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Serotonergicneuronribosomesregulatetheneuroendocrinecontrolof1
Drosophiladevelopment.2
3
LisaP.Deliu,DeeshpaulJadir,AbhishekGhosh,SavrajS.Grewal4
5
ClarkHSmithBrainTumourCentre,ArnieCharbonneauCancerInstitute,AlbertaChildren’sHospital6
ResearchInstitute,andDepartmentofBiochemistryandMolecularBiologyCalgary,UniversityofCalgary,7
AlbertaT2N4N1,Canada.8
9
Correspondence:[email protected]
11
ABSRACT12
13
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
27
INTRODUCTION28
29
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(which was not certified by peer review) is the author/funder.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
2
determinedsolelybyprocessesthatgoverncell-autonomousgrowth,butalsobyinter-organ36
communicationtoensurecoordinatedgrowthanddevelopmentacrossalltissuesandorgans(Boulanetal.,37
2015;DroujinineandPerrimon,2016;Grewal,2012).Hence,tissuespecificchangesinribosomefunction38
havethepotentialtomediatenon-autonomouseffectsonwhole-bodyphysiologytocontrolorganismal39
development.40
41
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
64
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
(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
3
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
84
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
102
Hereweprovidefurtherevidencefortissue-specificeffectsofRpinthecontroloflarvaldevelopment.We103
describehowlossofRpfunctionresultsindefectsinvesicle-mediatedsecretionspecificallyinthe5-HT104
neuronsthatinnervatethePGleadingtodevelopmentaldelayinMinuteanimals.105
(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
4
106
RESULTS107
108
Rps13/+animalsdonotshowaglobaldecreaseinribosomelevelsorproteinsynthesis109
110
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
127
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
(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
5
suggeststhattheMinutedelayeddevelopmentisnotduetoagloballossofribosomenumbersor140
translationalcapacity.141
142
rpS13/+animalsshowadefectinecdysonesignalling143
144
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
164
5-HTneuronalrpS13isrequiredforproperdevelopmentaltiming165
166
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
(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
6
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
194
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
(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
7
suggestthatexpressionoftheRpS13inneuronsthatcontrolPTTHsignalingdoesnotfullyaccountforthe210
rescueofMinutedevelopmentaltimingthatweobservedwithpan-neuronalRpS13expression.211
212
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
226
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
234
ThedevelopmentaldelayinrpS13/+animalsispartiallyreversebyserotonergicexpressionof235
synapticvesicleproteins.236
237
Wenextfocusedonwhatspecificaspectsofserotonergicneuronalbiologymightbealteredinrps13/+238
larvaetoexplaintheirdelayeddevelopment.Ithasbeenpreviouslyreportedthatthe5-HTneuronsthat239
projecttothePGareregulatedbynutrientavailabilityandthatinlownutrientconditionstheseneuronal240
projectionsarereduced,leadingtodiminished5-HTsignalingtothePGand,asaresult,reducedecdysone241
releaseanddelayeddevelopment.WethereforeinvestigatedwhetherrpS13/+animalsalsoshoweda242
reductioninaxonalprojectionsintothePG.Westained5-HTneuronsinbothcontrolandrpS13/+animals243
andinbothcases,weobservedprojectionstothePG.Whenwecountedboutonnumbersfrom5-HT244
(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
8
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
253
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
263
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
277
278
279
(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
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DISCUSSION280
281
SincethediscoverythatMinutesaremutantsforRps,aprevailinghypothesis(the“balancehypothesis”)282
hasbeenthattheirdevelopmentalphenotypesresultfromanimbalanceofcytoplasmicribosomalprotein283
concentrationsleadingtoincompleteribosomalsubunitassemblyandreducedoveralltranslation284
(Marygoldetal.,2007).However,ourdataindicatethatdespiteallcellsinaMinuteanimalbeing285
heterozygousforanRpgene,thisdoesnotresultinawhole-bodydecreaseinribosomenumbersorprotein286
synthesis.Rather,thedevelopmentaldelayphenotypesinMinuteanimalslikelyresultfrommoreselective287
effectsofRploss.Hereweidentify5-HTneuronsasoneimportantcell-typeinwhichRpfunctionis288
requiredforproperlarvaldevelopmentaltiming.289
290
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
306
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
(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
10
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
331
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
340
MATERIALSANDMETHODS341
342
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
(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
11
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
355
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
364
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
372
RpS13forward:AGGCAGTGCTCGACTCGTAT373
RpS13reverse:TTCCCGAGGATCTGTACCAC374
375
Beta-tubulinforward:ATCATCACACACGGACAGG376
Beta-tubulinreverse:GAGCTGGATGATGGGGAGTA377
378
Actin5Cforward:GAGCGCGGTTACTCTTTCAC379
Actin5Creverse:GCCATCTCCTGCTCAAAGTC380
381
18SrRNAforward:CCTGCGGCTTAATTTGACTC382
18SrRNAreverse:ATGCACCACCACCCATAGAT383
384
(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
12
28SrRNAforward:TGCCAGGTAGGGAGTTTGAC385
28SrRNAreverse:CAAGTCAGCATTTGCCCTTT386
387
spookierforward:TATCTCTTGGGCACACTCGCTG388
spookierreverse:GCCGAGCTAAATTTCTCCGCTT389
390
phantomforward:GGATTTCTTTCGGCGCGATGTG391
phantomreverse:TGCCTCAGTATCGAAAAGCCGT392
393
Puromycinassay394
Groupsof10wanderinglarvaeorearliertimepointlarvaewereinvertedinSchneider’smediaandthen395
transferredtoEppendorftubescontainingmediaplus5µg/mlpuromycin(Sigma),6differentreplicates396
wereusedforFigure1F.Thelarvalsampleswerethenlefttoincubateonamutatorfor40minutesatroom397
temperature.Followingincubation,theinvertedlarvaeweresnapfrozenforwesternblotanalyses.For398
experimentsonlarvalbrains,invertedlarvaewereplacedinice-coldPBSafterincubationwithpuromycin,399
andthebrainswereisolatedandlysedforwesternblotanalyses.400
401
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
415
20-HydroxyecdysoneFeeding416
Newlyhatchedw1118andrpS13/+larvaewerecollectedat24hrAELandplacedinfoodvials(50larvaeper417
vial)supplementedeitherwith20-hydroxyecdysone(Sigma-AldrichCASnumber5289-74-7)orequal418
volumeof95%ethanolforcontrols.20-hydroxyecdysonewasdissolvedin95%ethanoltoafinal419
(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
13
concentrationof0.3mg/ml.Aminimumoffourreplicateswasusedtocalculatethemeanvolumeforeach420
genotype.421
422
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
430
Boutonquantification431
Brainandattachedprothoracicglandsweredissectedfromw1118andrpS13/+larvaeduringthewandering432
L3stageandwerestainedwithanti-5HTandanti-shroudantibodies.ConfocalZstackimageswere433
acquiredand5-HTboutonsthatoverlappedthePG(basedonanti-shroudstaining)werecounted.The434
valuesforeachindividualbrainwererecordedandaveragesforeachgenotypewerecalculated.435
436
Statistics437
Forallexperiments,errorbarsrepresentstandarderrorofmean(SEM).DatawereanalyzedbyStudentst-438
testorMann-WhitneyUtest.AllstatisticalanalysisanddataplotswereperformedusingPrismsoftware.In439
allfigures,statisticallysignificantdifferencesarepresentedas*andindicatep<0.05.440
441
FIGURELEGENDS442
443
Figure1.DevelopmentaldelayofrpS13/+animalsisnotduetoadecreaseinribosomenumbersor444
reducedtranslationrates.445
446
(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
(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
14
(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
466
Figure2.DelayeddevelopmentinrpS13/+animalsisduetoimpairedecdysoneproduction.467
468
(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
482
483
Figure3.rpS13re-expressioninneuronspartiallyreversestherpS13/+developmentaldelay.484
485
(A)rpS13re-expressionintheprothoracicglandusingP0206-Gal4doesnotrescuethedevelopmental486
delayofrpS13/+larvae.Nodifferenceisseenbetween+/+controls(n=95)andUAS-rpS13overexpression487
(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
15
(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
rpS13/+n=176.Dataarepresentedas+/-SEM.*p<0.05,Mann-WhitneyUtest.499
(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
512
513
Figure4.rpS13re-expressionintheserotonergicneuronsthatinnervatetheprothoracicgland514
partiallyrescuesrpS13/+developmentaldelay.515
516
(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
(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
16
(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
rpS13/+n=230.Dataarepresentedas+/-SEM.*p<0.05,Mann-WhitneyUtest.526
(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
531
Figure5.Stimulationofproteinsynthesis,butnotXrp1knockdownintheserotonergicpartially532
rescuesrpS13/+developmentaldelay.533
534
(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
544
Figure6.rpS13/+animalsshownormalaxonalprojectionsintotheprothoracicgland,howeverare545
defectiveinsecretoryfunction.546
547
(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
(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
17
(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
568
Figure7.ExpressionofindividualSNAREcomplexproteinspartiallyrescuesrpS13/+569
developmentaldelayaswellastheotherrpS24/+andrpS26/+developmentaldelay.570
571
(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
587
SupplementalFigures.588
589
(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
18
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
602
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
610
FigureS3611
Fluorescentconfocalimagesofrepresentativetrh>GFP/+andtrh>GFP/rpS13wanderingL3larvalbrains.612
NucleiofneuronsstainedwithHoechst(magenta)while5-HTneuronsarelabelledwithGFP.Scalebars,613
50µm.614
615
616
(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
19
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Fig6
(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
)) (
� ������ ��� ��� ��� ���
)) (
� ���
��� ��� ��� ��� ���
A
B
+/+
UAS-nsyb/+
rpS13, UAS-nsyb/+
rpS13/+
trh>
Hours (AEL)
+/+
rpS13/+
UAS-syt1/+
rpS13, UAS-syt1/+
trh>
Hours (AEL)
C
trh>
� ���
��� ��� ��� ��� ��� ���Hours (AEL)
+/+
rpS13/+
UAS-SNAP29/+
rpS13, UAS-SNAP29/+
D
trh>
+/+
rpS24/+
rpS24, UAS-nsyb/+
Hours (AEL)
E
trh>
Hours (AEL)
+/+
rpS26, UAS-nsyb/+
rpS26/+
0 120 140 160 180 2000 120 140 160 180 200 220
0 120 140 160 180 2000 120 140 160 180 200 220
0 120 140 160 180 200 220
ns
*
ns
**
*
**
Fig7
(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
A M
outh
hoo
k m
ovem
ents
/m
inut
e
� ������
���
���
���
�����������
rpS13 mRNA
rpS13/+
w1118
0
0.5
1
1.5 *
rela
tive
leve
ls
rpS13/+w1118
Larv
al w
eigh
ts(m
g pe
r 10
anim
als)
*
0
20
40
60
80
rpS13/+w1118
*
rela
tive
larv
al s
ize
(pix
el a
rea
x 10
5 )
rpS13/+w1118
0
10
20
30
40
0 24 48 72 96 120 144 168 192Hours (AEL)
0
10
15
20
5
B C.
D
FigS1
(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
W111
8
W111
8
rpS13/+
rpS13/+
75 KDa
25 KDa
25 KDa
75 KDa
W111
8
W111
8
rpS13/+
rpS13/+
A
B
FigS2
(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
+/+
rpS13/+
Trh >
GFP
GFP
FigS3
(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