All adult zebrafish were housed in a recirculating aquaculture system maintained at 26–28 °C with a 14-hour light/10-hour dark cycle to support optimal biorhythms. Wild-type (AB strain) and specific transgenic lines—Tg(myl7:EGFP), Tg(fli1a:EGFP), Tg(fabp10a:dsRed), and Tg(ela3l:EGFP)—were procured from the China Zebrafish Resource Center (CZRC). The Tg(cmlc2:MitoTimer) line, which marks myocardial mitochondria, was developed in our lab to evaluate cardiac mitochondrial turnover. Adult zebrafish were maintained at a 1:1 male-to-female breeding ratio to ensure genetic diversity. All experiments were performed in triplicate under standardized conditions to ensure phenotypic reproducibility and consistency. All procedures were approved by the Animal Ethics Committee of AEC SFY 2025 058.

Antisense morpholino oligonucleotides (MOs) targeting the polr2i gene splice site (polr2i gene-MO: 5^′-ACTCATTTCAACTCACCATTCCTGA-3^′) were custom-designed by Gene Tools LLC (Philomath, OR, USA). A total of 400 one-cell stage embryos were microinjected, with 200 embryos receiving polr2i gene-MO and 200 injected with a standard control MO (control-MO: 5^′-CCTCTTACCTCAGTTACAATTTATA-3^′). All procedures strictly adhered to the Zebrafish Community Guidelines [22]. To validate MO specificity and establish the minimum effective dose, we conducted a dose-response analysis using escalating polr2i gene-MO concentrations. Embryos were injected with polr2i gene-MO at increasing concentrations (0.3, 0.5, 0.6, 0.8 mM; corresponding to 2.4, 4.0, 4.8, 6.4 ng/embryo) or control-MO (3.0 ng/embryo). Cardiac malformations (e.g., pericardial edema, ventricular hypoplasia) and intersegmental vascular (ISV) defects were quantified at 72 hours post-fertilization (hpf) using established morphological scoring criteria. MOs were delivered into the yolk of one-cell stage embryos at a final working concentration of 0.6 mM (4.8 µg/µL), with 1 nL injected per embryo, yielding a total dose of 4.8 ng/embryo. Embryos were maintained at 28 °C in E3 medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM MgSO₄, 0.33 mM CaCl₂), with microinjections performed using a TransferMan® 4R micromanipulator was coupled to a FemtoJet 4X microinjector (Eppendorf, Hamburg, Germany).

The polr2i gene cDNA was cloned into the pCS2+ vector for mRNA synthesis using the mMESSAGE mMACHINE T7 system (Ambion). Fertilized eggs were co-injected with polr2i gene MOs and capped polr2i gene mRNA (300 ng/µL) to assess rescue effects.

To analyse the effects on gene transcription levels under neomycin exposure, we collected groups of 72 hpf larvae with 50 embryos per group FastPure Cell/Tissue Total RNA Isolation Kit V2 (Vazyme, Nanjing, China, Cat No. RC112–01) to extract the total mRNA. subsequently, total mRNA was extracted using the PrimePure Cell/Tissue Total RNA Isolation Kit V2 (Thermo Fisher Scientific, Waltham, MA, USA, Cat No.12183) with a cDNA was subsequently prepared using a PrimeScript RT kit with gDNA Eraser (Takara, Kusatsu, Japan, Cat No.RR047A).

Quantitative real-time PCR (qRT-PCR) procedures were performed by SYBR® Green Realtime PCR Master Mix (TOYOBO, Osaka, Japan, No. QPK-201, QPK-201T) via the QuantStudio 5 Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA, Cat. A28574) was performed. The internal reference was $\beta$-actin, which was analysed using the 2^{-Δ⁢Δ⁢CT} method.

RNA expression analysis was accomplished using PCR with Rapid Taq DNA Polymerase (Vazyme, Cat. P223-01) at 95 °C for 3 min, 94 °C for 30 sec, 55 °C for 30 sec, 72 °C for 1 min (35 cycles) with primer sets of zebrafish polr2i gene, primer sets of zebrafish $\beta$-actin used as a control; forward; 5^′-AGCAGGATGCGGTTTTCTTTAT-3^′, reverse; 5^′-GCCATGCCAATGTTGTCGTT-3^′. PCR products were separated by electrophoresis on 2% agarose gels to analyze the expression.

Primers used in qRT-PCR and RT-PCR are listed in Supplementary Table 1.

To assess cardiac phenotypes, one-cell stage embryos of the Tg(myl7:EGFP) transgenic line were microinjected with polr2i gene-MO at a final concentration of 0.6 mM (equivalent to 4.8 ng/embryo). Embryos were harvested at 48 and 72 hpf for phenotypic analysis. Zebrafish larvae were anesthetized with 0.02% tricaine methanesulfonate (Sigma-Aldrich, St. Louis, MO, USA; Cat. E10521) and mounted on a 15-mm glass bottom cell culture dish (Nest, Wuxi City, Jiangsu Province, China; Cat.801002) in 3% methylcellulose (Sangon, Shanghai, China; Cat. A600616) and oriented laterally for imaging. Images were captured using a fluorescence microscope (ECLIPSE Ts2R-FL, Nikon, Chiyoda ku, Tokyo, Japan) for morphology observation.

Photographs were quantified using NIS-Elements D software (Nikon, Chiyoda ku, Tokyo, Japan) to determine the pericardial area and cardiac function per field of view. Heart rate was quantified by manual counting of ventricular contractions over three consecutive 15-second intervals per embryo and expressed as beats per minute (bpm). At the same time, images were extracted from these visual frequencies to measure the long (a) and short (b) axis lengths of the diastolic and systolic ventricles. And then calculate end-diastolic volume (EDV), end-systolic volume (ESV), stroke volume (SV), and cardiac output (CO) of the larvae. The common formula for EDV and ESV is: volume = 4/3$\pi$ ab². The calculation formula for SV is: SV = EDV – ESV, and the calculation formula for CO is: CO = SV $\times$ HR. The ejection fraction is (EDV-ESV)/EDV $\times$ 100%.

To assess vascular development, one-cell stage embryos of the Tg(fli1a:EGFP) transgenic line were microinjected with polr2i gene-MO (0.6 mM, equivalent to 4.8 ng/embryo). Embryos were harvested at 72 hpf and processed for analysis. Zebrafish were oriented on lateral side (anterior, left; posterior, right; dorsal, top), and mounted with 4% methylcellulose in a slide for observation by fluorescence microscopy. The morphology of the caudal vein plexus and the number of intersegmental vessels that connect the dorsal aorta to the dorsal longitudinal anastomotic vessels were analysed. Use ImageJ software (v1.53c, NIH, Bethesda, MD, USA) to analyze and measure the endpoints, number of vascular segments, and total skeleton length which can approximately represent the total length of blood vessels in the captured images.

For o-Dianisidine staining, the embryos were collected and fixed at room temperature for 4 h using 4% PFA. Following three 10-minute washes with phosphate-buffered saline containing 0.1% Tween-20 (PBST), embryos were incubated in o-dianisidine staining solution (40% ethanol, 0.65% H₂O₂, 10 mM sodium acetate, 0.6 mg/mL o-dianisidine; Sigma-Aldrich) under light-protected conditions for 30 minutes, then washed thrice with PBST. Embryos were subsequently treated with a depigmentation solution (1% KOH, 3% H₂O₂) for 2 minutes to reduce background pigmentation. Stained embryos were washed with PBST three times and stored in 80% glycerol (v/v) for imaging using a microscope (SMZ745T, Nikon).

Embryos and larvae were imaged with Nikon TS2R inverted fluorescence microscope and subsequently photographed with digital cameras. Quantitative image analyses processed using image based morphometric analysis (NIS-Elements D) with standardized thresholding parameters for all samples. Positive signals were defined by particle number using ImageJ. 10 animals for each group were quantified, and the total signal per animal was averaged.

All experiments included three independent biological replicates, with triplicate technical measurements per biological replicate. Data are presented as mean $\pm$ SD, as specified in figure legends. Statistical analyses were conducted using unpaired two-tailed Student’s t-tests or one-way ANOVA with Tukey’s post hoc test, as dictated by experimental design. Figure legends indicate the number of biological replicates (n) analyzed per experimental group. A value of p was considered statistically significant (*, p 0.05, **, p 0.01, ***, p 0.001).

After the experiment, zebrafish fry were euthanized in a buffered MS-222 solution (500 mg/L, pH ~7.0). Ensure that the fish fry are soaked in the solution for at least 30 minutes until their heartbeat completely stops, and then soak for an additional period of time to confirm death. This procedure follows the standards of the American Veterinary Medical Association (AVMA) Euthanasia Guidelines, and the MS-222 is also the most commonly used anesthetic in zebrafish research, which inhibits nerve impulse conduction by blocking neural sodium ion channels. The soaking method is usually used for administration, which can induce a rapid and reversible anesthesia state. When used, Tris or sodium bicarbonate should be buffered to neutral pH to avoid acidosis and ensure animal welfare [23].

All methods in this experiment were performed in accordance with relevant guidelines and regulations.

Bioinformatics analyses showed that POLR2I protein was correlated across vertebrate species (Fig. 1A). Expression patterns of the polr2i gene varied across different embryonic development stages of zebrafish, depicted in Fig. 1B. These findings underscore POLR2I protein’s conservation among vertebrates and validate the use of the zebrafish model to investigate the implications of polr2i gene mutations in CHD. RT-PCR analysis demonstrated that morpholino-mediated knockdown induced intron retention and exon extension, consistent with a splicing-through mechanism rather than exon skipping (Fig. 1C,D). Effective knockdown of polr2i gene was achieved by inhibiting e1i1 linkage leading to a direct read to the intron and confirmed by qRT-PCR (Fig. 1E). This validated knockdown model was utilized in subsequent experiments.

Fig. 1.

POLR2I protein is evolutionarily conserved and developmentally expressed. (A) A sequence alignment of zebrafish, human, mouse, rat, pig, dog, cattle, chimpanzee, horse, and xenopus laevis POLR2I proteins by Hiplot. (B) Quantitative real-time PCR (qRT-PCR) for ten embryo development stages (2 hpf, 4 hpf, 6 hpf, 10 hpf, 12 hpf, 24 hpf, 48 hpf, 72 hpf, 96 hpf, and 120 hpf) demonstrates different expression patterns of polr2i gene during embryonic development (n = 50, N = 3). (C,D) Schematic representation of precursor RNA, wild-type RNA, and polr2i gene morphant RNA, with RT-PCR results showing that splicing regulation at the MO-targeted e1i1 junction produces an intron retention effect. (E) qRT-PCR conformation that polr2i gene expression is significantly decreased in polr2i gene-MO embryos (n = 50, N = 3). Square: control group; Triangle: experimental group. ***, p 0.001. POLR2I proteins, RNA polymerase II subunit I; MO, morpholino oligonucleotides.

Following standard morpholino administration protocols, we performed dose-response testing through serial dilutions, and it was found that during zebrafish embryo development, the survival rate gradually decreased with the increase of the concentration while the malformation rate increased with the increase of the concentration, and the final concentration was determined to be 4.8 µg/µL, and the injection volume of each embryo was 1 nL, with a total dose of 4.8 ng/embryo (Supplementary Fig. 1A,B). polr2i gene morphants displayed significantly delayed developmental progression compared to the control group. Conspicuous morphological abnormalities in polr2i gene morphants at 2, 3, and 5 days post-fertilization (dpf) included reduced body and eye size, curvature of the body, pericardial edema, and diminished pigmentation (Fig. 2A–F).

Fig. 2.

Aberrant polr2i gene function causes developmental defects in zebrafish. (A–F) The bright-field images showed the overall morphology of 2 dpf, 3 dpf and 5 dpf zebrafish embryos injected with control-MO (A,C,E) and polr2i gene-MO (B,D,F). (G) Kaplan-Meier plot for control-MO (n = 200) and polr2i gene-MO (n = 200) zebrafish embryos. Statistical significance was determined using a log-rank test. The significant p value is shown. (H) Hatching rates at 72 hpf (n = 200, N = 3). (I) Malformation rates at 72 hpf (n = 200, N = 3). (J) Body length at 72 hpf (n = 15, N = 3). The red arrows indicate the pericardial edema. The scale bar represents 100 µm. **, p 0.01, ***, p 0.001.

To assess the survival of polr2i gene morphants, we monitored the survival of control and the polr2i gene morphants from the embryonic stage. More than 50% of polr2i gene morphants died at 3 days after birth (Fig. 2G). Hatching rate was significantly decreased in the polr2i gene morphants compared to the control group (Fig. 2H). In addition, 85% of polr2i gene-MO embryos died at 14 dpf. The onset of mortality was frequently preceded by progressive pericardial edema, implicating cardiac dysfunction as a primary factor in the early mortality observed in polr2i gene morphants. The rate of malformations increased significantly in the morphant (Fig. 2I) and the body length was significantly decreased in the polr2i gene morphants compared to the control group (Fig. 2J).

It is worth noting that when the polr2i gene was supplemented, the developmental abnormality related phenotype of zebrafish at 3 dpf was rescued. According to phenotype speculation, there may be a dose effect of mRNA. Using different concentrations of polr2i gene-MO:polr2i gene-mRNA (1:1; 1:2; 1:3), it was found that there was no significant difference in the phenotype of different concentrations of replenishment injection (Supplementary Fig. 2A). Compared with the knockout group, the total mortality rate (Supplementary Fig. 2B), malformation rate (Supplementary Fig. 2C), heart rate (Supplementary Fig. 2D), body length (Supplementary Fig. 2E), pericardial area (Supplementary Fig. 2F), and eye area (Supplementary Fig. 2G) of the supplementation group showed statistical differences, but there were no significant differences within each supplementation group. Overall, these results indicate that polr2i gene plays a crucial role in the developmental stages of zebrafish.

To assess structural heart abnormalities in polr2i gene knockdown embryos, we examined the hearts of 72 hpf embryos injected with control-MO and polr2i gene-MO, utilizing the Tg(myl7:GFP) transgenic strain that highlights the myocardium in green fluorescent protein (Fig. 3A–D). We observed elongated, string-like heart tubes with minimal overlap between the ventricle and atrium. And in the population of zebrafish larvae with low expression of polr2i gene, the incidence of cardiac abnormalities is much higher than that of the control group (Fig. 3E). Notably, by 72 hpf, pericardial edema was significantly exacerbated (Fig. 3F), indicating severe cardiac malformations or functional deficits in polr2i gene mutants.

Fig. 3.

Effects of polr2i gene knockdown on the heart of zebrafish. (A–D) Phenotypes of larvae of Tg(myl7:EGFP) lines. (E) Significantly more polr2i gene-MO embryos had abnormal cardiac development than control-MO embryos (n = 200, N = 3). (F) Pericardial area at 72 hpf (n = 10, N = 3). (G–K) Quantified analysis data after high-speed photography using ImageJ software. EDV (nL), ESV (nL), stroke volume (nL), ejection fraction, heart rate (per minute) and cardiac output (nL/min) in 72 hpf control-MO (n = 10, N = 3) and polr2i gene-MO (n = 10, N = 3) zebrafish embryos. (L) The expression levels of genes related to heart development in zebrafish embryos were detected using qRT-PCR technology after polr2i-MO knockdown or control-MO treatment (n = 50, N = 3). The scale bar represents 100 µm. A, atrium; V, ventricle. Student’s t-test, **, p 0.01, ***, p 0.001, ns: no statistical difference.

To evaluate polr2i gene-MO hearts for functional deficits, we measured ejection fraction and cardiac output in 72 hpf embryos. We used fluorescence microscopy to capture dynamic images of GFP+ hearts in control-MO and polr2i gene-MO Tg(myl7:GFP) transgenic embryos over several cardiac cycles. We extracted dynamic chamber volumes over time, including end-diastolic volume (EDV) and end-systolic volume (ESV). EDV and ESV were significantly reduced in polr2i gene morphant embryos (Fig. 3G). Three indices of ventricular function, all of which are calculated from EDV and ESV, were also significantly reduced. These include stroke volume SV = EDV – ESV, ejection fraction EF = SV/EDV and cardiac output (stroke volume $\times$ heart rate; Fig. 3H,I,J). The reduced ejection fraction is suggestive of systolic heart failure stemming from compromised cardiomyocyte contraction. In addition to these structural defects, decreased heart rate was also detected in polr2i gene morphant embryos at 48 and 72 hpf (Fig. 3K). Reduced ventricular and atrial contractility resulting in slower blood flow compared to control embryos.

Additionally, qRT-PCR analysis at 72 hpf revealed significant alterations in cardiovascular gene expression in polr2i gene morphant (Fig. 3L). Expressions of cardiac markers such as nppa, nkx2.5, and bmp4 were elevated, whereas gata4 showed reduced expression in polr2i gene-MO embryos, highlighting potential cardiovascular malformations resulting from the loss of polr2i gene function.

Vertebrates exhibit distinct left-right asymmetries in the structure and positioning of their cardiovascular and gastrointestinal systems. Abnormalities in these asymmetries lead to a wide range of congenital defects, including heart malformations.

We observed significant disturbances in cardiac left-right asymmetric patterning or looping in about 47% of polr2i gene-MO embryos at 72 hpf, including anomalies such as straight and D-looped heart tubes (Fig. 4A,B). Similar laterality abnormalities in the liver and pancreas were also noted in polr2i gene mutants compared to controls (Fig. 4C–F), emphasizing the important role of polr2i gene in the development of left-right asymmetry in zebrafish. The precise mechanisms underlying these defects require further investigation.

Fig. 4.

polr2i gene regulates the left-right asymmetry at early stage during zebrafish embryogenesis. (A) Dorsal view of embryonic hearts at 72 hpf in Tg(myl7:EGFP) lines (n = 10, N = 3). L, L-loop; S, Straight; D, D-loop. (B) Cardiac phenotype distribution of embryos injected with control-MO or polr2i gene-MO (n = 10, N = 3). (C) Dorsal view of embryonic livers at 72 hpf in Tg(fabp10a:dsRed) lines (n = 10, N = 3). L, Left; M, Middle; R, Right. (D) Hepatic phenotype distribution of embryos injected with control-MO or polr2i gene-MO (n = 10, N = 3). (E) Dorsal view of embryonic pancreas at 72 hpf in Tg(ela3l:EGFP) lines (n = 10, N = 3). L, Left; M, Middle; R, Right. (F) Pancreatic phenotype distribution of embryos injected with control-MO or polr2i gene-MO (n = 10, N = 3). The scale bar represents 100 µm. A, atrium; V, ventricle; Li, liver; Pa, pancreas.

Recent studies highlight the critical roles of mitochondrial dynamics and autophagy in cardiac health, emphasizing the balance between mitochondrial biogenesis and mitophagy to maintain mitochondrial number [24]. To evaluate mitochondrial quality and turnover rate, we utilized the MitoTimer—a mutant of the red fluorescent protein where fluorescence transitions from green to red as the protein matures—targeted to the mitochondrial matrix with a mitochondrial-targeting sequence. This construct was introduced into myocardial tissue using Tol2 technology to generate transgenic zebrafish expressing myocardium-specific mitochondrial markers.

We knocked down the polr2i gene in Tg(cmlc2:MitoTimer) transgenic zebrafish embryos to explore the relationship between polr2i gene expression and mitochondrial integrity (Fig. 5A–F). By 72 hpf, fluorescence microscopy revealed a significant reduction in mitochondrial quality and turnover rate in polr2i gene knockdown embryos compared to controls (Fig. 5G,H). This suggests that there is a correlation between polr2i gene knockdown, myocardial mitochondrial turnover and cardiac development.

Fig. 5.

Morpholino knockdown of polr2i gene impairs the myocardial mitochondrial quality in zebrafish. (A–F) The embryos injected with control or polr2i gene morpholino were analyzed under light (A,D) and fluorescence microscopy (B,C,E,F) for heart defects at 72 hpf. (G) Red fluorescence intensity of myocardium in Tg(cmlc2:MitoTimer) embryos injected with control-MO (n = 10) or polr2i gene-MO (n = 10). (H) (Red fluorescence intensity)/(green fluorescence intensity) of myocardium in Tg(myl7:EGFP;MitoTimer:dsRed) embryos injected with control-MO (n = 10) or polr2i gene-MO (n = 10). The scale bar represents 100 µm. Square: control group; Triangle: experimental group. The red box highlights the heart region. A, atrium; V, ventricle; ***, p 0.001.

Cardiac malformations are often associated with vascular abnormalities and compromised blood circulation. In control-MO injected Tg(fli1a:EGFP) zebrafish embryos, vascular structures, including natural intersegmental vessels (ISVs), appeared regular by 3 dpf (Fig. 6A,B). Conversely, polr2i gene-MO injected embryos exhibited significant angiogenesis impairment, characterized by a reduced number and length of ISVs (Fig. 6C–F). The caudal vein plexus (CVP), typically forming honeycomb-like structures at the tail by 3 dpf in controls, showed fewer loops in polr2i gene-MO embryos (Fig. 6B,D,G).

Fig. 6.

Morpholino knockdown of polr2i gene impairs the angiogenesis and CVP formation in zebrafish. (A–D) Representative fluorescent images of Tg(fli1a:EGFP) embryos at 72 hpf, with the vascular structures visualized by EGFP fluorescence. The red boxed regions of (A,C) are shown at a higher magnification in (B,D), respectively (Yellow arrow: Internodal blood vessels; White arrow: Tail vein plexus; White dashed box: vascular compensatory hyperplasia phenomenon. n = 10). (B) ISVs and CVP showed regular development in the embryos injected with control-MO (n = 10). (D) Compared with the control group, embryos injected with polr2i gene-MO presented a lower number of incomplete ISVs and specific defects in CVP formation (n = 10). (E) Quantification of the number of complete ISVs shows a decrease in polr2i gene morphants (n = 10). (F) Quantification of the total length of the ISVs shows a decrease in polr2i gene morphants (n = 10). (G) Quantification of loop formation at CVP shows a decrease in polr2i gene morphants (n = 10). ISV, intersegmental vessel; CVP, caudal vein plexus. The scale bar represents 100 µm. Square: control group; Triangle: experimental group. **, p 0.01, ***, p 0.001.

In addition, polr2i gene-mutated embryos often exhibit blood accumulation and clotting in the heart chamber and blood vessels. O-anisidine staining showed red blood cells (RBCs) accumulation. Compared with the control group (Fig. 7A–D), it was significantly enhanced in polr2i gene knockdown embryos (Fig. 7E–H), especially at 96 hpf, highlighting the serious vascular and circulatory damage caused by polr2i gene deletion. Morphometric quantification confirmed 1.8 $\pm$ 0.3-fold expansion of congested areas in morphants (Fig. 7I).

Fig. 7.

Morpholino knockdown of polr2i gene impairs the blood circulation in zebrafish. (A–D) The staining of hemoglobin in the control-MO group at 96 hpf. (E–H) The staining of hemoglobin in the polr2i gene-MO group at 96 hpf. (I) Quantify the areas of their erythrocyte aggregation. The red arrows indicate red blood cells accumulation in heart (n = 10). The scale bar represents 100 µm. Square: control group; Triangle: experimental group. ***, p 0.001.