Common Antigen Related Receptor Tyrosine Phosphatase PTP-3 Function Inde

12 Pages·2005·0.67 MB·English

TheJournalofNeuroscience,August17,2005•25(33):7517–7528•7517
Cellular/Molecular
The Two Isoforms of the Caenorhabditis elegans Leukocyte-
Common Antigen Related Receptor Tyrosine Phosphatase
PTP-3 Function Independently in Axon Guidance and
Synapse Formation
BrianD.Ackley,1,2RobertJ.Harrington,1MartinL.Hudson,1LisaWilliams,3CynthiaJ.Kenyon,3AndrewD.Chisholm,1
andYishiJin1,2
1SinsheimerLaboratories,DepartmentofMolecular,Cellular,andDevelopmentalBiology,UniversityofCalifornia–SantaCruz,SantaCruz,California
95064,2HowardHughesMedicalInstitute,SantaCruz,California95064,and3DepartmentofBiophysicsandBiochemistry,UniversityofCalifornia–San
Francisco,SanFrancisco,California94143
Leukocyte-commonantigenrelated(LAR)-likephosphatasereceptorsareconservedcelladhesionmoleculesthatfunctioninmultiple
developmentalprocesses.TheCaenorhabditiselegansptp-3geneencodestwoLARfamilyisoformsthatdifferintheextracellulardomain.
Weshowherethatthelongisoform,PTP-3A,localizesspecificallyatsynapsesandthattheshortisoform,PTP-3B,isextrasynaptic.
Mutationsinptp-3causedefectsinaxonguidancethatcanberescuedbyPTP-3BbutnotbyPTP-3A.Mutationsthatspecificallyaffect
ptp-3Adonotaffectaxonguidancebutinsteadcausealterationsinsynapsemorphology.Geneticdouble-mutantanalysisisconsistent
withptp-3Aactingwiththeextracellularmatrixcomponentnidogen,nid-1,andtheintracellularadaptor(cid:1)-liprin,syd-2.nid-1andsyd-2
arerequiredfortherecruitmentandstabilityofPTP-3Aatsynapses,andmutationsinptp-3ornid-1resultinaberrantlocalizationof
SYD-2.OverexpressionofPTP-3Aisabletobypasstherequirementfornid-1forthelocalizationofSYD-2andRIM.Weproposethat
PTP-3Aactsasamolecularlinkbetweentheextracellularmatrixand(cid:1)-liprinduringsynaptogenesis.
Keywords:synaptogenesis;axonguidance;celladhesion;(cid:1)-liprin;syd-2;LARreceptortyrosinephosphatase;nidogen
Introduction LAR–RPTPsbindlaminin–nidogenviathefifthFNIIIrepeat,
The type IIa family of receptor protein tyrosine phosphatases andRPTP(cid:2)bindsagrinandcollagenXVIIIviaitsfirstIgdomain
(RPTPs), which include leukocyte-common antigen related (O’Gradyetal.,1998;Aricescuetal.,2002).Thefunctionsofthe
(LAR)PTP-(cid:2)andPTP-(cid:3),arecelladhesionmoleculesthatregu- LAR and extracellular matrix (ECM) interaction are not clear.
latemultipledevelopmentalevents,includingneurogenesis(den Mutations in human collagen XVIII result in Knobloch’s syn-
Hertog et al., 1999; Johnson and Van Vactor, 2003; Paul and drome and defects in neural cell migrations (Kliemann et al.,
Lombroso, 2003; Ensslen-Craig and Brady-Kalnay, 2004). The 2003).Micelackingnidogen-1exhibitmotordeficitsthatsuggest
extracellulardomainsofLAR-likeRPTPscontainIg-likeandfi- aneurologicaldefect(Dongetal.,2002).LAR–RPTPsmayexert
bronectintypeIII(FNIII)repeats.AlternativesplicingofRPTP specific functions in neural development through distinct
genes result in isoforms that differ in the extracellular domain ligands.
andcanexhibittissue-specificexpressionandlocalization,sug- TheintracellulardomainsofLARinteractwithseveralmole-
gestingtheectodomainconfersfunctionalspecificity(O’Gradyet cules including the TRIO guanine nucleotide exchange factor
al.,1994;Pulidoetal.,1995;Honkaniemietal.,1998). (Debant et al., 1996), Enabled (Wills et al., 1999), (cid:4)-catenin
(Kypta et al., 1996), and liprins (Serra-Pages et al., 1998).
(cid:1)-Liprinshaveemergedaskeyregulatorsofsynaptogenesis.The
ReceivedMay18,2005;revisedJune24,2005;acceptedJune28,2005.
presynapticdensitiesofneuromuscularjunctions(NMJs)inCae-
B.D.A.wassupportedbytheHowardHughesMedicalInstitute(HHMI)andbyadevelopmentgrantfromthe
MuscularDystrophyAssociation.GrantsfromtheNationalInstitutesofHealthsupportedresearchinA.D.C.’slabo- norhabditiselegansandDrosophilaareabnormalin(cid:1)-liprinmu-
ratory(GM54657)andinY.J.’slaboratory(NS35546).Y.J.isanHHMIInvestigator.ptp-3(ok244)andptp-3(tm352) tants (Zhen and Jin, 1999; Kaufmann et al., 2002). Drosophila
weregeneratedbytheC.elegansgeneknock-outconsortiumandtheNationalBioresourceProjectfortheExperi-
LAR (DLAR) null mutations cause presynaptic density defects
mentalAnimalC.elegans(Tokyo,Japan),respectively.SomeofthestrainswereprovidedbytheCaenorhabditis
that are similar to, but weaker than, those in Dliprin mutants
GeneticsCenter(Minneapolis,MN).WethankM.Nonetfortheanti-UNC-10andanti-SNT-1antibodies,J.Bessereau
foranti-UNC-49antibodies,J.KramerforpJJ471plasmid,M.ZhenforhpIs3strain,andmembersofourlaboratories (Kaufmannetal.,2002).Dliprinloss-of-functionmutationsare
forcriticalreadingsofthismanuscript. epistatictoDLARoverexpressioneffects,suggestingthatDliprin
CorrespondenceshouldbeaddressedtoDr.YishiJin,329SinsheimerLaboratories,UniversityofCalifornia–Santa isrequiredforDLARfunctionatsynapses.Inmammals,LARand
Cruz,1156HighStreet,SantaCruz,CA95064.E-mail:[email protected]. (cid:1)-liprin are involved in the development and maintenance of
DOI:10.1523/JNEUROSCI.2010-05.2005
Copyright©2005SocietyforNeuroscience 0270-6474/05/257517-12$15.00/0 excitatorysynapses(Dunahetal.,2005).
7518•J.Neurosci.,August17,2005•25(33):7517–7528 Ackleyetal.•TheC.elegansLARRegulatesAxonalandSynapticPatterning
ptp-3,thesingleC.eleganstypeIIaRPTPgene,encodestwo frompCZ512andreplacingitwithaBglII-SpeIfragmentfrompJJ471,a
isoformsthatdifferintheextracellulardomain(seeFig.1A,C) transmembrane::GFPfusion(J.Kramer,personalcommunication).
(Harringtonetal.,2002).PTP-3Aismostsimilartovertebrate Transgenic animals were generated by germ-line transformation as
LAR,whereastheshorterisoform,PTP-3B,lackstheIgdomains describedpreviously(Melloetal.,1991).PTP-3A::GFP(pCZ521)and
PTP-3A(cid:3)phos::GFP(pCZ544)wereinjectedintoN2animalsat75and
and the first four FNIII repeats. PTP-3B regulates neuroblast
68ng/(cid:5)l,respectively,withpRF4[rol-6(SD)](Melloetal.,1991)at100
migrationduringembryogenesis(Harringtonetal.,2002).The
ng/(cid:5)l to generate juEx563 and juEx584. PTP-3B::GFP (pCZ512) was
functionofPTP-3Ahasnotbeendefined.InC.elegansmutations injectedat25ng/(cid:5)lwithPttx-3::RFP(Altun-Gultekinetal.,2001)at100
incollagenXVIII,cle-1ornidogen,nid-1,causesdistinctdefects ng/(cid:5)l to generate juEx686 and juEx687. The integrated transgenes
incellmigration,axonguidance,aswellasmorphologicaland PTP-3A::GFP(juIs194)andPTP-3B::GFP(juIs197)weregeneratedby
functionaldefectsinsynapses(KangandKramer,2000;Kimand UV/trimethylpsoralenmutagenesis.
Wadsworth,2000;Ackleyetal.,2001,2003). Whole-mountimmunostaining.Anti-UNC-10stainingwasdoneusing
Wereportheretheroleofptp-3inaxonguidanceandsynaptic the modified Bouin’s fixation as described previously (Nonet et al.,
morphology.PTP-3Aspecificallylocalizestosynapses,overlap- 1997).Forotherantibodies,animalswerefixedasdescribedpreviously
ping the presynaptic proteins SYD-2 and UNC-10. Isoform- (FinneyandRuvkun,1990),withthefollowingmodifications.Theani-
malswerefixedfor2honice,andthereductionstepwasperformed
specificmutantsandtransgenesdemonstratethatptp-3Bfunc-
using1(cid:4)boratebufferfor2hat37°C.Thefollowingprimaryantisera
tions in axon guidance and that ptp-3A contributes to synapse
wereusedinthisstudy:guineapiganti-PTP-3(1:20)(Harringtonetal.,
development. The synaptic defects of ptp-3 resemble those of
2002),mouseanti-GFP(1:1000;Roche,Indianapolis,IN),rabbitanti-
nid-1andsyd-2.Geneticdouble-mutantanalysesandcellbiology
UNC-10 (1:1000) (Koushika et al., 2001), chicken anti-UNC-10
studiessupportamodelwherebyPTP-3Aactsasalinkbetween
(1:2000), rabbit anti-SNT-1 (1:2000) (Nonet et al., 1993), and rabbit
extracellularcuesandasynapticorganizer. anti-SYD-2(1:2000)(ZhenandJin,1999).Thefollowingsecondaryan-
tibodieswerepurchasedfromMolecularProbes(Eugene,OR)andwere
MaterialsandMethods usedat1:2000:Alexa488-labeledgoatanti-guineapig,Alexa488-labeled
C.elegansstrains.AllC.elegansstrainsweremaintainedat20–22.5°Cas anti-mouse, Alexa 594-labeled anti-rabbit, Alexa 594-labeled anti-
describedpreviously(Brenner,1974).Thefollowingstrainswereusedin chicken,andAlexa647-labeledanti-rabbit.Allimageswerecollectedon
thisstudy:N2(var.Bristol),CH119[nid-1(cg119)](KangandKramer, aZeiss(Oberkochen,Germany)Pascalconfocalmicroscopeusingmulti-
2000),RB633[ptp-3A(ok244)],CZ333(juIs1),CZ1200(juIs76),CZ631 trackparameters,witheithera63(cid:4)(SNB-1::GFP)or100(cid:4)(immuno-
(juIs14),CZ900[syd-2(ju37)](ZhenandJin,1999),CZ1991(mnDf90/ fluorescence)objective.
mIn1mIs14),CZ3761[ptp-3(mu256)],CZ2857[ptp-3A(tm352);juIs1], Colocalization.Confocalstackswereprojectedintoasingleplaneand
CZ4660 [ptp-3A(ok244);juIs1], CZ3555 [ptp-3(mu256);juIs1], CZ4661 analyzedusingthehistogramtoolintheZeissLSM5software(version
[ptp-3A(ok244);nid-1(cg119); juIs1], CZ3997 [ptp-3(mu256); nid- 3.2).Aregionofinterestwasdrawnaroundthenervecord.Thresholding
1(cg119);juIs1],CZ3139[nid-1(cg119);syd-2(ju37);juIs1],CZ2981[syd- levelsweresetusingthe“determinefromregionofinterest”function.
2(ju37);ptp-3A(tm352);juIs1], CZ4929 [ptp-3(mu256);syd-2(ju37); The colocalization table was exported to Microsoft Excel (Redmond,
juIs1], CZ4918 [ptp-3(mu256); nid-1(cg119); syd-2 (ju37); juIs1], WA)forstatisticalanalysis.Thepercentageofcolocalizationwasdeter-
CZ4884(juIs194),CZ4913[ptp-3(mu256);juIs194],CZ4919(juIs197), minedasthetotalareaofregion3(pixelsatwhichbothchannelsare
CZ4914[ptp-3(mu256);juIs197],CZ4885[syd-2(ju37);juIs194],CZ3992 abovethreshold)dividedbythetotalareaforthatchannel(totalpixels
[ptp-3A(tm352);juEx584], CZ3839 (juEx563), CZ4249 (juEx686), abovethreshold).
DR2078 [mIn1 mIs14/bli-2(e678)unc-4(e120)] (Edgley and Riddle, GFP analysis. For axon morphology, animals were scored blind to
2001),andZM54(hpIs3)(Yehetal.,2005). genotype by examining cell type-specific GFP markers (juIs76 and
Themu256allelewasisolatedinascreenformutantswithdefective juIs14)withanAxioplan2microscopeusinga63(cid:4)Plan-apochromat
migrationsfortheQneuroblastasdescribedpreviously(Ch’ngetal., objective and a GFP long-pass filter set (Chroma, Battleboro, VT). A
2003). Double and triple mutants were constructed by crossing nid- defasciculationeventwascountedasaregionofthenervecordwheretwo
1(cg119)/(cid:1);mIn1mIs14/(cid:1)orsyd-2(ju37);mIn1mIs14/(cid:1)malestoho- ormoreprocessesappearedtobecomesplitfromonefascicleandwere
mozygousptp-3mutantanimals.ThemIn1mIs14balancestheptp-3re- visiblealongadistancethatwasgreaterthanthelengthoftwoneuronal
gionandcontainsagreenfluorescentprotein(GFP)marker(Edgleyand cellbodies,whichis(cid:5)20(cid:5)m.SynapsemorphologyofDtypeneurons
Riddle,2001).Non-GFPoffspringwereselectedandhomozygosed.The was visualized by juIs1 (Punc-25-SNB-1::GFP) and hpIs3 (Punc-25-
presence of the nid-1(cg119) allele was confirmed by PCR (Kang and SYD-2::GFP)usingaZeissPascalLSMconfocalmicroscope.Animals
Kramer,2000).Homozygotesofsyd-2(ju37)werechosenbythesluggish wereanesthetizedusing0.5%phenoxy-propanol(TCIAmerica,Port-
movementandegg-layingdefects(ZhenandJin,1999). land,OR)inM9andmountedon5%agarpads.Imageswereacquired
Molecularbiology.Theptp-3genespanstwocosmidclones,C09D8 using a 63(cid:4) Plan-apochromat lens and a 488 argon laser line at 3%
(GenBankaccessionnumberZ46811)andF38A3(GenBankaccession power.Themicroscopewassettouseasinglescanwitha505long-pass
numberZ49938).Allnumberinginthisreportisgivenrelativetothe filterset.
ATGforptp-3A(position7765inC09D8).Themu256lesionwasiden- Quantification. Measurements of SNB-1::GFP and UNC-10 puncta
tifiedbysequencingPCR-amplifiedregionsoftheptp-3exons(primer wereperformedonconfocalimagesasdescribedpreviously(Ackleyetal.,
sequencesavailableonrequest).Analysisofthesequencingresultsre- 2003),withminormodificationsandtheexperimenterblindtogeno-
vealedasingleadenosineinsertionatposition34784relativetotheATG type.Briefly,confocalimageswereprojectedintoasingleplaneusingthe
oftheptp-3Aisoform(correspondingtoposition9670inF38A3;Gen- maximumprojectionandexportedasatifffilewithascalebar.Using
BankaccessionnumberZ49938).Thetm352deletionremovesnucleo- Scion(Frederick,MD)Image,thefileswereconvertedtoabinaryimage
tides8494–9040andtheok244deletionremovesnucleotides8295–9639 usingthethresholdcommand,sothatthebinaryimageresembledthe
oftheptp-3Acodingregion. RGBimage.Aregionofinterestwasdrawnaroundtherelevantregionof
pCZ512(PTP-3B::GFP)hasbeendescribedpreviously(Harringtonet thenervecords.Thefollowingmeasurementoptionswereselected:area,
al.,2002).pCZ521(PTP-3A::GFP)wascreatedbyfusingtheGFPDNA X–Ycenter,perimeter/length,ellipsemajoraxis,ellipseminoraxis,in-
fromA.Fire’svectorpPD113.29toaptp-3AcDNAgeneratedbyreverse cludeinteriorholes,wandautomeasure,andheadings.Scalingwassetby
transcription-PCR(primersequencesavailableonrequest)ataunique measuringthescalebar;forGFP,images7pixelsequaled1(cid:5)m,andfor
PstI site in exon 27 (Fig. 1A). A genomic fragment corresponding to UNC-10puncta,11pixelsequaled1(cid:5)m.The“analyzeparticle”com-
nucleotide(cid:2)2565throughexon4wasgeneratedbyPCR,cutwithKpnI mand was used with a minimum size of 4 pixels and a maximum of
and NsiI, and fused to the ptp-3A cDNA–GFP fragment. pCZ544 10,000pixels.Thefollowingoptionswereselected:outlineparticles,ig-
(PTP-3A::GFP(cid:3)phos)wascreatedbyremovingaBglII-AvrIIfragment noreparticlestouchingedge,includeinteriorholesandresetcounter.
Ackleyetal.•TheC.elegansLARRegulatesAxonalandSynapticPatterning J.Neurosci.,August17,2005•25(33):7517–7528•7519
andresultsinastrongptp-3lossoffunc-
tionduringembryogenesis(Harringtonet
al.,2002).Weisolatedanewptp-3allele,
mu256,inascreenforanimalswithdefec-
tiveQcellmigration(Williams,2002).Se-
quencing of the ptp-3 locus from mu256
animalsidentifiedasinglenucleotidein-
sertion in exon 25 that would cause a
frame shift after amino acid Glu1756 in
thefirstphosphatasedomain,leadingtoa
premature stop (Fig. 1C). Immunostain-
ingusingantiseraraisedagainstthePTP-3
phosphatasedomainsshowedthat50%of
mu256animalshadnodetectableprotein
(datanotshown)and50%exhibitedonly
aweakreactivityinthesynapse-richnerve
ring(Fig.1F)(n(cid:6)100).mu256animals
displayed partially penetrant embryonic
(Emb)andlarvallethality(Lva)aswellas
variably abnormal (Vab) morphology
phenotypes (Table 1). The lethality and
morphologydefectsweresimilartothose
observed for op147 and in the broods of
animals injected with ptp-3 double-
stranded RNA (Harrington et al., 2002).
Thus, mu256 is a strong loss-of-function
mutationaffectingbothPTP-3isoforms.
To analyze the specific requirements
forthePTP-3Aisoform,weobtainedtwo
deletion alleles in the ptp-3A coding re-
gion, tm352 and ok244. The tm352 and
Figure1. Theptp-3locusandproteinlocalization.A,ptp-3genestructure:exonsareshownasshadedboxes,andintronsare ok244deletionsremoveoneorthreeFNIII
shownaslines.Largeintronsareindicatedasbrokenlineswiththesizelistedbelow.Thegenomicregionsusedtodriveisoform- repeats, respectively, and result in frame
specificexpressionareindicated.ThepositionsofrestrictionenzymesitesusedtomakeminigenesandGFPfusionsarealso shifts and premature terminations of
indicated.ptp-3A-specificexonsareshowninred,whereastheptp-3B-specificexonisshowninyellow.Theexonscommonto PTP-3A. Both ptp-3A alleles are likely to
ptp-3Aandptp-3Bareinblue.Thelocationsofthetm352,ok244,op147,andmu256lesionsareindicated.nt,Nucleotide.B,The eliminateptp-3Afunctionbutareunlikely
genestructureofthePTP-3::GFPisoformsisillustrated.Exonsareindicatedbyboxes,andintronsareindicatedaslines.The
toaffecttheexpressionofPTP-3Bbecause
placementoftheGFPcodingsequencesisindicatedbythegreenbox.C,PTP-3proteins:Ig-likedomainsareillustratedascircles,
thelesionsareoutsideoftheminimalres-
FNIIIdomainsareillustratedashexagons,thepredictedtransmembranedomainisillustratedasanoval,andthephosphatase
cuing fragment for ptp-3B (Harrington
domainsareillustratedasrectangles.TheasteriskindicatestheFNIIIrepeatmosthomologoustotheFNIIIrepeatcontainingthe
etal.,2002).Bywhole-mountimmuno-
laminin–nidogen-bindingsitefrommouseLAR.Theeffectsofthemutationsareshownbelow.TheX-headedlineindicatesthe
deletedportionoftheproteinandtheintroductionofastopcodon.D–F,Whole-mountimmunolocalizationofPTP-3inwild-type staining,areducedlevelofstainingwas
(D),ptp-3(ok244)(E),andptp-3(mu256)(F)animals.Stainingisobservedinthenervering(arrow)andventralnervecord observed in tm352 and ok244 animals
(arrowhead)ofwild-typeandptp-3(ok244)animals.Allmu256animalsexaminedlackedreactivityinthenervecord(F,arrow- (Fig.1E).Bothmutationscausesimilar
head).Approximately50%ofptp-3(mu256)animalsexhibitedweakPTP-3staininginthenervering(F,arrow).Scalebar,10(cid:5)m. phenotypes(seebelow),andtm352ani-
malsdidnotexhibitsignificantEmbor
Vab phenotypes (Table 1), suggesting
Table1.LethalityandVabinptp-3mutants that PTP-3A does not function in em-
%Lethalitya bryonicmorphogenesis.
Genotype n Emb Lva Vab
PTP-3expressioninthepostembryonic
Wildtype 823 0.1(cid:7)0.01 0.1(cid:7)0.01 0.1(cid:7)0.01
nervoussystem
ptp-3A(tm352) 994 0.5(cid:7)0.02 0.1(cid:7)0.01 0.1(cid:7)0.01
ptp-3(mu256) 726 5.0(cid:7)0.12 1.1(cid:7)0.03 4.5(cid:7)0.03 Wehavereportedpreviouslythatptp-3A
ptp-3(op147) 501 3.8(cid:7)0.01 0.4(cid:7)0.02 4.8(cid:7)0.08 and ptp-3B were highly expressed in the
postembryonic nervous system using
Dataareexpressedasmean(cid:7)SEM.
promoter-drivenGFPreportertransgenes
aPercentageofanimalsthatreachadultstagewithVabphenotype.
(Harrington et al., 2002). By modifying
the immunostaining procedure, we im-
TheresultingmeasurementswereexportedtoMicrosoftExcelforstatis-
proved the sensitivity of detecting endogenous PTP-3. We ob-
ticalanalysis.Significancewasdeterminedbyatwo-tailedStudent’sttest.
servedthatPTP-3wasconcentratedatthenerveringandalong
Results thenervecordsinlarvalandadultanimalsandshowedapunctate
Isolationofnewmutationsinptp-3 pattern (Fig. 1D). To determine the subcellular localization of
Thepreviouslyreportedptp-3mutation,op147,isatransposon PTP-3, we performed colocalization experiments with several
insertioninthephosphatasedomaincommontobothisoforms known synaptic proteins including UNC-49, a GABA receptor
7520•J.Neurosci.,August17,2005•25(33):7517–7528 Ackleyetal.•TheC.elegansLARRegulatesAxonalandSynapticPatterning
present on the postsynaptic muscle sur-
face(GallyandBessereau,2003),synapto-
tagmin(SNT-1),acomponentofsynaptic
vesicles(Nonetetal.,1993),andtwopre-
synaptic density proteins, SYD-2 and
UNC-10, the C. elegans RIM (Zhen and
Jin,1999;Koushikaetal.,2001).
Alongthenervecord,PTP-3wasadja-
centtobutdidnotoverlapUNC-49(Fig.
2A),consistentwithitbeingexpressedin
neurons.Withintheneurons,PTP-3par-
tially overlapped the SNT-1-containing
domainbutwasconcentratedattheedges
oftheSNT-1staining(Fig.2B).Therewas
a precise colocalization of PTP-3 with
SYD-2 and UNC-10 (Fig. 2C,D). More
specifically, we observed a smaller punc-
tumofPTP-3inornearthecenterofeach
ofthelargerSYD-2puncta.Thesedatain-
dicatethatPTP-3ispredominantlyasso-
ciatedwiththepresynapticdensity.
ToaddresswhetherPTP-3localization
wasdependentonsynapticvesicles,wean-
alyzedtheaccumulationofSNT-1,PTP-3,
and UNC-10 in unc-104(e1265) kinesin
mutants,whichcausessynapticvesiclesto
beretainedincellbodies(HallandHedge-
cock,1991).WeobservedthatPTP-3and
UNC-10punctashowedcolocalizationin
regionsofthenervecordlackingsynaptic
vesicles(Fig.2E,F).Thus,likeotherpre-
Figure2. PTP-3Alocalizestopresynapticregions.A–D,CostainingofPTP-3(green)withproteins(red)thatlabeldifferent
synaptic density components (Zhen and synapticdomains.Thebottompanelsarethemergedimages.A-A(cid:1),PTP-3(arrowheads)doesnotoverlapwithUNC-49(arrows).
Jin, 1999; Koushika et al., 2001), PTP-3 B–B(cid:1),PTP-3(arrowheads)isconcentratedattheedgesofSNT-1(arrows).C–C(cid:1),PTP-3colocalizeswithSYD-2.Notethesmaller
appearstobetraffickedtosynapsesinde- fociofPTP-3atthecenteroftheSYD-2puncta(arrows).D–D(cid:1),Seventy-fivepercentofUNC-10punctaareeitheroverlapped
pendentofsynapticvesicles. (arrowheads)orcoincident(arrows)withPTP-3.E,F,PTP-3istraffickedtosynapsesindependentofsynapticvesicles.Awild-type
animal(E)andanunc-104animal(F)areshownwithtriplelabelingofanti-PTP-3(green),anti-SNT-1(blue),andanti-UNC-10
PTP-3Aissynaptic,andPTP-3B (red).Themergedviewsareshowninthebottompanels.Inunc-104mutants,SNT-1ismostlyretainedincellbodies(arrow),
whereasPTP-3andUNC-10arepresentalongthenervecord(arrowheads).G,H,ThelocalizationofthePTP-3isoform-specificGFP
isextrasynaptic
fusionproteinsisshownrelativetoUNC-10.G–G(cid:1),PTP-3A::GFPappearedpunctateandoverlappedwiththeUNC-10puncta
Toaddressthesubcellularlocalizationof
(arrows), although the protein was also observed in larger clusters than the endogenous protein (arrowheads). H–H(cid:1),
the PTP-3A and PTP-3B isoforms, we
PTP-3B::GFPshoweddiffusedstainingthroughoutthenervecord(arrows)andrarelyoverlappedwithUNC-10puncta(arrow-
generated isoform-specific GFP fusion head).I–I(cid:1),Inptp-3A(ok244)mutants,PTP-3(arrowhead)stainingisreduced,andshowedlittleoverlapwithUNC-10(arrows).
transgenes.GFPwasinsertedin-framein Scalebars,5(cid:5)m.
the second phosphatase domain 53 aa
fromthestopcodon,andeachfusionpro-
teinwasexpressedundertheisoformen-
dogenouspromoter(Fig.1B).Bothtrans- These results suggest that PTP-3A is the major isoform that is
genes are functional (Harringon et al., 2002) (see below). localized to presynaptic domains. Consistent with this conclu-
PTP-3B::GFP showed a temporally regulated expression, with sion, in ptp-3A(ok244) mutant animals, (cid:8)25% of the UNC-10
highexpressionthroughoutembryogenesisandlarvaldevelop- puncta(n(cid:6)200)containedanyPTP-3,andtheamountofPTP-3
mentuntiltheL1–L2larvaltransition(datanotshown).Inthe presentatthoseUNC-10punctawasreduced(Fig.2I).
adultnervoussystem,PTP-3B::GFPwaspresentinthenervering
andalongaxonsoftheventralanddorsalnervecords(Fig.2H PTP-3Bhasamajorroleinmotoraxonguidance
and data not shown) and was also observed in the pharyngeal Previous analyses using a pan-neuronal morphological marker
epitheliumandthedevelopinguterusthroughoutdevelopment revealedthattheorganizationofthenervoussystemwasgrossly
(datanotshown).MostofthePTP-3B::GFPwasadjacenttothe normal in ptp-3(op147) animals (Harrington et al., 2002). To
UNC-10puncta,withonlyasmallamountofGFPoverlapping addresstheeffectsofptp-3mutationsonthenervoussystem,we
withUNC-10(Fig.2H).PTP-3A::GFPexpressionwasfirstde- focusedouranalysisontheventralcordmotoraxonsusingcell
tectedaroundthetwofoldstageofembryonicdevelopment(450 type-specific markers. The DA and DB neurons extend a den-
min after fertilization) in the nerve ring and nerve cords and driticprocessalongtheventralnervecordandanaxonalcom-
continuedataconstantlevelthroughlarvaldevelopment(data missurecircumferentiallyalongtheepidermistothedorsalnerve
notshown).PTP-3A::GFPwasseeninapunctatepatternalong cord(Fig.3A).TheDDandVDneuronsextendasingleprocess
nerveprocesses(Fig.2G)andoverlappedwithUNC-10inapat- alongtheventralnervecordthatbifurcatesandextendstothe
tern that was similar to the endogenous protein (Fig. 2D,G) dorsalnervecord(Fig.3F).Theseaxonprojectionpatternsare
Ackleyetal.•TheC.elegansLARRegulatesAxonalandSynapticPatterning J.Neurosci.,August17,2005•25(33):7517–7528•7521
nationpoint(Fig.3E)orstopprematurely
(Fig. 3H,J). The axon projection defects
of the DD and VD neurons in mu256 in
trans to mnDf90, a chromosomal defi-
ciencyfortheptp-3locus,wascomparable
withthatofmu256homozygousanimals,
consistentwithmu256beingastrongloss-
of-function mutation in ptp-3 (Fig. 3L).
TofurtheraddresshowthetwoPTP-3iso-
forms function in axon guidance, we in-
troducedtheisoform-specificGFPtrans-
genesintomu256animals.Expressionof
thePTP-3B::GFPisoformessentiallyres-
cued the DA and DB axon guidance de-
fectsinmu256animals(Table2,Fig.3K).
Specific expression of PTP-3A::GFP only
showed a weak partial rescue (Table 2).
ThesedatathereforearguethatPTP-3Bis
themajorisoformthatfunctionsinaxon
outgrowthandguidanceinC.elegans.
ptp-3Acontributesto
synapsedevelopment
BecausePTP-3Aislocalizedtopresynap-
ticregions,weaskedwhethermutationsin
ptp-3causedanydefectsinsynapticdevel-
opment.Wefirstexaminedthepatternof
synapses by staining animals with
UNC-10 antisera. In wild-type animals,
UNC-10wasdistributedinasetofevenly
sized and spaced puncta (Fig. 4A). We
measuredthepunctaareaandthenumber
ofpunctaalonga100(cid:5)msectionofnerve
cord (see Materials and Methods for de-
Figure3. Mutationsinptp-3causedefectsinaxonguidance.A,Schematicdiagramdescribingtheaxonoutgrowthpatternsfor tails).Inwild-typeanimals,theaverage
theDAandDBcholinergicmotorneurons.Thecellbodiesareillustratedasopencircles,whereastheprocessesareindicatedas area was 0.17 (cid:7) 0.01 (cid:5)m2 (mean (cid:7)
blacklines.Thepositionofthevulvaisindicatedbyaopendiamond.TheshadedboxistheregionshowninBandC.B,Inwild-type SEM), with an average of 87.3 (cid:7) 10.5
animals,theDB6andDB7neurons(indicatedbyarrows)extendprocessestotheright,whereastheadjacentneurons,DA6and punctaper100(cid:5)m(Fig.4E,Table3).In
DA7,extendprocessestotheleft.C,Inthisptp-3(mu256)animal,theDB6andDB7axons(arrows)incorrectlyextendontheleft ptp-3Amutants(tm352andok244),the
sideofthenervecord.D,Regionoftheventralnervecordthathasbecomedefasciculated(arrow)fromthemainbundle(arrow- overall UNC-10 puncta appeared to be
heads).E,Twoaxonsthatfailedtoterminateattheendofthedorsalnervecord(arrow)areobservedtoturnanteriorlyand
formedinlargeraggregatesthaninwild-
continueextending.F,SchematicdiagramoftheDDandVDGABAergicmotorneurons.G,Inwild-typeanimals,thedorsalnerve
type animals (Fig. 4B, Table 3). In ptp-
cord(arrows)isobservedasacontinuousbundle.H,Inptp-3mutants,weobservedgapsalongthenervecordwheretheaxons
3A((cid:2))animals,UNC-10punctashowed
appeartohaveterminatedtheirmigrationprematurely.I,Inwild-typeanimals,thecommissuralprocesses(arrows)extendfrom
anaverageareaof0.48(cid:7)0.03(cid:5)m2,with
theventralnervecordtothedorsalcord(arrowheads).J,Twoaxoncommissuresfailedtoreachthedorsalnervecord(arrow-
heads):onestoppedmigratingprematurely(arrow),whereasasecondturnedprematurely(dashedarrow).DAandDB(K)andDD anaverageof53.0(cid:7)4.0punctaper100
andVD(L)axonguidancedataaredisplayedasthepercentageofanimalsthatexhibitedasidechoiceerror((cid:2)),targetingerror (cid:5)m. In ptp-3(mu256) animals, the de-
(u),ordefasciculation(f).InformationonspecificaxonoutgrowthphenotypesisinTable2.L/R,Left/right. fectsseenwithUNC-10expressionwere
notworse(0.36(cid:7)0.03(cid:5)m2,43.0(cid:7)10.2
punctaper100(cid:5)m),suggestingthatthe
loss of PTP-3A would account for the
highlystereotyped.UsingaGFPmarkerdrivenfromtheunc-25 observeddefects.
promoterforDtypeneuronsandaGFPmarkerdrivenfromthe Toevaluatetheeffectonthemorphologyofindividualsyn-
acr-2promoterfortheAandBtypeneurons(Huangetal.,2002), apses,weusedthetransgeneexpressingsynaptobrevinfusedto
weobserved(cid:8)10%ofwild-typeanimalsthatexhibitedanydevi- GFP(SNB-1::GFP)drivenbytheunc-25promoter(Hallamand
ations(Table2). Jin,1998;Nonet,1999).ThisSNB-1::GFPmarkerprovidesspa-
Neitheroftheptp-3A-specificalleles,tm352orok244,exhib- tialresolutiontodetectchangesinGABApresynapticmorphol-
itedgrossdefectsinmotorneuronaxonoutgrowthandguidance ogy.Inwildtypeanimals,SNB-1::GFPappearsasasetofevenly
(Table 2). However, the mu256 and op147 mutations caused a sizedandspacedfluorescentpuncta,withanaverageareaof0.8(cid:7)
significant level of projection defects, such as axons that made 0.0(cid:5)m2(Fig.5A,Table4).Inptp-3Aandptp-3(mu256)mutants,
incorrectchoicesintheirexitfromtheventralmidline(Fig.3C). theSNB-1::GFPpunctashowedsignificantenlargement,withan
Themostnoticeabledefectwasthedefasciculationofmotorax- averageareaof1.5(cid:7)0.1(cid:5)m2.TheareaofSNB-1::GFPpunctain
onsalongthedorsalorventralnervecords(Fig.3D).Inaddition, tm352/mnDf90 animals (1.7 (cid:7) 0.2 (cid:5)m2) was not significantly
someaxonswereobservedtoovergrowtheirappropriatetermi- differentfromtheptp-3A(tm352)homozygousanimals,support-
7522•J.Neurosci.,August17,2005•25(33):7517–7528 Ackleyetal.•TheC.elegansLARRegulatesAxonalandSynapticPatterning
ingthattm352andok244representacom- Table2.Axonguidancedefectsinptp-3mutantanimals
pletelossoffunctionforptp-3A,andsug- Genotype n Left/righta Targetingb Defasciculationc
gesting that the defects in SNB-1::GFP
DA/DBcholinergicneurons
puncta are mostly caused by the loss of
Wildtype 60 8.3% 0.0% 1.7%
PTP-3A.Asanotherwayofassessingthe
ptp-3A(tm352) 15 13.3% 0.0% 6.7%
overall effect of ptp-3 mutations on ptp-3A(ok244) 20 5.0% 0.0% 0.0%
SNB-1::GFP puncta, we plotted the ob- ptp-3(op147) 40 22.5% 10.0% 20.0%
served areas using a box-and-whiskers ptp-3(mu256) 20 40.0% 10.0% 50.0%
plot(Fig.6).Thisanalysisrevealedthatthe ptp-3(mu256);juEx563(PTP-3A::GFP) 50 36.0% 8.0% 40.0%
populationofSNB-1::GFPpunctainptp-3 ptp-3(mu256);juEx686(PTP-3B::GFP) 60 12.3% 5.3% 8.3%
mutantshadagreaternumberofpuncta DD/VDGABAergicneurons
Wildtype 50 2.0% 0.0% 0.5%
that were larger than those in wild type.
ptp-3A(tm352) N/D
Wecalculatedthevaluesrepresentingthe
ptp-3A(ok244) N/D
10thand90thpercentilesinwild-typean-
ptp-3(op147) 50 22.0% 12.0% 50.0%
imalsasbeing0.2–1.5(cid:5)m2,respectively,
ptp-3(mu256) 50 30.0% 22.0% 52.0%
and found that 30% of the SNB-1::GFP ptp-3(mu256)/mnDf90 25 16.0% 28.0% 56.0%
punctainptp-3A((cid:2))mutantswere(cid:9)1.5
N/D,Notdetermined.
(cid:5)m2,whereasonly1.5%were(cid:8)0.2(cid:5)m2.
aPercentageofanimalswithaxonsthatmadeasidechoiceerrorinexitfromtheventralnervecord.
Overall,thedefectinSNB-1::GFPwaspar- bPercentageofanimalswithaxonsthatterminatedprematurelyorovergrewthetarget.
alleltothealterationsinUNC-10staining. cPercentageofanimalsthatexhibitedtwoormoreregionsofdefasciculatedaxonsalongthenervecords.
TofurtheraddresstheroleofPTP-3A
in synapse formation, we tested whether
expressing PTP-3A::GFP could specifi-
cally rescue the synaptic defects in ptp-
3(mu256) animals. Because of interfer-
encefromtheGFPtags,wewereunableto
use the SNB-1::GFP and instead used
UNC-10 immunostaining. Neither
PTP-3A::GFPnorPTP-3B::GFPcauseda
significantchangeinthesizeofUNC-10
punctainwild-typeanimals(Fig.4E,Ta-
ble3).ptp-3(mu256)animalsthatwereex-
pressing PTP-3A::GFP had an average
area that was equivalent to wild type
(0.17 (cid:7) 0.01 (cid:5)m2), whereas UNC-10
puncta in mu256 animals expressing
PTP-3B::GFPweresignificantlylargerthan
wildtype(0.35(cid:7)0.05(cid:5)m2)(Fig.4E,Table
3).ThedistributionofUNC-10punctawas
alsorescuedinPTP-3A::GFP-expressingan-
imals(96.0(cid:7)14.5punctaper100(cid:5)m)but
not in PTP-3B::GFP-expressing animals
(43.6(cid:7)3.5punctaper100(cid:5)m)(Table3).
We conclude from these analyses that
PTP-3Aisthemajorfunctionalisoformat
synapsesandthatlossofPTP-3Aresultsin
UNC-10andSNB-1::GFPaccumulatingin
larger-than-normalpuncta.
BecausePTP-3AandPTP-3Bonlydif-
ferintheextracellulardomain,weasked
whethertheextracellulardomainwassuf- Figure4. ExpressionofUNC-10inmutants.A–D,UNC-10punctaareindicatedbyarrows,whereasabnormallysizedgapsare
ficientforthefunctionatsynapses.Exper- indicatedbyarrowheads.A,Inwild-typeanimals,UNC-10punctawereevenlysizedandspacedalongthenervecords.B,Inptp-3,
iments from Drosophila have demon- UNC-10punctaappearedmisshapen.Inthisptp-3Amutant,weobservedsmallerandlargerpunctathatwereaberrantlyspaced.
strated that the extracellular portion of C,D,Similardefectswereobservedinnid-1(cg119)(C)andsyd-2(ju37)(D)animals.E,AreaofUNC-10punctainwildtypeandin
ptp-3,nid-1,andsyd-2mutants,plottedasthemean(cid:7)SEM.
LARmayacttosignaltoothercells,inde-
pendent of the phosphatase domains
(Maurel-Zaffranetal.,2001).Wegenerated
aversionofPTP-3A::GFPlackingthephos-
phatasedomains(PTP-3A(cid:3)phos::GFP).ptp-3A(tm352)transgenic ptp-3geneticallyinteractswithsyd-2/(cid:1)-liprinandnid-1
animals expressing this protein had an average UNC-10 area of duringsynapseformation
0.39(cid:7)0.08(cid:5)m2(Fig.4E,Table3),indicatingthatthefunctionof TounderstandhowPTP-3Acouldfunctionatsynapses,weex-
PTP-3A in synapse formation is dependent on the cytoplasmic amined whether ptp-3 mutants exhibited genetic interactions
domain. with other known synaptogenesis genes. LAR–RPTPs can bind
Ackleyetal.•TheC.elegansLARRegulatesAxonalandSynapticPatterning J.Neurosci.,August17,2005•25(33):7517–7528•7523
Table3.RIMpunctameasurementsbygenotype actionsbetweenptp-3andthesegenes,we
Numberof Numberof constructed double and triple mutants
Genotype synapses Area(mm2) animals Puncta/100mm amongthethreegenesandexaminedthe
Wildtype 804 0.17(cid:7)0.01 8 87.3 (cid:7)10.5 synapse morphology using the
ptp-3A((cid:2)) 613 0.49(cid:7)0.03** 8 51.2 (cid:7)4.5* SNB-1::GFPtransgene.
ptp-3(mu256) 442 0.36(cid:7)0.03** 4 43.0 (cid:7)10.2* TheSNB-1::GFPpunctainsyd-2(ju37)
juIs194(PTP-3A::GFP) 131 0.22(cid:7)0.02 3 71.3 (cid:7)2.5 animalshadanaverageareaof1.2(cid:7)0.1
ptp-3(mu256);juIs194 493 0.17(cid:7)0.01 3 96.2 (cid:7)14.5 (cid:5)m2andwerenotenhancedintheptp-3A;
juIs197(PTP-3B::GFP) 501 0.18(cid:7)0.01 3 89.7 (cid:7)2.2 syd-2 (1.0 (cid:7) 0.1 (cid:5)m2) or ptp-3(mu256);
ptp-3(mu256);juIs197 982 0.35(cid:7)0.05** 3 43.6 (cid:7)3.5* syd-2 (1.3 (cid:7) 0.1 (cid:5)m2) double mutants
ptp-3A(tm352);juEx584(PTP-3ADphos::GFP) 85 0.39(cid:7)0.08** 3 39.0 (cid:7)23.0* (Fig.5E,6;Table4).Thesyd-2;ptp-3dou-
nid-1(cg119) 345 0.39(cid:7)0.04** 3 66.8 (cid:7)2.8
ble mutants were significantly different
nid-1(cg119);juIs194 700 0.18(cid:7)0.01** 3 37.0 (cid:7)6.7** fromptp-3A((cid:2))alone(p(cid:8)0.05)butnot
syd-2(ju37) 563 0.43(cid:7)0.02** 5 67.03(cid:7)10.4
syd-2(ju37);juIs194 583 0.12(cid:7)0.01** 3 55.0 (cid:7)7.1* from syd-2(ju37) (p (cid:9) 0.05). Box-and-
whiskers plot analysis revealed that syd-2
Dataareexpressedasmean(cid:7)SEM.Valuessignificantlydifferentfromwildtype:*p(cid:8)0.05;**p(cid:8)0.001. mutants had more small puncta ((cid:8)0.2
(cid:5)m2; 30.0%) and more large puncta
((cid:9)1.5 (cid:5)m2; 19.6%) than the wild type.
syd-2;ptp-3A double mutants resembled
syd-2, having 42.3% smaller puncta and
18.4%largerpuncta.Thus,ourresultsin-
dicatethatthelossofsyd-2isepistaticto
themutationsinptp-3.
Loss-of-function mutations in nid-1
causeSNB-1::GFPpunctatoappearelon-
gated,withmoresevereeffectsintheven-
tralnervecordthanthedorsalcord(Ack-
leyetal.,2003).Intheventralnervecord,
nid-1(cg119) animals had an average
punctum area of 1.7 (cid:7) 0.1 (cid:5)m2, which
was significantly larger than in wild type
(p(cid:8)0.005).Thepunctasize,distribution,
and morphology in the ventral cords of
nid-1;ptp-3A(ok244) (1.9 (cid:7) 0.1 (cid:5)m2) or
nid-1;ptp-3(mu256)(1.7(cid:7)0.1(cid:5)m2)dou-
blemutantswerenotsignificantlydiffer-
entfromnid-1singlemutants(p(cid:9)0.05)
(Figs.5E,6;Table4).Theseresultssuggest
that mutations in ptp-3 do not enhance
theSNB-1::GFPaccumulationdefectsas-
sociatedwiththelossofnid-1.
In contrast, in either ptp-3A;cle-1 or
ptp-3(mu256);cle-1 double mutants, we
observedmoreseverephenotypesthanin
either single mutant alone. In cle-1 mu-
tants,SNB-1::GFPpunctaarelargerthan
Figure5. PatternofSNB-1::GFPinGABAergicsynapsesinmutants.A,Inwild-typeanimals,theSNB-1::GFPpatternwas inwildtype,withanaverageareaof2.3(cid:7)
observedasequallysizedandevenlyspacedfluorescentpuncta(arrow).B,Inptp-3A(tm352)mutants,SNB-1::GFPwaspresentin 0.1 (cid:5)m2, and are separated by long dis-
larger,irregularlyspacedpuncta(arrows).C,Insyd-2(ju37)mutants,SNB-1::GFPpunctawerediffusedandoftenappeared tances(Ackleyetal.,2003).Thesedefects
contiguous.D,SNB-1::GFPpunctainnid-1(cg119)mutantswereelongatedandaberrantlyspaced.Cellbodies(arrowhead)were aremorphologicallydistinctfromthosein
occasionallyvisiblealongtheventralnervecordinallgenotypes.E,TheareaofSNB-1::GFPpunctainthewild-typeandvarious the syd-2 or nid-1 mutants. In cle-1 mu-
mutants,plottedasthemean(cid:7)SEM.
tants, 57.1% of the puncta were larger
thanthe90thpercentileinwildtype,and
noneweresmallerthanthe10thpercentile
(cid:1)-liprinsviathesecondphosphatasedomain,andthetwopro- (Fig.6).Inthedoublemutants,weobservedanaverageareaof
teinshavebeenreportedtofunctiontogetherinsynapseforma- 1.7 (cid:7) 0.1 (cid:5)m2 for ptp-3A;cle-1 and 1.1 (cid:7) 0.1 (cid:5)m2 for ptp-
tion(Kaufmannetal.,2002;Dunahetal.,2005).Wehaveshown 3(mu256);cle-1, as well as a new phenotype such that 26.7 and
thatmutationsinC.elegans(cid:1)-liprin,syd-2,alterthemorphology 40.0%ofthepuncta,respectively,were(cid:8)0.2(cid:5)m2,aphenotype
ofsynapses(Figs.4D,5C)(ZhenandJin,1999).Theextracellular thatwasnotseenineitherofthesinglemutants(Table4).The
portion of LAR-like receptors can bind nidogen–laminin and combinedlossofcle-1andptp-3hasamoredetrimentaleffecton
collagenXVIII.WehaveshownthatmutationsinC.elegansni- SNB-1::GFP accumulation than the loss of either single gene.
dogen,nid-1,andcollagenXVIII,cle-1,resultindistinctchanges Thesedataarguethatcle-1andptp-3likelyaffectsynapticdevel-
insynapticmorphology(Ackleyetal.,2003).Toassaytheinter- opmentviadistinctmechanisms.
7524•J.Neurosci.,August17,2005•25(33):7517–7528 Ackleyetal.•TheC.elegansLARRegulatesAxonalandSynapticPatterning
Becausebothnid-1andsyd-2appeared Table4.SNB-1::GFPareabygenotype
epistatic to ptp-3, we asked whether re- Wild-typeoutliersa
Numberof
movingnid-1fromeitherthesyd-2single- synapses Area(mm2) (cid:8)0.22mm2 (cid:9)1.31mm2
mutantorthesyd-2;ptp-3double-mutant
Wildtype 874 0.8(cid:7)0.0 8.8% 10.1%
animalscausedanyfurtheralterationsin
ptp-3A(tm352) 289 1.6(cid:7)0.1** 1.5% 30.0%
SNB-1::GFP accumulation. The nid-1;
ptp-3A(ok244) 569 1.4(cid:7)0.1** 3.7% 28.1%
syd-2 double mutants and nid-1; syd-2; ptp-3A/mnDf90 202 1.7(cid:7)0.2** 9.4% 36.1%
ptp-3Aornid-1;syd-2;ptp-3(mu256)triple ptp-3(mu256) 418 1.5(cid:7)0.1** 2.9% 38.3%
mutants all resembled syd-2 single mu- syd-2(ju37) 852 1.2(cid:7)0.1** 30.0% 19.6%
tants in terms of the shape and distribu- syd-2(ju37);ptp-3A(tm352) 982 1.0(cid:7)0.1* 42.3% 18.4%
tion of SNB-1::GFP puncta (Figs. 5E, 6; syd-2(ju37);ptp-3(mu256) 501 1.3(cid:7)0.1** 26.1% 29.1%
nid-1(cg119) 331 1.7(cid:7)0.1** 10.9% 34.1%
Table4).nid-1;syd-2animalshadanaver-
nid-1(cg119);ptp-3A(ok244) 493 1.9(cid:7)0.1** 14.0% 44.2%
ageareaof1.5(cid:7)0.2(cid:5)m2,andthetriple nid-1(cg119);ptp-3(mu256) 717 1.7(cid:7)0.1** 3.5% 35.6%
mutantshadaverageareasof1.2(cid:7)0.1and cle-1(cg120) 205 2.3(cid:7)0.1** 0.5% 57.1%
1.3(cid:7)0.1(cid:5)m2,respectively.Allweresig- cle-1(cg120);ptp-3A(tm352) 146 1.7(cid:7)0.2** 26.7% 32.9%
nificantlydifferentfromwildtype(p(cid:8) cle-1(cg120);ptp-3(mu256) 100 1.1(cid:7)0.2 41.0% 28.0%
0.005)butnotfromsyd-2singlemutants syd-2(ju37);nid-1(cg119) 484 1.5(cid:7)0.2** 36.1% 20.2%
(p (cid:9) 0.05). In addition, all of the mu- syd-2(ju37);nid-1(cg119);ptp-3A(tm352) 366 1.2(cid:7)0.1** 33.6% 23.5%
syd-2(ju37);nid-1(cg119);ptp-3(mu256) 438 1.4(cid:7)0.1** 14.2% 28.5%
tant combinations with syd-2 mutants
were observed to have SNB-1::GFP Dataareexpressedasmean(cid:7)SEM.Valuessignificantlydifferentfromwildtype:*p(cid:8)0.05;**p(cid:8)0.001.
aPercentageofpunctabelowthe10thpercentileorabovethe90thpercentileofwildtype.
punctathatwereofasizeandmorphol-
ogymostsimilartosyd-2singlemutants
(Table4).Thesedatashowthattheloss
ofsyd-2isepistatictomutationsinnid-1
andptp-3.
PTP-3localizationisdependenton
nid-1andsyd-2
The genetic interactions between ptp-3,
nid-1,andsyd-2couldbeexplainedbysev-
eral different models. NID-1 and PTP-3
couldacttorecruitSYD-2tosynapses,or
SYD-2andPTP-3couldfunctiontoorga-
nize NID-1 in the ECM. To differentiate
between these models, we examined the
amount of PTP-3 immunofluorescence
thatcolocalizedwithUNC-10inthenid-1
andsyd-2singlemutantsandnid-1;syd-2
double-mutantanimals(Fig.7)(seeMa-
terials and Methods). In wild-type ani-
mals,41.7%ofPTP-3signalabovethresh-
old was colocalized with UNC-10 (n (cid:6)
13). In nid-1 mutants, PTP-3 levels ap-
peared reduced, and the protein was ab-
normally distributed into smaller and
largerpunctawithgapsinthestainingpat-
tern(Fig.7B).However,35.1%ofthere-
maining PTP-3 was colocalized with
UNC-10 (n (cid:6) 15). This suggests that Figure6. BoxplotsofSNB-1::GFPquantitation.TheareaofallSNB-1::GFPmeasurementsbygenotypewererepresentedasbox
plots.Theboxrepresentsthevalueswithinthe25thto50thpercentiles,andthewhitebar(orgrayarrowhead)indicatesthemean
NID-1isrequiredtomaintainPTP-3lev-
observedvalue.Theendsofthewhiskersshowthe10thto90thpercentiles,withthe5thand95thpercentilesindicatedbyblack
elsbutdoesnotseemtobenecessaryfor
circles.Thedottedlinesillustratethevaluesthatwesetaslimitstodefinepunctathatareoutliersfromwildtypeasdefinedbythe
theassociationofPTP-3withUNC-10.
10th(0.2(cid:5)m2)and90th(1.5(cid:5)m2)percentilevalues.Inwildtype,theboxandwhiskersarecompact,indicatingthemeasure-
In syd-2 animals, the levels of PTP-3
mentsfallintoareproduciblepattern.TheSNB-1::GFPpunctainmutantswasmorevariableinsize,resultinginlargerplots.The
proteinwerecomparablewiththatofwild shapeoftheboxandwhiskersissimilarinthesyd-2single,double,andtriplemutantsdemonstratingthatthesemutantsare
type,butPTP-3wasdistributeddiffusely phenotypicallysimilarintheireffectonSNB-1::GFPaccumulation.nid-1singleandnid-1;ptp-3Adoublemutantsexhibitasimilar
alongthenervecords.Only14.7%ofthe distribution.Incontrast,cle-1singleandcle-1;ptp-3Adoublemutantsarephenotypicallydistinct.
PTP-3 present was colocalized with
UNC-10(Fig.7C)(n(cid:6)8).PTP-3wasseen
insomecellbodiesinsyd-2animals(15of100).Thisphenotype effectwasseensuchthatPTP-3levelswerereduced,and(cid:5)19.9%of
wasnotobservedinwildtype(0of100)ornid-1mutants(0of the remaining PTP-3 protein was colocalized with UNC-10 (Fig.
100).TheseobservationsaresuggestiveofSYD-2actingasamo- 7D)(n(cid:6)10).Thus,NID-1andSYD-2havedistinctrolesinthe
lecularscaffoldatsynapsesandbeingimportantforPTP-3traf- synapticaccumulationofPTP-3.
ficking to synapses. In nid-1;syd-2 double mutants an additive ThedecreasedPTP-3levelsinnid-1mutantssuggestedthat
Ackleyetal.•TheC.elegansLARRegulatesAxonalandSynapticPatterning J.Neurosci.,August17,2005•25(33):7517–7528•7525
mutants, SYD-2 was abnormally spaced
andclusteredintolargeraggregates,with
anaverageof1.0(cid:7)0.1(cid:5)m2(p(cid:8)0.05)
(Fig.8K).Theseobservationssuggestthat
bothNID-1andPTP-3arerequiredtolo-
calizeormaintainSYD-2atsynapses.
ExpressionofPTP-3Abypassesnid-1
butnotsyd-2mutants
Tofurthertestwhethernid-1solelyfunc-
tionsinmaintainingsynapticPTP-3Alev-
els,weelevatedtheexpressionofPTP-3A
innid-1mutantsandexaminedUNC-10
expression.Althoughtheaveragenumber
ofpunctaper100(cid:5)m(41.9(cid:7)8.9)wasnot
restoredtowild-typelevels(87.3(cid:7)10.5),
measurementofUNC-10punctashowed
that the average area (0.17 (cid:7) 0.01 (cid:5)m2)
was restored to wild-type levels (p (cid:9)
0.05).TheUNC-10areawassignificantly
differentfromnid-1animalsnotexpress-
ingPTP-3A::GFP(0.39(cid:7)0.04(cid:5)m2)(Fig.
4E,Table3).Moreover,immunostaining
of anti-SYD-2 showed that SYD-2 accu-
mulation was more punctate in nid-1;
Figure7. NID-1andSYD-2havedistincteffectsinPTP-3localization.ThelocalizationofPTP-3(green)andUNC-10(red)in
juIs194[PTP-3A::GFP] than nid-1 alone
nid-1(cg119),syd-2(ju37),andnid-1(cg119);syd-2(ju37)doublemutantsisshown.Theboxedregionsaremagnifiedintheright
panels.A,A(cid:3),PTP-3andUNC-10exhibitedanoverlappedpatternofaccumulationinwild-typeanimals.B,B(cid:3),Innid-1animals, (Fig. 8H). In contrast to the effects with
PTPlevelswerereduced,butthepatternremainedpunctate.Whenpresent,PTP-3coincidedwithUNC-10puncta(arrowhead), nid-1, PTP-3A::GFP expression in syd-2
whereassomeUNC-10punctalackedPTP-3(arrow).C,C(cid:3),Insyd-2mutants,PTP-3wasdiffuseandhadareducedcoincidencewith mutant animals did not restore UNC-10
UNC-10(arrowhead).PTP-3andUNC-10hadaccumulatedindependently(arrows),aneventthatwasrarelyseeninwildtype punctatowild-typelevels(Fig.4E,Table
((cid:8)25%).D,D(cid:3),Innid-1;syd-2doublemutants,PTP-3detectionwasmoderatebutpresentinamorepunctatepatternthansyd-2 3).Theseresultssupportourconclusion
singlemutants.However,PTP-3andUNC-10onlyrarelycoincided(arrowhead);rather,eachappearedtobeexclusivelypunctate that NID-1 is required to maintain
(arrows).Scalebar,5(cid:5)m. PTP-3A protein levels at synapses and
that SYD-2 is required for PTP-3A to
regulateUNC-10accumulation.
PTP-3doesnotfunctiontoorganizeNID-1.Toconfirmthis,we Discussion
examinedtheaccumulationofnidogeninptp-3mutants.Inwild-
LAR–RPTPisoformsprovidefunctionalspecificity
type animals, NID-1 was present in the mantle of the mech-
LAR-likereceptortyrosinephosphataseshavebeenbroadlyim-
anosensoryneuronsandbetweenthemuscleedgeandnervepro-
plicated in the patterning of the nervous system by regulating
cesses(KangandKramer,2000)(Fig.8A).NID-1accumulation
eventssuchasneuriteoutgrowth,targetrecognition,andsynap-
wasnormalinptp-3mutants,indicatingthatPTP-3Adoesnot
togenesis(Clandininetal.,2001;JohnsonandVanVactor,2003;
acttoorganizeNID-1inthesynapticECM(Fig.8B).
Dunahetal.,2005).OnechallengeinunderstandingRPTPfunc-
tionisidentifyinghowthesemoleculesfunctioninmultipleas-
SYD-2synapticlocalizationisalteredinptp-3and pectsofneurogenesis.AlternativesplicingofRPTPgenesgener-
nid-1mutants atesreceptorisoformdiversity,suggestingthatdistinctisoforms
WesubsequentlyexaminedthelocalizationofSYD-2intheptp-3 could regulate specific RPTP functions (O’Grady et al., 1994;
andnid-1mutantanimalsusingSYD-2antibodies(ZhenandJin, Pulido et al., 1995). We have shown here that the two PTP-3
1999).Inwild-typeanimals,SYD-2localizationwasdetectedas isoforms exhibit distinct roles in axon guidance and synaptic
puncta along the nerve cords (Fig. 8C) and in the body wall organization.Thetypesofaxonoutgrowthandguidancedefects
muscles(ZhenandJin,1999).Inptp-3mutants,SYD-2puncta inptp-3mutantsresemblethosethathavebeenreportedforLAR
werelargeanddiffusewithfrequentgapsalongthenervecord homologs in Drosophila and vertebrates (Desai et al., 1996;
(Fig.8E,F).Innid-1mutants,SYD-2appeareddiffusebuthad Kruegeretal.,1996;Garrityetal.,1999;Clandininetal.,2001;Xieet
fewgaps(Fig.8G). al.,2001;Stepaneketal.,2005).Ourobservationsindicatethese
To quantitate the effect of the loss of ptp-3A and nid-1 on functionsofLARareevolutionarilyconservedandthatreceptor
SYD-2,weusedaSYD-2::GFPfusionproteinthatwasexpressed isoformsprovidefunctionalspecificity.
undertheunc-25promoter(Yehetal.,2005).Inwild-typeani-
mals, the SYD-2::GFP appears as a set of regularly sized and PTP-3isoformssignalviadistinctpathways
spacedpunctaalongthenervecords,withanaverageof0.7(cid:7)0.1 Inadditiontoaxonguidanceandsynapticdefects,mutationsthat
(cid:5)m2(Fig.8I).Inptp-3A(tm352)mutants,wefoundthepuncta affectbothptp-3isoformscancauseembryoniclethalityresulting
weresignificantlyenlargedandclusteredtogether(Fig.8J).The fromdefectsinneuroblastmigration.ptp-3mutationssynergize
average area of SYD-2::GFP puncta in ptp-3A(tm352) mutants strongly with mutations affecting other pathways involved in
was1.0(cid:7)0.1(cid:5)m2(p(cid:8)0.05).Similarly,wefoundthatinnid-1 neuroblast migration (VAB-1/Eph receptor, EFN-4/Ephrin)
7526•J.Neurosci.,August17,2005•25(33):7517–7528 Ackleyetal.•TheC.elegansLARRegulatesAxonalandSynapticPatterning
(Chin-Sangetal.,2002;Harringtonetal.,
2002). Both neuroblast migration and
axonguidanceappeartobefulfilledbythe
short isoform PTP-3B, because ptp-3A-
specific mutations do not exhibit axon
guidance or embryonic morphogenesis
defects, nor do they synergize with Eph
signaling mutations (our unpublished
results).
PTP-3A regulates synapse formation,
as does NID-1 and SYD-2. Mutations in
nid-1causeaxonguidancedefectsthatare
distinct from those in ptp-3 mutant ani-
mals(KimandWadsworth,2000;Ackley
et al., 2003). Likewise, mutations in Eph
signaling cause synaptic defects that are
distinctfromthoseinptp-3(B.D.Ackley
andY.Jin,unpublisheddata).Theseob-
servationssuggestthatventralneuroblast
migration and axon guidance are regu-
latedbysimilarmechanisms.
ThePTP-3Aproteincontainsallofthe
domainsofPTP-3B,butthetwoproteins
are not functionally equivalent. What
mightleadtothedifferencesobservedin
function for PTP-3B and PTP-3A?
PTP-3Bisexpressedearlier,andinmore
tissue types, than is PTP-3A, suggesting
that some of the differences in PTP-3
functionmaybeattributabletoexpression
differences.However,withinneurons,we
foundthatPTP-3AandPTP-3Barelocal-
izedtodistinctsubcellularcompartments. Figure8. PTP-3andNID-1regulateSYD-2accumulation.A,B,NID-1localizationinwild-type(A)andptp-3A(B)animalsis
comparable.NID-1(green)islocalizedinacontinuouspatternattheinterfaceoftheventralnervecordfasciclesandthemuscles
AninteractionbetweenPTP-3Aandali-
(arrowheads)andinapunctatepatterninthemantleofthemechanosensoryneurons(arrows).C–H,SYD-2(red)localization.C,
gand that is dependent on the PTP-3A-
Inwild-typeanimals,SYD-2waspresentalongthenervecordsinapunctatepattern(arrowheads).D,syd-2(ju37)animalslacked
specificdomainscouldexplainthediffer-
staining(thepositionoftheventralnervecordisindicatedbyarrowheads).E,F,Inptp-3mutants,SYD-2wasobservedinlarger
ences in subcellular accumulation.
puncta(arrows)andalsoretainedincellbodies(arrowhead).G,Innid-1animals,SYD-2wasdiffuse,andthepuncta(arrows)
Together,ourresultssuggestthatPTP-3A appearedenlarged.SYD-2wasalsofoundincellbodies(arrowheads).H,Innid-1mutantsexpressingPTP-3A::GFP,SYD-2expres-
and PTP-3B likely interact with distinct sionispunctate(arrows),andtheamountofSYD-2incellbodiesisreduced(arrowheads).I,SYD-2::GFPexpressedintheGABAer-
molecularpartnersincontrollingspecific gicneuronsofwild-typeanimalsappearspunctatewiththeclustersbeingevenlysizedandspaced(arrowheads).GFPisobserved
aspectsofneuronaldevelopment. inthecellbodiesinallgenotypes(arrows;I–K).Thiseffectislikelyaresultofoverexpressionandnotthegeneticbackground.J,
ptp-3A(tm352)mutantsexhibitanalteredpatternofSYD-2::GFPaccumulation,withlargerclustersofGFP(arrowheads).K,
nid-1(cg119)animalshaveenlargedSYD-2::GFPpuncta(arrowheads)thatareseparatedbylargegaps.L,Themeanareaof
PTP-3Afunctionsasamolecularlinker
SYD-2::GFPpunctawasplottedbygenotype.Scalebars,10(cid:5)m.
atsynapses
Thegeneticinteractionsweidentifiedbe-
tweenptp-3andsyd-2wereexpectedgiven
thatLARhasbeenfoundtofunctionwith
(cid:1)-liprin to regulate synaptic morphology in vertebrates and in cellularligandscouldregulatethedistinctfunctionswehaveob-
Drosophila(Kaufmannetal.,2002;Dunahetal.,2005).However, servedforthetwoPTP-3isoforms.
thecytoplasmicdomainsofPTP-3AandPTP-3Bareidentical, Thegeneticepistasisofptp-3andnid-1indicatesthetwogenes
suggestingbothshouldinteractwithSYD-2equally.PTP-3Ais have a common output in synaptic development. PTP-3 and
sufficient to rescue the synaptic defects of ptp-3 mutants, but SYD-2arepresentatpresynapticdensities(ZhenandJin,1999;
PTP-3Bisnot.Thus,aninteractionwithSYD-2doesnotexplain Yehetal.,2005;presentstudy).Wefoundthattheaccumulation
thespecificityofPTP-3Ainsynapseformation. of SYD-2 was aberrant in both ptp-3 and nid-1 mutants. The
Previousworkhasshownthat(cid:1)-liprinsarecapableofinduc- functionofNID-1inSYD-2localizationis,atleastpartially,de-
ingLARtoaccumulateatfocaladhesion-likeplaquesincultured pendent on PTP-3, because we could restore a normal SYD-2
cells(Serra-Pagesetal.,1998).Focaladhesionsaresitesofcell– accumulationpatterninnid-1mutantsbysupplyingadditional
matrix interactions, suggesting that (cid:1)-liprins may regulate an PTP-3A.Likewise,PTP-3localizationisalteredinnid-1mutants,
interactionofLARreceptorswiththeECM.SpecificECMmole- suggestingtheSYD-2mislocalizationmaybeadirectresultofthe
culesare,infact,ligandsforLARreceptors,andbiochemicaland PTP-3mislocalization.ThesedataindicatethattheECMisnec-
geneticworkhasshownthatLARisoformsexhibitligandspeci- essaryforPTP-3andSYD-2toremainatspecificfoci.Supplying
ficity(O’Gradyetal.,1998).Thedatasuggestthatspecificextra- additional PTP-3A also overcomes the UNC-10 accumulation