Wild type (WT) zebrafish stocks were obtained from the Model Animal Research Center of Nanjing University (Nanjing, Jiangsu, China). Embryos were obtained from natural spawning of wild type adults and were grown at 28.5°C in embryo medium which was changed twice per day, as previously described (15). Morphological features were used to determine the embryonic developmental stage, as described by Kimmel et al(16). Embryos older than 24 h post-fertilization (pf) were incubated in 0.003% phenylthiourea to inhibit pigment formation.

MOs were purchased from Gene Tools (Philomath, OR, USA). The FABP3-MO was designed against the ATG start site of FABP3 to block its translation (FABP3-MO; 5′-ACG TGC CGA TAA AAG CGT CTG CCA T-3′). A standard control MO (Wt-MO; 5′-CCT CTT ACC TCA GTT ACA ATT TAT A-3′) served as a negative control (17). MO oligomers were dissolved in 0.3X 2 mmol/l Danieaus’s solution [58 mmol/l NaCl, 0.7 mmol/l KCl, 0.4 mmol/l MgSO₄, 0.6 mmol/l Ca(NO₃)₂, 5.0 mmol/l N-(2-hydroxyethyl) piperazine-N′-(2-ethanesulfonic acid) (HEPES), pH 7.6] (18). The oligomers were diluted to their final concentration in 0.2 mol/l KCl and 0.5% (w/v) phenol red (Sigma-Aldrich, St. Louis, MO, USA). Injection of 5.0 ng MO into single to four-cell stage zebrafish embryos using back-filled fine borosilicate glass capillary needles (Corning Inc., Corning, NY, USA) was performed as previously described (18). Following MO injection, embryos were incubated at 28.5°C for up to 3 days pf.

Following microinjection of MOs into zebrafish embryos, the embryos were incubated at 28.5°C for up to 4 days pf. In order to determine the effect of time on FABP3-MO-related morphological abnormalities of the heart, histological sections were used to identify heart-specific phenotypes. Larvae were anesthetized with 0.05% tricaine (3-aminobenzoic acid ethylester) and fixed in 4% paraformaldehyde in 0.1 M Sorenson’s buffer (pH 7.4). Larvae were washed in 0.01 M phosphate-buffered saline (PBS; pH 7.2), dehydrated in a graded series of ethanol, infiltrated and embedded in blocks. Each block was serially transverse-sectioned at 5 μm. Sections were hydrated, stained with Harris’ hematoxylin and eosin Y, dried and mounted in neutral balsam. For larvae within the block, measurements were made for the heart when the central portion of the heart was visible. In each group, six larvae were assessed for heart layer thickness and cell density at 96 h pf (4 days pf). All other slides of the selected heart were imaged at a final magnification of ×1000 with a Pixera Pro 600ES digital camera (Los Gatos, CA, USA) attached to a JD-801 microscope (Olympus, Tokyo, Japan).

Following injection of 5.0 ng MO into single to four-cell stage zebrafish embryos, zebrafish were harvested using collagenase I (Sigma-Aldrich) at 24 and 48 h pf, and washed with PBS. The single-cell suspensions of zebrafish were centrifuged, resuspended in 1 ml binding buffer and stained with 10 μl Annexin V-fluorescein isothiocyanate (FITC; BioVision, Mountain View, CA, USA) and 10 μl propidium iodide at room temperature for 15 min. The stained cells were analyzed immediately using flow cytometry as described previously (19).

Zebrafish larvae were digested with collagenase I (Sigma-Aldrich) to obtain single-cell suspensions. The ATP content of the zebrafish was measured using a luciferase-based luminescence assay kit (Biyuntian, Nantong, Jiangsu, China). Briefly, the single-cell suspensions of zebrafish were homogenized in ice-cold ATP-releasing buffer and light emission was recorded for 30 sec using a photon-counting luminometer (Turner Biosystems, Sunnyvale, CA, USA) pf at 24 and 48 h pf. The relative ATP level was normalized to the protein concentration of each sample, as determined by a bicinchoninic acid assay.

Intracellular reactive oxygen species (ROS) generation was determined using 6-carboxy-2,7-dichlorodihydrofluorescein diacetate (H₂-DCFDA; Sigma-Aldrich), as previously described (20). Briefly, following injection with 5.0 ng MO into single to four-cell stage zebrafish embryos, the embryos were collected pf at 24 and 48 h pf. The digested single-cell suspensions of zebrafish were washed and incubated with H₂-DCFDA for 20 min. Subsequent to this, the suspensions were washed several times and harvested in PBS. The fluorescence of H₂-DCFDA was detected using a fluorescence-activated cell sorter (FACS; excitation, 488 nm; emission, 530 nm).

The relative quantities of mtDNA were determined by qPCR, which was performed using an Applied Biosystems 7500 Sequence Detection system (ABI 7500 SDS; Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions (21). In brief, DNA was isolated from the zebrafish following microinjection 24, 48, 72 h pf using the Wizard^® Genomic DNA purification kit DNA extraction kit (Promega, Madison, WI, USA) and quantified using a NanoDrop 2.0 spectrophotometer (Thermo Scientific, Foster City, CA, USA). Two primer sets (listed in Table I) were used for PCR analysis. A 125 bp mtDNA fragment within the cytochrome b (CYTB) gene was used for the quantification of mtDNA. We previously cloned the PCR product into the plasmid pMD18-T and verified it by DNA sequencing. Plasmid standards of known copy number were used to generate a log-linear standard curve, from which the CYTB copy number was determined in the samples by qPCR. The results were normalized to a 168 bp region of the nuclear gene for 28S rRNA using a standard curve generated from a plasmid containing the 28S fragment. The ratio of mtDNA to nuclear DNA was determined to reflect the concentration of mitochondrial DNA per cell.

Total RNA was extracted from zebrafish using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA), and the extracted RNA was quantified using a NanoDrop 2.0 spectrophotometer (Thermo Scientific). Complimentary DNA was synthesized from 2 μg total RNA by using an AMV Reverse Transcriptase kit (Promega A3500; Promega, Madison, WI, USA) according to the manufacturer’s instructions. qPCR was performed using SYBR-Green PCR Master mix (Applied Biosystems) on an Applied Biosystems 7500 Sequence Detection System (ABI 7500 SDS). qPCR reaction conditions were 95°C for 10 min followed by 40 cycles at 94°C for 20 sec, 60°C for 20 sec and 72°C for 30 sec with a final extension of 7 min at 72°C. NK2 homeobox 5 (Nkx2.5), Wnt1, Wnt5a and Wnt11 expression was normalized against β-actin using the comparative CT method to determine the relative changes in mRNA expression in the target sample. The sequences of the primers used for each gene are shown in Table I.

All data are presented as the mean ± standard deviation of three independent experiments. Statistical significance was assessed using the independent-samples t-test using the statistical software package SPSS 16.0 (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

To investigate the function of FABP3 in the establishment of cardiac development, a loss-of-function study was performed using a previously characterized anti-FABP3 (ATG) MO (in our previous original study: knockdown of FABP3 impaired cardiac development in zebrafish through the retinoic acid signaling pathway). The MO oligonucleotides were injected into single to four-cell stage zebrafish embryos directly and is effective up to 3–4 days pf (18). Thus, in the present study, embryos were injected with 0.5 ng MO targeting FABP3 translation. Cardiac development is complete at ~96 h pf (4 days pf) (22), and the thickness of the heart is normally ~30–40 μm at 4 days pf. By 4 days pf, we observed defective phenotypes of embryos resulting from FABP3-MO by using a series of continuous section slides by tissue cutting (thickness, ~5 μm). Compared with the Wt-MO group, transverse sections of the heart of zebrafish larvae at 4 days pf displayed defective phenotypes, including a thin atrial-ventricular wall, a change in the relative position of the atrium and ventricle, an expansion of the diameter of the atrioventricular cavity and an alteration in the morphology of the bulbus arteriosus (Fig. 1). In addition to these defects, pericardial edema, developmental delay and incomplete looping of the developing heart were also observed in FABP-MO zebrafish as we demonstrated previously (23).

To study the effect of FABP3-MO on zebrafish cell apoptosis, the zebrafish larvae were harvested at 24 and 48 h pf using collagenase I. The single-cell suspensions of zebrafish larvae were quantified by flow cytometric analysis subsequent to staining with Annexin V-FITC. The results demonstrated that FABP3-MO promoted zebrafish cell apoptosis compared with Wt-MO (Fig. 2; ^*P<0.05).

To further investigate whether FABP3 knockdown in zebrafish leads to impaired mitochondrion function, the ATP content was assayed in the FABP3-MO and Wt-MO groups in zebrafish at 24 and 48 h pf. As shown in Fig. 3, the zebrafish ATP content in the FABP3-MO group was significantly decreased compared with the Wt-MO group at 24 and 48 h pf (^*P<0.05). The results showed that FABP3 knockdown in zebrafish decreased the total cellular ATP production.

ROS, a by-product of the electron transport chain, is predominantly produced by mitochondria (24). As shown in Fig. 4, in contrast with Wt-MO injected zebrafish, embryos injected with FABP3-MO at 24 and 48 h pf induced a significantly increased concentration of intracellular ROS levels, as indicated by increased fluorescence in the presence of the compound DCFDA (2′,7′-dichlorofluorescein diacetate; ^*P<0.05). This result further demonstrated that FABP3-MO impaired mitochondrion function in zebrafish.

In addition to the ATP content and ROS production detection of FABP3-MO injected zebrafish, the mitochondrial DNA copy number is regarded to indicate the cellular mitochondrion number and may be a possible marker of mitochondrion function. We measured mitochondrial and genomic DNA in FABP3-MO and Wt-MO injected embryos using qPCR. The results demonstrated that the mtDNA copy number was decreased in the FABP3-MO group compared with the Wt-MO group at three time points, in particular, the mtDNA copy number was significantly decreased at 24 h pf in the FABP-3MO group (Fig. 5; ^*P<0.05). However, there was no significant difference in mtDNA copy number at 48 and 72 h pf between the two groups (Fig. 5; P>0.05).

To further investigate the effect of FABP3-MO on cardiac development, the expression level of the cardiac development-specific gene, Nkx2.5 (the earliest known marker of vertebrate heart development), was determined (25). Notably, as shown in Fig. 6A, qPCR results demonstrated that Nkx2.5 was upregulated in zebrafish embryos injected with FABP3-MO compared with those injected with Wt-MO.

In addition, the mechanism through which FABP3 knockdown may affect cardiac development was investigated by focusing on the Wnt signaling pathway. Wnt signals exhibit developmental stage-specific and biphasic involvement in cardiomyogenesis and are also involved in heart disease (26–28). Wnt signaling consists of the canonical (Wnt1) and non-canonical (Wnt5 and Wnt11) Wnt signaling pathways. Thus, Wnt1, Wnt5a and Wnt11 were chosen to investigate the involvement of Wnt signaling in zebrafish cardiac development in injected FABP3-MO zebrafish embryos using qPCR at 24, 48 and 72 h pf, respectively. As shown in Fig. 6B–D, the expression pattern of the three Wnt signaling molecules was enhanced in FABP3-MO injected zebrafish at 24, 48 and 72 h pf. These results indicated that FABP3-MO affected the Wnt signaling pathway which may also have contributed to abnormal cardiac development.