Opportunities and challenges for using the zebrafish to study neuronal connectivity as an endpoint of developmental neurotoxicity.

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UC Davis Previously Published Works

Title

Opportunities and challenges for using the zebrafish to study neuronal connectivity as an

endpoint of developmental neurotoxicity.

Permalink

https://escholarship.org/uc/item/05b707r0

Journal

Neurotoxicology, 67

ISSN

0161-813X

Authors

Miller, Galen W

Chandrasekaran, Vidya

Yaghoobi, Bianca

et al.

Publication Date

2018-07-01

DOI

10.1016/j.neuro.2018.04.016

Peer reviewed

eScholarship.org

Powered by the California Digital Library

Review

Opportunities

and

challenges

for

using

the

zebra

fi

sh

to

study

neuronal

connectivity

as

an

endpoint

of

developmental

neurotoxicity

Galen

W.

Miller

,

Vidya

Chandrasekaran

,

Bianca

Yaghoobi

,

Pamela

J.

Lein

*

a_{DepartmentofMolecularBiosciences,UniversityofCalifornia,Davis,CA,95616,USA} b

DepartmentofBiology,SaintMary’sCollegeofCalifornia,Moraga,CA,94575,USA

ARTICLE INFO

Articlehistory:

Received14February2018

Receivedinrevisedform23April2018 Accepted24April2018

Availableonline25April2018

Keywords: Axons Dendrites Developmentalneurotoxicity(DNT) Invivoimaging Neuronalconnectivity Screeningplatform Synapses Zebrafish ABSTRACT

Chemicalexposures havebeen implicatedasenvironmentalrisk factorsthat interactwithgenetic susceptibilitiestoinfluenceindividualriskforcomplexneurodevelopmentaldisorders,includingautism spectrumdisorder,schizophrenia,attentiondeficithyperactivitydisorderandintellectualdisabilities. Alteredpatternsofneuronalconnectivityrepresentaconvergentmechanismofpathogenesisforthese andotherneurodevelopmentaldisorders,andgrowingevidencesuggeststhatchemicalscaninterfere withspecificsignalingpathways thatregulate thedevelopmentofneuronal connections.There is, therefore,agrowinginterestindevelopingscreeningplatformstoidentifychemicalsthatalterneuronal connectivity.Cell-cell,cell-matrixinteractionsandsystemicinfluencesareknowntobeimportantin definingneuronalconnectivityinthedevelopingbrain,thus,asystems-basedmodelofferssignificant advantagesovercell-basedmodelsforscreeningchemicalsforeffectsonneuronalconnectivity.The embryoniczebrafishrepresentsavertebratemodelamenabletohigherthroughputchemicalscreening thathasprovenusefulincharacterizingconservedmechanismsofneurodevelopment.Moreover,the zebrafishisreadilyamenabletogeneeditingtointegrategeneticsusceptibilities.Althoughuseofthe zebrafishmodelintoxicitytestinghasincreasedinrecentyears,thediversetoolsavailableforimaging structuraldifferencesinthedevelopingzebrafishbrainhavenotbeenwidelyappliedtostudiesofthe influenceofgenebyenvironmentinteractionsonneuronalconnectivityinthedevelopingzebrafish brain.Here,wediscusstoolsavailableforimagingofneuronalconnectivityinthedevelopingzebrafish, reviewwhathasbeenpublishedinthisregard,andsuggestapathforwardforapplyingthisinformation todevelopmentalneurotoxicitytesting.

©2018ElsevierB.V.Allrightsreserved.

Contents

1. Introduction ... 103

2. Embryoniczebrafishasamodelsystemforstudyingneuronalconnectivity ... 103

3. Exvivoimagingtechniques ... 104

3.1. Examplesillustratingtheuseofexvivoimagingtoassessneuronalconnectivityindevelopingzebrafish ... 106

4. Invivoimagingtechniques ... 106

4.1. Directdyelabeling ... 106

4.2. Genetictoolsforinvivoimaging ... 106

4.3. Examplesillustratingtheuseofinvivoimagingtoassessneuronalconnectivityindevelopingzebrafish(summarizedinTable2) . 107 4.3.1. LabelingofdendritesintheCNS ... 107

4.3.2. Synapticmarkers ... 108

4.4. Synapticactivitymeasurement ... 108

*Correspondingauthorat:DepartmentofMolecularBiosciences,UniversityofCalifornia,Davis,1089VeterinaryMedicineDr,CA,95616,USA.

E-mailaddresses:[email protected](G.W. Miller),[email protected](V.Chandrasekaran),[email protected](B.Yaghoobi),[email protected](P.J.Lein).

1

Theseauthorscontributedequallytothismanuscript.

https://doi.org/10.1016/j.neuro.2018.04.016

0161-813X/©2018ElsevierB.V.Allrightsreserved.

ContentslistsavailableatScienceDirect

NeuroToxicology

5. Conclusions... 108

Conflictofinterest ... 109

Acknowledgements ... 109

References... 109

1.Introduction

There is increasing consensus that environmental factors interactwithgenetic susceptibilitiestodeterminetherisk and/ orseverityofdiverseneurodevelopmentaldisorders,rangingfrom autismspectrumdisorders(ASD)toattentiondeficithyperactivity disorder(ADHD)tointellectualdisabilitiestoschizophrenia(Lein, 2015; Lyall et al., 2017; Mandy and Lai, 2016). While diverse environmentalfactorshavebeenimplicatedinthepathogenesisof neurodevelopmentaldisorders,muchattentionhasfocusedonthe human chemosphere, including chemical contaminants in the environment,chemicalsinpersonalcareproducts,foodadditives, and drugs. However, the identity of specific chemicals that influencetheriskand/or severityof neurodevelopmental disor-ders,andthemechanism(s)bywhichtheyinteractwithgenetic susceptibilities to contribute to adverse neurodevelopmental outcomes remain outstandingdata gaps. There are compelling reasonstoidentifychemicalriskfactorsforneurodevelopmental disorders.Notably,becauseincontrasttogeneticrisks,whichare currentlyirreversible,environmental factorsaremodifiablerisk factors.Therefore,identifyingspecificenvironmentalfactorsthat increase risk for neurodevelopmental disorders may provide rationalapproachesfortheprimarypreventionofthesymptoms associatedwiththesedisorders.

Genetic, histologic, in vivo imaging and functional data are converging onaltered patternsof neuronal connectivity as the biological basis underlying the behavioraland cognitive abnor-malitiesassociatedwithmanyneurodevelopmentaldisordersand intellectualdisabilities(Bourgeron,2009;Garey,2010,Geschwind andLevitt,2007;Penzesetal.,2011;RubensteinandMerzenich, 2003; Svitkina etal., 2010).Thecandidategenesmoststrongly implicated in the causation of neurodevelopmental disorders encodeproteinsthatregulatethepatterningofneuronalnetworks during development and in_fluence thebalance of excitatory to inhibitorysynapses(BelmonteandBourgeron,2006;Bourgeron, 2009; Delorme et al., 2013; Stamou et al., 2013). Neuronal connectivity refers to the structural and chemical interactions that connect neurons to form a functional circuit. Critical determinantsofneuronalconnectivityincludethenumber,length andbranchingpatternsofaxonsanddendrites,whichconstitute theneuron’sprimarysiteof efferentoutputand afferentinput, respectively,aswellastheformationandstabilizationofexcitatory andinhibitorysynapses(Chiuetal.,2014;Lein,2015;Stamouetal., 2013).Thesefindings suggest thatscreening for chemicalsthat interferewithaxonalgrowth, dendriticarborization orsynapse formation/stabilization is a feasible approach for identifying potentialchemicalriskfactorsforneurodevelopmentaldisorders, andforelucidatingthemechanismsbywhichchemicalsinteract withgeneticsusceptibilities.

Chemicaleffectsonneuronalconnectivityhavebeenreported using primary neuronal cell culture, typically derived from developingrodents(Bal-Priceetal.,2010;Chenetal.,2017;Sethi etal.,2017;Waymanetal.,2012a,b), andmorerecently,using neuralprecursorcells derivedfromhumaninducedpluripotent stemcell(iPSC)cultures(Druweetal.,2015; Ryanetal.,2016). While human iPSC-derived neuronal cell models may better predict the effects of neurotoxic chemicals on the developing humanbrainthanprimaryrodentneuronalcellculture(Ryanetal., 2016), human iPSC-derived neuronal cell cultures present

challenges in that they do not yet reliably differentiate into mature neurons with distinguishable axons and dendrites and functionalsynapses.Regardlessofspecies,invitromodelsdonot fullyrecapitulatethecomplexcell-cellandcell-matrixinteractions or systemic influencesknown toinfluence development in the vertebrate nervous system (Lein et al., 2005). Moreover, it is difficulttocorrelatechangesinneuronalconnectivitywithdeficits inbehavior(Leinetal.,2005).Invivodevelopmentalneurotoxicity (DNT)studies,predominantlyusingrodentmodels,havealsobeen usedtodemonstratechangesindendritic complexityand brain morphology due todevelopmentaltoxicantexposure (Wayman etal.,2012b;Yangetal.,2009).However,invivorodentstudiesare time-andcost-prohibitiveforscreening,andgeneeditingcanbe challenging (Bal-Price et al., 2012, 2010; Lein et al., 2005). Embryoniczebrafishovercomemanyofthechallengesassociated withinvitromodelsand traditionalinvivorodent models.The inherentadvantagesofthismodelcoupledwithrecentadvancesin imaging provide a powerful approach for in vivo studies of chemical effects on neuronal connectivity in the developing nervous system.Thus, thegoal ofthis review istopresent the toolboxthatcouldbeadaptedtostudyneuronalconnectivityinthe developingnervoussystemofthelarvalzebrafish,andtoreview publishedexamplesillustratingtheiruse.

2.Embryoniczebrafishasamodelsystemforstudyingneuronal connectivity

Thezebrafishhasbecomeapowerfulresearchtoolinthefieldof developmentalneurobiologyanddevelopmentalneurotoxicology (Bradyetal.,2016;Garciaetal.,2016;Kalueffetal.,2016;Leinetal., 2005;Nishimuraetal.,2016;PattonandZon,2001;Wileyetal., 2017).Inherentadvantagesofthezebrafishmodelincludeoptical transparency,exuterodevelopment,whicheliminatesconfounds associatedwithmaternaltoxicity,rapidnervoussystem develop-ment, significantly lower costs than traditional in vivo rodent models,and adaptability for higher throughputscreening com-paredtorodentmodels.Zebrafishexpresshomologsfor>70%of human genes (Howe et al., 2013), and their genome is fully sequenced (http://www.ensembl.org/Danio_rerio/Info/Index), whichfacilitatesgeneticmanipulationsforbothshort-(transient) and long-term (stable) transgene and mutant expression. This genetictractabilityenablesnotonlymechanisticstudies,butalso integrationofrelevanthumangenemutationsintoscreensofgene byenvironmentinteractions.

AnimportantconsiderationinthecontextofDNTisthatthe fundamental mechanismsof neurodevelopmentarehighly con-servedbetweenzebra_fish,humansandothervertebratemodels (Gilbert,2010).Likemammals,zebrafishhaveathree-partbrain structure(telencephalon,mesencephalonandrhombencephalon). Zebrafish and mammals have similar mechanisms of early developmental patterning (homeobox gene gradients etc.) and cellular differentiation/proliferation (radial glial cells, etc.), and they express a similar range of neurochemical phenotypes, includingGABA(Higashijimaetal.,2004),glutamate(Higashijima et al., 2004), serotonin (McLean and Fetcho, 2004), dopamine (McLeanandFetcho,2004),norepinephrine(McLeanandFetcho, 2004),glycine(Higashijimaetal.,2004)andacetylcholine(Panula et al., 2010). Zebrafish also possess all of the classical sense modalities,includingvision, olfaction,taste,touch,balance,and

hearing;and theirsensorypathwaysshareanoverallhomology withhumans.

Developmentally, there is one major structural difference between mammalian and zebrafish neurodevelopment: during earlyneurulation, thezebrafish neuraltube ischaracterized by eversion,whereasthemammalianneuraltubeundergoes evagi-nation (Wullimann and Mueller, 2004; Wullimann and Rink, 2002).Therefore,zebrafishbrainstructuresareinvertedrelativeto rodents and humans, with the ventricle forming around the outsideofthebraininsteadofinternally.Whilethischangesthe placementofbrainstructuresinthezebrafishbrainrelativetothe mammalianbrain,otheraspects,includingthemRNAexpression patterns,and cellproliferative zones,remain synonymouswith thatofmammalianbrains(MuellerandWullimann,2005).Ofnote, forebrainstructuresareconservedbetweenzebrafishand mam-malian models: thezebrafish subpallium is homologoustothe mammalianbasalganglia(Aokietal.,2013;Muelleretal.,2008); the dorsal and ventral pallium are synonymous with the hippocampusand amygdala, respectively (Mueller et al., 2011; Rodriguezetal., 2002;Wullimann, 2009);and thehabenula is homologoustothedorsaldiencephalon(Hendricksand Jesutha-san, 2007a). Cognitive behavioral tests suggest that anatomic substratesofcognitivebehaviorarealsoconservedbetweenfish andothervertebrates.Thus,similartoobservationsof hippocam-pallesionsinmammals,lesionsofthestructuralhomologofthe hippocampusinfishselectivelyimpairspatialmemory(Rodriguez etal.,2002).

Becauseofthehomologiesbetweenzebrafishand mamma-lianneurodevelopment,zebrafishhavebeenusedextensivelyto study mechanisms of neurodevelopment (Chapouton et al., 2010; Wullimann, 2009; Wullimann and Mueller, 2004)and, morerecently,tomodelhumanneurodevelopmentaldisorders (Hoffmanetal.,2016;IjazandHoffman,2016;Meshalkinaetal., 2017;Noyesetal.,2015;Stewartetal.,2014).Zebrafisharealso beingincreasinglyusedforDNTstudies.TheendpointsofDNT oftenassessedin zebrafish studiesinclude grossteratological deficits, transcriptional changes, and behavioral readouts (Chuehet al.,2016; LevinandTanguay,2011; Mandrelletal.,

2012; Truong et al., 2011, 2014). Notably absent from DNT studiesinzebrafishareoutcomesfocusedonstructuralchanges in neuronal connectivity, particularly in the central nervous system(CNS).Relevantendpointsofneuronalconnectivitythat havebeenexaminedinthedevelopingzebrafishnervoussystem includeoutgrowthof axonsanddendrites, synapseformation and synaptic activity (Fig. 1). Below, we review the tools currentlyavailableforexvivoandinvivoimagingtechniquesthat canbeleveragedtoquantifyneuronalconnectivityindeveloping zebrafish,andwediscusstheirrelativeadvantagesand disadvan-tages(Table1).

3.Exvivoimagingtechniques

Two ex vivo techniques commonly used in other model organisms–insituhybridizationtolocalizeRNAand immunohis-tochemistrytolocalizeproteinantigens–havebeensuccessfully adaptedforuseinembryonicandlarvalzebrafish.Bothtechniques requirethattheembryosorlarvaebefixed,thus,dynamicchanges in neuronal connectivity are difficult to capture. Of the two techniques,insituhybridizationisusedmorewidelyinzebrafish becauseof thehigher specificityand relative easeof designing probes specific for target RNA.The Thisse lab hasdeveloped a libraryofinsituhybridizationdatafromzebrafishlarvae,including manyneural-specifictargets(ThisseandThisse,2004).Thesedata, andtheprobesusedtogeneratethem,arepublicallyavailableon www.zfin.org.In2005,MuellerandWullimannpublishedan in-depth atlas of early zebrafish brain development using in situ

probestoidentifytranscriptomicprofilesinspecificbrainregions andcelltypes(MuellerandWullimann,2005).Despiteawealthof toolsandinformation,therearesomedrawbacksofusingRNAin situ hybridizationtoobtaindetails ofstructuralchanges during DNT.Thegeneexpressioninformationobtainedusingstandardin situhybridizationprotocolsisoftenoflowerresolutionthanother labelingtechniques. Moreover, becausemRNA translation often takesplaceproximaltothecellbody,insituhybridizationtypically does not enable visualization of structural determinants of neuronalconnectivity,e.g.,axons,dendritesandsynapticcontacts

(Wuetal.,2016;Yoonetal.,2016).Thesedisadvantagesmaybe overcomebycombiningRNAinsituhybridizationwith immuno-histochemistry.

Immunohistochemistryhasbeen usedboth in whole-mount (Fig. 2)and sectioned larval zebrafish preparationstoexamine specificsubsetsofneuronsinthetail,optictectumandareasofthe brain (Easley-Neal et al., 2013; Yang et al., 2011). There are numerousvalidated,zebrafish-specific,neural-targeted antibod-ies,many of which are available through, or described on, the Zebrafish International Resource Center (ZIRC) website (http:// zebrafish.org). However, in comparison to mammalian models, immunohistochemistryhasbeenlimitedinzebrafishembryosdue topoorcrossreactivityofantibodiesdevelopedagainst mammali-an antigens with zebrafish antigens. As newer antibodies are developedspecificallyagainstzebrafishantigens,itislikelythat

immunohistochemistry will become increasingly valuable for studyingneuralconnectivityinthedevelopingzebrafishnervous system.

Array tomography, which combines immunohistochemistry, plasticized ultra-thinsectionsand high-resolution three-dimen-sional immunofluorescence imaging,couldprovetobeaviable method for visualizing neural connectivityin zebrafish ex vivo

(Leungetal.,2013;Michevaetal.,2010;MichevaandSmith,2007; Robles et al., 2011; Wang and Smith, 2012). This approach enhances image resolution, thereby allowing for collection of informationregardingneuronalconnectivityandotherstructural changes to thenervous system (Robles et al., 2011). The main drawbacks ofusingarraytomographyincluderesource require-ments (equipment and trained personnel), and increased time requiredtoprocessbothsamplesandimages.

Table1

Techniquesforneuronalimaginginlarvalzebrafish. Approach Whole mount Strain In/ Ex vivo Timeto implementation

Consistency Advantages Disadvantages

Immunohistochemistry(IHC) Yes Any Ex vivo

<1weekfrom

sampling

Good Wholemountorsections,validated antibodies

Limitedcross-speciesreactivityfor zebrafishproteinswithavailable antibodies

RNAinsituhybridization Yes Any Ex vivo

<1weekfrom

sampling

Good Largebodyofcomparablework, wholemountorsections,highly specifictargeting

TargetsRNAnotprotein(doesnot accountforpost-translational modifications)

Arraytomography No Any Ex vivo

<1weekfrom

sampling

Good High-resolutionimagingofIHC,ISH orgeneticlabelingtechniques

Increasedprocessingofbothsamples andimages

Transgeniclines:Enhancer trapFLIPtrapBrainbow,etc.

Yes Specific totarget

In vivo

6+months Good Stableandreliableexpression patterns,fluorescentlabelsallowfor highresolutionimaging

Relativelylongtimetocreate,need separatelinesforexaminingdifferent expressionpatterns Injectionand/or electroporationfor expressionoftransient geneticmarkers Yes Any In vivo

Immediate Variable Mosaicexpression,rapid

implementation,possibilityofsingle celllabeling

Highlevelsofmortality,mosaic expression,invasive,intersample variationinlabeling

Dyes/indicators Yes Any In vivo

Immediate Variable Rapidimplementation,mosaic expression,possibilityofsinglecell labeling

Highlevelsofmortality,invasive, inter-samplevariationinlabeling

Fig.2.Immunohistochemicalstainingofzebrafishlarvaeusinganantibodyagainstacetylatedtubulin.Zebrafishlarvaeat5dayspost-fertilizationwerefixedandstained withanantibodyagainstacetylatedtubulin,andthenimagedusingaLeicaSTEDconfocalmicroscope.Representativephotomicrographsillustratetubulinimmunoreactivity inthebrain(A)andtail(B)ofzebrafishlarvae.

3.1.Examplesillustratingtheuseofexvivoimagingtoassessneuronal connectivityindevelopingzebrafish

In general, most published work using ex vivo imaging has focusednotonvisualizingneuronalcellmorphologyorstructural parameters of neuronal connectivity, but rather on identifying neuronalcelltypes(Higashijimaetal.,2004;McLeanandFetcho, 2004)andbrainregions(Muelleretal.,2011,2008;Wullimannand Rink,2002)inthedevelopingzebrafishbrain.Anotableexception is a study that combined fluorescence in situ hybridization, immunohistochemistryandgeneticmarkerswitharray tomogra-phytoidentifyandcharacterizeGABAergiccellsinthetectumand periventricularneurons(Roblesetal.,2011).Additionally,exvivo

imaging techniques have been employed to study effects of chemicalson:(i)motorneuronmalformation(Babinetal.,2014; Svobodaet al.,2002; Welshetal., 2009; Yanget al.,2011);(ii) ganglioncell,Schwanncellandoligodendrocytepatterning(Parng etal.,2007);(iii)axonalpatterninginthebrainandspinalcord(Li etal.,2009);and(iv)developmentalRNAexpressionpatternsin theopticnerve(Royetal.,2016).

4.Invivoimagingtechniques

Manyinvivogeneticandimagingtoolshavebeendevelopedto studythegeneticregulationofzebrafishnervoussystem develop-ment.Thesegenetictoolsareusedinconjunctionwithadvanced imagingapproaches,suchasconfocalmicroscopyorlightsheet microscopy(Fig.3), andcomputationalmethodstoexaminethe structureandfunctionofthedevelopingzebrafishnervoussystem. Inrecentyears,lightsheetmicroscopyhasbecomeavaluabletool forvisualizingnotonlymorphologicalprocesses,butalsoneuronal activityinthewholebrainofembryoniczebrafish.Advantagesof lightsheetmicroscopyincludeits lowembryonicphototoxicity, highsignal-to-noiseratio, aswell ashighspatiotemporalresolution, whichisnecessaryforvisualizingfastmorphologicaleventswith highresolution[reviewedin(Ahrensetal.,2013;Ichaetal.,2016; Keller and Ahrens, 2015; Panier et al., 2013)]. These tools and techniquesareadaptableforDNTstudiesofchemicaleffects on neuronalconnectivity,asdiscussedinthissection.

4.1.Directdyelabeling

Lipophilic carbocyanine dyetracing (DiI, DiD, DiO,etc.)has beensuccessfullyusedforanterogradeandretrogradeneuronal

cell labelingin zebrafish (Jonteset al., 2000;Zou etal., 2014). These dyes diffusealong thelipid membrane of injectedcells, resultinginisolated whole-celllabeling. Theyareversatileand canbeusedinbothliveandfixedtissues.Althoughcarbocyanine dyesallowforvisualizationofcellularmorphology,labelingisnot precise,andinter-samplevariationiscommon.Similarly, neuro-nalactivitycanbemeasuredbyinjectingcalcium-sensingdyes intothebrainoflivefish(Kassingetal.,2013;NiellandSmith, 2005).WhilethistechniqueallowsliveCa2+_{imaging,itisquite}

invasive, sample to sample variation is common and acute toxicityand/ortraumaisaconcern.Thus,fordetectingsignificant differencesbetweenexperimentalgroups,alargesamplesizeis oftenneeded.Becauseofthisandthelabor-intensivenatureof theseprocedures,thesetechniquesmaybeusefulformechanistic studies, but currentlyare not feasible for mediumthroughput chemicalscreeningapplications.

4.2.Genetictoolsforinvivoimaging

AlargenumberofcDNAplasmidsandtransgenicfishlineshave beengeneratedtoexaminethemorphologyofdiversepopulations ofneuronsbyfluorescence.Enhancertrapscreeningandbacterial artificial chromosome transgenesis, sometimes in combination with the Tol2 transposon system, have been used to create fluorescent taggedenhancer traplines that showpan-neuronal expressionorexpressionlimitedtoneuronalcellsubpopulations (AsakawaandKawakami,2008;Asakawaetal.,2008;Satouetal., 2013; Scottand Baier,2009;Susteret al., 2009; Yoshidaet al., 2010).TheTol2transposableelementhasbeenusedtogenerate multiple enhancer trap and gene trap constructs, which are included in the zTrap database (http://kawakami.lab.nig.ac.jp/ ztrap/)(Kawakamiet al.,2010).Thisdatabaseprovidesa useful resourceforidentifyingfishlinesthathavethedesiredembryonic patternsforevaluationsofneurotoxicologiceffectsofchemicals. Manyoftheenhancertraplinesusegreenfluorescentprotein (GFP) as their fluorophore, which limits visualization of the interactionsbetweendifferentsubsetsofneurons.Thislimitation hasbeenaddressedbygeneratingtransgenicfishlinesthatenable combinatorial gene expression strategies. Two of the most commonlyusedcombinatorialexpressionstrategiesare:(i)binary systems such as GAL4-UAS, Lex A and Tet systems; and (ii) recombination-basedsystemssuchasCre/loxPorFlp/FRTsystems. These two strategies are reviewed in greater detail elsewhere (Hockingetal.,2013,Scott,2009,WeberandKoster,2013).Briefly,

Fig.3.Representativeimagesobtainedusinglightsheetmicroscopyoftransgeniczebrafishexpressingfluorescentproteinslinkedtoneural-specificpromoters.(A)Dorsal viewofbrainillustratingGABAergicneurons(red)andbloodvessels(green).(B)LateralviewofbrainshowingGABAergicneurons(green),bloodvessels(yellow)andcell nuclei(blue).Embryoswerefixedat120hpost-fertilization,brainsextractedandthenstainedovernightwith40_{,6-diamidino-2-phenylindole(DAPI).Brainswerethen}

mountedincapillarieswith1.5%low-meltagaroseandopticallyclearedin2,20_{-thiodiethanol(TDE)beforeimagingwithZEISSLightsheetZ.1at20Xmagnification.(For}

intheGAL4-UASbinarysystem,insteadofcouplingtheenhancer regionoraminimalpromotertoGFP,thepromoterorenhancer elementisplacedupstreamoftheGAL4gene.ThisensuresGAL4 proteinexpressioninthetissueofinterest.OncetheGAL4protein isexpressed,theGAL4bindstotheUASsequenceanddrivesthe expressionofanygenedownstreamoftheUASsequence.Many transgeniclinesexpressingthesebinarysystemsarelistedinthe ZebrafishInternationalResourceCenter(ZIRC)database(http:// zebrafish.org).

OfthemanypublicationstodatedescribingtheuseoftheGal4/ UAS driversystem in thezebrafish, oneof themostinfluential utilizedtheyeastGal4-DNAbindingdomainfusedtotheherpes simplexvirustranscriptionalactivationdomainVP16(Gal4-VP16) toincreaseexpressionoftheUAS-driventarget(KosterandFraser, 2001). The Gal4-VP16 system significantly amplifies transgene expression,andcandrivetheexpressionofmultipletargetgenes. Thishasproventobeavaluableresourceforinvivoimaging.Over 150transgenicenhancertraplinesandtheirexpressionpatterns arelistedinastudybyScottandBaier,withexpressionpatterns describedin manyspecificregionsof thebrainandspinal cord (Scott and Baier, 2009). Similarly, approximately 100 Gal4 enhancer trap lines that show brain-specific expression have recentlybeenidentified,andthesearepubliclyavailableviaZIRC (Otsunaetal.,2015).UsingacombinationofGal4-VP16and Cre-Loxwithbacterialartificialchromosomerecombineering,22 Gal4-andCre-baseddriverscontainingupstreamanddownstreamgene regulatoryregionsforcell-typespecificneurotransmitters, neuro-transmittersynthesizingenzymesorneuropeptideswere gener-atedwitheachofthetransgeniclineslabelingaspecificsubsetof neurons(Forsteretal.,2017).Inaddition,theFLIPtraplinescreated andmaintainedbytheCaltechCenterforExcellenceinGenomic Sciences provide additional fluorescently tagged enhancer trap lines with genes expression in the zebrafish nervous system (http://fliptrap.usc.edu/static/anatomies.html). These enhancer trap zebrafish lines provide a valuable resource for examining theeffectsofchemicalexposureonaspecificneuronalpopulation. ItisimportanttonotethatsomeoftheavailableGAL4linesare maintainedasfrozensperm,whichmayrequire6+monthsprior leadtimetogeneratelarvalzebrafishformechanisticstudiesor screeningapplications.

Oneofthedrawbacksoftheenhancertrappingstrategyisthat manyoftheselineshavenon-neuronalexpressioninadditionto expressioninthenervoussystem,whichsometimeslimitstheir utilityfor studyingcellular morphologyof developing neurons. Theuseofphotoconvertiblereporters,suchasUAS-Kaede,allows examinationofspecificneurons/neuronalprocessesina popula-tionoflabeledcells(Scottetal.,2007).Incorporationoftheneuron restrictivesilencingelementin thetransgenehasbeen demon-strated to limit non-neuronal expression in transgenic lines, therebymakingthesetransgeniczebrafishextremelyvaluablefor examiningneuronalmorphology(Bergeronetal.,2012).Arecent imagingstudy(Marquartetal.,2015)ofabout100GAL4,Creand enhancertraplinesshowed thatusingUAS–reporter transgenes with 30_{UTR sequences that bound non-neuronal microRNAs} significantlylimitednon-neuronalexpressionofthesetransgenes. ThismakesmanyoftheknownGAL4linesmoreusefulforanalysis of neuronal cell populations. The expression patterns of these transgenes are available ona 3-D database of gene expression known as Brain Browser (https://science.nichd.nih.gov/confl u-ence/display/burgess/Brain+Browser), as well as the zebrafish information network (Zfin, http://zfin.org) and the zebrafish enhancer trapdatabase maintainedby the Burgess lab (http:// burgesslab.nichd.nih.gov/).

One of the most exciting advances of these combinatorial systemsfor visualizingneuronshasbeenthegenerationof the multi-labeled Zebrabow/Brainbow fish lines and plasmids

(Panetal.,2013).ThesefishexpressaDNAconstructthatencodes for three fluorophores – red fluorescent protein (RFP), cyan-fluorescentprotein(CFP)andyellowfluorescentprotein(YFP)– underthecontrolofaubiquitouspromoterormultiplecopiesof UAS,witheachfluorophoreflankedbytwouniqueLoxPsites.The UAS-Zebrabowconstructscanbeexpressedindistinctsubsetsof neurons by using tissue specific GAL4 drivers. Under basal conditions,thefirstfluorophore(RFP)intheconstructisexpressed in allcells. WhenCre recombinase is introduced(either as an injectedmRNA/proteinorbycrossingtoaCre-transgenicline),the fluorophoresarestochasticallyexpressedthroughouttheaffected celltypesduetotheactivityoftheCrerecombinaseonthe_flanking LoxPsites.Theendeffectisarangeofcolorsobservedthroughout target tissues with neighboring cells often displaying distinct colors(Panetal.,2013,2011;WeberandKoster,2013).Thus,these lines provide an excellent method for imaging not only the neuronalmorphologyofindividualcellswithinapopulation,but alsotheinteractionsbetweenconnectingneurons.

Mosaicexpressionofreporterconstructscanalsobegenerated byinjectingcDNAplasmidsintotheembryo.Earlystage(1–2cells) injectionwithplasmidsencodingBrainbowfluorophores(driven byaCMVpromotersinceneuron-specificThypromotersdonot workinzebrafish)intoCre-transgeniclines,orco-injectionof Cre-recombinase mRNA/protein into other lines, results in mosaic expression of the fluorophores, thus allowing for non-specific tissueexpressioninanyfishline(Panetal.,2013).Alternatively, plasmid/mRNA canbe injecteddirectlyintothe thirdor fourth ventricleat24hpost-fertilization.Iftheseinjectionsareclosely followedbyelectroporationacrossthebrain,theplasmidswillbe incorporatedintothedevelopingneurons(Cerdaetal.,2006;Dong etal.,2011;HendricksandJesuthasan,2007b;Tawketal.,2009). Injectingat24hpost-fertilizationcatchesneuronsduring prolif-eration (radial glia, neuronal stem cells, etc.), and facilitates labelingofneuronsindifferentregionsofthebrain.

4.3.Examplesillustratingtheuseofinvivoimagingtoassessneuronal connectivityindevelopingzebrafish(summarizedinTable2) 4.3.1.LabelingofdendritesintheCNS

Dendritic arbor formation is a dynamic process and critical structural determinant of neuronal connectivity. Neurotoxic chemicalshavebeenshowntointerferewithnormalpatternsof dendriticarborization,andaltereddendriticcomplexityislinked tomultipleneurodevelopmentaldisorders(Copf,2016;Lein,2015; Stamouetal.,2013).Imagingofcompletedendriticarborsinthe intactzebrafishbrainisdifficult,buttoolshavebeendevelopedto make these measurements possible. For example, dendritic arborization and synapse formation hasbeen quantified in the optic tectum of developing zebrafish by injecting a GAL4/UAS plasmid(Niellet al.,2004).The plasmidusedfor thesestudies containedapan-neuronalgold_fishalpha-1tubulindriver(Koster and Fraser, 2001), and expressed both dsRed, a whole cell biomarker,and GFP-taggedPSD95,a postsynapticmarker(Niell et al., 2004). Using a membrane-targeted yellow _fluorescent proteindrivenbythealdoca(aldolasec,fructose-biphosphatea) promoter, neuronal cell polarization and dendrite formation of Purkinjecellswasvisualizedinthezebrafishcerebellum(Tanabe et al., 2010). Using membrane-bound fluorescent proteins, dendrite formation in retinal ganglion cells was imaged using two-photon confocal microscopy (Choi et al., 2010). Similarly, severalpopulationsofretinalganglioncellslabeledwithseparate fluorophores driven by cell-type specific promoters have been imaged(Mummetal.,2006).Anotherstudyusedacombinationof membrane targeted Brainbow with bacterial artificial chromo-sometransgeniclinesusingDsRedandGFPtolabelglycinergicand glutamatergic neurons in the hindbrain of zebrafish larvae

(Kinkhabwalaetal.,2011).Thisapproachallowedvisualizationof notonlytheuniquemorphologiesofglycinergicvs.glutamatergic neurons,butalsotheirphysicalassociationstoeachother.While thesestudiesfocusedonthepolarization,initiationandformation ofdendriticarbors,thetechniquesusedcouldbereadilyappliedto assesstheeffectsofchemicalsondendriticcomplexitythroughout theCNS.

4.3.2.Synapticmarkers

Neuronalsynapsesarespecializedcell-celljunctionsthatallow communicationbetweenneurons.Thedistributionandprocessing ofinformationinthenervoussystemisdeterminedbythepattern ofsynapticconnectionsformedbetweenneuronsduring develop-ment.Imbalancesinthetypesand numberof synapsesformed duringdevelopmentcancontributetoadverseclinicaloutcomes. Whenvisualizingsynaptic connections,it is importanttomark boththepresynapticcell(i.e.theaxon)andthepostsynapticcell (i.e.thedendriteorsoma),astheseconnections,especiallyduring development,aredynamic.Whilepre-andpostsynapticmolecules are conserved in the zebrafish genome (Bayes et al., 2017), relativelyfewofthesehavebeentargetedforinvivoimaging.Of the available studies, the predominant focus has been on presynaptic markers. Axonal growth cones and early synapto-genesishavebeenvisualizedusingthedyesDiD(1,10 -dioctadecyl-3,3,30,30-tetramethylindocarbocyanine)andDiO(3,30 -dioctadecy-loxacarbocyanineperchlorate) to mark growing neuronsin the zebrafishspinalcord(Jontesetal.,2000).Thesamegrouplater identifiedpresynapticterminalsusingGFP-linkedN-cadherinand verified these data by counterstaining for the synaptic vesicle proteinSV2(Jontesetal.,2004).Expressionofthefusionprotein was drivenin neuronal tissue usinga Gal4/UAS system (Jontes et al., 2004; Koster and Fraser, 2001). A combination of GFP-synaptophysinand cytosolic DsRed (bothdrivenbya Gal4/UAS system)wasusedtodemonstratethedynamicprocessofsynaptic formationandstabilizationin thedeveloping zebrafishnervous system(MeyerandSmith,2006), andfluorescentmarkershave beenusedtostudysynapsintraffickingduringsynapseformation inspinalneurons(Easley-Nealetal.,2013).Whilemanyofthese techniquesweredevelopedtodeterminethekineticsofsynapse formation in zebrafish spinal neurons, they could be easily repurposedfor usein thezebrafish brain,andfor studyingthe

effects of chemicals onsynapse formation and number in the developingbrain.

4.4.Synapticactivitymeasurement

Thesmallsizeofthezebrafishembryonicbrain,coupledwith the linearorganization of thebrain and the inversion of brain structure (i.e.the“deeper”neuronsareonthesurface)enables imaging of activity within the whole brain using current microscopy techniques (Feierstein et al., 2015; Leung et al., 2013).Injectionofacalciumindicatordyeintothetectalneuropil hasbeenusedtoanalyzevisualresponsesinlivelarvalzebrafish (Niell and Smith, 2005). A similar, non-invasive method for measuringbrainactivityinvolvestheuseofgeneticallyencoded Ca2+ _{indicators (GECIs). The GECI lines, when driven by}

pan-neuronalpromotersandimagedusingtwophotonmicroscopyor light sheetmicroscopy, allowvisualization of synaptic activity, evenatthelevelofsingleactionpotentialwithinthedeveloping zebrafish brain (Akerboom et al., 2012). The newer generation calciumsensorshavebeenusedtomeasureactivityinthewhole zebra_fishbrain,andinresponsetospeci_ficbehavioraltraitssuchas optomotorbehaviorandfictivebehaviorinlarvalzebrafish(Ahrens etal.,2012;Randlettetal.,2015).Inaddition, immunohistochemi-cal staining forextracellular signal–regulated kinase (ERK)was used as another readout for neuronal activity (Randlett et al., 2015).Thesedatawereusedtogenerateazebrafishbrainatlas (Z-brain - http://engertlab.fas.harvard.edu/Z-Brain/) that correlates neuronal activity and neuroanatomical information. GECIs in combinationwithadvanced imaging and processingtechniques caneven allowforCa2+imaging infreelyswimming larvalfish (Congetal.,2017;Kimetal.,2017;Knafoetal.,2017).

5.Conclusions

Thelarvalzebrafishprovidesanunparalleledopportunityfor medium throughput invivoneuronal imagingof structuraland functionalparametersofneuronalconnectivity.Bothexvivoandin vivotechniquesforimagingneuronalconnectivityatthecellular levelhavebeendevelopedforthedevelopingzebrafishbrainbut have yet to be applied to DNT research. Additionally, new visualizationtools,microscopymethodsandcomputationaltools

Table2

Examplesofliveimaging.

Target Method Brainregionor

celltype

Reference

Dendrites Transgeniclinescontrolledbyisl2borbrn3cRGCspecificpromotersandtransientexpressiondrivenbya Gal4/UASsystemcontrolledbythesamepromoters

RGCs (Mummetal., 2006) Transgeniclinesexpressingmembraneboundfluorescentproteinsdrivenbyeitherisl2borbrn3cRGC

specificpromoters

RGCs (Choietal., 2010) Membrane-targetedyellowfluorescentproteindrivenbythePurkinjecellspecificaldoca(aldolasec,

fructose-biphosphatea)promoter

Purkinjecells (Tanabeetal., 2010) Dendritesandsynaptic

connections

GAL4drivercontrolledbythepan-neuronalgoldfishalpha-1tubulinpromoterandUASactivatorexpresses dsRed(wholecell)andPSD95:GFP(presynapticmarker)

Tectum (Nielletal., 2004) Synapticconnections LivestainingusingDiD(1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine)andDiO(3,3’

-dioctadecyloxacarbocyanineperchlorate)

Spinalneurons (Jontesetal., 2000) MarkedpresynapticterminalsusingGFP-linkedN-cadherin,andverifiedbycounterstainingforthe

synapticvesicleproteinSV2

Spinalneurons (RB)

(Jontesetal., 2004) GFP-SynaptophysinandcytosolicDsRed,bothdrivenbyaGal4/UASsystemandtargetedusingan

RGC-specificbrn3Cpromoter

Optictectum andRGCs

(Meyerand Smith,2006) Transgeniclineneurogenin1:GFPandfluorescentlytaggedsynapticproteinsusingtheGal4/UASsystem

drivenbyaRBneuronspecificpromoter

Spinalneurons (RB)

(Easley-Neal etal.,2013) Synapticactivity Calciumindicatordyedirectlyinjectedintothetectalneuropil Tectum (Nielland

Smith,2005) Thegeneticallyencodedcalciumindicator(GECI)transgeniclinesundercontrolofvariousneuronal

promoters

Various (Akerboom etal.,2012) RGC=retinalganglioncells.

arecontinuouslybeingdevelopedacrossdiversemodelorganisms thatcouldbeadaptedtothezebrafish[reviewedby(Leeetal., 2016)]. Ex vivo techniques are a good starting point, since generating or acquiring transgenic lines is time consuming (Table 1). This approach is facilitated by the availability of extensive reviews describing resources available to study the nervoussystemofzebrafishlarvae[reviewedby(Chapoutonetal., 2010);alsoseeTable3.Usingthesetechniques,researcherscan identify affected regions/cell types and find/create appropriate transgeniclines. Alternatively,mosaic expression(e.g. transient geneticmarkers)underthecontrolofapan-neuronaldrivercan helpidentifyDNTeffectsinspecificbrainregions.

Incorporatingtheseimagingtechniqueswillrequiremodifi ca-tionstoincreasethroughputandreproducibility.Inourexperience, useofavailablenon-stabletransgenicmarkersproducesunreliable expression patterns and is not currently practical for DNT screening applications.Additionally, thesetechniques areoften equipment intensive, requiring sophisticated and expensive microscopesandimageanalysis toolstoobtainand processthe datagenerated.Theseproblemsaresurmountableandtheaddition of neuronal morphogenic data to the extensive databases of genetic, “omic”, teratological and behavioral phenotypic data alreadypublishedwillbeasignificantadvanceforunderstanding DNTandgenebyenvironmentinteractionsthatcontributetothe pathogenesisofneurodevelopmentaldisorders.

Conflictofinterest None.

Acknowledgements

WethankIngrid Brust-Mascherand ColinReardonoftheUC Davis Health Science District Advanced Imaging Facility for assistance in generating the representative photomicrographs showninFigs.2and3.ThisworkwassupportedbytheNational Institute of Environmental Health Sciences (grants ES014901, ES011269 and ES023513) and the UnitedStates Environmental Protection Agency (grant R833292). GWM was supportedby a postdoctoralfellowship from theNationalInstitute of Environ-mentalHealthSciences(F32ES024070).

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Zebrafishresourcesforgeneexpressioninformation.

Database Descriptionofavailabletools Websitelink

zTrap FishEnhancertrapandgenetraplineswithdiverseembryonicexpressionpatterns http://kawakami.lab.nig.ac.jp/ztrap

ZIRC ZebrafishInternationalResourceCenterwebsitehasinformationaboutfishlinessuchas GAL4lines,EST/cDNAs,Antibodiesetc.

http://zebrafish.org

BrainBrowser 3-Ddatabasewithgeneexpressionpatternsforaround100enhancertraplineswith neuronalexpressionpatterns.

https://science.nichd.nih.gov/confluence/display/ burgess/Brain+Browser

ZFIN Zebrafishmodelorganismdatabasecontainingfishlines,geneexpressiondata,antibodies etc.

http://zfin.org

ZebrafishEnhancer TrapDatabase

Thedatabasecontainsinformationfortheneuronalenhancertraplinesgeneratedbythe Burgesslab

http://burgesslab.nichd.nih.gov/

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