Amigo Adhesion Protein Regulates Development of Neural Circuits in Zebrafish Brain

19 Pages·2014·7.6 MB·English

THEJOURNALOFBIOLOGICALCHEMISTRY VOL.289,NO.29,pp.19958–19975,July18,2014
©2014byTheAmericanSocietyforBiochemistryandMolecularBiology,Inc. PublishedintheU.S.A.
Amigo Adhesion Protein Regulates Development of Neural
Circuits in Zebrafish Brain*
Receivedforpublication,December23,2013,andinrevisedform,June3,2014Published,JBCPapersinPress,June5,2014,DOI10.1074/jbc.M113.545582
XiangZhao‡1,JuhaKuja-Panula‡,MariaSundvik‡§,Yu-ChiaChen‡§,VilmaAho¶,MarjaanaA.Peltola‡,
TarjaPorkka-Heiskanen¶,PerttiPanula‡§,andHeikkiRauvala‡2
Fromthe‡NeuroscienceCenter,§InstituteofBiomedicine/Anatomy,and¶InstituteofBiomedicine/Physiology,Universityof
Helsinki,HelsinkiFIN-00014,Finland
Background:Amigosaretransmembraneproteinssuggestedtomediateadhesiveinteractionsofcells.
Results:Down-regulationofAmigoproteinexpressionorinhibitionofitsfunctionresultsindefectsoffiberpathwaydevelop-
mentanddisturbedlocomotorfunctionsinzebrafish.
Conclusion:TheneuralformofAmigoisrequiredforconstructionoffunctionalneuralcircuitries.
Significance:Amigoisidentifiedasanovelfactorthatregulatesformationofneuronalconnections.
TheAmigoproteinfamilyconsistsofthreetransmembrane haverecentlygainedincreasinginterestaskeyfactorsguiding
proteinscharacterizedbysixleucine-richrepeatdomainsand developmentofneuronalconnectivity(forarecentreview,see
one immunoglobulin-like domain in their extracellular moi- Ref. 1). LRR domains are protein-protein interaction motifs
eties.Previousinvitrostudieshavesuggestedaroleashomo- thathavebeenimplicatedindevelopmentandplasticityoffiber
philicadhesionmoleculesinbrainneurons,buttheinvivofunc- tracts,recognitionofaxontargets,synaptogenesis,anddisor-
tionsremainunknown.Herewehaveclonedallthreezebrafish dersofthenervoussystem.Slitproteins(Slit1–3),Trkrecep-
amigosandshowthatamigo1isthepredominantfamilymem- tors(TrkA,TrkB,andTrkC),andNogoreceptor1(NgR1)are
berexpressedduringnervoussystemdevelopmentinzebrafish. among the most studied LRR proteins in the nervous system
Knockdown of amigo1 expression using morpholino oligonu- development(2,3).
cleotides impairs the formation of fasciculated tracts in early AMIGO(amphoterin-inducedgeneandopenreadingframe)
fiberscaffoldsofbrain.Asimilardefectinfibertractdevelop- wasfoundbyordereddifferentialdisplayasatranscriptup-reg-
mentiscausedbymRNA-mediatedexpressionoftheAmigo1 ulatedinrathippocampalneuronsthatextendneuritesupon
ectodomain that inhibits adhesion mediated by the full- ligation of the transmembrane receptor RAGE (receptor for
length protein. Analysis of differentiated neural circuits advanced glycation end products) by amphoterin (HMGB1;
reveals defects in the catecholaminergic system. At the high mobility group box-1) or by anti-RAGE antibodies (4).
behaviorallevel,thedisturbedformationofneuralcircuitryis Together with two homologous proteins, the three AMIGOs
reflectedinenhancedlocomotoractivityandintheinability (AMIGO1–3)formanovelfamilyoftransmembraneLRRpro-
ofthelarvaetoperformnormalescaperesponses.Wesuggest teins.ThethreeAMIGOsdisplayabout50%sequencesimilar-
that Amigo1 is essential for the development of neural cir- itycomparedwitheachother,andtheyhavethesamedomain
cuitsofzebrafish,whereitsmechanisminvolveshomophilic organization,withsixextracellularLRRmotifsandoneimmu-
interactionswithinthedevelopingfibertractsandregulation noglobulin-like (Ig) domain, another protein motif that is
of the Kv2.1 potassium channel to form functional neural widelyexpressedinneuralrecognitionmolecules(5).InBLAST
circuitrythatcontrolslocomotion. searches using the AMIGO1 ectodomain to identify homolo-
gous proteins outside of the AMIGO family, the best fits are
found for the axon-guiding Slit proteins and the Nogo-66
Developmentoffunctionalneuralcircuitsdependsonspa- receptor(NgR1).
tiallyandtemporallyprecisecellularinteractionsmediatedbya AMIGO1andAMIGO2arehighlyexpressedinthemamma-
widevarietyofsecretedandmembrane-boundfactors.Proteins liannervoussystem,whereasAMIGO3displaysabroadexpres-
containing extracellular leucine-rich repeat (LRR)3 domains sion pattern in different tissues (4, 6, 7). In vitro studies have
suggestedthatAMIGO1actsasahomophilicadhesionmole-
*ThisworkwassupportedbytheAcademyofFinlandandtheSigridJusélius cule that induces outgrowth and fasciculation of neurites in
Foundation. central neurons (4). The crystal structure of the AMIGO1
1Supported by the Doctoral Program in Brain and Mind of the Doctoral dimer has recently revealed that homophilic binding of
SchoolinHealthSciences.
AMIGO1 occurs through its LRR domains (8). In addition,
2Towhomcorrespondenceshouldbeaddressed:NeuroscienceCenter,P.O.
Box56(Viikinkaari4),FIN-00014UniversityofHelsinki,Finland.Tel.:358-9- AMIGO1andAMIGO2(alsodesignatedasAlivin1)havebeen
19157621;Fax:[phone];E-mail:[email protected]. reportedtoenhancesurvivalofneuronsinculture(9,10).Fur-
3Theabbreviationsusedare:LRR,leucine-richrepeat;CA,catecholaminergic;
thermore, AMIGO1 has recently been found to bind to the
DiV,diencephalonventricle;dpf,day(s)postfertilization;hpf,hour(s)post-
fertilization;Igdomain,immunoglobulin-likedomain;MBP,maltose-bind-
ing protein; MLCT, medial longitudinal catecholaminergic tract; MLF,
medial longitudinal fasciculus; MO, medulla oblongata; POC, postoptic PCR, quantitative RT-PCR; SLC, short latency C-start; LLC, long latency
commissure;TH,tyrosinehydroxylase;vcc,ventralcaudalcellcluster;qRT- C-start;ANOVA,analysisofvariance.
This is an Open Access article under the CC BY license.
19958 JOURNALOFBIOLOGICALCHEMISTRY VOLUME289•NUMBER29•JULY18,2014
AmigoinBrainDevelopment
Kv2.1 potassium channel and to affect excitability of central and 5(cid:2)-AGC GAC AGG GAG TCA AGT AG-3(cid:2). The zebra
neuronsviatheKv2.1interactions(11). fish (cid:2)-actin (AF057040.1, GI:3044209), elongation factor 1
Despiteseveralinvitrofindingsprovidingcluestodevelop- (cid:3)1 (zgc:109885, GI:90652818), and ribosomal protein L13a
mental roles of the AMIGOs, their in vivo functions remain (BC047855.1,GI:28838761)weresetastemplatequantitycon-
unknown. In the current study, we have used the zebrafish trols,andthesameprimerswereusedasdescribedpreviously
model to study Amigo functions in nervous system develop- (12).ThePCRswereprocessedwiththeBio-RadCFX96real-
ment. We have cloned all three amigo genes of zebrafish and time PCR machine using the CFX96TM real-time PCR detec-
focusedourfunctionalstudiesonamigo1,whichwasfoundto tionsystem.Thethreeconstructedamigoplasmidspreparedin
bethemajormemberofthegenefamilyexpressedinzebrafish differentdilutions(from1mg/mlto0.1(cid:4)g/ml)inmilli-Qwater
duringthenervoussystemdevelopment.Usingthemorpholino wereusedasstandardsforqPCR.Theamigo1expressionlevel
knockdown approach and expression of the Amigo1 ectodo- normalizedto(cid:2)-actinin1dpflarvaewassetas1.
mainasadominantnegativereceptor,weshowthatAmigo1is Cloningofamigo1andkv2.1mRNAs—Thefull-lengthopen
essential for the development of fiber tracts in the zebrafish reading frame mRNA construct encoding Amigo1 was pre-
brain. paredbyRT-PCRwithprimers5(cid:2)-ATAAGATCTATGCCC
CCTTCCATTAATTG-3(cid:2)and5(cid:2)-AAGAATTCCCGGTCA
EXPERIMENTALPROCEDURES AAAGATACACATCCTC-3(cid:2).TheconstructincludesBglII
Animals—Anoutbredzebrafish(Daniorerio)strainfroma and EcoRI restriction sites on both sides of the amigo1 first
local resource, the Turku line, was used in this study for its strand cDNA in the full-length transcript. These restriction
steadyyieldofembryos(12).Fishfeeding,breeding,andmain- siteswereusedforinsertionintothepMCexpressionvector,
tenance were done according to an established protocol (13). whichcapstheinsertedfragmentwiththesequenceelements
TheexperimentpermitswereobtainedfromtheUniversityof ofthe(cid:2)-globin5(cid:2)-UTRand3(cid:2)-UTRonbothofitssidesandin
HelsinkiCommitteeforanimalexperimentsandtheNational additiontheSV40poly(A)signalatthe3(cid:2)-tail.Thiselevatesthe
AnimalExperimentBoardinagreementwiththeethicalguide- transcribed mRNA stability and activity about 100-fold in
linesoftheEuropeanConvention.Embryosofeithersexwere mRNA injection experiments (14). The mRNA encoding the
stagedaccordingtothenumberofhourspostfertilization(hpf) Amigo1ectodomainwaspreparedwithprimers5(cid:2)-ATAAGA
ordayspostfertilization(dpf).Topreventpigmentformation, TCTATGCCCCCTTCCATTAATTG-3(cid:2)and5(cid:2)-ATGAAT
0.2mM1-phenyl-2-thiourea(Sigma)wasaddedtothemedium TCT CAG CCA TTG CCG TTG AGG-3(cid:2). GFP mRNA was
ofembryosafterspawning. used as another control in mRNA rescue experiments, and it
Cloning of amigo Genes and Quantitative RT-PCR—Ze- was prepared using the pEGFP-C1 vector. All mRNAs were
brafish orthologs were found by BLASTing the peptide preparedusingthemMessagemMachinekit(Ambion,Austin,
sequencesofthemammalianAMIGOproteinsusingtheUCSC TX)accordingtothemanufacturer’sinstructions.
genome browser and Ensembl zebrafish ZV9 database. Total The full-length kv2.1 cDNA (ZDB-GENE-090831-3) was
RNA from 1–8-dpf larvae was extracted and reverse-tran- prepared by RT-PCR with primers 5(cid:2)-ATA AGC TTC CCT
scribed.ThesubsequentPCRwasperformedasdescribedpre- CGGCAGGAATGAGTAA-3(cid:2)and5(cid:2)-ATGGATCCTCAA
viously(12).RNAqualityandamountwereanalyzedspectro- AGGCCCTTATCAAAAG-3(cid:2)andclonedintopMCexpres-
photometrically by a NanoVue Plus spectrophotometer (GE sion vector. A cDNA fragment encoding the N terminus and
Healthcare).Theprimers5(cid:2)-ATGCCCCCTTCCATTAAT the transmembrane loops of the Kv2.1 protein (432 amino
TG-3(cid:2) and 5(cid:2)-CCG GTC AAA AGA TAC ACA TCC TC-3(cid:2) acids)wasclonedintopMal-c2Evector(NewEnglandBiolabs
were used for cloning the full-length coding sequence of Inc.) for Kv2.1 ectodomain-maltose-binding protein (MBP)
amigo1(ENSDARG00000079620).Theprimers5(cid:2)-ATGACC recombinantexpressionwithprimers5(cid:2)-ATGGATCCCCCT
TCG ACA TCT TGC ATG GTT-3(cid:2) and 5(cid:2)-GAT TCA AAC CGGCAGGAATGAGTAA-3(cid:2)and5(cid:2)-ATAAGCTTCTTG
AAG CAG GAT TTT AAG G-3(cid:2) were used for cloning the ATGGCCTTCTCTTGT-3(cid:2).
full-length coding sequence of the second gene that displays Antibodies against Amigo1 and Kv2.1—Chicken polyclonal
clear homology compared with the amigo genes (ENS- antibodies were produced against the zebrafish Amigo1
DARG00000079569;designatedhereasamigo3a).Theprimers (Agrisera,Sweden)usingAmigo1ectodomain-GST(glutathi-
5(cid:2)-ATGCTGTGTGCTCAGGGTGTGGC-3(cid:2)and5(cid:2)-TCA one S-transferase) fusion protein as the antigen. In order to
TCTCTCTGATTCTATGTGCTTTCCT-3(cid:2)wereusedfor makeahighqualityantigen,theAmigo1extracellularcoding
cloningthefull-lengthcodingsequenceofthethirdhomologous sequence was cloned into pGEX-2TK GST fusion vector (GE
gene(ENSDARG00000074469;designatedhereasamigo3b).The Healthcare)usingtheprimers5(cid:2)-ATAGGATCCTGCGCC
PCRproductswerepurifiedwiththeMinEluteGelExtractionkit AGCAACATTGTCAG-3(cid:2)and5(cid:2)-ATGAATTCTCAGCCA
(Qiagen,Hilden,Germany),ligatedtothepGEM-Teasyplasmid TTGCCGTTGAGG-3(cid:2)toaddtheBamHIandEcoRIrestric-
(Promega),andsequenced. tion sites on both sides of the coding sequence. The Amigo1
The quantitative RT-PCR (qRT-PCR) primers of amigo1 ectodomain-MBPfusionproteinwasproducedbycloningthe
were5(cid:2)-TCGCCGTGAGTGAATACCTC-3(cid:2)and5(cid:2)-TGC codingsequenceintopMAL-c2Evector.Theprimers5(cid:2)-ATG
CAA GCA ACC CAC CAA A-3(cid:2). The qPCR primers of AATTCTGCGCCAGCAACATTGTCAG-3(cid:2)and5(cid:2)-ATA
amigo3awere5(cid:2)-GGCTGTGTTGTGACCCTTGT-3(cid:2)and GGATCCTCAGCCATTGCCGTTGAGG-3(cid:2)wereusedfor
5(cid:2)-GATGAGATGGCTGGAGATGGA-3(cid:2).TheqPCRprim- addingEcoRIandBamHIonbothsidesofthecodingsequence.
ersofamigo3bwere5(cid:2)-ACACTGGCTTCACCACACT-3(cid:2) TheplasmidsweretransformedintoEscherichiacoliXL-1blue
JULY18,2014•VOLUME289•NUMBER29 JOURNALOFBIOLOGICALCHEMISTRY 19959
AmigoinBrainDevelopment
strain by electroporation (Bio-Rad) for Amigo1 ectodomain- Whole Mount in Situ Hybridization—Whole mount in situ
GST/MBPrecombinantexpression.Therecombinantprotein hybridization was carried out as described previously (15),
waspurifiedusingtwodifferentaffinitycolumns(GSTorMBP usingthespecificprobes(16)ofpax2a(theZIRCcb378),pax6a
tag).ThepurifiedAmigo1ectodomain-GST/MBPfusionpro- (ZIRC cb280), and krox20 (ZIRC cb427). Larvae at 28 hpf
teinwasanalyzedonSDS-PAGEstainedwithCoomassieBlue (Prim-5)and2dpf(Long-pec)stageswereusedintheexperi-
(Invitrogen). ments. The probe for the detection of amigo1 expression
TheIgYantibodiesintheimmunizedeggyolkwerefirstpuri- was obtained from the cDNA clone encoding the Amigo1
fiedbyammoniumsulfateprecipitation.Inaffinitypurification, ectodomain.
thefirststepwascarriedoutonanaffinitycolumnloadedwith Detection of Apoptosis and Proliferation—A fluorometric
Amigo1 ectodomain-GST fusion protein. The second step in TUNELsystem(DeadEndTM,Promega)wasusedforstaining
affinitypurificationwascarriedoutonanotheraffinitycolumn ofapoptosisinwholemounts,andEdustaining(Click-iTEdu
loaded with Amigo1 ectodomain-MBP fusion protein to AlexaFluor555,Invitrogen)wasusedfordetectionofcellpro-
excludetheproteinsthatbindnonspecificallytotheGSTtag. liferation in whole mounts. Apoptosis and proliferation were
The purified zebrafish Amigo1 antibodies were diluted to 1 exploredaccordingtotheprotocolrecommendedbytheman-
mg/mlaliquotscontaining0.01%NaN andstoredat(cid:3)80°C. ufacturer(12).
3
Monoclonal anti-Kv2.1 antibodies (K39/25) were obtained MorpholinoOligonucleotideandmRNAInjections—Knock-
from the NeuroMab facility (University of California, Davis/ downexperimentsofamigo1werecarriedoutwithtranslation-
NationalInstitutesofHealth). blockingantisensemorpholinooligonucleotides(MOs)(Gene
Western Blotting and Co-immunoprecipitation—Zebrafish Tools)targetedtothe5(cid:2)-upstreamsequenceflankingthetrans-
Amigo1antibodies(1:1000correspondingto1(cid:4)g/ml)andthe lation start site (MO1, 5(cid:2)-GGC ATT TCT GAC ACG CAG
purifiedmonoclonalKv2.1antibodies(2(cid:4)g/ml)wereusedfor TTA AAA T-3(cid:2); MO2, 5(cid:2)-TGT GGT TGT AGC ACA AGT
Western blotting and for co-immunoprecipitation that was CAT AAA C-3(cid:2)) of theamigo1 transcript. To knock down
carriedoutaccordingtoapreviouslydescribedprotocol(11). amigo3bexpression,thetranslation-blockingMOwas5(cid:2)-GGC
Dithiobis(succinimidyl propionate) cross-linking (Pierce) was CAC ACC CTG AGC ACA CAG CAT T-3(cid:2). The 5-mispair
appliedasbefore(11).Anti-HNK-1antibodies(C0678,1:500; oligonucleotide5misMO,GcCATTTgTGAgACcCAcTTA
mousemonoclonal,Sigma-Aldrich)andanti-acetylatedtubu- AAAT-3(cid:2)(mispairsareinlowercasetype),wasusedasanMO
lin(6-11B-1mousemonoclonal,1:1000;Sigma-Aldrich)were injection control. Cloning of the mRNAs encoding the full-
usedtodetectzebrafishaxonogenesisandaxonaldevelopment length Amigo1 and the ectodomain of Amigo1 (mRNA and
inWesternblottingandwholemountimmunostaining.Mouse EmRNA,respectively)isexplainedabove.
monoclonal anti-(cid:2)-actin antibody (A2228, 1:1000; Sigma-Al- MicroinjectionsoftheMOsandthemRNAsintozebrafish
drich)wasusedasthecontrolofsampleloading.Thezebrafish embryoswerecarriedoutasdescribedpreviously(12).Anali-
tissue preparation and Western blotting were carried out as quotof4nlofthemixture(correspondingto4ngofMOswhen
describedpreviously(12,13). 100Msolutionwasused)wasinjectedintotheyolkofa1–4-cell
Immunocytochemistry—Mouse monoclonal anti-tyrosine embryo that was allowed to develop at 28.5°C. For Amigo1
hydroxylase(TH)antibody(Diasorin,Stillwater,MN)wasused mRNAandGFPmRNAinjections,0.1–0.2(cid:4)g/(cid:4)lmRNAwas
forthewholemountstainingofthecatecholaminergicsystem mixedwithinjectionsolutionafterheating.Theinjectioncon-
in3dpflarvalbrainsasdescribedearlier(12).Themouseanti- centration was tested for eliminating ubiquitous overexpres-
3A10 (1:100; Developmental Studies Hybridoma Bank), sioneffects(12).ForKv2.1mRNAinjections,0.1(cid:4)g/(cid:4)lmRNA
chicken anti-Amigo1 antibodies (5 (cid:4)g/ml), and anti-HNK-1 wasmixedwiththeinjectionsolution.
antibodies(1:100)wereusedforwholemountimmunostaining MicroscopyandImageAnalysis—Confocalimagingoflarvae
ofzebrafishlarvae.TheAmigo1ectodomain-MBPfusionpro- stainedwithanti-THandanti-Amigo1antibodieswascarried
teinwaspurifiedandusedasacompetingantigeninantibody out as described (12). Zeiss LSM710 Pascal confocal micros-
binding(100(cid:4)g/ml).Forimmunohistochemistryofthelarvae, copysystemwasusedforimaginglarvalsamplesstainedwith
thesampleswerefixedin2%paraformaldehydefor2hatroom anti-3A10 antibodies and anti-HNK1 antibodies. Stacks of
temperatureandthenwashedandpreincubatedinphosphate- imagestakenat0.6–1.2-(cid:4)mintervalswerecompiledtomake
bufferedsalinecontaining0.1%Tween20(PBS-T,pH7.4)with maximumintensityprojectionimages.Specimensfrominsitu
1%dimethylsulfoxide(DMSO)and4%normalgoatserumat hybridization were examined with inverted light microscopy
4°Covernightorlonger.Thespecimenswereincubatedwith usingOlympusIX70connectedthroughaCCDcameratothe
the primary antibodies in the preincubation solution (PBS-T Analysis(cid:2)software.Highresolutionimageswereobtainedwith
with2%normalgoatserum)forover12hat4°Cunderslow a digital MicroFire S99808 camera (Optronics) attached to
stirring.ThesampleswerethenwashedthoroughlywithPBS-T OlympusBX51epifluorescencemicroscope(Olympus,Tokyo,
and incubated with the Alexa(cid:2)-conjugated goat anti-chicken Japan).TheacquiredimageswerefurtherprocessedwithCore-
(Alexa488 and Alexa546) or goat anti-mouse (Alexa488 and lDRAW(cid:2) Graphics Suite X6 (Corel Corp.). Analysis of cell
Alexa568)secondaryantibodies(diluted1:2000)intheprein- numbersfromthewholestackofimagesscannedthroughthe
cubation solution for over 12 h at 4°C. The samples were sampleswascarriedoutusingImageJtocountthestainedcells
washedwithPBS-Ttwicefor30min,oncewithPBSfor30min, (17).
andoncewith50%glycerolinPBSfor1handtheninfiltrated Staining intensity of axonal tracts was measured by Zeiss
overnightin80%glycerolinPBSbeforemounting. EfficientNavigation(ZEN2012)software(CarlZeissMicroIm-
19960 JOURNALOFBIOLOGICALCHEMISTRY VOLUME289•NUMBER29•JULY18,2014
AmigoinBrainDevelopment
aging GmbH) with single maximum intensity z-stacking pro- AMIGOorthologsofdifferentspecieswerecarriedoutusing
jections containing the whole image stacks. To quantify the GeneiousProR6(BiomattersLtd.).
degreeofaxonaltractdevelopment,theaxonaltract/cellbody
ratiowasmeasuredoneachmaximumintensityz-stackingpro- RESULTS
jection(18).Forthedeterminationofmediallongitudinalfas-
ZebrafishAmigoProteinsandDetectionofZebrafishAmigo
cicle(MLF),averagepixelintensityofanti-HNK-1immunore-
1—AccordingtothecharacteristicLRRandIgdomains,three
active MLF descending from the ventral caudal cluster (vcc)
putativeamigotranscriptsarefoundinthezebrafishZv9data-
cellswasmeasured(50(cid:4)2(cid:4)m)andcomparedwiththatofthe
base(Ensembl,searchzebrafish).Thefull-lengthcDNAswere
vcccellbodies(5(cid:4)5(cid:4)m)inthesamez-stackingprojection.For
cloned by using mRNA from zebrafish larvae and primers
thedeterminationofthemediallongitudinalcatecholaminer- designed according to the putative homologous sequences
gictract(MLCT),averagepixelintensityofanti-TH1-immuno- foundintheZv9database.Comparisonofthededucedamino
reactive MLCT descending from the locus coeruleus was acid sequences of the zebrafish Amigos with the human,
measured (30 (cid:4) 3 (cid:4)m) and compared with that of the locus rodent,chicken,medaka,andXenopusproteinswascarriedout
coeruleuscellbodies(3(cid:4)3(cid:4)m)inthesamez-stackingprojec- using the Geneious software. Within the 17 Amigo proteins
tion.Theaveragevalueof5misMOlarvaewasusedfornormal- quoted, the zebrafish Amigo1 shares over 50% identity in its
izationinthetractanalyses.Thebackgroundfluorescencewas aminoacidsequencecomparedwiththeAmigo1proteininall
subtractedfromeachmeasurement.Allimageacquisitionand otherspecies(Fig.1A).Thetwootherzebrafishsequences(des-
analysiswasperformedblindtotheexperimentalgroup. ignatedasAmigo3aand-3b)displayahigherdegreeofsimilar-
Anatomicalstructuresoflarvalbrainwerenamedandnum- itytoAMIGO3thantoAMIGO2.ItappearsthatnoAMIGO2
beredusingtheneuroanatomicalatlasofdevelopingzebrafish orthologhasyetbeenidentifiedinteleostspecies.
brain(19)andtheatlasbasedonlocationofTHneuronsin5dpf A phylogenetic tree view of the alignment result clearly
larvalfish(20,21). showedthatwithintheAmigofamily,Amigo1hasthehighest
Behavioral Assays—Two behavioral assays were used. degree of homology when compared with the orthologs
First, the locomotor activity of 6-dpf larvae was observed expressedindifferentspecies(Fig.1A).ZebrafishAmigo3aand
during10minin24-wellplatesasdescribedpreviously(20, Amigo3b are both similar to mammalian AMIGO3 and
22). The analysis included 80 fish (20/group in four inde- groupedintoadifferentbranchcomparedwithAMIGO2ofthe
pendentexperiments). other species (Fig. 1A). Zebrafish Amigo3a and -3b might be
In the second behavioral assay, we assessed the startle encodedbyduplicatecopiesofthesamegene.
response(C-startresponse)of9-dpflarvaeelicitedbyelectrical In order to detect expression of the Amigos in larval
stimuli.Wechoseelectricalstimulationbecauseitallowedusto zebrafish, the primers for qRT-PCR of amigo1, amigo3a, and
provideafixedandstablestartlestimulusineachgroupoflar- amigo3bweredesignedandusedforexpressionanalysisinlar-
vaeduringconstantillumination(23).Fortheelectricalstimu- vaeatthedevelopmentalstages1–8dpf.TheqRT-PCRresults
lationexperiments,recordingchambers(60ml,x(cid:5)5cm,y(cid:5)4 revealedthatallthreeamigoexpressionsstartfromthebegin-
cm,z(cid:5)3cmsharedfromthelaboratoryofH.A.Burgess)were ning of early developmental stages (Fig. 1B). Expression of
fittedwithstainlesssteelsidewallsasbipolarstimulatingelec- amigo1 was found to be the highest of the three amigo tran-
trodes(5V,0.01s).Foreachexperiment,20larvaewereaccli- scripts.Expressionofamigo1alreadystartedtoincreaseafter
matedintherecordingchamberfor3minbeforetesting.The 24hpfandwasincreasedinalogarithmicmannerduringthe
MotionPro X3 fast recording camera (Redlake Imaging) was stages1–8dpf.Expressionofamigo3aandamigo3bstartedto
controlledbyascriptwritteninMatlab(MathWorks).Record- increase from 3 dpf and was always lower than expression of
ingwasstarted200msbeforetheelectricalstimuluswasgiven amigo1.Expressionofamigo3bwasfoundtobethelowestof
(via the MotionPro XTM data acquisition hardware module, the three transcripts. Before 4 dpf, amigo1 expression was
RedlakeImaging)andwascontinuedfor1s.Thiswasrepeated foundtobeover5-foldhigherthanthatofamigo3aoramigo3b.
threetimeswiththesamelarvaeinthechamber,witha3-min After4dpf,amigo1expressionwasstillover3-foldhigherthan
intervalsetbetweeneachelectricalstimulus.Sequentialimages expressionofthetwoothertranscripts.
oftheC-startresponsewerecapturedevery1ms.Latencywas Becauseamigo1wasfoundtobethemajoramigotranscript
definedasthetimefromthebeginningofthestimulationtothe inzebrafishearlyembryos,wedecidedtofocusonthisfamily
C-bend formation. The captured images were analyzed with membertoelucidateputativeamigofunctionsinnervoussys-
Floteversion2.1.ShortlatencyC-start(SLC;initiatedwithin20 temdevelopment.Becausetheantibodiesthatwehaveprevi-
msafterstimulus)andlonglatencyC-start(LLC;initiatedlater ouslygeneratedagainstthemouseAMIGO1(11)didnotspe-
than20msafterstimulus)shareidenticalmovementtrajecto- cifically detect the zebrafish Amigo1 protein in Western
riesandwereclassifiedaccordingtothepreviousreport(24). blotting of SDS extracts of larvae, we generated antibodies
Foreachgroup,theexperimentwasrepeatedfourtimeswith against the zebrafish Amigo1 sequence. We constructed a
fishfromdifferentinjections. fusionplasmidcloneencodingAmigo1ectodomain-GSTand
AnalyticalSoftware—Student’sttestandone-wayANOVA purifiedtheexpressedproteintobeusedasanimmunogenin
analyseswerecarriedoutusingOriginProversion8.0.Intensi- chicken(4).Westernblottingrevealedthattheaffinity-purified
ties of the Western blot bands were examined with Quantity antibodies detect specifically the Amigo1 ectodomain-MBP
One version 4.6.2 (Bio-Rad). Sequence alignments of the fusionproteinandtheendogenouslyexpressedproteininlarval
JULY18,2014•VOLUME289•NUMBER29 JOURNALOFBIOLOGICALCHEMISTRY 19961
AmigoinBrainDevelopment
FIGURE1.AMIGOproteinsindifferentspecies,expressionoftheamigosinzebrafish,andproductionofantibodiestodetectAmigo1ofzebrafish.A,
alignmentoftheaminoacidsequencesofthezebrafishAmigoswith14AMIGOproteinsfoundinotherspecies(ontheleft).Identityoftheaminoacid
sequencesisindicatedinthetable.Aphylogenetictreeof17AMIGOproteinsfoundinXenopus,chicken,rat,mouse,human,zebrafish,andmedakawas
generatedbytheClustalmultiple-sequencealignmentprogram(ontheright).ThesimilaritiesandidentitieswerecomputedbyGeneious.Scalebar,numberof
aminoacidsubstitutions/site.B,expressionofthethreeamigosduringzebrafishdevelopmentdetectedbyqRT-PCR.Full-lengthcDNAplasmidclonesof
amigo1,amigo3a,andamigo3bwereconstructedandusedforthestandardizationofeachqRT-PCR.Templateswerenormalizedbythreereferencegenesas
describedunder“ExperimentalProcedures.”amigo1expressioncomparedwith(cid:2)-actinin1-dpflarvaewassetas1.amigo1expressionincreasesover30-fold
duringdevelopmentfrom1to8dpf.ForeachqRT-PCRtest,totalRNAwasextractedfromapoolof20larvae.Thedataarebasedonthreeindependent
experiments.Meanvalues(cid:6)S.E.(errorbars)areindicated.C,antibodiesagainstzebrafishAmigo1.Specificityoftheanti-Amigo1antibodiesisshownby
Westernblottingontheleft.ThepurifiedantibodiesstaintheAmigo1ectodomain-MBPfusionprotein(E-MBP)band((cid:7)75kDa)clearlywithweakbackground.
TheantibodiesalsodetectspecificallytheAmigo1band((cid:7)60kDa)intheSDSextractoftheadultzebrafishbrain.CoomassieBluestainingontherightconfirms
highexpressionoftheAmigo1ectodomain-MBPfusionproteinusedforpurificationandbindingtestsoftheantibodies.D,wholemountimmunostainingof
5-dpflarvalbrainswiththeanti-Amigo1antibodies.Goatanti-chickenAlexa546(orangetowhite)wasusedasthesecondaryantibodyforfluorescentlabeling.
Themaximumz-stackingprojectionimages(maximumdepth,130(cid:4)m)werescannedfrombothventralanddorsalsidestoshowtheAmigo1expression
patternthroughoutthebrain(thethicknessofthesamplesover200(cid:4)m).Comparedwiththeuninjectedorthe5misMO-injectedlarvae,immunostainingis
clearlyinhibitedintheMO1-injectedlarvaeorintheuninjectedlarvaeinthepresenceofthecompetingantigen(E-MBPcomp).CC,cerebellarcrest;CeP,
cerebellarplate;Di,diencephalon;Pr,pretectum;SR,superiorraphe;Tel,telencephalon;TeO,tectumopticum.Scalebar,100(cid:4)m.E,domainstructureof
zebrafishAmigo1basedonthesequenceofthetranscript.Recognitionsitesofthedesignedmorpholinooligonucleotides(MO1andMO2)aroundthe5(cid:2)-UTR
ofthetranscriptaremarkedbyshortredlines.ThepartofthetranscriptencodingtheAmigo1ectodomainwasusedfordominantnegativeexpression(Am1
EmRNA).IG-domain,immunoglobulin-likedomain;LRRCT,leucine-richrepeatC-terminalflankingdomain;LRRNT,leucine-richrepeatN-terminalflanking
domain;TM-domain,transmembranedomain.
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SDSextractsthatcorrespondstothemolecularmassofAmigo1
(Fig.1C).
InadditiontothezebrafishAmigo1specificantibodies,we
designedaknockdownapproachtostudyexpressionandfunc-
tionsofAmigo1inzebrafish.Twogene-specificantisenseMOs
(MO1andMO2;Fig.1E)weredesignedforamigo1translation
inhibition (25). Because the amigo1 transcript contains only
oneintegratedexonwithoutanyintrons,MO1andMO2were
targeted for binding to the 5(cid:2)-UTR sites near the start codon
(Fig.1E).Theyarenotexpectedtoinhibitproteintranslation
from capped amigo1 mRNA injected in rescue experiments.
Uninjected larvae and larvae injected with 5mis MO (having
fivenon-pairingnucleotidescomparedwithMO1)wereusedas
controls.
ToverifythespecificityoftheAmigo1immunostaining,we
usedtwodifferentapproaches.First,immunostainingusingthe
anti-Amigo1 antibodies was strongly reduced in the knock-
down morphants compared with the uninjected or the 5mis
MO-injected controls (Figs. 1D and 2A). To restore Amigo1
expressioninknockdownexperiments,wetestedinjectionsof
Amigo1mRNAatthedosesrangingfrom0.2to0.8ng.Amigo1
wasimmunostainedatanintensitycomparablewiththeendog-
enousexpressioninlarvaeinjectedwithabout0.4ngofAmigo1
mRNA (Fig. 2A; see also Western blotting results of Fig. 3,A
FIGURE 2. Immunohistochemistry of Amigo1 expression during early
and B). As an additional control, Amigo1 ectodomain-MBP
developmentofthezebrafishbrain.A,wholemountimmunostainingof
fusion protein was premixed with the antibodies for antigen Amigo1in28-hpflarvae.Anti-Amigo1antibodieswereusedastheprimary
antibodies,andgoatanti-chickenAlexa488antibodieswereusedasthesec-
competition in immunostaining. Amigo1-specific immuno-
ondaryantibodiesforfluorescentlabeling.Controlgroupsarethe5misMO-
stainingwasblockedintheprebindingexperimentsinasimilar injected and the uninjected larvae. amigo1 knockdown morphants are
manner as in the knockdown experiments (Fig. 1D, E-MBP markedasMO1andMO2.Rescuedgroupsarecoinjectedwiththefull-length
mRNA(MO1(cid:8)mRNAandMO2(cid:8)mRNA).DT,dorsalthalamus;P,pallium;Pr,
comp).
pretectum;Po,preopticarea.Theconfocalimageswerescannedandstacked
Amigo1 Expression in Larval Brain—The affinity-purified asshownbytheinsetontheright.Thearrowineachframeindicatestheoptic
recessofDiVasthelandmark.Scalebar,60(cid:4)m.B,doublestainingof28-hpf
antibodiesdetectedAmigo1inwholemountimmunostaining
larvaewithchickenanti-Amigo1andmouseanti-HNK1antibodies;thepri-
already at 28 hpf in the larval brain (Fig. 2A). Amigo1 was maryantibodiesweredetectedwithanti-chickenAlexa546(red;anti-Amigo1
intensely stained in the neuronal progenitor cell layers lining antibodies)andgoatanti-mouseAlexa488(green;anti-HNK1antibodies).B1,
anti-Amigo1antibodiesshowedstainingonthesuperficiallayerofthehind-
thediencephalicventricle(DiV)andtheopticrecessofthefore-
brainventricle(HbVe).Thearrowindicatesthedirectionfromposteriorto
brain(26,27).Furthermore,theanteriordiencephalonwasalso anterior.B2andB3,Z-stackingconfocalimagesscannedunderhighermag-
nificationshowthatanti-Amigo1andanti-HNK1antibodiesstainthetracts.
specificallystainedonthealarplateprosomere,fromwhichthe
TherectanglesinB1indicatetheareasofB2andB3.H,hindbrain;TPOC,tractof
anteriorthalamicnucleiderive(28),andontheupperlayerof thepostopticcommissure.Scalebars,25(cid:4)m(B1)and4(cid:4)m(B2andB3).C,the
posteriortuberculum.Essentiallynodetectionwasobservedin ascendingPOCintelencephalon(Tel)stainedwithmouseanti-HNK1antibod-
ies(left)andchickenanti-Amigo1antibodies(middle)andoverlayofthestain-
the amigo1 knockdown morphants compared with the unin- ings(right).Thearrowshowstheleadingpartofthetractinthedirectionofits
jectedorthe5misMO-injectedcontrols(Fig.2A).Expression extension.Scalebar,5(cid:4)m.
of amigo1 was partially rescued by coinjection of the amigo1
full-lengthmRNAwiththeMOs,asevidencedbyintensiveand Gross Morphology of amigo1 Knockdown Morphants—In
ubiquitous Amigo1 staining in DiV cells (Fig. 2A, MO1 (cid:8) injections with different MO doses, the death rates in larvae
mRNAandMO2(cid:8)mRNA). injected with MO1, MO2, or 5mis MO were essentially the
At 28 hpf, Amigo1 was found to be expressed in both cell same(about5%)atthedosesof2–4ngbutweresignificantly
layers and the early developing fiber tracts. MLF and the increased at higher doses (data not shown). We therefore
postopticcommissure(POC)belongtoearlydevelopingfiber selected 4 ng as the dose for the injections in further experi-
scaffoldsofzebrafishthatwereimmunostainedwiththeanti- mentsbecauseitwasfoundtobethehighestdoseoftheMOs
Amigo1andtheanti-HNK-1antibodies.Amigo1wasdetected allowinghighproportionsoflivelarvae.
at the leading front and along the fascicle of the developing WeusedWesternblottingof3dpflarvallysatestoevaluate
tracts(Fig.2,BandC). the effects of the MOs at a dose of 4 ng on Amigo1 protein
The early embryonic brain development in zebrafish is expression. Amigo1 expression essentially disappeared in the
largelyaccomplishedat5dpf(29,30).Atthisstage,Amigo1is MO1-andMO2-injectedlarvae,whereasnomajorchangewas
widely distributed in most nuclei in telencephalon and com- observed in amigo3a MO-injected morphants or in larvae
missuresindiencephalon(fortheexpressionpatternat5dpf, injectedwith5misMO.Coinjectionoftheamigo1mRNAwith
seeFig.1D). MO1 or MO2 displayed a clear rescue effect on the protein
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FIGURE3.KnockdownofAmigo1proteinexpressionandphenotypicfeaturesoftheknockdownlarvae.A,Westernblottingof3-dpflarvaelysateswith
anti-Amigo1antibodies.NoAmigo1bandisdetectedontheMO1andMO2lanes.B,quantificationofAmigo1expressionbyplotdensityanalyses.In3-dpf
amigo1knockdownmorphants(MO1andMO2),amigo1expressionissignificantlyinhibitedcomparedwiththe5misMO-injectedcontrols.Theplotdensityof
thebandsfromuninjectedlarvalSDSextractswassetas1fornormalization.Westernblotswererepeatedthreetimesinthreeindependentexperiments(n(cid:5)
3;***,p(cid:10)0.001inone-wayANOVAfollowedbyTukey’sposthoctest).C,Edustaining(red)andfluorescentTUNELstaining(green)donotdisplaydifferences
betweentheamigo1knockdownmorphants(MO1)andtheuninjectedcontrol.CeP,cerebellarplate;Di,diencephalon;H,hindbrain;MHB,midbrainhindbrain
boundary;Tel,telencephalon;TeO,tectumopticum.Scalebar,50(cid:4)mintheEdu-stainedand40(cid:4)mintheTUNEL-stainedpanel.D,numbersofapoptoticcells
basedonTUNELstaininginprosencephalonandmesencephalonof28-hpflarvae.Theamigo1morphantsdonotdisplayasignificantdifferencecompared
withthe5misMO-injectedcontrols.Thedataarebasedonthreeindependentexperiments;apoptoticcellnumbersin15larvaeofeachgroupwerecounted
(n(cid:5)15,one-wayANOVAfollowedbyTukey’sposthoctest).Errorbars,S.E.
expression(Fig.3A).QuantificationoftheWesternblotresults defects during development (Fig. 4A). The knockdown mor-
revealed (cid:9)85% decrease in Amigo1 expression in the MO1- phantsdidnoteitherdisplayanydifferenceinexpressionlevels
injectedmorphantsand(cid:9)90%intheMO2-injectedmorphants of regional transcription factors (pax2a, pax6a, and krox20)
(Fig. 3B). The 4-ng dose in the MO injections thus appears thatareknowntobeimportantforbraindevelopment(Fig.4,B
optimal to avoid nonspecific effects and to obtain efficient and C). Closer inspection, however, suggested that the mor-
knockdown effects of the Amigo1 protein expression. Immu- phantshaveshorterlengthandwidthofhead(Fig.4B,pax6a
nostaining of Amigo1 in the larvae injected with 4 ng of the and krox20 stainings), and smaller tectum opticum (Fig. 3C,
MOs (Figs. 1D and 2A) was found to agree with the protein TeO) and midbrain-hindbrain boundary (MHB; cell layer
expressiondata. betweencerebellarplateandmidbraintectum;Figs.3Cand4B)
Whentheendogenousamigo1expressionwasinhibiteddur- comparedwiththecontrols.
ing the development using injections of MO1 and MO2 at a Amigo1 knockdown might perturb brain development by
doseof4ng,themorphantsdidnotshowgrossmorphological causingproblemsincellproliferationandsurvival.Wethere-
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AmigoinBrainDevelopment
FIGURE4.Amigo1morphantsdonotdisplaydefectsingrossmorphologyorinexpressionoftranscriptionfactorsthatregulateneuronaldevelop-
ment.A,lightmicroscopyimagesof3-dpflarvae.Scalebar,1mm.B,wholemountinsituhybridizationofpax2aandpax6ain2-dpflarvae(thepharyngula
periodfromHigh-pectoLong-pec)andkrox20in24hpflarvae(thepharyngulaperiodfromPrim5toPrim15).Thefirstlaneshowsalateralviewofpax2ain
2-dpflarvaeasamarkerofthemidbrain-hindbrainboundary(MHB)development.Scalebar,200(cid:4)m.Thesecondlaneshowsadorsalviewofpax6ain2-dpf
larvaeasamarkerofforebraindevelopment.Scalebar,100(cid:4)m.Thelastlaneshowsadorsalviewofkrox20in24-hpflarvaeasamarkerofrhombomere3(r3)
andrhombomere5(r5)inhindbrain.Scalebar,80(cid:4)m.Theasterisksindicatethesmallermidbrain-hindbrainboundaryandrhombomere3ofMO1larvae.C,
quantificationofpax2a,pax6a,andkrox20expressionbyqRT-PCRusingtotalRNAextractedfrom2-dpflarvae.EachqRT-PCRsamplewasobtainedfromapool
of20larvae.Templateswerenormalizedbythreereferencegenes,asdescribedunder“ExperimentalProcedures.”Thedataarebasedonthreeindependent
experiments.Meanvalues(cid:6)S.E.(errorbars)areindicated.
fore carried out EdU and TUNEL stainings in whole mount Amigo1 knockdown morphants and whether Amigo1 and
preparations of larvae but did not find any apparent changes Kv2.1interactinzebrafishneurons.
(Fig.3C).Countingofapoptoticcellsintheareacoveringthe Based on the sequences in the zebrafish Zv9 database
wholeprosencephalonandmesencephalon(31)didnotreveal (Ensembl, search D.rerio), a putative kv2.1 transcript is
any differences in different experimental settings used in the expressedinzebrafishdisplaying66%identityinitssequence
study(Fig.3D). compared with the mouse Kv2.1 transcript. We produced an
Amigo1RegulatesExpressionoftheKv2.1PotassiumChannel N-terminal fragment of the zebrafish Kv.2.1 containing the
and Binds to Kv2.1—We have recently shown that AMIGO1 extracellulardomainandthetransmembraneloopsasanMBP
bindstoKv2.1inadultmouseneuronsandregulatesvoltage- fusion protein ((cid:7)80 kDa). Monoclonal antibodies against
gated potassium currents through Kv2.1 (11). We therefore mouse Kv2.1 (K39/25) were found to recognize the recombi-
decidedtostudywhetherexpressionofKv2.1ischangedinthe nantKv2.1fragment(Fig.5A).WesternblottingofSDSextracts
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19966 JOURNALOFBIOLOGICALCHEMISTRY VOLUME289•NUMBER29•JULY18,2014
AmigoinBrainDevelopment
from3dpflarvaeshowedthattheKv2.1proteinisexpressedin embryos. Coinjection of the full-length amigo1 mRNA (Am1
the larvae, and its expression is strongly decreased in the mRNA)withMO1displayedarescuingeffect(Fig.6,AandB).
amigo1knockdownlarvae(MO1),displayingabout10%ofthe WehavepreviouslyshownthattheAMIGOproteinsdisplay
expressionfoundincontrollarvae(Fig.5,AandB).Coinjection homophilicbindingthatappearsimportantforfasciculationof
of amigo1 mRNA with MO1 (MO1 (cid:8) Am1 mRNA) restored neuritesinculturesofratbrainneurons.Furthermore,theect-
theexpressionto30–40%ofthatfoundinthecontrols.Coin- odomainofAMIGO1insolutionwasshowntoactasadomi-
jectionofkv2.1mRNAwithMO1restoredKv2.1expressionto nantnegativereceptorandtoinhibitAMIGO1-mediatedadhe-
80%ofthelevelseeninthecontrollarvae(Fig.5,AandB). sionandfasciculationinbrainneuronsinvitro(4).Inorderto
Double immunostaining of brain sections from adult provideanassaythatdoesnotdependonMOs,wepreparedan
zebrafishshowedcolocalizationofKv2.1andAmigo1through- mRNA encoding the extracellular part of Amigo1 (Am1
outthebrain,shownfordiencephalicneuronsinFig.5C.Coex- EmRNA,codingforthesixLLRdomainsandoneIgdomainof
pressionwasalsoobservedinthehindbrainventriclecelllayers Amigo1;Fig.1E)andinjecteditintofertilizedembryos.Expres-
(HbVe) and the edge of the cerebellar plate (CeP) in 28-hpf sion of the dominant negative protein in the injected larvae
larvae (Fig. 5D), but the staining patterns did not suggest couldbedistinguishedbyWesternblottingwithanti-Amigo1
expressioninthesamecells. antibodies(Fig.6C).Asintheamigo1MO1morphants,devel-
Because the immunostaining experiments suggested colo- opment of the early tracts was severely disturbed in larvae
calizationofAmigo1andKv2.1inadultzebrafishneurons,we injectedwiththeAm1EmRNA.Forexample,intheconfocal
carried out co-immunoprecipitation experiments using adult stacking projections, MLF could hardly be discerned in the
brain samples. These experiments suggested binding of Am1EmRNA-injectedlarvae(Fig.6A).IncontrasttotheAm1
Amigo1toKv2.1(Fig.5E).Thebindingwasmoreprominentin EmRNA, larvae injected with the full-length mRNA did not
dithiobis(succinimidylpropionate)-cross-linkedsamplescom- display such inhibition (Fig. 6A, Am1 mRNA) but even dis-
paredwithsampleswithoutanycross-linker.Theapparentrea- playedrescueofaxonalgrowth(Fig.6A,MO1(cid:8)Am1mRNA).
sonforthisisthatthetransmembranedomainsoftheproteins The larvae coinjected with MO1 and kv2.1 mRNA (MO1 (cid:8)
mediatebindingarelargelylostindetergentextractionusedin kv2.1 mRNA) showed MLF defects, as did the amigo1 MO1
the co-immunoprecipitation experiments, which was previ- morphantsandtheAm1EmRNA-injectedlarvae(Fig.6A).
ouslyshownintheexperimentsusingbrainextractsfrommice Statistics of the 28-hpf larvae immunostained with anti-
(11). HNK-1 antibodies showed that significantly lower numbers
Amigo1 Is Required for the Development of Early Axonal of the amigo1 knockdown morphants and amigo1 EmRNA-
ScaffoldsinZebrafishBrain—At28hpf,theinitialaxonscaffold injected larvae had intact MLF than 5mis MO-injected or
ofthezebrafishbrainissimple,consistingofafewlongitudinal amigo1mRNA-injectedlarvae(Fig.6D).IntensityoftheMLF
tractsconnectedbycommissures(32,33).Antibodiesagainst staining confirmed the defective tract development in the
thecellsurfacemarkerHNK-1(34)specificallylabelneurons knockdownmorphantsandintheEmRNA-injectedlarvae(Fig.
andaxonsintheearlyembryonicnervoussystem(35).Weused 6E). A clear rescuing effect in the development of MLF was
immunocytochemistry with anti-HNK-1 antibodies to visual- foundincoinjectionoftheamigo1mRNAwiththeMOs(Fig.6,
ize the possible role of Amigo1 in the development of early D and E), which could not be achieved by kv2.1 mRNA
nervoussystemstructures(Fig.6,AandB). coinjection.
Confocal stacking projections of the lateral side of larvae Thevccandthehindbraincellclusterareimportantforthe
clearly showed disturbed fiber tract development in amigo1 developmentofearlyascendinganddescendinglongtracts(35,
knockdownmorphantsat28hpf(Fig.6A).TheMLFandtract 36)andwerefoundtobecorrectlylocatedintheknockdown
of the postoptic commissure belong to the very early major morphants(Fig.6A,vccandhc).Underhighermagnification,
tractsandonlyappearasthinfibersintheamigo1MO1mor- theMO1morphantsdisplayedaclearlyreducedHNK-1stain-
phants compared with the wild-type or 5mis MO-injected ingofaxonsfromtheventralcaudalcellclusters(Fig.6B,top
FIGURE5.Amigo1regulatesKv2.1expressionandbindstoKv2.1.A,monoclonalanti-Kv2.1antibodiesdetecttheectodomainofzebrafishKv2.1(Kv2.1
E-MBP)inWesternblotting.Westernblottingof3-dpflarvalSDSextractsusingtheanti-kv2.1antibodiesshowsstrongdown-regulationofKv2.1inAmigo1
knockdownmorphants(MO1)comparedwiththe5misMO-injectedortheuninjectedlarvae.WesternblottingsuggeststhatKv2.1expressionisenhancedby
coinjectionofamigo1orkv2.1mRNAwithMO1.B,quantificationofKv2.1expressionintheamigo1MO1morphantsshowssignificantdown-regulationofthe
expressioncomparedwiththe5misMO-injectedortheuninjectedlarvae.IntheMO1/Amigo1mRNA-coinjectedlarvae,Kv2.1expressionisincreasedto30%
oftheuninjectedcontrols,whichisstillsignificantlylessthantheexpressioninthe5misMOgroup.IntheMO1/Kv2.1mRNA-coinjectedlarvae,Kv2.1expression
isincreasedtoover60%oftheuninjectedcontrols.Westernblotswererepeatedfourtimesinfourindependentexperiments(n(cid:5)4;**,p(cid:10)0.01inone-way
ANOVAfollowedbyTukey’sposthoctest).C,doublestainingofadultbrainsectionswithanti-Amigo1andanti-Kv2.1antibodiesshowstheircoexpressioninthe
diencephalicneuronalcellsintheadult(over1-year-old)zebrafish.Theprimaryantibodiesweredetectedwithgoatanti-chickenAlexa488(green;anti-Amigo1
antibodies)andgoatanti-mouseAlexa568(red;anti-Kv2.1antibodies).ThelightmicroscopyimageontherightshowsAmigo1wholemountinsituhybrid-
izationinabrainsagittalsection.Rectangle,theAmigo1-expressingbrainregionthatwasusedforthedoubleimmunostainingofAmigo1andKv2.1.Scalebar,
10(cid:4)m.CP,centralposteriorthalamicnucleus;PGZ,periventriculargrayzoneofoptictectum;PPv,ventralperiventricularpretectalnucleus;TeV,tectum
ventricle;TL,toruslongitudinalis;TPp,periventricularnucleusofposteriortuberculum;Val,vlavulacerebelli.D,doublestainingof28-hpflarvaewithanti-
Amigo1andanti-Kv2.1antibodiesshowsthatKv2.1andAmigo1arebothexpressedinthehindbrainventricleareafromanearlydevelopmentstagebutare
notexpressedinthesamecells.Therectangularareainthelightmicroscopyimageontherightistheregionshowninthefluorescentimages.Scalebar,20(cid:4)m.
CeP,cerebellarplate;H,hindbrain;HbVe,hindbrainventricle;MHB,midbrain-hindbrainboundary.E,coimmunoprecipitation(IP)ofKv2.1andAmigo1from
adultbrainSDSextractsusingthemonoclonalanti-Kv2.1andthepolyclonalchickenanti-Amigo1antibodies.Dithiobis(succinimidylpropionate)(DSP)-treated
SDSextractsshowenhancedcoimmunoprecipitation.Non-immuneIgYandIgGwereusedasnegativecontrolsinWesternblottingofAmigo1andKv2.1,
respectively.Intheanti-Amigo1andanti-Kv2.1controls,immunoprecipitationandWesternblottingwerecarriedoutwiththesameantibodies.Errorbars,S.E.
JULY18,2014•VOLUME289•NUMBER29 JOURNALOFBIOLOGICALCHEMISTRY 19967