Research Article 565

IntroductionMuscle cells possess an elaborate cytoskeletal structure, which isessential for the coordination of contraction and the generation offorce. The basic machinery of contraction is organised into bundlesof long myofibrils, which are each made of stacks of sarcomeres.Sarcomeres consist of thin filaments, attached to Z discs, and thickfilaments, attached to M lines. Filamentous actin associates withtropomyosin, troponin and other proteins to form thin filaments,which intercalate with thick filaments that are predominantly madeof myosin. The contraction of striated muscle is regulated by thetroponin protein complex, which mediates the Ca2+-dependentinteraction of actin and myosin within the sarcomere. The troponincomplex possesses three subunits: troponin C, troponin I andtroponin T. Troponin T anchors the complex to the thin filamentby binding to tropomyosin. Following stimulation, calcium ionsreleased from the sarcoplasmic reticulum bind to troponin C andthis leads to conformational changes of the complex. Troponin Iconsequently relieves its inhibition, which allows the binding ofmyosin to actin. Simultaneously, troponin T and tropomyosin alterconformation, allowing myosin-binding sites on actin to be exposed(Alberts et al., 2007; Galinska-Rakoczy et al., 2008; Phillips et al.,1986; Zot and Potter, 1987).

In humans, loss of function of the slow-twitch-muscle-specifictroponin T isoform TNNT1 causes a recessive form of Nemalinemyopathy (OMIM 605355), a genetically heterogeneous diseaseresulting from mutations in thin filament genes and characterisedby muscle weakness and rod-like inclusions in muscle fibres(Johnston et al., 2000). Dominant mutations in the fast-twitchskeletal troponin T isoform TNNT3 or the troponin I isoformTNNI2 are responsible for a disorder called distal arthrogryposistype 2B (OMIM 601680) (Sung et al., 2003), characterised by

clenched fist, overlapping fingers, ulnar deviation and positionalfoot deformities. Mutations affecting the three cardiac subunits ofthe complex (TNNC1, TNNT2 and TNNI3) are also associated witha variety of cardiomyopathies (Kimura et al., 1997; Landstrom etal., 2008; Mogensen et al., 2003; Mogensen et al., 2004; Murphyet al., 2004; Peddy et al., 2006; Thierfelder et al., 1994).

In addition to the known role in the regulation of contraction,there is some evidence that troponin is required for formation andmaintenance of sarcomeres. In zebrafish, mutations of the silentheart locus, which encodes cardiac troponin T2, lead to sarcomereloss and myocyte disarray, attributed to coordinate reduction ofthin filament gene expression (Sehnert et al., 2002). A similardisruption of sarcomere assembly was observed in Tnnt2–/– mice,which die during embryogenesis due to failure of cardiaccontraction (Ahmad et al., 2008; Nishii et al., 2008). Body wallmuscle contraction and sarcomere organisation are disrupted inCaenorhabditis elegans mutants defective for genes encodingtroponin T or troponin I (Burkeen et al., 2004; Myers et al., 1996).In Drosophila melanogaster, severe mutant alleles of troponin Tlead to a failure of sarcomere assembly, whereas hypomorphictroponin mutant alleles initially develop normal muscle and thenundergo muscle damage (Fyrberg et al., 1990).

We have used zebrafish to investigate the role of thin filamentproteins in the process of sarcomere assembly. Zebrafishdevelopment is rapid, with muscle fully differentiated by 48 hourspost-fertilisation (hpf) (Kimmel et al., 1995). Zebrafish muscle hasa similar composition to human muscle, in comparison withinvertebrates (Bassett et al., 2003). Moreover, the optical propertiesof the embryo make it highly amenable to light microscopy. Likein mouse and human, the troponin genes in zebrafish exist inseveral isoforms, differentially expressed in cardiac, slow-twitch

SummaryIn striated muscle, the basic contractile unit is the sarcomere, which comprises myosin-rich thick filaments intercalated with thinfilaments made of actin, tropomyosin and troponin. Troponin is required to regulate Ca2+-dependent contraction, and mutant forms oftroponins are associated with muscle diseases. We have disrupted several genes simultaneously in zebrafish embryos and have followedthe progression of muscle degeneration in the absence of troponin. Complete loss of troponin T activity leads to loss of sarcomerestructure, in part owing to the destructive nature of deregulated actin–myosin activity. When troponin T and myosin activity aresimultaneously disrupted, immature sarcomeres are rescued. However, tropomyosin fails to localise to sarcomeres, and intercalatingthin filaments are missing from electron microscopic cross-sections, indicating that loss of troponin T affects thin filament composition.If troponin activity is only partially disrupted, myofibrils are formed but eventually disintegrate owing to deregulated actin–myosinactivity. We conclude that the troponin complex has at least two distinct activities: regulation of actin–myosin activity and,independently, a role in the proper assembly of thin filaments. Our results also indicate that sarcomere assembly can occur in theabsence of normal thin filaments.

Key words: Muscle, Sarcomere, Troponin, Zebrafish

Accepted 6 October 2010Journal of Cell Science 124, 565-577 © 2011. Published by The Company of Biologists Ltddoi:10.1242/jcs.071274

Troponin T is essential for sarcomere assembly inzebrafish skeletal muscleMaria I. Ferrante*, Rebecka M. Kiff, David A. Goulding and Derek L. Stemple‡

Wellcome Trust Sanger Institute, Hinxton, Cambridge, CB10 1SA, UK*Present Address: Stazione Zoologica Anton Dohrn, Villa Comunale, 80121 Naples, Italy‡Author for correspondence (ds4@sanger.ac.uk)

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or fast-twitch skeletal muscle (Fu et al., 2009; Hsiao et al., 2003).Additionally, troponin genes have been duplicated in the zebrafishgenome relative to mammalian genomes. The opportunity tosimultaneously knockdown expression of different genes usingcombinations of antisense morpholino oligonucleotides (MOs) inzebrafish allows us to overcome this genetic redundancy(Nasevicius and Ekker, 2000).

We disrupted expression of different members of the troponin Tfamily. In fast-twitch muscle, redundant expression of threedifferent troponin T genes (tnnt2c, tnnt3a and tnnt3b) guaranteesnormal assembly in single loss-of-function experiments, but theconcomitant knockdown of all three genes leads to a block innormal myofibrillogenesis. We demonstrate that it is the loss ofregulation of the actin–myosin activity that leads to failure ofsarcomere assembly. When we block myosin function with themyosin inhibitor blebbistatin, striations are recovered but anadditional effect of the loss of troponin T function on thin filamentassembly is unmasked. Finally, we have identified a second classof phenotype that results when only single troponin genes aredisrupted. Depletion of tnnt3a or tnnt3b by MOs, or loss of tnni4a.2through mutation, allows assembly but leads to sarcomeredisintegration, which is completely suppressed by inhibition ofmyosin activity.

ResultsDepletion of troponin T activity in fast-twitch muscledisrupts sarcomere assemblyIn the zebrafish trunk skeletal musculature, slow-twitch fibres andfast-twitch fibres are separated. Mononucleate slow-twitch fibresare superficially located, immediately under the skin, and areparallel to the anteroposterior axis, whereas multinucleated fast-twitch fibres are located deeper in the trunk, are more abundantand have an oblique orientation (Bassett et al., 2003; Devoto et al.,1996). Three troponin T genes, tnnt2c, tnnt3a and tnnt3b, areexpressed in fast-twitch muscle during zebrafish development(Table 1; supplementary material Fig. S1). The tnnt2c gene isstrongly expressed in myotomes from the beginning of

somitogenesis and is downregulated from 36 hpf, whereas thetnnt3a and the tnnt3b genes are expressed from 18 hpf and 24 hpf,respectively (Hsiao et al., 2003; Thisse and Thisse, 2005). Weknocked down expression of all three genes simultaneously withtranslation-blocking MOs. Embryos injected with triple tnnt MOswere shorter than embryos injected with a single MO and wereoccasionally bent, with abnormally shaped somites and pericardialoedema but with beating hearts, which indicated that the cardiacmuscle in these embryos was functional. At 28 hpf, embryos wereimmotile, and myofibrils in fast-twitch muscle depleted of tnnt2c,tnnt3a and tnnt3b were markedly disorganised and very rarelypossessed any striations (Fig. 1A,B), indicating that functionaltroponin T is required for sarcomere assembly in fast-twitch muscle.When analysed at 48 hpf, fast-twitch muscle in the triple MO-injected embryos showed a reduced level of filamentous actin,which was distributed in small foci. This was coupled with a lossof striation, as indicated by the anti-titin antibody T12, whichrecognises an epitope in proximity of the Z line (Fig. 1C,D).Phalloidin staining for actin, coupled with antibody staining for -actinin, showed colocalisation of the two proteins in foci, althoughactin also appeared in aggregates without -actinin (Fig. 2C;supplementary material Fig. S2C). Interestingly, other markersindicated that, although myofibrils were substantially disorganised,not all sarcomeric components were completely delocalised. Weused a second anti-titin antibody T11, which recognises an epitopeat the boundary between the I-band and A-band (Trombitas andPollack, 1993), and detected some regions with short stretches ofperiodic T11 staining in the embryos injected with triple tnnt MOs(Fig. 1E,F). Similarly, an antibody directed against myomesin, amajor component of M lines, showed that M lines also partiallymaintained regular spaced distribution in some regions (Fig. 1G,H).Tropomyosin failed to appear in striations but showed normallevels of expression in troponin-T-depleted muscles (Fig. 1I,J).Because the tropomyosin antibody that we had used in singlestaining did not work well in combination with phalloidin, wecloned tropomyosin 3 in frame with the red fluorescent proteinmCherry and injected the mRNA encoding for this fusion protein

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Table 1. Troponin genes described in this studyOriginal zebrafish

gene nameNew gene

name Zebrafish ensembl ID Expression patternHuman

orthologueHumanprotein Associated disease

zgc:193865 tnnt1 ENSDARG00000037954 Expressed in embryos, tissuedistribution stilluncharacterised

TNNT1 Troponin T1 Nemalinemyopathy

tnnt2 tnnt2a ENSDARG00000020610 Cardiac TNNT2 Troponin T2 CardiomyopathiesLOC562296 tnnt2b ENSDARG00000031920 Unknown TNNT2tnnt1 tnnt2c ENSDARG00000032242 Somites early, slow and fast

muscles laterTNNT2

zgc:114184 tnnt2d ENSDARG00000002988 Slow muscle from 24 hpf TNNT2si:ch211-136g2.1 tnnt2e ENSDARG00000045822 Expressed in embryos, tissue

distribution stilluncharacterised

TNNT2

tnnt3a tnnt3a ENSDARG00000030270 Fast muscle, head muscles,pectoral fin muscles,hypaxial muscles

TNNT3 Troponin T3 Distalarthrogryposistype 2B

tnnt3b tnnt3b ENSDARG00000068457 Fast muscles, pectoral fin,hypaxial muscles, headmuscles

TNNT3

tnni2a.4 tnni2a.4 ENSDARG00000029069 Musculature TNNI2 Troponin I2 Distalarthrogryposistype 2B

Listed are the names currently used to designate genes encoding zebrafish troponin T and troponin I and the new gene names that we propose on the basis of the orthology analysis. For each gene we also list the corresponding ensembl ID, the profile of expression, the human homologue gene, the human protein product and the associated human disease.

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in wild-type and triple tnnt MO-injected embryos. We then fixedthe embryos and stained them with phalloidin. Our results showthat tropomyosin and actin colocalised in the controls but did notoverlap in the triple tnnt MO-injected embryos (supplementarymaterial Fig. S3). Transmission electron microscope (TEM) analysisconfirmed these results, showing that a large part of troponin-T-depleted muscle was filled with dispersed thick filaments (Fig.1M). We also observed areas with disorganised and expanded Zlines (Fig. 1N), areas with thin-filament-free sarcomeres with no Z

lines (Fig. 1O), and areas with accumulations of material, which,on the basis of light microscope analysis, we believe to be actin(Fig. 1P).

To prove that the observed phenotype was specific, we clonedone of the three troponins, tnnt2c, in frame with mCherry. A DNAconstruct encoding the fusion protein downstream of thecytomegalovirus (CMV) promoter was injected into one-cell wild-type embryos. The exogenous DNA was expressed mosaically ina small subset of cells. At 28 and 48 hpf, isolated muscle cells

567Sarcomere assembly requires troponin

Fig. 1. Disruption of sarcomere assembly of fast-twitch muscle of embryos simultaneously depleted of tnnt2c, tnnt3a and tnnt3b. (A,B)Phalloidin stainingshows forming and mature striated myofibrils in control embryos at 28 hpf (A) but completely disrupted myofibrils in triple tnnt MO-injected embryos at the samestage (B). MJ, myotendinous junctions; HM, horizontal myoseptum. (C,D)At 48 hpf, the titin epitope T12 (C�,D�; green) localises to normal Z lines, and actin(C�,D�; red) appears in striations in control embryos (C), whereas the two proteins are randomly dispersed in the cytoplasm in triple tnnt MO-injected embryos (D).DAPI stains the nuclei (C�,D�; blue). Single channels are shown in grey. (E–H) The titin epitope T11 localises to the I-band–A-band boundaries and myomesin tothe M lines in control embryos (E,G). They are still seen in discrete structures, although these are scattered around in the cytoplasm, in triple tnnt MO-injectedembryos (F,H). (I,J)Tropomyosin localisation to thin filaments observed in the control (I) is lost in the triple tnnt MO-injected embryos (J). Scale bars: 25m,insets are 2� greater magnifications of corresponding panels. (A,B)Lateral views of rostral somites of 28 hpf embryos. (C–J) Lateral views of a single myotome.Confocal images were obtained from planes deep in the trunk musculature. (K–P) TEM images of fast-twitch muscles in 48 hpf control (K,L) and in triple tnntMO-injected embryos (M–P). White arrowheads indicate Z lines and black arrows indicate M lines. In M, clusters of thick filaments can be observed.Accumulations of Z line proteins can be seen in N; in O, thin filament-free sarcomeres can be observed. The white asterisk marks the area where the Z line shouldhave been. Accumulations of actin can be observed in P (black asterisk). Scale bars: 500 nm.

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producing the red fluorescent troponin T could be observed. Inthese cells, the protein localised correctly to thin filaments(supplementary material Fig. S4D–F). When this DNA constructwas co-injected with the triple tnnt MOs, those cells producing thefusion protein recovered striations, whereas surrounding musclecells not expressing the troponin-T–mCherry fusion protein showeddisorganised distribution of actin and lacked striations(supplementary material Fig. S4G–I).

To determine the extent of troponin T downregulation, we usedtwo different anti-troponin T antibodies (Ct3 and JLT-12) in whole-mount immunofluorescence assays. Both antibodies recognisedtroponin T on thin filaments in control embryos (supplementarymaterial Fig. S5A,C and not shown) but did not react in triple tnntMO-injected embryos (supplementary material Fig. S5B,F and notshown), indicating that troponin T expression was abolished by thetnnt MOs.

Because expression of thin filament genes has been reported tobe reduced in troponin-T-depleted cardiac muscle (Sehnert et al.,2002), we stained triple tnnt MO-injected embryos for expressionof tropomyosin 3, troponin C, troponin I2 and troponin T3b by insitu hybridisation. We did not detect any change in the level orpattern of expression of the first two, but found tnni2 to be slightlyupregulated and tnnt3b, one of the genes targeted with the MO, tobe downregulated (data not shown).

Inhibition of myosin function suppresses loss of striatedorganisation but does not restore proper thin filamentassemblyTo assess whether the effect of troponin T depletion on myofibrilswas due to deregulation of actin–myosin activity, we analysed

the phenotype of embryos injected with tnnt2c, tnnt3a and tnnt3bMOs while simultaneously inhibiting myosin function. We usedblebbistatin, a myosin inhibitor (Straight et al., 2003) known toinhibit contraction in skeletal muscle and cardiomyocytes(Skwarek-Maruszewska et al., 2009). We added blebbistatin tocontrol MO-injected and triple tnnt MO-injected embryos at 26hpf, and analysed embryos at 48 hpf. At this stage, both controlMO- and triple tnnt MO-injected embryos were immotile anddisplayed heart oedema. Myofibrils in control MO-injectedembryos treated with blebbistatin had become less compact,although sarcomere markers had not lost their striated appearance(Fig. 2B,F,J,N). We observed a recovery in myofibril structure inblebbistatin-treated triple tnnt MO-injected embryos, withimmature sarcomeres that were, however, regularly aligned alongmyofibrils that had recovered periodicity of Z lines, thickfilaments and M lines (Fig. 2D,H,L; supplementary material Fig.S2) but that showed no recovery of tropomyosin localisation tosarcomeres (Fig. 2P).

In electron micrographs, we observed misaligned myofibrilsand sarcomeres with less-defined Z and M lines in blebbistatin-treated control embryos (Fig. 3B). The loss of sarcomeric integrityobserved in DMSO-treated triple tnnt MO-injected embryos (Fig.3C) was partially restored in blebbistatin-treated triple tnnt MO-injected embryos, where regularly spaced, enlarged Z lines withintervening thick filaments could be observed (Fig. 3D). In cross-sections, control muscles displayed regular, hexagonal arrangementof thick filaments surrounded by thin filaments (Fig. 3E), whereasthe triple tnnt MO-injected embryos showed bundles of thickfilaments with no intercalating thin filament, regardless of theblebbistatin treatment (Fig. 3F,G).

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Fig. 2. Recovery of striated myofibrils intriple tnnt-depleted embryos uponinhibition of myosin activity. (A–L)Phalloidin (red in A–D), -actinin (greenin A–D), fast myosin (white in E–H) andmyomesin (white in I–L) staining ofembryos incubated with DMSO revealsnormal myofibrils in a control embryo(A,E,I) and the expected disruption indistribution of actin, Z lines, thickfilaments and M lines in triple tnnt MO-injected embryos (C,G,K). When embryosare treated with blebbistatin, less compactmyofibrils are observed in controls (B,F,J)and striated myofibrils can be seen in tripletnnt MO-injected embryos (D,H,L).(M–P) Tropomyosin does not recover itslocalisation to thin filaments in tnnt MO-injected embryos treated with blebbistatin(P). DAPI (blue) stains the nuclei. Scalebars: 25m, insets are 2� magnificationsof corresponding panels.

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To examine the role of unregulated actin–myosin activity, weindependently disrupted myosin assembly using sloth mutants. Insloth mutants, the chaperone protein Hsp90a.1 is defective,preventing efficient myosin folding and thick filament assembly(Granato et al., 1996; Hawkins et al., 2008). We injected the tripletnnt MOs into slou45 embryos. Wild-type sibling embryos injectedwith triple tnnt MOs displayed the expected phenotype withdisrupted sarcomeres (supplementary material Fig. S6C). Bycontrast, slou45 mutant embryos had wavy myofibrils with I–Z–Ibrushes (supplementary material Fig. S6B). Injection of triple tnntMOs in slou45 embryos did not lead to the formation of actinaggregates, and myofibrils recovered periodic distribution of actinand -actinin (supplementary material Fig. S6D), confirming thatrudimentary sarcomeres can form in the absence of myosin activity.

569Sarcomere assembly requires troponin

Fig. 3. Blebbistatin treatment does not rescue the loss of normal thinfilaments in troponin-T-depleted sarcomeres. (A–G) Electron microscopyimages of 48 hpf embryos, in tangential (A–D) and cross-sections (E–G).Control embryos (A,E) show normal sarcomeres with well-defined Z lines(white arrowheads) and M lines (black arrows) and regular hexagonalarrangement of thick filaments with intercalating thin filaments (circle in E).In blebbistatin-treated control embryos (B), Z lines are less sharp and M linesare not clearly defined. In tnnt-depleted muscles (C,F), sarcomeric componentsare disorganised (C) and bundles of thick filaments in cross-sections do notappear to be intercalating with thin filaments (F). Upon blebbistatin treatment,wider and regularly spaced Z lines can be observed in triple tnnt MO-injectedembryos (D) and virtually no thin filaments are visible inside thick filamentsbundles (circle in G). Scale bars: 1m (A–D); 150 nm (E–G).

Fig. 4. Myofibrils degrade in fast-twitch muscles of tnnt3-depletedembryos. (A–C) 3 dpf control (A), tnnt3a MO-injected (B) and tnnt3b MO-injected (C) embryos. (D–F) When trunks of the same embryos are observedunder polarised light, embryos injected with tnnt3a MO (E) and tnnt3b MO(F) show total and partial loss of birefringence, respectively. (G–I) Lateralviews of rostral somites in 3 dpf embryos. Confocal images from a plane deepin the trunk musculature show that actin is highly organised in fast muscle ofcontrol embryos (G), but appears completely disorganised in tnnt3a MO-injected embryos (H) and partially disorganised in tnnt3b MO-injectedembryos (I; the inset shows a magnification of the myotendinous junctionwhere actin localised in vertical Z lines or in horizontal stretched, non-striatedfilaments gives rise to a characteristic chequered appearance). Phalloidin iswhite and nuclei are counterstained with DAPI (blue). Scale bars: 50m;10m in insets. (J–L) TEM images of fast-twitch muscles in 40 hpf control(J), tnnt3a MO- (K) and tnnt3b MO-injected embryos (L) reveal similar loss ofsarcomeric integrity in the MO-injected embryos, with stacks of thickfilaments and accumulations of Z line proteins (white arrowheads). Scale bars:1m. (M,N) Forming fast-twitch fibres appear striated in both in control (M)and tnnt3a MO-injected (N) embryos at 28 hpf. Fast myosin is white andnuclei are blue. Scale bars: 20m. (O,P) At 48 hpf, M lines stained bymyomesin (green) are regularly intercalated with thin filaments (phalloidin,red) in control embryos (O), whereas they cluster in groups and are excludedfrom actin-rich areas in tnnt3a MO-injected embryos (P, inset shows clusters).Scale bars: 15m; 10m in inset. M–P are lateral views of a single myotome,nuclei are stained with DAPI (blue).

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In these embryos, however, tropomyosin did not recover regulardistribution and appeared to be less abundant and unlocalisedthroughout the cytoplasm (supplementary material Fig. S6H).

Partial loss of troponin activity in fast-twitch muscle leadsto degradation of myofibrilsSingle injection of tnnt3a or tnnt3b MOs impaired motility ofembryos at 3 days post-fertilisation (dpf) and did not cause othermajor disruptions of embryonic development (Fig. 4A–C). Striatedmuscles possess a property known as form birefringence, whicharises as a result of the parallel packing of long fibres, giving themthe ability to polarise light. Larvae at 3 dpf showed complete(tnnt3a) or partial (tnnt3b) loss of muscle birefringence (Fig. 4D–F), indicating loss of myofibrillar organisation. Phalloidin stainingshowed a distinctive distribution of actin in round aggregates intnnt3a MO-injected embryos (Fig. 4H). In tnnt3b MO-injectedembryos, we found partial disruption, with striations preserved inthe central part of myotubes but lost at the myotendinous junctions(Fig. 4I), indicating that at this stage tnnt3a can partially compensatefor tnnt3b and that myotube ends are the first areas to be affectedby a diminished availability of troponin T. At 5 dpf, however,myofibril disintegration progressed in tnnt3b MO-injected embryosand diffuse aggregates of actin could also be observed in the fast-twitch muscle of these embryos (supplementary material Fig. S7).

At the ultrastructural level, we observed sarcomeres with severaldefects, including wider and wavier Z lines, and areas withdisintegrated sarcomeres, dispersed thick filaments andaccumulations of Z line proteins (Fig. 4J–L).

In fast-twitch muscle, striations first become visible at 28 hpf.Phalloidin and myosin staining at this stage showed that sarcomeresinitially form normally in tnnt3 MO-injected embryos (Fig. 4N andnot shown) and that double knockdown of tnnt3a and tnnt3b hadthe same phenotype as single tnnt3a disruption (data not shown).In tnnt3a MO-injected embryos, sarcomeric proteins later becameabnormally localised and separated into distinct cellular domains.For example, in muscle co-stained for actin and myomesin, groupsof discrete and evenly spaced M lines could be seen distinctlyseparated from the actin aggregates (Fig. 4P).

We obtained independent confirmation of the effect of partialdisruption of troponin function by analysing the knockout of atroponin I isoform in the same complex. A nonsense allele oftnni2a.4, a fast-twitch troponin I gene, was isolated in collaborationwith the Zebrafish Mutation Resource at the Sanger Institute(http://www.sanger.ac.uk/cgi-bin/Projects/D_rerio/mutres/mutation.pl?project_id966&mutation_id170). The mutation is predictedto truncate the 176 amino acid protein after the 66th amino acid(Q67X) (Fig. 5B), eliminating more than half of the protein,including the C-terminus, which is thought to interact with actin

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Fig. 5. Progressive loss of fast-twitch fibre myofibrillar integrity in tnni2sa0058 mutant embryos. (A,B)Sequence traces from a wild-type fish (A) and atnni2a.4sa0058 carrier fish (B) show the nucleotide change (asterisk) that converts C to T, generating a stop codon. (C–F) In a 5-day-old wild-type sibling observedunder polarised light (C,E, at two different light intensities) trunk muscles are birefringent, whereas in a mutant embryo they are not (D,F). (G,H)Lateral views ofsingle myotomes. Confocal images were obtained from planes deep in the trunk musculature. At 48 hpf, tnni2a.4 mutant embryos show myofibrils that are losingtheir integrity, as highlighted by phalloidin staining for thin filaments (G�,H�; red) and -actinin staining for Z lines (G�,H�; green). DAPI stains the nuclei (G�,H�;blue). Single channels are shown in grey; overlap of signals shows in yellow in the merged panels (G,H). Scale bars: 10m. (I,J)At 3 dpf, fast myosin (white) iscompletely delocalised in tnni2a.4 mutants (J) whereas wild-type siblings show normal fast muscle (I). Nuclei are stained in blue. Scale bars: 20m. Insets are 2�magnifications of corresponding panels.

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monomers and tropomyosin on the thin filament (Galinska-Rakoczyet al., 2008) to inhibit actin–myosin interactions.

Starting from 3 dpf, tnni2a.4sa0058 mutants were unable to swimproperly and at 5 dpf failed to inflate the swim bladder. At thisstage, trunk muscles showed no birefringence (Fig. 5C–F). As withtnnt3a MO-injected embryos, fast-twitch myofibrils showed a lossof striation and diffuse actin and -actinin staining by 48 hpf (Fig.5G,H). Myosin staining at 3 dpf also revealed severe disruption ofthick filaments (Fig. 5I,J). When analysed at 28 hpf, mutants couldnot be distinguished from wild-type siblings, indicating thatmyofibrils had formed normally and therefore that the fast-twitchmuscle disintegration was progressive.

The partial loss of troponin phenotype is suppressed byloss of myosin activityWe tested whether the phenotype is due to unregulated actin–myosin activity. In tnnt3 MO-injected embryos, myofibrildegradation proceeds through a first stage in which actin isdistributed in sharp vertical bands and stretched horizontal fibresin enlarged areas of the myotubes, to a second stage in which actinbecomes localised to round aggregates (Fig. 4H,I; supplementarymaterial Fig. S7B), again suggesting that deregulated actin–myosinactivity might have a role in the genesis of this phenotype.

We added blebbistatin to control MO- and tnnt3a MO-injectedembryos at 26 hpf and analysed embryos at 48 hpf. Untreatedtnnt3a MO-injected embryos showed the expected disrupted actinlocalisation with no striations (Fig. 6C), whereas blebbistatin-treated embryos had wavy but clearly striated myofibrils (Fig. 6D).This indicated that, in the absence of actin–myosin contraction, thelack of troponin T function does not lead to sarcomeredisintegration. Because the tnni2a.4 mutant phenotype is the sameas the tnnt3a MO phenotype, we used blebbistatin to verify whetherinhibition of actin–myosin contraction could suppress the disruptionof sarcomeres in the tnni2a.4 mutants. We found that four out of20 control DMSO-treated offspring from heterozygous parentsdisplayed the expected disrupted actin phenotype at 48 hpf, whereasnone of the 20 observed blebbistatin-treated embryos from thesame clutch displayed obvious actin accumulations.

We confirmed these results independently using sloth mutants.We injected the tnnt3a MO into slou45 embryos. Wild-type siblingembryos injected with tnnt3a MO displayed the expected phenotypewith degrading myofibrils (Fig. 6F), whereas slou45 mutant embryoshad wavy myofibrils with I–Z–I brushes (Fig. 6G). Injection oftnnt3a MO in slou45 embryos did not lead to the formation of actinaggregates, and myofibrils were indistinguishable from uninjectedslou45 embryos of the same stage (Fig. 6H), confirming that normalinteractions between myosin and actin are required for the Tnnt3loss of function to affect sarcomere maintenance.

Disruption of tnnt2c abolishes sarcomere assembly inslow-twitch muscleThe tnnt2c gene is expressed in fast-twitch but also in slow-twitchmuscle (http://zfin.org/cgi-bin/webdriver?MIvalaa-markerview.apg&OIDZDB-GENE-030520-1). Embryos injected with tnnt2cMO alone developed normally and did not show any morphologicalabnormalities, but they were completely immotile at 24 hpf andlacked the normal twitching movements typical of this stage. Wefirst observed spontaneous twitching at around 28 hpf, when fast-twitch muscle becomes functional. By 48 hpf, we found that embryoshad recovered their ability to move, hatch and swim normally. Slow-twitch myofibrils of tnnt2c MO-injected embryos at 24 hpf were

thin, the filamentous actin was completely dispersed or distributedin regularly spaced foci but not in properly formed sarcomeres (Fig.7A,B), and myosin was found in blocks but also dispersed in thecytoplasm or in thin, loose myofibrils (Fig. 7C,D). Electronmicroscopic analysis of slow-twitch muscle ultrastructure confirmedthe absence of normal sarcomeres at 24 hpf in tnnt2c MO-injectedembryos and showed accumulation of Z line proteins and dispersedthick filaments (Fig. 7F, arrowheads and arrow, respectively). At 30

571Sarcomere assembly requires troponin

Fig. 6. Suppression of the tnnt3a progressive myofibrillar disorganisationphenotype in the absence of functional thick filaments. (A–D) Incubationwith the myosin inhibitor blebbistatin: phalloidin staining of embryosincubated with DMSO reveals normal myofibrils in a control embryo (A) andthe expected disruption in actin distribution in a tnnt3a MO-injected embryo(C). When embryos are treated with blebbistatin, loose myofibrils are observedin a control embryo (B) and wavy but striated myofibrils can be seen in atnnt3a MO-injected embryo (D). Scale bars: 25m, insets are 2�magnifications of corresponding panels. (E–H) Analysis in the sloth mutant: a48 hpf wild-type sibling displays normally striated fast fibres (E). In a wild-type sibling injected with the tnnt3a MO (F), myofibrils undergo degradationand actin becomes diffuse. In a sloth mutant embryo (G), actin is distributed inthick filament-free sarcomere-like blocks along myofibrils. In a sloth mutantinjected with tnnt3a MO (H), actin remains in blocks rather than dispersed inthe cytoplasm. Scale bars: 10m, insets are 2� magnifications ofcorresponding panels. All images are lateral views of a myotome. Confocalimages were obtained from planes deep in the trunk musculature. Phalloidinstaining is white and DAPI for nuclei is blue.

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hpf, actin staining in tnnt2c MO-injected embryos showed sarcomeresthat had grown laterally and were decorated with titin T12 (Fig.7G,H), although myofibrils were still wavy. Fast-twitch muscle atthis stage was normal. At 5 dpf, phalloidin staining showed a normaldistribution of filamentous actin in the slow-twitch muscle of tnnt2cMO-injected embryos (Fig. 7J).

We found that the recovery of striations in myofibrils of tnnt2cMO-injected embryos after 30 hpf (supplementary materialFig. S8) is due to the expression of another slow-twitch-muscle-specific troponin T encoded by a second, slow-twitch-muscle-specific troponin T gene, tnnt2d (http://zfin.org/cgi-bin/webdriver?MIvalaa-markerview.apg&OIDZDB-GENE-050626-97) (for nomenclature see Materials and Methods, Table 1 andsupplementary material Fig. S1). We designed a translation-blockingMO for tnnt2d and found that disruption of this gene on its own did

not lead to any gross morphological abnormalities or developmentaldefect (data not shown). Immunostaining for myosin, and phalloidinstaining for actin at 48 hpf in tnnt2c MO-injected embryos showedthat slow-twitch muscle had almost completely recovered striationsalong their length, although myofibrils were thinner than in controls(Fig. 8B,B�,F). Depletion of tnnt2d led to mild alterations of musclecell morphology, with some small myofibrils detaching from themain bundles (Fig. 8C,C�,G; also observed at 5 dpf, Fig. 8J). Whentnnt2c and tnnt2d were simultaneously disrupted, we found thatslow-twitch muscle did not recover at 48 hpf (compare Fig. 8D,D�,Hwith 8B,B�,F), demonstrating that from 30 hpf tnnt2d is able toprovide enough troponin T to the slow-twitch muscle to compensatefor tnnt2c loss of function.

We examined the earlier steps of sarcomere formation in slow-twitch muscle at 14–16 somite stages. In control embryos, a few

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Fig. 7. Sarcomere assembly is delayed in slow-twitch muscle depleted of tnnt2c. (A–D) Mature sarcomeres can be seen in 24 hpf control embryos (A,C), butnot in stage matched tnnt2c MO-injected embryos (B,D) stained for filamentous actin (A,B) and slow myosin (C,D). Scale bars: 25m, insets are 2�magnifications of corresponding panels. (E,F)Dysmorphology at the ultrastructural level is revealed by TEM images of slow muscle in 24 hpf control (E) andtnnt2c MO-injected embryos (F). Accumulations of Z line proteins (black arrowheads) and dispersed thick filaments (black arrow) can be found in the MO-injectedembryos (F). Scale bars: 500 nm. (G,H)30 hpf control embryos stained with an anti-titin antibody (G�,H�; green, T12), and phalloidin (G�,H�; red), show normalsarcomeres (G), whereas tnnt2c MO-injected embryos do not (H). Nuclei are counterstained with DAPI (G�,H�; blue). Single channels are shown in grey. Theasterisk in H marks clustering nuclei in a lateral line rosette. Scale bars: 30m, insets are 2� magnifications of corresponding panels. (I,J)Slow-twitch muscleappeared normal at 5 dpf in control (I) and tnnt2c MO-injected (J) embryos. Scale bars: 50m.

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myofibrils could be detected in the anterior somites, whereas incaudal somites rare striations were just becoming detectable(supplementary material Fig. S9A,C). In tnnt2c MO-injectedembryos we never observed similar structures, myosin appeared toaggregate in rods or foci, actin appeared more diffuse, and overlapof the signal was less frequent (supplementary material Fig. S9B,D),indicating that knockdown of tnnt2c impairs sarcomere assemblyfrom the start.

DiscussionIn this study, we have demonstrated that troponin is required fornormal myofibrillogenesis. First, it is required to regulate actin–myosin interactions, and second it affects proper thin filamentassembly. We also show that in the absence of normal thin filaments,assembly of rudimentary sarcomeres can occur.

Sarcomeres do not form in the absence of functionaltroponinsDespite the observation that sarcomeric substructures can formand align in the absence of main sarcomere components, such asthick filaments (Hawkins et al., 2008; Wohlgemuth et al., 2007),we did not find normal sarcomeres in muscles of embryos depletedof troponin T, instead aggregates of Z line proteins and dispersedthick filaments were observed from the very beginning of muscledifferentiation. In silent heart mutants, defective for the cardiacisoform troponin T2a, sarcomere assembly is impaired anddispersed thick filaments were reported (Sehnert et al., 2002). Inthat study, it was proposed that a common regulation of thinfilament genes would be altered in the absence of troponin T and

that lack of assembly is due to downregulation of thin filamentgenes and the consequent dearth of thin filament proteins (Huanget al., 2009; Sehnert et al., 2002). Similar observations, however,have not been reported for mouse troponin mutants (Nishii et al.,2008). In D. melanogaster, downregulation of troponin I but notof other major thin filaments genes has been reported in flightmuscles of a troponin T mutant (Nongthomba et al., 2007).

The consequences of tnnt2a disruption in the zebrafish hearthave been further investigated with confocal microscopy (Huanget al., 2009). These authors reported loss of periodicity for thin andthick filaments and for Z and M lines, similar to that observed inskeletal muscle. They concluded that tnnt2a is required for thestriations of thin filaments, which in turn affects the periodicity ofZ bodies, M lines and thick filaments.

We show that the failure of assembly is principally due to thederegulated actin–myosin activity rather than to changes in theabundance of thin filament components or to lack of thin filamentstriations, because the regular distribution of major sarcomerecomponents, lost in embryos depleted of the three troponin Tproteins, is restored by blocking myosin activity.

In the absence of troponin, actin and myosin are not foundtogether because their unregulated interaction is destructive. Instead,actin accumulates in aggregates and in abnormal Z lines, andmyosin remains as dispersed thick filaments or appears in stacksof thick filaments in which myomesin can localise. Altered actindynamics in this context probably lead to abnormal accumulationof Z line proteins in substantially enlarged Z lines (Fig. 9B).

Additionally, our data indicate that the loss of a functionaltroponin complex independently affects proper thin filament

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Fig. 8. tnnt genes are required for sarcomere assembly in slow-twitch muscle. (A–D) Slow myosin staining (white) in 48 hpf control embryos (A,A�) and inembryos injected with tnnt2c MO (B,B�), tnnt2d MO (C,C�) or co-injected with tnnt2d and tnnt2c MOs (D,D�) shows that myofibrillar organisation is lost in thedouble-injected embryos. Nuclei are counterstained with DAPI (blue). A–D are lateral views of rostral somites and A�–D� are higher magnification images showingmuscle cells. In both cases, confocal images are from the most superficial layer of trunk muscles. Scale bars: 25m (A–D); 10m (A�–D�). (E–H) Phalloidinstaining (white) in 48 hpf control embryos (E) and in embryos injected with tnnt2c MO (F), tnnt2d MO (G) or co-injected with tnnt2d and tnnt2c MOs (H)confirms loss of sarcomeres in double-injected embryos. Nuclei are counterstained with DAPI (blue). Scale bars: 10m. (I–J) Slow myosin staining (white) ofcontrol (I) and tnnt2d MO-injected (J) embryos shows fairly normal slow fibres at 5 dpf in the MO-injected embryo, with some myofibrils detached from the mainbundles (white arrowheads). Scale bars: 30m, insets are 2� magnifications of corresponding panels.

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assembly (Fig. 9C). Although we found that the periodicity offilamentous actin is restored upon blocking actin–myosin activityand that actin filaments extend from expanded Z lines towardsthick filaments, importantly we also found that filamentous actinis not associated with tropomyosin in the absence of troponinactivity. Probably, actin filaments that are not decorated withtroponin and tropomyosin cannot be detected in our electronmicroscopy preparations (Fig. 3). The association of tropomyosinwith actin is stabilised by the binding of troponin T (Hitchcock-DeGregori and Heald, 1987), and it is likely that association oftropomyosin with actin becomes unstable in the troponin-T-depletedmuscle. Our interpretation is that overall thin filament assembly isimpaired in the absence of troponin; however, further analysis ofthe distribution of other proteins associated with thin filaments(e.g. nebulin) will be needed to further support this conclusion.Our in situ hybridisation experiments did not reveal major changesin tropomyosin expression in the triple tnnt MO-injected embryosand the troponin gene tnni2 was only slightly upregulated whiletnnt3b was slightly downregulated. Therefore, we suggest thattranscriptional changes are not major determinants of the phenotypearising from the loss of troponin activity.

Disruption of single fast-twitch muscle tnnt genes causes onlypartial loss of troponin activity because they are partially redundant.When we disrupt expression of a gene that is expressed early, suchas tnnt2c in slow-twitch muscle, then sarcomere assembly isimpaired. When we disrupt expression of genes that becomefunctional later, after tissue differentiation is initiated, then the firststeps of myofibril formation occur but deregulation of actin–myosin activity eventually leads to sarcomere disintegration, withsarcomeric components becoming abnormally redistributed todistinct cellular domains. Thus, in the absence of functional troponinT, different sarcomeric substructures disintegrate independentlybefore the complete loss of sarcomere integrity rather thandisintegrating simultaneously.

We were able to examine the function of another component ofthe fast-twitch-muscle-specific troponin complex by using tnni2a.4mutants. These mutants showed myofibril degradation with thesame progression and timing as observed for tnnt3a MO-injectedembryos. Furthermore, similar to observations for tnnt3a, themyosin inhibitor blebbistatin was able to suppress the tnni2a.4mutant phenotype. As in the case of tnnt3a and tnnt3b, generedundancy might explain the lack of early phenotype in tnni2a.4mutants. Six tnni2 genes are indeed reported in ZFIN, some ofwhich are expressed in myotomes during somitogenesis.

Studies in D. melanogaster have shown that the hypercontractionmuscle phenotype caused by mutations in the troponin T or troponinI genes is suppressed by myosin mutations that reduce actin–myosin force production (Nongthomba et al., 2003). Our data areconsistent with the D. melanogaster data and validate them in avertebrate animal. Furthermore, our ability to resolve the sarcomerecomposition with several markers at different time points and topartially suppress the defect with chemical inhibition of myosinprovides further insights into the mechanism. We have shown thattroponin is required for normal myofibrillogenesis and for properthin filament assembly, and that partial loss of troponin activityallows sarcomere assembly but that unregulated actin–myosinactivity leads progressively to loss of sarcomeric integrity.

Sarcomere assembly modelsIt has been shown that sarcomeric components can acquire periodicityin the absence of thick filaments (Hawkins et al., 2008; Wohlgemuth

et al., 2007). Similarly, one interpretation of our finding that normalthin filaments are absent in the rudimentary sarcomeres restoredupon blocking unregulated actin–myosin activity in troponin-T-depleted muscle could be that sarcomere assembly occursindependently of thin filament formation (Fig. 3D and Fig. 9C).

Recent work has suggested that myofibrillogenesis in zebrafishmight proceed through stages that include the initial formation ofpre-myofibrils containing Z bodies and non-muscle myosin, nascentmyofibrils with non-aligned muscle myosin, and eventually maturemyofibrils with aligned thick filaments, consistent with previousobservations using cultured avian muscle cells (Sanger et al., 2005;Sanger et al., 2009). Alternative models imply the existence ofactin stress-fibre-like structures as the first template for sarcomereassembly (Dlugosz et al., 1984) or the stitching of independentlyforming thick filaments and I–Z–I bodies (Holtzer et al., 1997).

We found that slow muscle myosin becomes localised tosarcomeres in very young somites (supplementary material Fig.S9) and that sarcomeres appear as early as the 15-somite stage,which is earlier than previously reported (Sanger et al., 2009). Atthis stage, in wild-type embryos, filamentous actin is mainlylocalised in unsegmented bundles running along the myocytelength, although some is localised to forming sarcomeres, where itoverlaps with slow myosin. Our analysis of tnnt2c MO-injectedembryos indicates that filamentous actin does not become periodicif the troponin complex is defective, and that muscle myosinremains in rodlets or aggregates. Despite a difference in the timingof muscle myosin appearance, our results indicate a transition fromsmaller immature sarcomeres to mature sarcomeres during recoveryin tnnt2c MO-injected embryos (Fig. 7). This is consistent with thepre-myofibril model. Unregulated actin–myosin contraction that isdue to troponin loss of function is able to interfere with the layingof pre-myofibrils because actin and Z lines are randomly distributedin troponin-T-depleted embryos but align in blebbistatin-treated

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Fig. 9. Consequences of the loss of troponin function. (A)Schematicdepiction of sarcomeres in the wild-type condition in which troponin (blueovals), on thin filaments (thick blue lines) together with tropomyosin,regulates actin–myosin interactions. Thick filaments are shown in green.(B)In the absence of troponin, sarcomeres do not form, instead stacks of thickfilaments, expanded Z lines (black ovals) and aggregates of filamentous actin(blue clouds) can be observed. (C)If in the absence of troponin, the actin–myosin activity is blocked, then sarcomeres form although filamentous actin(thin blue lines) does not associate with tropomyosin and Z lines are expanded.

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embryos (Fig. 2). By contrast, thick filaments appear to be able toform as independent units, even in the absence of organised thinfilaments. This is shown by the striated distribution of T11, whichrecognises a titin epitope proximal to the ends of thick filaments,and of myomesin, which marks the M lines (Fig. 2), and by thepresence of thin filament-free, Z-line-free sarcomeres in electronmicroscopy micrographs (Figs 1 and 3). In the absence of thinfilaments and -actinin, titin might hold together the thick filament-rich substructures, possibly maintaining the attachment at theposition of the Z lines thanks to the telethonin protein (Bertz et al.,2009). The T12 antibody that targets a portion of the protein at theZ line level appears randomly distributed, although this might beexplained by the requirement of an intact Z line for recognition ofthe epitope. Our data do not disprove the pre-myofibril model butindicate that the role of non-muscle myosin and the dynamics ofthick filament assembly might still require further definition.

Relevance to the human diseasesWe found an excess number of zebrafish troponin genes ascompared with mammalian orthologues and have proposed a newnomenclature for this group of genes, which is summarised inTable 1. Notably, in human and rodent skeletal slow muscle, thefoetal troponin T function is provided by the cardiac gene TNNT2,which is then replaced by the slow-twitch-muscle-specific TNNT1gene after birth (Jin, 1996; Jin et al., 2003). Consistently, we foundthat embryonic genes expressed in zebrafish skeletal slow-twitchmuscle are more similar to the human TNNT2 than to the slow-twitch-muscle-specific TNNT1gene.

Mutations in the human skeletal troponin T subunit are linked totwo different pathologies, recessive Nemaline myopathy anddominant distal arthrogryposis. In the case of Nemaline myopathy,a stop mutation in the slow-twitch-specific troponin T described inan Amish family generates a truncated protein that is rapidly degraded(Wang et al., 2005), suggesting that slow muscles in these patientsare completely deficient in the troponin function. A hallmark ofNemaline myopathy is the accumulation of Z line proteins inaggregates called nemaline rods, which resemble the accumulationsof Z line proteins that we observed in micrographs from tnnt2c MO-or triple tnnt MO-injected embryos, indicating that the troponin Tloss of function leads to a similar myopathy in human and zebrafish.

Missense or stop mutations in the fast-twitch-specific troponinT and troponin I genes cause distal arthrogryposis, a syndromecharacterised by limb contractures with muscular weakness but nomuscle atrophy (Ochala, 2008). We showed that there are nointrinsic differences in the mechanisms of sarcomere assembly andloss between slow and fast fibres in the absence of normal troponinand, therefore, we believe that the differences in the human TNNT1versus TNNT3 or TNNI2 phenotypes can be explained by thenature of the mutations. In the case of distal arthrogryposis, theseare often dominant missense mutations that lead to a reduction inthe contractile force rather than causing major changes in thedistribution of myofibrillar components.

Materials and MethodsOrthology analysisWe have used TreeFam, a database of phylogenetic trees of animal genes(www.treefam.org) (Li et al., 2006; Ruan et al., 2008), to verify the number oftroponin genes present in the zebrafish genome. We found that there are four genesencoding troponin C (TreeFam TF318191), 13 genes encoding troponin I (TreeFamTF313374) and eight genes encoding troponin T (TreeFam TF313321).

We aligned the sequences of the human, mouse, zebrafish and stickleback troponinT proteins using ClustalW2 (http://www.ebi.ac.uk/Tools/clustalw2/index.html). Wefound five zebrafish genes in the human TNNT2 branch, one is the cardiac-specific

gene known as tnnt2, the silent heart locus, which we suggest should be renamedtnnt2a; therefore the gene most closely related to tnnt2a, LOC562296, should becalled tnnt2b. Genes tnnt2a and tnnt2b both show obvious synteny to the humanTNNT2 gene. There are three other genes encoding zebrafish troponin T2 isoformsas defined by orthology. These were known as tnnt1, zgc:114184 and si:ch211-136g2.1. The tnnt1 gene was described in literature as the slow-twitch-specific gene(Hsiao et al., 2003); however, it does not branch together with the human TNNT1gene and it appears to be a teleost-specific gene. It shows conserved synteny withcryptochrome 1a, between zebrafish and stickleback, and we suggest it should berenamed tnnt2c. The other two are duplicated genes in zebrafish, with apparentlyonly one copy in stickleback and we suggest that these should be renamed tnnt2d(zgc:114184) and tnnt2e (si:ch211-136g2.1), respectively. The zebrafish gene thatclusters together with the human TNNT1 gene is presently called zgc:193865, whichwe suggest should be called tnnt1. We have obtained approval for the proposed newnomenclature and summarise the changes in Table 1.

Maintenance of zebrafishBreeding zebrafish (Danio rerio) lines were maintained at 28°C on a cycle of 14hours light and 10 hours dark. Fertilised eggs were obtained from natural spawningand grown in incubators at a temperature between 24°C and 28°C, depending on thestage required. Embryos were staged according to standard references (Kimmel etal., 1995). For imaging purposes, live embryos were manually dechorionated with#5 watchmaker’s forceps, when necessary anaesthetised with 0.02% tricaine (ethyl3-aminobenzoate methanesulphonic acid), and mounted for viewing in 3%methylcellulose in embryo medium (Westerfield, 2000). Pictures were taken using aZeiss Axio Imager M1 microscope and a Zeiss AxioCam HRc digital camera.

Morpholino injectionsThe following morpholino-modified antisense oligonucleotides were ordered fromGenetools: tnnt2c MO, 5�-CCACAAACTCTTCGGTGTCGCACAT-3� (ATG MO);tnnt2d MO, 5�-GAAGTAAACACTCACGATTCTGTTG-3� (ATG MO); tnnt3aMO, 5�-CTCAATGTCCTCTGTGTCTGACATG-3� (ATG MO); tnnt3b MO, 5�-AACATCCTCAGTATCAGACATGATG-3� (ATG MO); and standard control oligo,5�-CCTCTTACCTCAGTTACAATTTATA-3�.

Oligonucleotides were diluted in morpholino buffer (5 mg/ml phenol red, 4 mMHEPES pH 7.2, 160 mM KCl) and injected in 1–2 cell stage embryos. We used 6ng for tnnt2c MO, 6 ng for tnnt2d MO, 3 ng for tnnt3a MO, 6 ng for tnnt3b MOand equal amounts of the control MO. Needles were pulled from glass capillarytubes using a needle puller (Kopf Instruments) and injections were performed usinga PV 820 Pneumatic Picoump micro-injector (World Precision Instruments).

Plasmids, mRNA and DNA injectionsThe coding sequence for the mCherry fluorescent protein (Shaner et al., 2004) wasamplified with the following primers: FmCheClav2, 5�-ggcggccatcgatATGGTC -TCTAAGGGCGAG-3� and RmCheEcoRInostop, 5�-ggcggccGAATTCaTATTTGTA -CAGTTCATC-3�.

The second primer was a modified reverse primer that abolished the stop codon.This insert was cloned in pCS2 using ClaI and EcoRI to generate the pCS2mCherryvector. The tropomyosin 3 (tpma) and troponin T2c (tnnt2c) coding sequences wereamplified by RT-PCR from 48 hpf embryo RNA with the following primers:FTpmaEcoRI, 5�-ggcggccGAATTCATGGATGCCATTAAGAAG-3�; RTpmaXbaI,5�-ggcggccTCTAGATTAAATGGAGGTCATGTC-3�; FTnnt2cEcoRInew, 5�-ggc -ggccGAATTCATGTGCGACACCGAAGAG-3� and RTnnt2cXhoInew, 5�-ggcggcc -CTCGAGTTACTTCCAGGACTTCCT-3�.

PCR fragments were digested with EcoRI and XbaI or EcoRI and XhoI and cloneddownstream of the mCherry coding sequence in the pCS2mCherry vector, andplasmid were sequence verified. The pCS2mCherry–Tpma plasmid was linearisedwith NotI and used as a template to obtain mRNA in an in vitro transcription reactionwith the RNA polymerase SP6 (Biolabs). Typically, 150 pg of mRNA were injectedin one-cell embryos. For DNA injections, the coding sequence for the fusion proteinmCherry–Tnnt2c was amplified from the pCS2mCherry–Tnnt2c vector with thefollowing primers: FNheI–mCherry, 5�-ggcggccGCTAG CatgGTCTC TAAGGG -CGAG-3� and RBglII–Tnnt2c, 5�-ggcggccAGATCTTTA CTTCCAGGACTTCCT-3�.

The PCR fragment was digested with NheI and BglII and cloned in the backboneof the pECFP–ER vector (Clontech) digested with the same restriction enzymes toremove the 846 bp CFP–ER sequence. The pEmCherry–Tnnt2c vector was linearisedwith StuI and purified with phenol/chloroform; 60 pg was injected into one-cellembryos. Both in the mRNA and DNA injections experiments, fluorescence wasdetected in live 24 and 48 hpf embryos using a 40� water immersion lens on a LeicaSP5 confocal microscope. Fluorescent embryos were fixed in 4% paraformaldehydeand processed as described in the following subsection.

Immunolabelling and imagingFor phalloidin staining, embryos were fixed at the appropriate stage in 4%paraformaldehyde in PBS overnight at 4°C, washed with PBS containing 2% TritonX-100, incubated with Alexa Fluor 488 or Alexa Fluor 568 conjugated to phalloidin(Invitrogen), resuspended according to the manufacturer’s instructions and diluted1:100 in PBS, washed in PBS containing 0.1% Tween and mounted with Vectashield

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Mounting Media with DAPI (Vector Laboratories). For fluorescenceimmunohistochemistry, embryos were fixed at the appropriate stage overnight inmethanol at –20°C or in 4% paraformaldehyde in PBS at 4°C. B4 (myomesin), F59(slow myosin), EB165 (fast myosin), CH1 (tropomyosin) and Ct3 (troponin T)antibodies were obtained from the Developmental Studies Hybridoma Bank and used1:5 (if obtained as supernatant) or 1:100 (if obtained as concentrate). Anti-titin T12, agift from Yaniv Hinits (King’s College, London, UK) was used 1:10. Anti -actinin,anti-titin T11 and anti-troponin JLT-12 were purchased from Sigma (A7811, T9030and T6277) and used 1:100. The secondary antibody used was Alexa-Fluor-488-labelled goat anti-mouse IgG (A-11001; Invitrogen). Embryos were washed severaltimes in PBS containing 0.1% Triton X-100 and 0.2% BSA, incubated overnight inthe same buffer with 5% of goat serum and primary antibodies, washed again,incubated with secondary antibody overnight, then washed and mounted withVectashield Mounting Media with DAPI (Vector Laboratories). For double-staining,phalloidin was added together with the secondary antibody. Samples were imaged withthe Leica SP5 confocal microscope using a 40� or a 63� oil immersion objective.

Electron microscopyEmbryos were immersed in freshly prepared primary fixative containing 2%paraformaldehyde with 2% glutaraldehyde in 0.1M sodium cacodylate buffer (pH7.42) containing 0.1% magnesium chloride and 0.05% and calcium chloride at 20°Cfor 10 minutes before transferring to an ice bath for the remainder of 2 hours.Samples were rinsed three times for 10 minutes each in sodium cacodylate bufferwith added chlorides on ice. Secondary fixation with 1% osmium tetroxide insodium cacodylate buffer only was carried out at room temperature for 1 hour. Allfollowing steps were performed at room temperature. Embryos were rinsed threetimes in cacodylate buffer over 30 minutes and mordanted with 1% tannic acid for30 minutes followed by a rinse with 1% sodium sulphate for 10 minutes. Sampleswere dehydrated through an ethanol series of 20, 30 (staining en bloc with 2% uranylacetate at this stage), 50, 70, 90 and 95% for 20 minutes each then 100% for 3�20minutes. Ethanol was exchanged for propylene oxide (PO) for 2�15 minutesfollowed by 1:1 PO to Epon resin mixture for at least 1 hour, and neat Epon (witha few drops of PO) overnight. Embryos were embedded in a flat moulded tray withfresh resin and cured in an oven at 65°C for 24 hours. We cut 40-nm sections on aLeica UCT ultramicrotome, stained the sections with uranyl acetate and lead citratefor contrast, and imaged on an FEI 120kV Spirit Biotwin using an F415 Tietz CCDcamera.

GenotypingWe genotyped tnni2sa0058 embryos using the following nested primers: tnni2-4-1, 5�-GAGAACAAACATCTGACAATGG-3�; tnni2-4-2, 5�-GGTCAATTAA ATGATT -TGAAGC-3�; tnni2-4-3, 5�-TTCTTGAACTTGCCCTTCAG-3�; and tnni2-4-4,5�-CAGCAGAGCCTGAAGCATAG-3�. Whole 5 dpf embryos were fixed inmethanol and then dried on a heat block. Genomic DNA was extracted by incubatingwith proteinase K at 55°C overnight. Mutant slou45 embryos are easily distinguishedfrom wild-type siblings because they are completely immotile.

Blebbistatin treatmentBlebbistatin was purchased from Sigma (B0560-1MG) and resuspended in DMSO.A final concentration of 6 M was added to embryo medium at 28 hpf, and fish werecollected at 48 hpf. DMSO only was added to untreated controls.

We are grateful to Elisabeth Busch-Nentwich, Gavin Wright, StevenHarvey, Bill Saxton and Simon Hughes for helpful discussion andcritical reading of this manuscript, and to Laurel Lam Hung for herassistance. We also thank the Sanger Zebrafish Mutation Resource.This work was supported by the Wellcome Trust (grant numbers WT077037/Z/05/Z, WT 077047/Z/05/Z). Deposited in PMC for releaseafter 6 months.

Supplementary material available online athttp://jcs.biologists.org/cgi/content/full/124/4/565/DC1

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SummaryKey words: Muscle, Sarcomere, Troponin, ZebrafishIntroductionResultsDepletion of troponin T activity in fast-twitch muscle disrupts sarcomereInhibition of myosin function suppresses loss of striated organisation butPartial loss of troponin activity in fast-twitch muscle leads toThe partial loss of troponin phenotype is suppressed by lossDisruption of tnnt2c abolishes sarcomere assembly in slow-twitch muscle

Table 1.Fig. 1.Fig. 2.Fig. 3.Fig. 4.Fig. 5.Fig. 6.Fig. 7.DiscussionSarcomeres do not form in the absence of functional troponinsSarcomere assembly modelsRelevance to the human diseases

Fig. 8.Fig. 9.Materials and MethodsOrthology analysisMaintenance of zebrafishMorpholino injectionsPlasmids, mRNA and DNA injectionsImmunolabelling and imagingElectron microscopyGenotypingBlebbistatin treatment

Supplementary materialReferences