Modern genomic tools reveal the structural and cellular diversity of cnidarian nervous systems

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Reference

Modern genomic tools reveal the structural and cellular diversity of cnidarian nervous systems

RENTZSCH, Fabian, JULIANO, Celina, GALLIOT, Brigitte

Abstract

Cnidarians shared a common ancestor with bilaterians more than 600 million years ago. This sister group relationship gives them an informative phylogenetic position for understanding the fascinating morphological and molecular cell type diversity of bilaterian nervous systems.

Moreover, cnidarians display novel features such as endodermal neurogenesis and independently evolved centralizations, which provide a platform for understanding the evolution of nervous system innovations. In recent years, the application of modern genomic tools has significantly advanced our understanding of cnidarian nervous system structure and function. For example, transgenic reporter lines and gene knockdown experiments in several cnidarian species reveal a significant degree of conservation in the neurogenesis gene regulatory program, while single cell RNA sequencing projects are providing a much deeper understanding of cnidarian neural cell type diversity. At the level of neural function, the physiological properties of ion channels have been described and calcium imaging of the nervous system in whole animals has allowed for the [...]

RENTZSCH, Fabian, JULIANO, Celina, GALLIOT, Brigitte. Modern genomic tools reveal the structural and cellular diversity of cnidarian nervous systems. Current Opinion in

Neurobiology , 2019, vol. 56, p. 87-96

DOI : 10.1016/j.conb.2018.12.004

Available at:

http://archive-ouverte.unige.ch/unige:112883

Disclaimer: layout of this document may differ from the published version.

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Modern genomic tools reveal the structural and cellular diversity of cnidarian nervous systems

Fabian Rentzsch

^1,2

, Celina Juliano

³

and Brigitte Galliot

⁴

Cnidarianssharedacommonancestorwithbilateriansmore than600millionyearsago.Thissistergrouprelationshipgives themaninformativephylogeneticpositionforunderstanding thefascinatingmorphologicalandmolecularcelltypediversity ofbilateriannervoussystems.Moreover,cnidariansdisplay novelfeaturessuchasendodermalneurogenesisand independentlyevolvedcentralizations,whichprovidea platformforunderstandingtheevolutionofnervoussystem innovations.Inrecentyears,theapplicationofmoderngenomic toolshassignificantlyadvancedourunderstandingofcnidarian nervoussystemstructureandfunction.Forexample, transgenicreporterlinesandgeneknockdownexperimentsin severalcnidarianspeciesrevealasignificantdegreeof conservationintheneurogenesisgeneregulatoryprogram, whilesinglecellRNAsequencingprojectsareprovidingamuch deeperunderstandingofcnidarianneuralcelltypediversity.At thelevelofneuralfunction,thephysiologicalpropertiesofion channelshavebeendescribedandcalciumimagingofthe nervoussysteminwholeanimalshasallowedforthe identificationofneuralcircuitsunderlyingspecificbehaviours.

Cnidarianshavearrivedinthemoderneraofmolecular neurobiologyandareprimedtoprovideexcitingnewinsights intotheearlyevolutionofnervoussystems.

Addresses

1SarsCentreforMarineMolecularBiology,Norway

2DepartmentforBiologicalSciences,UniversityofBergen,Norway

3DepartmentofMolecularandCellularBiology,UniversityofCalifornia Davis,CA95616,UnitedStates

4DepartmentofGeneticsandEvolution,InstituteofGeneticsand GenomicsinGeneva(iGE3),UniversityofGeneva,Switzerland Correspondingauthors:Rentzsch,Fabian(fabian.rentzsch@uib.no), Juliano,Celina(cejuliano@ucdavis.edu),

Galliot,Brigitte(Brigitte.galliot@unige.ch)

CurrentOpinioninNeurobiology2019,56:87–96

ThisreviewcomesfromathemedissueonNeuronalidentity EditedbySachaNelsonandOliverHobert

https://doi.org/10.1016/j.conb.2018.12.004 0959-4388/ã2019ElsevierLtd.Allrightsreserved.

Introduction

Cnidariannervoussystemshaveattractedtheinterestof zoologistsandneurobiologistssincethemiddleofthe19th century, when first ‘neuro-muscular-epithelialcells’ and nematocytes[1,2],andafewdecadeslaterneurons[3–5]

wereidentified.Centraltothisinterestwastheapparent simplicity of the cnidarian body plan, which was soon realized to reflect a long evolutionary distance to other animalgroups.Itisnowwellestablishedthatcnidariansare thesistergrouptothebilaterians[6–8]andthusoccupya key positionintheanimal treeoflifeforunderstanding early stagesin nervous system evolution.Cnidarians are carnivoresthatarefoundintwostrikinglydifferentmor- photypes:sessilepolyps,generallyattachedtoasubstrate, and free-swimming medusae. In atypical cnidarian life cycle, a swimming planula larva is derived from sexual reproductionandcontinuestodevelopintoasessilepolyp (Figure 1). Cnidarians comprise two principle groups, anthozoansandmedusozoans;thepolypsofthesegroups shareasimilaranatomybutdifferintheirdevelopmental potential and theirrolein the lifecycle. Inanthozoans, polypsarethesexuallymaturestagethatcompletesthelife cyclebygeneratinggametesthatgiverisetonewplanulae (Figure 1a). In medusozoans, polyps asexuallygenerate medusae,andthemedusaearethesexuallymaturestage thatproducesgametestoclosethelifecycle(Figure1b).

However,thereisvariationinthelifecycleamongmedu- sozoanspecies,manyofwhichhaveatruncatedlifecycle, havinglostoneormultiplestages[9].

Nervous system complexity and organization differ betweenthesessilepolypandfree-swimmingmedusae, whichislikelyduetodifferencesinlifestyle.Polypshave a bi-layered, tube-shaped body with a single opening, surroundedbyaringoftentacles.Thetentaclesareused for catching preyand thesingle openingserves as both mouthandanus.Thenervoussystemintheouterlayer, namedepidermisorectoderm,isorganizedasanervenet, thoughthedensityofneuronsistypicallyhigheratboth theoralandaboralends.Insomespecies,anerveringat the oral end coordinates the feeding response, which involves tentacle motility and the ‘mouth’ opening.

Theinnertissuelayer,namedgastrodermisorendoderm, canbeaplainepithelialsheet(asinmanymedusozoans), or itcanbear folds(themesenteries)thatcarrygonadal tissueandlongitudinalmusculature(asinmanyanthozo- ans).Thegastrodermalnervoussystemisalsoanervenet, butinsomespeciesprominenttractsofneuritesrunalong thebaseof themesenteries [10–13].

Availableonlineatwww.sciencedirect.com

ScienceDirect

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Medusaedisplayamuchhigherdegreeofnervoussystem centralizationascomparedtopolyps.Aprominentnerve ringisoftenlocatedatthemarginoftheirbellandmany medusae contain well-developed eyes thatcan be inte- gratedintosophisticatedlightandgravity-sensingorgans, therhopalia[14,15].Thenerveringandthesensorystruc- turescontributetothecontrolofswimmingbehavior,for exampletheavoidanceof obstacles or escapefrom pre- dators [16–18]. Studies of different medusae revealed interestingfeaturesintheregulationoftheirlocomotion.

Inscyphomedusae,contractionofthebellmusculatureis coordinatedwiththehelpofbidirectionalsynapsesinthe motor nerve net [19,20]. In thehydromedusae Aglantha digitale,slow swimming and escape swimmingaremediated bythesamemotorneurons,whichcangeneratetwotypes ofactionpotentials.Weakdepolarizationtriggerssmalland slow,calcium-drivenspikes,whereasstrongdepolarization causes the largeand fast, sodium-driven spikes that result in escapeswimming[21,22].Theongoingcharacterizationof neuronalionchannelsinseveralcnidarianspecieswillhelp torelatesuchphysiologicalpropertiesofneuronstotheir molecularconstitution[23–26].

The cells that comprise cnidarian nervous systems are traditionally grouped into three broad classes: sensory/

sensory-motorneurons,ganglionneurons,andmechano- sensory cells called cnidocytes. Sensory neurons are definedbytheir upright positionin theepitheliumand thepresence of an apical cilium.Ganglion neurons are considered a morphological equivalent of interneurons;

their somata are located basally within the epithelium.

Consistentwiththeirclassificationasneurons,bothsen- soryandganglioncellsextendneuritesontheirbasalside

thatformabasi-epithelialmeshwork(Figure2).Distinc- tion of neurites as axons and dendrites has not been shown yet. Cnidocytes (‘stinging cells’) are cnidarian- specificcellsthatcancontainstructuresresemblingpre- synapticsites[27,28],acnidocilandahighlysophisticated extrusiveorganelle,thecnidocyst,thatdischargesbyfast Ca²⁺-dependent exocytosis for catching prey [29,30].

Thesefeatureshaveledtothesuggestionthatcnidocytes are highly derived neural cells (reviewed in Ref. [11]).

Morphological and molecular analyses reveal that each neuronalclasscontainsseveralsubpopulations,character- izedfor example bytheexpression of particular neuro- peptidesorbythepresenceofaspecifictypeofcnidocyst [31–34].Inrecentyears, thegenomes of severalcnidar- ians have been sequenced [35–39], transgenic reporter lines have been established [40,41,42], and gene- knockdown or genome-editing technologies have been implemented for Hydra ([43,44], Hydractinia [45,46], Clytia[47,48] and Nematostella[49,50,51].These tech- nologiesopenthedoortoacomprehensiveunderstanding ofthecomposition,thedevelopment,andthefunctionof cnidariannervoussystems[9,52,53].

Transgenic reporterlinesand singlecell RNA sequencingas new toolsfor studyingthe diversityofcnidarian neurons

Histologicalandultrastructuralobservationshaveprovided manyinsightsintothemorphologyofneuronsindifferent cnidarians[54–57],butidentifyingdefinedsubpopulations ofneuronsandcapturingthedynamicsof theirdevelop- ment has been a major challenge. The generation of transgenic reporter lines in several cnidarian species [40,41,42] was an important step in overcoming this

Figure1

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Current Opinion in Neurobiology

Cnidarianlifecycles.

(a)LifecycleofananthozoanexemplifiedbyNematostellavectensisinclockwiseorder.Maturepolyps(ontheleft)releasegametesintothe water.Afterblastulaandgastrulastages,theanimalsdevelopintofree-swimmingplanulae,whichsettleontheiraboralpoleanddevelopinto sessilepolypsthatstartfeeding.(b)Lifecycleofamedusozoan,exemplifiedbythehydrozoanClytiahemispherica.Gametesarereleasedby medusaeanddevelopafterfertilizationintoplanulae.Theplanulaesettleandformcoloniesofdifferentpolyps,includingfeedingpolypsand reproductivepolyps.Thereproductivepolypsasexuallygeneratemedusaetocompletethelifecycle.Notethat,therearemanyvariationsto medusozoanlifecycles.Forexample,freshwaterHydrapolypspropagateasexuallybybuddingorthepolypsreproducesexuallybyforming gametes;fertilizedHydraembryosdevelopdirectlyintopolypswithoutproducingaswimminglarvalform.Therefore,Hydralackboththeplanula andmedusastages.ArtworkbyJohannaKraus.

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problem.Throughtheuseofgeneralneuronalpromoters, thefirsttransgeniclineswereusedtoobtainabroadpicture of cnidariannervous systemstructure and development [58,59]. However, there is now a growing number of transgenic lines with fluorescent proteins expressed in specific neural subpopulations [60,61,62,63,64].

Theserevealed,forexample,thatneuronswithstereotypic projection patterns and positions in the body column contribute to theNematostellanervous system, thus sug- gestingthattheseeminglydiffuse andrandomcnidarian nervenetshavereproducibleelements[60].

Understandingthecomplexityandorganizationofcnidar- ian nerve nets requires uncovering neuronal diversity, whichisnowpossibleusingsinglecell RNAsequencing (scRNA-seq).Thistechnologycombinedwithtransgenic reporterlineshasthepotentialtoprovideadetailedpicture

of cnidariannervous systems. Thusfar, scRNA-seqhas beenappliedtoboththeNematostellaandHydraadultpolyp tocharacterizeneuronaldiversity[63,64].Usingtrans- geniclineswithneuronalGFPexpression,severalthou- sand neurons were collected and sequenced from each animal,givingsignificant insightsintocnidariannervous systemsandprovidingrichdatasetsforfutureexploration.

Analysis of sequenced Nematostella neurons revealed 32 clusters with unique gene markers. In addition, the Nematostellaneuronscouldbesplitbroadlyintotwounique transcriptionalstatesdefinedbytheexpressionofunique setsoftranscriptionfactors.Transgeniclineswerecreated tostudythesetwoneuronalstatesandthisrevealeddiffer- encesinmorphologyandposition[63].InHydra,12neu- ronal subtypes were identified with distinct molecular signatures.Usingbothtransgenicreporterlinestohighlight neuralsubtypesandinsituhybridization,thelocationof

CnidariannervoussystemsRentzsch,JulianoandGalliot 89

Figure2

(a) (b)

(c) (d)

Labelingofneuronsintransgeniccnidarians.

(a)AGFP-labeledbipolarneuronafterdissociationofaHydrapolyp.(b)AsensorycellinthebodywallofaNematostellalateplanula(green).The apicalsurfaceoftheectodermisorientedtothetop.Thecapsulesofcnidocytes(magenta)arelabeledwithadifferenttransgene[62].(c)A multipolarneuronclosetotheoralopeningofayoungNematostellapolyp,labeledbyanNvElav1::mOrangetransgene.(d)Thegastrodermal nervoussystemofaNematostellapolypincludesprominenttractsofneuritesalongthemesenteriesandanervenetbetweenthesetracts.The transgeneisNvElav1::mOrange.Scalebarsin(b,d)20mm,in(C)10mm.Imagecredits:(a)StefanSiebert;(b–d)Oce´aneTournie`re.

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eachneuronalsubtypewasidentifiedthuscreatingaspatial andmolecularmapoftheHydranervoussystem,including the identification of distinct neuronal subtypes in the epidermalandgastrodermalnervenets[64].

TheapplicationofscRNA-seqtouncoverneuronaldiver- sityincnidariansisverypromising;however,moreworkis required to determine whether the full transcriptional diversity has been uncovered in Nematostella and Hydra. Importantly, the clustering parameters, number ofcellssequenced,andsequencingdepthareallimpor- tant factors contributing to the number of clusters reported in a given study. In addition, the degree of similarity between cell types is an important consider- ation. Sequencing a small number of transcripts from relatively few cells is sufficient to discern two very different cells types due to large transcriptional differ- ences.Delineatingcelltypesbyclusteranalysisismore difficult when considering cell types with similar transcriptional profiles; we don’t yet fully understand the level of sampling required to distinguish two neuronal subtypes,whichlikelyhaveahighleveloftranscriptional overlap.Finally,itisunclearwhatleveloftranscriptional differencesbetweentwocelltypeswarrantsclassification astwodifferentneuronalsubtypes.Unlikeincnidarians, the full diversity of Caenorhabditis elegans neurons is knownfromalargebodyofpreviousworkandtherefore attemptsto identify thetranscriptional signatures of C.

elegansneuronsusingscRNA-seqareinformativeindefin- ingbenchmarks.InC.elegans,nearly7000singleneurons weresequencedwithamedianofapproximately700tran- scriptspercell;thisrevealed40ofthe118knownneuro- nalsubtypes[65].Itislikelythatincreasingthenumberof sequencedcellsand/orincreasingthesequencingdepth wouldultimatelyuncoverthetranscriptionalsignaturesof all118subtypes.TheNematostellaandHydrasingle cell datasetsweresequencedatdifferentdepths–amedianof approximately550transcriptspercellforNematostellaand amedian of approximately5650 transcripts percell for Hydra. In addition, different clustering methodologies wereused,soitislikelytooearlytomakedirectcompar- isonsbetweentheneuronaldiversityofNematostellaand Hydra [63,64]. Ultimately, scRNA-seq experiments describingtheneuronaldiversityofanyorganismshould be validated at the bench. Regardless, scRNA-seq is clearlyaveryvaluabletoolthatwillallowustouncover theneuronaldiversityinalargearrayofcnidarianspecies andlifestages.Thisinformationcanbeusedto gainan understandingoftheorganizationalprinciplesunderlying thecnidariannervoussystemandwillprovidemolecular handlesforthefunctionalmanipulationsrequiredtotest nervoussystemdevelopmentand function.

Thedevelopmentalbasisforthegenerationof neuralcell types

Approachestomanipulategenefunctionusingmorpho- linos, CRISPR/Cas9, RNAi, and shRNAs have been

implemented in several cnidarian species [43–

45,47,48,49,50,51,66,67]. In combination with trans- geniclinesanddatacollectedfromscRNA-seq,theability to test gene function allows for detailed analysis of cnidariannervoussystemdevelopmentandfunction,thus providingabasisforevolutionarycomparisons.

Surprisingly,thestemcellsthatgiverisetoneuronsand cnidocytesmightbequitedifferentbetweenmedusozo- ansandanthozoans.Inhydrozoans(aclassofmedusozo- ans),themultipotentinterstitialstemcellsgiverisetoall cellsofthenervoussystem,aswellasglandcellsandgerm cells[68–72].Bycontrast,inanthozoans,interstitialstem cellshave notbeen foundand thenervoussystemmay instead arise from epithelial-like stem cells [59,73], suggestingthat interstitialstemcells mightbe ahydro- zoan-specificor medusozoan-specificinnovation[74].

Candidategeneapproachesusedtostudythemolecular controlofneurogenesissuggestasignificant degreeof conservation in the broad specification of neurons between cnidarians andbilaterians (reviewed inRefs.

[13,73]).Forexample,NotchandWntsignalling,soxB, atonal/neurogenin and achaete-scute family genes play central roles inNematostella neurogenesis [59,75,76–

78] and soxB and nanos genes function in Hydractinia neurogenesis[79,80]. However,incontrast toNema- tostella and most bilaterians, Notch signaling appears not be involved in the regulation of neurogenesis in bothadultHydraandembryonic Hydractinia,inwhich neuronsderivefromthenon-epithelialinterstitialcells [46,81,82]. Currently, only a fewfunctional studies haveaddressedthedevelopmentofspecificneuralcell types.In adultHydra,apicalneuronsrequirethePara- Hoxgenegsx/cnox-2 fortheirdevelopment [83]andin Nematostella,PaxAandMef2areinvolvedintheforma- tion of cnidocytes [84,85]. Now, with new tools and resources, like those provided by scRNA-seq, it is possible to move beyond candidate gene approaches toobtainalessbiasedviewoftheregulatorynetworks thatunderliecnidarian neurogenesis.

In adult cnidarian organisms, the nervous system is continuously replaced in homeostatic animals and is capableof regeneration, thus providingaplatform for understandingtheregulationofadultneurogenesis.In polyps,the whole nervous system readilyregenerates after significant loss of the body column, while in medusae,regenerationisrestrictedtosomeorganssuch as the manubrium or the eyes [86–88]. Comparative analysis of progenitor behaviours between cnidarians andbilaterianmodelorganismsshowingsimilarregen- erativepotential(e.g.planarians),willallowustodeter- mine possible common molecular mechanisms [89].

Suchcomparisonscanbemadeusingamputationpara- digmsorablationofspecificstructuresliketheeyesof jellyfishandplanarians [88,90].

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Conserved molecularbasisfor neurotransmissionin cnidariansand bilaterians

Even thoughneuroidconduction, that iselectricalcon- duction across non-neuronal cells, is observed across a wide repertoire of organisms, that is plants, protists, porifers,cnidariansandbilaterians[91],electrophysiolog- icalstudiesperformedonthegiantaxonfromthehydro- medusaeAglantha,havedemonstratedthatcnidarianand bilaterian synapsesexhibit similarproperties,with their activity relying on the formation of presynaptic and postsynaptic potentials [92,93]. Genomic sequencing from choanoflagellates, poriferans and placozoans has actually demonstrated that the molecular components ofthepost-synapticdensityarealreadyalmostcomplete in phyla that do not differentiate nerve cells [94,95].

Genomic sequencing and transcriptomic sequencing fromcnidarians haveconfirmedpharmacologicalstudies showing that most chemical neurotransmitters used in bilateriansarealsoactiveincnidarianneurons,acetylcho- line, glutamate, GABA, glycine for fast transmission, catecholamines and serotonin for slow transmission [96,97,98]. Signaling through these neurotransmitters is requiredfor coordinated behaviorssuchas therather complex feedingresponse[99].

The role ofpeptidesin neuralsignaling Neuropeptides and epitheliopeptides are a prominent featureofthecnidariannervoussystem.Theexpression ofnumerousGprotein-coupledreceptorsandthediscov- ery of peptide-gated ion channels suggests that these peptidesplayakeyroleinbothslowandfastneurotrans- mission[31,100,101].Inmammals,peptidergicsignaling is neuromodulatory,involvedin slowneurotransmission and interacting with fast neurotransmission driven by small molecules such as GABA and glutamate. This neuromodulatory roleof neuropeptidesmight represent a typical synapomorphy of nervous systems or alterna- tively, aconvergentevolutionary traitin cnidarians and bilaterians.

Recent studieshighlight thepower of functional geno- micstotestthefunctionofneuropeptidesincnidarians.

For example, disruptionof theexpression of aGLWa- mide neuropeptide in Nematostella [102] resulted in a subtledelayintheprogressionfromswimmingtosessile lifestagesunderlaboratoryconditions.Twootherrecent studies analyzed how light cues regulate therelease of gametes,afeaturecommontomanyanimals.Agroupof cells required for spawning in the gonad of the Clytia hemispherica medusaare bothlightsensitiveand secrete neuropeptides. Opsin9 is expressed in these cells and mutating the Opsin9 gene blocks light induced oocyte maturation and spawning. The same cells express a neuropeptide that functions as thematuration-inducing hormone (MIH), and secretion of this neuropeptide requires the stimulation of Opsin9 by blue-cyan light

[103,104]. These elegant experiments provide an interesting example of direct coupling of sensory and neurosecretory functions in one cell, a situation that mayhavebeenmorecommonearlyinanimal evolution [105].Future studies should address the role of neuropeptides in nervous system function using similar approaches.

In Hydra, epitheliopeptides can act either positivelyor negatively on neurogenesis and/or neurotransmission [101].Thisepitheliopeptidesignalingpointstoatightly regulatedcross-talkbetweenthemyoepithelialcellsand thenervoussystem.The physiologicalfunction andthe regulationof thiscrosstalkarecurrentlynotwellunder- stood.Theeliminationof theinterstitialstemcells,and consequentlyneurons,leadstotheupregulationoftaxon- specificepitheliopeptidesintheepithelialcellsinHydra [97].Thismaysuggestaproto-neuronalfunctionforthe epithelialcells,whicharewidelyrecognizedasthecells fromwhichsynapticconductionlikelyemerged[106].A differentiated nervous system could repress the proto- neuronalfunctionof theepithelialcells, whereasin the absence of neurogenesis, this potential could be expressed. While this hypothesis remains to be tested, onepossibleinterpretationisthatpeptidesplayedakey role in the emergence of neurons from epithelialcells, thatiscellsthatintegratedthreemajorfunctions:secretion,integration,andconductivity(Figure3).

The wiring ofcnidariannervoussystems Electrophysiological recordings have beeninstrumental for understanding conduction andfunction in cnidarian nervoussystems[22,107,108].Geneticallyencodedrepor- tersofneuralactivityandtoolsforoptogeneticmanipula- tions now also allow system-wide analyses. This has recently been accomplished in Hydra by transgenic expression of the fluorescent calcium sensor protein GCaMP6s in the entire nervous system [109]. This allowedforimagingofnervoussystemactivityinawhole animal and the identification of neuronal populations whoseactivitiescorrelate withtwomaintypes ofprevi- ously described electrical activity – contraction bursts (CBs) and rhythmic potentials (RPs) [107,110,111]. As previously postulated, CBs are associated with longitu- dinal contraction. Unexpectedly, two non-overlapping RP networkswereidentified, one in theepidermisand one in the gastrodermis. While the gastrodermal RP is related to radial contraction as previously thought, the epidermal RP is related to longitudinalelongation as a responsetolightstimulation.Apartfromtheirfunctional and spatial separation, the two RP neuron populations alsodisplayeddifferencesincellularmorphology[109].

These findings-coupled with the new molecular and spatialmapoftheHydranervoussystem[64],therecent classification of the Hydra behavioral repertoire [112], and the development of new technologies to measure Hydranervoussystemactivity[113],meanswearenow

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poised to gain a comprehensive understanding of the Hydranervoussystemformmoleculestobehavior.

Conclusions andoutlook

Cnidarian neurobiology is enjoying a renaissance with severalspeciesbeingamenabletogeneticmanipulations, thus allowing for the visualization and interrogation of nervous system development and function. Calcium imagingwill likelysoon beused together with optoge- netictoolsfortheactivationandinhibitionofindividual neurons,whichwillleadtonewinsightsintothelogicof neuralcircuitsinnervenet-basednervoussystems.Stud- iesofneuraldevelopmentcurrentlyfocusonthespecifi- cationofdifferentneuralcelltypes;inthenearfuture,we expectthesestudiestoexpandtocellularaspectsofthe formationofneuralconnectivityvianeurites andsynap- ses.Basic questions aboutthe nature of cnidarian neurons,includingtheidentificationofdistinctdendritesand axonsandthemolecularcompositionofchemicalsynap- sesremainunanswered.Thedevelopmentofcellculture protocols has so far eluded cnidarian researchers, but wouldbeusefulforaddressingthesequestions.Another emergingtopicisthecrosstalkbetweenneuronsandthe microbiomethatrecentlyhasbeen evidencedinHydra, pointing to previously overlooked functions of the

nervoussystem[114],possiblymaintainedinbilaterians [115,116]. At the organismal level, adding genetically tractablemodelsystemsfromotherclasses ofcnidarians wouldallow,for example,studyingthedevelopment of convergently evolved eyes and centralizations of the nervoussystem (in scyphozoansand cubozoans), or the neuralbasisoftheexquisitebehaviouralrepertoireofbox jellies (cubozoans). Extrapolating fromtherecent prog- resssummarizedhere,itislikelythatmanynewinsights intothefascinatingneurobiologyofthisdiversegroupof animalsareonthehorizon.

Conflictofintereststatement Nothingdeclared.

Acknowledgements

WethankJohannaKrausfortheartworkinFigure1;StefanSiebertforthe imageinFigure2a;andOce´aneTournie`refortheimagesinFigure2b–d.

WorkinFR’slabissupportedbygrantsfromtheResearchCouncilof NorwayandtheUniversityofBergen(NFRgrant251185/F20),andbythe SarsCentrecorebudget.TheworkintheGalliotlaboratoryissupportedby theSwissNationalFoundation(SNFgrant31003_169930)andbythe CantonofGeneva.CelinaJuliano’slabreceivesfundingfromthe UniversityofCalifornia,Davis.

Figure3

“Nerve cells arose by the coupling of electrical activity with the secretion of biologically active substances so that a chain of events in response to stimuli in alteration of effector activity” (Lentz, 1968)

“A nervous system is an organized constellation of cells (neurons) specialized for the repeated conduction of an excited state from receptor sites or other neurons to effectors or other neurons.” (Bullock and Horridge, 1965)

“Inputs from many receptors must merge on common “coordinators” before integration is achieved. Integration is as fundamental as conduction to any nervous system.” (Passano, 1963)

Which property came first?

How did these processes get connected?

How many times?

Thethreepropertiesthatarenecessaryandsufficienttobuildanervoussystem.

QuotationsarefromRefs.[117–119].

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