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Cardiovascular Research Advance Access originally published online on June 25, 2008
Cardiovascular Research 2008 80(2):200-208; doi:10.1093/cvr/cvn177
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Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2008. For permissions please email: journals.permissions@oxfordjournals.org

Heart-specific isoform of tropomyosin4 is essential for heartbeat in zebrafish embryos

Long Zhao1,{dagger}, Xinyi Zhao1,{dagger}, Tian Tian1,{dagger}, Quanlong Lu2, Nirma Skrbo-Larssen1, Di Wu1, Zheng Kuang1, Xiaofeng Zheng1, Yanchao Han1, Shuyan Yang1, Chuanmao Zhang2 and Anming Meng1,*

1 Protein Science Laboratory of the Ministry of Education, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, People's Republic of China
2 Laboratory of Cell Proliferation and Differentiation of the Ministry of Education, College of Life Sciences, Peking University, Beijing 100871, People's Republic of China

* Corresponding author. Tel: +86 10 62772256; fax: +86 10 62794401. E-mail address: mengam{at}mail.tsinghua.edu.cn

Received 12 February 2008; revised 17 May 2008; accepted 19 June 2008

Time for primary review: 34 days


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 
Aims: Tropomyosin (Tpm) proteins, encoded by four Tpm genes (Tpm1–4), are associated with the stabilization of the F-actin filaments and play important roles in modulating muscle contraction. So far, little is known about Tpm4 function in embryonic heart development and its involvement in the cardiovascular diseases. In this study, we investigated functions of different isoforms of tpm4 in embryonic heartbeat in zebrafish.

Methods and results: The transgenic zebrafish line, T2EGEZ8, was generated by insertion of a Tol2 transposon gene trap vector, and homozygous mutants (T2EGEZ8m/m) of this line showed failure of embryonic heartbeat without other detectable phenotypes. Observation by transmission electron microscopy revealed that the ventricular myocytes of mutant fish contained fewer, disorganized myofibrillar filaments. The transposon genome in T2EGEZ8 fish was found by thermal asymmetric interlaced-polymerase chain reaction (TAIL-PCR) and reverse transcription-polymerase chain reaction to have inserted into the ninth intron of the tpm4 locus, which resulted in production of Tpm4-GFP fusion proteins and loss of normal transcripts tpm4-tv1 and tpm4-tv2. Whole-mount in situ hybridization indicated that tpm4-tv1, encoding a peptide of 284 residues, is specifically expressed in the heart of zebrafish embryos, while tpm4-tv2, encoding a peptide of 248 residues, is mainly present in the vasculature but absent in the heart. Knockdown of tpm4-tv1 and tpm4-tv2 within wild-type embryos led to the failure of heartbeat, which could be rescued by coinjection with tpm4-tv1 mRNA but not with tpm4-tv2 mRNA.

Conclusion: Tpm4-tv1 is a heart-specific isoform of Tpm4 and is essential for heartbeat in zebrafish embryos.

KEYWORDS Zebrafish; Heart; Tropomyosin; Transposon; Thin filament


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 
The pump function of the heart relies on mechanical properties of myofibrils, each of which consists of thin and thick filaments. Tropomyosin (Tpm) dimers in the form of {alpha}-helical coiled-coil wrap around filamentous actin (F-actin) along the thin filament to stabilize the filament.1–3 Tpm is also bound by troponin proteins to form complexes that play an essential role in the regulation of myofilament contractile activation.4 So far, four Tpm genes, Tpm1–4, have been identified in vertebrates. Each Tpm gene can express multiple isoforms by alternative uses of exons and promoters.5 Among these Tpm genes, Tpm1 is the only one that has been found to play an important role in cardiac muscle function and has been shown to associate with familial hypertrophic cardiomyopathy in human.6,7 In contrast, mutations in the TPM2 or TPM3 locus have been found to associate with nemaline myopathy or cap myopathy in skeletal muscle.5,8 In cardiac lethal mutant Mexican axolotl, the embryonic heart has lower levels of Tpm proteins and myosin and fails to beat.9–11 Lack of sarcomeric myofilbrils and heartbeat of this axolotl mutant can be rescued by overexpression of murine {alpha}-Tpm/Tpm1 or ATmC-3/Tpm4,12,13 suggesting that lack of either Tpm1 or Tpm4 is responsible for the heartbeat failure. Interestingly, the 5'UTR and the coding regions of Tpm4 cDNA isolated from the mutant axolotl hearts do not carry any mutations,13 which questions the fundamental role of Tpm4 in the heartbeat of the axolotl embryos. Thus, it remains unclear whether Tpm4 is required for the heart development and function. The involvement of Tpm2 and Tpm3 in the heartbeat of vertebrate embryos is unknown.

Chemical mutagenesis in zebrafish has allowed clarification of functions of several cardiac filament components.14–18 Recently developed Tol2 transposon technology has been successfully applied to make transgenic lines, and to trap tissue-specific enhancers or developmentally regulated genes in zebrafish.19 The Tol2 transposon-mediated enhancer and gene trap approaches have been successfully used to generate insertional mutants,20,21 opening alternative ways to create developmental mutants in zebrafish.

In this paper, we report a zebrafish mutant with no heartbeat that was generated by Tol2 transposon-mediated insertional mutagenesis. The heartbeat failure in the mutant embryos is due to reduced amounts and disorganization of myofibrillar filaments in ventricular myocytes. We demonstrate that the mutant phenotype is caused by the absence of functional variant 1 of Tpm4, the longer isoform of 284 amino acids, which is specifically expressed in the embryonic heart.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 
The investigation conformed with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

2.1 Fish and gene trapping
Gene trap vector T2EGE was modified from T2KSAG19 by replacing the original splicing acceptor (second intron of rabbit β-globin gene) and the SV40 poly(A) signal with the splicing acceptor of the first intron and the poly(A) signal of zebrafish ef1{alpha} gene, respectively. One-cell embryos from AB strain were injected with 30–50 pg T2EGE DNA and 30–100 pg transposase mRNA. The injected founder (F0) fish were crossed to wild-type fish of AB strain, and their progeny (F1) were observed for GFP expression using Leica M16F microscope. The GFP-positive transgenic embryos, in which GFP expression pattern was presumably identical to that of the trapped gene, were grown up to establish transgenic lines. In this pilot experiment, we established 11 transgenic lines that expressed GFP in various tissues, 2 of which, including T2EGEZ8, gave rise to mutant phenotypes when bred to homozygotes. The genotypes of heterozygous and homozygous fish of this line were indicated by T2EGEZ8+/m and T2EGEZ8m/m, respectively.

Tg(flk1:RFP);T2EGEZ8+/m double transgenic embryos were generated by crossing Tg(flk1:RFP) homozygous fish, which express RFP in vascular vessels, to T2EGEZ8+/m fish. Tg(flk1:RFP);T2EGEZ8m/m were obtained by crossing Tg(flk1:RFP);T2EGEZ8+/m fish.

2.2 TAIL-PCR, RT–PCR, and tpm4 cDNA cloning
To identify genomic sequences flanking integrated transposon, TAIL-PCR was performed essentially as described by Parinov et al.22 Total RNA was extracted from 35 wild-type or transgenic embryos using the Trizon RNA isolation kit (Invitrogen) and used to make the first-strand cDNA by reverse transcription using M-MLV reverse Transcriptase (Promega). Specific primer sets for RT–PCR included V1D5 (5'-GAGAGGCATGAAGGTAATCGAG-3') and V1B3 (5'-AGTAGAGCGAGACAGATGGATC-3') for tpm4-tv1; V2D5 (5'-ATGAAGGACGAGGAGAAGATGGAG-3') and V2D3 (5'-GCATTCCCAGGTTTTCTTCTTTTG-3') for tpm4-tv2; V1D5 and EGFPr3 (5'-GCTGAACTTGTGGCCGTTTAC-3') for tpm4-tv1-GFP fusion transcript; V2D5 and EGFPr3 for tpm4-tv2-GFP fusion transcript; actin5 (5'-ATGGATGATGAAATTGCCGCAC-3') and actin3 (5'-ACCATCACCAGAGTCCATCACG-3') for β-actin. PCR reactions were carried out as follows: denaturation for 3 min at 94°C; 33 cycles (94°C for 45 s; 56°C for 45 s; 72°C for 60 s); final extension for 7 min at 72°C. PCR products were analysed on 2% agarose gel.

The coding sequences of tpm4-tv1 (GenBank accession number NM_001024467), tpm4-stv1 lacking the exon 9 of tpm4-tv1 and tpm4-tv2 (GenBank accession number NM_213158 [GenBank] ) were amplified by RT–PCR using primers V1-B5 (5'-AGATCAACCTGCAACCATGGAG-3') and V1-B3 (5'- AGTAGAGCGAGACAGATGGATC-3') for tpm4-tv1 or V1-B5 and sV1E3 (5'-TTAAATGGAGGTCATGTCGTTG-3') for tpm4-stv1 or V2-B5 (5'- ATGACAGGTGTGACTTCTTTGGAC-3') and V2-3B (5'- ACGTCGTTTTCTCTCATCGTCTTG-3') for tpm4-tv2. The resulted products were cloned into the vector pXT7 for in vitro synthesis of mRNA. Amplification of tpm4-tv1-specific, tpm4-tv2-specific and consensus cDNA sequences used the following primer sets V1S5 (5'-CGGGATCCATTTCTGGCAGCTCCTGGTTGCATG-3') and V1S3 (5'- CCCAAGCTTGTCAGCAGCTTTTTTCTCAGACAG-3'), V2S5 (5'- CGGGATCCAAAATTATCTTCTGCAAAAGAAG-3') and V2S3 (5'- CCCAAGCTTATAAAAATTTATTACACAGAAAGTG-3'), and C5 (5'-AAGCTGCTGACGCGGAGGGTGATG-3') and C3 (5'-GAGTTCATCTTCAAGGTCATCAATG-3'), respectively. These sequences were individually cloned into pXT vector, which lack β-globin-derived untranslated regions, for making in situ hybridization probes.

2.3 Whole-mount in situ hybridization, morpholinos, microinjection and genotyping
Digoxigenin-UTP-labelled antisense RNA probes and capped mRNA were synthesized by in vitro transcription (Roche). The used morpholinos (MO) were V1MO1 (5'-ATCTGGTGTTCTGACGAGAAATT-3'), V1MO2 (5'-TTGAAAGTGTGGTCAGACTAAT-3'), V2MO1 (5'-AGGCTAAAAACTCTATTGTACCCCG-3'), V2MO2 (5'-GTCACACCTGTCATTTTAACTAGCT-3'), and sMO1 (5'-GTAGACGATACCATTCAGCGACTTC-3'). MOs or mRNAs were injected into yolk of embryos at the one-cell stage.

T2EGEZ8+/m and T2EGEZ8m/m embryos were separated from wild-type embryos based on the presence of GFP fluorescence. For separating T2EGEZ8m/m from T2EGEZ8+/m at or before 24 h post-fertilization (hpf), genomic DNA was extracted from single embryos after whole-mount in situ hybridization and photography. Wild-type tpm4-tv1 allele was amplified using primers Tpm4IN5 (5'-ATGCTTCCTTCAGATGACTCAAAC-3') and Tpm4IN3 (5'-GGACTAAACTTTGTGATCTCGTAC-3'). The presence or the absence of a 592 bp fragment identified T2EGEZ8+/m or T2EGEZ8m/m embryos, respectively.

2.4 Transmission electron microscopy
The whole embryos or isolated hearts at 3-day post-fertilization (dpf) and 5 dpf were fixed, embedded in the Spur resin, and sectioned. The sections were stained with uranyl acetate and lead citrate and observed under a transmission electron microscope (JEM-1010).


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Supplementary material
 Funding
 References
 
3.1 Expression pattern of the trapped gene in the T2EGEZ8 trapped line
We performed an insertional mutagenesis using a modified Tol2-based transposon trap vector, T2EGE, which contained a promoterless GFP reporter. One of our trapped transgenic lines, T2EGEZ8, showed GFP expression in the heart and other tissues. Because a proportion of T2EGEZ8m/m homozygous embryos exhibited heartbeat failure, we characterized this line in detail.

Observation of 1,454 embryos from 15 pairwise crosses of T2EGEZ8+/m heterozygous fish identified 26.5% of embryos with brighter GFP (homozygous insertion), 49.8% with weaker GFP (heterozygous insertion) and 23.7% without GFP (no insertion), showing an inheritance fashion in accordance with Mendel's law of segregation. When heterozygous fish were mated to wild-type fish, half of their progeny displayed an identical GFP expression pattern and the other half had no GFP, suggesting that a single insertion is directly associated with GFP expression.

GFP expression in T2EGEZ8 embryos becomes detectable in the head and lateral plate mesoderm, the notochord and the neural tube during mid-segmentation (Figure 1A). At 1 dpf, GFP is prominent in various head and trunk vasculature, including metencephalic artery, middle cerebral vein, optic veins, dorsal aorta, cardinal vein, and intersegmental vessels, as well as in somitic boundaries, horizontal myoseptum, and the presumptive floor plate (Figure 1B). At Day 2, GFP expression pattern is not altered (Figure 1C and E–H), but GFP is more clearly seen in branchial arteries (Figure 1E) and in both the atrium and ventricle of the heart (Figure 1F). Around Day 5, hepatic vessels, swim bladder arteries and intestinal vessels are also GFP-positive (Figure 1L–N). In Tg(flk1:RFP);T2EGEZ8 double transgenic embryos, RFP-positive vascular endothelial cells are also GFP-positive (Figure 1I), suggesting the expression of the trapped gene in these cells. In addition, we noted that some circulating blood cells, presumably macrophages, were GFP-positive after 3 days of development (Supplementary material online, Movie S1). These observations suggest that the trapped gene in T2EGEZ8 fish may be primarily involved in the development of the heart and the vasculature.


Figure 1
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  		Figure 1 Expression pattern of the trapped gene in T2EGEZ8 transgenic fish and phenotypes of mutant embryos. (A–C, and L) Fluorescent images of wild-type (+/+), heterozygous (+/m) and homozygous (m/m) siblings at the 15-somite stage (A), 36 hpf (B), 48 hpf (C), and 5 dpf (L), showing GFP expression. Arrowheads and arrows in (B and C) indicate the middle cerebral vein/metencephalic artery and the optic vein, respectively. (D) Bright-field images of 48-hpf live embryos. (E–G) Boxed areas in (C) at a higher magnification, showing GFP in branchial arteries (E), heart (F), the floor plate (G, red arrows), horizontal myoseptum (G, red arrowheads), and dorsal aorta (G, yellow arrows), respectively. (H) A cross-section at the posterior region of a 48-hpf heterozygote, showing GFP locations as indicated in (G). (I) A posterior trunk region of a Tg(flk1:RFP);T2EGEZ8m/m double transgenic embryos. The superimposed image showed partially overlapping of flk1 expression (red) and the trapped gene (green) in the dorsal aorta and intersegmental vessels. The arrowheads indicate the somitic boundaries. (J and K) Hearts of wild-type (J) and homozygous (K) embryos at a higher magnification, which were boxed in (D). The ventricle and the atrium in (F, J, and K) were indicated by V and A, respectively. (M and N) Boxed areas in (L) at a higher magnification, showing GFP in aortic arches (M) and in intestinal vessels (N) of the heterozygous embryo, respectively.

 
3.2 Failure of the heartbeat in T2EGEZ8 homozygous mutant embryos
In wild-type zebrafish, the embryonic heart beats at 26–28 hpf, resulting in circulation of haematopoietic cells. We found that the heart of T2EGEZ8 homozygous embryos, which were separated from heterozygotes by their higher levels of GFP, never beat visibly (Supplementary material online, Movies S2 andS3, and see Supplementary material online, Figure S1) and died at 7–9 dpf. Homozygous mutants started to display pericardial oedema, an enlarged heart and smaller eyes at 2 dpf (Figure 1D, J and K). The number of blood cells in homozygous mutants appeared to be comparable to that in wild-type or heterozygous siblings (Figure 1D), but they did not visibly flow (Supplementary material online, Movie S4 and see Supplementary material online, Figure S1), suggesting normal haematopoiesis in the mutants. At 5 dpf, 31 of 116 mutants had blood cells in the atrium and another 31 of 116 mutants had these cells in both the atrium and ventricles, indicating that the vasculature might have functioned normally. Considering that the mutants had no heartbeats, we speculated that the flow of blood cells could be driven by mechanical movement of the whole body. This hypothesis was confirmed by the observation that touch-induced movements of 3-day mutant embryos caused immediate rostral flow of blood cells in the posterior caudal vein (data not shown).

The homozygous embryos at 48 hpf moved normally responding to a mechanical touch (Supplementary material online, Movies S5 and S6), implying that the neuronal networks and the muscular system may not been affected. T2EGEZ8 heterozygous embryos survived to adulthood. Taken together, our data suggest that the transposon insertion in T2EGEZ8 fish disrupts a gene essential for early heart function.

3.3 Normal expression of heart-, vasculature-, or musculature-specific markers in T2EGEZ8 mutant embryos
To confirm defects in T2EGEZ8 mutants, we examined the expression of genes that are involved in the development of the heart, vasculature, and musculature. The expression level of the cardiac mesoderm marker nkx2.5, the heart tube marker cmlc2, the atrial marker amhc, and the ventricular marker vmhc was unaltered at various stages in the mutant embryos (Figure 2), suggesting that the specification of the cardiac mesoderm and the formation of the heart chambers are not affected. Similarly, the haemangioblast marker scl1, the haematopoietic stem cell marker gata2, and the vascular endothelial markers fli1a, flk1, and flt4 all exhibited normal expression patterns in mutants (see Supplementary material online, Figure S2), indicating that vasculogenesis, angiogenesis, and haematopoiesis took place normally in these embryos. The erythroid/myeloid progenitor marker gata1 was expressed normally in mutants before circulation started. When the gata1-positive cells in wild-type siblings all had entered the circulation at 36 hpf, however, these cells in the mutants were still present in posterior region of the intermediate cell mass (ICM) (see Supplementary material online, Figure S2C), indicative of a secondary effect of the heartbeat failure. Additionally, we examined the expression of the muscle marker myoD and the somite marker tgfbi, and failed to detect abnormal expression (see Supplementary material online, Figure S3A and B). The organization of actin filaments in fast muscle, slow muscle, and myoseptum, which were detected by phalloidin staining, appeared to be normal in 3-day mutants (see Supplementary material online, Figure S3C and D). Thus, the disruption of the trapped gene in T2EGEZ8 may not interfere with the formation of the skeletal musculature. We hypothesize that loss of Tpm4 function in the developmental processes/tissues discussed here might have been compensated by other members of the Tropomyson family in the mutants.


Figure 2
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  		Figure 2 Normal expression of the heart-specific markers in T2EGEZ8 mutant embryos. (A) Expression of the cardiac mesoderm marker nkx2.5. In the top panel were dorsal views, and in the lower panel were lateral views. (B) Expression of the heart tube marker cmlc2. Anteroventral views. (C) Expression of the atrial marker amhc. (D) Expression of the ventricular marker vmhc. (C and D) Anteroventral views in the top panel; anterodorsal views in the lower panel. Note that the expression level of these markers in mutant (m/m) embryos was comparable to that in wild-type (+/+) embryos. The ventricle and the atrium were indicated by yellow arrows and red arrowheads, respectively.

 
3.4 Interruption of tpm4 gene by the transposon insertion in T2EGEZ8 transgenic line
We then went on to identify the gene trapped in T2EGEZ8 transgenic fish. A total of eight fragments were obtained through TAIL-PCR. Sequencing analysis revealed that five of these fragments were derived from the tpm4 locus, two represented vector integration without transposon excision, and one was a sequence of unknown characteristics. Further sequencing and bioinformatics analyses disclosed that the transposon was integrated into the ninth intron between exons 8 and 9a of the tpm4 locus (Figure 3A). The tpm4 gene is predicted to express two transcript variants, tpm4-tv1 and tpm4-tv2, by using alternative promoter and exons. tpm4-tv1 and tpm4-tv2 share exons 3, 4, 5, 6b, 7, and 8, and encode 284- and 248 amino acids proteins, respectively. The integration of the transposon downstream the common eighth exon was expected to result in production of fusion transcripts tpm4-tv1-GFP (v1G) and tpm4-tv2-GFP (v2G) in T2EGEZ8 transgenic fish (Figure 3B). We confirmed the presence of v1G and v2G transcripts in both T2EGEZ8 homozygotes and heterozygotes by RT–PCR analysis using specific primers (Figure 3C). Importantly, wild-type tpm4-tv1 and tpm4-tv2 were not detected in the homozygotes by RT–PCR; and their amounts in heterozygotes were lower than that in wild-type siblings. This suggests that the transposon insertion completely eliminates the expression of wild-type tpm4 mRNAs, and that their absence is related to the heartbeat failure in T2EGEZ8 mutants.


Figure 3
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  		Figure 3 Zebrafish tpm4 gene organization and interruption by transposon insertion. (A) Illustration of tpm4 genomic organization and its two transcript variants and their protein products in wild-type fish. V1, tpm4-tv1 transcript; V2, tpm4-tv2 transcript; pV1, Tpm4-tv1 protein; pV2, Tpm4-tv2 protein. (B) Illustration of organization of the interrupted tpm4 locus and its two transcript variants and their protein products in T2EGEZ8 fish. V1G, tpm4-tv1-GFP fusion transcript; V2G, tpm4-tv2-GFP fusion transcript; pV1G, Tpm4-tv1-GFP fusion protein; pV2G, Tpm4-tv2-GFP fusion protein. (C) Identification by RT–PCR of transcript variants in wild-type (+/+), heterozygous (+/m), and homozygous (m/m) embryos at 36 hpf. The tpm4-tv1, tpm4-tv2, tpm4-tv1-GFP, and tpm4-tv2-GFP transcripts were detected with primer sets V1D5/V1B3, V2D5/V2D3, V1D5/EGFPr3, and V2D5/EGFPr3, respectively. The relative positions of primers were shown in (A and B). The reaction control (c) had all reaction components except the template. Note that wild-type transcripts were absent in homozygous embryos.

 
3.5 Distinct expression patterns of two variants of tpm4 during embryogenesis
We examined spatiotemporal expression of tpm4-tv1 and tpm4-tv2 during zebrafish embryogenesis by whole-mount in situ hybridization using variant-specific probes (Figure 4A). tpm4-tv1 transcript became detectable around the 20-somite stage in the presumptive myocardial cells (data not shown). This variant was expressed evenly in the whole heart tube before 40 hpf (Figure 4B–D). Its expression level was higher in the ventricle than in the atrium at 48 hpf (Figure 4E) and became very low at 72 hpf (Figure 4F). Thus, tpm4-tv1 is a heart-specific isoform in zebrafish.


Figure 4
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  		Figure 4 Differential expression patterns of tpm4-tv1 and tpm4-tv2 during embryogenesis detected by in situ hybridization. (A) Locations of the sequences used for making variant-specific probes. Between vertical red lines was the corresponding coding sequence and the black region is shared by both variants. tv1s, tpm4-tv1-specific probe; tv2s, tpm4-tv2-specifc probe; and tvs, common probe for both variants. (B–F) Expression of tpm4-tv1, detected by the tv1s probe, in the heart at 20 hpf (B) (lateral view), 24 hpf (C) (lateral view), 30 hpf (D), 48 hpf (E), and 72 hpf (F). Embryos in (B–D and F) were lateral views, and embryo in (E) was ventral view. (G–K) Expression of tpm4-tv2, detected by the tv2s probe, in the head mesoderm, lateral plate mesoderm, and the neural tube at the 10-somite stage (G–I), and in the vasculature at 24 hpf (J) and 30 hpf (K). (H and I) Dorsal views of the head and middle trunk, respectively; others, lateral views. Arrows indicated the middle cerebral vein and metencephalic artery. (L–P) Combined expression pattern detected by the tvs probe at the 10-somite stage (L), 20 hpf (M), 24 hpf (N), 48 hpf (O), and 72 hpf (P). All embryos were lateral views with the heart indicated by arrowheads. (Q–V) Ventral head views showed expression of tpm4-tv1, detected by the tv1s probe, in the hearts of wild-type (+/+), heterozygous (+/m), and homozygous (m/m) embryos at 24 hpf (Q–S) and 48 hpf (T–V). The ventricle and atrium were indicated by arrows and arrowheads.

 
The expression pattern of tpm4-tv2 differs from that of tpm4-tv1. tpm4-tv2 transcripts became detectable at the 3-somite stages in the cephalic mesoderm (presumptive rostral blood island) and lateral plate mesoderm (presumptive ICM precursors) (data not shown). At the 10-somite stage, its expression in the head and lateral plate mesoderm was more prominent with appearance of a midline domain (neural tube) (Figure 4G–I). The expression of tpm4-tv2 retained in the head and trunk vessels and dorsal neural tube at 24 hpf (Figure 4J) and 30 hpf (Figure 4K), and it declined below detectable levels after 2 days of development. Thus, tpm4-tv2 appears to be a vasculature-specific isoform. The GFP expression pattern in T2EGEZ8 embryos, as described earlier, resembled a combination of tpm4-tv1 and tpm-v2 expression patterns, supporting a notion that the former results from a fusion of endogenous tpm4-tv1 and tpm4-tv2 to GFP by splicing. Comparing with GFP pattern in T2EGEZ8 embryos, neither of tpm4 transcript variants was detected by in situ hybridization in the floor plate, horizontal myoseptum, and somitic boundaries, which could be ascribed to sensitivity limit of the probes.

We also examined tpm4 expression pattern using a consensus sequence, which was shared by tpm4-tv1 and tpm4-tv2, as a probe. This probe detected strong expression in myotomes in addition to in the heart at various stages (Figure 4L–P), which was identical to that described in ZFIN expression database (http://zfin.org) but different from the GFP pattern in T2EGEZ8 embryos. We speculated that the detected expression in myotomes resulted from cross-hybridization to other Tpm family genes.

We compared the expression level of tpm4-tv1 in the heart by whole-mount in situ hybridization in wild-type, heterozygous, and homozygous embryos (Figure 4Q–V). Prior to or at 24 hpf, no change in tpm4-tv1 expression level was detected (Figure 4Q–S). At 36 hpf, its expression level in the homozygous mutants was higher than wild-type embryos (data not shown). This difference in the ventricle of the heart become more obvious at 48 hpf, with the highest level in mutant, middle level in heterozygote, and the lowest level in wild-type embryos. This suggests that a feedback regulatory mechanism exists for maintaining an appropriate amount of functional Tpm4-tv1 protein.

3.6 Heartbeat failure due to absence of Tpm4-tv1
We next investigated functions of different tpm4 variants during zebrafish embryogenesis by MO knockdown and overexpression approaches. We designed five antisense MO oligonucleotides: two translational MOs targeting tpm4-tv1 (v1MO1 and v1MO2), two translational MOs targeting tpm4-tv2 (v2MO1 and v2MO2), and one splicing MO (sMO1) targeting the junction sequence between the fifth exon and the sixth intron. The targeting efficiency of these MOs was tested in T2EGEZ8 embryos by observing GFP expression following injections. We found that v1MO1 and v1MO2 were unable to block GFP production of transgenic embryos and that injected embryos developed normally with normal heartbeat (data not shown), suggesting ineffectiveness of these two MOs. Injections of v2MO1 and v2MO2 completely inhibited GFP expression in the vasculature but not in the heart and caused no detectable defects (data not shown), suggesting that tpm4-tv2 is dispensable during fish embryogenesis. sMO1 injection abolished GFP expression in the heart and vasculature of T2EGEZ8 embryos (Figure 5A and B), suggesting that it is able to block production of both Tpm4-tv1 and Tpm4-tv2 proteins. Wild-type embryos injected with 20 ng sMO1 manifested no heartbeats and no circulation with a pericardial oedema and smaller eyes, but otherwise normal appearance (Figure 5C and Supplementary material online, Movies S7 and S8). sMO1-induced defects phenocopied those of T2EGEZ8 mutants. Importantly, coinjection with tpm4-tv1 mRNA, but not with tpm4-v2 mRNA or tpm4-stv1 mRNA, which is identical to tpm4-tv1 except for the deletion of exon 9, rescued sMO1-induced defects in wild-type embryos (Figure 5D). Taking these data together, we propose that tpm4-tv1 rather than tpm4-tv2 is essential for the heartbeat of zebrafish embryos. We also noted that overexpression of tpm4-stv1 mRNA or tpm4-tv1-GFP fusion mRNA in wild-type embryos did not affect the heartbeat and blood circulation. This implies that the truncated protein lacking C-terminal of Tpm4-tv1 and Tpm4-tv1-GFP fusion protein may not have a dominant-negative effect on wild-type Tpm4-tv1.


Figure 5
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  		Figure 5 Phenotypes induced by knockdown of tpm4-tv1. (A) Fluorescent image showing GFP expression in T2EGEZ8 transgenic embryos at 24 hpf. (B) Fluorescent image of 24-hpf T2EGEZ8 transgenic embryos injected with 20 ng sMO1, showing absence of GFP expression. (C) sMO1-injected wild-type embryos (top four) at 48 hpf showed pericardial oedema and smaller eyes, which were similar to T2EGEZ8 mutant phenotypes, compared with uninjected embryos (bottom two). Arrowheads indicated the heart locations. (D) Statistical data showing the percentage of embryos with heartbeats and circulation at 36 hpf. Note that sMO1 (20 ng)-induced heartbeat failure could be rescued by coinjection of 700 pg tpm4-tv1 (v1) mRNA but not by coinjection of 650 pg tpm4-tv2 (v2) mRNA or 700 pg tpm4-stv1 (sv1) mRNA. Injection with tpm4-tv1-GFP (v1G) fusion mRNA or tpm4-stv1 (sv1) in wild-type embryos had no effect on the heartbeat. The number of observed embryos was shown in parentheses. The tpm4-stv1 (sv1) mRNA was the truncated isoform lacking exon 9 of tpm4-tv1 (v1).

 
3.7 Sarcomeric defects in myocytes of T2EGEZ8 mutant heart
Vertebrate dimeric Tpm molecules form head-to-tail polymers and binds to actin along thin filaments, which are believed to stabilize the filaments.23–25 Tpm proteins also regulate muscle contraction by binding to other components of the contractile apparatus.26 The important role of Tpm in muscle contraction and relaxation and the heart-specific expression of tpm4-tv1 prompted us to examine heart filament structures in the heart of T2EGEZ8 mutant embryos by transmission electron microscopy. As shown in Figure 6, ventricular cardiomyocytes in wild-type embryos at 3 and 5 dpf contained abundant myofibrils with distinct sarcomeric structures such as A-band, I-bands, and Z-discs (Figure 6A, A', C and C'). In contrast, in cardiomyocytes of T2EGEZ8 homozygous mutant embryos the amount of myofibrils was reduced; the arrays of existing sarcomeres were disorganized; and Z-discs of sarcomeres were absent (Figure 6B, B', D and D'). The sarcomeric defects in the T2EGEZ8 mutant heart resemble those found in zebrafish sih/TNNT2 and pik/titin mutants that exhibit loss of cardiac contractility.15,16 The fact that loss of the C-terminal region of Tpm4-tv1 in the mutant embryos affects sarcomere assembly and stability supports the idea that the C-terminal region of Tpm4-tv1 is necessary for forming the head-to-tail array of Tpm molecules.


Figure 6
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  		Figure 6 Abnormal organization of cardiac thin filaments in T2EGEZ8 mutant embryos. (A–D and A'–D') Transmission electronic microscopic views of myofibrillar filaments in ventricular myocytes of 3-day (A–B') or 5-day (C–D') embryos. Pictures in (A–D) were originally taken at a magnification of x10 000. Pictures in (A'–D'), which corresponded to the boxed areas in (A–D), were originally pictured at a magnification of x20 000 and enlarged subsequently. Large amounts of long filaments with distinguishable Z-discs (arrowhead) were present in wild-type (+/+) hearts (A, A', C and C'), while the mutant (m/m) myocytes (B, B', D and D') only contained fewer, disorganized filaments (indicted by arrows).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Supplementary material
 Funding
 References
 
In summary, we generated a zebrafish mutant with heartbeat failure, T2EGEZ8 mutant, using Tol2 transposon-mediated mutagenesis. The heartbeat failure of T2EGEZ8 mutant embryos was due to instability of cardiac filaments. We further demonstrated that the 284-amino acids variant of tpm4 gene was specifically expressed in the heart and its loss caused embryonic heartbeat failure in zebrafish.

Structural studies mainly on skeletal muscle Tpm indicate that {alpha}-Tpm dimers form long linear polymers in a head-to-tail fashion and the polymers wind around and protect the actin filaments.1,2,25 The head-to-tail linear aggregation of parallel-aligned Tpm dimers absolutely requires their nine N-terminal and nine C-terminal residues.27,28 The longer form of Tpm4 should have the same structural characteristics. In T2EGEZ8 mutant embryo, the insertion of the transposon genome into the tpm4 locus led to production of Tpm4-tv1-GFP and Tpm4-tv2-GFP fusion proteins (Figure 3). In Tpm4-tv1-GFP, N-terminal 257-amino acids region of Tpm4-tv1 was fused to GFP, resulting in loss of C-terminal 27 residues of Tpm4-tv1. We postulate that the absence of C-terminal 27 residues of Tpm4-tv1 in the mutants could lead to loss of the ability of head-to-tail intermolecular aggregation of Tpm4-tv1-GFP in the mutant myocytes, and as a result the filamentous actin fibres become instable. It will be interesting to test if Tpm4-tv1-GFP is indeed unable to form head-to-tail aggregates.

The tpm4 gene in zebrafish expresses two variants, 284-amino acid Tpm4-tv1 and 248-amino acid Tpm4-tv2. As shown above, the tpm4-tv1 is a heart-specific variant and plays an essential role in cardiac contractility. The Tpm4 locus has also been found to express two isoforms, 284- and 248-amino acid proteins, in chick,29 frog,30 and pufferfish.31 In these species, the 284-amino acid Tpm4 is predominantly expressed in the heart, whereas the 248-amino acid isoform is expressed in more types of tissues. We would expect that Tpm4 also play an important role in cardiac muscle contraction and relaxation in these species. By exploring NCBI databases, we found at least two human cDNA clones (with GenBank accession numbers CR599958 [GenBank] and AK023385 [GenBank] ) that encode the 284-amino acid isoform of TPM4, and noted that TPM4 ESTs are present in the human heart tissue. It is likely that the human TPM4 is also a component of cardiac contractile apparatus and that its mutations may cause heart diseases. tpma, another Tpm family gene in zebrafish, has been reported to express in the heart of zebrafish embryos, and its expression is significantly reduced in troponin T mutant.15 The expression of tpma in T2EGEZ8 mutant embryos appeared to be slightly reduced at 24 hpf (see Supplementary material online, Figure S4), suggesting that tpma expression requires Tpm4-tv1 function. Unlike tpm4-tv1, however, tpma expression was not detected in wild-type or T2EGEZ8 mutant embryos at 36 hpf, implying that they might have distinct activities at different developmental stages.

Tpm4-tv2 is expressed in the vasculature and the dorsal neural tube. However, its interruption in T2EGEZ8 homozygous embryos or its MO knockdown in wild-type embryos fails to cause detectable abnormalities in the vasculature and motility, suggesting that other Tpm proteins can compensate for loss of Tpm4-tv2. The potential candidates in zebrafish that may have redundant functions with tpm4-tv2 include tpm3 and tpm4-like, which have an expression pattern similar to tpm4-tv2 (http://zfin.org).


   Supplementary material
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
Funding
 References
 
Supplementary material is available at Cardiovascular Research online.


   Funding
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
References
 
This work was supported by grants from the ‘863’ Program (#2006AA02Z167), from the Major Science Programs of China (#2006CB943401), and from the National Basic Research Program of China (#2005CB522502).


   Acknowledgements
 
We are grateful to Dr K. Kawakami for providing Tol2 reagents, Dr S. Lin for Tg(flk1:RFP) fish, and Dr Ning Yan for helpful discussion. We thank other members of Meng Lab for helps and discussions.

Conflict of interest: none declared.


   Notes
 
{dagger} These authors contributed equally to this work. Back


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 

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