The ancestral retinoic acid receptor was a low-affinity sensor triggering neuronal differentiation

17 Pages·2017·2.34 MB·English

SCIENCE ADVANCES | RESEARCH ARTICLE
DEVELOPMENTAL BIOLOGY Copyright©2018
TheAuthors,some
The ancestral retinoic acid receptor was a low-affinity
rightsreserved;
exclusivelicensee
sensor triggering neuronal differentiation
AmericanAssociation
fortheAdvancement
Mette Handberg-Thorsager,1*† Juliana Gutierrez-Mazariegos,2† Stefan T. Arold,3 ofScience.Noclaimto
originalU.S.Government
Eswar Kumar Nadendla,4 Paola Y. Bertucci,1 Pierre Germain,4 Pavel Tomançak,5
Works.Distributed
Keely Pierzchalski,6 Jace W. Jones,6 Ricard Albalat,7 Maureen A. Kane,6 William Bourguet,4 underaCreative
Vincent Laudet,2द Detlev Arendt,1,8ঠMichael Schubert9ঠCommonsAttribution
NonCommercial
Retinoic acid (RA) is an important intercellular signaling molecule in vertebrate development, with a well- License4.0(CCBY-NC).
established role in the regulation of hox genes during hindbrain patterning and in neurogenesis. However,
the evolutionary origin of the RA signaling pathway remains elusive. To elucidate the evolution of the RA
signaling system, we characterized RA metabolism and signaling in the marine annelid Platynereis dumerilii,
apowerfulmodelforevolution,development,andneurobiology.Bindingassaysandcrystalstructureanalyses
showthattheannelidretinoicacidreceptor(RAR)bindsRAandactivatestranscriptionjustasvertebrateRARs,
yet with a different ligand-binding pocket and lower binding affinity, suggesting a permissive rather than in-
structiveroleofRAsignaling.RARknockdownandRAtreatmentofswimmingannelidlarvaefurtherrevealthat D
the RA signal is locally received in the medial neuroectoderm, where it controls neurogenesis and axon ow
outgrowth,whereasthespatialcolinearhoxgeneexpressionintheneuroectodermremainsunaffected.These nlo
findingssuggestthatoneearlyroleofthenewRARinbilaterianevolutionwastocontrolthespatiallyrestricted ad
e
onset of motor and interneuron differentiation in the developing ventral nerve cord and to indicate that the d
regulationofhox-controlledanterior-posteriorpatterningaroseonlyatthebaseofthechordates,concomitantwith fro
m
ahigh-affinityRARneededfortheinterpretationofacomplexRAgradient. h
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INTRODUCTION Invivo,RAissynthesizedfromvitaminAintwodifferentoxida- ://a
d
Invertebrates,retinoicacid(RA)regulatesawiderangeofbiological tivesteps.Thefirstone,therate-limitingdehydrogenationofretinolto va
n
processesindevelopmentandadultlife.Duringembryogenesis,RA retinal,iscatalyzedbyretinoldehydrogenases(RDHs)oftheshort-or c
e
s
controlspatterningalongtheanteroposteriorbodyaxisandisrequired medium-chaindehydrogenase/reductasesuperfamilies[forexample, .s
c
forthe proper formation ofvarious organs, including heart, liver, SDR-RDH10orMDR-ADH3(alcoholdehydrogenase3)](3).Alter- ie
n
kidney,limbs,andthenervoussystem(1,2).Inthedevelopingcentral natively,retinalcanbefurtherproducedfromtheenzymaticcleavage c
e
nervoussystem,RAmediatesanteroposteriorregionalizationviathe ofb-carotenebycarotenoidcleavageoxygenase(CCO)enzymes.The m
a
directcontrolofhoxtranscriptionandsubsequentlystimulatesneuro- secondstep,theproductionofRAfromretinal,iscatalyzedbythe g.o
genesisandpromotesneuronaldifferentiationviatheactivationof retinaldehydedehydrogenases(RALDHs).Althoughdifferentisomers rg
pax6,ngn2,anddbx1inspecificregionsoftheneuroectoderm(1,2). ofRAexist,suchasall-transRA(ATRA),9-cisRA(9cRA),and13-cis on/
RA(13cRA),themostimportantbiologicallyactiveRAisomerinver- A
p
1DevelopmentalBiologyUnit,EuropeanMolecularBiologyLaboratory,Meyerhofstrasse tebratesisATRA(3,4). ril 5
1,69012Heidelberg,Germany.2MolecularZoologyTeam,InstitutdeGénomique ThemajormediatorsofRAsignalingaretheretinoicacidreceptor , 2
FonctionnelledeLyon,UniversitédeLyon,UniversitéLyon1,CNRS,InstitutNationalde (RAR)andtheretinoidXreceptor(RXR),whichareligand-dependent 01
laRechercheAgronomique,EcoleNormaleSupérieuredeLyon,46Alléed’Italie,69364 9
LyonCedex07,France.3KingAbdullahUniversityofScienceandTechnology,Center transcriptionfactorsbelongingtothenuclearreceptorsuperfamily.
forComputationalBioscienceResearch,DivisionofBiologicalandEnvironmental RARandRXRheterodimerizeonspecificDNAstretches,calledRAre-
SciencesandEngineering,Thuwal23955-6900,SaudiArabia.4CentredeBiochimie sponseelements(RAREs),presentinthepromoterregionsoftarget
Structurale,Inserm,CNRS,UniversitédeMontpellier,29RuedeNavacelles,34090 genes(5–7).TheRARligand-bindingdomain(LBD)mediatesthe
Montpellier,France.5MaxPlanckInstituteofMolecularCellBiologyandGenetics,
Pfotenhauerstrasse108,01307Dresden,Germany.6DepartmentofPharmaceutical high-affinitybindingofATRAwithadissociationconstantinthenano-
Sciences,SchoolofPharmacy,UniversityofMaryland,20NorthPineStreet,Baltimore, molarrange(K =0.4nM)(8).TheLBDcontains12helicesthatform
MD21201,USA.7DepartamentdeGenètica,MicrobiologiaiEstadística,Institutde theligand-binddingpocket(LBP)(9),andbindingoftheligandmodifies
RecercadelaBiodiversitat(IRBio),FacultatdeBiologia,UniversitatdeBarcelona,
AvingudaDiagonal643,08028Barcelona,Spain.8CentreforOrganismalStudies, thepositionofhelix12,allowingtherecruitmentofcoactivators.This
UniversityofHeidelberg,ImNeuenheimerFeld230,69120Heidelberg,Germany. associationofcoactivators,inturn,leadstotheinitiationoftranscription
9SorbonneUniversités,UniversitéPierreetMarieCurie(UPMC)UniversitéParis06, ofRAtargetgenes(7,10,11).
CNRS,LaboratoiredeBiologieduDéveloppementdeVillefranche-sur-Mer,Observatoire
AlthoughRAsignalingistraditionallydescribedasachordateinno-
OcéanologiquedeVillefranche-sur-Mer,181CheminduLazaret,06230Villefranche-
sur-Mer,France. vation,keyelementsoftheRAmachinery,suchasRARandRXR,have
*Presentaddress:MaxPlanckInstituteofMolecularCellBiology andGenetics, beenidentifiedinseveralnonchordategenomes(12,13).Forexample,
Pfotenhauerstrasse108,01307Dresden,Germany.
genesencodingRAR,RXR,andRALDH,aswellastheRA-catabolizing
†Theseauthorscontributedequallytothiswork.
‡Theseauthorscontributedequallytothiswork. enzymeCYP26(cytochromeP450subfamily26),havebeenidenti-
§Presentaddress:SorbonneUniversités,UniversitéPierreetMarieCurie(UPMC)Uni- fiedintheannelidCapitellateleta(12,13).Incontrast,intheannelid
versitéParis06,CNRS,ObservatoireOcéanologiquedeBanyuls-sur-Mer,AvenuePierre Helobdellarobusta,RARandCYP26havebeenlost(13),andtheonly
Fabre,66650Banyuls-sur-Mer,France.
reportofrolesofRAinannelidsassessedtheeffectsofveryhighcon-
¶Correspondingauthor.Email:[email protected](M.S.);arendt@embl.
de(D.A.);[email protected](V.L.) centrations of retinal on juvenile and regenerating worms (14).
Handberg-Thorsageretal.,Sci.Adv.2018;4:eaao1261 21February2018 1of16
SCIENCE ADVANCES | RESEARCH ARTICLE
Furthermore,thefunctionalcharacterizationofRARsfromtwogastropod (3). These included genes encoding the nuclear receptors RAR and
mollusks,ThaisclavigeraandNucellalapillus,hasestablishedthatthesere- RXR, enzymes involved in RA synthesis and degradation [CCO,
ceptorsareunabletobindRA(15,16).Therefore,albeitthepresenceofthe SDR,MDR,RALDH—thatis,membersofthealdehydedehydrogenase
mainRAsignalingcomponentsinvariousbilateriantaxa,RAR-dependent 1a (ALDH1a) and ALDH8a subfamilies—and CYP26], retinoid tran-
RAsignalingstillretainsitsstatusasachordate-specificfeature. sport proteins and plasma membrane–associated receptors [cellular
Here,wedescribethecharacterizationofanovelRARclonedfrom RA-bindingprotein(CRABP),cellularretinol-bindingprotein(CRBP),
theannelidPlatynereisdumerilii.WeshowthattheP.dumeriliiRAR retinol-binding protein 4 (RBP4), transthyretin (TTR), and stimulated
(PduRAR)actsasligand-activatedtranscriptionfactorinthepresence by RA 6 (STRA6)], and enzymes involved in the synthesis and deg-
ofATRAand13cRA,bothofwhicharepresentinembryos,larvae,and radation of retinoid storage products [lecithin-retinol acyltransferase
adults.However,RAactivatestheannelidreceptoronlyinthemicro- (LRAT), acyl-CoA:retinol acyltransferase (DGAT), and retinoid iso-
molarrange,indicatingthatPduRARisalow-affinityRAsensorand merohydrolase(RPE65)](Fig.1).
notachordate-typehigh-affinityRAR.Furthermore,thecrystalstruc- Phylogeneticanalysesoftheobtainedgenesequencesallowedusto
tureofthePduRARLBDrevealsthatdifferentialbindingofRAmole- identifysinglerarandrxrhomologsinP.dumerilii.Furthermore,we
culestotheLBPsoftheP.dumeriliiandvertebratereceptorsdefinesthe characterizedfivealdh1a,onealdh8a,threecyp26,onesdr-rdh10,one
structural basis for these affinity differences. During development, mdr-adh3, and three cco genes, demonstrating that the basic RA
PduRARdoesnotcontrolanteroposteriorpatterningoftheembryo signalingmachinery,composedofreceptorsaswellasRA-synthesizing
anddoesnotregulatehoxgeneexpression.Incontrast,RAsignaling andRA-degradingenzymes,ispresentinP.dumerilii(Fig.1andfig.
functionsduringannelidneurogenesistocontroldifferentiationandax- S1).Avertebrate-typeRAmetabolismandRAR-dependentsignaling
onaloutgrowthofmedialneurontypesintheventralnervecord.Our werethusalreadypresentatthebaseofbilaterians. D
o
workisthefirsttounravelRARfunctionoutsidechordatesandsuggests Tomaintainretinoidhomeostasis,vertebratescanaccumulatereti- w
n
thatoneearlyroleofRAR-dependentRAsignalingwasthespatio- noidsintheliverandmobilizethemtosatisfytheRArequirementsof lo
a
temporalcontrolofmotorandinterneurondifferentiation.Ourcom- peripheraltissues.Retinolisesterifiedtoretinylestersbytheactionof d
e
pseanrasotirveanddattahiantdhicigahte-athffaintitthye,iannsctreustcrtaivleRARARRliskeelvyoalvceteddoanslaypatertmheisbsaivsee tthraenvsefortrembreadtebLacRkAtToorertDinGolAbTy1reentiznyymleess,tearndhyrdertionlyalseesst(eirnsccluadninbge d fro
m
ofchordates,concomitantwitharoleforRAsignalinginglobalantero- RPE65)(18).P.dumeriliiseemstolacklratandrpe65orthologs,al- h
posteriorpatterningthroughhoxgeneregulation. thoughanorthologofdgathasbeenidentifiedinP.dumerilii,suggest- ttp
ingthatdenovosynthesisofretinylestersispossible(Fig.1andfig.S1). ://a
d
Retinoidsaregenerallyboundtoproteinsthatsolubilizeandstabilize va
n
RESULTS theminaqueousenvironments.P.dumeriliilackshomologsofthetyp- ce
s
Identification of RA signaling pathway components in icalvertebrateretinoidbindingproteins,thatis,oftheintracellularcrbp .s
P. dumerilii andcrabp,aswellasoftheextracellularrbp4andttr.Concomitantly, cie
n
TogaininsightsintotheevolutionoftheRApathway,wescreenedtran- stra6, the membrane receptor required for rbp4-dependent retinol c
e
m
scriptomic and genomic databases of the marine annelid P. dumerilii uptakeintotargetcellsinvertebrates(3),isalsoabsentfromP.dumerilii.
a
(17)formolecularcomponentscharacterizingchordateRAsignaling These data suggest that, although the core components of the RA g.o
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Fig.1. Molecularcomponentsofretinoidmetabolismandsignaling.Schematicrepresentationofretinoidmetabolism,storage,transport,andsignalinginverte-
brates.Metabolicenzymes,bindingproteins,andnuclearreceptorsareshowninblue.Namesinboldlettersindicatethepresenceofageneencodingtheorthologofa
givenvertebrateproteinintheP.dumeriliigenome.ItalicletteringindicatestheabsenceofthecompletecorrespondinggenefamilyinP.dumerilii.Namesinregular
lettersindicatethepresence,inP.dumerilii,ofmembersofagivengenefamilybuttheabsenceofaspecificP.dumeriliiorthologofthecorrespondingvertebrate
gene.Ofnote,theinvolvementofADHenzymesinretinalsynthesisinvertebrateshasbeenchallenged(3).
Handberg-Thorsageretal.,Sci.Adv.2018;4:eaao1261 21February2018 2of16
SCIENCE ADVANCES | RESEARCH ARTICLE
pathwayareconservedbetweenP.dumeriliiandvertebrates(thatis,
ligandproduction/degradationandreceptors),keymechanismsforret-
inoidstorage,intercellulartransport,cellularuptake,andintracellular
bindingofretinoidsarenot.Thisisconsistentwiththeideathatthe
vertebratecomponentsforhepaticstorage,mobilization,andtransport
ofretinoidswerelineage-specificinnovations(18).
Molecular characterization of the PduRAR
TocharacterizethePduRARandPduRXRproteinsinvitro,wecloned
andexpressedtherespectivegenes.TheDNAbindingdomainofthe
annelidRARsharesapproximately88%sequenceidentitywiththat
ofthethreehumanRARs(RARa,RARb,andRARg)and92%withthat
ofarecentlyidentifiedmolluskRAR(N.lapillus)(16).Incontrast,the
LBDsharesonlyabout55%identitywiththatofthehumanRARsand
60%withthatoftheN.lapillusRAR,andofthe25aminoacidsknown
tointeractwithATRAintheLBPofthehumanRARa(19),5aredif-
ferentintheannelidortholog(fig.S2).
TheDNAbindingpropertiesoftheannelidRAR/RXRheterodimer
werevalidatedusingelectrophoreticmobilityshiftassays(EMSAs) D
o
(Fig. 2A). Binding was assessed on consensus direct repeat (DR) w
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elementsasdefinedforvertebrates(8).UsingaradiolabeledDR2probe, lo
a
nobindingofRARorRXRalonecouldbedetected.Incontrast,the d
e
RwAasRs/pReXciRfichbeteecraoudsiemitewrsatsroonugtclyombopuenteddtbhyeaDnRe2xceelesmsoefnut.nTlahbiselbedinDdiRn2g d fro
m
probebutnotbyanonspecificunlabeledprobe.Bindingwasalsolostin h
thepresenceofunlabeledDR1andDR5.Otherelements,suchasDR0, ttp
DR3,andDR4,inducedonlymarginalcompetition.Theseresultsindi- ://a
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catethattheannelidRAR/RXRheterodimerbindsspecificallytoarange va
n
ofRAREsverysimilartothoseofitsvertebrateorthologs(thatis,DR1, c
e
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DR2,andDR5). .s
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WesubsequentlytestedtheabilityofthePduRARtobinddifferent ie
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retinoidsandactivatetranscriptioninaligand-dependentmanner. c
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First, we performed transient transactivation assays using a Gal4
a
LBDreceptorchimera.OurresultsshowthattheRARLBDisableto g.o
activatetranscriptionofaluciferasereportergeneinadose-dependent rg
m13acnRnAereliincitthinegparesstreonncgeeorfrAesTpRonAs,e1t3hcaRnA9,caRnAd(9FciRg.A2,Bw).itHhoAwTeRveAr,aitnids PFdigu.R2X.RMheotelerocudlimarecr.hBaortahctreecriezpattoiorsnwoefrePsdyunRthAeRsi.z(eAd)iDnNvAitrroe,caongdntithioenhbetyetrhoediPmdeurRwAaRs/ on Ap/
importanttonotethatforanyofthethreeligands,anactivationofthe allowedtobindtoa32P-labeledDR2RAREprobe.Coldcompetitorscorrespondto10-or ril 5
receptorwasobservedonlywithrelativelyhighligandconcentrations 100-foldexcessofunlabeledoligonucleotides(DR0toDR5).Anonspecificelement(NS) , 2
(1to10mM).Thisisinstarkcontrasttothesituationinvertebrates, wasusedasanegativecontrol.Unprogrammedreticulocyteswereusedascontrol(Ctrl). 01
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whereRARactivationbyATRAisalreadyobservedataconcentration Proteinandlabeledprobecomplexesareindicatedbyanarrow(shownonlyinthePduRAR/
of1nM(20). PduRXRlane).(B)TheabilityofPduRARtoactivatethetranscriptionoftheluciferasereporter
Direct binding analyses of PduRAR to different ligands using genewastestedintransfectedhumanembryonickidney(HEK)293Tcellsinthepresence
ofincreasingconcentrations(0.1,1,and10mM)ofATRA,9cRA,and13cRA.TheGal4DNA
limitedproteolysisassays(LPAs)confirmedtheseresults(Fig.2C).We
bindingdomainconstructalonewasusedasanegativecontrol(Ctrl).Barsaremeans±SD
observedthat,startingataconcentrationof1mM,theRARLBDwas
(n=3).(C)TheabilityofPduRARtobinddifferentligandswastestedbyLPA.Theligands
protectedfromproteolysisbyallthreeligands.Thespecificityofthese
(ATRA,9cRA,and13cRA)wereusedatincreasingconcentrations(0.1,1,and10mM).
effectswasvalidatedusingtheRARantagonistBMS493(21).Inthepres- Ethanolwasusedasanegativecontrol(lane−),andprotectedbandsareindicatedby
enceofATRA,highconcentrationsofBMS493(10mM)inducedade- arrows.(D)CompetitionassayusingincreasingconcentrationsoftheRARantagonist
creaseinthe transcriptionoftheluciferase reportergene(Fig.2D). BMS493(0.1,1,and10mM)inthepresenceof10mMATRA.TheGal4DNAbinding
Furthermore,LPAsshowedaBMS493-inducedprotectionfromproteol- domainconstructalonewasusedasanegativecontrol(Ctrl).Barsaremeans±SD
ysisstartingataconcentrationof1mM,indicatingthatBMS493binding (n=3).(E)BindingofPduRARtoincreasingconcentrationsofBMS493(0.1,1,and
totheLBDantagonizedtheATRA-dependenttranscriptionalactivityof 10mM)asassessedbyLPA.Ethanolwasusedasanegativecontrol(lane−),and
RAR(Fig.2E).Ofnote,theannelidRXRactivatedtranscriptionofalu- protectedbandsareindicatedbyarrows.(F)Titrationofthefluorescein-labeledinterac-
tiondomains(ID1andID2)ofthecorepressorsNCOR(nuclearreceptorcorepressor)and
ciferasereportergeneinaconcentration-dependentmannerusingeither
SMRT(silencingmediatorforretinoidandthyroidhormonereceptors)byunliganded
9cRAortheRXR-specificagonistBMS649(fig.S3A)(22).Moreover,the
PduRARmonitoredbyfluorescenceanisotropy.Assayswereperformedinthree
useoftheRXR-specificantagonistUVI3003(23)inhibitedRXRtran-
independentexperiments,anddataareexpressedasmeans±SEM.(G)Titrationof
scriptionalactivity(fig.S3B).
thefluorescein-labeledinteractiondomain1(ID1)ofthecorepressorNCORbyPduRAR
Afterthecharacterizationoftheligand-bindingproperties,weas- inthepresenceofdifferentretinoids:ATRA,9cRA,and13cRA.Assayswereperformedin
sessedthecapacityoftheannelidRARtointeractwithreceptorcofactors, threeindependentexperiments,anddataareexpressedasmeans±SEM.
Handberg-Thorsageretal.,Sci.Adv.2018;4:eaao1261 21February2018 3of16
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usingafluorescence-basedassaythatmonitorstheligand-inducedas- H3andtheC-terminalpartofthedomaincontaininghelixH11andthe
sociationofcofactorfragments.Twocorepressors,NCOR(nuclearre- activationhelixH12(Fig.3A).InmoleculesBandD,theNterminiof
ceptorcorepressor)andSMRT(silencingmediatorforretinoidand helixH3andhelixH11wereshiftedoutwardoftheLBPtoaccommo-
thyroidhormonereceptor),wereanalyzedbecausetheyareknownto datetheATRAmolecule.Unexpectedly,helices H12oftheligand-
interactwithunligandedvertebratereceptorsthroughtwointeraction bound molecules B and D were not stabilized in the so-called
domains(calledID1andID2)(24).Usingfluorescein-labeledpeptides agonist-boundoractiveconformationbutdockedontothesurfaceof
derivedfromNCORandSMRT,wefirstevaluatedthebindingprefer- asymmetry-relatedligandedLBD,resultinginanarm-exchangedcon-
ences of RAR in the absenceof ligand.WefoundthatRARbound formation(Fig.3Aandfig.S4C).Wewereunabletoobserveanyinter-
mostavidlytoNCORID1(Fig.2F).WethentitratedtheNCORID1 pretableelectrondensityfortheligandinmoleculesAandC,whose
peptidewithRARinthepresenceofdifferentretinoids(ATRA,13cRA, helicesH12wereintheagonist-andantagonist-boundconformations,
and9cRA)(Fig.2G).Asexpected,weobservedthatthethreeretinoids respectively(Fig.3A).BothsubunitsBandDboundATRAinanori-
areabletobindtoRARandinduceNCORID1release,withATRA entationthathasneverbeenobservedinmammalianRARstructures
beingmorepotentthan13cRAand9cRAininducingthedissociation (Fig.3B).AlthoughmoleculesBandDwereinvolvedindifferentcrystal
(Fig.2G). packinginteractions,theirATRAmoleculeswerefullysuperimposable,
TogaininsightsintothemolecularmechanismsofannelidRARlig- stronglysupportingthefactthattheunusualbindingmodeoftheligand
andbinding,wedeterminedthecrystalstructureoftheLBDofthe wasnotinfluencedbycrystalpackingconstraints.
PduRARincomplexwithATRAataresolutionof2.7Å(tableS1). InhumanRARa,theLBPextendsfromhelixH11ononesidetothe
TheasymmetricunitcontainedfourmoleculesorganizedastwoLBD twoconservedpolarresiduesR276inhelixH5andS287inthetipofthe
homodimers(fig.S4A).Electrondensityfortheligandwasonlyvisible b-turnS1/S2,whichformahydrogenbondnetworkwiththecarboxyl- D
o
insubunitsBandD,eachofwhichformeddimerswithanonliganded atemoietyoftheretinoidligandontheotherside(Fig.3B).Incontrast, w
n
LBD(subunitsAandC,respectively)(fig.S4,AandB).Allfoursub- inthePduRAR,theligandisrotatedbyalmost90°,resultinginthe lo
a
unitssuperimposedverywell,exceptintheN-terminalregionofhelix carboxylgroupofATRAlyingbetweentheN-terminalregionofhelices d
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Fig.3. CrystalstructureofthePduRARLBD.(A)SuperimpositionofthefourPduRARLBDs(MolAtoMolD)containedintheasymmetricunit.aHelices(H1toH12)
arelabeled.ThetwoATRAmoleculespresentinsubunitsBandDareshownasyellowsticks.(B)SuperimpositionoftheLBPsofhumanRARaboundtotheagonist
AM580(yellow)andofthePduRAR(MolB)boundtoATRA(blue).TheATRApresentinMolD(whichwassuperimposedonMolBandisnotshown)isdisplayedas
greensticks.HumanRARaR276andS287thatareengagedinpolarinteractionswiththecarboxylatemoietyofAM580areshown.(C)Close-upviewoftheLBPof
PduRARboundtoATRA.Residuesincontactwiththeligandareshownandlabeled.(D)SuperimpositionoftheLBPsofhumanRARaboundtotheagonistAM580
(yellow)andofthePduRAR(MolB)boundtoATRA(blue)showinghowtheV356Fmutationaffectsligandbindingtotheannelidreceptor.(E)Transcriptionalactivityof
thePduRARV356Fmutant,intransfectedHEK293Tcells,inthepresenceofincreasingconcentrationsofATRA(1,5,and10mM).TheGAL4DNAbindingdomainalone
wasusedasanegativecontrol(Ctrl).Barsaremeans±SD(n=3).(F)SuperimpositionoftheLBPsofhumanRARaboundtotheagonistAM580(yellow)andofthe
PduRAR(MolB)boundtoATRA(blue).ThevanderWaalsinteractionsbetweenAM580andhelixH12residuesI410andL414areindicatedasdottedlines.(G)Su-
perimpositionoftheLBPsofhumanRARaboundtotheagonistAM580(yellow)andofthePduRAR(MolB)boundtoATRA(blue),highlightingthesmallerdistances
betweenequivalentresiduepositionsinhelicesH3andH11intheannelidreceptorwhencomparedtovertebratereceptors.
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SCIENCE ADVANCES | RESEARCH ARTICLE
H3andH6(Fig.3,AandB).Thevastmajorityofthecontactsmadeby Thus,PduRARlikelybehavesasasensorratherthanasahigh-affinity
ATRAwiththeLBPwereloosevanderWaalsinteractions,andthe receptor.
carboxylatemoietyoftheliganddidnotformanynoticeablehydro-
genbondswithsurroundingLBPresidues(Fig.3C).Thisstructure Retinoids in developing and adult P. dumerilii
suggestedthatthecarboxylatemoietyplayslittleroleinthebinding TotestwhetherRAispresentinP.dumerilii,wenextdeterminedthe
ofretinoidstoPduRAR,whichstronglycontraststhesituationinhu- retinoidcontentsineggs,embryos,larvae,andadultsusingliquidchro-
manRARs.Thisobservationwasinagreementwithourcell-based matography–tandemmassspectrometry(LC-MS/MS)forRA(4,25)
resultsshowingsimilar bindingefficienciesofATRA, 13cRA, and andhigh-performanceliquidchromatographywithultravioletdetection
9cRAfortheannelidreceptor(Fig.2B).Tovalidatethealternative (HPLC-UV)forretinolandretinylesters(4).Wewereabletoidentify
bindingmodeofPduRAR,wemutatedthevalineatposition356in ATRA,13cRA,retinol,andretinylestersbutfailedtodetect9cRAabove
helixH3intoaphenylalanine(V356F),whichisthecorresponding thelimitofdetectionofourassay(Table1)(25).Theconcentrationsof
residueatthispositioninhumanRARs(F228inRARa)(Fig.3D). ATRAand13cRAwereinthesamerange,andoften,13cRAwaspre-
Onthebasisofthestructure,thissubstitutionshouldspecificallyaffect sentathigherlevelsthanATRA(Table1).Forexample,inP.dumerilii
thePduRARbindingmode.Inagreementwiththishypothesis,weob- adults,wedetected38.3±4.03pmol/gtissueofATRAand46.8±3.89
served a marked decrease in the capacity of ATRA to activate the pmol/gtissueof13cRA(Table1),withoneunitofpmol/gtissueofa
V356Fmutant(Fig.3E). givenretinoidtranslating,approximately,intoanaverageconcentration
ComparisonsoftheATRA-boundPduRARLBDstructuretothat of1nM(26).Thisfindingcontrastswiththesituationinvertebrates,
ofthehumanRARaLBDboundtotheagonistAM580(24)revealed whereATRAlevelsgenerallytendtobehigherthanthoseof13cRA(26).
another striking difference with possible functional implications. Retinoidswerefurtherquantifiedinunfertilizedeggsandatdifferent D
o
Whensuperimposingthetwostructures,weobservedthat,inthehu- developmentalstages:18hourspost-fertilization(hpf)(gastrulating w
n
manreceptor,AM580insertsitstetramethyl-cyclohexylringbetween protrochophore),51hpf(earlymetatrochophorelarva),and74hpf lo
a
helicesH3andH11,whereasintheannelidRAR,theb-iononeringof (earlynectochaetelarva)(17).TherelativelevelsofATRAand13cRA d
e
AheTliRceAsHre3sidaneddHde1e1pearrewciltohsienrttoheeaLcBhPot(hFeigr.in3Ft)h.eAlisgaancdoendsPedquuRenAcRe,, wanecreewgeitnherthalelynlootwioenrtdhuartinemgebmrybornyiocgreentiensoisidthsaanreinsytnhtehaedsiuzletd,ifnroamccoprrde-- d fro
m
hencegeneratingapossiblysuboptimaldockingsurfaceforhelixH12 cursormoleculesstoredintheegg,whereas,inadults,precursorsare h
inthetranscriptionallyactiveconformation(Fig.3,FandG).More- obtainedfrom the diet. During embryogenesis, ATRAlevels were ttp
over,althoughdirectcontactsexistbetweenagonistligandsandhelix highestduringgastrulation(4.58±0.16pmol/gtissue),whereas13cRA ://a
d
H12residues(I410andL414)inhumanRARa,nosuchinteractions levelswerehighestin51hpflarvae(7.47±4.63pmol/gtissue)andgas- va
n
wereobservedintheATRA-boundPduRAR(Fig.3F).Theseobserved trulatingembryos(6.34±2.21pmol/gtissue)(Table1).Theseresultssug- ce
s
structuraldifferencesbetweentheannelidandhumanRARLBDssug- gestthat,asinvertebrates,activeretinoidlevelsaretightlycontrolled, .s
c
gestedthatATRAonlyweaklystabilizestheactiveconformationof althoughthekineticsofATRAand13cRAlevelsduringP.dumerilii ie
n
helixH12inthePduRAR.Thisfindingexplainedboththedissociation embryogenesisdiffer,suggestingdistinctcontrolmechanisms.Further- c
e
m
ofhelixH12fromitsLBDcoreintheBandDsubunitstructuresand more,giventhatATRAand13cRAlevelswereassessedinwholeanimals,
a
thesignificantlyweakerATRA-inducedtranscriptionalactivityofthe weexpectthatRAconcentrationsaresignificantlyhigherintargettissues, g.o
annelidreceptorincell-basedassays,whencomparedtotheactivityof henceleadingtotheligand-dependentactivationofPduRAR. rg
atypicalhumanRAR(fig.S4D). RetinolandretinylestersserveasprecursorsforthesynthesisofRA on/
Together,ourfunctionalandstructuraldataindicatethatPduRAR invertebrates(3,27).InP.dumerilii,weobservedmuchhigherlevelsof A
p
isabletobinddifferentRAisomers(atleastATRA,13cRA,and9cRA) retinolandretinylestersthanofATRAor13cRA(Table1),similarto ril 5
with a micromolar affinity by using distinctive ligand-binding whatisobservedinvertebrates(3,4,26),whichsuggeststhatthese , 2
0
modesandstructuralresponseswhencomparedtovertebrateRARs. retinoidsalsoserveasprecursorsforRAsynthesisintheannelid.The 1
9
Table1.EndogenousretinoidsinP.dumerilii.Valuesaremeans±SD.Serumvaluesarepmol/gtissue.n.d.,notdetected.Developmentalstagescorrespond
togastrulationat18hpf,theearlymetatrochophorelarvaat51hpf,andtheearlynectochaetelarvaat74hpf.
Stage ATRA 9cRA 13cRA Retinol Retinylesters
Unfertilizedegg 0.92±0.74 n.d. 0.73±0.41 50±31.25 180±68.66
(n=4)
Gastrulatingprotrochophore 4.58±0.16 n.d. 6.34±2.21 420±200 1000±417.81
(n=2)
Earlymetatrochophorelarva 2.74±1.52 n.d. 7.47±4.63 830±489.34 1880±1004.92
(n=9)
Earlynectochaetelarva 3.39±1.10 n.d. 2.53±1.17 300±125.65 900±567.46
(n=6)
Adultworm 38.3±4.03 n.d. 46.8±3.89 878.5±102.3 22170±4581
(n=4)
Handberg-Thorsageretal.,Sci.Adv.2018;4:eaao1261 21February2018 5of16
SCIENCE ADVANCES | RESEARCH ARTICLE
fact that retinol and retinyl ester levels are higher in gastrulating rescentprotein(GFP)expressionoftheirrespectivemRNAs(fig.S6)
embryosthanineggssuggeststhatretinoidsarestoredinotherforms andnotedageneraldevelopmentaldelayforallMOinjections(Fig.5),
intheoocyteandthat,whenembryonictranscriptionstarts,retinoland whichisconsistentwithpreviousreportsontheuseofMOsindevel-
retinylestersaresynthesizedfromthesealternativelystoredretinoids. opmentalsystems(30).
LarvaeinjectedwithrarMOdevelopedregularlyintolarvalstages
Expression of genes involved in RA synthesis, signaling, (98%,n=363),comparabletocontrols(98%,n=196).Toscorefor
and degradation morphologicaleffects,welabeledfixedlarvaewith4′,6-diamidino-2-
ThebiosynthesisofRAisbesttrackedbystudyinggenesinvolvedinits phenylindole(DAPI)andwithanantibodydirectedagainstacetylated
metabolism.Invertebrates,RALDHenzymesareenrichedinsomatic tubulin that labels axons and cilia (29, 31). Morphological defects
tissuesattheinterfacebetweentheyolksacandtheembryo(28).In becameapparentinthefullydevelopedmetatrochophorelarva,with
P.dumerilii,threeoffivealdh1aparalogs(aldh1a_1,aldh1a_4,and aberrantaxonalscaffoldsinthedifferentiatingventralnervecordthat
aldh1a_5),whicharelikelyinvolvedinRAsynthesis,werefoundinthe wereclearlydistinctfromcontrolMO-injectedscaffolds(Fig.5,A,C,
yolkymacromeres(fig.S5,A,B,C,E,F,H,andJtoM)andinthead- E,andI).Therar morphantsexhibitedoverallreducedconnectives
jacentmesodermalbandsthatstretchoutbetweenthemacromeresand (50%,n=22),whereascommissuralaxonsappearedlessaffected(Fig.
theoverlyingneuroectoderm(fig.S5,FandG).Thisexpressionisinline 5, C and E). Although commissural trajectories were partially
withthenotionthatRAmetabolismtakesplaceintheannelidyolkand disorganized,thecapacityofaxonstocrossthemidlinewasnotimpaired
thatoxidationtoRAisperformedeitherintheyolkitselforinthe (Fig.5,CandE).WealsoanalyzedlarvaeinjectedwithrxrMOandfound
overlyingearly-formingsomites.Oncethemidgutisformed,thethree themtobeoverallmorphologicallyverysimilartotherarMO–injected
aldh1ageneswereexpressedintissuessurroundingthemidgut,suggest- ones,characterizedbyaspecificreductionofconnectivesandaltered D
o
ingafunctionrelatedtofoodintakeandthecontrolofmetabolism commissuralbundling(75%,n=60)(Fig.5,GandI).Inaddition,we w
(fig.S5,D,I,andN).Afourthaldh1agene,aldh1a_3,wasexpressedin observedabehavioraleffectinrxrMO–injectedlarvaeinthatsome nlo
a
othertissues(fig.S5,OtoR).Theinsituhybridizationexperiments didnotswimbutratherstayedonthebottomofthedish,althoughthey d
e
atanrygerteisnuglttsh,ewfhifitchhPis.ldikuemlyerdiulieiatoldlho1wadpeavrealloopgm,aelndhta1lae_x2p,rdesidsionnotleyvieellsd. werAenagaillyesiasnodfasypnpaepartoedtangmorimnaelxlyprdeesvsieolonp,eadm(5a5rk%e,rnfo=rp2r8e6s)y.napticdif- d fro
m
ThethreeP.dumeriliicyp26genes(cyp26_1,cyp26_2,andcyp26_3) ferentiation,inrarmorphantsrevealedaspecificdecreaseofneuronsin h
showedexpressioninthemidgut,whichisconsistentwithapossible themedialtrunkdomainthatdifferentiatesbetween22and56hpf(29), ttp
modulationofRAactivityinthistissue(fig.S5,StoU). whereasearlierforminglarvalneuronswereunaffected(100%,n=4) ://a
d
TodeterminewheretheRAsignalisreceived,weanalyzedtheex- (Fig.5,LandM).ThissuggestsaspecificrequirementforRAsignaling va
n
pressionoftheannelidRARandRXR.Thefirstprominentexpression inlarvalneuronaldifferentiationinthemedialneuroectoderm.To c
e
s
oftherargenewasinthelarvalneuroectoderm(Fig.4,AtoC),andat corroborate this point, because serotonin is an abundant neuro- .s
c
48hpfitwasspatiallyrestrictedtothemedialdomain(Fig.4C),where transmitterinboththelarvalnervoussystemandtheventralnervecord ie
n
pax6andnk6expressionoverlapandhb9+motorneuronsdifferenti- (29),weinvestigatedthedevelopmentofserotonergicneuronsin c
e
m
ate(29).Lateralneuroectodermwasdevoidofexpression.Atthesame P.dumeriliirarandrxrmorphants.Wefoundthat,afterbothrarand
a
time,rarwasalsoexpressedinthebrain(Fig.4,AtoD)andinthe rxrMOinjections,theearlydeveloping,embryonicserotonergicneu- g.o
developingmesodermalbandsinthetrunk(Fig.4,AtoC).Startingbe- ronswereunaffected,whereasthenumberofthelaterdifferentiating, rg
tween24and30hpf,therargenewasfurtherdetectableinthedeveloping larvalserotonergicneuronswasreduced(Fig.5,B,D,F,H,J,andK). on/
stomodeumandproctodeum,whereitremainedexpresseduntil72hpf Thisreductionwasmorepronouncedinterminallydifferentiated A
p
(Fig.4,BtoD).Furthermore,intheyoungworm,rar wasexpressedin serotonergicneurons(withaxons)(Fig.5K)thanindifferentiating ril 5
themidgut(Fig.4E).CoherentwithapossibledimerizationofRAR serotonin-positivecells(withoutaxons)(Fig.5J). , 2
0
andRXR,therxrgenewasexpressedatsimilarstagesinthesametissues Wenextassessedtheexpressionofthetranscriptionfactorhb9, 1
9
(neuroectoderm,brain,mesoderm,stomodeum,andmidgut)(Fig.4,Fto whichspecificallymarksmotorneuronsdifferentiatingfromthemedial
I).Together,theexpressionpatternsofthemainRAsignalingcompo- neuroectoderminbothannelids(29)andvertebrates(32).InP.dumerilii
nentsinP.dumerilii(Fig.4,JtoM)areconsistentwiththenotionthat larvaeinjectedwithrarMO,wefoundreducedhb9-expressingcells
endogenouslyproducedRAactivatesRAR/RXRheterodimersinatissue- (55%,n=9)(Fig.5,NandO).Finally,wetestedwhetherthefailure
specificmannertofunctionduringdevelopmentofthenervoussystem todifferentiatemedialnervecordneuronsisduetodisturbedlarval
andthealimentarycanal. patterningmediatedbyhoxgenes,which,inP.dumerilii,showspatial
colinearexpressionalongthetrunkneuroectoderm(33).Contradicting
Medial neuron differentiation and connective formation thisnotion,thesegmentalexpressionofhox1intheneuroectodermwas
defects in RAR and RXR knockdowns notaffectedbyrarMOinjections(100%,n=20)(Fig.5,PandQ).
Theoverlappingneuroectodermalexpressionofrarandrxrgenesin Inlinewithrarandrxrexpressioninthestomodeum,whichlater
conjunctionwiththeabilityofbothreceptorstoformheterodimers developsintothelarvalforegut,wefurtherobservedaduplication(18%,
suggestsafunctionalinteractioninvivotocontrolnervoussystemde- n=22)orenlargement(27%,n=22)ofthestomodeumafterrarMO
velopment.Totestthishypothesis,wetargetedrarandrxrtranslation injections(Fig.5,A,C,E,andI),indicatingthatthereceptionofanen-
byamorpholinooligonucleotide(MO)knockdownapproach.Asafirst dogenousRAsignalisalsorequiredforproperstomodealdevelopment.
testforthecapacityandspecificityofthesynthesizedMOstoinhibit Comparablestomodeumduplications(8%,n=60)orenlargements
translationinP.dumerilii,weinjected,intoone-cell–totwo-cell–stage (16%,n=60)werealsoobservedinrxrknockdownspecimens(Fig.5,
embryos, h2b-gfp mRNA with an upstream MO target sequence GandI).
togetherwiththerarMO,rxrMO,or5-mismatchcontrolMOs.We Finally,coinjectionoftherarandrxrMOs(atlowerconcentrations)
foundthatboththerarandrxrMOsspecificallyabolishedgreenfluo- resulted in phenotypes identical to those obtained by single MO
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Fig.4. Expressionofretinoidreceptors(rarandrxr)inP.dumerilii.Geneexpressionisshowninblue.(AtoE)P.dumeriliirarexpression.(AtoC)rarexpressionin es
.s
formingneuroectoderm(ne),mesodermalprecursorcells[dorsallongitudinalmuscle(dlm)andmesodermalbands(mdb)],posteriorgrowthzone(blackarrowheads), c
ie
andthebrainregion(br).(C)At48hpf,rarisexpressedinneuroectoderm,inaregiongivingrisetomotorneurons(yellowdashedcircles),intheventralmidline(arrow, n
c
left)andsubjacentcells(whitearrowhead,right).(BtoD)Stomodealrarexpressionat30to72hpf(circles).(D)rarisexpressedintwodorsallylocateddomainsofthe e
m
brainat72hpf(whitearrowheads).(E)At6dpf(dayspost-fertilization),rarisexpressedinthemidgut(mg)andposteriorgrowthzone(blackarrowhead).(FtoI) a
g
P.dumeriliirxrexpression.(F)At24hpf,rxrisexpressedintheventralmidline(arrow)andtwodomainsofthebrain(blackarrowheads).(GandH)Introchophoreand .o
nmeecstoodcherametaelplarrevcauer,sroxrrcisellesx(pmredsbse)danindsthtoembordaienum(br()cairncdle)t.h(Ie)Beynt5iredpnfe,urxroreecxtpordeessrmion(nise)r,eisntrcilcutdedingtomtwotoordonmeuarionnsodfotmheainbsra(iyne(lalorrwowdhasehaedds)oauntdlintoe),tiassnudesinsuthrreouunnddienrglytinhge onrg/
midgut(mg).(JtoM)SummaryofthedevelopmentalexpressioninP.dumeriliiofRAmetabolismandsignalingcomponents.At24hpf(J),48hpf(K),and72hpf(L),aldh1a A
p
(green)genesareexpressedintheblastoporalregionandmacromeres,whereasrar(purple)isinneuroectodermandstomodeum.aldh1aandrarexpressionoverlapinthe ril 5
formingmesodermalbands.(M)Inthelatenectochaetelarva(5to6dpf),aldh1a,cyp26,andrarexpressionoverlapinthemidgut(red).Ventralviewsoflarvaeareshown.Scale , 2
bars,50mm.*orcircle,foregut;whitedashedline,ciliatedband;yellowdashedcircles,motorneurondomain. 0
1
9
injectionsinbothventralnervecordandstomodeum(Fig.5I),indicat- theventralnervecord,whichisconsistentwiththenotionthat,during
ingthatRARandRXRcooperativelyfunctioninthesamesignaling neurogenesis,endogenousRAR-dependentsignalingisrequiredatlar-
cascadeasRAR/RXRheterodimersmediatingRAsignalingduring valratherthanprelarvalstages(Fig.5,R,S,andV).Together,theresults
P.dumeriliidevelopment. oftheraroverexpressionandknockdownexperimentsareconsistent
witharoleforRARinneuronaldifferentiationandaxonformation
Enhanced commissural axon formation induced by ectopic withinthemedialpartofthelarvalP.dumeriliineuroectoderm.
RAR activity
TocomplementtheMO-basedknockdownresults,weinjectedmRNAs Impaired medial neuron differentiation by neuroblast
encodingthefull-lengthRARproteinintoP.dumeriliizygotes.Ectopic depletion following ATRA and 13cRA treatments
expressionofrarledtoalterationsofaxonalscaffoldsattheendofthe TofurtherinvestigatetherolesofRAsignalinginlarvalventralnerve
larvaldifferentiationphase(Fig.5,TtoV),inlinewiththephenotypes corddevelopment,weassessedtheeffectsofexogenousATRAand
ofrarMO–injectedlarvae.InrarmRNA–injectedlarvaefixedat48hpf, 13cRA,thetwoactiveRAisomerspresentduringP.dumeriliidevelop-
we observed an increase in the formation of commissural axons, ment. Larvae were treated with increasing concentrations of both
whereastheformationoflongitudinalconnectivesappearedunaffected compoundsinthemicromolarrange.BothATRAand13cRAtreat-
(Fig.5,TandU).Theectopiccommissuralaxonsmostlikelyemerge mentsatlarvalstagessloweddownthelocomotionofthelarvaeand
fromthemorelateralneuroectodermalregions(29),wherecommissur- completelyabolishedcrawlingmovements(movieS1),whichisindic-
alneuronsarelocatedinwild-typelarvae.Notably,rarmRNA–injected ativeofaspecificeffectonthedifferentiationandpossiblyprojectionof
larvaefixedatearlylarvalstages(at41hpf)didnotshowaphenotypein motorneuronsinnervatingthesomaticmusculature.Forcomparison,
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Fig.5. PerturbationofRARandRXRfunctionsinP.dumerilii.(AtoQ)KnockdownofrarandrxrbyMOinjectionsintoP.dumeriliizygotes.(A,C,E,andG)rarandrxr A
p
MstoOminojdeecatilodnesfceacutsseinaclrueddinucgtidounpolifcactoinonnsec(wtivheitse(cdoansnh;ewdhciitreclea)r.ro(Bw,sD),aFn,danmdisHg)uMidiasnpclaeceomfceonmtmanisdsureredsuc(ctoiomnmof;ylaerlvloawlsearrorotownse)ringicthneevuernontrsal(anrerorvwesc),obrdut(VnNotCo)fasnewuerlolnass ril 5
oftheearlynervoussystem(arrowheads),uponrarandrxrMOinjections.(I)Histogramshowingproportionsofobtainedknockdownphenotypes:normaldevelopment , 20
(“normal”),delayeddevelopment(“delay”),andVNCandstomodeumdefects(“VNC&stomodeumdefects”).(JandK)Boxplotsshowingthenumberofserotonergic 19
cellsandneuronsinthetrunkofP.dumeriliilarvae.Datadistribution(circles),medianvalues(boldline),andTukeywhiskersareshown.wt,wildtype.Unpairedtteston
themeanvaluewasusedforstatisticalanalyses(n.s.,nonsignificant).P=0.3352andP=0.5842in(J)andP=0.0566andP=0.6064in(K)for,respectively,rxr-5mm
controlMOversusrarMOandrxr-5mmcontrolMOversusrxrMO.(LtoO)Expressionoftheneuronaldifferentiationmarkersynaptotagmin(syt)(LandM)andofthe
motorneuronmarkerhb9(NandO),showingthatMO-inducedrarknockdowndisruptsdifferentiationinthelarvalnervoussystem(arrows),butnotintheearlylarval
nervoussystem(arrowheads).(PandQ)hox1expressionisunaffectedbyrarMOinjections(arrowheads).(RtoU)raroverexpressionbymRNAinjectionintoP.dumerilii
zygotes.(RandS)Earlylarvalnervoussystemisnotaffectedbyraroverexpression(arrows).(TandU)InjectionofrarmRNAinducesanincreaseofcommissuresinthe
larvalnervoussystem(arrows).(V)OverviewofembryonicandlarvalneurogenesisinP.dumeriliiwithrespecttothedifferentiation(diff.)offunctionalneurons(29).
Ventralviewsoflarvaeareshown.(AtoHandRtoU)Acetylatedtubuliningreen,serotonininred,andnucleiinblue.(LtoQ)Geneexpressioninblue.Numberof
affectedovertotalnumberofassayedspecimensisindicated.Scalebars,50mm.
similartreatmentsdidnotslowdowntheswimmingspeedofprelarvae serotonergicneuronsthatarelongitudinallyprojecting,aneffect,again,
(movieS2). morepronouncedfor13cRAthanforATRAtreatments(Fig.6,FtoJ
Concerningaxonalscaffoldformation,treatmentswithbothcom- andQ).ATRAand13cRAfurtherdecreasedthenumberofhb9-positive
poundsfrom48to80hpfledtoaclearconcentration-dependentreduc- cellsintheneuroectodermbutnotinthestomodeum(fig.S7,AtoF).In
tioninlengthanddensityoflongitudinalconnectives,withtheoverall contrast,theanteriorspatialboundaryoftheneuroectodermalexpres-
numberofcommissuralprojectionsbeinglessaffected(Fig.6,AtoE sionofhox1andhox4wasunaffected(fig.S7,GtoIandMtoO),whereas
andP).Ofnote,13cRAtreatmentsresultedinmoreseverephenotypes thatofhox1atthebaseofthecirriandinthestomodeumwasdown-
thanATRAtreatmentsatthesameconcentration.Corroboratingthis regulated(fig.S7,JtoL).Therefore,andstrikingly,rarMO–injected
further,treatedlarvaegenerallyshowedareductionofdifferentiated andRA-treatedlarvaeshowedverysimilarphenotypes.
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Counterintuitiveatfirst,wereasonedthatifRAactedasamotor RBP4,TTR,CRBP,andCRABP),allofwhichareabsentfromannelids.
neurondifferentiationsignal,itmightinterferewiththemaintenance Thissuggeststhattheancientmodeofsignalpropagationwasbymere
ofasymmetricallydividingneuroblaststemcellsintheneuroectoderm. diffusion(bothextra-andintracellularly)orthatretinoidbindingand
Consistentwiththisnotion,wenotedamarkedreductionofcelldivi- transporthaveprimitivelybeenassumedbyotherfactorsandthatthis
sionintheneuroectodermaltissueinbothATRA-and13cRA-treated primitivesystemhassecondarilybeenreplacedinchordates.Anintra-
larvaeafter4hoursof5-ethynyl-2′-deoxyuridine(EdU)incubation, cellularretinoidbindingcapacity,forexample,hasalreadybeende-
whereas cell proliferation in the brain appeared largely unaffected scribedformembersoftheILBP(intracellularlipidbindingprotein)
(Fig.6,KtoO).Tomorespecificallytestforthepresenceofneuroblasts superfamily in other protostomes, such as insects and crustaceans
intreatedyoungworms,weassessedneuroectodermalnk6andpax6 (35,36).
expressionatearlylarvalstages,whichdemarcatesbothproliferating Third,anotherpivotaldifferencebetweentheannelidandchordate
stemcell–likeneuroectodermalprecursorsanddifferentiatingneurons RAsystemsistheabsenceofanaxialpatterningfunctioninannelids,
(29).Inlinewiththeaboveobservations,theexpressionofbothmarkers togetherwiththelackofadirectregulationofhoxgenes.Thisclassical
wasstronglyreducedinaconcentration-dependentmanneraftertreat- andwell-knowndevelopmentalfunctionofRAsignalinghassofaronly
mentsatearlylarvalstages(Fig.6,RtoTandVtoX).Opticalcross- beendemonstratedinchordates,althoughitstillremainstobead-
sectionsrevealedthattheexpressionofbothgeneswasreducedindeep dressed inechinoderms and hemichordates. Therefore,thisroleof
tissuelayers,wheredifferentiatingneuronsarelocatedandcompletely RAcanbeconsideredeitheradeuterostomeorchordatesynapomorphy,
abolishedinsuperficiallayerscontainingdividingneuroblasts(Fig.6,U anditmayhaveevolvedconcomitantlywiththeelaborationofhigh-
andY).Therefore,thereducednumberofdifferentiatingmotorneurons affinityRARs.TheechelonedresponseofdifferenthoxgenestoRA
appearstobeparalleledbyadepletionofneuroblastsgivingrisetothese wouldthusbetheconsequenceofagainofregulatoryprecisionbya D
o
neurons,whichsuggeststhattheeffectofRAtreatmentisonthedividing high-affinityreceptorthatiscapableofinterpretingminuteligandcon- w
n
stemcellsratherthantheirdifferentiatingdescendants. centrationdifferencesalongananteroposteriormorphogengradient.In lo
a
chordates,thisgradientprovidespositionalinformationalongthe d
e
DISCUSSION astnrtuecrtouproesstienrioalrlbgoedrmyalxaiysetross(p2)e.cIifnyztheberlaofcisahti,ofnoranexdafmatpelseo,fitphaarsticbueleanr d fro
m
WehaveexhaustivelycharacterizedRAsignaling,bothbiochemically shownthatRAcandirectlyconveytieredpositionalinformationover h
and functionally, in the developing marine annelid P. dumerilii. Our longdistances,functioninginagradientthatisrobusttofluctuationsof ttp
work establishes firmly that RAR-mediated RA signaling evolved in RAsynthesisandwhoseestablishmentrequiresCYP26-dependentRA ://a
d
early bilaterians. However, our data also suggest an early role of this degradation(37).Together,weproposethatthetransformationofRAR va
n
pathwayverydifferentfromthecontrolofhoxanteroposteriorpatterning fromasensortoahigh-affinityreceptor,whichlikelytookplaceatthe ce
s
thathas been describedforvertebrates. baseofthechordates,wasaprerequisitefortheacquisitionofaglobal .s
c
First,theaffinityofthePduRARforitsligands(ATRAor13cRA) axialpatterningfunctionofRAasagradient-formingmorphogen. ie
n
anditslevelofactivationbytheseligandsaremuchlowerthanthose Onthebasisofthesefindingsandadditionalcomparativestudies,a c
e
m
knownforvertebratereceptors.Fortheannelidreceptor,micromolar coherentviewofRAsignalingevolutionisemerging.Earlyinmetazoan
a
retinoiddoseswerenecessarytoreach1/10ofthetranscriptionalactivity evolution,retinoidsappeartohavebeenrecruitedassignalingmole- g.o
measuredforhumanRARaatnanomolarretinoidconcentrations(8). cules.AlthoughRAR itself is absentincnidarians, theexistence of rg
OurstructuralanalysesestablishedthatPduRARaccommodatesATRA RXRorthologsactivatedby9cRAindicatestheexistenceofnuclearrecep- on/
viaabindingmodethatisdifferentfromtheoneusedbyvertebrate torscapableofmediatingretinoidactions(38),possiblyashomodimers A
p
RARs.IntheP.dumeriliiorientation,theliganddoesnotgenerate orheterodimerswithothernuclearreceptors(39).Incnidarians,two ril 5
thenecessarycontactswithresiduesoftheRARLBPtoensurehigh- major roles have emerged that may be derived from ancient RA , 2
0
affinitybindingandstrongstabilizationofthetranscriptionallyactive signalingfunctions.First,RAcontrolsthestrobilationprocessinthe 1
9
receptorconformation.Giventhatendogenousretinoidlevelsdonot jellyfishAureliaaurita,thatis,themetamorphosisfromthepolypto
seem to be overtly elevated in both developing and adult worms, themedusastage(39).Duringthisprocess,RAappearstobeproduced
whencomparedtothoseinvertebrates(3,4,26),theannelidRAR bytheinnerlayerbecausetheexpressionofraldhisstrongestinthe
thusappearstoactasaretinoidsensorratherthanasavertebrate-type endodermofthemouthregion,wherestrobilationisinitiated.Incon-
high-affinityreceptor.Suchasensorroleisexertedintissuesthatare trast,rxrispresentintheoverlyingectoderm.Second,RAsignaling
characterized by high exposure to RA, such as the neuroectoderm appearstoplayanancientroleinneuronaldifferentiation.AnRXRan-
overlyingtheRAproducingmesodermalbandsandmidgut.Itisfurther tibodylabelsdifferenttypesofneuronsinthepolypsoftheseapansy
supportedbythefactthatPduRARisactivatedbyATRAand13cRA, RenillakoellikeriandthecoralAcroporamillepora(40),suggestinga
whicharepresentinequivalentamountsinembryosandadults.Inline roleinneurogenesis.Inlinewiththishypothesis,RAinducesneuronal
withourdata,theRARoftheseaurchin,adeuterostome,ischaracter- differentiationinR.koellikeriprimarycellcultures(41),andRA-treated
izedbyaligandaffinitycomparabletothatofPduRAR(34).Together, planulalarvaeofthehydrozoanClavamulticornisshoweddefectsinthe
theseresultsindicatethattheancestralRAR,whichevolvedatthebase differentiationandpositioningofpeptidergicneuronsand,interesting-
ofthebilaterians,mighthavebeenaretinoidsensorandthathigh- ly,adisorganizedarrangementofnervenetaxons,indicativeofanRA
affinityRARsprobablyevolvedonlylater,specificallyinthechordate functioninaxonalpathfinding(42).
lineage. TwomajornoveltiesinRAsignalingthenoccurredinthebilaterian
Second,theaffinityrefinementofchordateRARswasverylikely stem line.First, RXR was complemented by asecond RAR, which
correlatedwithothermodificationsoftheRApathway.Forexample, formsheterodimerswithRXR(8).Weproposethat,viaopportunistic
ourstudyrevealedthatannelidsdiffer fromchordatesinthecom- evolution,RARevolved asanRAsensorby exploitingpreexisting
plementsofretinoidbindingandtransportproteins(thatis,STRA6, retinoids.Thisinvolvedchangesinspecificity,from9cRA(asreported
Handberg-Thorsageretal.,Sci.Adv.2018;4:eaao1261 21February2018 9of16
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Fig.6. EffectsofexogenousRAondevelopingP.dumerilii.(AtoQ)EffectsofATRAand13cRAonlarvaldevelopment(48to80hpf).(AtoE)Reductionofdifferentiationwithin
theVNC(ingreen,acetylatedtubulin),particularlyinlongitudinalconnectives(conn;whitearrows)andlessincommissuralprojections(comm;yellowarrows),wasobservedwith
increasingRAconcentrations.(FtoJ)Treatmentsreducethenumberofdifferentiatingserotonergicneurons(inred,serotonin)alongtheVNC(arrows).(KtoO)Numberofproliferating
cells(inpurple,4-hourEdUincubation)inlarvalneuroectoderm(circles)isreducedbyATRAand13cRA,whereascellproliferationinthelarvalbrainislessaffected(dashedcircles).(A,F,
andK)Thefirst(I),second(II),andthird(III)larvalsegmentsareindicated.(PandQ)BoxplotsofVNClengthandwidthandofserotonergicneuronnumbersindimethylsulfoxide(DMSO)
controlsandaftertreatmentswith4mMATRAor4mM13cRA.Datadistribution(circles),medianvalues(boldline),andTukeywhiskersareshown.Unpairedttestonthemeanvaluewas
usedforstatisticalanalyses(**P<0.01).(P)RatioofVNClengthandtotalbodylength,asshownin(A),andcomparisonofVNCwidthinthefirsttwobodysegments,indicatedaswidthI
andIIin(A).Leftboxplot:P=0.4048forDMSOversusATRAandP=0.0006forDMSOversus13cRA.Rightboxplot:P=0.002andP=0.0001(widthI)andP=0.0017andP=0.0001(width
II)for,respectively,DMSOversusATRAandDMSOversus13cRA.(Q)SerotonergicneuronnumbersinDMSO-,ATRA-,and13cRA-treatedembryos.P=0.6504forDMSOversusATRAand
P=0.0001forDMSOversus13cRA.(RtoY)Treatmentsreducenk6(RtoT)andpax6(VtoX)expressionintheVNCduringlarvaldevelopment(34to40hpf).Geneexpressioninpink.(U
andY)Orthogonalopticalsectionsshowingtheabsenceofnk6andpax6fromproliferatingneuroectoderm(superficiallayer,whiteoutline)anddecreaseofnk6andpax6expressionin
thepostmitoticlayersbelowtheneuroectoderm(middleanddeeplayers,purpleandyellowoutlines,respectively).VentralviewsofdevelopingP.dumeriliiareshown.Numberof
affectedovertotalnumberofassayedspecimensisindicated.Scalebars,50mm[except(U)and(Y),20mm].
Handberg-Thorsageretal.,Sci.Adv.2018;4:eaao1261 21February2018 10of16