Dual-color Fluorescence Research Articles

A rapid and closed-tube method based on the dual-color fluorescence loop-mediated isothermal amplification for visual detection of Atlantic salmon (Salmo salar)

Loop-mediated isothermal amplification (LAMP) visual detection based on hydroxyl naphthol blue (HNB) and SYTO 9 is often confounded by the narrow color variation window and the requirement of empirical preset of cutoff intensity value. To improve the suitability for naked-eye inspection, the present work proposed a strategy based on the fluorescence property of SYTO 9 and HNB to enlarge the contrast and a novel dual-color fluorescence LAMP (dfLAMP) assay was developed for visual detection of Atlantic salmon. Specifically, HNB of 26.25 μM, blended with SYTO 9 of 0.75–1.5 μM, was added in the mixture before amplification, producing light green fluorescence for both positive and negative samples. After amplification, green or yellow-green fluorescence was observed for positive samples, while only orange-red fluorescence emitted for negative ones, enabling an easy and rapid distinguish. The optimized dfLAMP assay has proved its specificity and can detect as little as 1 fg Atlantic salmon DNA.

Rational construction of sulfur dioxide and viscosity bifunctional fluorescent probe and its applications in water, food and bioimaging

Abnormal sulfur dioxide (SO2) and microenvironmental viscosity are detrimental to human health, so the effective monitoring of these indicators in relevant foods and organisms is of great significance. The dual-functional fluorescent probes that are sensitive to active sulfur and microenvironment at the subcellular organelle level can link the spatiotemporal changes of different species, which are crucial for understanding complex biological processes. In this work, a high-performance bifunctional red fluorescent probe named Probe 2 was rationally designed and constructed by screening different electron-donating groups. The probe exhibits good chromogenic and turn-on fluorescence responses to SO2 and viscosity, and can target mitochondria well. The probe has been successfully applied for accurate and rapid determination of sulfite content in water and food samples with good recovery (93.2–98.1 % and 94.2–100.4 %). Furthermore, Probe 2 enables real-time tracking and dual-color fluorescence imaging of SO2 and viscosity in cells and zebrafish, especially its high sensitivity to changes in these two parameters during mitochondrial dysfunction and apoptosis.

Single-Particle Analysis of the Interaction Between Molecules and Protein Aggregated Species by Dual-Color Time-Resolved Fluorescence Spectroscopy.

Amyloid protein aggregation is widely involved in a number of neurodegenerative diseases for which novel therapeutic and diagnostic strategies are still needed. Owing to the complex and heterogeneous nature of the aggregated species responsible for toxicity in these disorders, a detailed characterization of the interaction of molecules of interest with the amyloid aggregates is a challenging endeavor. Here, we present the experimental and analytical steps of a protocol which combines dual-color fluorescence cross-correlation spectroscopy and dual-color single-particle fluorescence spectroscopy to quantify the binding affinity and stoichiometryof an inhibitor of α-synuclein amyloid aggregation. This approach allows studying the interaction in detail and through two independent analytical methods, thus yielding a remarkably robust tool that could be extended to investigating the interaction of molecules of interest to other pathogenic protein aggregates as well as multi-ligand/multi-receptor complexes.

Cost-effective high-speed, three-dimensional live-cell imaging of HIV-1 transfer at the T cell virological synapse

The availability of cost-effective, highly portable, and easy to use high-resolution live-cell imaging systems could present a significant technological break-through in challenging environments, such as high-level biosafety laboratories or sites where new viral outbreaks are suspected. We describe and demonstrate a cost-effective high-speed fluorescence microscope enabling the live tracking of virus particles across virological synapses that form between infected and uninfected Tcells. The dynamics of HIV-1 proteins studied at the cellular level and the formation of virological synapses in living Tcells reveals mechanisms by which cell-cell interactions facilitate infection between immune cells. Dual-color 3D fluorescence deconvolution microscopy of HIV-1 particles at frames rates of 100 frames per second allows us to follow the transfer of HIV-1 particles across the Tcell virological synapse between living Tcells. We also confirm the successful transfer of virus by imaging Tcell samples fixed at specific time points during cell-cell virus transfer by super-resolution structured illumination microscopy.

Differentiation Capacity of Bone Marrow-Derived Rat Mesenchymal Stem Cells from DsRed and Cre Transgenic Cre/loxP Models

Cre/loxP recombination is a well-established technique increasingly used for modifying DNA both in vitro and in vivo. Nucleotide alterations can be edited in the genomes of mammalian cells, and genetic switches can be designed to target the expression or excision of a gene in any tissue at any time in animal models. In this study, we propose a system which worked via the Cre/loxP switch gene and DsRed/emGFP dual-color fluorescence imaging. Mesenchymal stem cells (MSCs) can be used to regenerate damaged tissue because of their differentiation capacity. Although previous studies have presented evidence of fusion of transplanted MSCs with recipient cells, the possibility of fusion in such cases remains debated. Moreover, the effects and biological implications of the fusion of MSCs at the tissue and organ level have not yet been elucidated. Thus, the method for determining this issue is significant and the models we proposed can illustrate the question. However, the transgenic rats exhibited growth slower than that of wild-type rats over several weeks. The effects on the stemness, proliferation, cell cycle, and differentiation ability of bone marrow–derived rat MSCs (BM-rMSCs) from the models were examined to ensure our design was appropriate for the in vivo application. We demonstrated that MSC surface markers were maintained in DsRed and Cre transgenic rMSCs (DsRed-rMSCs and Cre-rMSCs, respectively). A WST-8 assay revealed decreased proliferative activity in these DsRed-rMSCs and Cre-rMSCs; this result was validated through cell counting. Furthermore, cell cycle analysis indicated a decrease in the proportion of G1-phase cells and a concomitant increase in the proportion of S-phase cells. The levels of cell cycle–related proteins also decreased in the DsRed-rMSCs and Cre-rMSCs, implying decelerated phase transition. However, the BM-rMSCs collected from the transgenic rats did not exhibit altered adipogenesis, osteogenesis, or chondrogenesis. The specific markers of these types of differentiation were upregulated after induction. Therefore, BM-rMSCs from DsRed and Cre transgenic models can be used to investigate the behavior of MSCs and related mechanisms. Such application may further the development of stem cell therapy for tissue damage and other diseases.

Three-dimensional visualization of heart-wide myocardial architecture and vascular network simultaneously at single-cell resolution.

Obtaining various structures of the entire mature heart at single-cell resolution is highly desired in cardiac studies; however, effective methodologies are still lacking. Here, we propose a pipeline for labeling and imaging myocardial and vascular structures. In this pipeline, the myocardium is counterstained using fluorescent dyes and the cardiovasculature is labeled using transgenic markers. High-definition dual-color fluorescence micro-optical sectioning tomography is used to perform heart-wide tissue imaging, enabling the acquisition of whole-heart data at a voxel resolution of 0.32 × 0.32 × 1 μm3. Obtained structural data demonstrated the superiority of the pipeline. In particular, the three-dimensional morphology and spatial arrangement of reconstructed cardiomyocytes were revealed, and high-resolution vascular data helped determine differences in the features of endothelial cells and complex coiled capillaries. Our pipeline can be used in cardiac studies for examining the structures of the entire heart at the single-cell level.

Fusion between Glioma Stem Cells and Mesenchymal Stem Cells Promotes Malignant Progression in 3D-Bioprinted Models.

The interaction between glioma stem cells (GSCs) and mesenchymal stem cells (MSCs) in the glioma microenvironment is considered to be an important factor in promoting tumor progression, but the mechanism is still not fully elucidated. To further elucidate the interaction between GSCs and MSCs, two 3D-bioprinted tumor models (low-temperature molding and coaxial bioprinting) were used to simulate the tumor growth microenvironment. Cell fusion between GSCs and MSCs was found by the method of Cre-LoxP switch gene and RFP/GFP dual-color fluorescence tracing. The fused cells coexpressed biomarkers of GSCs and MSCs, showing stronger proliferation, cloning, and invasion abilities than GSCs and MSCs. In addition, the fused cells have stronger tumorigenic properties in nude mice, showing the pathological features of malignant tumors. In conclusion, GSCs and MSCs undergo cell fusion in 3D-bioprinted models, and the fused cells have a higher degree of malignancy than parental cells, which promotes the progression of glioma.

Probing Iron-Mediated Synergistic Change of Cl- and HClO in Liver Cancer Cells with a Dual-Color Fluorescence Reporter.

The ambiguous molecular mechanism remains a leading cause for the high mortality rate of liver cancer. An evident iron overload has been unveiled in liver cancer cell proliferation, which is closely related to oxidative stress. However oxidative stress-regulated chloride intracellular channel protein 1 (CLIC1) obviously increases in liver cancer cells. Cl- is also involved in cell proliferation, and its downstream product, HClO, can induce cell carcinoma when over-generated. However, whether iron overload could mediate the variation of intracellular Cl- and HClO is still uncharted. Herein, we present a dual-responsive fluorescence reporter MQFL-NH2 for simultaneously visualizing the fluctuation of Cl-/HClO at the same spot in living cells. Electrostatic binding to Cl- effectively gave an attenuated signal with blue fluorescence, and HClO induced a sharp green fluorescence. In HL-7702 cells stimulated with iron, the blue/green dual fluorescence of MQFL-NH2 displayed that Cl- and HClO were elevated. In contrast, they were both reduced in iron-removed SMMC-7721 cells. Further results revealed that iron overload could promote the levels of Cl- and HClO by up-regulating CLIC1 and myeloperoxidase. Altogether, the work will energetically contribute to grasp the molecular mechanism in iron overload-mediated pathogenesis of liver cancer.

2D MOF Nanosensor-Integrated Digital Droplet Microfluidic Flow Cytometry for In Situ Detection of Multiple miRNAs in Single CTC Cells.

Current circulating tumor cells (CTCs) detection strategies based on surface epithelial markers suffer from low specificity in distinguishing between CTCs and epithelial cells in hematopoietic cell population. Tumor-associated miRNAs within CTCs are emerging as new biomarkers due to their high correlation with tumor development and progress. However, in-situ simultaneous analysis of multiple miRNAs in single CTC cell is still challenging. To overcome this limitation, a digital droplet microfluidic flow cytometry based on biofunctionalized 2D metal-organic framework nanosensor (Nano-DMFC) is developed for in situ detection of dual miRNAs simultaneously in single living breast cancer cells. Here, 2D MOF-based fluorescent resonance energy transfer (FRET) nanosensors are established by conjugating dual-color fluorescence dye-labeled DNA probes on MOF nanosheet surface. In the Nano-DMFC, 2D MOF-based nanoprobes are precisely microinjected into each single-cell encapsulated droplets to achieve dual miRNA characterization in single cancer cell. This Nano-DMFC platform successfully detects dual miRNAs at single-cell resolution in 10 mixed positive MCF-7 cells out of 10 000 negative epithelial cells in serum biomimic samples. Moreover, this Nano-DMFC platform shows good reproductivity in the recovery experiment of spiked blood samples, which demonstrate the high potential for CTC-based cancer early diagnosis and prognosis.

Mechanisms of phase-separation-mediated cGAS activation revealed by dcFCCS.

Cyclic GMP-AMP synthase (cGAS), as a DNA sensor, plays an important role in cGAS-STING pathway, which further induces expression of type I interferon as the innate immune response. Previous studies reported that liquid-liquid phase separation (LLPS) driven by cGAS and long DNA is essential to promote catalytic activity of cGAS to produce a second messenger, cyclic GMP-AMP (cGAMP). However, the molecular mechanism of LLPS promoting cGAS activity is still unclear. Here, we applied dual-color fluorescence cross-correlation spectroscopy (dcFCCS), a highly sensitive and quantitative method, to characterize phase separation driven by cGAS and DNA from miscible individual molecule to micronscale. Thus, we captured nanoscale condensates formed by cGAS at close-to-physiological concentration and quantified their sizes, molecular compositions and binding affinities within condensates. Our results pinpointed that interactions between DNA and cGAS at DNA binding sites A, B, and C and the dimerization of cGAS are the fundamental molecular basis to fully activate cGAS in vitro. Due to weak binding constants of these sites, endogenous cGAS cannot form stable interactions at these sites, leading to no activity in the absence of LLPS. Phase separation of cGAS and DNA enriches cGAS and DNA by 2 to 3 orders of magnitude to facilitate these interactions among cGAS and DNA and to promote cGAS activity as an on/off switch. Our discoveries not only shed lights on the molecular mechanisms of phase-separation-mediated cGAS activation, but also guided us to engineer a cGAS fusion, which can be activated by 15bp short DNA without LLPS.

Typical, atypical and cryptic t(15;17)(q24;q21) (PML::RARA) observed in acute promyelocytic leukemia: A retrospective review of 831 patients with concurrent chromosome and PML::RARA dual-color dual-fusion FISH studies.

The diagnosis of acute promyelocytic leukemia (APL) relies on the identification of PML::RARA fusion. While the majority of APL cases harbor a typical t(15;17)(q24;q21), atypical genetic mechanisms leading to the oncogenic PML::RARA fusion have been reported yet their frequency and scope remain poorly characterized. We assessed the genetic findings of 831 cases with APL investigated with concurrent chromosome banding analysis and dual-color dual-fusion fluorescence in situ hybridization (D-FISH) analysis at our institution over an 18.5-year timeframe. Seven hundred twenty-three (87%) cases had a typical balanced t(15;17) with both testing modalities. Atypical karyotypic results including complex translocations, unbalanced rearrangements and insertional events occurred in 50 (6%) cases, while 6 (0.7%) cases were cryptic by conventional chromosome studies despite PML::RARA fusion by D-FISH evaluation. Atypical FISH patterns were observed in 48 (6%) cases despite apparently balanced t(15;17) on chromosome banding analysis. Two hundred fifty (30%) cases displayed additional chromosome abnormalities of which trisomy/tetrasomy 8 (37%), del(7q)/add(7q) (12%), and del(9q) (7%) were most frequent. Complex and very complex karyotypes were observed in 81 (10%) and 34 (4%) cases, respectively. In addition, 4 (0.5%) cases presented as an apparently doubled, near-tetraploid stemline clone. This report provides the largest appraisal of cytogenetic findings in APL with conventional chromosome and PML::RARA D-FISH analysis. By characterizing the frequency and breadth of typical and atypical results through the lens of these cytogenetic testing modalities, this study serves as a pragmatic source of information for those involved in the investigation of APL in both the clinical and research laboratory settings.

Encoding with a fluorescence-activating and absorption-shifting tag generates living bacterial probes for mammalian microbiota imaging.

The mammalian microbiota plays essential roles in health. A primary determinant to understand the interaction with the host is the distribution and viability of its key microorganisms. Here, a strategy of encoding with a fluorescence-activating and absorption-shifting tag (FAST) is reported to prepare living bacterial probes for real-time dynamic, dual-modal, and molecular oxygen-independent imaging of the host microbiota. Carrying FAST endows bacteria with rapid on-demand turn on-off fluorescence by adding or removal of corresponding fluorogens. Encoded bacteria are able to reversibly switch emission bands for dual-color fluorescence imaging via fluorogen exchange. Due to molecular oxygen-independent emission of FAST, encoded bacteria can emit fluorescence under anaerobic environments including the gut and tumor. These living probes demonstrate the applicability to quantify the vitality of bacteria transplanted to the gut microbiota. This work proposes a unique fluorescence probe for investigating the dynamics of the host microbiota.

Aptamer functionalization and high-contrast reversible dual-color photoswitching fluorescence of polymeric nanoparticles for latent fingerprints imaging

Exploiting the intelligent stimuli-responsive fluorescent materials, which can selectively recognize lysozyme in fingerprints and enable high-contrast imaging, is still a challenge in latent fingerprints (LFPs) detection. Herein, based on Fluorescence Resonance Energy Transfer (FRET) mechanism, aptamer functionalization and high-contrast reversible dual-color photoswitching fluorescence of polymeric nanoparticles (LBA-RPFPNs) have been successfully constructed for efficient imaging of LFPs. Experiments results showed that LBA-RPFPNs displayed good water dispersibility, fast photo-responsiveness, favorable photoreversibility and good long-term stability. Of note, the aptamer possessed the ability of LBA-RPFPNs to target lysozyme in LFPs, thus realizing the selectivity of LFPs. Furthermore, the fluorescence of LBA-RPFPNs was reversibly switched between green and red through UV/visible light, achieving for high-contrast imaging of LFPs. This strategy provides good potential in disclosing the valuable information within LFPs.

Comprehensive Analysis of miRNA Regulation of MGAT3 Using the miRFluR Assay

Studies related to miRNAs and their ability to tune protein expression have gained much attention in recent years. In the field of glycobiology, miRNAs are now given special consideration as they have been shown to be key regulators of many glycosylation pathways. The enzyme MGAT3 (β‐1,4‐mannosyl‐glycoprotein 4‐beta‐N‐acetylglucosaminyltransferase) is responsible for the addition of a bisecting GlcNAc to the core mannose residue of complex or hybrid N‐glycans. This is a unique modification of complex N‐glycans that has been reported to play important roles in signal transduction, growth factor signaling, tumor progression and metastasis. In this project, we generated a comprehensive map of the regulation of MGAT3 by miRNA using a newly developed high throughput assay: miRFluR. This assay uses ratiometric analysis of a genetically encoded dual‐color fluorescence reporter (cerulean/mCherry) to identify regulatory miRNA:mRNA interactions. A dataset for the interaction between the entire human miRome (~2700 miRs) and MGAT3 has been collected and analysis of the miR regulatory map is presented. Our data provides insight into the regulation of this important glycan and its functions in disease.

Assessment of the Role of NuclearENDOGGene and mtDNA Variations on Paternal Mitochondrial Elimination (PME) in Infertile Men: An Experimental Study.

In humans and most animals, maternal inheritance of mitochondria and mitochondrial DNA (mtDNA) is considered as an universal assumption. Recently, several lines of evidence suggest that different species seem to employ distinct mechanisms to prevent the inheritance of paternal mtDNA. There are few studies in the literature on the molecular basis of sperm mtDNA elimination in mammals and paternal mtDNA transmission in humans. Endonuclease G (ENDOG) is a mitochondrial nuclease encoded by nuclear ENDOG gene. The critical importance of ENDOG gene on paternal mitochondrial elimination (PME) has been previously demonstrated in model organisms such as C. elegans and D. melanogaster. However, its mechanism in human is still unclear. Therefore, we aimed to evaluate whether nuclear ENDOG gene copy number could be a potential marker of paternal mtDNA transmission or not.Male factor infertility patients diagnosed with different infertility subgroups such as azoospermia, oligoteratozoospermia, astheno-teratozoospermia were included in this study: 13 infertile men and 25 healthy men as control group. Quantitative real-time polymerase chain reaction (qPCR) analysis and dual-color Fluorescence in situ hybridization (FISH) method were used to compare the groups. FISH method was applied to verify qPCR results and two signals were observed in nearly all patients. ENDOG gene copy number data were evaluated by comparing them with entire human mtDNA next-generation sequencing (NGS) analysis results obtained through bioinformatics and proteomics tools. Mitochondrial whole genome sequencing (WGS) data allowed determination of novel and reported variations such as single nucleotide polymorphisms (SNPs), multiple nucleotide polymorphism (MNP), insertion/deletion (INDEL). Missense variants causing amino acid substitution were filtered out from patients' mtDNA WGS data.Relative copy number of target ENDOG gene in male infertility patients [0.49 (0.31 - 0.77)] was lower than healthy controls [1.00 (0.66 - 1.51)], and statistical results showed significant differences between the groups (p < 0.01). A total of 38 missense variants were detected in the genes encoding the proteins involved in the respiratory chain complex. Moreover, we detected paternal mtDNA transmissions in the children of these patients who applied to assisted reproductive techniques.In conclusion, this study reveals that ENDOG gene may be an important factor for the PME mechanism in humans. To the best of our knowledge, this is the first study in humans about this topic and assessment of ENDOG gene sequencing and gene expression studies in a larger sample size including patients with male factor infertility would be our future project.

A TICS-fluorophore based probe for dual-color GSH imaging

Cascading reactions in fluorophores accompanied by the replacement of different fluorescence wavelengths can be used to develop luminescent materials and reactive fluorescent probes. Based on multiple signal channels, the selectivity of probes can be improved and the range of response to guest molecule recognition can be expanded. By regulating the position, number, and activity of active sites in fluorophores, fluorescent probes that successively react with thiol and amino groups in cysteine (Cys), homocysteine (Hcy) have been developed, which can only react with the thiol group of GSH. In this paper, we report the first probe capable of cascading nucleophilic substitution reaction with the thiol group and amino group of GSH at a single reaction site, and showed the dual-color recognition of GSH, which improved the selectivity of GSH also was an extension of GSH probes. The probe Rho-DEA was based on a TICS fluorophore, and the intramolecular cascade nucleophilic substitution reaction occurs with Cys/Hcy. The thiol substitution of the first step reaction with Cys/Hcy was quenched due to intersystem crossing to triplet state, so GSH can be selectively recognized from the fluorescence signal. Rho-DEA has the ability of mitochondrial localization, and finally realized in situ dual-color fluorescence recognition of GSH in mitochondria.

Cationic Conjugated Polyelectrolytes with Aggregation-Induced Ratiometric Fluorescence.

The molecular diversity of aggregation-induced emission remains a challenge due to the limitation of conventional synthesis methods. Here, a series of novel neutral and cationic conjugated polymers composed of various ratios of tetraarylethylene (TAE) containing a bridged oxygen (O) and fluorene (F) units is designed and synthesized via the geminal cross-coupling (GCC) of 1,1-dibromoolefins. The incorporation of TAE segments into the conjugated backbone of polyfluorene produces pronounced aggregation-induced ratiometric fluorescence, i.e., aggregation-induced emission (AIE) at 520-600nm and grows synergistically with aggregations-caused quenching (ACQ) at 400-450nm. The content of fluorene unit in the polymer backbones determines the intensity of the initial fluorescence in the blue light region. The huge distinction (about 150nm) in dual emission wavelengths caused by the environment change makes these conjugated polyelectrolytes particularly suitable for ratiometric fluorescence sensing. Based on electrostatic interaction mechanism, the gradual addition of heparin into the cationic conjugated polymers aqueous solutions can induce dual-color fluorescence changes with a detection limit of 9× 10-9 m. This work exhibits the great facility of using GCC reaction to synthesis the conjugated TAE polymers with superior AIE properties and special functions.

Dual-Color Fluorescence Switch-On Probe for Imaging G-Quadruplex and Double-Stranded DNA in Living Cells.

A tripodal quinone-cyanine dye having one donor and three acceptors, that is, one quinone and three N-methylbenzothiazolium moieties, QCy(MeBT)3, was synthesized by simple Knoevenagel condensation between 2-hydroxybenzene-1,3,5-tricarbaldehyde and N-methyl-2-methylbenzothiazolium iodide. The 700 nm (λex, 570 nm) and 600 nm (λex, 470 nm) fluorescence emission of QCy(MeBT)3 was significantly and individually enhanced with the addition of G-quadruplex (G4) DNA and double-stranded DNA (dsDNA), respectively. The results of docking simulations and the response against the viscosity change revealed that the dual-fluorescence response was caused by the difference in the binding mode of QCy(MeBT)3 depending on the DNA structure. The results of fluorescence microscopy imaging experiments using QCy(MeBT)3 suggested that G4 DNAs and dsDNAs in the cell nucleus can be imaged with near-infrared (NIR, 700 nm) and red (600 nm) fluorescence emissions. Furthermore, pyridostatin-induced G4 formation in the living cells can be imaged with NIR fluorescence. The results indicated that QCy(MeBT)3 has huge potential to be a NIR-fluorescent molecular probe for analyzing the structural dynamics of nucleic acids in living cells with a normal fluorescence microscope.

Lensless dual-color fluorescence imaging device using hybrid filter

In this study, a dual-band hybrid filter that achieves high excitation light rejection performance in a lensless imaging system was fabricated and incorporated into an imaging device. The hybrid filter consisted of interference and absorption filters, and a fiber optic plate (FOP). The interference filters were attached to both sides of the FOP, which was placed on top of the absorption filter to suppress the decrease in spatial resolution. In addition, the lamination order was optimized to achieve a high fluorescence observation performance. The fabricated hybrid filter was mounted on an image sensor and had the ability to indicate the green and red fluorescence components.

Neuron navigator 3 (NAV3) is required for heart development in zebrafish.

As a tightly controlled biological process, cardiogenesis requires the specification and migration of a suite of cell types to form a particular three-dimensional configuration of the heart. Many genetic factors are involved in the formation and maturation of the heart, and any genetic mutations may result in severe cardiac failures. The neuron navigator (NAV) family consists of three vertebrate homologs (NAV1, NAV2, and NAV3) of the neural guidance molecule uncoordinated-53 (UNC-53) in Caenorhabditis elegans. Although they are recognized as neural regulators, their expressions are also detected in many organs, including the heart, kidney, and liver. However, the functions of NAVs, regardless of neural guidance, remain largely unexplored. In our study, we found that nav3 gene was expressed in the cardiac region of zebrafish embryos from 24 to 48h post-fertilization (hpf) by means of in situ hybridization (ISH) assay. A CRISPR/Cas9-based genome editing method was utilized to delete the nav3 gene in zebrafish and loss of function of Nav3 resulted in a severe deficiency in its cardiac morphology and structure. The similar phenotypic defects of the knockout mutants could recur by nav3 morpholino injection and be rescued by nav3 mRNA injection. Dual-color fluorescence imaging of ventricle and atrium markers further confirmed the disruption of the heart development in nav3-deleted mutants. Although the heart rate was not affected by the deletion of nav3, the heartbeat intensity was decreased in the mutants. All these findings indicate that Nav3 was required for cardiogenesis in developing zebrafish embryos.