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Reduce, Reuse, Recycle - Developmental Signals in Spinal CordRegeneration

Citation for published version:Cardozo, MJ, Mysiak, K, Becker, T & Becker, CG 2017, 'Reduce, Reuse, Recycle - Developmental Signalsin Spinal Cord Regeneration', Developmental Biology. https://doi.org/10.1016/j.ydbio.2017.05.011

Digital Object Identifier (DOI):10.1016/j.ydbio.2017.05.011

Link:Link to publication record in Edinburgh Research Explorer

Document Version:Peer reviewed version

Published In:Developmental Biology

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Download date: 25. Mar. 2020

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Title:Reduce,Reuse,Recycle-DevelopmentalSignalsinSpinalCordRegeneration

MarcosJulianCardozo,KarolinaS.Mysiak,ThomasBecker,CatherinaG.Becker*

*Correspondingauthorcatherina.becker@ed.ac.uk

CentreforNeuroregeneration,EMS:BiomedicalSciences,49LittleFranceCrescent,EdinburghEH164SB

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Abstract

Anamniotes, fishes andamphibians, have the capacity to regenerate spinal cord tissue afterinjury,generatingnewneuronsthatmatureandintegrateintothespinalcircuitry.Elucidatingthe molecular signals that promote this regeneration is a fundamental question inregenerationresearch.Modelsystems,suchassalamandersandlarvalandadultzebrafishareusedtoanalysesuccessfulregeneration.Thisshowsthatmanydevelopmentalsignals,suchasNotch, Hedgehog (Hh), BoneMorphogenetic Protein (BMP), Wnt, Fibroblast Growth Factor(FGF), Retinoic Acid (RA) and neurotransmitters are redeployed during regeneration andactivateresidentspinalprogenitorcells.Herewecomparetherolesofthesesignals inspinalcorddevelopmentandregenerationofthemuchlargerandfullypatternedadultspinalcord.Understanding how developmental signalling systems are reactivated in successfullyregeneratingspeciesmayultimatelyleadtowaystoreactivatesimilarsystemsinmammalianprogenitorcells,whichdonotshowneurogenesisafterspinalinjury.

Keywords

neuraltube,regeneration,development,signallingpathways,spinalcordinjury,CNS

Highlights

Regeneratingspeciesupregulatedevelopmentalsignallingsystemsduringrepair.

Spinalprogenitorcellsintegrateamultitudeofsignalstospecificoutputpathways.

Developmentalsignalsdirectregenerationofthefullypatternedadultspinalcord.

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1.Introduction

How animals are capable of reconstructing a functional tissue after a disruptive injury is afundamentalquestion in regenerativebiology.Oneprincipleappears tobe thatmechanismsthat regulate cell proliferation, differentiation or death during development are redeployedafterinjury.

Mammalscanfunctionallyregenerateafeworgans,suchastheliver,butthecentralnervoussystem (CNS) regenerates very poorly [e.g.(Hugnot and Franzen, 2011)]. In contrast, someadultvertebratescanregeneratemanyorgansystemssuccessfully,includingheart,limbsandCNS(Gemberlingetal.,2013).Teleostfishes,larvalXenopusandsalamandersaretheleadingmodels thatcansuccessful regeneratespinalcordafteran injury (DiazQuirozandEcheverri,2013;Edwards-Faretetal.,2017).Recently,zebrafish larvalmodelsofspinalcord injury(SCI)have been introduced, taking advantage of tissue transparency, and transgenic andmutantlines that allow for time-lapse and drug screening analyses (Briona and Dorsky, 2014;Ohnmachtetal.,2016).

Because regeneration of the spinal cordmay recapitulate steps in its the development, webriefly summarise major events in spinal cord development. The construction of the spinalcord during initial development requires specific signalling pathways that regionalize andpatternthewholetissuethatthengrowsandgainscomplexity.Primaryneurulationorneuraltube (NT) closure occurs during early development, after the specification of the germinallayers.Theflatneuralplateundergoesaseriesofmorphogeneticchangesthatendinadorsalfusion resulting in a closed NT in amniotes. In amphibians and teleosts this process differs(Arayaetal., 2016; LoweryandSive,2004). The cellsof theneuralplate fuse in themidlinegivingrisetoastructurecalledneuralkeel,whichreorganisesinaneuralrod,thusgivingriseto a NT by a process of cavitation. Independently of this difference in neurulation amongspecies, themolecular instructions used forNT patterning are evolutionarily conserved to ahighdegree(Arayaetal.,2016;LoweryandSive,2004).

Wnt,FGFandRAsignalgradientsestablishtheantero-posterioraxisoftheCNS(Kudohetal.,2002; Storey et al., 1998). These gradients also serve as axon guidance cues duringdevelopment and lead to axonal regeneration along the antero-posterior axis after SCI(RasmussenandSagasti,2016).

Alongthedorso-ventral(DV)axis,thepatterningoftheNTismainlycontrolledbyHh,WntandBMP signal gradients (Fig. 2) (Le Dreau andMarti, 2012). These gradients establish specificdomains of progenitor cells which express distinct combinations of homeodomain proteins(Briscoeetal.,2000).Theseactivatespecifictranscriptionalprogrammesinprogenitorcells,sothat distinct progenitor domains give rise to different population of neurons (Gouti et al.,2015).

ProliferationanddifferentiationofprogenitorcellsalsorequiresactivityofmanyoftheabovesignalsaswellasactivityofNotch(YeoandChitnis,2007),RA(Wilsonetal.,2004),FGF(Diez

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del Corral and Storey, 2004) signalling and the action of different neurotransmitters likedopamineorserotonin(Barreiro-Iglesiasetal.,2015;Reimeretal.,2013).

During successful spinal cord regeneration, molecular pathways initially used to build thetissue/organduringdevelopmentareredeployed.IntheCNS,Wnt,BMPorHhpathways(Fig.1),whichareregulatingdevelopmentalneurogenesis,aredramaticallyreactivatedafterinjury(Vergaraetal.,2005).Duringadultregeneration,distancesoverwhichsignalgradientshavetobeestablishedaremuchlarger.Forexample,proliferationofventricularprogenitorcellsleadstoanexpansionofthespinalcordcentralcanal inzebrafish,rats,andnewts(Radojicicetal.,2007;Zukoretal.,2011) (Fig.2).Newneuronsand theirprocessesalsohave tocover to re-establishconnectionscanbemuchgreaterthaninembryos.Moreover,theadultcellularandextracellularmatrixenvironmentdiffersfromthatduringdevelopment.Afterinjury,processes,such as such inflammation (Kizil et al., 2015) and scar formation contribute to this differentenvironment(RaposoandSchwartz,2014).In this review we compare the involvement of the different signalling pathways inneurogenesisduringdevelopmentandregenerationofthespinalcordafteraninjury.

2.Pathways

2.1Notch

Notchsignallingisknownasaregulatorofneuralprogenitorcellfate,controllingneurogenesisandgliogenesis (Ablesetal.,2011).Notch is important in juxtracrinecell-cellcommunication(Fig.1).However,Notchsignallingcanalsooccurbetweennon-neighbouringcellsbytransientbasalactin-basedfilopodialcontacts(Cohenetal.,2010).

Delta-Notch signalling participates in NT formation from early developmental stages.DisruptionofNotchsignalingpreventsprogenitorscellsinHensen'snodeinthechickenfrompopulating the floorplate (GrayandDale,2010).Notchactivity isnecessary tomaintain theidentityofthefloorplateitselfinchickhindbrainandspinalcord(leRouxetal.,2003).Mutantstudies inzebrafishshowthatDelta-Notchsignaling is required for lateral floorplateandP3domaindevelopment(Schaferetal.,2007).Conditionalknock-outofthegeneencodingtheE3ligaseMindBomb-1,whichregulatesendocytosisofNotchligands,showsthatNotchcontrolsneurogenesis and gliogenesis during NT development in mice (Fig. 2) (Kang et al., 2013).notch1 or notch3 are expressed in precursors and immature neurons of the spinal cord inmammalsandareimportantforneuronaldifferentiationandmaturationoftheSC(Yamamotoet al., 2001). Indeed,notch1 expression is enhanced in the SCafter injury in theependymaland parenchymal cells and is thought to suppress neurogenesis (Fig. 2) (Yamamoto et al.,2001).

In thezebrafishspinalcord,differentproliferativeprogenitorsexpressnotch receptorgenes,whereasprecursors andpost-mitotic neurons expresseddeltaA and jagged2 ligand genes.AdisruptionofDeltafunctioncausesoverproductionofearly-specifiedmotorneurons(MNs)inzebrafishwhich leads to a depletion of progenitors in themotor neuron progenitor domain(pMN)overtime(AppelandEisen,1998).Thisisalsoobservedindelta1andhes1;hes5double-mutantmice(Hatakeyamaetal.,2004;Marklundetal.,2010).jagged2,expressedinthepMN,seemstomaintainthiscellpopulationinanundifferentiatedstate.TheinhibitionofJagged2-

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mediated signalling bymorpholinos (MO) in zebrafish or short-hairpin RNA (shRNA) in chickcause variations in thenumberofMNandGABAergic Kolmer-Agduhr (KA'') cells cells in theventral spinal cord (Yeo andChitnis, 2007). This particular activity ofNotch signalling seemsimportant for SC regeneration in adult zebrafish, asnotch1a/b, notch2,her4.1, her4.5, her9,jagged1b and deltaC genes are upregulated after SCI (Fig. 2). Indeed, the transgenic over-activationofNotch signalling inependymo-radial glial (ERG) cells, theprogenitor cells in thezebrafishspinalcord(Reimeretal.,2008),duringregenerationreducesproliferationofERGsandthenumberofnewmotorneuronsgenerated.Conversely,theblockingofNotchsignallingusing the gamma-secretase inhibitor DAPT increases motor neuron generation after SCI.Similarly, Notch activity in progenitor cells in the adult zebrafish telencephalon duringconstitutiveneurogenesisalsoattenuatesneurogenesis(Chapoutonetal.,2010).TheseresultshighlightthepotentialofmodulatingNotchsignallinginprogenitorcellsbydrugtreatmentsinordertopromotegenerationofmotorneurons(Diasetal.,2012).TheexpressionpatternandrelationshipamongNotchsignallingproteins inpMNcellsandMNsduringdevelopmentandadulthoodandthefactthatNotchsignallingdynamicscontrolasymmetriccelldivision intheNT (Das and Storey, 2012) raise the question whether altering the mode of division ofprogenitorcellscouldleadtomotorneurogenesisafterspinalcordinjuryalsoinmammals.

Notch interacts with other pathways during development and regeneration. For example,Notch1inactivationspecificallyinneuralprogenitorcellsresultsinanincreaseinthenumberof V2 interneurons at the expense of MNs and in a gradual disappearance of the ventralcentral canal inmice (Peng et al., 2007). This is similar tomanipulations of theHhpathway(belowandFig.3)(Yangetal.,2006).Indeed,NotchsignallingmaintainsHhresponsivenessinneural progenitors duringdevelopment of the spinal cord, shown in zebrafish (Huang et al.,2012).NotchisassociatedwithHhsignallingattheleveloftheprimarycilium(Stasiulewiczetal., 2015). This cellular structure plays important roles in Hh, Wnt and Notch signaltransduction and pathway activity (Sasai and Briscoe, 2012) (Fig. 3). A relation with BMPsignallinghasbeenobservedduringfrogtadpoletailregenerationthatincludestheSC.Inthiscontext, the activation of Notch signalling is sufficient to reactivate regeneration afteramputationduringarefractiveperiodduringwhichtailregenerationnormallydoesnotoccur.Conversely,regeneration isabolished ifNotch isblockedduringtheregenerativeperiod.ThenotcheffectisapparentlydownstreamofBMPsignalling(Fig.3)(Becketal.,2003).

It is still unknownwhetherNotchparticipates in the regenerationof interneuronsduring SCregenerationaswellaswhetherandhowNotchactivityinteractswiththatofothersignallingpathwaysthatareredeployedduringregeneration,suchasHhorBMPs.

2.2Hedgehog

Signallingmediatedbythemorphogengradientsetupbythesecretedproteinhedgehog(Hh)determines the ventral patterning of the CNS and therefore plays pivotal roles in nervoussystem development. Its activity is crucial for early specification, regionalization anddifferentiation of the NT and for subsequent axon guidance and connectivity (Briscoe andTherond,2013)(Fig.1).

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DuringNTdevelopment,Shh,expressedinthenotochord,activatesthesignallingpathwayintheNTfloorplate(Echelardetal.,1993).Thefloorplatecells,sourceofmanyinductivesignals,thenstarttoexpressShhthemselves.Theproteindiffusesdorsally,thussignallinginagradedmannerfromventraltodorsal.Thisleadstoestablishingdifferentprogenitordomains(Fig.2)(Chamberlain et al., 2008). Shh spreading and responsiveness of neural progenitor cells arebothcriticalforpatterning(Dessaudetal.,2008).IthasbeenalsoshownmorerecentlythatinadditiontodiffusionHhligandcanbetransportedthroughfilopodia-likestructures(Sandersetal., 2013), but it is still unknown if thismodeof transport plays a significant role duringNTdevelopment.

DuringNTdevelopment,thereceptorandnegativefeedbackregulatorptch1/2isexpressedinthe domains immediately dorsal to the source of Shh, thus inhibiting / balancing themorphogenactivity.Ligandconcentrationandtheexposuretimetotheligandaffectthefinalfateof thereceivingcells (Dessaudetal.,2007). FailureofHhsignallingstronglyaffects theventralandalsodorsalpatterningoftheCNS(Chiangetal.,1996).TheShhsource,adjacenttopMNandP3domains,iscriticalforanormalbalanceofgenerationofMNs,oligodendrocytesandventral interneurons(RibesandBriscoe,2009).ZebrafishfloorplatesecretesShha,Shhband also Ihhb which seems particularly important for oligodendrocyte differentiation inzebrafish(Chungetal.,2013).

During regeneration of the axolotl tail, the activity of Hh proteins as ventral organiser isimportant.ShhexpressionintheventralmostcellsoftheSCisrequiredfortheestablishmentof DV progenitor domains in the newly forming ependymal tube. The proliferation of theblastemaandtheoveralltailregenerationalsodependsonHhsignalling(Schnappetal.,2005).

Afteratransectionlesion,ashasbeendoneinthelesionedspinalcordofadultzebrafish,thepatterning activity of the hedgehog pathway may not be required, as the spinal cordprogenitorcellsmaintaintheirprogenitordomainidentityfromdevelopment.However,duringregeneration after SCI, shha,ptch2, smo andHh target genes, likeolig2, are upregulated inependymalcells(Fig.2)andthis is importantfor injury-inducedregenerationofneurons.Forexample, the generation of new MNs (Reimer et al., 2009) or serotonergic interneurons(Kuscha 2012) after injury is reduced by blocking Hh signalling with cyclopamine. Drugtreatmentexperiments suggest thatduringMNdevelopmentand regeneration, thestrengthofHhsignallingcanbemodulatedbydopamine through thedopaminereceptorD4a (Fig.3)(Reimeretal.,2013).Interestingly,shhisalsoupregulatedafterSCcompressioninjuryinmice;expressinghighlevelsforuptoatleast1monthafterinjury(Fig.2)(Chenetal.,2005).

FutureresearchisneededtounderstandhowHhpathwaycomponentsareconfiguredinadultprogenitorcellsandhowshhexpressionisupregulatedandtheproteinspreadsduringspinalcord regeneration. In other tissues like the heart the outflow tract (epicardial layers of theatrium and the bulbous arteriosus) works as a signalling centre and source of Hh ligand topromoteepicardialregenerationaftercardiacdamage(Wangetal.,2015).

During spinal cord development, Hh signalling controls regulators of the cell cycle in neuralprogenitors (Cayusoetal., 2006), themodeofdivisionof thepMNcells (Saadeetal., 2013)andthesizeandproportionsoftheNT(Uyguretal.,2016). Itremainstobefullyestablished

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which of the different actions of Hh signalling are recapitulated in the progenitor pool ofependymalcellsduringSCregeneration.

2.3BMP

TheBMPpathwayregulatesmanyfundamentalprocessesduringdevelopmentandadulthoodand for that, itwas suggested to call it “body”morphogeneticproteinpathway, rather than“bone”morphogeneticprotein, according towhere ithadbeenoriginallydiscovered (Reddi,2005).

The BMP pathway (Fig. 1) is critical for CNS development (Hegarty et al., 2013). Duringgastrulation,neuralinductionfromtheSpemannorganizerispromotedbyNoggin(Lambetal.,1993)whichbindsBMP4withhighaffinityblockingBMPsignallingactivity(Zimmermanetal.,1996).Once theneural tissue isdetermined,BMPsignallingplays important rolesduringNTdevelopmentfrompatterningtogenerationofpost-mitoticneurons,axonguidance,synapseformationandgliogenesis(LeDreauandMarti,2013).

BMPgradientformationiscrucialforDVbodyaxispatterning, includingDVpatterningoftheNT, and this action is highly conserved among animals (Bier and De Robertis, 2015). Theexpression of bmp4 and bmp7 in the epidermal ectoderm is important for the dorsal fateacquisitionoftheprospectiveNTatneuralplatestage(Fig.2)(Liemetal.,1995).BmpactivityisrequiredfortheentirepatterningalongtheDVaxisaffectinggenerationofneurons(Barthetal., 1999). After SCI in adult rats, bmp7 expression is upregulated in glial cells and MNs(Setoguchietal.,2001).bmp2 isalsoupregulatedafterSCI inmouse(Setoguchietal.,2004).bmp4isupregulated1monthafterinjuryinmouse.bmp4appearsupregulatedinneuronsandGFAP-positive astrocytes. High levels of bmp4 are also found in the ependymal cells, thepresumptivespinalprogenitorcells,within1mmofthelesionsite(Fig.2)(Chenetal.,2005).TheseresultssuggestanactiveroleofBMPsafterinjury.Indeed,inhibitionofBMPsignallinginXenopus tadpoles prevents regeneration of the tail after amputation. Conversely, theactivationof thepathwayduringanaturallyoccurringrefractiveperiodduringwhichthetailcannormallynotregenerate,restoresregenerativecapacity(Becketal.,2003).

During development, the release of BMP inhibitors like Chordin, Flik, Follistatin and Nogginfrom the notochord is critical for the ventral cell fate regulation of the developing NT. Forinstance, noggin mutant mice are characterized by a lack of floor plate cells and lack orreduced number of MNs in the NT (McMahon et al., 1998). During regeneration of theXenopus tail, Noggin induction reduces the rate of cell division in the SC, suggesting amitogenicroleofBMPs(Becketal.,2006).Noggininductionbycelltransplantexperimentsinthe injured SC of mice promotes the differentiation of ß-tubulin positive neurons,oligodendrocytesandastrocytesincorrelationwithapartialfunctionalrecoveryoflocomotoractivity(Setoguchietal.,2004).HoweverBMPinhibitionalsoincreasesthelesionvolumeandthe number of macrophages in the injured SC (Enzmann et al., 2005). Moreover, BMPsignallingalsoparticipatesingliosisandastrocytedifferentiationduringSCI(Sahnietal.,2010;Xiao et al., 2010). BMP receptor localizes into lipid rafts when β1-integrin is ablated in the

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ependymal stem cells,which is associatedwith increased astrogliosis after SCI (North et al.,2015).Hence,BMPsignallingmayhavedifferentpro-andanti-regenerativerolesafterSCI.

DespitethecentralroleofBMPsfornervoussystemdevelopmentandregeneration inmanyspecies, very little is known about its expression and roles in the zebrafish during SCregeneration. Data from an expression profile indicate that BMP signalling components aredifferentially expressed after spinal cord crush injury, suggesting specific roles for BMPsignallinginregeneration(Huietal.,2014).Fluorescentfusionproteinscouldbeusedinfutureto visualise BMP transports and gradients of the secreted protein. The genetic toolkitsavailable in zebrafish plus the use of models for neuron ablation using theNitroreductase/Metronidazole(NTR/Mtz)system(Curadoetal.,2007;Ohnmachtetal.,2016)couldhelp to elucidatehow this pathway is functionally involved in the regenerationof thespinalcord.

2.4Wnt

Wntsignalling,aregulatorofcellproliferation,growthandfateiscriticalfordevelopmentandtissue homeostasis (MacDonald et al., 2009). Its roles during tissue regeneration have beenstudiedindifferentanimalmodels(Cleversetal.,2014;WehnerandWeidinger,2015). IntheCNS,Wntsignallingcomponentsareactivatedasa responseto injury (Lambertetal.,2016),suggestingtheimportanceofWntsignallinginCNSregeneration.

Wnt signalling (Fig. 1) participates in CNS development fromneural induction (Stern, 2001).Once the neural tissue is established, Wnt/β-catenin acts as a morphogen patterning theantero-posterioraxisof theCNS (KieckerandNiehrs,2001).Wntsignalling inducesposteriormarkers (Kudoh et al., 2002). During subsequent development,Wnt, together with Hh andBMPsignallingisresponsibleforthecorrectdorso-ventralNTpatterning(Fig.2)(LeDreauandMarti,2012).WntsareexpresseddorsallyintheNTinanopposinggradienttotheHhventralsignalling(Alvarez-Medinaetal.,2008).Theseoppositegradientsareimportanttopatternthedifferentdomainsofprogenitorsalongthedorso-ventralaxisandalsotodeterminethesizeoftheNTstructurebycontrollingthedifferentiationrateoftheprogenitors(Kichevaetal.,2014).DorsalWnt is important forproliferationofventricularprogenitorcellsandthe formationofdorsal neurons (Megason and McMahon, 2002). Indeed β-catenin, which is essential forcanonicalWnt signalling, is required for themaintenance and proliferation of the neuronalprogenitors in the entire spinal cord (Zechner et al., 2003). A prominent activity of Wntsignalling inneurogenesis is thecontrolof thecell cycle (NiehrsandAcebron,2012).Mousemodels carryinga stabilizedβ-catenin inneuralprecursorsdevelopbiggerbrains, associatedwith the control of cell cycle exits of the progenitors (Chenn and Walsh, 2002). Duringcorticogenesis, Wnt modulates the spindle-size asymmetry which lead the asymmetric celldivision; a necessary step for neurogenesis (Delaunay et al., 2014). In adult animals, Wntsignallingcontrolsself-renewal,proliferationanddifferentiationofstemcellnichesindifferentorgans and tissues (Clevers et al., 2014). In the adult CNS, Wnt signalling is necessary forneurogenesis, differentiation and survival of neurons and for synaptogenesis (Inestrosa andArenas,2010).

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Studiesintailregenerationinurodeleamphibianshaveshownthatwnt5a,wnt5b,wnt7a,andwnt10aareexpressed indifferentdomainsalongtheanteroposterioraxisof the intactadultCNS and after an injury (Caubit et al., 1997a). wnt10a is strongly expressed after tailamputation,suggestingitsimportanceforthenewtissueproliferationandpatterning(Caubitet al., 1997b). In Xenopus it has been shown by grafting experiments and the Tet-onconditional transgenic system blocking Wnt-β-catenin in a tissue specific manner, that theWnt-β-catenin pathway is important for SC regeneration (Lin et al., 2012). In rodents, theexpressionofWntligandsandinhibitorycomponentsofthepathwayaredifferentlyregulatedafterSCIincomparisontotheunlesionedcondition(Gonzalez-Fernandezetal.,2014).

During SCI in zebrafish larvae, spinal radial glia exhibit Wnt/ß-catenin signalling, which isimportantforneurogenesisbutmayalsoplayanimportantroleforaxonalregrowth(Brionaetal., 2015). After SCI in adult zebrafish, an increase ofWnt/β-catenin signalling is observed,whichisnecessaryforlocomotorrecovery(Strandetal.,2016).

StudiesinzebrafishusingconditionalknockoutsorinduciblesystemslikeTetON(Wehneretal.,2015)couldhelptoelucidatecelltypesandmechanismsbywhichWntanditsregulatorsaffectSCregeneration.ThestrengthofimaginginthezebrafishCNScouldbeusedtovisualizeWntprotein transport through filopodial structures (Luz et al., 2014) or axons. These technicalpossibilities and the regenerative ability of the zebrafishmakes it particularly interesting toexploreWntsignallinginzebrafishSCI.

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2.5FGF

FGFsignalling(Fig.1)playsimportantrolesindevelopmentfromcellfatespecificationtothedeterminationofaxisandmorphogeneticprocesses(DoreyandAmaya,2010).TheformationofanFGFgradient iscrucial for itsmorphogeneticactivity(BokelandBrand,2013).WhetherFGF plays a role early during neural induction is still a controversy (discussed in (Dorey andAmaya,2010))DuringtheestablishmentoftheanteroposterioraxisoftheCNS,FGFactsasaneuralmorphogen (KengakuandOkamoto, 1995).High levelsof FGFpromoteexpressionofposteriormarkers and conversely, lowdoses, anteriormarkers (Storey et al., 1998). Indeed,FGFreceptorsignalling is required for theelongationof thespinalcordprimordium inchick,where FGF works as a posteriorising signal (Fig. 2). FGF signalling maintains the neuralprogenitor population close to the regionof theHensen´s node (Mathis et al., 2001). Later,FGFiscrucialforCNSdorso-ventralpatterning,contributingparticularlytothespecificationofthe ventral progenitor domains (Lupo et al., 2006) and neurogenesis. FGF promotesproliferationandsurvivalofneuroepithelialcells invitroandtheirdifferentiationintomatureneuronsandgliadependingonligandconcentration(Qianetal.,1997).InthemouseSC,MNsexpress the FGF ligand FGF1 (Elde et al., 1991) while human and rat MNs express FGF9(Nakamura et al., 1997). Indeed, the treatment of forebrain-derived neural stem cells withFGF2inducesthegenerationofMNswhichexpresstheMNmarkerHB9(Jordanetal.,2009).Inaddition,differentFGFproteinscanactaspotentsurvivalfactorsforspinalMNsinculture(Ford-Perrissetal.,2001).

The activity of FGF in glial cells seems important during regenerative processes in the CNS.Brain or SC injury in the salamander Pleurodeleswaltl increase FGF2 expression in reactiveastrocytes at the lesion site (Fahmy and Moftah, 2010). Tail amputation experiments inPleurodeles, have shown that during regeneration FGF2 expression is upregulated in theependymalcells,promotingproliferationandneuraldifferentiationfromthisprogenitorcells(ZakyandMoftah,2014)InotheramphibianslikeAmbystomamexicanumorXenopustadpoles,FGFsignalling isalso importantduringtheregenerativeprocessof thespinalcord (Linetal.,2012;Makanae et al., 2016). In zebrafish, different FGF signalling pathway components areupregulated inneuronsandgliaafterSCI.ThisupregulationoftheFGFpathway is importantfortheformationofaglialbridgeafterSCI,whichhasbeenproposedtoactsasascaffoldfortheaxonscrossingthelesionsite(Goldshmitetal.,2012).EvidencefrommammalianmodelsshowsthatFGFplaysimportantrolesinglialcellsduringbrainorSCinjury(Kangetal.,2014;TripathiandMcTigue,2008). Inmice, theadministrationofFGF2 followingSCI increases thenumber of progenitor cells at the lesion site and favours glial bridge formation and axonalregeneration(Goldshmitetal.,2014).

Itwouldbe interestingtodeterminewhethercross-regulatory interactionsofFGFwithothersignallingpathwayslikeRAorShh,whichcontributetocoordinatetranscriptionalprogramsasthespinalcordprogressivelymatures(BriscoeandNovitch,2008;DiezdelCorralandStorey,2004),arerecapitulatedduringregeneration.Forexample,FGFsignallingpromotesspinalcordelongation by controlling cell cycle length through the regulation of cyclins D1 and D2 inconcertwithHhsignalling(Lobjoisetal.,2004).Duringthecoordinationofventralpatterningandcaudalaxisextension,FGFandHhsignallingactinanegativefeedbackloopbymodulatingtheexpressionof theShhreceptorPatched2(Fig.3) (Moralesetal.,2015). In thecontextof

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limbregeneration,ithasbeenobservedrecentlyhowthecrosstalkbetweenFGFandShhleadsregeneration(Nacuetal.,2016).

Duringneuraldifferentiation,FGFsignallingcanmodulatechromatinorganisation(Pateletal.,2013) pointing to another potential mechanism of FGF action in regeneration. During thetransition from multi-potent progenitor to neural progenitor cell in the chick spinal cord,differentchromatinmodifiersaredownregulated.Amongthem isHDAC1,whichdependsonFGFsignallingactivity.(Olivera-Martinezetal.,2014).Interestingly,HDAC1isabletoregulateproliferation in radial glial cells in theXenopus optic tectum (Tao et al., 2015), indicating apotentialmechanismbywhichFGF controls cell differentiation in theCNS. The capacitiesofFGF to control chromatin modulators may have implication for the activation of theregeneration-associatedgeneexpressionprogramme inprogenitorcellsalsoduringneuronalregeneration.

2.6Retinoicacid

The RA pathway (Fig. 1) is important for cell communication during development andadulthood. Its functions are crucial for spinal cord development (Lara-Ramirez et al., 2013;Maden, 2006). RA has been proposed as an important signal for regeneration of differentorgans,suchasthelungs,limbs,peripheralnervoussystem(PNS)andCNS(Maden,2007).

During development, RA is known as a posteriorising agent (Maden andHolder, 1992). Thetreatment ofXenopus tadpoleswith RA promotes the expression of posterior neural genes,such ashoxb3, and suppresses anteriormarkers, likeXotx2 (Papalopulu and Kintner, 1996).DuringNTpatterning,theRAreceptors(RXRα,RARαandRARβ)areexpressedintheNTwhileRaldh2,theenzymegeneratingRA,ispresentinthepresomiticmesodermandsomites.TheRAsignal fromthe somites isnecessary forneuraldifferentiationandventralpatterning (Fig.2)(DiezdelCorraletal.,2003).TheRAsignalling functionduringNTpatterning isconserved inzebrafish (Englandetal.,2011;Lara-Ramirezetal.,2013). Indeed,adecreaseofRAreceptoractivity is associated with the activation of neural fate (Blumberg et al., 1997). For thisposteriorisingactivity,RAinteractswithWntandFGFsignalling,whichismediatedbytheRAhydroxylaseCyp26inzebrafish.WntandFGFsignallinginhibitcyp26expression,whichinturninhibitsRAsignalling(Kudohetal.,2002).

TheRApathwayisalsoimportantforthedorsoventralpatteringofthespinalcord(Wilsonetal.,2004).InavitaminA-deficientquailmodelinwhichRAisabsent,V1interneuronandMNmarkergenesaredownregulated.RAsupportsthegenerationofMNs(SockanathanandJessell,1998)byactivatinggenesnecessaryforventralNTpatterningandMNspecification(Novitchetal.,2003).Oneretinoid-induciblegene isGDE2 (glycerophosphodiesterphosphodiesterase2)which inducesMNdifferentiation (RaoandSockanathan,2005) inhibitingNotch signalling inthe pMN cells from the differentiatedMNs (Sabharwal et al., 2011). RA also promotes thedifferentiationofaneuronalfatefromdifferenttypesofstemcell lines invitroand invivo inthespinalcord(Duester,2013;Janesicketal.,2015).

AfterSCIinrats,RALDH2activitywasfoundupregulatedincellsaroundthelesionsite(Kernetal., 2007) andRARßagonist treatment reduces the formationof theglial scar andenhances

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axonaloutgrowthcapacity(Goncalvesetal.,2015).ThissuggestspotentialbeneficialeffectsofRAactivityafterSCI.

In adult newts, retinoid signalling controls the expression of specific microRNAs, which areexpressed in ependymoglial cells during tail and spinal cord regeneration. In turn, thesemicroRNAscontroltheexpressionoftheRAreceptor,RARβ2(LeppandCarlone,2015).Theseobservationsareopeningnewinsightsintohowthispathwayisbalancedandfunctionsduringsuccessfulspinalcordregeneration.

Little is known about the effects of RA signalling on neurogenesis during spinal cordregeneration in zebrafish. Expression data indicates that the RA signalling pathway isupregulatedaftera spinal cord lesion (Reimeretal., 2009). There isnoapparentneed forafunctionofRAinpatterningofthelesionedadultspinalcord,asthisretainsitsdorso-ventralpattern from development (see above). Further functional experiments, including thevisualisationandperturbationofRAgradients(Schillingetal.,2016)willhavetoclarifytheroleof RA in spinal cord regeneration. In thedeveloping spinal cord, RA seems tomaintain highlevels of FGFandNotch signalling inorder todrive stemcell differentiation (Paschaki et al.,2012).RA isalsonecessaryforthemodulationofcomponentsofneurotransmitterpathwayslikedopamine(Samadetal.,1997)orserotonin,discussedfurtherdown(O'Reillyetal.,2007)(Fig. 3). These observations open the question if this crosstalk plays an active role duringregeneration.

2.7Dopamine/Serotonin

Neurotransmitters,inadditiontotheirroleintransmittingspecificneuronalinformation,alsopromote neurogenesis in the CNS during development and adulthood. Acetylcholine,dopamine (DA),GABA, glutamate, nitric oxide, neuropeptide Y, noradrenaline and serotonin(5-HT) influence neural cell proliferation and differentiation (Berg et al., 2013).Neurotransmitters are released from producing cells, acting in target cells throughtransmembraneG-Protein-CoupledReceptors (GPCRs).Thesecommonanddiverse receptorscontrolcellularprocesses likeproliferation,differentiationandadultneurogenesis (DozeandPerez,2012).

DifferentcomponentsofDAsignallingareexpressedinprogenitorcellsintheCNS(Diazetal.,1997;Ohtanietal.,2003).Aminergic(DA,5-HT,noradrenaline)projectionstothespinalcordcontribute to the maturation of locomotor networks (Sharples et al., 2014). In zebrafish,dopaminefromthedopaminergicdiencephalo-spinaltractisabletoactdirectlyonthespinalprogenitorcellsduringdevelopmentbypromotingthegenerationofMNsbyaugmentingShhsignalling (Reimer et al., 2013). Similarly, the depletion of monoamines in vivo affects Hhsignallingintheadultrathippocampus(Rajendranetal.,2009).5-HTplaysasimilarroleduringdevelopment;5-HTdepletion inratsdelaysthedifferentiationofneurons(LauderandKrebs,1978). In zebrafish, 5-HT promotes the embryonic development of MNs by acting onprogenitorcellproliferation(Barreiro-Iglesiasetal.,2015).TheeffectandmechanismofactionappearstobeindependentofShhandthereforedifferentfromthatofDA.

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Inthesalamanderbrain,DAcontrolsneurogenesisandregenerationofdopaminergicneurons.DA keeps the stem cells that give rise toDAneuronsquiescent in a negative feedback loop(Bergetal.,2011).Intheadultspinalcordofzebrafish,dopamineandserotoninaugmentthenumberofMNsregeneratedafteran injury,which issimilartotheirdevelopmental function(Barreiro-Iglesiasetal.,2015;Kuschaetal.,2012;Reimeretal.,2013).

Tobetterunderstandhowneurotransmittersactonneurogenesis, thesecouldbe tracked invivo. Fluorescent false neurotransmitters (FFN), which can be tracked by imaging to studymonoamineexocytosishaverecentlybeendescribed(Pereiraetal.,2016).Forfuturestudies,FFN could be applied in the transparent developing and regenerating zebrafish embryo tounderstand how neurotransmitters control neurogenesis and modulate regeneration. Also,decipheringthelocalizationandinteractionofthedifferentreceptorswouldhelptoelucidatethemolecularcodethatactinprogenitors.Thecrosstalkwithsignallingpathways,suchasthehedgehogpathway,andchangesingeneexpressiondownstreamofDAor5-HTsignallingareofinterestforfuturestudies(Fig.3).

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3.Discussion/Conclusions

Progenitors in the developing and regenerating spinal cord express amultitude of differentreceptors and are therefore able to receive a large number of signals. In the regeneratingspinal cord, developmental signals derived from neighbouring cells, axons, and thecerebrospinal fluid (CSF) are “reused” and direct the regenerative response of spinalprogenitors.While each receptor acts on its own signalling cascade, there is also significantcross-talkbetweenpathways,oftenintegratedontofeweffectors(asillustratedforHhinFig.3).Itwillbeinterestingtodeterminehowtheseinteractionsarealteredduringregeneration,giventhattheylikelyintegratesignalsuniquetoregeneration,suchasinflammatorymediatorsfrom immunecells.The recentprogress ingeneticand imaging technologiesnowallows thedetailedinvestigationofthesesubcellularsignallingprocessesinvivousingthelarvalzebrafishtoinvestigatesuccessfulcentralnervoussystemrepair.

15

Acknowledgements

WethankDrDanielWehnerforcriticalinput.ThisworkwassupportedbyanEMBOLong-TermFellowship(ALTF946-2014),co-fundedbytheEuropeanCommission(MSCALTFCOFUND2013,GA-2013-609409) (MJC),and theBBSRC (BB/L021498/1,CGB,TB)andNC3Rs (NC/l001063/1,CGB, TB). We apologize to the colleagues whose work was not cited because of spacelimitations.

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Fig.1.Molecularbasicsofsignallingpathways.Notch:Notchreceptor,uponbindingtoligandsDelta or Serrate, undergoes cleavage by γ-secretase and its intracellular domain (NICD)translocates to the nucleus where it associates with the effectors suppressor of hairless(CBF1/RBPjk)andmastermind (MAM) toactivatecanonicalNotch targetgenes,which in thenervous system include hes1/5, the HES related genes hesr1/2, glial fibrillary acidic protein(GFAP)andthebrainlipid-bindingprotein(BLBP).Inthesignal-producingcell,mindbomb(Mib)promotes ubiquitination and endocytosis of Notch ligand.Hedgehog (Hh): The Hh ligandsSonicHedgehog(Shh),DesertHedgehog(Dhh), IndianHedgehog(Ihh) [inzebrafishtherearetwomoreligands,ShhbandIhhb],aretranslatedandsecretedfromproducingcells.Hhligandsdiffuseorare transported through the tissuesoverdifferentdistances from the source,alsothrough filopodial-like structures, to their receiving cells. In the receiving cells, Hh proteinbindstothetransmembranereceptorPatched(Ptch),whichinturnleadstothedisinhibitionof Smoothened (Smo) and its translocation to the primary cilium. Smo activates a series ofevents involving reduction of cAMP/PKA activity, which promotes the accumulation of anactivatorformofthetranscriptionfactorGli(GliA)overtherepressorform(GliR).GliAentersthe nucleus and initiates the transcription of Hh target genes.BMP: BMP ligands compriseapproximately30secretedcytokines,whichbelongtothesuperfamilyoftransforminggrowthfactor β (TGF-β). The binding of BMP ligands to the Type I/II receptors in receiving cells,promotes receptorphosphorylation,which in turn leads to thephosphorylationof theSmadproteins. Phosphorylated Smads form a complex that translocates to the nucleus to initiatetranscription. The kinase activity of type I receptor requires ligand binding, ligand–receptoroligomerization,andtransphosphorylationviathetypeIIreceptor.ThereceptorscanactivateSmad-dependent or independent pathways like P38 mitogen-activated protein kinase (p38MAPK)orc-JunN-terminalkinases(JNK).EndogenousBMPinhibitorssuchasNoggin,Chordin

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orCerberusact todiminish thebindingofBMP to its receptorandhence theactivityof thepathway.Wnt:WntsaresecretedCys-richproteins,conservedinmetazoananimals,encodedby19genesinmammalsand25identifiedgenesinzebrafish.Wntproteinsaresecretedfromproducingcellsortransportedthroughfilopodial-likestructurestotargetcells.Theinteractionof Wnt ligands with the receptor Frizzled activates several interconnected downstreampathways,generallyknownascanonicalandnon-canonical.Inthecanonicalpathway,Frizzled,through the activation of Dishevelled (Dvl) protein, promotes the dissociation of β-cateninfromtheAxin/APC/GSK3βcomplex,leadingtothenucleartranslocationofβ-catenin,whereitassociates with the T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) proteins toregulate the transcriptionofWnt targetgenes. In thenon-canonicalWnt/planarcellpolarity(PCP) pathway, the activation of Frizzled and Dvl leads to a signalling cascades resulting incytoskeleton modifications. The non-canonical Wnt/Ca2+ pathway causes an increase incalcium flux across the plasma membrane and/or from the intracellular stores, ultimatelyleading to theactivationof signals affecting cell fate.Formoredetailswe suggest the “Wnthomepage” (http://web.stanford.edu/group/nusselab/cgi-bin/wnt/). FGF: FGF ligands areextracellularproteins,whichsignal inanautocrine,paracrineorendocrinemanner.SecretedFGF ligands bind to their receptors (FGFR), a single-pass transmembrane tyrosine kinasemolecule, through the interaction with heparin sulphate proteoglycan (HSPG). FGFRs areencoded by 4 genes (FGFR1-4) which, by being alternatively spliced, confer ligand-receptorbindingspecificity.TheyactivateFRS2-Ras-MAPK,PLCγ-NFATandPI3K-AKTsignallingpathwaystomediateFGFtargetgenetranscriptionthroughtheactivityofthetranscriptionfactorsFOS,NFAT and FOXO. Retinoic Acid (RA): Retinol, a form of vitamin A, is transported from thebloodstreamtotheRA-generatingcellbyretinolbindingprotein(RBP)andtransthyretin(TTR),entering into the cell through STRA6 receptor. Enzymatic processing of retinol byretinaldehyde dehydrogenases (RALDHs) and retinol dehydrogenases (RDHs) results in theformationofRA. ThisRA-generating cell releasesRA to theneighbouring cells,whichentersthatcell’snucleusandassociateswithnuclearRAreceptor(RAR)andretinoidXreceptor(RXR)toinitiategenetranscription.Forreferencesseetext.

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Fig. 2. Signalling pathways during spinal cord development and regeneration.Development:Duringthepatterningofthespinalcord,thenotochordandthefloorplate(FP)areestablishedasasignallingcentreforShh(blue),whichdiffusesthroughthetissueinaventral-high,dorsal-low gradient. In contrast, BMP andWnt (green) are released from the roof plate (RP) in adorsal-high,ventral-lowgradient.Attheventricularzone(VZ)oftheneuraltube,thecellsthatmaintainhighNotchexpressionpreserve theirproliferative state (orange),whereas thecellswith higher Delta/Serrate expression differentiate into mature neurons (brown). Distinctventral progenitor zones (p0-p3 (green) and pMN (red)) are set up by the morphogensdiffusing from FP and RP. Along the anterio-posterior axis, the spinal cord is patterned byanterior-high expression of RA (purple) released from the somiteswhich forms an opposinggradient to the posterior-high expression of FGF and Wnt (pink). Regeneration: In theunlesioned adult spinal cord, the VZ around the narrow central canal (*) is lined withependymo-radial glia (ERGs),which show littleornoactivityof thedevelopmental signallingpathways.Afteralesion,theERGsproliferate,whichexpandsthediameterofthecentralcanalin zebrafish, rats and newts, and ERGs upregulate the expression of genes present duringspinal cord development, such as shh, notch1a/1b and bmp4. The inserts in the top rightcorners of each expression pattern indicate the model organism (zebrafish (blue)/mouse(brown))usedtoobtainthedata.Forreferencesseetext.

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Fig.3.Exampleoftheconvergenceofsignallingpathwaysintheneuralprogenitorcellsrelatedto Hh - cAMP/PKA modulation. During spinal cord development, Notch augments Shhsignalling by increasing the accumulation of Smo in the primary cilium. Notch signalling isinhibited,ifthereceptorisblockeddownstreamofBMPsignalling.Duringthecoordinationofventralpatterningandcaudalaxisextension,FGFandHhsignallingactinanegativefeedbackloopbymodulatingtheexpressionofShhreceptorPatched2.RAdownstreampathwaysinhibitNotchsignallinginthepMNcellstoinduceMNspecification.Thecross-regulationbetweenRA,FGF and Wnt acts to determine the anterio-posterior identity of the progenitors andcoordinate the transcriptional programs as the spinal cord matures. In addition, RA isnecessaryforthemodulationofcomponentsofneurotransmitterpathwayslikedopamineorserotonin. Dopamine signalling alters the sensitivity of progenitors to Hh signalling duringspinal neurogenesis though the dopamine receptor 4a (D4a) during development andregenerationofthespinalcord.Forreferencesseetext.

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