tactile stimulation.Thefrequency ofmusclecontractionsduring bends ofthetrunkandtail.By26hpf,embryosswiminresponse to respond tomechanosensorystimulationwithtwo orthree rapidC- stimulation andobserved from17to26hpf.After21hpf,embryos of thetrunkandtail.Thiscoilingisindependentsensory earliest behavior consistsofrepetitive, slow andalternatingcoiling hours post-fertilization(hpf)(Saint-AmantandDrapeau,1998). The 2007). (Bassett andCurrie,2003;Kunkel etal.,2006;Lieschke andCurrie, potentially serve asanimalmodelsofhumanmotor disorders Saint-Amant andDrapeau,1998).Fifth,zebrafish mutantscan assayable andwell-characterizedbehaviors (EatonandFarley, 1973; et al.,2002;Fetcho,2006).Fourth, zebrafish embryos exhibit readily analyze thephysiologyofembryonicneuronsandmuscles(Drapeau al., 2002).Third,electrophysiologicaltechniquescanbeusedto calcium transients,inlive fish (FetchoandBhatt,2004;Wilson et of dynamicevents, suchascellmigration,axonaloutgrowth and 1996). Second,embryosaretransparent,facilitating thevisualization identify genesthatareessentialforproperbehaviors (Granatoetal., disorders. First,forward geneticscanbeappliedtothisorganism to Zebrafish areusefulforthestudyofmotordevelopment and INTRODUCTION Montréal, Montréal, Québec,H3T1J4,Canada *Present address: DépartementdePathologieetBiologieCellulaire, Universitéde Genetics, KumamotoUniversity, Kumamoto860-0811,Japan. Accepted 29May 2007 † 2 1 Zebrafish,Ryanodinereceptor, Muscle,Calcium,Multi-minicore disease KEY WORDS: in mutants,suggestingadefectthemuscle.CalciumimagingrevealedthatCa to weakcontractionsoftrunkmuscles.ElectrophysiologicalrecordingsfrommuscleshowedthatoutputtheCNSwasnormal Wild-type zebrafishembryosswimawayinresponsetotactilestimulation.Bycontrast, Hiromi Hirata of multi-minicoredisease receptor defect,showslowswimmingandprovideamodel Zebrafish (2007)doi:10.1242/dev.004531 Development 134,2771-2781 Immunostaining demonstratedthatryanodineanddihydropyridinereceptors,whichareresponsibleforCa Michigan, AnnArbor, MI48109-1048,USA. insertion inanintronofthe ryanodine receptor1b( slow swimmingiscausedbyweakmusclecontractionsduetoimpairedexcitation-contractioncoupling.Indeed,mostofthe membrane depolarization,wereseverelyreducedattransverse-tubule/sarcoplasmicreticulumjunctionsinmutantfastmuscle.Thu yk Nagashima Ayako zebrafish oligonucleotides thatrestoredthenormalsplicingof micrographs showedminicorestructuresinmutantfastmuscles.Furthermore,followingtheintroductionofantisensemorpholino congenitalmyopathy, multi-minicore disease(MmD),whichisdefinedbyamorphouscoresinmuscle.Electron a leadto humans Author forcorrespondence (e-mail:[email protected]) Department ofMolecular, CellularandDevelopmentalBiology, Universityof Graduate SchoolofScience,NagoyaUniversity, Nagoya464-8602,Japan. Zebrafish embryosdisplaythreestereotypedbehaviors by36 relatively relaxed 1,2,† , Takaki Watanabe relatively relaxed 2 , WilsonW. Cui ryr1b mutants maybeusefulforunderstandingthedevelopmentandphysiologyofMmD. ryr1b ) mRNAinmutantscarriedanonsensemutationthatwasgeneratedbyaberrantsplicingduetoDNA 3 , leadingtoahypomorphicconditionin Institute ofMolecularEmbryologyand 2 , Weibin Zhou 1 , JunHatakeyama ryr1b 2 , swimmingwasrecoveredinmutants.Thesefindingssuggestthat mutants havearyanodine and JohnY. Kuwada 3 , ShawnM.Sprague intracellular modulatory , thelargest known protein,weighs560kDa channel, whichisformedwith fourRyR1monomers.The a clusteroffourDHPRs,associates withaCa subunits (Catterall,2000; Flucheretal.,2005).Atetrad,whichis composed ofthevoltage-sensing andpore-forming muscle (Blocketal.,1988). Theskeletal muscleDHPRis direct physicalinteractionsbetweenDHPRandRyR1inskeletal actin/myosin toproducemusclecontraction. 1994). Elevated cytoplasmic Ca (SR) toallow Ca ryanodine receptor1(RyR1)intheadjacentsarcoplasmicreticulum located inthet-tubule membrane.DHPRsthentrigger theopeningof changes ofthedihydropyridine receptor(DHPR),avoltage sensor invaginations oftheplasmamembrane,andcauseconformational membrane spreaddown thetransverse-tubules (t-tubules), whichare Armstrong andProtasi,1997).Depolarizationsoftheplasma muscle movement byexcitation-contraction (E-C)coupling(Franzini- Brehm, 2005)andthechangeofmembranepotentialisconverted to depolarize themusclemembrane(BussandDrapeau,2001;Wen and release acetylcholineattheneuromuscularjunction(NMJ)to spinal cord(Fetcho,1992;Gahtanetal.,2002).Motorterminals pattern thatalternatelyactivates motorneuronsineachsideofthe located inthehindbrainandspinalcordcreateappropriatemotor et al.,2002).Oncetriggeredbysensoryinputs,interneuronalnetworks the trunkandtailortrigeminalsensoryneuronsinhead(Drapeau in contraction ofmuscles.Touch issensedbyRohon-Beardneurons steps, startingwiththesensingoftactilestimuliandending and Drapeau,2001). being similartothefrequency ofswimmingbyadultzebrafish (Buss swimming increasesfrom7Hzat26hpfto3036hpf,thelatter The membranesoft-tubules andSRarejuxtaposed and permit The processoftouch-inducedswimminginvolves anumberof relatively relaxed 2+ transients werereducedinmutantfastmuscle. 2 2+ relatively relaxed release fromtheSRtocytosol (Meissner, 2 , LouisSaint-Amant ␤ 1 subunit, andauxiliary mutants. RYR1 mutationsin 2+ ntr,atvtsthesliding of , inturn,activates RESEARCH ARTICLE mutants swimslowlydue 2+ release following 2+ 2, *, -releasing RyR1 ␣ ␣ 2 1S subunit, ␦ 1 and 2771 ␥ s, 1

DEVELOPMENT MATERIALS ANDMETHODS physiology ofMmD. understanding thedevelopment ofamorphous coresandthe (Lyfenko etal.,2004). ryr guidelines setforthbytheUniversity ofMichigan and Nagoya University. (Nüsslein-Volhard andDahm,2002;Westerfield, 2000),whichmeetthe Zebrafish werebredandraisedaccording toestablishedprocedures Animals Ca as asecondarycellularresponsetoisolateregions of defective development ofcores,ithasbeenproposedthatcoresareformed histological sections.Althoughlittleisknown aboutthe MmD isdefined bythepresenceofmultiplesmallcoresin recessive RYR1 mutation(Engel,1967;Jungbluthetal.,2004). scoliosis andrespiratoryinsufficiency, but isinheritedthrougha of CCDmuscle.MmDischaracterizedbymuscleweakness, axis ofthemusclefibers, canbeobserved in histological sections et al.,1993).Amorphouscentralcores,whichrunalongthelong characterized byinfantile hypotoniaandmuscleweakness(Zhang dominant RYR1 mutation,withtwo recessive exceptions, andis halothane (MacLennanandPhillips,1992).CCDiscausedbya susceptible individual isexposed tocertainanesthetics,suchas dominant trait,MHappearsasahypermetaboliccrisiswhen disease (CCD)andmulti-minicore(MmD).Inheritedasa hyperthermia (MH),andtwo congenitalmyopathies,central-core involved inapharmacogeneticmuscledisorder, malignant impaired inRyR1-deficient myotubes(Takekura etal.,1995). (Takeshima etal.,1994).TheformationofDHPRtetradsisalso dysfunction ofthediaphragmmusclesshortlyafterbirth move becauseoftheabsenceE-Ccouplinganddiefrom tissues, but atrelatively low levels. RyR1-deficient micedonot functions incardiacmuscleandRyR3isexpressed bymany abundant isoforminskeletal muscle.RyR2predominantly inmammals(FillandCopello,2002).RyR1isthemost RyR isoforms(RyR1,RyR2andRyR3)areencodedbydifferent number andpositionofmembranedomainsisnotknown. Three transmembrane domainsaswellthechannelpore,exact hydrophobic C-terminusdomainmightcontainseveral density (Franzini-ArmstrongandProtasi,1997).Althougha observed inelectronmicrographsasdotsofhighelectronic (Takeshima etal.,1989)and,thus,RyR1channelscanbe 2772 zebrafish. that analysisofthe in mutantsrestorednormalswimming.Thesefindings suggest oligonucleotides against fibers. Interestingly, applicationofantisensemorpholino relaxed a prematurestopcodon.SimilartohumanMmD, an intronofthe relaxed mRNA, encodingRyR1,carriedanonsensemutationin suggesting adefectinE-Ccoupling.Infact, mostofthe tubules weredramaticallydecreasedinmutantfast muscles, CNS. Ca due toweakmusclecontractionsdespitenormaloutputfromthe breeding stockofzebrafish. Mutantsdisplayedslow swimming mutant, whichwas identified asaspontaneousmutationinour In thispaper, wecharacterizedthe Mutations inRyR1,encodedbytheRYR1 geneinhumans,are mi340 2+ regulation fromregions ofnormalCa was identified asaspontaneousmutationinourbreeding stockof RESEARCH ARTICLE mutants. AnalysisofgenomicDNA foundaninsertionin mutants displayedsmallamorphouscoresinmuscle 2+ transients inthemusclecytosol andRyR1atthet- ryr1b relatively relaxed gene thatresultedinaberrantsplicingand ryr1b that blocked theaberrantsplicing mutant maybeusefulfor relatively relaxed 2+ homeostasis relatively relatively ryr1b ( ryr ) primer 5,5 primer 4,5 forward primer4,5 reverse primer3,5 primer 5,5 2004). Briefly, weinjectedCalciumGreen-1dextran (10,000 Corporation) toinducefictive swimming. tail ofthepinnedembryousingaPicospritzerIII(Parker Hannifin solution (20psi,20millisecondspulse)fromapipettewith Instruments). Mechanosensorystimulationwas delivered byejectingbath collected withClampex 8.2andanalyzedwithClampfit 9.0(bothAxon Instruments), low-pass filtered at5kHzandsampled10kHz.Datawere Recordings wereperformedwithanAxopatch200Bamplifier (Axon NaCl, 2.9mMKCl,2.1CaCl Sigma), pinnedonaSylgarddishandimmersedinEvans solution(134mM anaesthetized in0.02%tricaine(ethyl3-aminobenzoatemethanesulfonate, elsewhere (BussandDrapeau,2000).Briefly, 48hpfzebrafish embryoswere The dissectionprotocolsforinvivo patchrecordingshave beendescribed Muscle recording Image onaG4Macintosh(Apple). frame grabber(LG-3,ScionCorporation),andwereanalyzedwith Videos werecapturedwithaCCDcamera(WVBP330,Panasonic) anda microscopy. Mechanosensorystimuliweredelivered tothetailwithforceps. Embryonic behaviors wereobserved andvideo-recordedusingdissection Video-recording ofzebrafishbehavior forward primer3,5 reverse primer2,5 forward primer2,5 forward primer1,5 were clonedbyreverse transcriptase(RT)-PCR withthefollowing primers: Twelve overlapping cDNA fragmentscovering thecodingregion of Cloning of for thephysicalmapping(Hukriedeetal.,1999). previously (Baharyetal.,2004).TheLN54radiationhybridpanelwas used microsatellites (Gatesetal.,1999;Shimoda1999),asdescribed carriers thatwerecrossedtoidentifymutantsformeioticmapping to ryr Mapping behavioral assay. diluted to0.5mg/mlinEvans solutionandappliedtoembryos1hourbefore Ruthenium red(Sigma),aninhibitorofRyR1(Pessahetal.,1985),was Pharmacological treatment The protocolsforCa Ca Hepes, 10mMEGTA and4mMNa genotype oftheembryoswas determined bygenomicPCR. performed byconfocalmicroscopy (FV-500, Olympus).Afterimaging,the scanning ofaCalciumGreen-1dextran-labeled musclecellat800Hzwas bath solutionusingaPicospritzerIIItothetailofpinnedembryos.Line- immobilize embryos.Mechanosensorystimulationwas appliedbyejecting inhibitor, N-benzyl-p-toluene sulphonamide(Sigma)(Cheungetal.,2002)to embryos werebathedinEvans solutionwith5mMofthemusclemyosin pinned toaSylgarddishwithtungstenwires.Thetricainewas washed outand crosses. At48hpf,embryoswereanaesthetizedwith0.02%tricaineand Probes) intooneblastomereof8-to16-cell-stageprogeny of consisted of105mMpotassiumgluconate,16KCl,2MgCl modulation optics(40 The electrodewas visuallyguidedtopatchmusclecellsusingHoffman borosilicate glass(Narishige)toyieldelectrodeswithresistancesof3-10M d-tubocurarine (Sigma)withouttricaine.Patch electrodeswerepulledfrom embryos werepartiallycurarizedinEvans solutioncontaining3 access totheunderlyingmuscles.For electrophysiologicalrecordings, mMHepesat290mOsmandpH7.8).Theskinwas peeledoff toallow 10 reverse primer1,5 2+ carrier fish werecrossedwithwild-typeWIKfish togeneratemapping imaging inmuscle Ј Ј Ј -ACACACGGCGCAATAAAGCATCAGAGTGTG-3 -TCCTGGAACTATCTGAGCAACATGACCTGC-3 -AAGACTGAACATGAGTCGCACCAGCTCTG-3 ryr1b Ј Ј Ј cDNA Ј -TCAATGTGGTTGTGATCAGTGGGAACGGGA-3 Ј -CATCTGTGGCCTCCAAGAGGGCTTTG-3 -CTGTGTAAAAAGGCCTGTGGTGCTGTAGAT-3 -AGCAGTGTCAACAGGACAGGGAGTGAATG-3 2+ -GAGAAAACGCACGGATTTTCTGATTTCTCC-3 Ј -CCTCCTCCTGGTTATGCACCATGTTATGAG-3 ϫ imaging have beendescribedpreviously (Hirataetal., Ј water immersionobjective). Theelectrodesolution -CTGCTCTCGCTCTCAGACAGAAGAGTC-3 2 , 1.2mMMgCl 3 ATP at273mOsmandpH7.2. Development 134(15) 2 , 10mMglucoseand M ryr ␮ r , Molecular m tiptothe Ј carrier in- Ј Ј ; forward ; forward 2 Ј ; reverse ; reverse , 10mM ryr1b ␮ ⍀ M Ј Ј Ј Ј Ј Ј ; ; ; ; ; ; .

DEVELOPMENT AGGAGAGTTAAGTCAAGGCTACAGTCGGTC-3 5 CTGGCCCGTAAACTGGAGTTTGATGGTC-3 11, 5 10, 5 primer 10,5 primer 9,5 primer 8,5 primer 7,5 primer 9,5 primer 8,5 primer 7,5 primer 6,5 BioReagents, 1/200(MortonandFroehner, 1989).Alexa Fluor488- 1/2(Leungetal.,1988);anti-DHPR Bank, anti-DHPR IgG1 isotype,Affinity BioReagents,1/200(MortonandFroehner, 1987); 34C, IgG1isotype,Sigma,1/1000(Airey etal.,1990);anti-DHPR (Hirata etal.,2005).Thefollowing primaryantibodieswereused:anti-RyR: minutes andthensubjectedtoimmunostainingasdescribedpreviously embryos werefixed in4%paraformaldehydeatroomtemperaturefor20 Sylgard dishwithtungstenwires.Afterpeelingoff theskinattrunkregion, Zebrafish embryoswereanaesthetizedin0.02%tricaineandpinnedona Immunostaining aberrant exon: MO3 wererespectively designedagainstacceptoranddonorsitesofthe ATTTATA-3 was usedforcontrolMO:MO, 5 exon48: antisense morpholino(MO)1was designed againstsplicedonorsiteof To knockdown RyR1bproteinsynthesis(Nasevicius andEkker, 2000), Knockdown bymorpholino AACAGTGGGGCACATTTAGTGAGCAGAGG-3 5 The following primerswereusedforRT-PCR andgenomicPCR:Primer#1, PCR primer 6,5 Zebrafish ryanodinereceptor mutation Dahm, 2002). ryr1b Ј Ј -GTGTATTAAGGACTAGTCGGTCCCATTGG-3 -GTGGGTTTCTTGCCCGATATGAGAGCTTCA-3 For inhibitionofaberrantsplicing Standard controlMO(randomizedsequenceavailable fromGeneTools) Injections wereperformedasdescribedpreviously (Nüsslein-Volhard and Ј Ј -CTTGCTGATCTGGAACACTCTCTTTGGAG-3 -ACGTCATCTTTTTAGCTCCCTCCACGAGAC-3 MO3, 5 ryr1b Ј Ј Ј Ј Ј ␤ -GGAAGAGTTGAAAAATCCCCACACGAACAG-3 Ј Ј Ј -TGCAGTCTGAGCAGAAAGTCTACTGTGCAG-3 -CTTAAAGTACTGGTTTATGAGCGGGAGCAG-3 -ATCACCACAAAGTTCTGCTCCTCTCGCTTG-3 -AACAGTGGGGCACATTTAGTGAGCAGAGG-3 -GTTGATATCCCACACAATGATCCACTGGGC-3 -AACTACCGGCGCACACAAACAGGCAG-3 -GAAGTCTTCATCTTCTGGTCTAAATCGCAC-3 Ј : VD2 Ј . -CACCACAGAAATGGCTCTGGCTCTGAAC-3 Ј ryr1b -TATCTTACACTTACCTTTAAATAAG-3 MO1, 5 1 B12, IgG1isotype,Developmental StudiesHybridoma MO2, 5 Ј -ATGATTGAGTTTACCGTATCCAGAG-3 Ј -ATTGGTTGACTCCTGATACTCAATG-3 ryr1b ␣ Ј -CCTCTTACCTCAGT TACA - 2: 20A,IgG2aisotype,Affinity mRNA, antisenseMO2and Ј ; reverse primer12,5 Ј Ј ; forward primer12,5 Ј . . Ј Ј ; reverse primer11, ; Primer#2,5 Ј Ј ; forward primer . Ј ; reverse primer Ј Ј Ј Ј Ј Ј Ј ; reverse ; forward ; forward ; forward ; forward Ј ; reverse ; reverse ; reverse ␣ Ј . 1: 1A, Ј Ј Ј Ј - - - ; primer, 5 ATGGTGAGAAGGCTGAGAAGGAAGTGGAGG-3 cloned withthefollowing primers: hybridization. An C-terminus aminoacidsand148bpofthe3 OsO in 0.1Msodiumcacodylatebuffer, theembryoswerepost-fixed with1% M sodiumcacodylatebuffer, pH7.2,overnight at4°C.Afterbeingwashed embryos werefixed with6%glutaraldehyde-2%paraformaldehydein0.1 elsewhere (Hatakeyama etal.,2004;Schredelseker etal.,2005).Briefly, The protocolsfortransmissionelectronmicroscopy have beendescribed Transmission electron microscopy PBS at37°Candthenembeddedinit–80°C.Sections(10 development, embryoswereequilibratedin15%sucrose/7.5%gelatin described previously (Hirataetal.,2004).For sectioningaftercolor In situhybridizationstowholemountedzebrafish wereperformedas In situhybridization antibodies (1/1000,MolecularProbes). and Alexa Fluor555-conjugatedanti-mouseIgG2awereusedassecondary conjugated anti-mouseIgG,Alexa Fluor488-conjugatedanti-mouseIgG1 An autosomal,recessive mutation, weak musclecontractions ryr RESULTS recorded withtheirheadsrestrainedonaSylgarddish,leaving the contractions duringswimming,48hpfembryoswerevideo- supplementary material).To examine thefrequency andstrengthof Student’s 1.61±0.44 cm/s, embryos swimmingaway muchlessefficiently (wild-typeat36hpf: embryos swimmingaway rapidlyupontactilestimulationand phenotype was examined byvideo-microscopy, withwild-type bends at24hpfwereunperturbed(datanotshown). Theswimming n spontaneous coilingat22hpf(wild-typesiblings:0.21±0.11Hz, exhibited significantly slower swimmingafter36hpf,but identified fromourbreedingstockofzebrafish. Mutantembryos microscope (H-7000,Hitachi)operatedat75kV. Ultrathin sections(80nm)werecutandexamined usinganelectron with acryostat(CM3050S,Leica).An =34; 4 mutants showslowswimmingbecauseof for 60minutes,andthendehydratedembeddedinEpon812. ryr Ј -CGTCATCATGTCGTCACACTTCATGTCCGG-3 mutants: 0.19±0.09Hz, t -test, P ryr1a n <0.001; Fig.1A-O,andseeMovies 1,2inthe =12; probe covering 1157bpoftheC-terminuswas ryr at 36hpf:0.71±0.16cm/s, most ofthetrunk andtailfree. pinned onaSylgard dishleaving and Q,theheadsofembryoswere with thatofwild-typesiblings.For P ryr trunk andtailduringswimmingin sibling. ( by tactilestimulationofawild-type during aswimmingepisodeinduced displacement ofthetrunkandtail from avideoshowingnormal ( hpf) toswim,butonlyslowly. Fig. 1. induced an to swimawayrapidly. ( induced awild-typeembryo(48hpf) ( response totouch. weak musclecontractionsin exhibit slowswimmingand P A-E n ryr1b relatively relaxed Superimposition ofalltheframes ) =14) andtouch-inducedfast C- RESEARCH ARTICLE mutants issmallercompared Mechanosensorystimulation ) ryr1a ryr probe covering 1089bpofthe Q Ј -UTR was usedforinsitu ) Displacementofthe mutant embryos ryr forward primer, 5 mutant embryo(48 Ј ; ( ryr1a ryr F-O ␮ Ј m) werecut mi340 . ) Touch reverse n ), was 2773 =12; ryr Ј -

DEVELOPMENT in cytoplasmic Ca Depolarization ofthemusclemembrane causesatransientincrease The fact thatmusclecontractionsin Output oftheCNSisnormalin swimming in contractions bytrunkandtailmusclesarethecauseofslow inability tofeedeffectively. Theseobservations suggestthatweak around 7-15dayspost-fertilization(dpf),possiblyfromtheir anatomical defectsin with wild-typesiblings(Fig.1P,Q). Therewerenoobvious ryr Armstrong andProtasi,1997). Because musclesweredefective in actin/myosin slidingandthe contraction ofmuscle(Franzini- was perturbed inmutantmusclesbyinjecting live embryos with from bothwild-type-siblingand by tactilestimulationwererecorded(Fig.2A).Voltage recordings the CNSand/orNMJ,voltage responsesinmusclesevoked muscle defect.To seewhethertherewas adecreaseinsignalingfrom or thatcontractileresponsesofmuscleswerecompromisedduetoa muscle was decreasedbecauseofadefectintheCNSand/orNMJ, those inwildtypesuggestedthattheoutputfromCNSonto tail movements was significantly smallerin and (38.7±5.2Hz, was comparablebetweenwild-typesiblings trunk andtailfreetomove. Thefrequency ofmusclecontractions 2774 (wild type:1.02±0.25mV, 1.21±0.14 mV, muscles werecomparable(wildtype:1.16±0.07mV, rhythmic depolarizationsfromwild-type-siblingandmutant fast swimming inresponsetotouch(Fig.2B-E).Theamplitudesof the showed rhythmicdepolarizationsindicative ofnormalfictive Ca downstream oftheNMJ. mutants andsuggestthatthey harboradefectinmuscles, results indicatethattheCNSandNMJfunctionnormallyin 2+ mutants, weexamined whether theincreaseincytosolic Ca ryr transient issmallerin RESEARCH ARTICLE embryos (35.9±4.5Hz, ryr n . =4), aswerethedepolarizationsinslow muscles 2+ ryr mediated byE-Ccouplingthat resultsin embryos andlarvae, but mutantsdiedat n =5; n =10), but theamplitudeoftrunkand ryr ryr ryr ryr : 0.90±0.17mV, mutant fast andslow muscles mutant fastmuscle mutants wereweaker than ryr ryr mutants mutants compared n =5). These n =4; n =10) ryr ryr 2+ : Fig. 3C).Furthermore,Ca mutants (wild-type contractions in coupling infast musclesappearstobethebasisforweak responded normallytotactilestimulation.Thus,adefectinE-C whenallmutants with wild-typesiblingsat24hpf(datanotshown), Ca n relative level ofCalciumGreen-1fluorescence( ryr amplitude ofCa 4U) but notin were presentatt-tubule–SR junctionsinwild-typefast muscles(Fig. structures thatpresumablyrepresent juxtaposedRyRsandDHPRs the immunohistochemistryresults. Patterned electron-dense mutation inslow muscles (Fig.4K-T).Electronmicrographsverified distribution ofRyRsand ofDHPRsubunits wereunperturbedbythe reduced inmutantfast muscles (Fig.4D,F,H,J). However, the DHPR presumptive t-tubule–SR junctions (Fig.4I).Ontheotherhand, DHPR fast muscles(Fig.4B).Similarly, anti-DHPR (Fig. 4A),whereasRyRlabelingwas significantly reducedinmutant presumablyrepresentedthet-tubule–SR junctions muscles, which that RyRsweredistributed inastriatedpatternwild-type fast examined in in mutantfast muscles,thedistribution ofRyRs andDHPRswere t-tubule–SR junctions.To seehow E-Ccouplingmightbedefective RyRs, bothofwhichareclusteredatthejuxtaposedmembranes E-C couplingismediatedbydirectinteractionbetweenDHPRsand tubule–SR junctionsin forE-Ccouplingare notclustered att- By contrast,Ca anti-DHPR type fast muscles(Fig.4C,E,G).Doublelabelingwithanti-RyR and anti-DHPR =7; 2+ uat comparedwithwild-typesiblingsat48hpf[wild-type mutants ryr indicator dye,CalciumGreen-1dextran (Fig.3A). The ␣ ␣ 2 wereprobablylocalizedtot-tubule–SR junctionsin wild- 1, DHPR ⌬ F/F: 0.13±0.05, ␣ ␣ 2 confirmed thatRyRsandDHPRswerecolocalized at 2, respectively, showed thatDHPR ryr ryr 2+ muscle ofawild-typesibling(D)andan (fictive swimming).( ofdepolarizations mutant (C)displayedsimilarpatterns muscle fibersofawild-typesibling(B)andan indicate thetimeofstimulation. display similarrhythmicdepolarizations.Arrows patch electrode. ( puff ofbathsolutiontothetailandare recorded witha evoked bymechanosensorystimulationdelivered bya to themusclecells.Musclevoltageresponses are damage theCNS.Theskinispeeledoff toallowaccess tungsten wires through thenotochord soasnotto procedure. Embryos(48hpf)are pinnedonadishwith mutants. Fig. 2.TheCNSandNMJfunctionnormallyin ryr 2+ mutant muscles.Labelingwithanti-RyRshowed transients inslow musclewerenotperturbedin mutant fast muscles(Fig.4V).Thedistribution of transients infast musclewas 3.3-timessmallerin ␤ mutant muscles. ⌬ F/F: 0.35±0.08, and DHPR ( A 2+ n ) Schematicsummaryoftheexperimental =7; Student’s transients inmutantswerecomparable ryr B , C ␣ D ) Voltage recordings from fast mutants 2 labelingweresignificantly , E n ) Voltage recordings from slow =5; ryr t -test, Development 134(15) ⌬ ␣ F/F: 0.35±0.07, 1, anti-DHPR P ⌬ <0.001, Fig.3B]. ␣ F/F): 0.43±0.13, 1, DHPR ryr mutant (E) ryr ␤ ␤ ryr n and and =5; ryr

DEVELOPMENT relative fluorescence represents thetransientincrease ofCa scanning withaconfocalmicroscopy. ( Green-1 fluorescence ( stimulation byapuff ofliquidtothetail.Therelative levelofCalcium Embryos (48hpf)were pinnedonadishandgivenmechanosensory 1 dextranwasinjectedintoonecellof8-to16-cell-stageembryos. ( that ofan muscle followingstimulation(arrow) ofawild-typeembryo(black)and between wildtypeand presumptive RyR/DHPRparticlesinslow muscleswas comparable Zebrafish ryanodinereceptor mutation gene, candidate forthe phenotypeinmutantssuggestedthat RyR1 meioses).Thus, both geneticmappingandthe 1110 recombinants in recombination withapolymorphic marker in assembly ofthezebrafish genome.Furthermore,therewas no was foundinbetweenthese two markers intheZv6Ensemble (Fig. 5A).Ageneencodingformuscleryanodinereceptor1( 1110meioses) meioses) andz8343(6.9cM,77recombinantsin two microsatellites,z737(2.1cM,23recombinants in 1110 was meioticallymappedtoaregion ofchromosome18defined by To identifythegeneresponsible forthe Fig. 3.Ca (black). muscle isunperturbedin The mutatedgenein of clusteringbyRyRsandDHPRsatthet-tubule–SR junctions. are deficient inE-Ccouplingtheirfast muscles becauseofalack A Schematic summaryoftheexperimentalprocedure. CalciumGreen- ) ryr1b 2+ ryr transients are smallerin cDNA was clonedandsequenced fromwild-typeand mutant (red). ( ryr ⌬ mutation. To seewhether F/F) inmusclefiberswasassayedbyline- ryr ryr C mutants (red) compared towild-type mutants (Fig.4W,X). Thus, ) Thetransientincrease ofCa ryr mutants encodesRyR1 B ryr ) Thetransientincrease in ryr mutant fastmuscles. phenotype, themutation ryr1b ryr1b ryr1b (<0.09 cM,0 was agood 2+ 2+ ryr was the in slow in thefast mutants ryr1b ryr ) (GenBank #AB247454,Fig.5B).The mutant embryos.Wild-type stop codoninthemiddleoffull-lengthproteinthatwas 5 contained a32-bpinsertion.Thisinsertiongeneratedpremature type 5C, lane3),confirming thattheshorterbandcorrespondedtowild- wild-type embryosofanotherstrain,theABzebrafish (Fig. lane 2).Onlytheshorterfragmentwas amplified fromthecDNA of whereas thelongerfragmentwas predominantinmutants(Fig.5C, from agroupofwild-typesiblingsastemplate(Fig.5C,lane1), longer andshorterPCRfragmentswereamplified usingthecDNA insertion was specifically foundin primers flankingtheinsertionwas performedtoconfirm thatthe C-terminus (Meissner, 1994).Reverse transcriptase(RT)-PCR with sequences forthepredictedtransmembranedomainslocatedat lanes 1,4),whereas wt/ only awild-type(short)RT-PCR fragment atbothstages(Fig.5F, hpf (Fig.5F).Wild-type embryosofthewt/wtgenotypeshowed PCR, respectively, toassaythegenotype andsplicingat2448 cross oftwo this possibility, theheadandtrunkofindividual embryosfroma 24 hpf,suggeststhatdefective splicingwas stage-dependent.To test in which after 36hpfbut notearlierthan30hpf,whereaswild-type embryos mammals. RyR1 isessentialfornormalmusclefunctioninzebrafish, asitisin phenocopied touch-inducedslow swimmingat36hpf( Ruthenium red,aninhibitorofRyR(Pessahetal.,1985), also knockdown byMO1.Treatment ofwild-typeembryoswith control MO-injectedembryos(Fig.5E),confirming the efficacy of and 48hpfinMO1-injectedembryoswas reducedcompared with defects, theamountof as forswimmingatlaterstages.Correlatedwiththebehavioral RyR1b isrequiredfortouch-inducedcoilingatearlierstagesaswell five trials)ratherthanthenormalfast, vigorouscoils,suggestingthat tail following tactilestimulationat24hpf(82.0±7.8%, MO1-injected embryosalsoexhibited weakcoilsofthe trunkand P the that the32-bpinsertionwas foundonlyinthelongerproduct.Thus, n MO-injected wild-typeembryos(MO-1injected:0.81±0.20cm/s, injected wild-typeembryosswam moreslowly thancontrol wild-type embryosandassayedtouchresponsesat36hpf.MO1- were complementarytothesplicedonorsiteofexon 48,into We injectedantisensemorpholinooligonucleotides(MO1),which knockdown ofRyR1bandapplicationaspecific inhibitorofRyR. phenotype, weattemptedtophenocopy slow swimming byantisense majority offast muscleRyR1bwould probablybenon-functional. that predictsatruncatedRyR1blackingthechanneldomains,great 2005). Becausetheaberrantsplicingresultsinaprematurestopcodon family transposons(datanotshown) (Ivicset al.,2004;Kawakami, repeated motifatbothendsthatarecharacteristicofTc1/mariner might representatransposableelement,becauseitcontained additional exon inthemutant and donorsites,confirming thatthe32-bpsequenceactsasan flanking the32bpingenomicinsertcontainedsplicingacceptor between exon 48and49ofthe DNA insertion,includingthe32-bpcDNA insertion,intheintron =12; controlMO-injected:1.78±0.35cm/s, 001,mc iemtn mro.Interestingly, mostofthe <0.001), muchlike mutantembryos. The fact thatweakmusclecontractionsinmutantswereobvious To confirm whetheralossofRyR1bisresponsibleforthe Genomic sequencingrevealed that ryr ryr1b phenotype isvery likely tobeduemutationin ryr1b . Sequencingofthelongerandshorterfragmentsverified ryr was knocked down exhibited weak contractionsat carriers weresubjectedtogenomic PCRandRT- ryr1b ryr embryos exhibited bothwild-type(short) mRNA withnormalsplicingat24,36 ryr1b ryr1b ryr1b RESEARCH ARTICLE gene. Thisgenomicinsertion ryr1b ryr encodes 5076aminoacids ryr gene (Fig.5D).Sequences mutants (Fig.5C).Both mutants carrya4046-bp cDNA from n =12; Student’s n =20). Thus, ryr ryr1b n Ϸ mutants Ј 43-76, t to the 2775 . -test, ryr

DEVELOPMENT sequences. This classification suggested thattherewereatleastfive RyR1, RyR2orRyR3based onhomologywiththehuman radiation hybridpanel(Hukriede etal.,1999)andidentified as zebrafish RyRsequences werephysicallymappedwiththeLN54 that includedsequencesencoding forRyRs(Table 1). Eachofthe for humanRYR1, RYR2 and RYR3, andfound14genomic contigs Ensemble assemblyofthezebrafish genomewithproteinsequences To identifyotherzebrafish genesencodingRyRs,weblastedtheZv6 duplicated The zebrafishgenomecontainspotentially to stage-dependentaberrantsplicinginmutants. These resultssuggestthatthemutantbehavioral phenotype was due mutant productbecamepredominantat48hpf(Fig.5F, lane6). with comparableintensityat24hpf(Fig.5F, lane3),whereasthe Mutant embryos( and mutant(long)fragmentsatbothstages(Fig.5F, lanes2,5). 2776 RESEARCH ARTICLE ryr ryr/ryr genes ) gave bothwild-typeandmutantfragments hpf (Fig.6B-E).Bycontrast,expression of deep axialmusclesprobablyrepresenting fast musclesat24and48 RyR1b isexpressed byfast muscles. duplicates ofRyR1wereboth expressed bymusclesbut thatonly et al.,1996)(Fig.6F-I).These resultsindicatethatthepotential superficial musclesunder theskin,representingslow muscles(Devoto fast muscle.Insituhybridization revealed that muscles, however, showed that trunk (Fig.6A).RT-PCR usingcDNA fromdissected adult fast ryr1b ryr1b Because ryr1b in LG12and in linkagegroup(LG)10and different ryanodinereceptorgenesinzebrafish: two forRyR1[ and should beexpressed byfast muscles.RT-PCR showed thatboth is expressed byfastmuscle ryr ryr1a mutants weredefective inE-Ccoupling fast muscle, ryr2b were expressed from1to5dpfaswellbytheadult in LG17)andoneforRyR3( slow muscles.SR,sarcoplasmic reticulum. present inbothwild-type(W)andmutant(X) aggregates aremuscle (V).TheRyR-DHPR type fastmuscle(U)butnotin between t-tubuleandSRmembranesinwild- DHPR aggregates are visibleasdenseparticles junctions inmusclesat48hpf.PutativeRyR- ( show ahighermagnificationofmusclefibers. but notin slow (S)muscleandinmutant(T) (purple) are colocalizedinwild-typefast(I)and pattern. ( pattern. (A,B,K,L),anti-DHPR hpf withanti-RyR were assayedinfastandslowmusclesat48 ( DHPR wild-type fastmuscles. dramatically decreased in Fig. 4.E-C-couplingcomponentsare muscles also express RyR (L),DHPR muscles alsoexpress RyR (O) andDHPR (K),DHPR muscles express RyR mutant fastmuscles(B,D,F,H). slow Wild-type junctions, whereas clusteringisnotobservedin presumably corresponds tothet-tubule–SR DHPR (A),DHPR express RyR DHPR (C,D,M,N), anti-DHPR U-X A-H ) Electron micrographs oft-tubule–SR , K-R ␤ ␣ ␣ 2 (G) in a striated pattern that 2 (G)inastriatedpattern fastmuscles 2 (G,H,Q,R).Wild-type ryr1b (P) andDHPR ryr1b ) The distribution of RyRs andDHPRs ) ThedistributionofRyRs I , ryr J , S , ␣ mutant fastmuscle(J).Insets in LG18],two forRyR2( T , but not 2 (Q) in a striated pattern, asin 2 (Q)inastriatedpattern, ) RyR (green) andDHPR ) RyR ␣ ␤ ␣ 2 (R)inastriated Development 134(15) ryr1a ryr1a ryr1b ryr 1 (C),DHPR (E,F,O,P) andanti- mutant slow ryr , was expressed in was expressed by was observed in ␣ ryr 1 (M),DHPR fast muscles. in LG20). mutant fast ␣ ␤ 1 (N), (E) and ␣ ␣ 1 2 ryr2a ryr1a ␤

DEVELOPMENT mapping placedthe was diminishedat48hpf(lane6). mutants, theintensityofwild-type(short)bandamplifiedbyRT-PCR wascomparabletothatof themutant(long)bandat24 RT-PCR withprimers#1and#2usingindividualembryosasatemplate.Genotypes,shown,were identifiedbygenomicPCR.Noti The Zebrafish ryanodinereceptor mutation seen inMmDmuscles. mutant fast musclesdisplayed ultrastructuraldefectssimilartothose at 7dpfand,insomecases,the SR was missingat14dpf.Thus, nm at14dpf;Fig.7E,F).Disorganization oftheSRwas alsoevident muscles increasedwithdevelopment (50-500nmat7dpf;100-800 diameter) at2dpf(Fig.7D).The diameterofthecoresinmutantfast muscle, however, displayedsmallamorphous cores(50-100nmin in wild-typefast muscleat2,7and14dpf(Fig.7A-C).Mutant fast shown). Well-formed actin/myosinbundles andSRwereobserved appeared comparablebetweenwildtypeandmutant(data not transmission electronmicroscopy. Superficial slow muscles transverse sectionsoflarval axialmuscles wereanalyzedby whether in musclefibers (Engel,1967;Jungbluthetal.,2004). To examine human andispathologicallydefined bymultipleamorphous cores MmD, arecessive myopathy, iscausedbyRYR1 mutationsin whereas control MOdoesnot.Afragmentofnormal antisense morpholino(MO1,seeD)intorecently fertilizedwild-typeembryoseffectively decreases normal bp) from agroup ofwild-typesiblings(lane1),whereas themutantbandwaspredominant in ( (0/1110).( gene Fig. 5. in theintron betweenexons48and49of faintly inthemutantlane.Onlyshorterbandwasamplifiedfrom wild-typeABembryos(lane3).( C ) Reverse transcriptase (RT)-PCR usingprimers#1and#2(seeB)amplifiedbothalongermutantband(169bp)shorterwil ) Reversetranscriptase(RT)-PCR ryr ryr ryr mutant isadiseasemodelofMmD mutant embryoshaveamutationin mutants displayedmorphologicaldefectsinmuscle, B ) ryr ryr mutants containeda32-bpinsertion(red) in locus betweenz737andz8343inchromosome 18.Norecombination wasfoundwithapolymorphicmarkerinthe ryr1b that includestheadditional32-bpsequencefoundinmutantcDNA.( ryr1b ryr1b due toaDNAinsertionthatleadsaberrantsplicingof mRNA wasexaminedbyRT-PCR withprimers#1and#2(seeD).( ryr1b ryr mRNA thataddedanonsensecodon5 control MO-injected: 0.74±0.22cm/s;Student’s recovery (>1.0cm/s) inswimming(MO2injected:1.34±0.32cm/s; However, 49%(23/47)ofMO2-injected normal (short)andmutant(long) fragments(Fig.7H,lane5). wt/ only thewild-type,shortfragment (Fig.7H,lane4).MO2-injected injected wt/wtembryosexhibited normalswimmingandexpressed effect eitheronswimmingorsplicing(Fig.7H,lanes1-3).MO2- genotype andsplicing(Fig.7H).InjectionofcontrolMOhad no subjected togenomicPCRandRT-PCR, respectively, toassay tactile stimulationat36hpf,theheadandtrunkofembryoswere progeny of the spliceacceptorsiteof32-bpinsertintorecentlyfertilized injection ofantisensemorpholinooligonucleotides(MO2)against by preventing aberrantsplicing.Thistreatment was examined by mRNA, wewondered whethernormalswimmingcouldberestored Because the ryr embryos exhibited normal swimmingandexpressed boththe ryr ryr carriers (Fig.7G).Aftertestingtheirresponseto ryr mutation was duetodefective splicing of mutants (lane2).Thewild-typebandcanbeseen D ) ryr ryr1b mutants carrya4046-bpinsertion(red) Ј mRNA at24,36and48hpf, to thetransmembranedomains. RESEARCH ARTICLE ryr/ryr ryr1b E ) Injectionofan F ) GenomicPCRand mRNA. embryos exhibited hpf(lane3)but t d-type band(137 -test, ce that,in ( A P ) Meiotic <0.001). ryr1b ryr1b 2777

DEVELOPMENT that expressed bymuscleat24 (B,F)and48(D,H)hpf.Cross-sections show E) and expressed byadultfastmuscles. ( and r mRNA from deepmuscles indicates that expressed bysuperficial,slowmusclesbutnot byfastmuscles(G,I). respectively. Fig. 6. 18.4 14.6 18.4 9) muchlike controlMO-injectedmutants(Fig.7H,lane3). 18.6 9.8 mutant, longfragment,withlittlenormalproduct(Fig.7H,lanes8, 17 swimming (<1.0cm/s,51%,24/47)predominantlyexpressed the Gene contrast, MO2-injectedmutantembryosthatexhibited slow 12.0 that incontrolMO-injected 15.0 mRNA tomutant,longmRNA (Fig.7H,lanes6,7)comparedwith 17.6 12 17 17.9 They expressed 17.6 anincreaseintheproportionofwild-type,short 17 20 13.3 15.5 18 different loci.TheLODscore indicatesreliability ofphysicalmappingwithscores higherthan5considered reliable. LG,link containing partialsequencesimilartohumanryanodinereceptors. Eachgenomiccontigwasphysicallymappedbyprobing theLN54 12 A blastsearch ofthezebrafishgenomicdatabase(Zv6Ensemble)withprotein sequencesofhumanryanodinereceptor 1,2and3i LODscore 17 Zv6_LG20_scaffold2945_BX682544 18 17 Zv6_NA2171 18 15.9 Zv6_LG12_scaffold2462 18 15.9 Zv6_LG12_scaffold2444 Zv6_LG12_scaffold2525_BX005070 LG Zv6_LG12_scaffold2517_BX323986 Zv6_NA172 Zv6_LG12_scaffold1783 10 Zv6_LG10_scaffold1543 10 Zv6_NA1631 Zv6_LG18_scaffold2704 Zv6_LG18_scaffold2701 Zv6_LG10_scaffold1488_BX323802 Zv6_LG10_scaffold1488_BX088525 Zv6 genomiccontig Table 1.Zebrafishhasfiveryanodinereceptor genes 2778 ryr1b yr1a ryr1a ryr1b RESEARCH ARTICLE are expressed at1, 2 and3dpf,inadults.RT-PCR with is expressed bydeep,fastmuscles (C,E),whereas (F-I) probes. Wholemounts showthatbothgenesare and ( A ) Reverse transcriptase (RT)-PCR showsthatboth ) Reversetranscriptase(RT)-PCR ryr1a are expressed byfastandslowmuscle, ryr/ryr B-I ) Insituhybridizationwith embryos (Fig.7H,lane3).By ryr1b but not ryr1a ryr1a is ryr1b ryr1b is (B- RyR atthet-tubule–SR junctionsandtoadecreasein Ca reduction ofRyR1binfast musclesleadstoadecreaseinDHPR and condition duetoreducedsynthesisofnormalmRNA. Therefore,a were aberrantlysplicedinthemutants,leadingtoahypomorphic two zebrafish mutationscorroborate thefinding that theformation and theSRwerenotdamaged.Taken together, thephenotypesof dramatic decreaseinbothDHPR andRyR1even whenthet-tubules tubule–SR junctions. In significantly reduced, but RyR1was correctlytargeted tothet- junctions in components, suchasRyRsandDHPRs,atthet-tubules–SR muscles. Third,therewas adramaticdecreaseinE-C-coupling ryr from theSRintocytosol, leadingtoweakmusclecontractionsin Zhou etal.,2006).In because ofanullmutationinDHPR Zebrafish InformationNetwork), isdeficient inE-Ccoupling following thedepolarizationofmusclesweredecreasedin MO2-injected mutants. Furthermore,electronmicrographsofthefast musclesof injected mutantsdiedataround7-15dpf,muchlike uninjected by 60hpf,presumablyduetobreakdown oftheMO.TheseMO2- embryos thatexhibited recovery at36hpfexhibited slow swimming However, rescueofswimmingwas transient,becausethe23 CNS was normalin depolarization ofthemusclemembraneevoked byoutputfromthe contractions duetoanE-C-couplingdefectinfast muscles.First, Several findings demonstratethat muscles ryr DISCUSSION mediated treatmentinvivo. indicates that the aberrantsplicingisresponsiblefor treatment cantransientlyrestorenormalswimming,confirming that shown). Thus,prevention ofaberrantsplicingwithantisenseMO complementary tothedonorsplicesiteof32-bpinsert(datanot another antisensemorpholinooligonucleotides(MO3)thatwas at 7dpf( The zebrafish immotilemutant, mutants. mutants haveanE-C-couplingdefectinfast n ryanodine receptor3 ryanodine receptor2b ryanodine receptor2a ryanodine receptor1b ryanodine receptor1a =4, datanotshown). Similarresultswereobtainedwith ryr ryr ryr fast muscles.Indeed,mostofthe mutants mightbeusefulforexamining antisense- mutants displayedamorphouscoresinfast muscle ryr relaxed age group; LOD,logofodds. ryr fast muscles.Second,Ca mutants, bycontrast,therewas a (slow muscle) (fast muscle) dentified 14different genomiccontigs mutant muscles,DHPR radiationhybridpaneltooneoffive ryr relaxed ␤ mutants exhibit weakmuscle (Schredelseker etal.,2005; 1 (also known as Development 134(15) ryr phenotype. Italso ryr1b 2+ 2+ transcripts transients cacnb1 released ␣ ryr 1 was fast ryr –

DEVELOPMENT determines exon preference,mightbe differentially regulated were aberrantlysplicedat48 hpf.Asplicingfactor, which normally processedat24hpf,whereas most occur duringlaterstages.Infact, halfofthe developmentally regulated andthattheaberrantsplicingmayonly explained bythepossibility thatsplicingof after 36hpf.Thislackofamutant phenotypeat24hpfcanbe touch withnormalC-bendsat24hpfandexhibited slow swimming Although family oftransposons(Ivicsetal.,2004;Kawakami, 2005). insertion revealed inverted repeatscharacteristicoftheTc1/mariner transposable elementinzebrafish. Indeed,sequencing ofthe mutant The 32-bpexon was includedina4046-bpDNA insertioninthe transcripts, whichresultedinthegenerationofanonsensecodon. In ryr1b targeted totheSRin is probablyadirectconsequenceofthefailure ofRyR1btobe 2002). Thefailure ofDHPRtolocalizethet-tubule–SR junctions cultured mammalianmyotubes(Kugler etal.,2004;Proenza etal., physical interactionwithanintracellularloopofDHPR 1995). Indeed,acytoplasmic domainofRyR1isessential forthe of DHPRtetradsrequiresthepresenceRyR1(Takekura etal., Zebrafish ryanodinereceptor mutation essential forE-Ccouplingatlaterstages, ryr mutants, wefounda32-bpinsertioninmostofthe is the ryr1b ryr1b gene. Thislarge insertionmight bederived froma ryr was expressed byfast musclesat24hpfandwas gene ryr mutants. ryr ryr1b ryr1b mutants respondedto mRNA transcripts ryr1b transcripts were might be ␣ ryr1b 1 in weakness. Fourth, both Third, both ryr responsible forboththe with humanMmD.First,mutations ingenesencodingRyR1are sections. Theamorphous coresinMmD(2-25 display myopathycharacterized byminicoresinhistological The zebrafish The differences mayhave evolved. of thetwo muscletypesdictatedivergent RyR1sandhow these coupling betweenslow andfast musclestoseehow therequirements would beinterestingtoexamine potentialdifferences inE-C functional difference inE-Ccouplinghasnotbeenexamined. It higher thanthatinslow muscle(Morrissetteetal.,2000),any analysis indicatesthatchannelactivity ofRyR1infast muscleis et al.,2000;Morrissette2003).Althoughsingle-channel yellowfin tuna( appears inotherfish, suchasbluemarlin( division oflaborbetweenduplicatedRyR1sinslow andfast muscles expressed byslow musclesand at 24hpfisconsistentwiththishypothesis. translation inwild-typeembryosleadstoweakmusclecontractions between 1and2dpf.Thefinding thatknockdown ofRyR1b We foundthatzebrafish have two genesencodingRyR1; phenotype andMmDareinherited asautosomalrecessives. ryr recovery (lanes6,7). lower (lanes8,9)compared tomutantsthatexhibited swimming, theproportion ofwild-typemRNAwasmuch On theotherhand,inmutantsthatexhibitedslow (short band)wasincreased (lanes6,7compared tolane3). ( and expressed bothfragments(lane5).Inmutantembryos MO2-injected wt/ and expressed onlythewild-type,shortfragment(lane4). MO2-injected wt/wtembryosexhibitednormalswimming predominantly expressed themutantfragment(lane3). MO-injected type shortandthemutantlongfragments(lane2).Control exhibited normalswimmingandexpressed boththewild- by RT-PCR (lane1).Control MO-injectedwt/ swimming andexpressed onlythewild-type,shortfragment Control MO-injectedwt/wtembryosexhibitednormal were subjectedtogenomicPCRandRT-PCR, respectively. at 36hpf,andtheheadtrunkofindividualembryos carrier incross. Swimmingofinjectedembryoswasexamined MO2 (lanes4-9)wasinjectedintotheprogeny ofan of themutantgeneisshown.( designed againstthespliceacceptorsiteofaberrantexon genes withthelocationofmorpholino(MO2)thatwas mRNA. Adiagramshowingthewild-typeandmutant antisense morpholinothatblocksabnormalsplicingof ( The SRisdisorganizedat7dpfandmissing14dpf. 50-500 nmand100-800at2,714dpf,respectively. (yellow arrowheads). Diametersofthecores are 50-100nm, and 14dpf.( black arrowheads), andbundlesofactinmyosinat2,7 wild-type fastmusclesshowingsarcoplasmic reticulum (SR; splicing of phenotype canberescued bypreventing aberrant to thatobservedinMmDandthebehavioral Fig. 7. mutant isananimalmodelforMmD ryr/ryr G Thebehavioraldefectsof ) ryr ryr Thunnus albacares ) showingrecovery ofswimming,normalsplicing mutants andindividuals withMmDexhibit muscle ryr mutant phenotypesharesseveral crucialfeatures mutants exhibitaminicore phenotypesimilar ryr1b D-F ryr/ryr ryr ) Amorphouscores are seenin ryr ryr mRNA. phenotype andMmD.Second,both the embryos exhibitedslowswimmingand embryos exhibitednormalswimming mutants andindividuals withMmd ryr1b ) (Francketal.,1998;Morrissette ( A-C RESEARCH ARTICLE ryr H ) Electron micrographs of by fast muscles.Asimilar ) Control MO(lanes1-3)or mutants are treatable byan Makaira nigricans ␮ m indiameter)are ryr embryos ryr mutants ryr ryr1b ryr1b ryr1a 2779 ) and

DEVELOPMENT (Monnier etal.,2003),similartothezebrafish hypomorph inhumanRYR1 thatwas responsibleforMmD 2006). Interestingly, Monnierandhercolleaguesreporteda is underclinicaltrials(Muntonietal.,2005;Wilton andFletcher, represent aneffective treatmentforthehumangeneticdiseaseand Deutekom etal.,2001).Thisnovel therapeuticstrategy might individuals harboringsplicingdefects(Goyenvalle etal.,2004;van cultured musclecellsfromDuchennemusculardystrophy dystrophin expression incertaindystrophinmutantmiceand finding, antisense-mediatedexon skippingrestorednormal blocking aberrantsplicingwithantisensereagents. similarity to a nonsensecodoninmostofthehumanRYR1 transcripts.Given the in anintron,resultingincorrectsplicingandthegenerationof case, anaberrantsplicedonorsitewas generatedbyapointmutation splicing of application ofanantisensemorpholinothatincreasedthenormal pathological analysisoftheconsequencesMmD. accessibility mightbeusefulfordetailedphysiologicaland Airey, J.A.,Beck, C.F., Murakami,K.,Tanksley, S.J.,Deerinck, T. J.,Ellisman, References http://dev.biologists.org/cgi/content/full/134/15/2771/DC1 Supplementary materialforthisarticleisavailableat Supplementary material Organization. HumanFrontierInternational ScienceProgram wassupportedbyLongTerm Fellowshipofthe Fellowship from FRSQ.H.H. Promotion ofScienceforYoung Scientists.L.S.-A.waspartlysupportedbya J.H. wassupportedbyaResearch FellowshipoftheJapanSocietyfor toJ.Y.K. Fund.SupportedbygrantsfromLife SciencesInternational theNINDS Memorial FoundationforMedicalandPharmaceuticalResearch andtheGoho Sumitomo Foundation,theKowaLifeScienceMochida Foundation,theNarishigeNeuroscience Research Foundation,the Science Science Promotion Foundation,theBrainScienceLife Culture, Sports,ScienceandTechnology ofJapanandtheHoriInformation by aGrant-in-AidforYoung Scientists(A)from theMinistryofEducation, Research onPriorityAreas ‘SystemGenomics’and‘Glia-Neuron Network’and fish care, respectively. Thisworkwassupported byaGrant-in-AidforScientific and Tokuko Niwa(NagoyaUniversity)fordiscussion, technicalsuggestionand Ikuya Nonaka(MusashiHospital,NationalCenterofNeurology andPsychiatry) equipment. We alsothankKoichiKawakami(NationalInstituteofGenetics), University) forhelpfuldiscussionandgenerous useofelectrophysiology We thankRichard I.Hume(UniversityofMichigan)and Yoichi Oda(Nagoya zebrafish as ananimalmodelofMmD(Takeshima etal.,1994).Bycontrast, RyR1-deficient micedieonthedayofbirthlimitstheirusefulness showed thatthey areevident in embryonicmuscles.Thefact that demonstrated amorphouscoresinzebrafish Matsuoka andIwai, 1984).However, electronmicroscopy clearly staining appearstonotbeusefulinfish (Johnstonetal.,1975; 1986; Swash andSchwartz, 1981).Unfortunately, NADH-TR NADH-TR (tetrazoliumreductase)(Engel,1967;Martinetal., associated withlabelingforreductaseactivity inmitochondria with 2780 Buss, R.andDrapeau, P. Block, B.A.,Imagawa,T., Campbell,K.P. andFranzini-Armstrong, C. Bassett, D.I.andCurrie,P. D. Bahary, N.,Davidson,A.,Ransom,D.,Shepard,H., Trede, J.,Stern, N.,Zhou, Biol. Chem. junctional footprotein isoforms inmature avianfasttwitchskeletalmuscle. M. H.andSutko,J.L. Biol. the transversetubule/sarcoplasmic reticulum junctioninskeletalmuscle. Structural evidencefordirect interactionbetweenthemolecularcomponents of dystrophy andcongenitalmyopathy. cloning inzebrafish. Y., Barut,B.and Zon,L.I. We succeededintreatingmuscleweakness 107 RESEARCH ARTICLE , 2587-2600. ryr ryr1b 265 ryr mutants die7-15dpf,but theirfast development and , 14187-14194. mutants, thiscaseofMmDmightbetreatedby and restorednormalswimming.Germanetoour Methods CellBiol. (1990). Identificationandlocalizationoftwotriad (2000). Physiological properties ofzebrafish (2004). TheZonlaboratoryguidetopositional 20) Thezebrafishasamodelformuscular (2003). Hum. Mol.Genet. 77 , 305-329. ryr 12 mutant musclesand ryr ryr R265-R270. , mutants. Inthis mutants bythe J. Cell (1988). J. 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