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Article

High-Salt Exposure Disrupts Cardiovascular Development in Zebrafish Embryos, Brachyodanio rerio, via Calcium and MAPK Signaling Pathways

by
Ebony Thompson
1,
Justin Hensley
1 and
Renfang Song Taylor
2,*
1
Division of Natural Sciences, Southwest Baptist University, 1600 University Rd, Bolivar, MO 65613, USA
2
Biology Department, School of Physical Sciences and Engineering, 1100 5th Street, Anderson, IN 46012, USA
*
Author to whom correspondence should be addressed.
J 2025, 8(3), 26; https://doi.org/10.3390/j8030026
Submission received: 7 May 2025 / Revised: 27 June 2025 / Accepted: 8 July 2025 / Published: 14 July 2025
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Abstract

Cardiovascular disease and hypertension are major global health challenges, and increasing dietary salt intake is a known contributor. Emerging evidence suggests that excessive salt exposure during pregnancy may impact fetal development, yet its effects on early embryogenesis remain poorly understood. In this study, we used zebrafish (Danio rerio) embryos as a model to investigate the developmental and molecular consequences of high-salt exposure during early vertebrate development. Embryos subjected to elevated salt levels exhibited delayed hatching, reduced heart rates, and significant alterations in gene expression profiles. Transcriptomic analysis revealed over 4000 differentially expressed genes, with key disruptions identified in calcium signaling, MAPK signaling, cardiac muscle development, and vascular smooth muscle contraction pathways. These findings indicate that early salt exposure can perturb crucial developmental processes and signaling networks, offering insights into how prenatal environmental factors may contribute to long-term cardiovascular risk.

1. Introduction

Cardiovascular diseases are the leading cause of mortality each year worldwide [1]. With today’s fast-paced lifestyle, a high dietary intake of sodium is common in both adults and even younger populations. High salt intake is associated with elevated blood pressure, a major risk factor for cardiovascular disease. From lab-based research to clinical data analysis, studies consistently indicate a strong positive relationship between high sodium intake and adverse cardiovascular outcomes [2,3,4,5,6].
Gestational hypertension may contribute to an increased risk of developing chronic hypertension later in life, and recent studies have suggested that both gestational hypertension and preeclampsia are linked to long-term cardiovascular complications [7]. High salt intake has been shown to reduce implantation sites in rats by altering metabolic pathways and disrupting homeostatic balance [8]. Additionally, salt intake during pregnancy affects fetal renal development and alters the expression of key elements of the renin–angiotensin system, as demonstrated in studies with pregnant ewes [9]. A high-salt maternal diet has also been associated with developmental changes in cardiac cells and the renin–angiotensin system in offspring [10].
The zebrafish (Danio rerio) is an important model organism widely used to study metabolic processes, developmental biology, and genetics due to its small size, optical transparency, ease of treatment, and low-cost embryo care [11,12,13,14,15]. Both embryonic and post-embryonic stages of zebrafish development are well characterized, allowing for detailed studies linking genetic changes to observable morphological outcomes [16,17].
Environmental salt stress affects zebrafish development, influencing hatch rate, survival, brain and heart formation, bladder development, and the reproductive system. Pathways such as Ataxia Telangiectasia Mutated (ATM) and Sonic Hedgehog (Shh) are altered under high-salt conditions [18,19,20,21,22]. High-salt diets during pregnancy are known to induce elevated blood pressure and increase the risk of hypertension in offspring by altering the nitric oxide (NO)/protein kinase G (PKG) pathway [23]. Salt exposure also disrupts metabolic pathways, the renin–angiotensin system, and endocrine signaling, all of which can contribute to cardiovascular disease [24].
The intracellular mitogen-activated protein kinase (MAPK) signaling pathway is a crucial signaling cascade that transmits external signals to the cell interior and regulates key processes such as growth, differentiation, and apoptosis [25]. The MAPK signaling pathway plays a central role in the development of cardiac and vascular disease [26,27] and is especially important in salt-sensitive hypertension [28]. These mechanisms—along with alterations to the renin–angiotensin and endocrine systems—underline the pathogenesis of salt-related cardiovascular disorders. In a previous study, we demonstrated that high glucose exposure affected zebrafish development via the Wnt signaling pathway [29]. In this study, we apply a similar methodology to investigate how salt exposure influences zebrafish embryonic development, with a particular focus on the MAPK and calcium signaling pathways.

2. Materials and Methods

2.1. Materials

Pure sodium chloride (NaCl, Sigma made to 2% w/v) was used to prepare the treatment media. E3 embryonic medium was obtained from Carolina Biological Supply Company, Burlington, NC, USA.

2.2. Animal Husbandry

Adult wild-type zebrafish were obtained from breeding facilities at Carolina Biological Supply Co. Fish maintenance, breeding conditions, and egg production were performed according to internationally accepted standards protocol [30]. Embryos were obtained from Carolina Biological Supply. Adults were raised in embryonic media (E3) under standard conditions at 28.5 °C with a 14 h light/10 h dark cycle, and the embryonic stages are in accordance with standard procedures [11].

2.3. Salt Exposure Protocol

A total of 125 embryos were randomly distributed into five Petri dishes, with 25 embryos per dish. Two dishes (n = 50 embryos) were exposed to E3 medium supplemented with 2% NaCl (salt-treated groups), and three dishes (n = 75 embryos) served as untreated controls in standard E3 medium. Salt exposure began at ~3 hpf and continued until 120 hpf. Hatch rates and heart rates were assessed at defined time points, as described in the Section 3.

2.4. Heart Rate Measurement and Cardiac Imaging

To assess heart rate in developing zebrafish embryos, we recorded short video clips and manually counted heartbeats over a 10 s interval, then converted the values to beats per minute (bpm). Heart rate was measured in five individual embryos per group at each designated time point, and the average heart rate was reported. For morphological analysis, images were captured using an inverted microscope at 4× and 10× magnification, with a focus on the cardiac region to allow for comparison of heart structure between control and salt-treated embryos.

2.5. RNA Extraction and Sequencing

At 120 hpf, whole embryos from the two salt-treated groups and three control groups were pooled into 5 samples (2 salt-treated, 3 controls) for total RNA extraction. RNA was isolated using the Zymo Research RNA Extraction Kit (Irvine, CA, USA), following the manufacturer’s instructions. RNA concentration and quality were assessed using a NanoDrop 2000 spectrophotometer (DeNovix Inc., Wilmington, DE, USA).
RNA sequencing was conducted by Novogene Corporation Inc. (Sacramento, CA, USA). Detailed protocols for library preparation, sequencing, and quality control are described in a previous study [29].

2.6. RNA-Seq Data Processing and Analysis

Raw reads in FASTQ format were quality filtered to remove adaptors, poly-N sequences, and low-quality reads. Clean reads were mapped to the zebrafish reference genome using HISAT2 v2.0.5. StringTie (v1.3.3b) [31] was used for transcript assembly, and Feature Counts v1.5.0-p3 was used for gene-level quantification. Gene expression levels were normalized to FPKM (Fragments Per Kilobase of transcript per Million mapped reads).
Differential gene expression analysis was performed using the DESeq2 R package (v1.20.0), applying Benjamini–Hochberg correction for false discovery rate. Genes with adjusted p ≤ 0.05 were considered significantly differentially expressed.

2.7. Pathway and Functional Enrichment Analysis

Gene Ontology (GO) and KEGG pathway enrichment analyses were conducted using the cluster Profiler R package, with significance set at adjusted p < 0.05. Gene set enrichment analysis (GSEA-2.0) was also performed using the Broad Institute’s software to assess subtle expression changes across predefined gene sets.

2.8. Statistical Analysis

Hatch rate and heart rate data were analyzed using one-way ANOVA, followed by Tukey’s post hoc test for pairwise comparisons. A p-value < 0.05 was considered statistically significant.

3. Results

3.1. Effect of Salt on Zebrafish Embryo Development

3.1.1. The Effect of Salt on Morphology and Physiology Changes

Salt exposure significantly impacted the developmental timeline of zebrafish embryos. Notably, the hatch rate was delayed by approximately 24 h in the salt-exposed group compared to the control group, indicating a delay in embryonic development associated with salt exposure (Figure 1A, ** p < 0.01).
The evaluation of heart rate revealed significant alterations induced by salt exposure. On both day 3 and day 4 post-fertilization, embryos in the salt group exhibited a significantly reduced heart rate compared to those in the control group. The mean heart beat per minute was 100 and 74 in the control and salt groups, respectively, on day 3 and 132 and 84 on day 4, as shown in Figure 1B, * p < 0.05.
Analysis of morphological parameters revealed distinct differences between the salt-exposed and control groups. At 120 hpf, embryos in the salt group exhibited smaller brain, heart, and tail morphology compared to those in the control group. The arrow focuses on the heart size of zebrafish at 120 hpf, as shown in Figure 1C. This suggests that salt exposure may interfere with the normal growth and development of these vital organs during early embryogenesis.

3.1.2. RNA-Seq Analysis Displayed Significant Gene Dysregulation in the Salt-Treated Embryos

In this study, we investigated the global transcriptome changes in salt-treated zebrafish embyos using RNAseq. Biological replicates were performed for the RNA-seq. A Venn diagram shows the overlap genes and unique genes in these two groups of data (Figure 2A). Figure 2B shows the number of genes identified in the gene function classification groups. With 25K transcripts, data analysis identified 4324 (2%) differentially expressed genes (DEGs) with 2133 upregulated and 2190 downregulated, respectively (fold change > 2 and p < 0.05) (Figure 2C,D). Gene expression differs between the control and high-salt treatment groups, as shown in the heatmap in Figure 2E.
To determine changes in expresson levels of individual genes, we identified the top 10 most significant up- and downregulated genes, listed in Table 1 and Table 2. The major GO biological precesses and molecular functions enriched from the top 10 upregulated genes included enabling beta-N-acetylglucosaminidase activity, arresting domain involved in protein transport, pancreatic progenitor cell differentiation and proliferation factor b, monocarboxylic acid transmembrane transport, monocarboxylic acid transmembrane transport, enabling double-stranded DNA binding activity and nucleosomal DNA binding activity and Ino80 complex component located in the nucleus.
Also, glyceophosphocholine and phosphodiesterase activity were measured, and within the top 10 downregulated genes, nucleotidyltransferase activity and tRNA binding activity were examined, as well as the following:
Aconitate decarboxylase activity in mitochondria, taurine/sodium symporter activity;
L-glutamine transmembrane transporter activity, involved in protein coding;
Taurine/sodium symporter activity; intracellular signal transduction; organic cation transmembrane transporter activity; protein coding activity; ATP binding activity; protein serine kinase activity; and protein serine/threonine kinase activity.

3.1.3. Functional Analysis of DEGs Using Gene Ontology (GO) Analysis

In order to identify the functional associations of the DEGs, Gene Ontology (GO) enrichment analysis was performed using the cluster Profiler R package, in which gene length bias was corrected. GO terms with corrected p-values less than 0.05 were considered significantly enriched by differentially expressed genes. Among GO analysis, 24 Biological processes (BPs), 14 Cellular Components (CCs) and 8 Molecular Functions (MFs) were identified. Figure 2B shows the GO analysis for both up- and downregulated DEGs. Figure 3A shows the top 30 GO in a dot graph.

3.1.4. KEGG Pathway Analysis

We analyzed the biological pathways using Kyoto Encyclopedia of Genes and Genomes (KEGG-v5). KEGG is a database resource for understanding high-level functions and utilities of the biological system, such as the cell, the organism and the ecosystem, from molecular-level information, especially large-scale molecular datasets generated by genome sequencing and other high-throughput experimental technologies (http://www.genome.jp/kegg/). Further, 1101 out of 2190 downregulated DEGs were found in KEGG, where 149 pathways were involved in at least one DEG. There was significant enrichment for pathways in KEGG pathways, including metabolite, DNA repair and development. Also, 1470 genes out of 2133 upregulated DEGs were found in KEGG, where 140 pathways were involved in at least one DEG. The significant KEGG enrichment has calcium, MAPK, Wnt, Notch, cardiac muscle, and cardiac smooth muscle signaling pathways. These pathways are highly consistent with the development of Zebrafish embryos, especially during heart development. Figure 3B shows the top 20 enriched pathways in up- and downregulated DEGs. Figure 4 shows the calcium signaling pathway, indicating a total of 20 up- and downregulated genes are involved in this pathway. Figure 5 shows the MAPK pathway, with altered genes in color. Figure 6 shows cardiac muscular and vascular smooth muscle signaling pathways, with identified altered genes.

4. Discussion

Zebrafish embryos offer a valuable model for studying the developmental effects of high-salt exposure. A key advantage is that zebrafish embryos can be treated directly with salt solutions, bypassing the placental–fetal barrier that complicates similar studies in mammalian models. This direct exposure enables precise control of the extracellular environment and allows for real-time observation of developmental processes, making zebrafish particularly suited for investigating environmental stressors such as excess sodium on early organogenesis. Given the high degree of conservation in cardiovascular and signaling pathways, findings from salt-exposed zebrafish embryos may provide meaningful insight into how maternal high salt intake impacts fetal development in humans [32,33].
Globally, high salt consumption is on the rise, largely driven by increased intake of processed and fast foods. Excessive dietary salt intake is strongly associated with elevated risk for hypertension and cardiovascular diseases, including during pregnancy. Maternal high salt intake has been implicated in adverse outcomes for both mothers and offspring, contributing to long-term health risks such as gestational hypertension and the development of preeclampsia [8,10,24,34,35].
Emerging evidence suggests that high-salt exposure during pregnancy can lead to increased circulating inflammatory mediators within the placenta, disrupting normal fetal development. These placental changes may contribute to the intrauterine programming of cardiovascular dysfunction, thereby increasing susceptibility to chronic disease later in life [7,22].
In this study, we investigated the developmental impact of high-salt exposure using a 2% NaCl concentration in zebrafish embryos. Following salt treatment, we observed significant alterations in heart rate, hatching success, and cardiac morphology. Transcriptomic analysis via RNA sequencing further revealed salt-induced differential gene expression, highlighting potential molecular pathways, including calcium signaling and MAPK cascades, that may mediate these phenotypic effects.
While early cardiac development is a critical window of sensitivity to environmental stressors, our study focused specifically on transcriptomic changes at a later stage (120 hpf), when embryos had completed major organogenesis and exhibited measurable phenotypic effects. We acknowledge that evaluating earlier time points or incorporating methods such as in situ hybridization would provide additional insight into the temporal dynamics of salt-induced cardiac alterations. However, our primary aim was to characterize pathway-level changes at a stage when salt-induced developmental phenotypes, including cardiac morphology and function, were clearly distinguishable. This approach enabled us to identify candidate molecular mechanisms potentially driving the observed defects.
The observed delays in hatch rate and alterations in morphological features, particularly in the heart, suggest that salt exposure exerts a disruptive effect on zebrafish embryonic development. These findings align with previous studies, indicating the susceptibility of developing embryos to cardiovascular disturbances [18,19,36,37,38,39].
The reduced heart rate observed in the salt-exposed group further underscores the impact of salt on cardiovascular function during early development [19,26].
The molecular mechanisms underlying salt-induced developmental abnormalities require further investigation. It is plausible that salt-mediated disruptions in metabolic processes and signaling cascades contribute to the phenotypic changes observed in zebrafish embryos. In our study, pathway enrichment analysis using RNA-Seq data revealed significant alterations in several pathways known to regulate embryonic development, particularly those related to cardiac formation and function.
Notably, multiple general developmental pathways were dysregulated in salt-treated embryos, including metabolism, Wnt, Notch, cell cycle regulation, apoptosis, and DNA repair. These findings suggest that high-salt exposure broadly influences essential cellular processes critical for normal development.
Among the 149 differentially regulated KEGG pathways identified in the high-salt treatment group, 9 of the top 20 were metabolism-related. These included biosynthesis of amino acids, fatty acids, and cytochrome P450-mediated metabolism. Additionally, several signaling pathways involved in developmental regulation were among the most significantly affected, including the apelin, adipocytokine, GnRH, and FoxO signaling pathways. Pathways involved in cell fate determination and communication, such as apoptosis, peroxisome function, and SNARE interactions in vesicular transport, were also impacted. Two biosynthesis-related pathways were prominently enriched in the top 20 results (Figure 3). These findings underscore the broad systemic effects of high-salt exposure on embryonic development and cellular homeostasis.
Together, these pathway alterations highlight the complex and multifaceted nature of salt-induced developmental disruption. They also provide a broader context for our focused investigation into specific signaling cascades. Among these, the calcium signaling and mitogen-activated protein kinase (MAPK) pathway (Figure 4 and Figure 5) adrenergic signaling in cardiomyocytes and regulation of actin cytoskeleton pathways play important roles for normal heart functions. Therefore, we next examined how high-salt exposure modulated gene expression within these pathways.
The MAPK signaling pathway is a highly conserved cascade that plays a pivotal role in regulating numerous cellular processes, including proliferation, differentiation, apoptosis, and stress responses. In the context of cardiac development, MAPK signaling is essential for the proper formation and maturation of the heart. During embryogenesis, it contributes to the specification of cardiac progenitor cells, the proliferation of cardiomyocytes, and the morphogenesis of cardiac chambers and valves. MAPK signaling also facilitates communication between myocardial and endocardial layers, ensuring coordinated development of heart structure and function. In postnatal and adult hearts, the MAPK pathway remains active and is involved in mediating the cardiac response to physiological and pathological stress, such as hypertrophy, ischemia, and pressure overload. Dysregulation of this pathway has been implicated in a variety of cardiovascular diseases, including congenital heart defects, dilated cardiomyopathy, and heart failure [25,26,28,40,41,42,43,44].
In zebrafish, components of the MAPK pathway are expressed early in development, and previous studies have demonstrated that perturbations in MAPK activity can lead to abnormal cardiac morphology and function. Thus, alterations in MAPK signaling in response to environmental stressors such as high-salt exposure may underlie some of the developmental cardiac abnormalities observed in this study [27,44,45,46,47].
Our data also point to significant involvement of the calcium signaling pathway, which is central to cardio genesis, excitation–contraction coupling, and intracellular signaling in developing embryos. Perturbations in calcium homeostasis—likely triggered by ionic imbalance due to excess sodium—may account for the observed cardiac abnormalities. Our data indicate that the upregulation of genes such as GPCR and IP3R in calcium signaling pathway supports the hypothesis that salt stress disrupts normal calcium dynamics, potentially altering cardiac muscle development and function. The cardiac muscle contraction and vascular smooth muscle contraction signaling pathways were significantly altered in the salt-treated group, supporting the conclusion that high-salt exposure affects key signaling mechanisms involved in heart development. These changes, along with disruptions observed in the MAPK signaling pathway, suggest a molecular basis for the impaired cardiac development seen in zebrafish embryos under salt stress [27,48,49,50,51,52]. While our transcriptomic analysis highlights MAPK signaling as a significantly enriched pathway following NaCl exposure, we acknowledge the limitation that we did not perform functional experiments (e.g., genetic manipulation or pathway inhibition) to directly test its role in cardiac development. Due to resource and time constraints, further mechanistic validation is beyond the scope of the current study. However, the identification of MAPK signaling, supported by prior studies linking this pathway to heart development, provides a strong foundation for future investigations.
Many of the genes differentially expressed in response to high-salt exposure are involved in pathways critical to zebrafish heart development, including calcium signaling, MAPK, adrenergic signaling, and regulation of actin cytoskeleton. These pathways govern essential processes, such as cardiac progenitor specification, chamber morphogenesis, myocardial contraction, and stress responses. Among the top 10 significantly downregulated genes identified in our RNA-Seq dataset, alpk2 (Alpha protein kinase 2) stands out due to its known role in epicardium morphogenesis and regulation of Wnt signaling—a pathway central to early heart formation. The downregulation of alpk2 may contribute to the observed defects in cardiac morphology and function in salt-treated embryos, providing a molecular link between environmental salt stress and disrupted cardiovascular development. Given the evolutionary conservation of these pathways, these findings may have implications for understanding how prenatal salt exposure impacts fetal heart development in humans [53,54].
Beyond developmental biology, our findings have broader implications for environmental toxicology and public health. Zebrafish serve as a sensitive and scalable model for assessing the developmental impact of environmental stressors, including dietary components such as salt. The observed disruption of conserved signaling pathways, particularly MAPK and calcium signaling, suggests potential mechanistic links between high-sodium environments and adverse developmental outcomes. These insights may help elucidate how maternal salt consumption during pregnancy could influence fetal organogenesis and predispose offspring to long-term cardiovascular risks. Our model provides a valuable platform for future toxicological screening of other environmental and dietary factors that may affect early developmental programming in humans.
Together, these findings suggest that high-salt exposure affects zebrafish embryonic development through a combination of morphological and molecular mechanisms. The disruption of MAPK and calcium signaling pathways provides a plausible link between environmental salt exposure and developmental programming of cardiovascular outcomes. Given the conservation of these pathways across vertebrates, these results may offer insight into similar mechanisms in mammalian models and underscore the importance of regulating maternal salt intake during pregnancy.
We acknowledge that our externally applied 2% NaCl (≈341 mM Na+) in E3 medium does not precisely match human plasma sodium concentrations (≈140–150 mM). However, several prior zebrafish studies have shown that 1–3% NaCl treatments effectively induce osmoregulatory and cardiovascular stress responses relevant to human high-salt exposure and hypertension [55,56,57]. In the absence of direct sodium ion measurements in our embryos, we rely on these established protocols to model a high-salt challenge. Future work should include quantitative assays (e.g., ion-selective electrodes or ICP-MS) to measure internal sodium concentrations and strengthen the translational relevance.

5. Conclusions

Taken together, our findings highlight the vulnerability of early vertebrate development to high-salt exposure and suggest that perturbations in calcium and MAPK signaling pathways may underlie long-term cardiovascular outcomes associated with maternal salt intake.

Author Contributions

Conceptualization, R.S.T.; methodology, R.S.T.; investigation, R.S.T.; resources, R.S.T.; data curation, E.T. and J.H.; writing—original draft preparation, R.S.T.; writing—review and editing, R.S.T., E.T. and J.H.; visualization, J.H.; supervision, R.S.T.; project administration, R.S.T.; funding acquisition, R.S.T. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

The study was conducted according to the guidelines of the Organization for Research Review Board (RRB) No. 21 according to the regulations of Public Health Service (PHS), the Animal Welfare Act (AWA) and IACUC.

Informed Consent Statement

Not applicable.

Data Availability Statement

We would like to share the data.

Acknowledgments

We would like to thank Novogene company for conducting the RNA seq and seq data analysis.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
MAPKmitogen-activated protein kinase
hpfhours post-fertilization
RNAribonucleic acid
DEGsdifferentially expressed genes

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Figure 1. Effects of high-salt exposure on zebrafish embryonic development. (A) Hatch rate comparison between control and 2% NaCl-treated embryos at 72 and 96 h post-fertilization (hpf). (B) Heart rate (beats per minute) measured in 5 individual embryos per group at 120 hpf; values are presented as mean ± standard deviation (SD). (C) Cardiac morphology of representative embryos at 96 hpf in control and salt-treated groups, imaged using an inverted microscope at 4×. Arrows indicate visible abnormalities in heart structure in the salt-exposed embryos, including pericardial edema and altered heart shape. (D) Cardiac morphology at 10× in control and salt-treated groups at 120 hpf. Statistical significance was assessed using an unpaired t-test; asterisks denote significance levels (p < 0.05, p < 0.01). Sample size: n = 5 per group.
Figure 1. Effects of high-salt exposure on zebrafish embryonic development. (A) Hatch rate comparison between control and 2% NaCl-treated embryos at 72 and 96 h post-fertilization (hpf). (B) Heart rate (beats per minute) measured in 5 individual embryos per group at 120 hpf; values are presented as mean ± standard deviation (SD). (C) Cardiac morphology of representative embryos at 96 hpf in control and salt-treated groups, imaged using an inverted microscope at 4×. Arrows indicate visible abnormalities in heart structure in the salt-exposed embryos, including pericardial edema and altered heart shape. (D) Cardiac morphology at 10× in control and salt-treated groups at 120 hpf. Statistical significance was assessed using an unpaired t-test; asterisks denote significance levels (p < 0.05, p < 0.01). Sample size: n = 5 per group.
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Figure 2. Transcriptomic analysis of control and high-salt-treated zebrafish embryos using RNA-Seq. (A) Venn diagram showing the number of shared and unique genes expressed in control and 2% NaCl-treated embryos. (B) Gene Ontology (GO) functional classification analysis categorizing differentially expressed genes (DEGs) into biological processes, molecular functions, and cellular components. (C) Volcano plot depicting differential gene expression between salt-treated and control embryos. Significantly upregulated genes are shown in red, and significantly downregulated genes are shown in green (cutoff: |log2 fold change| > 1, adjusted p < 0.05). (D) Bar graph summarizing the total number of upregulated and downregulated genes in the salt-treated group compared to controls. (E) Heatmap of DEGs showing hierarchical clustering of gene expression profiles. Red indicates higher expression and green indicates lower expression levels across replicates. Statistical thresholds for DEGs were set at a false discovery rate (FDR) < 0.05. Each group included biological replicates.
Figure 2. Transcriptomic analysis of control and high-salt-treated zebrafish embryos using RNA-Seq. (A) Venn diagram showing the number of shared and unique genes expressed in control and 2% NaCl-treated embryos. (B) Gene Ontology (GO) functional classification analysis categorizing differentially expressed genes (DEGs) into biological processes, molecular functions, and cellular components. (C) Volcano plot depicting differential gene expression between salt-treated and control embryos. Significantly upregulated genes are shown in red, and significantly downregulated genes are shown in green (cutoff: |log2 fold change| > 1, adjusted p < 0.05). (D) Bar graph summarizing the total number of upregulated and downregulated genes in the salt-treated group compared to controls. (E) Heatmap of DEGs showing hierarchical clustering of gene expression profiles. Red indicates higher expression and green indicates lower expression levels across replicates. Statistical thresholds for DEGs were set at a false discovery rate (FDR) < 0.05. Each group included biological replicates.
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Figure 3. GO terms for significantly enriched DEGs based on molecular functions. (A) Scatter plot showing top 30 enriched GO; (B) 20 enriched KEGG pathways in control and salt groups.
Figure 3. GO terms for significantly enriched DEGs based on molecular functions. (A) Scatter plot showing top 30 enriched GO; (B) 20 enriched KEGG pathways in control and salt groups.
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Figure 4. Calcium signaling pathway analysis based on RNA-Seq data from control and high-salt-treated zebrafish embryos. Differentially expressed genes (DEGs) involved in the calcium signaling pathway were mapped using KEGG pathway analysis. Red bars indicate significantly upregulated genes, while green bars represent significantly downregulated genes in salt-treated embryos compared to controls. The color of each bar reflects the magnitude of the log2 fold change.
Figure 4. Calcium signaling pathway analysis based on RNA-Seq data from control and high-salt-treated zebrafish embryos. Differentially expressed genes (DEGs) involved in the calcium signaling pathway were mapped using KEGG pathway analysis. Red bars indicate significantly upregulated genes, while green bars represent significantly downregulated genes in salt-treated embryos compared to controls. The color of each bar reflects the magnitude of the log2 fold change.
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Figure 5. Genes involved in the mitogen-activated protein kinase (MAPK) signaling pathway were analyzed for differential expression using RNA-Seq data. Red bars indicate significantly upregulated genes, and green bars indicate significantly downregulated genes in the salt-treated group compared to controls. The color of each bar reflects the log2 fold change in gene expression.
Figure 5. Genes involved in the mitogen-activated protein kinase (MAPK) signaling pathway were analyzed for differential expression using RNA-Seq data. Red bars indicate significantly upregulated genes, and green bars indicate significantly downregulated genes in the salt-treated group compared to controls. The color of each bar reflects the log2 fold change in gene expression.
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Figure 6. Cardiac and vascular muscle contraction signaling pathways altered by high-salt exposure in zebrafish embryos. Cardiac muscle contraction signaling pathway and vascular smooth muscle contraction signaling pathway were analyzed based on differentially expressed genes identified by RNA-Seq in salt-treated vs. control embryos. Genes significantly upregulated in the salt group are shown in red, and downregulated genes are shown in green. The bar color represents the log2 fold change in gene expression.
Figure 6. Cardiac and vascular muscle contraction signaling pathways altered by high-salt exposure in zebrafish embryos. Cardiac muscle contraction signaling pathway and vascular smooth muscle contraction signaling pathway were analyzed based on differentially expressed genes identified by RNA-Seq in salt-treated vs. control embryos. Genes significantly upregulated in the salt group are shown in red, and downregulated genes are shown in green. The bar color represents the log2 fold change in gene expression.
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Table 1. Top 10 upregulated genes in salt-treated zebrafish embryos identified by RNA-Seq. This table presents the ten most significantly upregulated genes in zebrafish embryos exposed to 2% NaCl compared to controls. Genes are listed alongside their associated biological processes and molecular functions, based on Gene Ontology (GO) annotations.
Table 1. Top 10 upregulated genes in salt-treated zebrafish embryos identified by RNA-Seq. This table presents the ten most significantly upregulated genes in zebrafish embryos exposed to 2% NaCl compared to controls. Genes are listed alongside their associated biological processes and molecular functions, based on Gene Ontology (GO) annotations.
GeneBiological ProcessMolecular Function
mgea5Glycoprotein metabolic processEnables beta-N-acetylglucosaminidase activity
arrdc3bProtein transportArrestin domain involved in protein transport
ppdpfbCell differentiation in nervous system, pancreatic system, and pronephric ductPancreatic progenitor cell differentiation and proliferation factor b
slc16a9bSolute carrier, integral component of plasma membraneMonocarboxylic acid transmembrane transport
slc16a6bSolute carrier, integral component of plasma membraneMonocarboxylic acid transmembrane transport
h1f0Negative regulation of DNA recombination and nucleosome positioning, chromosome condensationEnable double stranded DNA binding activity and nucleosomal DNA binding activity
h1fxChromosome condensation, negative regulation of DNA recombination and nucleosome positioningEnable double stranded DNA binding activity and nucleosomal DNA binding activity
histh1lChromosome condensation, negative regulation of DNA recombination and nucleosome positioningEnable double stranded DNA binding activity and nucleosomal DNA binding activity
ino80eBrain developmentIno80 complex component located in the nucleus
gpcpd1Glycerophospholipid catabolic process, lipid metabolismGlyceophosphocholine and phosphodiesterase activity
Table 2. Top 10 downregulated genes in salt-treated zebrafish embryos identified by RNA-Seq. This table lists the ten most significantly downregulated genes in zebrafish embryos following exposure to 2% NaCl, compared to untreated controls. Each gene is annotated with its known biological processes and molecular functions, based on Gene Ontology (GO) classifications.
Table 2. Top 10 downregulated genes in salt-treated zebrafish embryos identified by RNA-Seq. This table lists the ten most significantly downregulated genes in zebrafish embryos following exposure to 2% NaCl, compared to untreated controls. Each gene is annotated with its known biological processes and molecular functions, based on Gene Ontology (GO) classifications.
GeneBiological ProcessMolecular Function
si:ch211-153b23.3Regulation of translational fidelityNucleotidyltransferase activity and tRNA binding activity
irg1lImmunoresponse; inflammatory response to wounding and bacterium expressed in cloaca, liver, oral region, and pharynxAconitate decarboxylase activity in mitochondria
SLC6A6 (2
of 2)		Taurine transport component in plasma membraneTaurine/sodium symporter activity
slc38a2Amino acid transmembrane transport and sodium ion transportL-glutamine transmembrane transporter activity
si:dkey-162h11.3Protein coding in nucleoplasmInvolved in protein coding
slc6a6aTaurine transport component in plasma membraneTaurine/sodium symporter activity
si:ch211-153b23.4Apoptotic processIntracellular signal transduction
slc22a16Organic cation transport, transmembrane transportOrganic cation transmembrane transporter activity
plekhs1Protein coding, mild elevation of blood glucose levels and insulin resistanceProtein coding activity
alpk2Epicardium morphogenesis, heart development, negative regulation of Wnt signaling pathwayATP binding activity, protein serine kinase activity, protein serine/threonine kinase activity
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Thompson, E.; Hensley, J.; Taylor, R.S. High-Salt Exposure Disrupts Cardiovascular Development in Zebrafish Embryos, Brachyodanio rerio, via Calcium and MAPK Signaling Pathways. J 2025, 8, 26. https://doi.org/10.3390/j8030026

AMA Style

Thompson E, Hensley J, Taylor RS. High-Salt Exposure Disrupts Cardiovascular Development in Zebrafish Embryos, Brachyodanio rerio, via Calcium and MAPK Signaling Pathways. J. 2025; 8(3):26. https://doi.org/10.3390/j8030026

Chicago/Turabian Style

Thompson, Ebony, Justin Hensley, and Renfang Song Taylor. 2025. "High-Salt Exposure Disrupts Cardiovascular Development in Zebrafish Embryos, Brachyodanio rerio, via Calcium and MAPK Signaling Pathways" J 8, no. 3: 26. https://doi.org/10.3390/j8030026

APA Style

Thompson, E., Hensley, J., & Taylor, R. S. (2025). High-Salt Exposure Disrupts Cardiovascular Development in Zebrafish Embryos, Brachyodanio rerio, via Calcium and MAPK Signaling Pathways. J, 8(3), 26. https://doi.org/10.3390/j8030026

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