),ptth-GAL4(GiftfromM.349

O’Connor),trh-GAL4(38388),GMR29H01-GAL4(47343),UAS-dmyc-dp110(25914),UAS-rheb(9689),UAS-350

NaChBac(9469),UAS-trh(27638),UAS-nsybGFP(6921,6922),UAS-syt1(6925),UAS-SNAP29(56817),351

P0206-GAL4(GiftfromM.O’Connor),lgr3-GAL4(66683),UAS-Xrp1RNAi(34521),w1118;PBac[w[+m]]=WH]352

rpS24[f06717]/cyoGFP(19002),P[ry[+t7.2]=PZ]rpS26[04553]/cyoGFP(12048),UAS-sGFP(GiftfromM.353

Gonzlez-Gaitan).354

MeasurementofDrosophiladevelopmentandbodysize.356

Formeasuringdevelopmenttimingtopupalstage,newlyhatchedlarvaewerecollectedat24hrAELand357

placedinfoodvials(50larvaepervial)andkeptat25oC.Thenumberofpupaewascountedtwiceaday.358

Foreachexperimentalcondition,aminimumoffourreplicateswasusedtocalculatethemeantimeto359

developintopupae.Tomeasurepupalvolume,pupaewereimagedusingaZeissDiscovery.V8360

StereomicroscopewithAxiovisionimagingsoftware.Pupallengthandwidthweremeasured,andpupal361

volumewascalculatedusingtheformula,volume=4/3π(L/2)(l/2)2.Aminimumoffourreplicateswas362

usedtocalculatethemeanvolumeforeachgenotype.363

QuantitativePCR.365

TotalRNAwasextractedfromlarvae(groupsof10)usingTRIzolaccordingtomanufacturer’sinstructions366

(Invitrogen;15596–018).RNAsampleswerethensubjectedtoDNasetreatmentaccordingto367

manufacturer’sinstructions(Ambion;2238G)andreversetranscribedusingSuperscriptII(Invitrogen;368

100004925).ThegeneratedcDNAwasusedasatemplatetoperformqRT–PCRs(ABI7500realtimePCR369

systemusingSyBrGreenPCRmix)usingspecificprimerpairs.PCRdatawerenormalizedtoeitheractinor370

alpha-tubulinlevels.Thefollowingprimerswereused:371

RpS13forward:AGGCAGTGCTCGACTCGTAT373

RpS13reverse:TTCCCGAGGATCTGTACCAC374

Beta-tubulinforward:ATCATCACACACGGACAGG376

Beta-tubulinreverse:GAGCTGGATGATGGGGAGTA377

Actin5Cforward:GAGCGCGGTTACTCTTTCAC379

Actin5Creverse:GCCATCTCCTGCTCAAAGTC380

18SrRNAforward:CCTGCGGCTTAATTTGACTC382

18SrRNAreverse:ATGCACCACCACCCATAGAT383

28SrRNAforward:TGCCAGGTAGGGAGTTTGAC385

28SrRNAreverse:CAAGTCAGCATTTGCCCTTT386

spookierforward:TATCTCTTGGGCACACTCGCTG388

spookierreverse:GCCGAGCTAAATTTCTCCGCTT389

phantomforward:GGATTTCTTTCGGCGCGATGTG391

phantomreverse:TGCCTCAGTATCGAAAAGCCGT392

Puromycinassay394

Groupsof10wanderinglarvaeorearliertimepointlarvaewereinvertedinSchneider’smediaandthen395

transferredtoEppendorftubescontainingmediaplus5µg/mlpuromycin(Sigma),6differentreplicates396

wereusedforFigure1F.Thelarvalsampleswerethenlefttoincubateonamutatorfor40minutesatroom397

temperature.Followingincubation,theinvertedlarvaeweresnapfrozenforwesternblotanalyses.For398

experimentsonlarvalbrains,invertedlarvaewereplacedinice-coldPBSafterincubationwithpuromycin,399

andthebrainswereisolatedandlysedforwesternblotanalyses.400

WesternBlotting402

Wholeinvertedlarvaeorisolatedbrainswerelysedwithabuffercontaining20mMTris-HCl(pH8.0),137403

mMNaCl,1mMEDTA,25%glycerol,1%NP-40andwithfollowinginhibitors:50mMNaF,1mMPMSF,1404

mMDTT,5mMsodiumorthovanadate(Na3VO4)andproteaseinhibitorcocktail(Rochecat.no.405

04693124001)andphosphataseinhibitor(Rochecat.no.04906845001),accordingtothemanufacturer’s406

instruction.ProteinconcentrationsweremeasuredusingtheBio-RadDcProteinAssaykitII(5000112).407

Foreachexperiment,equalamountsofproteinlysatesforeachsample(40µg)wereresolvedbySDS-PAGE408

andelectrotransferredtoanitrocellulosemembrane.BlotswerethenbrieflystainedwithPonceauSto409

visualizetotalproteinandthensubjectedtowesternblotanalysiswithspecificantibodies.Proteinbands410

werethenvisualizedbychemiluminescence(enhancedECLsolution,PerkinElmer).Primaryantibodies411

usedwereanti-puromycin(3RH11)antibody(1:1000,Kerafast,Boston,USA,cat.no.EQ0001),anti-412

eIF2alpha(1:1000,AbCam#26197).SecondaryantibodieswerepurchasedfromSantaCruz144413

Biotechnology(sc-2030,2005,2020,1:10,000).414

20-HydroxyecdysoneFeeding416

Newlyhatchedw1118andrpS13/+larvaewerecollectedat24hrAELandplacedinfoodvials(50larvaeper417

vial)supplementedeitherwith20-hydroxyecdysone(Sigma-AldrichCASnumber5289-74-7)orequal418

volumeof95%ethanolforcontrols.20-hydroxyecdysonewasdissolvedin95%ethanoltoafinal419

concentrationof0.3mg/ml.Aminimumoffourreplicateswasusedtocalculatethemeanvolumeforeach420

genotype.421

Immunostainingandmicroscopy423

Drosophilalarvaewerefixedin8%paraformaldehyde/PBSatroomtemperaturefor30min.Afterblocking424

for2hin1%BSAinPBS/0.1%Triton-X100,invertedlarvaewereincubatedovernightinanti-5HT(Sigma-425

AldrichAB125)andanti-shroudantibody(giftfromR.Niwa)(1:2000,1:500dilutions).Primaryantibody426

stainingwasdetectedusingAlexaFluor488(MolecularProbes)goat-antirabbitsecondaryantibodies.427

Brainswithattachedprothoracicglandswerethendissectedoutandmountedoncoverslipsusing428

mountingmedia(Vectashield).429

Boutonquantification431

Brainandattachedprothoracicglandsweredissectedfromw1118andrpS13/+larvaeduringthewandering432

L3stageandwerestainedwithanti-5HTandanti-shroudantibodies.ConfocalZstackimageswere433

acquiredand5-HTboutonsthatoverlappedthePG(basedonanti-shroudstaining)werecounted.The434

valuesforeachindividualbrainwererecordedandaveragesforeachgenotypewerecalculated.435

Statistics437

Forallexperiments,errorbarsrepresentstandarderrorofmean(SEM).DatawereanalyzedbyStudentst-438

testorMann-WhitneyUtest.AllstatisticalanalysisanddataplotswereperformedusingPrismsoftware.In439

allfigures,statisticallysignificantdifferencesarepresentedas*andindicatep<0.05.440

FIGURELEGENDS442

Figure1.DevelopmentaldelayofrpS13/+animalsisnotduetoadecreaseinribosomenumbersor444

reducedtranslationrates.445

(A)Developmentaltimingfromlarvalhatchingtopupationofw1118andrpsS13/+animals,n=147and170447

respectively.rpS13/+animalsareonaverage35hoursdelayedwhencomparedtotheirwild-typecontrols.448

Dataarepresentedas+/-SEM.*p<0.05,Mann-WhitneyUtest.449

(B)RepresentativeimagesofW1118andrpS13/+pupae.Scalebar,1mm.450

(C)ChangeinpupalvolumeofrpS13/+(n=212)whencomparedtow1118controls(n=229).rpS13/+451

animalsgrowonaverage11%largerthancontrols.Dataarepresentedas+/-SEM.*p<0.05,Mann-452

WhitneyUtest.453

(D)Transcriptlevelsof18Sand28SrRNAinw1118andrpsS13/+shownoreductioninribosomalrRNAin454

rpsS13/+heterozygotes.mRNAwasisolatedfromthirdinstarwanderinglarvae.TotalRNAwasisolated455

andmeasuredbyqRT-PCR,n=4independentsamplespergenotype.Dataarepresentedas+/-SEM.p>456

0.05,Student’st-test.457

(E)Relativetranslationratesbasedonquantificationofpuromycinstaining,n=6independentsamplesper458

genotype.Dataarepresentedas+/-SEM.p>0.05,Student’st-test.459

(F)PuromycinlabellingassayshowsnodifferenceinrpS13/+translationalratesinthirdinstarwandering460

larvaewhencomparedtocontrols.Left,PonceauSstainingshowingtotalprotein.Right,anti-puromycin461

andanti-eIF2∝(loadingcontrol)immunoblots.462

(G)Relativeproteinconcentrationlevelsfromthirdinstarwanderingw1118andrpsS13/+larvae.463

Absorbancewasmeasuredat465nmusingtheBradfordassay,n=5independentsamplespergenotype.464

Dataarepresentedas+/-SEM.p>0.05,Student’st-test.465

Figure2.DelayeddevelopmentinrpS13/+animalsisduetoimpairedecdysoneproduction.467

(A)TranscriptlevelsoftwoHalloweengenes,phantomandspookierexpressionaredelayedinrpS13/+469

larvaewhencomparedtowild-typecontrols.mRNAwasisolatedfromthirdinstarlarvaeevery24hours470

beginningwiththe96hoursAELtimepointuntilpupationofeachrespectivegenotype.TotalRNAwas471

isolatedandmeasuredbyqRT-PCR,n=4independentsamplespergenotype.Dataarepresentedas+/-472

SEM.473

(B)Ecdysone(20HE)wassupplementedintofoodata0.3mg/mLconcentrationinbothrpS13/+andw1118474

animals,n=92,n=116,respectively.20HEpartiallyrescuedrpS13/+developmentaldelayby10hours475

(~33%).Controlsforbothw1118andrpS13/+werefedthesameconcentration(0.3mg/mL)of95%ethanol476

infood,n=142,n=148,respectively.Dataarepresentedas+/-SEM.*p<0.05,Mann-WhitneyUtest.477

(C)UAS-rhebexpressionintheprothoracicglandusingP0206-Gal4partiallyrescuesthedevelopmental478

delayofrpS13/+larvaeby15hours(~36%).Nodifferenceisseenbetween+/+controlsandUAS-rpS13479

overexpression.+/+n=152,UAS-rheb/+n=157,rpS13/+n=151,UAS-rheb/rpS13n=144.Dataare480

presentedas+/-SEM.*p<0.05,Mann-WhitneyUtest.481

Figure3.rpS13re-expressioninneuronspartiallyreversestherpS13/+developmentaldelay.484

(A)rpS13re-expressionintheprothoracicglandusingP0206-Gal4doesnotrescuethedevelopmental486

delayofrpS13/+larvae.Nodifferenceisseenbetween+/+controls(n=95)andUAS-rpS13overexpression487

(n=95)orbetweenrpS13/+(n=98)andrpS13,UAS-rpS13/+(n=87)animals.Dataarepresentedas+/-488

SEM.p>0.05,Mann-WhitneyUtest.489

(B)rpS13re-expressionintheimaginaldiscsusingesgts-Gal4doesnotrescuethedevelopmentaldelayof490

rpS13/+larvae.Thereisaminorincreaseindevelopmentaltimingbetween+/+controls(n=96)andUAS-491

rpS13overexpression(n=29)andanenhanceddelaybetweenrpS13/+(n=99)andrpS13,UAS-rpS13/+(n492

=86)animals.Dataarepresentedas+/-SEM.*p<0.5,Mann-WhitneyUtest.493

(C)Pan-neuronalexpressionofrpS13inrpS13/+animalsusingelav-Gal4partiallyrescuesthe494

developmentaldelayby10hours(~33%).+/+n=180,rpS13/+n=167,UAS-rpS13/+n=182,rpS13,UAS-495

rpS13/+n=226.Dataarepresentedas+/-SEM.*p<0.5,Mann-WhitneyUtest.496

(D)ExpressionofrpS13usingasecondpan-neuronaldriver,nSyb-Gal4alsopartiallyrescuesrpS13/+497

developmentaldelayby10hours(~33%).+/+n=182,rpS13/+n=180,UAS-rpS13/+n=190,rpS13,UAS-498

(E)Pan-neuronalexpressionofrpS13inrpS13/+animalsusingelav-Gal4partiallyrestoresrps13/+500

overgrowthphenotype.+/+n=313,rpS13/+n=468,UAS-rpS13/+n=456,rpS13,UAS-rpS13/+n=501

475.Dataarepresentedas+/-SEM.*p<0.05,Mann-WhitneyUtest.502

(F)Pan-neuronalexpressionofrpS13inrpS13/+animalsusingnSyb-Gal4partiallyrestoresrps13/+503

overgrowthphenotype.+/+n=588,rpS13/+n=484,UAS-rpS13/+n=586,rpS13,UAS-rpS13/+n=504

536.Dataarepresentedas+/-SEM.*p<0.05,Mann-WhitneyUtest.505

(G)Ventralnervecord(VNC)width(µm)wasusedtocomparebrainsizesinthirdinstar,wanderinglarvae506

of+/+controls(n=12)andrpS13/+(n=12)animals.Dataarepresentedas+/-SEM.p>0.05,Student’st-507

test.508

(H)Puromycinlabellingassayshowsnodecrease,butratherasmallincreaseinrpS13/+translationrates509

inthirdinstarwanderinglarvaebrainswhencomparedtocontrols,+/+.Left,PonceauSstainingshowing510

totalprotein.Right,anti-puromycinimmunoblot.511

Figure4.rpS13re-expressionintheserotonergicneuronsthatinnervatetheprothoracicgland514

partiallyrescuesrpS13/+developmentaldelay.515

(A)rpS13expressionintheptthneuronsusingptth-Gal4hasaverymildeffectonthedevelopmentaldelay517

ofrpS13/+larvae.Nodifferenceisseenbetween+/+controls(n=233)andUAS-rpS13overexpression(n=518

288)orbetweenrpS13/+(n=282)andrpS13,UAS-rpS13/+(n=282)animals.Dataarepresentedas+/-519

SEM.*p<0.05,Mann-WhitneyUtest.520

(B)rpS13expressionintheLgr3neuronsusingLgr-GalhasnoeffectondevelopmentaltimingofrpS13/+521

orcontrolanimals.+/+n=192rpS13/+n=176,UAS-rpS13/+n=177,rpS13,UAS-rpS13/+n=166.Data522

arepresentedas+/-SEM.*p<0.05,Mann-WhitneyUtest.523

(C)rpS13expressionintheserotonergicneuronsusingTrh-Gal4partiallyrescuesdevelopmentaldelayof524

rpS13/+larvaeby14hours(~36%).+/+n=241rpS13/+n=176,UAS-rpS13/+n=234,rpS13,UAS-525

(D)rpS13expressioninasubsetofserotonergicneuronsthatdirectlyinnervatetheprothoracicgland,527

usingGMR29H01-Gal4partiallyrescuesdevelopmentaldelayofrpS13/+larvaeby9hours(~30%).+/+n=528

225rpS13/+n=196,UAS-rpS13/+n=192,rpS13,UAS-rpS13/+n=248.Dataarepresentedas+/-SEM.*p529

<0.05,Mann-WhitneyUtest.530

Figure5.Stimulationofproteinsynthesis,butnotXrp1knockdownintheserotonergicpartially532

rescuesrpS13/+developmentaldelay.533

(A)dmycover-expressionintheserotonergicneuronsusingTrh-Gal4partiallyrescuesdevelopmental535

delayofrpS13/+larvaeby13hours(~31%).+/+n=198,UAS-dmyc/+n=196,rpS13/+n=173,rpS13,536

UAS-dmyc/rpS13n=186.Dataarepresentedas+/-SEM.*p<0.05,Mann-WhitneyUtest.537

(B)rhebover-expressionintheserotonergicneuronsusingTrh-Gal4partiallyrescuesdevelopmentaldelay538

ofrpS13/+larvaeby14hours(~33%).+/+n=172,UAS-rheb/+n=235,rpS13/+n=132,UAS-dmyc/rpS13539

n=185.Dataarepresentedas+/-SEM.*p<0.05,Mann-WhitneyUtest.540

(C)RNAiknockdownofXrp1inserotonergicneuronsusingTrh-Gal4hasnoeffectondevelopmentaltiming541

ofrpS13/+larvae.+/+n=200,UAS-Xrp1RNAi/+n=160,rpS13/+n=179,UAS-Xrp1RNAi/rpS13n=203.542

Dataarepresentedas+/-SEM.*p<0.05,Mann-WhitneyUtest.543

Figure6.rpS13/+animalsshownormalaxonalprojectionsintotheprothoracicgland,howeverare545

defectiveinsecretoryfunction.546

(A)Fluorescentconfocalimagesofrepresentative+/+andrpS13/+prothoracicglandsshowinginnervation548

ofserotonergicneurontermini.Anti-5-HTPislabelledwithGFP,whiletheprothoracicglandislabelledin549

redusingananti-shroudantibody.NucleiofboththebrainandprothoracicglandarestainedwithHoechst.550

Scalebars,50µm.551

(B)Brainsof+/+(n=11)andrpS13/+(n=11)wanderinglarvaeweredissectedandstainedfor5-HT.552

Boutonsofserotonin(5-HT)neuronterminithatoverlaptheprothoracicglandwerecounted.Dataare553

presentedas+/-SEM.p>0.05,Student’st-test.554

(C)UAS-NaChBacexpressionintheserotonergicneuronsusingtrh-Gal4doesnotrescuethedevelopmental555

delayofrpS13/+larvae.Nodifferenceisseenbetween+/+controls(n=154)andUAS-NaChBac556

overexpression(n=135)orbetweenrpS13/+(n=125)andUAS-NaChBac/rpS13(n=108)animals.Data557

arepresentedas+/-SEM.p>0.05,Mann-WhitneyUtest.558

(D)UAS-trhexpressionintheserotonergicneuronsusingtrh-Gal4doesnotrescuethedevelopmentaldelay559

ofrpS13/+larvae.+/+n=93,rpS13/+n=71,UAS-trh/rpS13n=91.Dataarepresentedas+/-SEM.*p<560

0.05,Mann-WhitneyUtest.561

(E)Fluorescentconfocalimagesofrepresentativetrh>sGFP/+andtrh>sGFP/rpS13wanderingL3larvae562

brains.Anti-5-HTisshowninred,whilethesecretedGFPisgreen.Imagesareof5-HTneuronsaxons563

projectingintothePG.564

(F)Fluorescentconfocalimagesofrepresentativetrh>sGFP/+andtrh>sGFP/rpS13prothoracicglands565

showinginnervationofserotonergicneuronterminiintothePG.Nucleioftheprothoracicglandarestained566

withHoechst(magenta)whilesecretedGFPfrom5-HTneuronsareingreen.Scalebars,50µm.567

Figure7.ExpressionofindividualSNAREcomplexproteinspartiallyrescuesrpS13/+569

developmentaldelayaswellastheotherrpS24/+andrpS26/+developmentaldelay.570

(A)nsybover-expressionintheserotonergicneuronsusingTrh-Gal4partiallyrescuesdevelopmentaldelay572

ofrpS13/+larvaeby12hours(~30%).+/+n=182,UAS-nsyb/+n=180,rpS13/+n=190,rpS13,UAS-573

nsyb/rpS13n=176.Dataarepresentedas+/-SEM.*p<0.05,Mann-WhitneyUtest.574

(B)syt1over-expressionintheserotonergicneuronsusingTrh-Gal4partiallyrescuesdevelopmentaldelay575

ofrpS13/+larvaeby17hours(~43%).+/+n=168,UAS-syt1/+n=151,rpS13/+n=137,rpS13,UAS-576

syt1/rpS13n=136.Dataarepresentedas+/-SEM.*p<0.05,Mann-WhitneyUtest.577

(C)SNAP29over-expressionintheserotonergicneuronsusingTrh-Gal4partiallyrescuesdevelopmental578

delayofrpS13/+larvaeby15hours(~39%).+/+n=168,UAS-SNAP29/+n=140,rpS13/+n=137,rpS13,579

UAS-SNAP29/rpS13n=135.Dataarepresentedas+/-SEM.*p<0.05,Mann-WhitneyUtest.580

(D)nsybover-expressionintheserotonergicneuronsusingTrh-Gal4partiallyrescuesdevelopmentaldelay581

ofrpS24/+larvaeby6hours(~26%).+/+n=168,rpS24/+n=105,UAS-nsyb/rpS24n=124.Dataare582

presentedas+/-SEM.*p<0.05,Mann-WhitneyUtest.583

(E)nsybover-expressionintheserotonergicneuronsusingTrh-Gal4partiallyrescuesdevelopmentaldelay584

ofrpS26/+larvaeby11.5hours(~42%).+/+n=168,rpS13/+n=98,rpS26,UAS-nsyb/rpS26n=120.Data585

arepresentedas+/-SEM.*p<0.05,Mann-WhitneyUtest.586

SupplementalFigures.588

FigureS1590

(A).TranscriptlevelsoftherpS13heterozygotesarereducedtohalfthewild-typelevelspresentin591

controls.mRNAwasisolatedfromthirdinstarwanderinglarvae.TotalRNAwasisolatedandmeasuredby592

qRT-PCR,n=4independentsamplespergenotype.Dataarepresentedas+/-SEM.*p<0.05,Student’st-593

test.594

(B)RelativelarvalsizeofW1118andrpS13/+animalsthroughoutdevelopment.Larvalareawasmeasured595

every24hoursafterhatchinguntilwanderingandrecordedaspixelarea.Dataarepresentedas+/-SEM.596

(C)Larvalweight(mg)atwanderingL3stageofW1118andrpS13/+animals.Larvaeweremeasuredin597

groupsof10,n=10independentsamplespergenotype.Dataarepresentedas+/-SEM.*p<0.05,Student’s598

t-test.599

(D)Mouthhookmovementsrecordedinoneminuteoffeedingfor96-hourL3larvaeofW1118andrpS13/+600

animals.W1118n=20,rpS13/+n=20.Dataarepresentedas+/-SEM.*p<0.05,Student’st-test.601

FigureS2603

Puromycinlabellingassayshowsnodecrease,butratherasmallincreaseinrpS13/+translationalratesin604

wholelarvaewhencomparedtoagematchedcontrols.605

(A)96-hourW1118andrpS13/+larvae.Left,PonceauSstainingshowingtotalprotein.Right,606

anti-puromycinimmunoblot.607

(B)120-hourW1118andrpS13/+larvae.Left,PonceauSstainingshowingtotalprotein.Right,anti-608

puromycinimmunoblot.609

FigureS3611

Fluorescentconfocalimagesofrepresentativetrh>GFP/+andtrh>GFP/rpS13wanderingL3larvalbrains.612

NucleiofneuronsstainedwithHoechst(magenta)while5-HTneuronsarelabelledwithGFP.Scalebars,613

50µm.614

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