Introduction

The myocyte enhancer factor 2 (Mef2) proteins belong to the ancient MADS family of transcription factors (Shore and Sharrocks 1995) and play an important role in morphogenesis and myogenesis of skeletal, cardiac, and smooth muscle cells (Olson et al. 1995). The N-terminal end of MEF2 factors contains a Mef2 domain and a highly conserved MADS-box, which together mediate DNA binding, dimerization, and co-factor interactions (Potthoff and Olson 2007; Black and Olson 1998; McKinsey et al. 2002), while the C-terminal regions of Mef2 proteins function as transcriptional activation domains (Potthoff and Olson 2007; Martin et al. 1994). There are four vertebrate Mef2 genes, Mef2a, Mef2a-b, Mef2a-c, and Mef2a-d, which are expressed in distinct, but overlapping patterns during embryogenesis through adulthood (McKinsey et al. 2002). Zebrafish is a popular model system for studying early embryo development and human disease (Lieschke and Currie 2007). It has also become an attractive model for studying regeneration of various tissues and organs, such as fins, heart, spinal cord, brain, and muscle (Gemberling et al. 2013). To fully exploit zebrafish in these studies, great emphasis has been placed on generation of fish lines that express desired transgenes, usually the fluorescence proteins, in either specific subsets of cells and tissues or a wide range of cells (Burket et al. 2008). These lines allow application of high-resolution imaging in vivo on zebrafish model. In addition, studies in zebrafish demonstrated that mef2aa is highly expressed in the heart (Wang et al. 2005) and somites (Wang et al. 2006) during zebrafish embryogenesis. Currently, we aimed to construct a transgene mef2aa:EGFP to induce the expression of EGFP in cardiomyocytes and muscle cells under the control of mef2aa promoter. The generation of Tg(mef2aa:EGFP) line will provide the new insights of understanding the mef2a expression in embryo and facilitate further analysis of the roles of mef2a in vivo during the development and tissue regeneration in adults.

Materials and methods

Zebrafish

Zebrafish (AB strain) were raised and maintained at 28.5 °C as previously described (Huang et al. 2013; Wang et al. 2015, 2016). Embryos were collected through natural mating. Embryonic stages were determined according to time post-fertilization and characteristics described by Kimmel et al. (1995). Embryos after 24 h post-fertilization (hpf) were treated with 0.2 mM 1-phenyl-2-thio-urea (PTU, Sigma) to prevent the pigmentation for further observation and imaging. Zebrafish embryos used for whole-mount in situ hybridization were collected at various stages, fixed with 4 % paraformaldehyde (PFA) in phosphate-buffered saline (PBS) overnight at 4 °C or 2 h at room temperature, washed with PBST, dehydrated in methanol, and stored at −20 °C until use. Embryos at stages earlier than 24 hpf were dechorionated after fixation, prior to storage.

Construction of mef2a:EGFP plasmid

Mef2aa promoter fragments (2044 bp) were amplified from the adult zebrafish genomic DNA by polymerase chain reaction (PCR) (Primers are listed in Table S1). Subsequently, the fragments were sub-cloned into pENTR™5′-TOPO® vector (Life Technologies) to produce 5′ Entry clone. pME-EGFP plasmid and p3E-polyA plasmid were obtained from Tol2Kit (Kwan et al. 2007). Three entry clones and the pDestTol2pA2 destination vector were used to generate the expression construct by LR recombination reaction as described in the Multisite Gateway Manual (Life Technologies).

RNA synthesis

Transposase RNA was generated using the pCS2-transposase plasmid as a template. Plasmid DNA was linearized with restriction endonuclease KpnI (NEB) and purified using the Zymoclean™ Gel DNA Recovery kit from agarose gels. Then, the mMESSAGE mMACHINE (Ambion, Life Technologies) was used to synthesize the capped RNA in vitro. The synthesized transposase RNA was purified by the RNeasy® MinElute® Cleanup kit (Qiagen).

RNA extraction, reverse transcription, and PCR

Tissue was homogenized and frozen in TRIzol Reagent (Life Technologies) and stored at −80 °C. The RNA was extracted following the manufacturer’s instruction. 1 µg of RNA was reverse transcribed into cDNA by the use of Transcriptor First Strand cDNA Synthesis Kit (Roche) according to the manufacturer’s instructions. Synthesized cDNA was stored at −20 °C. All PCR amplifications were carried out in a total volume of 50 µl using specific primers and Advantage 2 Polymerase Kit (Clontech). The RT-PCR primers for zebrafish mef2aa and ef1α are listed in Table S1.

Microinjection of plasmid DNA and transposase RNA

The plasmid DNA and transposase RNA were mixed and microinjected into eggs at single-cell or two-cell stage. Approximately, 75 pg of plasmid DNA and 25 pg of transposase RNA were injected per embryo. Post-injection embryos were incubated at 28 °C in incubator.

Transgenic zebrafish screening

Injected embryos were screened through examining fluorescence expression using the stereo fluorescence microscopy (Olympus, MVX10; Zeiss, SteREO Discovery V20). Founders were raised until sexually mature and individually mated with wild-type (WT) fish, and the offspring were examined for fluorescence expression in muscle cells. F1 embryos were obtained from one of the positive founders and raised. Eight surviving positive male F1 fish were mated with wild-type females to generate F2 fish. F2 males were mated with F2 females to breed F3 generation.

Preparation of mef2aa RNA probe and whole-mount in situ hybridization (WISH)

The cDNA fragment (approximately 500 bp) of zebrafish mef2a was amplified by PCR. The primers used are listed in Table S1. Digoxigenin-labeled RNA probe was transcribed in vitro using T7 RNA polymerase, with the linearized pGEM®-T Easy vector (Promega) containing the DNA fragment of zebrafish mef2aa. The WISH procedure to detect mef2aa expression was carried out as described previously (Huang et al. 2013).

Imaging

Embryos subjected to whole-mount in situ hybridization were mounted in 75 % glycerol/PBST and photographed under a stereomicroscope (Olympus, MVX10) with DP71 camera. A Leica TCS SP5 was used for confocal imaging. Embryos were embedded in 0.8 % low melting point agarose dissolved in E3 medium with 0.03 % tricaine methanesulfonate (MS222, Sigma). All images acquired were compiled and edited using Adobe Photoshop and Illustrator software.

Results

Establishment of Tg(mef2aa:EGFP) zebrafish

There are two mef2a genes in zebrafish, mef2aa and mef2ab. It was reported that mef2aa was expressed in developing heart and somites (Wang et al. 2005, 2006), whereas mef2ab gene was not detected in zebrafish heart and somites at the corresponding stages (Yogev et al. 2013). Therefore, we choose to generate Tg(mef2aa:EGFP) transgenic zebrafish line that expresses EGFP in muscle cells. Firstly, we generated the mef2aa:EGFP transgenic construct (Fig. 1) containing a pair of Tol2 elements with mef2aa regulatory elements and the gene encoding EGFP using the multisite gateway system (Fig. 1; Figure S1). Then, the synthetic transposase mRNA and the expression construct were co-injected into zebrafish fertilized eggs at single-cell stage (Fig. 1). A fragment of 2044-bp DNA sequence was identified to drive muscle-specific expression of EGFP in zebrafish embryos. Founders were raised until sexually mature and individually mated with WT fish line. Of the 67 founders, 20 germ-line transmitted transgenic fish were identified. F1 embryos obtained from one of the positive founders, which were screened through examining the expression of fluorescence under a fluorescence stereomicroscope, and the positive F1 fish were raised. Eight surviving positive male F1 fish were mated with WT females to generate F2 fish. About 50 % of F2 offspring embryos carried EGFP expression, suggesting that the germline transmission rate of transgene complied with Mendelian inheritance law. F2 males were mated with F2 females to breed F3 generation. Finally, the Tg:(mef2aa:EGFP) ntu803 transgenic line was confirmed by around 75 % fluorescence induction in the F3 generation.

Fig. 1
figure 1

Establishment of Tg(mef2aa:EGFP) zebrafish. The synthetic transposase mRNA and the expression construct containing a Tol2 construct with a mef2aa promoter, a ~2-kb DNA upstream of the translational start site of mef2aa, and EGFP are co-injected into zebrafish fertilized eggs

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EGFP expression in Tg(mef2aa:EGFP) ntu803 zebrafish embryo in vivo

We examined the green fluorescence in Tg(mef2aa:EGFP) ntu803 zebrafish embryos through confocal imaging analysis. It was observed that GFP was weakly expressed at 75 % epiboly stage (Fig. 2a). In 5-somite and 20-somite stage embryos, we found GFP was present in telencephalon and myotomes (Fig. 2b, c). The transgene expression at these stages was similar to the endogenous expression of mef2aa shown through whole-mount in situ hybridization analysis (Fig. 2d). At 24 hpf stage, GFP was strongly expressed in myotomes and heart (Fig. 2e, e′). The endogenous expression of mef2aa was also detected in myotomes and heart at the corresponding stage (Fig. 2f). At 36 hpf, 3 dpf, and 4 dpf stage, the GFP expression in heart and myotomes increasingly remained (Figs. 2g, h, 3a, b, c, c′, c″). In addition, GFP expression was detected in head muscle including intermandibularis anterior and posterior (INTM-A and INTM-P), interhyoideus (INTE), hyoideus inferior (HH-INF), sternohyoideus (SH), ocular muscles (OM), levator arcus palatini (LEV-AP), levator operculi (LEV-OP), dilatator operculi (DIL-OP), adductor hyomandibulae (AD-HYO) (Fig. 3d, d′). The pectoral fin muscles were also clearly labeled with EGFP including abductor muscle (ABM) and adductor muscle (ADM) (Fig. 3e, e′, e″).

Fig. 2
figure 2

Confocal images showing expression of EGFP transgene in Tg(mef2aa:EGFP ntu803 embryos. a 75 % epiboly, lateral view, weak expression of GFP, b 5-somite stage, lateral view, weak expression of GFP in head, c 24-somite stage, lateral view, weak expression of GFP in somite and head, d 20-somite stage, lateral view, in situ hybridization analysis of mef2aa expression, weak expression in somite and head, e 24 hpf, lateral view, GFP expression in somite and heart, e′ GFP expression in heart, arrowhead indicates heart, f In situ hybridization analysis of mef2aa expression at 24 hpf, lateral view, g 36 hpf, lateral view, GFP expression in somite and heart, arrowhead indicates heart, h in situ hybridization analysis of mef2aa expression at 36 hpf, arrowhead indicates heart

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Fig. 3
figure 3

Confocal images showing expression of EGFP transgene in Tg(mef2aa:EGFP) ntu803 embryos at late stages. a 3 dpf, lateral view, GFP expression in somite and heart, b 4 dpf, lateral view, GFP expression in somite, heart and pectoral fins muscles, c, c″ 3 dpf, Ventral view, GFP expression in heart co-localized with mCherry in Tg(cmlc2:mCherry). V ventricle, A atrium, d, d′ 3 dpf, Ventral view and lateral view, GFP expression in head muscles. INTM-A and INTM-P intermandibularis anterior and posterior, INTE interhyoideus, HH-INF hyoideus inferior, SH sternohyoideus, OM ocular muscles, LEV-AP levator arcus palatine, LEV-OP levator opercula, DIL-OP dilatator operculi, AD-HYO adductor hyomandibulae, e, e″ 4 dpf, Ventral view and lateral view, GFP expression in pectoral fins muscles. White arrowhead indicates abductor muscle (ABM), Red arrowhead indicates adductor muscle (ADM). (Color figure online)

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EGFP expression in adult Tg(mef2aa:EGFP) ntu803 zebrafish heart and myotomes

Next, we examined GFP expression in adult Tg(mef2aa:EGFP) ntu803 zebrafish heart and myotomes. We dissected the heart by surgery and found that EGFP was highly present in adult zebrafish heart (Fig. 4a, b), but not in bulbus arteriosus (Fig. 4b). Cryostat sections of the Tg(mef2aa:EGFP) ntu803 zebrafish heart confirmed GFP expression in cardiomyocytes (Fig. 4c). In the transection analysis of zebrafish trunk, we demonstrated that GFP expression in myotome was weakly distributed in restricted region (Fig. 4d, e). RT-PCR analysis revealed that the endogenous expression of mef2aa in adult zebrafish heart is higher than that in brain and myotome (Fig. 4f), which supports that mef2aa is highly expressed in adult heart but not in skeleton muscle.

Fig. 4
figure 4

Mef2aa expression in adult zebrafish heart and myotome. a Adult Tg(mef2aa:EGFP) ntu803 zebrafish heart in bright field, b morphology of adult Tg(mef2aa:EGFP) ntu803 zebrafish heart under fluorescence stereo microscope. Red arrowhead indicates bulbus arteriosus, c cross section of adult Tg(mef2aa:EGFP) ntu803 zebrafish heart under fluorescence microscope, d transverse section analysis of adult Tg(mef2aa:EGFP) ntu803 zebrafish myotome in bright field, e transverse section analysis of adult Tg(mef2aa:EGFP) ntu803 zebrafish myotome under fluorescence microscope. Arrowheads indicate GFP expression sites, f reverse transcription polymerase chain reaction analysis of mef2aa expression in brain, heart, and myotome. (Color figure online)

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Discussion

Currently, we generated a Tg(mef2aa:EGFP) ntu803 transgenic zebrafish line that expresses EGFP in muscle cells. Mef2a transcripts are expressed in a wide array of tissues in mammals, while MEF2A protein appears to be considerably more restricted and is abundant in skeletal muscle, heart, and brain tissues (Suzuki et al. 1995) Accumulating evidence indicates that Mef2a is associated with cardiovascular and muscle development (Estrella et al. 2015). In addition, knockdown of mef2a in zebrafish embryos impairs the cardiac contractility (Wang et al. 2005) and posterior somite development (Wang et al. 2006). Our data are consistent with these findings that mef2a is highly expressed in developing heart and muscles. A loss-of-function mutation in the human Mef2a causes an autosomal dominant form of coronary artery disease, including its most serious complication myocardial infarction (Wang et al. 2003; Bhagavatula et al. 2004). Most mice lacking Mef2a die suddenly within the immediate perinatal period and exhibit pronounced dilation of the right ventricular, myofibrillar disarray and activation of a fetal cardiac gene program. The few Mef2a knock out mice that survives to adulthood is also susceptible to sudden cardiac death with mitochondrial deficiency (Naya et al. 2002). It was revealed that the level of mef2aa transcripts in adult zebrafish heart is higher than that in myotome. However, mef2aa was expressed in both heart and myotome in zebrafish embryos. Taken together, these observations suggest that mef2a is vital to the function of adult heart in vertebrates. Interestingly, the cranial muscles and pectoral fin muscles were clearly labeled with EGFP in Tg(mef2aa:EGFP) ntu803 line embryos, which allows high-resolution imaging of these muscles in vivo and promotes further studies of the role of mef2a in the development of these muscles. In addition, Mef2a was revealed to involve skeletal muscle regeneration in mice (Liu et al. 2014). It will be of interest to explore the expression and function of mef2a during skeletal muscle regeneration in the established Tg(mef2aa:EGFP) ntu803 line adults.