a. L’adressage des récepteurs à la membrane ne coïncide pas forcément avec leur activation
La traversée de la PP par les axones commissuraux est intrigante puisque des axones naviguent dans un champ de signaux répulsifs. Les différents travaux de l’équipe, dont les miens, permettent d’améliorer notre compréhension de ce processus contre-intuitif. Ainsi nous comprenons mieux la régulation spatio-temporelle des récepteurs, permettant la sensibilisation aux signaux répulsifs, mais également la topographie physico-chimique du domaine que traversent les axones. Cependant, beaucoup d’inconnues subsistent. Parmi celles-ci, on retrouve la spatio-temporalité de l’activation des signalisations. En effet, différents mécanismes vont inhiber les récepteurs à la membrane. Comme présenté dans l’introduction, les récepteurs Robo possèdent une capacité d’auto-inhibition structurale, mais Robo1 va également être inhibé en formant des homodimères ou des hétérodimères avec Robo2 ou Robo3 (Long et al., 2004; Evans et al., 2015; Barak et al., 2019). On retrouve une capacité d’auto-inhibition chez les PlxnA. Ainsi, leur domaine extracellulaire va interagir en cis avec un autre domaine extracellulaire de PlxnA, en « tête-à-queue », pour inhiber les deux récepteurs et cette inhibition est levée en présence de Sema3B (Kong et al., 2016). Il est donc nécessaire de savoir si l’adressage des récepteurs à la membrane coïncide avec leur activation. PlxnA1 illustre bien l’importance de cette information. En effet, ce récepteur est partagé entre Sema3B et SlitC. Sachant que ces deux signalisations n’ont pas le même rôle dans la traversée, il est possible d’imaginer qu’elles
67 ne soient pas activées avec la même spatio-temporalité. Une autre hypothèse est que l’activation de ces signalisations, au sein du CC, soit compartimentée.
Les travaux d’Aurora Pignata ont montré que l’adressage des récepteurs à la membrane s’accompagne de changements comportementaux. Dans le cas de l’adressage de Robo1 ou Robo2, le CC va respectivement augmenter son comportement exploratoire ou tourner. L’étude de McConnell et al. suggère que les signalisations SlitN/Robo sont essentielles pour ces comportements (McConnell et al., 2016). Pour confirmer ces observations in vivo, les auteurs altèrent des effecteurs des signalisations. Cependant, ces effecteurs sont partagés par de nombreuses signalisations. L’étude des interactions ligands-récepteurs est donc nécessaire pour renforcer le lien entre récepteurs et comportements.
b. L’utilisation de la BiFC dans le contexte de la traversée est prometteuse
Nous avons essayé d’aborder la question en utilisant la BiFC, technique d’analyse d’interaction protéine-protéine. Malgré des premiers résultats encourageants, le développement de cette technique aurait nécessité un sujet entier. L’un des défis à relever provient de la sécrétion des ligands de guidage. En effet, les seules études de BiFC concernant des ligands sécrétés ont été effectuées au niveau de la synapse, où la distance entre la source du ligand et récepteur est très faible (entre 20 et 40nm) (Feinberg et al., 2008; Yamagata and Sanes, 2012; Macpherson et al., 2015; Shearin et al., 2018).
Dans le cas d’un paradigme expérimental basé sur l’électroporation, le contingent de ligands disponibles est constitué à la fois de ligand BiFC et de ligand sauvage. Ainsi, à moins d’être suffisamment proche d’une source de ligand BiFC, la quantité de ce dernier sera trop faible pour induire un signal détectable. Cependant, l’étude de la morphologie révèle le contact étroit entre CC et pieds basaux. Cette information suggère que l’étude des interactions ligand-récepteur de guidage, dans la PP, devrait être possible.
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Annexes
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SeminarsinCell&Developmental Biology
jo u r n al hom ep a g e :w w w . e l s e v i e r . c o m / l o c a t e / s e m c d b
Review
Commissuralaxonnavigation inthespinalcord: Arepertoire of repulsiveforcesisin command
HugoDucuing,ThibaultGardette,AuroraPignata,ServaneTauszig-Delamasure, ValérieCastellani∗
UniversityofLyon,UniversityofLyon1ClaudeBernardLyon1,NeuroMyoGeneInstitute,CNRSUMR5310,INSERMU1217,16rueRaphaelDubois,F-69000 Lyon,France
Thenavigationofcommissuralaxonsinthedevelopingspinalcordhasattractedmultiplestudiesover theyears.Manyimportantconceptsemergedfromthesestudieswhichhaveenlightenthegeneral mechanismsofaxonguidance.Thenavigationofcommissuralaxonsisregulatedbyaseriesof cel-lularterritorieswhichprovidesthediverseguidanceinformationnecessarytoensurethesuccessive stepsoftheirpathfindingtowards,across,andawayfromtheventralmidline.Inthisreview,wediscuss howrepulsiveforces,bypropelling,channelling,andconfiningcommissuralaxonnavigation,bringkey contributionstotheformationofthisneuronalprojection.
©2017ElsevierLtd.Allrightsreserved.
Contents
EarlytheoriesofchemotropismandchemoaffinitybyRamon YCajalandSperryprovidedthebasisformorethanacenturyof researchonaxonguidancemechanisms[1,2].Thesetheories
pos-∗Correspondingauthor.
E-mailaddress:valerie.castellani@univ-lyon1.fr(V.Castellani).
tulatedtheexistenceofmoleculesactingatlongandshortdistances toattracttheaxonterminal,thegrowthcone.Theirrolewas pos-tulatedtokeeptheaxons along theirproperpathand toguide themtowardstheirtargets.Unanticipatedlyfromthesetheories, repulsiveeffectsofaxonguidancemoleculesturnedouttoprovide majorforcesdrivingaxonnavigation.In1984,Haydonand collab-orators,usingvideo-timelapsemicroscopyinneuronalcultures, reportedthatserotoninhasaneuron-typespecificinhibitoryeffect https://doi.org/10.1016/j.semcdb.2017.12.010
1084-9521/©2017ElsevierLtd.Allrightsreserved.
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Fig.1.Modesofactionofrepulsiveforcesinaxonguidance.
Astheygrow,axonsareorientedbyacombinationofattractiveandrepulsiveforces.Amongtherepulsiveones,wecandistinguish3mainmodesofaction.A.Propelling.
Axonsperceivediffusiblechemorepulsivecuesemanatingfromagroupofguidepostcellsandturnawayfromthissource.B.Confinement.Agroupofguidepostcellsact asabarrierandconfinetheaxonswithinaterritory.Theynotnecessarilydeflectthemawaybutpreventthemfromexitingit,partlybytheemissionofdiffusiblecues.C.
Channeling.Severalgroupsofphysicallyseparatedguidepostcellsconstraincommissuralaxonswithinanarrowpath,byreleasingrepulsivecues.
ongrowthcones[3].Thepropertyofgrowthconestructuresto retractaftercontactwithsomeotheraxonalmembranesurfaces wasthendiscoveredbyKapfhammerandRaper[4].These obser-vationsechoedthoseofVerna,whowrotethatdorsalrootsensory neurons“interact differently withdermal than with epidermal cells.Whilenervefibresreadilyextendoverdermalcells,forming closemembraneassociationswithsomeofthem,theydemonstrate astrongavoidancereactionwithepidermalcellsbychangingtheir directionofextension”[5].Hepostulatedthatmoleculesreleased byepidermalcellsmightdeflectawaynervefibregrowth trajec-tory,oppositelytothosewhichwerefoundtoattracttheaxons, namelyatthattime,theneurotrophicfactorNGF[6].Fromthese pioneerfindings,repulsiveforceshavebeendemonstratedtoplay instrumental rolesin a largerange of developing neuronal cir-cuits. Asevidenced by numerous studies[7,8], repulsive forces canconstrainaxonnavigationinvariousways,channellingaxonal bundles,deflectingawaythegrowthconetrajectory,andcreating sharpboundariestodelineatenon-permissiveterritories(Fig.1).
Asinapinball,repulsiveforceswouldactaslaunchpad,bumpers, andslingshotstopropelanddynamicallyimpactontheaxon/ball trajectory.The developmentof commissural axons providesan appealingcontext toinvestigate how suchrepulsiveforces can directaxonnavigation.Wereviewheretheprincipalyet identi-fiedsourcesofrepulsivecues,thenatureoftheirinfluencesand themolecularsignalsmediatingtheiractionduringcommissural axonnavigationinthespinalcord.
1.1. Formationofcommissuralcircuits
InBilateria,commissuralneuronsformcomplexcircuitsthat interconnectbothsidesofthecentralnervoussystem(CNS).They areessentialforthecorrectprocessingandcoordinationof vari-oussensorymodalities,motorresponses,andotherbrainfunctions [9]. Theseinterneurons extendtheiraxonacrossthemidlineat variousaxiallevelsoftheCNS.For instance,thecorpuscallosum enablescommunicationbetweentheleftandrightcorticalareas, theopticchiasmallowsorganismswithbilateralvisiontocorrectly integratevisualcues,andspinalcommissuresensurethecorrect coordinationofvariousmotorcommands.Thesecommissuresare establishedduringembryonicandearlypost-nataldevelopment inahighlyspecificspatialandtemporalmanner[10].Defectsin thecorrectwiringofcommissuralcircuitshavebeenobservedin manyneurodevelopmentaldisorders.However,ifmalformationsof
thecorpuscallosumhavebeenwellcorrelatedwithvarioushuman disorders,littleisknownoftheconsequencesofspinal commis-suresdefects.Indeed,patientshavingmutationsinROBO3gene, affectingcommissuresofthehindbrainandthespinalcord,have nolargesensorimotordeficit.Rather,theyexhibitaveryspecific diseasereferredtoashorizontalgazepalsywithprogressive sco-liosis(HGPPS)[11].Thissuggestshighdegreeofcompensationof commissuraldefectswithdevelopmentalorigin.
1.1.1. Developmentofthedorsalcommissuraltract
Thespinalcommissuralneuronsareaheterogeneous popula-tionsubdivided in severalpools, differingin theirlocation and timingof birth,each ofthem specifiedbyvarious transcription factors[12,13].Amongthem,dI1interneuronssettleearlyinthe mostdorsalpartofthespinalcord,closetotheRoofPlate(RP).
TheyarisefromaMATH1-positivepoolofprogenitors,that gener-atesbothipsilateralandcommissurallineagesandarespecifiedby LHX2/LHX9transcriptionfactors[14,15].dI1commissural(dI1c) neurons trajectory is highly stereotyped and has been exten-sivelystudiedinthemouse,notablybyusingMATH1::LacZand MATH1::GFPtransgenicmice [16]. dI1c axons firstextend ven-trally,turningawayfromtheRPandlayingclosetothepialsurface (Fig.2).Ataroundmid-distanceoftheventralbordertheybreak awayfromthelateralborder tore-orientmediallytowardsthe centralFloorPlate(FP)byrunningalongthemotoneurondomain.
Suchbreakoftrajectoryisalsotypicalofchickcommissuralaxons, apartfromthepioneeroneswhichcoursewithcircumferential tra-jectory.Incontrast,inxenopusandzebrafishembryos,theaxons coursebyfollowingthecircumferenceofthetubeuntilreaching theFP[17,18].Next,commissuralaxonsentertheFP,crossitand turnrostrallywithoutevercrossingthemidlineagaintoconnect theirfinaltargets.CommissuralneuronsarisearoundE9.5inthe mouseandnavigatetowardstheFPfromthisstage.ByE10.5,some ofthemhavealreadycrossedthemidlineandbyE12.5,mostof themhave.ByE13.5,theyarenavigatingtowardstheirfinaltarget followinglongitudinalroutes[19–21].
1.2. Guidepostterritoriesinstructingcommissuralaxon navigationinthespinalcordthroughrepulsiveaction
Historically,themainintermediatetargetandcrucialsignaling hubforcommissuralaxonnavigationhasbeenfoundtobetheFP.
ItheavilyinfluencesthedI1cguidance,andwecanthusreferto
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Fig.2.Guidepostterritoriesinstructingcommissuralaxonsnavigationinthespinal cord.
Spinalcommissuralaxontrajectoryishighlystereotyped.dI1caxons(inblack)arise fromadorsalterritory.Theyextendventrally,crossthemidlinethroughthefloor plate(FP),andthenturnrostrallywithoutevercrossingthemidlineagaintoconnect theirfinaltarget.Thistrajectoryisinfluencedbyvariousguidepostcellsalongthe way.First,axonsarepushedawaybychemorepulsivecuesemanatingfromtheroof plate(RP)(inblue)andfollowthepialsurface(ingreen).Meninges(ingrey)andthe DREZ/DRBZ(inred)keeptheaxonsawayfromtheCNS/PNSboundarythrough dif-fusiblecues.Ataroundmid-distancefromtheventralside,axonsturntowardsthe FP(inpurple),thenrunalongthemotoneuronsdomain(inred).Axonsneverenter themotoneurondomainnortheventricularzone(inlightgreen),theseterritories channelingthecommissuraltracttowardstheFP.AstheyreachtheFP,axons inter-actwiththebasallamina(ingreen)andnavigatethroughtheFPglialcellsradial processes.Uponcrossing,theygainsensitivitytorepulsivecuesemanatingfrom theFP,thattheydidnotperceivebeforeandthusexittheFP,accomplishasharp turningintherostraldirectionandnavigatelongitudinallyinbundles,guidedby variousgradientsofguidancecues,includingrepulsiveones.
FPcellsasguidepostcells.TheFPhasbeenextensivelystudiedand manyofitsmolecularmechanismshavebeenunveiled.However, avarietyofothercellswithinthespinalcordbringcontributionsto thenavigationofcommissuralaxons,includingglialcells,neurons andprogenitors.Thesedifferentcell-typescontributetogetherto sharplydelineatethepathofcommissuralneurons.
1.2.1. Kickoffrepulsiveforcestoorientcommissuralaxon navigation
1.2.1.1. Theroofplate. TheRPisprobablythesecondmoststudied groupofguidepostcellsaftertheFP.Itiscomposedofglialcells thatlayonthedorsalmidlineofthespinalcord.Thesecellscome fromprogenitorsthatareinducedinthemostlateralregionsofthe neuralfolds[22].ThisinductionreliesheavilyonBMPsignalling, mediatedbythetranscriptionfactorsLMX1A/B[23].Uponneural tubeclosure,theyarenoteasilydistinguishablefromothercells, inparticularneuralcrestcells,butastheydifferentiatetheystart toexpressspecificmarkers,notablyBMPsandWNTs[22].TheRP isthefirstdorsalstructuretodifferentiateandthenimpactsall otherdorsalpopulationsdifferentiation.Electro-microscopy stud-iesrevealed that, when differentiated, RP cells have two small processesextendingradiallytowardsthepialsurfaceandthe cen-tralcanal[24,25].TheRPactsasabarrierthatnoaxoncancross
beforeE16.5,whenadorsalcommissureisestablished[26]. Inter-estingly,theRPitselfundergoesratherimportantmorphological changesbetweenE11.5andE16.5,fromanarchstructuretoathin wall-likestructure[25].TheRPisacrucialorganizingcentreofthe differentdorsallineages.NotablythroughBMPsandWNTs,it spec-ifiesseveralclassesofadjacentdorsalinterneuronsandregulates theirproliferation,migration,andguidance[27].
Beyondthispatterning function,RPcells providethedriving forcewhichpropelsdl1caxonsemergingfromtheirsomaaway fromthedorsalside.Thiseffectwasshowntobemediatedbythe morphogensBMPs,namelybyGDF7:BMP7heterodimersactingvia theBMPRIBreceptor[28,29](Fig.3).Notonlytheirdirectionbut theirgrowthratealsoappearsregulatedbyBMPs[30].An addi-tionalrepellentproteinwasfoundproducedbytheRP,asecreted factor named Draxin.Draxin mutant micedisplay commissural axonalmigrationandfasciculationdefectsconsistentwitha repul-sivemodeofaction[31].DraxinsharesUNC5,DCC,andNeogenin receptorswithNetrin-1,asecretedmoleculeinitiallydiscoveredto actasachemoattractant[32,33].AlthoughDraxinhasbeenshown tobindUNC5andNeogenininvitro[34],itsrepellentroleinvivo wasreportedtobetriggeredviaitsbindingtoDCC[35](Fig.3).It canalsobenotedthattherepulsivefactorSlit2ishighlyexpressed attheRPatE13[36].Mostdorsalcommissuralaxonsarealready ontheirwayinthecontralateralsideatthisstage,whetherthis sourcecontributestothekickoffofcommissuralaxonsistherefore questionable.
1.2.2. Repulsiveforcestoconfinecommissuralaxonnavigationin thecentralnervoussystem
Commissural axons are destined to connect neurons of the centralnervoussystem(CNS)andmustbeconsequentlystrictly confinedwithinthespinalcord.Thisisnottrueforallspinalcord axons,sinceonthecontrary,thoseofthemotoneuronsprojectout oftheCNSthroughtheMotorExitPoint(MET).Moreover,inthis case,onlytheaxonsexittheCNS,theneuronalsomabeingconfined withintheCNS.Conversely,sensoryaxonsofthedorsalroot gan-gliapenetratethespinalcordviatheDorsalRootEntryZone(DREZ), whiletheirsomaremainoutside.Incontrast,somenon-neuronal cellsentertheCNS,suchasendothelialcellswhichinfiltratethe CNStissuetobuildthebloodvessels.Thus,cellsandneurites traf-fickingacrosstheCNS/PNSfrontierisstrictlycontrolled,fromthe onsetandthroughoutlife.
1.2.2.1. Confinementby themeninges. Meningesarea protective multi-layered structure that envelops the brain and the spinal cord.Theyaremainlycomposedoffibroelasticcellsandblood ves-sels.Meningesoriginatefromsomaticmesodermthatcoversthe neuraltube shortly afterneuraltube closure, aroundE9 in the mouseembryo[37,38].Theyactasbarriersthroughoutlife, con-trollingexchangesbetweenthecentralnervoussystemandwhat laysoutside.Inthebrain,meningeshavebeenshowntoinitiate amorphogenicsignalingcascadethatregulatesthedevelopment ofamajordorsalcommissure,thecorpuscallosum.Themeninges inhibitcallosalaxonoutgrowththroughBMP7.WNT3,expressedby pioneercallosalaxons,latercountersthiseffect.WNT3expression isregulatedbyanothermemberoftheBMPfamily,GDF5,expressed byadjacentCajal-Retziusneurons,whichinturnisregulatedby asolubleinhibitor,DAN,expressedbythemeninges[37].Inthe spinalcord,invivostudieslacktohighlighttheroleofthemeninges oncommissuralneurondevelopment.However,arecentinvitro studyshowedthatthemeningesareabletoproducesecretedcues thatcaneitherattractorrepulsedifferentneuronalpopulations.
Consistentwithinvivobehaviours,theseexperiencesshowedthat motoneuronsandsensoryneuronsareattractedbymeningeswhile ipsilateralandcommissuralneuronsarerepelledbythem[38].
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Fig.3.Kickoffrepulsiveforcesattheroofplate.
TheBMPfamilymembersGDF7andBMP7aresecretedbyroofplategliacellsandbindtotheaxonalBMPR-IBreceptorandrepelaxonstowardtheventralpartofthespinal cord.Inparallel,thesecretedfactorDraxinpropelsaxonsviaaxonalDCC.
1.2.2.2. Closing the CNS/PNS gate: confinementby the dorsalroot entryzone (DREZ)/ dorsalroot bifurcationzone (DRBZ). Commis-suralaxonsnavigateatcloseproximityofthepia.Earlystudies establishedthattheirgrowthconesrarely,ifever,contactthebasal lamina(Holley&Silver1987,Yaginumaetal1991).Thebasal lam-inais punctuatedbytheDREZand theMET, which ensurethe circulationbetweenthecentralNervousSystem(CNS)andthe sur-roundingtissues.TheDREZconsistsofabreakinthelaminaanda clusterofspecializedcellsarisingfromtheneuralcrest,the bound-arycapcells,whichpreventbothcellbodiesandtheiraxonsfrom leavingtheCNS,andgapsbetweentheglialend-feet[39].Around E11,theperipheralsensoryneuronssendaxonstowardsthespinal
1.2.2.2. Closing the CNS/PNS gate: confinementby the dorsalroot entryzone (DREZ)/ dorsalroot bifurcationzone (DRBZ). Commis-suralaxonsnavigateatcloseproximityofthepia.Earlystudies establishedthattheirgrowthconesrarely,ifever,contactthebasal lamina(Holley&Silver1987,Yaginumaetal1991).Thebasal lam-inais punctuatedbytheDREZand theMET, which ensurethe circulationbetweenthecentralNervousSystem(CNS)andthe sur-roundingtissues.TheDREZconsistsofabreakinthelaminaanda clusterofspecializedcellsarisingfromtheneuralcrest,the bound-arycapcells,whichpreventbothcellbodiesandtheiraxonsfrom leavingtheCNS,andgapsbetweentheglialend-feet[39].Around E11,theperipheralsensoryneuronssendaxonstowardsthespinal