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Research Article 565 T is essential for assembly in zebrafish

Maria 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 ([email protected])

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

Summary In striated muscle, the basic contractile unit is the sarcomere, which comprises -rich thick filaments intercalated with thin filaments made of , and troponin. Troponin is required to regulate Ca2+-dependent contraction, and mutant forms of are associated with muscle . We have disrupted several simultaneously in zebrafish and have followed the progression of muscle degeneration in the absence of troponin. Complete loss of activity leads to loss of sarcomere structure, in part owing to the destructive of deregulated actin–myosin activity. When troponin T and myosin activity are simultaneously disrupted, immature are rescued. However, tropomyosin fails to localise to sarcomeres, and intercalating thin 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, are formed but eventually disintegrate owing to deregulated actin–myosin activity. 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 the absence of normal thin filaments.

Key words: Muscle, Sarcomere, Troponin, Zebrafish

Introduction clenched fist, overlapping , ulnar deviation and positional Muscle cells possess an elaborate cytoskeletal structure, which is deformities. affecting the three cardiac subunits of essential for the coordination of contraction and the generation of the complex (TNNC1, TNNT2 and TNNI3) are also associated with . The basic machinery of contraction is organised into bundles a variety of (Kimura et al., 1997; Landstrom et

Journal of Cell Science of long myofibrils, which are each made of stacks of sarcomeres. al., 2008; Mogensen et al., 2003; Mogensen et al., 2004; Murphy Sarcomeres consist of thin filaments, attached to Z discs, and thick et al., 2004; Peddy et al., 2006; Thierfelder et al., 1994). filaments, attached to M lines. Filamentous actin associates with In addition to the known role in the regulation of contraction, tropomyosin, troponin and other to form thin filaments, there is some evidence that troponin is required for formation and which intercalate with thick filaments that are predominantly made maintenance of sarcomeres. In zebrafish, mutations of the silent of myosin. The contraction of striated muscle is regulated by the , which encodes cardiac troponin T2, lead to sarcomere troponin complex, which mediates the Ca2+-dependent loss and myocyte disarray, attributed to coordinate reduction of interaction of actin and myosin within the sarcomere. The troponin thin filament expression (Sehnert et al., 2002). A similar complex possesses three subunits: , and disruption of sarcomere assembly was observed in Tnnt2–/– mice, troponin T. Troponin T anchors the complex to the thin filament which die during embryogenesis due to failure of cardiac by binding to tropomyosin. Following stimulation, contraction (Ahmad et al., 2008; Nishii et al., 2008). Body wall released from the bind to troponin C and and sarcomere organisation are disrupted in this leads to conformational changes of the complex. Troponin I Caenorhabditis elegans mutants defective for genes encoding consequently relieves its inhibition, which allows the binding of troponin T or troponin I (Burkeen et al., 2004; Myers et al., 1996). myosin to actin. Simultaneously, troponin T and tropomyosin alter In Drosophila melanogaster, severe mutant of troponin T conformation, allowing myosin-binding sites on actin to be exposed lead to a failure of sarcomere assembly, whereas hypomorphic (Alberts et al., 2007; Galinska-Rakoczy et al., 2008; Phillips et al., troponin mutant alleles initially develop normal muscle and then 1986; Zot and Potter, 1987). undergo muscle damage (Fyrberg et al., 1990). In , loss of function of the slow-twitch-muscle-specific We have used zebrafish to investigate the role of thin filament troponin T isoform TNNT1 causes a recessive form of Nemaline proteins in the process of sarcomere assembly. Zebrafish (OMIM 605355), a genetically heterogeneous development is rapid, with muscle fully differentiated by 48 hours resulting from mutations in thin filament genes and characterised post- (hpf) (Kimmel et al., 1995). Zebrafish muscle has by muscle and rod-like inclusions in muscle fibres a similar composition to muscle, in comparison with (Johnston et al., 2000). Dominant mutations in the fast-twitch (Bassett et al., 2003). Moreover, the optical properties skeletal troponin T isoform TNNT3 or the troponin I isoform of the make it highly amenable to light microscopy. Like TNNI2 are responsible for a disorder called distal arthrogryposis in mouse and human, the troponin genes in zebrafish exist in type 2B (OMIM 601680) (Sung et al., 2003), characterised by several isoforms, differentially expressed in cardiac, slow-twitch 566 Journal of Cell Science 124 (4)

or fast-twitch skeletal muscle (Fu et al., 2009; Hsiao et al., 2003). and is downregulated from 36 hpf, whereas the Additionally, troponin genes have been duplicated in the zebrafish tnnt3a and the tnnt3b genes are expressed from 18 hpf and 24 hpf, relative to mammalian . The opportunity to respectively (Hsiao et al., 2003; Thisse and Thisse, 2005). We simultaneously knockdown expression of different genes using knocked down expression of all three genes simultaneously with combinations of antisense oligonucleotides (MOs) in -blocking MOs. Embryos injected with triple tnnt MOs zebrafish allows us to overcome this genetic redundancy were shorter than embryos injected with a single MO and were (Nasevicius and Ekker, 2000). occasionally bent, with abnormally shaped and pericardial We disrupted expression of different members of the troponin T oedema but with beating , which indicated that the cardiac family. In fast-twitch muscle, redundant expression of three muscle in these embryos was functional. At 28 hpf, embryos were different troponin T genes (tnnt2c, tnnt3a and tnnt3b) guarantees immotile, and myofibrils in fast-twitch muscle depleted of tnnt2c, normal assembly in single loss-of-function experiments, but the tnnt3a and tnnt3b were markedly disorganised and very rarely concomitant knockdown of all three genes leads to a block in possessed any striations (Fig. 1A,B), indicating that functional normal myofibrillogenesis. We demonstrate that it is the loss of troponin T is required for sarcomere assembly in fast-twitch muscle. regulation of the actin–myosin activity that leads to failure of When analysed at 48 hpf, fast-twitch muscle in the triple MO- sarcomere assembly. When we block myosin function with the injected embryos showed a reduced level of filamentous actin, myosin inhibitor , striations are recovered but an which was distributed in small foci. This was coupled with a loss additional effect of the loss of troponin T function on thin filament of striation, as indicated by the anti- T12, which assembly is unmasked. Finally, we have identified a second class recognises an epitope in proximity of the Z line (Fig. 1C,D). of that results when only single troponin genes are staining for actin, coupled with antibody staining for - disrupted. Depletion of tnnt3a or tnnt3b by MOs, or loss of tnni4a.2 , showed colocalisation of the two proteins in foci, although through , allows assembly but leads to sarcomere actin also appeared in aggregates without -actinin (Fig. 2C; disintegration, which is completely suppressed by inhibition of supplementary material Fig. S2C). Interestingly, other markers myosin activity. indicated that, although myofibrils were substantially disorganised, not all sarcomeric components were completely delocalised. We Results used a second anti-titin antibody T11, which recognises an epitope Depletion of troponin T activity in fast-twitch muscle at the boundary between the I-band and A-band (Trombitas and disrupts sarcomere assembly Pollack, 1993), and detected some regions with short stretches of In the zebrafish trunk skeletal musculature, slow-twitch fibres and periodic T11 staining in the embryos injected with triple tnnt MOs fast-twitch fibres are separated. Mononucleate slow-twitch fibres (Fig. 1E,F). Similarly, an antibody directed against myomesin, a are superficially located, immediately under the , and are major component of M lines, showed that M lines also partially parallel to the anteroposterior axis, whereas multinucleated fast- maintained regular spaced distribution in some regions (Fig. 1G,H). twitch fibres are located deeper in the trunk, are more abundant Tropomyosin failed to appear in striations but showed normal and have an oblique orientation (Bassett et al., 2003; Devoto et al., levels of expression in troponin-T-depleted muscles (Fig. 1I,J). 1996). Three troponin T genes, tnnt2c, tnnt3a and tnnt3b, are Because the tropomyosin antibody that we had used in single

Journal of Cell Science expressed in fast-twitch muscle during zebrafish development staining did not well in combination with phalloidin, we (Table 1; supplementary material Fig. S1). The tnnt2c gene is cloned in frame with the red fluorescent protein strongly expressed in myotomes from the beginning of mCherry and injected the mRNA encoding for this fusion protein

Table 1. Troponin genes described in this study Original zebrafish New gene Human Human gene name name Zebrafish ensembl ID Expression pattern orthologue protein Associated disease zgc:193865 ENSDARG00000037954 Expressed in embryos, TNNT1 Troponin T1 Nemaline distribution still myopathy uncharacterised tnnt2a ENSDARG00000020610 Cardiac TNNT2 Troponin T2 Cardiomyopathies LOC562296 tnnt2b ENSDARG00000031920 Unknown TNNT2 tnnt1 tnnt2c ENSDARG00000032242 Somites early, slow and fast TNNT2 muscles later zgc:114184 tnnt2d ENSDARG00000002988 Slow muscle from 24 hpf TNNT2 si:ch211-136g2.1 tnnt2e ENSDARG00000045822 Expressed in embryos, tissue TNNT2 distribution still uncharacterised tnnt3a tnnt3a ENSDARG00000030270 Fast muscle, head muscles, TNNT3 Troponin T3 Distal pectoral fin muscles, arthrogryposis hypaxial muscles type 2B tnnt3b tnnt3b ENSDARG00000068457 Fast muscles, pectoral fin, TNNT3 hypaxial muscles, head muscles tnni2a.4 tnni2a.4 ENSDARG00000029069 Musculature TNNI2 Troponin I2 Distal arthrogryposis type 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. Sarcomere assembly requires troponin 567 Journal of Cell Science

Fig. 1. Disruption of sarcomere assembly of fast-twitch muscle of embryos simultaneously depleted of tnnt2c, tnnt3a and tnnt3b. (A,B)Phalloidin staining shows forming and mature striated myofibrils in control embryos at 28 hpf (A) but completely disrupted myofibrils in triple tnnt MO-injected embryos at the same stage (B). MJ, myotendinous junctions; HM, horizontal myoseptum. (C,D)At 48 hpf, the titin epitope T12 (CЈ,DЈ; green) localises to normal Z lines, and actin (CЉ,DЉ; red) appears in striations in control embryos (C), whereas the two proteins are randomly dispersed in the in triple tnnt MO-injected embryos (D). DAPI stains the nuclei (Cٞ,Dٞ; blue). Single channels are shown in grey. (E–H) The titin epitope T11 localises to the I-band–A-band boundaries and myomesin to the 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-injected embryos (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: 25m, insets are 2ϫ 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 tnnt MO-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 should have been. Accumulations of actin can be observed in P (black asterisk). Scale bars: 500 nm.

in wild-type and triple tnnt MO-injected embryos. We then fixed lines (Fig. 1O), and areas with accumulations of material, which, the embryos and stained them with phalloidin. Our results show on the basis of light microscope analysis, we believe to be actin that tropomyosin and actin colocalised in the controls but did not (Fig. 1P). overlap in the triple tnnt MO-injected embryos (supplementary To prove that the observed phenotype was specific, we cloned material Fig. S3). Transmission (TEM) analysis one of the three troponins, tnnt2c, in frame with mCherry. A DNA confirmed these results, showing that a large part of troponin-T- construct encoding the fusion protein downstream of the depleted muscle was filled with dispersed thick filaments (Fig. cytomegalovirus (CMV) was injected into one-cell wild- 1M). We also observed areas with disorganised and expanded Z type embryos. The exogenous DNA was expressed mosaically in lines (Fig. 1N), areas with thin-filament-free sarcomeres with no Z a small subset of cells. At 28 and 48 hpf, isolated muscle cells 568 Journal of Cell Science 124 (4)

producing the red fluorescent troponin T could be observed. In the phenotype of embryos injected with tnnt2c, tnnt3a and tnnt3b these cells, the protein localised correctly to thin filaments MOs while simultaneously inhibiting myosin function. We used (supplementary material Fig. S4D–F). When this DNA construct blebbistatin, a myosin inhibitor (Straight et al., 2003) known to was co-injected with the triple tnnt MOs, those cells producing the inhibit contraction in skeletal muscle and cardiomyocytes fusion protein recovered striations, whereas surrounding muscle (Skwarek-Maruszewska et al., 2009). We added blebbistatin to cells not expressing the troponin-T–mCherry fusion protein showed control MO-injected and triple tnnt MO-injected embryos at 26 disorganised distribution of actin and lacked striations hpf, and analysed embryos at 48 hpf. At this stage, both control (supplementary material Fig. S4G–I). MO- and triple tnnt MO-injected embryos were immotile and To determine the extent of troponin T downregulation, we used displayed heart oedema. Myofibrils in control MO-injected two different anti-troponin T (Ct3 and JLT-12) in whole- embryos treated with blebbistatin had become less compact, mount assays. Both antibodies recognised although sarcomere markers had not lost their striated appearance troponin T on thin filaments in control embryos (supplementary (Fig. 2B,F,J,N). We observed a recovery in structure in material Fig. S5A,C and not shown) but did not react in triple tnnt blebbistatin-treated triple tnnt MO-injected embryos, with MO-injected embryos (supplementary material Fig. S5B,F and not immature sarcomeres that were, however, regularly aligned along shown), indicating that troponin T expression was abolished by the myofibrils that had recovered periodicity of Z lines, thick tnnt MOs. filaments and M lines (Fig. 2D,H,L; supplementary material Fig. Because expression of thin filament genes has been reported to S2) but that showed no recovery of tropomyosin localisation to be reduced in troponin-T-depleted (Sehnert et al., sarcomeres (Fig. 2P). 2002), we stained triple tnnt MO-injected embryos for expression In electron micrographs, we observed misaligned myofibrils of tropomyosin 3, troponin C, troponin I2 and troponin T3b by in and sarcomeres with less-defined Z and M lines in blebbistatin- situ hybridisation. We did not detect any change in the level or treated control embryos (Fig. 3B). The loss of sarcomeric integrity pattern of expression of the first two, but found to be slightly observed in DMSO-treated triple tnnt MO-injected embryos (Fig. upregulated and tnnt3b, one of the genes targeted with the MO, to 3C) was partially restored in blebbistatin-treated triple tnnt MO- be downregulated (data not shown). injected embryos, where regularly spaced, enlarged Z lines with intervening thick filaments could be observed (Fig. 3D). In cross- Inhibition of myosin function suppresses loss of striated sections, control muscles displayed regular, hexagonal arrangement organisation but does not restore proper thin filament of thick filaments surrounded by thin filaments (Fig. 3E), whereas assembly the triple tnnt MO-injected embryos showed bundles of thick To assess whether the effect of troponin T depletion on myofibrils filaments with no intercalating thin filament, regardless of the was due to deregulation of actin–myosin activity, we analysed blebbistatin treatment (Fig. 3F,G). Journal of Cell Science

Fig. 2. Recovery of striated myofibrils in triple tnnt-depleted embryos upon inhibition of myosin activity. (A–L) Phalloidin (red in A–D), -actinin (green in A–D), fast myosin (white in E–H) and myomesin (white in I–L) staining of embryos incubated with DMSO reveals normal myofibrils in a control embryo (A,E,I) and the expected disruption in distribution of actin, Z lines, thick filaments and M lines in triple tnnt MO- injected embryos (C,G,K). When embryos are treated with blebbistatin, less compact myofibrils are observed in controls (B,F,J) and striated myofibrils can be seen in triple tnnt MO-injected embryos (D,H,L). (M–P) Tropomyosin does not recover its localisation to thin filaments in tnnt MO- injected embryos treated with blebbistatin (P). DAPI (blue) stains the nuclei. Scale bars: 25m, insets are 2ϫ magnifications of corresponding panels. Sarcomere assembly requires troponin 569

Fig. 3. Blebbistatin treatment does not rescue the loss of normal thin

Journal of Cell Science filaments in troponin-T-depleted sarcomeres. (A–G) Electron microscopy images 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 hexagonal arrangement of thick filaments with intercalating thin filaments (circle in E). Fig. 4. Myofibrils degrade in fast-twitch muscles of -depleted In blebbistatin-treated control embryos (B), Z lines are less sharp and M lines embryos. (A–C) 3 dpf control (A), tnnt3a MO-injected (B) and tnnt3b MO- are not clearly defined. In tnnt-depleted muscles (C,F), sarcomeric components injected (C) embryos. (D–F) When trunks of the same embryos are observed are disorganised (C) and bundles of thick filaments in cross-sections do not under polarised light, embryos injected with tnnt3a MO (E) and tnnt3b MO appear to be intercalating with thin filaments (F). Upon blebbistatin treatment, (F) show total and partial loss of birefringence, respectively. (G–I) Lateral wider and regularly spaced Z lines can be observed in triple tnnt MO-injected views of rostral somites in 3 dpf embryos. Confocal images from a plane deep embryos (D) and virtually no thin filaments are visible inside thick filaments in the trunk musculature show that actin is highly organised in fast muscle of bundles (circle in G). Scale bars: 1 m (A–D); 150 nm (E–G).  control embryos (G), but appears completely disorganised in tnnt3a MO- injected embryos (H) and partially disorganised in tnnt3b MO-injected embryos (I; the inset shows a magnification of the myotendinous junction To examine the role of unregulated actin–myosin activity, we where actin localised in vertical Z lines or in horizontal stretched, non-striated independently disrupted myosin assembly using sloth mutants. In filaments gives rise to a characteristic chequered appearance). Phalloidin is sloth mutants, the protein Hsp90a.1 is defective, white and nuclei are counterstained with DAPI (blue). Scale bars: 50m; preventing efficient myosin folding and thick filament assembly 10m in insets. (J–L) TEM images of fast-twitch muscles in 40 hpf control (Granato et al., 1996; Hawkins et al., 2008). We injected the triple (J), tnnt3a MO- (K) and tnnt3b MO-injected embryos (L) reveal similar loss of tnnt MOs into slou45 embryos. Wild-type sibling embryos injected sarcomeric integrity in the MO-injected embryos, with stacks of thick with triple tnnt MOs displayed the expected phenotype with filaments and accumulations of Z line proteins (white arrowheads). Scale bars: disrupted sarcomeres (supplementary material Fig. S6C). By 1m. (M,N) Forming fast-twitch fibres appear striated in both in control (M) contrast, slou45 mutant embryos had wavy myofibrils with I–Z–I and tnnt3a MO-injected (N) embryos at 28 hpf. Fast myosin is white and nuclei are blue. Scale bars: 20m. (O,P) At 48 hpf, M lines stained by brushes (supplementary material Fig. S6B). Injection of triple tnnt u45 myomesin (green) are regularly intercalated with thin filaments (phalloidin, MOs in slo embryos did not lead to the formation of actin red) in control embryos (O), whereas they cluster in groups and are excluded aggregates, and myofibrils recovered periodic distribution of actin from actin-rich areas in tnnt3a MO-injected embryos (P, inset shows clusters). and -actinin (supplementary material Fig. S6D), confirming that Scale bars: 15m; 10m in inset. M–P are lateral views of a single myotome, rudimentary sarcomeres can form in the absence of myosin activity. nuclei are stained with DAPI (blue). 570 Journal of Cell Science 124 (4)

In these embryos, however, tropomyosin did not recover regular At the ultrastructural level, we observed sarcomeres with several distribution and appeared to be less abundant and unlocalised defects, including wider and wavier Z lines, and areas with throughout the cytoplasm (supplementary material Fig. S6H). disintegrated sarcomeres, dispersed thick filaments and accumulations of Z line proteins (Fig. 4J–L). Partial loss of troponin activity in fast-twitch muscle leads In fast-twitch muscle, striations first become visible at 28 hpf. to degradation of myofibrils Phalloidin and myosin staining at this stage showed that sarcomeres Single injection of tnnt3a or tnnt3b MOs impaired of initially form normally in tnnt3 MO-injected embryos (Fig. 4N and embryos at 3 days post-fertilisation (dpf) and did not cause other not shown) and that double knockdown of tnnt3a and tnnt3b had major disruptions of (Fig. 4A–C). Striated the same phenotype as single tnnt3a disruption (data not shown). muscles possess a property known as form birefringence, which In tnnt3a MO-injected embryos, sarcomeric proteins later became arises as a result of the parallel packing of long fibres, giving them abnormally localised and separated into distinct cellular domains. the ability to polarise light. Larvae at 3 dpf showed complete For example, in muscle co-stained for actin and myomesin, groups (tnnt3a) or partial (tnnt3b) loss of muscle birefringence (Fig. 4D– of discrete and evenly spaced M lines could be seen distinctly F), indicating loss of myofibrillar organisation. Phalloidin staining separated from the actin aggregates (Fig. 4P). showed a distinctive distribution of actin in round aggregates in We obtained independent confirmation of the effect of partial tnnt3a MO-injected embryos (Fig. 4H). In tnnt3b MO-injected disruption of troponin function by analysing the knockout of a embryos, we found partial disruption, with striations preserved in troponin I isoform in the same complex. A nonsense of the central part of myotubes but lost at the myotendinous junctions tnni2a.4, a fast-twitch troponin I gene, was isolated in collaboration (Fig. 4I), indicating that at this stage tnnt3a can partially compensate with the Zebrafish Mutation Resource at the Sanger Institute for tnnt3b and that myotube ends are the first areas to be affected (http://www.sanger.ac.uk/cgi-bin/Projects/D_rerio/mutres/mutation. by a diminished availability of troponin T. At 5 dpf, however, pl?project_id966&mutation_id170). The mutation is predicted myofibril disintegration progressed in tnnt3b MO-injected embryos to truncate the 176 amino protein after the 66th and diffuse aggregates of actin could also be observed in the fast- (Q67X) (Fig. 5B), eliminating more than half of the protein, twitch muscle of these embryos (supplementary material Fig. S7). including the C-terminus, which is thought to interact with actin Journal of Cell Science

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 a tnni2a.4sa0058 carrier fish (B) show the change (asterisk) that converts C to T, generating a stop codon. (C–F) In a 5-day-old wild-type sibling observed under 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 of single myotomes. Confocal images were obtained from planes deep in the trunk musculature. At 48 hpf, tnni2a.4 mutant embryos show myofibrils that are losing ;their integrity, as highlighted by phalloidin staining for thin filaments (GЉ,HЉ; red) and -actinin staining for Z lines (GЈ,HЈ; green). DAPI stains the nuclei (Gٞ,Hٞ blue). Single channels are shown in grey; overlap of signals shows in yellow in the merged panels (G,H). Scale bars: 10m. (I,J)At 3 dpf, fast myosin (white) is completely delocalised in tnni2a.4 mutants (J) whereas wild-type siblings show normal fast muscle (I). Nuclei are stained in blue. Scale bars: 20m. Insets are 2ϫ magnifications of corresponding panels. Sarcomere assembly requires troponin 571

and tropomyosin on the thin filament (Galinska-Rakoczy thin, the filamentous actin was completely dispersed or distributed et al., 2008) to inhibit actin–myosin interactions. in regularly spaced foci but not in properly formed sarcomeres (Fig. Starting from 3 dpf, tnni2a.4sa0058 mutants were unable to swim 7A,B), and myosin was found in blocks but also dispersed in the properly and at 5 dpf failed to inflate the swim bladder. At this cytoplasm or in thin, loose myofibrils (Fig. 7C,D). Electron stage, trunk muscles showed no birefringence (Fig. 5C–F). As with microscopic analysis of slow-twitch muscle ultrastructure confirmed tnnt3a MO-injected embryos, fast-twitch myofibrils showed a loss the absence of normal sarcomeres at 24 hpf in tnnt2c MO-injected of striation and diffuse actin and -actinin staining by 48 hpf (Fig. embryos and showed accumulation of Z line proteins and dispersed 5G,H). Myosin staining at 3 dpf also revealed severe disruption of thick filaments (Fig. 7F, arrowheads and arrow, respectively). At 30 thick filaments (Fig. 5I,J). When analysed at 28 hpf, mutants could not be distinguished from wild-type siblings, indicating that myofibrils had formed normally and therefore that the fast-twitch muscle disintegration was progressive.

The partial loss of troponin phenotype is suppressed by loss of myosin activity We tested whether the phenotype is due to unregulated actin– myosin activity. In tnnt3 MO-injected embryos, myofibril degradation proceeds through a first stage in which actin is distributed in sharp vertical bands and stretched horizontal fibres in enlarged areas of the myotubes, to a second stage in which actin becomes localised to round aggregates (Fig. 4H,I; supplementary material Fig. S7B), again suggesting that deregulated actin–myosin activity might have a role in the genesis of this phenotype. We added blebbistatin to control MO- and tnnt3a MO-injected embryos at 26 hpf and analysed embryos at 48 hpf. Untreated tnnt3a MO-injected embryos showed the expected disrupted actin localisation 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, the lack of troponin T function does not lead to sarcomere disintegration. Because the tnni2a.4 mutant phenotype is the same as the tnnt3a MO phenotype, we used blebbistatin to verify whether inhibition of actin–myosin contraction could suppress the disruption of sarcomeres in the tnni2a.4 mutants. We found that four out of

Journal of Cell Science 20 control DMSO-treated offspring from heterozygous parents displayed the expected disrupted actin phenotype at 48 hpf, whereas none of the 20 observed blebbistatin-treated embryos from the same clutch displayed obvious actin accumulations. We confirmed these results independently using sloth mutants. We injected the tnnt3a MO into slou45 embryos. Wild-type sibling embryos injected with tnnt3a MO displayed the expected phenotype with degrading myofibrils (Fig. 6F), whereas slou45 mutant embryos had wavy myofibrils with I–Z–I brushes (Fig. 6G). Injection of tnnt3a MO in slou45 embryos did not lead to the formation of actin aggregates, and myofibrils were indistinguishable from uninjected slou45 embryos of the same stage (Fig. 6H), confirming that normal Fig. 6. Suppression of the tnnt3a progressive myofibrillar disorganisation phenotype in the absence of functional thick filaments. (A–D) Incubation interactions between myosin and actin are required for the Tnnt3 with the myosin inhibitor blebbistatin: phalloidin staining of embryos loss of function to affect sarcomere maintenance. incubated with DMSO reveals normal myofibrils in a control embryo (A) and the expected disruption in actin distribution in a tnnt3a MO-injected embryo Disruption of tnnt2c abolishes sarcomere assembly in (C). When embryos are treated with blebbistatin, loose myofibrils are observed slow-twitch muscle in a control embryo (B) and wavy but striated myofibrils can be seen in a The tnnt2c gene is expressed in fast-twitch but also in slow-twitch tnnt3a MO-injected embryo (D). Scale bars: 25m, insets are 2ϫ muscle (http://zfin.org/cgi-bin/webdriver?MIvalaa-markerview. magnifications of corresponding panels. (E–H) Analysis in the sloth mutant: a apg&OIDZDB-GENE-030520-1). Embryos injected with tnnt2c 48 hpf wild-type sibling displays normally striated fast fibres (E). In a wild- MO alone developed normally and did not show any morphological type sibling injected with the tnnt3a MO (F), myofibrils undergo degradation abnormalities, but they were completely immotile at 24 hpf and and actin becomes diffuse. In a sloth mutant embryo (G), actin is distributed in thick filament-free sarcomere-like blocks along myofibrils. In a sloth mutant lacked the normal twitching movements typical of this stage. We injected with tnnt3a MO (H), actin remains in blocks rather than dispersed in first observed spontaneous twitching at around 28 hpf, when fast- the cytoplasm. Scale bars: 10m, insets are 2ϫ magnifications of twitch muscle becomes functional. By 48 hpf, we found that embryos corresponding panels. All images are lateral views of a myotome. Confocal had recovered their ability to move, hatch and swim normally. Slow- images were obtained from planes deep in the trunk musculature. Phalloidin twitch myofibrils of tnnt2c MO-injected embryos at 24 hpf were staining is white and DAPI for nuclei is blue. 572 Journal of Cell Science 124 (4) Journal of Cell Science

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), but not in stage matched tnnt2c MO-injected embryos (B,D) stained for filamentous actin (A,B) and slow myosin (C,D). Scale bars: 25m, insets are 2ϫ magnifications of corresponding panels. (E,F)Dysmorphology at the ultrastructural level is revealed by TEM images of slow muscle in 24 hpf control (E) and tnnt2c MO-injected embryos (F). Accumulations of Z line proteins (black arrowheads) and dispersed thick filaments (black arrow) can be found in the MO-injected embryos (F). Scale bars: 500 nm. (G,H)30 hpf control embryos stained with an anti-titin antibody (GЈ,HЈ; green, T12), and phalloidin (GЉ,HЉ; red), show normal sarcomeres (G), whereas tnnt2c MO-injected embryos do not (H). Nuclei are counterstained with DAPI (Gٞ,Hٞ; blue). Single channels are shown in grey. The asterisk in H marks clustering nuclei in a lateral line rosette. Scale bars: 30m, insets are 2ϫ magnifications of corresponding panels. (I,J)Slow-twitch muscle appeared normal at 5 dpf in control (I) and tnnt2c MO-injected (J) embryos. Scale bars: 50m.

hpf, actin staining in tnnt2c MO-injected embryos showed sarcomeres not lead to any gross morphological abnormalities or developmental that had grown laterally and were decorated with titin T12 (Fig. defect (data not shown). Immunostaining for myosin, and phalloidin 7G,H), although myofibrils were still wavy. Fast-twitch muscle at staining for actin at 48 hpf in tnnt2c MO-injected embryos showed this stage was normal. At 5 dpf, phalloidin staining showed a normal that slow-twitch muscle had almost completely recovered striations distribution of filamentous actin in the slow-twitch muscle of tnnt2c along their length, although myofibrils were thinner than in controls MO-injected embryos (Fig. 7J). (Fig. 8B,BЈ,F). Depletion of tnnt2d led to mild alterations of muscle We found that the recovery of striations in myofibrils of tnnt2c cell morphology, with some small myofibrils detaching from the MO-injected embryos after 30 hpf (supplementary material main bundles (Fig. 8C,CЈ,G; also observed at 5 dpf, Fig. 8J). When Fig. S8) is due to the expression of another slow-twitch-muscle- tnnt2c and tnnt2d were simultaneously disrupted, we found that specific troponin T encoded by a second, slow-twitch- slow-twitch muscle did not recover at 48 hpf (compare Fig. 8D,DЈ,H muscle-specific troponin T gene, tnnt2d (http://zfin.org/cgi-bin/ with 8B,BЈ,F), demonstrating that from 30 hpf tnnt2d is able to webdriver?MIvalaa-markerview.apg&OIDZDB-GENE-050626- provide enough troponin T to the slow-twitch muscle to compensate 97) (for nomenclature see Materials and Methods, Table 1 and for tnnt2c loss of function. supplementary material Fig. S1). We designed a translation-blocking We examined the earlier steps of sarcomere formation in slow- MO for tnnt2d and found that disruption of this gene on its own did twitch muscle at 14–16 stages. In control embryos, a few Sarcomere assembly requires troponin 573

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,AЈ) and in embryos injected with tnnt2c MO (B,BЈ), tnnt2d MO (C,CЈ) or co-injected with tnnt2d and tnnt2c MOs (D,DЈ) shows that myofibrillar organisation is lost in the double-injected embryos. Nuclei are counterstained with DAPI (blue). A–D are lateral views of rostral somites and AЈ–DЈ are higher magnification images showing muscle cells. In both cases, confocal images are from the most superficial layer of trunk muscles. Scale bars: 25m (A–D); 10m (AЈ–DЈ). (E–H) Phalloidin staining (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: 10m. (I–J) Slow myosin staining (white) of control (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 main bundles (white arrowheads). Scale bars: 30m, insets are 2ϫ magnifications of corresponding panels.

myofibrils could be detected in the anterior somites, whereas in that lack of assembly is due to downregulation of thin filament caudal somites rare striations were just becoming detectable genes and the consequent dearth of thin filament proteins (Huang

Journal of Cell Science (supplementary material Fig. S9A,C). In tnnt2c MO-injected et al., 2009; Sehnert et al., 2002). Similar observations, however, embryos we never observed similar structures, myosin appeared to have not been reported for mouse troponin mutants (Nishii et al., aggregate in rods or foci, actin appeared more diffuse, and overlap 2008). In D. melanogaster, downregulation of troponin I but not of the signal was less frequent (supplementary material Fig. S9B,D), of other major thin filaments genes has been reported in flight indicating that knockdown of tnnt2c impairs sarcomere assembly muscles of a troponin T mutant (Nongthomba et al., 2007). from the start. The consequences of tnnt2a disruption in the zebrafish heart have been further investigated with (Huang Discussion et al., 2009). These authors reported loss of periodicity for thin and In this study, we have demonstrated that troponin is required for thick filaments and for Z and M lines, similar to that observed in normal myofibrillogenesis. First, it is required to regulate actin– skeletal muscle. They concluded that tnnt2a is required for the myosin interactions, and second it affects proper thin filament striations of thin filaments, which in turn affects the periodicity of assembly. We also show that in the absence of normal thin filaments, Z bodies, M lines and thick filaments. assembly of rudimentary sarcomeres can occur. We show that the failure of assembly is principally due to the deregulated actin–myosin activity rather than to changes in the Sarcomeres do not form in the absence of functional abundance of thin filament components or to lack of thin filament troponins striations, because the regular distribution of major sarcomere Despite the observation that sarcomeric substructures can form components, lost in embryos depleted of the three troponin T and align in the absence of main sarcomere components, such as proteins, is restored by blocking myosin activity. thick filaments (Hawkins et al., 2008; Wohlgemuth et al., 2007), In the absence of troponin, actin and myosin are not found we did not find normal sarcomeres in muscles of embryos depleted together because their unregulated interaction is destructive. Instead, of troponin T, instead aggregates of Z line proteins and dispersed actin accumulates in aggregates and in abnormal Z lines, and thick filaments were observed from the very beginning of muscle myosin remains as dispersed thick filaments or appears in stacks differentiation. In silent heart mutants, defective for the cardiac of thick filaments in which myomesin can localise. Altered actin isoform troponin T2a, sarcomere assembly is impaired and dynamics in this context probably lead to abnormal accumulation dispersed thick filaments were reported (Sehnert et al., 2002). In of Z line proteins in substantially enlarged Z lines (Fig. 9B). that study, it was proposed that a common regulation of thin Additionally, our data indicate that the loss of a functional filament genes would be altered in the absence of troponin T and troponin complex independently affects proper thin filament 574 Journal of Cell Science 124 (4)

assembly (Fig. 9C). Although we found that the periodicity of filamentous actin is restored upon blocking actin–myosin activity and that actin filaments extend from expanded Z lines towards thick filaments, importantly we also found that filamentous actin is not associated with tropomyosin in the absence of troponin activity. Probably, actin filaments that are not decorated with troponin and tropomyosin cannot be detected in our electron microscopy preparations (Fig. 3). The association of tropomyosin with actin is stabilised by the binding of troponin T (Hitchcock- DeGregori and Heald, 1987), and it is likely that association of tropomyosin with actin becomes unstable in the troponin-T-depleted muscle. Our interpretation is that overall thin filament assembly is impaired in the absence of troponin; however, further analysis of the distribution of other proteins associated with thin filaments (e.g. ) will be needed to further support this conclusion. Our in situ hybridisation experiments did not reveal major changes in tropomyosin expression in the triple tnnt MO-injected embryos and the troponin gene tnni2 was only slightly upregulated while tnnt3b was slightly downregulated. Therefore, we suggest that Fig. 9. Consequences of the loss of troponin function. (A)Schematic transcriptional changes are not major determinants of the phenotype depiction of sarcomeres in the wild-type condition in which troponin (blue arising from the loss of troponin activity. ovals), on thin filaments (thick blue lines) together with tropomyosin, Disruption of single fast-twitch muscle tnnt genes causes only regulates actin–myosin interactions. Thick filaments are shown in green. partial loss of troponin activity because they are partially redundant. (B)In the absence of troponin, sarcomeres do not form, instead stacks of thick When we disrupt expression of a gene that is expressed early, such filaments, expanded Z lines (black ovals) and aggregates of filamentous actin as tnnt2c in slow-twitch muscle, then sarcomere assembly is (blue clouds) can be observed. (C)If in the absence of troponin, the actin– myosin activity is blocked, then sarcomeres form although filamentous actin impaired. When we disrupt expression of genes that become (thin blue lines) does not associate with tropomyosin and Z lines are expanded. functional later, after tissue differentiation is initiated, then the first steps of myofibril formation occur but deregulation of actin– myosin activity eventually leads to sarcomere disintegration, with et al., 2007). Similarly, one interpretation of our finding that normal sarcomeric components becoming abnormally redistributed to thin filaments are absent in the rudimentary sarcomeres restored distinct cellular domains. Thus, in the absence of functional troponin upon blocking unregulated actin–myosin activity in troponin-T- T, different sarcomeric substructures disintegrate independently depleted muscle could be that sarcomere assembly occurs before the complete loss of sarcomere integrity rather than independently of thin filament formation (Fig. 3D and Fig. 9C). disintegrating simultaneously. Recent work has suggested that myofibrillogenesis in zebrafish

Journal of Cell Science We were able to examine the function of another component of might proceed through stages that include the initial formation of the fast-twitch-muscle-specific troponin complex by using tnni2a.4 pre-myofibrils containing Z bodies and non-muscle myosin, nascent mutants. These mutants showed myofibril degradation with the myofibrils with non-aligned muscle myosin, and eventually mature same progression and timing as observed for tnnt3a MO-injected myofibrils with aligned thick filaments, consistent with previous embryos. Furthermore, similar to observations for tnnt3a, the observations using cultured avian muscle cells (Sanger et al., 2005; myosin inhibitor blebbistatin was able to suppress the tnni2a.4 Sanger et al., 2009). Alternative models imply the existence of mutant phenotype. As in the case of tnnt3a and tnnt3b, gene actin -fibre-like structures as the first template for sarcomere redundancy might explain the lack of early phenotype in tnni2a.4 assembly (Dlugosz et al., 1984) or the stitching of independently mutants. Six tnni2 genes are indeed reported in ZFIN, some of forming thick filaments and I–Z–I bodies (Holtzer et al., 1997). which are expressed in myotomes during somitogenesis. We found that slow muscle myosin becomes localised to Studies in D. melanogaster have shown that the hypercontraction sarcomeres in very young somites (supplementary material Fig. muscle phenotype caused by mutations in the troponin T or troponin S9) and that sarcomeres appear as early as the 15-somite stage, I genes is suppressed by myosin mutations that reduce actin– which is earlier than previously reported (Sanger et al., 2009). At myosin force production (Nongthomba et al., 2003). Our data are this stage, in wild-type embryos, filamentous actin is mainly consistent with the D. melanogaster data and validate them in a localised in unsegmented bundles running along the myocyte . Furthermore, our ability to resolve the sarcomere length, although some is localised to forming sarcomeres, where it composition with several markers at different time points and to overlaps with slow myosin. Our analysis of tnnt2c MO-injected partially suppress the defect with chemical inhibition of myosin embryos indicates that filamentous actin does not become periodic provides further insights into the mechanism. We have shown that if the troponin complex is defective, and that muscle myosin troponin is required for normal myofibrillogenesis and for proper remains in rodlets or aggregates. Despite a difference in the timing thin filament assembly, and that partial loss of troponin activity of muscle myosin appearance, our results indicate a transition from allows sarcomere assembly but that unregulated actin–myosin smaller immature sarcomeres to mature sarcomeres during recovery activity leads progressively to loss of sarcomeric integrity. in tnnt2c MO-injected embryos (Fig. 7). This is consistent with the pre-myofibril model. Unregulated actin–myosin contraction that is Sarcomere assembly models due to troponin loss of function is able to interfere with the laying It has been shown that sarcomeric components can acquire periodicity of pre-myofibrils because actin and Z lines are randomly distributed in the absence of thick filaments (Hawkins et al., 2008; Wohlgemuth in troponin-T-depleted embryos but align in blebbistatin-treated Sarcomere assembly requires troponin 575

embryos (Fig. 2). By contrast, thick filaments appear to be able to gene known as tnnt2, the silent heart locus, which we suggest should be renamed tnnt2a; therefore the gene most closely related to tnnt2a, LOC562296, should be form as independent units, even in the absence of organised thin called tnnt2b. Genes tnnt2a and tnnt2b both show obvious synteny to the human filaments. This is shown by the striated distribution of T11, which TNNT2 gene. There are three other genes encoding zebrafish troponin T2 isoforms recognises a titin epitope proximal to the ends of thick filaments, as defined by orthology. These were known as tnnt1, zgc:114184 and si:ch211- and of myomesin, which marks the M lines (Fig. 2), and by the 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 TNNT1 presence of thin filament-free, Z-line-free sarcomeres in electron gene and it appears to be a -specific gene. It shows conserved synteny with microscopy micrographs (Figs 1 and 3). In the absence of thin cryptochrome 1a, between zebrafish and stickleback, and we suggest it should be filaments and -actinin, titin might hold together the thick filament- renamed tnnt2c. The other two are duplicated genes in zebrafish, with apparently only one copy in stickleback and we suggest that these should be renamed tnnt2d rich substructures, possibly maintaining the attachment at the (zgc:114184) and tnnt2e (si:ch211-136g2.1), respectively. The zebrafish gene that position of the Z lines thanks to the protein (Bertz et al., clusters together with the human TNNT1 gene is presently called zgc:193865, which 2009). The T12 antibody that targets a portion of the protein at the we suggest should be called tnnt1. We have obtained approval for the proposed new Z line level appears randomly distributed, although this might be nomenclature and summarise the changes in Table 1. explained by the requirement of an intact Z line for recognition of Maintenance of zebrafish the epitope. Our data do not disprove the pre-myofibril model but Breeding zebrafish (Danio rerio) lines were maintained at 28°C on a cycle of 14 indicate that the role of non-muscle myosin and the dynamics of hours light and 10 hours dark. Fertilised eggs were obtained from natural spawning and grown in incubators at a temperature between 24°C and 28°C, depending on the thick filament assembly might still require further definition. stage required. Embryos were staged according to standard references (Kimmel et al., 1995). For imaging purposes, live embryos were manually dechorionated with Relevance to the human diseases #5 watchmaker’s forceps, when necessary anaesthetised with 0.02% tricaine (ethyl 3-aminobenzoate methanesulphonic acid), and mounted for viewing in 3% We found an excess number of zebrafish troponin genes as methylcellulose in embryo medium (Westerfield, 2000). Pictures were taken using a compared with mammalian orthologues and have proposed a new Zeiss Axio Imager M1 microscope and a Zeiss AxioCam HRc digital camera. nomenclature for this group of genes, which is summarised in Table 1. Notably, in human and rodent skeletal slow muscle, the Morpholino injections The following morpholino-modified antisense oligonucleotides were ordered from foetal troponin T function is provided by the cardiac gene TNNT2, Genetools: tnnt2c MO, 5Ј-CCACAAACTCTTCGGTGTCGCACAT-3Ј (ATG MO); which is then replaced by the slow-twitch-muscle-specific TNNT1 tnnt2d MO, 5Ј-GAAGTAAACACTCACGATTCTGTTG-3Ј (ATG MO); tnnt3a gene after birth (Jin, 1996; Jin et al., 2003). Consistently, we found MO, 5Ј-CTCAATGTCCTCTGTGTCTGACATG-3Ј (ATG MO); tnnt3b MO, 5Ј- AACATCCTCAGTATCAGACATGATG-3Ј (ATG MO); and standard control oligo, that embryonic genes expressed in zebrafish skeletal slow-twitch 5Ј-CCTCTTACCTCAGTTACAATTTATA-3Ј. muscle are more similar to the human TNNT2 than to the slow- Oligonucleotides were diluted in morpholino buffer (5 mg/ml phenol red, 4 mM twitch-muscle-specific TNNT1gene. HEPES pH 7.2, 160 mM KCl) and injected in 1–2 cell stage embryos. We used 6 ng for tnnt2c MO, 6 ng for tnnt2d MO, 3 ng for tnnt3a MO, 6 ng for tnnt3b MO Mutations in the human skeletal troponin T subunit are linked to and equal amounts of the control MO. Needles were pulled from glass two different pathologies, recessive and tubes using a needle puller (Kopf Instruments) and injections were performed using dominant distal arthrogryposis. In the case of Nemaline myopathy, a PV 820 Pneumatic Picoump micro-injector (World Precision Instruments). a stop mutation in the slow-twitch-specific troponin T described in , mRNA and DNA injections an Amish family generates a truncated protein that is rapidly degraded The coding sequence for the mCherry fluorescent protein (Shaner et al., 2004) was (Wang et al., 2005), suggesting that slow muscles in these patients amplified with the following primers: FmCheClav2, 5Ј-ggcggccatcgatATGGTC- TCTAAGGGCGAG-3Ј and RmCheEcoRInostop, 5Ј-ggcggccGAATTCaTATTTGTA- Journal of Cell Science are completely deficient in the troponin function. A hallmark of CAGTTCATC-3Ј. Nemaline myopathy is the accumulation of Z line proteins in The second primer was a modified reverse primer that abolished the stop codon. aggregates called nemaline rods, which resemble the accumulations This insert was cloned in pCS2 using ClaI and EcoRI to generate the pCS2mCherry of Z line proteins that we observed in micrographs from tnnt2c MO- vector. The tropomyosin 3 (tpma) and troponin T2c (tnnt2c) coding sequences were amplified by RT-PCR from 48 hpf embryo RNA with the following primers: or triple tnnt MO-injected embryos, indicating that the troponin T FTpmaEcoRI, 5Ј-ggcggccGAATTCATGGATGCCATTAAGAAG-3Ј; RTpmaXbaI, loss of function leads to a similar myopathy in human and zebrafish. 5Ј-ggcggccTCTAGATTAAATGGAGGTCATGTC-3Ј; FTnnt2cEcoRInew, 5Ј-ggc - Missense or stop mutations in the fast-twitch-specific troponin ggccGAATTCATGTGCGACACCGAAGAG-3Ј and RTnnt2cXhoInew, 5Ј-ggcggcc- T and troponin I genes cause distal arthrogryposis, a syndrome CTCGAGTTACTTCCAGGACTTCCT-3Ј. PCR fragments were digested with EcoRI and XbaI or EcoRI and XhoI and cloned characterised by limb with muscular weakness but no downstream of the mCherry coding sequence in the pCS2mCherry vector, and muscle (Ochala, 2008). We showed that there are no were sequence verified. The pCS2mCherry–Tpma plasmid was linearised intrinsic differences in the mechanisms of sarcomere assembly and with NotI and used as a template to obtain mRNA in an reaction with the RNA polymerase SP6 (Biolabs). Typically, 150 pg of mRNA were injected loss between slow and fast fibres in the absence of normal troponin in one-cell embryos. For DNA injections, the coding sequence for the fusion protein and, therefore, we believe that the differences in the human TNNT1 mCherry–Tnnt2c was amplified from the pCS2mCherry–Tnnt2c vector with the versus TNNT3 or TNNI2 can be explained by the following primers: FNheI–mCherry, 5Ј-ggcggccGCTAG CatgGTCTC TAAGGG - Ј Ј nature of the mutations. In the case of distal arthrogryposis, these CGAG-3 and RBglII–Tnnt2c, 5 -ggcggccAGATCTTTACTTCCAGGACTTCCT- 3Ј. are often dominant missense mutations that lead to a reduction in The PCR fragment was digested with NheI and BglII and cloned in the backbone the contractile force rather than causing major changes in the of the pECFP–ER vector (Clontech) digested with the same restriction to distribution of myofibrillar components. remove the 846 bp CFP–ER sequence. The pEmCherry–Tnnt2c vector was linearised with StuI and purified with phenol/chloroform; 60 pg was injected into one-cell embryos. Both in the mRNA and DNA injections experiments, fluorescence was Materials and Methods detected in live 24 and 48 hpf embryos using a 40ϫ water immersion lens on a Leica Orthology analysis SP5 confocal microscope. Fluorescent embryos were fixed in 4% paraformaldehyde We have used TreeFam, a database of phylogenetic trees of animal genes and processed as described in the following subsection. (www.treefam.org) (Li et al., 2006; Ruan et al., 2008), to verify the number of troponin genes present in the zebrafish genome. We found that there are four genes Immunolabelling and imaging encoding troponin C (TreeFam TF318191), 13 genes encoding troponin I (TreeFam For phalloidin staining, embryos were fixed at the appropriate stage in 4% TF313374) and eight genes encoding troponin T (TreeFam TF313321). paraformaldehyde in PBS overnight at 4°C, washed with PBS containing 2% Triton We aligned the sequences of the human, mouse, zebrafish and stickleback troponin X-100, incubated with Alexa Fluor 488 or Alexa Fluor 568 conjugated to phalloidin T proteins using ClustalW2 (http://www.ebi.ac.uk/Tools/clustalw2/index.html). We (Invitrogen), resuspended according to the manufacturer’s instructions and diluted found five zebrafish genes in the human TNNT2 branch, one is the cardiac-specific 1:100 in PBS, washed in PBS containing 0.1% Tween and mounted with Vectashield 576 Journal of Cell Science 124 (4)

Mounting Media with DAPI (Vector ). For fluorescence Caenorhabditis elegans muscle structure and function caused by mutation of troponin , embryos were fixed at the appropriate stage overnight in I. Biophys. J. 86, 991-1001. methanol at –20°C or in 4% paraformaldehyde in PBS at 4°C. B4 (myomesin), F59 Devoto, S. H., Melancon, E., Eisen, J. S. and Westerfield, M. (1996). Identification of (slow myosin), EB165 (fast myosin), CH1 (tropomyosin) and Ct3 (troponin T) separate slow and fast muscle precursor cells , prior to somite formation. antibodies were obtained from the Developmental Studies Hybridoma Bank and used Development 122, 3371-3380. 1:5 (if obtained as supernatant) or 1:100 (if obtained as concentrate). Anti-titin T12, a Dlugosz, A. A., Antin, P. B., Nachmias, V. T. and Holtzer, H. (1984). The relationship gift from Yaniv Hinits (King’s College, London, UK) was used 1:10. Anti -actinin, between stress -like structures and nascent myofibrils in cultured cardiac myocytes. anti-titin T11 and anti-troponin JLT-12 were purchased from Sigma (A7811, T9030 J. Cell Biol. 99, 2268-2278. and T6277) and used 1:100. The secondary antibody used was Alexa-Fluor-488- Fu, C. Y., Lee, H. C. and Tsai, H. J. (2009). The molecular structures and expression patterns of zebrafish troponin I genes. Gene Expr. Patterns 9, 348-356. labelled goat anti-mouse IgG (A-11001; Invitrogen). Embryos were washed several Fyrberg, E., Fyrberg, C. C., Beall, C. and Saville, D. L. (1990). Drosophila melanogaster times in PBS containing 0.1% Triton X-100 and 0.2% BSA, incubated overnight in troponin-T mutations engender three distinct syndromes of myofibrillar abnormalities. the same buffer with 5% of goat serum and primary antibodies, washed again, J. Mol. Biol. 216, 657-675. incubated with secondary antibody overnight, then washed and mounted with Galinska-Rakoczy, A., Engel, P., Xu, C., Jung, H., Craig, R., Tobacman, L. S. and Vectashield Mounting Media with DAPI (Vector Laboratories). For double-staining, Lehman, W. (2008). Structural basis for the regulation of muscle contraction by phalloidin was added together with the secondary antibody. Samples were imaged with troponin and tropomyosin. J. Mol. Biol. 379, 929-935. the Leica SP5 confocal microscope using a 40ϫ or a 63ϫ oil immersion objective. Granato, M., van Eeden, F. J., Schach, U., Trowe, T., Brand, M., Furutani-Seiki, M., Haffter, P., Hammerschmidt, M., Heisenberg, C. P., Jiang, Y. J. et al. (1996). Genes Electron microscopy controlling and mediating locomotion behavior of the zebrafish embryo and larva. Embryos were immersed in freshly prepared primary fixative containing 2% Development 123, 399-413. paraformaldehyde with 2% glutaraldehyde in 0.1M sodium cacodylate buffer (pH Hawkins, T. A., Haramis, A. P., Etard, C., Prodromou, C., Vaughan, C. K., Ashworth, 7.42) containing 0.1% magnesium chloride and 0.05% and calcium chloride at 20°C R., Ray, S., Behra, M., Holder, N., Talbot, W. S. et al. (2008). The ATPase-dependent for 10 minutes before transferring to an ice bath for the remainder of 2 hours. chaperoning activity of Hsp90a regulates thick filament formation and integration Samples were rinsed three times for 10 minutes each in sodium cacodylate buffer during skeletal muscle myofibrillogenesis. Development 135, 1147-1156. with added chlorides on ice. Secondary fixation with 1% osmium tetroxide in Hitchcock-DeGregori, S. E. and Heald, R. W. (1987). Altered actin and troponin binding sodium cacodylate buffer only was carried out at room temperature for 1 hour. All of amino-terminal variants of chicken striated muscle alpha-tropomyosin expressed in following steps were performed at room temperature. Embryos were rinsed three Escherichia coli. J. Biol. Chem. 262, 9730-9735. times in cacodylate buffer over 30 minutes and mordanted with 1% tannic acid for Holtzer, H., Hijikata, T., Lin, Z. X., Zhang, Z. Q., Holtzer, S., Protasi, F., Franzini- Armstrong, C. and Sweeney, H. L. (1997). Independent assembly of 1.6 microns long 30 minutes followed by a rinse with 1% sodium sulphate for 10 minutes. Samples bipolar MHC filaments and I-Z-I bodies. Cell Struct. Funct. 22, 83-93. were dehydrated through an ethanol series of 20, 30 (staining en bloc with 2% uranyl ϫ Hsiao, C. D., Tsai, W. Y., Horng, L. S. and Tsai, H. J. (2003). Molecular structure and acetate at this stage), 50, 70, 90 and 95% for 20 minutes each then 100% for 3 20 developmental expression of three muscle-type troponin T genes in zebrafish. Dev. Dyn. ϫ minutes. Ethanol was exchanged for propylene oxide (PO) for 2 15 minutes 227, 266-279. followed by 1:1 PO to Epon resin mixture for at least 1 hour, and neat Epon (with Huang, W., Zhang, R. and Xu, X. (2009). Myofibrillogenesis in the developing zebrafish a few drops of PO) overnight. Embryos were embedded in a flat moulded tray with heart: A functional study of tnnt2. Dev. Biol. 331, 237-249. fresh resin and cured in an oven at 65°C for 24 hours. We cut 40-nm sections on a Jin, J. P. (1996). Alternative RNA splicing-generated cardiac troponin T isoform switching: Leica UCT ultramicrotome, stained the sections with uranyl acetate and lead citrate a non-heart-restricted genetic programming synchronized in developing cardiac and for contrast, and imaged on an FEI 120kV Spirit Biotwin using an F415 Tietz CCD skeletal muscles. Biochem. Biophys. Res. Comm. 225, 883-889. camera. Jin, J. P., Brotto, M. A., Hossain, M. M., Huang, Q. Q., Brotto, L. S., Nosek, T. M., Morton, D. H. and Crawford, T. O. (2003). Truncation by Glu180 nonsense mutation Genotyping results in complete loss of slow skeletal muscle troponin T in a lethal nemaline We genotyped tnni2sa0058 embryos using the following nested primers: tnni2-4-1, 5Ј- myopathy. J. Biol. Chem. 278, 26159-26165. GAGAACAAACATCTGACAATGG-3Ј; tnni2-4-2, 5Ј-GGTCAATTAA ATGATT - Johnston, J. J., Kelley, R. I., Crawford, T. O., Morton, D. H., Agarwala, R., Koch, T., TGAAGC-3Ј; tnni2-4-3, 5Ј-TTCTTGAACTTGCCCTTCAG-3Ј; and tnni2-4-4, Schaffer, A. A., Francomano, C. A. and Biesecker, L. G. (2000). A novel nemaline 5Ј-CAGCAGAGCCTGAAGCATAG-3Ј. Whole 5 dpf embryos were fixed in myopathy in the Amish caused by a mutation in troponin T1. Am. J. Hum. Genet. 67, methanol and then dried on a heat block. Genomic DNA was extracted by incubating 814-821. u45 Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B. and Schilling, T. F. (1995). Journal of Cell Science with proteinase K at 55°C overnight. Mutant slo embryos are easily distinguished Stages of embryonic development of the zebrafish. Dev. Dyn. 203, 253-310. from wild-type siblings because they are completely immotile. Kimura, A., Harada, H., Park, J. E., Nishi, H., Satoh, M., Takahashi, M., Hiroi, S., Sasaoka, T., Ohbuchi, N., Nakamura, T. et al. (1997). Mutations in the cardiac Blebbistatin treatment troponin I gene associated with hypertrophic . Nat. Genet. 16, 379-382. Blebbistatin was purchased from Sigma (B0560-1MG) and resuspended in DMSO. Landstrom, A. P., Parvatiyar, M. S., Pinto, J. R., Marquardt, M. L., Bos, J. M., Tester, A final concentration of 6 M was added to embryo medium at 28 hpf, and fish were D. J., Ommen, S. R., Potter, J. D. and Ackerman, M. J. (2008). Molecular and collected at 48 hpf. DMSO only was added to untreated controls. functional characterization of novel hypertrophic cardiomyopathy susceptibility mutations in TNNC1-encoded troponin C. J. Mol. Cell. Cardiol. 45, 281-288. 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