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Abstract Perfluorinated compounds (PFAS) are well recognized toxic pollutants for humans, but if their effect is equally harmful for healthy and fragile people is unknown. Addressing this question represents a need for ensuring global health and wellbeing to all individuals in a world facing the progressive increase of aging and aging related diseases. This study aimed to evaluate the impact of perfluorooctane sulfonate (PFOS), perfluorooctanoic acid (PFOA) and perfluorohexanoic acid (PFHxA) exposure on development and skeletal phenotype using the osteogenesis imperfecta (OI) zebrafish model Chihuahua (Chi/+), carrying a dominant glycine substitution in the α1 chain of collagen I and their wild-type (WT) littermates. To this purpose Chi/+ and WT zebrafish expressing the green fluorescent protein under the early osteoblast marker osterix were exposed from 1 to 6 days post fertilization to 0.36, 1.5 and 3.0 mg/L PFOS, 0.005 and 0.5 mg/L PFOA and 0.01, 0.48 and 16.0 mg/L PFHxA, and their development and skeletal phenotype investigated. Morphometric measurements, confocal microscopy evaluation of operculum area delimited by the fluorescent preosteoblasts and mineral deposition analysis following alizarin red staining were employed. PFOS and the highest concentration of PFHxA significantly impaired standard length in both genotypes. Osteoblast differentiation was significantly compromised by PFOS and by PFOA only in Chi/+. Limited to WT exposed to PFOA a reduced mineralization was also observed. No effect was detected after PFHxA exposure. Apoptosis was only activated by PFOA, specifically in Chi/+ mutant operculum osteoblasts. Interestingly, an altered lipid distribution in both WT and mutant fish was revealed after exposure to both pollutants. In conclusion, our data demonstrate that PFAS impair operculum development mainly compromising cell differentiation in mutant fish whereas alter lipid hepatic distribution in both genotypes with a more severe effect on Chi/+ preosteoblast survival. These results represent a first warning sign of the negative impact of PFAS exposure in presence of genetically determined skeletal fragility.

Objectives: Guo Min decoction (GMD) is a Chinese traditional medicine that can regulate allergy-related symptoms. Although GMD treatment was reported to treat allergy-associated symptoms by regulating the immune response, the rationale between GMD treatment and angiogenesis has not been reported yet. Our objective is to investigate the angiogenesis-modulating activity of GMD. Materials and Methods: In this study, we used fluorescence recording, alkaline phosphatase (AP) activity staining, and real-time polymerase chain reaction (PCR) experiments to examine the effects of GMD on angiogenesis in a zebrafish model. Results: GMD-treated zebrafish embryos exhibited more intercapillary spaces in the caudal vein plexus (Mock: 11.1 ± 1.8 [n = 20; n: numbers of embryos]; GMD-treated: 16.2 ± 1.9 [n = 20]). AP activity staining showed that treatment with GMD and liquorice (Gan Cao, a component of GMD) induced subintestinal vein outgrowth. However, glycyrrhizin (a component of Gan Cao) had no obvious pro-angiogenic effects on zebrafish. Furthermore, real-time PCR experiments indicated that GMD exposure might be through regulating angiogenesis-related genes (cdh5, nrp1a, and flt1) expressions. Conclusion: Based on these observations, we proposed that GMD had pro-angiogenic activity in a zebrafish model, and it might partially be contributed by one of the components, liquorice.

Abstract Ischemic stroke (IS) is the most recorded case of stroke that is caused by decreased blood flow to the brain. Nowadays, therapeutical agents for IS are limited and they have not shown maximum clinical results. Therefore, the exploration of new candidates for IS treatment continues to be done. Zebrafish as one of the animal models has its advantages and currently is being developed to be incorporated into the drug discovery pipeline of IS. This review explores the latest applications of the zebrafish model in screening potential therapeutic agents for IS. Key factors related to the experimental design such as developmental stage and strain, routes of drug administration, induction methods, and experimental parameters are also elaborated. Finally, this review offers future recommendations for the use of zebrafish in the pre-clinical study of IS. This review is beneficial as a reference for establishing drug screening protocols using the zebrafish IS model.

Abstract In vertebrates, skeletal muscle comprises fast and slow fibers. Slow and fast muscle cells in fish are spatially segregated; slow muscle cells are located only in a superficial region, and comprise a small fraction of the total muscle cell mass. Slow muscles support low-speed, low-force movements, while fast muscles are responsible for high-speed, high-force movements. However, speed and strength of movement are not binary states, but rather fall on a continuum. This raises the question of whether any recruitment patterns exist within fast muscles, which constitute the majority of muscle cell mass. In the present study, we investigated activation patterns of trunk fast muscles during movements of varying speeds and strengths using larval zebrafish. We employed two complementary methods: calcium imaging and electrophysiology. The results obtained from both methods supported the conclusion that there are spatially-ordered recruitment patterns in fast muscle cells. During weaker/slower movements, only the lateral portion of fast muscle cells is recruited. As the speed or strength of the movements increases, more fast muscle cells are recruited in a spatially-ordered manner, progressively from lateral to medial. We also conducted anatomical studies to examine muscle fiber size. The results of those experiments indicated that muscle fiber size increases systematically from lateral to medial. Therefore, the spatially ordered recruitment of fast muscle fibers, progressing from lateral to medial, correlates with an increase in fiber size. These findings provide significant insights into the organization and function of fast muscles in larval zebrafish, illustrating how spatial recruitment and fiber size interact to optimize movement performance.

Abstract Flavors are widely utilized in the food and oral pharmaceutical industry, particularly in products for children, to enhance palatability and promote ingestion willingness. The complex compositions of flavors potentially induce severer toxicity especially in children. In this study, zebrafish embryos are applied for toxicity screening of flavor and its compounds by immersing in flavor solutions followed by the assessment of morphology of zebrafish larvae. Geraniol is identified as the prominent toxic compound and is considered highly toxic to zebrafish embryo. In further toxicology study, geraniol demonstrates the concentration‐dependent developmental toxicity as the obvious reduction of body and eye lengths, as well as the increased prevalence of tail deformities, pericardial edema, and spine deformation. Zebrafish larvae treated with geraniol exhibit reduction in liver area and exocrine pancreas length, increase in yolk sac area, as well as elevation of triglycerides and total cholesterol, which indicate the inhibited nutrient utilization. Transcriptome analysis reveals that under geraniol treatment, 248 differentially expressed genes (DEGs) are downregulated, whereas 23 DEGs are upregulated, and 110 DEGs are related to metabolic process. Biological processes of lipid metabolism, carbohydrate metabolism, protein hydrolysis, and transmembrane transport, including their involved functional genes, are all downregulated. These findings reveal the developmental toxicity of geraniol by affecting the nutrient utilization‐related organs development and biological processes. This study establishes an efficient screening model for identifying toxic flavor compounds during developing stages, thereby elucidating the potential safety risks of geraniol exposure in zebrafish and providing a comprehensive understanding of its potential toxicity mechanism.

Carbendazim is an agricultural fungicide and a widely present ecotoxic pollutant, but its damaging effects on the nervous system are not fully understood. Here, we assessed effects of short-term exposure to carbendazim on zebrafish brains. It has been revealed firstly that carbendazim can accumulate in the brain after a 7-day exposure, with the maximum concentration of 68.22 ± 9.84 μg/kg, which caused tissue vacuolization and neuronal damage, then led to a decline in motor behavioral abilities, especially a significant reduction in the distance moved. These changes may be attributed to metabolism abnormalities. Based on metabolomics and lipidomics, substantial alterations were observed in 264 lipids and 135 metabolites, with notable enrichment in 5 metabolic pathways, including glycerophospholipid metabolism, glycerolipid metabolism, and sphingolipid metabolism. To investigate system-level variations, weighted correlation network analysis was utilized to screen for 4 biomarkers strongly associated with carbendazim exposure: citric acid, guanosine-5′-monophosphate, sphingosine, and monoacylglycerol. The alterations of these markers confirmed damages of carbendazim to zebrafish nervous system, further elucidating that metabolic disorders caused by carbendazim in brains led to tissue damage and subsequent changes in motor behavior. This study provides scientific evidence for neurotoxicities of carbendazim and offers new insights into ecological risks.

Objective: In our wireless-centric world, evaluating the health effects of radio frequency electromagnetic radiation (RF-EMR) is crucial. An existing research gap pertains to the replication of real-world specific absorption rates (SAR) for RF-EMR, especially within aquatic environments. We aimed to bridge this gap using an innovative TEM cell platform to replicate realistic SAR conditions in water and assess RF-EMR's impact on neuroanatomical and behavioral changes. Results: We examined RF-EMR effects on zebrafish embryos exposed to RF-EMR during the 4-58 hours post-fertilization phase. Temporary neuroanatomical enlargements and minor behavioral shifts were observed, diminishing by day 8 post-fertilization. Conclusion: Short-term RF-EMR exposure at tested levels did not yield significant long-term effects. Nevertheless, investigating prolonged exposure remains imperative. Our study serves as a pioneering model for future investigations into the biological consequences of RF-EMR exposure, highlighting the importance of assessing its health implications in our wireless-centric world

Objective: The study aimed to evaluate the effect of Lawsonia inermis flower extract in an Alzheimer’s disease (AD)-induced zebrafish model. Materials and Methods: A total of 60 adult fishes were segregated into five groups (n = 12) and treated with various dilutions of L. inermis extract, respectively. The study was conducted in two phases. The effective dilution range was identified in Phase I (7 days), and the respective dilution was used in Phase II (14 days) for sodium valproate-induced AD. The treatment groups were screened for anxiety, motor coordination, and memory using a novel tank diving test, swim velocity, and maze test, respectively. Treated fishes from each group were investigated for histopathology examination. The results were analyzed with a one-way analysis of variance followed by a post hoc Tukey multiple comparison test using GraphPad Prism. Results: In Phase I, a dose-dependent reduction of degeneration was observed in the pathology of the brain with no significant changes in cognition. In Phase II, the effective dose (8 μg/day/0.5 mg fish) of L. inermis flower extract has shown a significant reduction in anxiety, degeneration, and motor dysfunction with a significant increase in memory and learning. Conclusions: The current investigation has shown that the L. inermis flower extract has a neuroprotective impact on AD zebrafish.

Abstract Background Polycomb group proteins, known for their role in epigenetic gene silencing, are critical regulators of cell fate and tissue development across species. These highly conserved proteins are assembled into two multi-protein complexes: Polycomb Repressive Complex 1 and Polycomb Repressive Complex 2. Main body In zebrafish, a prominent vertebrate model, the roles of PRC1 and PRC2 have been extensively studied, particularly in the context of organogenesis. This review focuses on the emerging functions of PcG proteins in zebrafish development, with an emphasis on the involvement of PRC1 and PRC2 in the formation and differentiation of the heart and pectoral fins. By integrating findings from recent studies, we aim to provide a comprehensive overview of how PcG function contributes to the intricate processes underlying zebrafish heart and pectoral fin development. Conclusion This review highlights the zebrafish model as a powerful system for unravelling the complex roles of PcG proteins in vertebrate development.

Juntao Chen,1,2,* Chunjiao Lu,2,* Wenjie Xie,1,2 Xiaoqian Cao,1 Jiannan Zhang,1 Juanjuan Luo,2 Juan Li1 1Key Laboratory of Bioresources and Eco-Environment of Ministry of Education, College of Life Science, Sichuan University, Chengdu, People’s Republic of China; 2Engineering Research Center of Key Technique for Biotherapy of Guangdong Province, Shantou University Medical College, Shantou, People’s Republic of China*These authors contributed equally to this workCorrespondence: Juan Li, Key Laboratory of Bioresources and Eco-environment of Ministry of Education, College of Life Science, Sichuan University, No. 24 South Section 1, Yihuan Road, Chengdu, 610065, People’s Republic of China, Email lijuanscuhk@163.com Juanjuan Luo, Engineering Research Center of Key Technique for Biotherapy of Guangdong Province, Shantou University Medical College, 22 Xinling Road, Shantou, 515041, People’s Republic of China, Email 15jjluo1@stu.edu.cnIntroduction: The proliferation of nanoplastics (NPs) has emerged as a significant environmental concern due to their extensive use, raising concerns about potential adverse effects on human health. However, the exact impacts of NPs on the early development of hematopoietic organs remain poorly understood.Methods: This investigation utilized fluorescence microscopy to observe the effects of various NP concentrations on the caudal vein plexus (CVP) development in zebrafish embryos. Subsequent RNA sequencing (RNA-seq) identified genes related to CVP deformities and hematopoietic stem/progenitor cells (HSPCs) in zebrafish embryos exposed to NPs. Additionally, single cell RNA sequencing (scRNA-seq) analysis identified genes associated with the development of CVP and HSPCs. RT-qPCR assessed changes in expression of these genes in zebrafish embryos exposed to different NP concentrations.Results: The impact of NPs on zebrafish embryos was investigated, revealing significant reductions in survival and hatching rates and decreases in body length alongside increased heart rates. Exposure to NPs at 8 mg/L severely impaired zebrafish CVP development. RNA-seq revealed that NPs exposure altered the activity of oxidative enzymes, hydrolases, and the extracellular matrix in zebrafish embryos. Treatment with 10 μM NAC effectively rescued the CVP defects induced by NPs. Additionally, scRNA-seq identified genes associated with EC and HSPC development, and subsequent RT-qPCR validation confirmed significant expression changes in these genes.Conclusion: The results of this study suggest that NPs induce oxidative stress in vascular ECs and HSPCs, which mediates CVP damage and impairs hematopoiesis in zebrafish embryos.Keywords: nanoplastics, caudal vein plexus, oxidative stress, endothelial cells, hematopoietic stem/progenitor cells, zebrafish, single cell RNA sequencing

Exposure to valproic acid (VPA) during embryogenesis has become a valuable tool for modeling neurodevelopmental disorders in animal models such as zebrafish (Danio rerio). This article examines the effects of embryonic exposure to VPA in zebrafish on the basis of 39 articles sourced from PubMed and Google Scholar. We conducted a systematic review and meta-analysis to elucidate the common impacts of VPA exposure and reported that VPA significantly altered development at various levels. Behaviorally, zebrafish exposed to VPA exhibit notable changes in their social interaction patterns. Physiologically, VPA exposure leads to significant alterations, including decreased heart rates, increased mortality rates, and pronounced morphological abnormalities. Pharmacological exposure has been linked to neuroanatomical and neurochemical changes. At the genetic level, VPA exposure is associated with the differential expression of genes involved in neurodevelopment and neuronal function. The synthesized data from these studies underscore the utility of zebrafish as a model organism for investigating the effects of teratogen exposure on neurodevelopment.

Abstract Background Non‐alcoholic fatty liver disease (NAFLD) has become the most common chronic liver disease in recent years, but the pathogenesis is not fully understood. Therefore, it is important to establish an effective animal model for studying NAFLD. Methods Adult zebrafish were fed a normal diet or a high‐fat diet combined with egg yolk powder for 30 days. Body mass index (BMI) was measured to determine overall obesity. Serum lipids were measured using triglyceride (TG) and total cholesterol (TC) kits. Liver lipid deposition was detected by Oil Red O staining. Liver injury was assessed by measuring glutathione aminotransferase (AST) and glutamic acid aminotransferase (ALT) levels. Reactive oxygen species (ROS) and malondialdehyde (MDA) were used to evaluate oxidative damage. The level of inflammation was assessed by qRT‐PCR for pro‐inflammatory factors. H&E staining was used for pathological histology. Caspase‐3 immunofluorescence measured apoptosis. Physiological disruption was assessed via RNA‐seq analysis of genes at the transcriptional level and validated by qRT‐PCR. Results The high‐fat diet led to significant obesity in zebrafish, with elevated BMI, hepatic TC, and TG. Severe lipid deposition in the liver was observed by ORO and H&E staining, accompanied by massive steatosis and ballooning. Serum AST and ALT levels were elevated, and significant liver damage was observed. The antioxidant system in the body was severely imbalanced. Hepatocytes showed massive apoptosis. RNA‐seq results indicated that several physiological processes, including endoplasmic reticulum stress, and glucolipid metabolism, were disrupted. Conclusion Additional feeding of egg yolk powder to adult zebrafish for 30 consecutive days can mimic the pathology of human nonalcoholic fatty liver disease.

Abstract Diquat (DQ) is a non-selective, fast-acting herbicide that is extensively used in aquatic systems. DQ has been registered as the substitute for paraquat due to its lower toxicity. However, the widespread presence of DQ in aquatic systems can pose an ecological burden on aquatic organisms. In addition, DQ can degrade into its metabolites, diquat-monopyridone (DQ-M) and diquat-dipyridone (DQ-D) in the environment, while the ecological risks of the metabolites remain uncertain. Herein, the aquatic ecological risks of DQ and its metabolites were compared using zebrafish as model non-target organisms. Results indicated that DQ and its metabolites did not induce significant acute toxicity to zebrafish embryos at environmentally relevant levels. However, exposure to DQ and DQ-D resulted in oxidative stress in zebrafish larvae. DQ treatment led to increased levels of reactive oxygen species (ROS), malondialdehyde (MDA), and glutathione (GSH) in the larvae, while DQ-D increased internal MDA and GSH levels. Moreover, the activities of the antioxidative enzymes, superoxide dismutase (SOD) and catalase (CAT) were significantly suppressed by DQ and DQ-D. Besides, the expression levels of oxidative stress-related genes (Mn-SOD, CAT, and GPX) were disturbed accordingly after DQ and DQ-D treatments. These findings highlighted the importance of a more comprehensive understanding of the ecological risks of agrochemical substitutions as well as agrochemical metabolites. Such knowledge is crucial for significant improvements in agrochemical regulation and policy-making in the future.

Abstract One important functional food ingredient today, valued for its health properties and ability to prevent disease, is bee pollen, which comprises a combination of nectar, pollen from plants, and the secretions of bees. In this research, the tyrosinase (TYR) inhibiting abilities of the peptides derived from bee pollen protein hydrolysates are investigated. Various proteases were utilized to generate these peptides, followed by testing at different concentrations. Tyrosinase inhibition activity was detected in all cases, while the hydrolysate drawn from 5.0% w/v neutrase exhibited the best IC50 value and was thus investigated further via ultrafiltration to separate the active fractions. The highest potential for tyrosinase inhibition was recorded for the fractions below 0.65 kDa. Subsequent purification steps via SEC and RP-HPLC led to the identification of the VDGYPAAGY (named VY-9) peptide via LC-Q-TOF-MS/MS in fraction F1–2, known for its non-toxic and hydrophobic characteristics albeit poor water solubility. The synthesized VY-9 peptide demonstrated competitive inhibition, with IC50 values of 0.55 ± 0.03 µM for mono-phenolase and 2.54 ± 0.06 µM for di-phenolase activities, as confirmed by molecular docking analysis revealing dominant hydrogen bond interactions with TYR. Effective concentrations of 0.2–1.6 µM of VY-9 showed negligible cytotoxicity in B16F10 cells. Melanin synthesis suppression was examined via qRT-PCR, and western blot in MITF, TYR, TRP-1, and TRP-2. Cell death in zebrafish embryos was evaluated in vivo using a toxicity assay which revealed no significant influence from VY-9, while anti-melanogenic effects were observed when the concentration was 4 µM, suggesting bee pollen-derived peptides’ potential in cosmetic and pharmaceutical depigmentation applications.

The phenolic endocrine disruptor molecule 4-nonylphenol (4-NP) is prevalent in several industrial goods and the environment, adversely affecting the health of people, animals, and aquatic organisms. It is necessary to assess the harmful impact of this contaminant on the ocular systems of aquatic animals. The investigation was done on adult male zebrafish exposed to 4-nonylphenol for 21 days. At the conclusion of the research, the expression of oxidative tress-related genes (sod, cat, nrf2) was assessed in the zebrafish eye. One hundred twenty mature zebrafish were categorized into four categories. The zebrafish in the first group were maintained in standard reverse osmosis (RO) water, whilst the second group was subjected to ethanol at a concentration of 10 µl/L of water and designated as the vehicle group. Fish from two toxicity groups were subjected to 4-nonylphenol at concentrations of 100 and 200 µg per liter of water. The mRNA expression levels of superoxide dismutase (SOD), catalase (CAT), and nuclear factor erythroid 2 related factor 2 (Nrf2) in the eyes of zebrafish exposed to 200 µg of 4-nonylphenol per liter of water exhibited considerable downregulation.

Abstract Diabetes is a pandemic disease that causes the loss of control of glucose regulation in the organism, in consequence of dysfunction of insulin production or functionality. In this work, the antidiabetic bioactivity of 182 fractions from 19 cyanobacteria strains derived from the LEGE Culture Collection were analysed using the 2-NBDG assay in zebrafish larvae. From this initial screening, two fractions (57 (06104_D) and 107 (03283_B)) were identified as promising insulin mimetics. These were further characterized by measuring glucose levels in whole larvae, the expression of glucose transporters (GLUT 1–3) using western blot, and the mRNA expression levels of the glut2, pepck, and insa genes using real-time qPCR. Both fractions showed a decrease in free glucose levels. Furthermore, exposure to fraction 06104_D decreased GLUT1 and increased insa mRNA levels. The chemical composition of these fractions was determined using LC-HRESIMS/MS and compared to inactive fractions of the same polarity in order to identify the unique bioactive molecules. The molecular networks constructed using the GNPS platform revealed that fraction 06104_D contained mass clusters primarily composed of chlorins, lipids, and terpenoids, while fraction 03283_B contained xanthophylls, peptides, and terpenoids. To correlate the observed activity with the chemical composition of fraction 06104_D, pheophorbide a was chosen as a representative of chlorophyll derivatives. Exposure to zebrafish larvae at 10 and 20 µM confirmed the increased glucose uptake on the 2-NBDG assay. These findings highlight the bioactivity of chlorophyll derivatives as insulin mimetic compounds, as well as cyanobacteria as a source of potential therapeutic diabetes applications.

Abstract Background Lovastatin, the main lipid-lowering component in red yeast rice, is a golden anti-lipid drug, but its long-term application is continuously challenged by potential skeletal muscle atrophy. Daidzein, an isoflavone derived from soybeans and many Chinese medicines, shows therapeutic potential in treating muscle-related diseases and metabolic disorders. However, whether daidzein can improve lovastatin-induced muscle atrophy and the specific mechanism needs to further study. Methods Lovastatin-induced mice and zebrafish muscle atrophy models were used to validate the protective effect of daidzein in vivo. And the lovastatin-induced C2C12 myotube atrophy model was employed to validate the therapeutic efficacy and investigate the specific mechanism of daidzein in vitro. We combined specific siRNA targeting FOXO3a and AMPK-selective inhibitor, agonist to elucidate AMPK/FOXO3a-dependent muscle-protective mechanism of daidzein. The anti-atrophy effects of daidzein through blockage of abnormal activation of AMPK/FOXO3a was presented in Immunofluorescence, H&E staining, Western blot, qRT-PCR. Serum creatine kinase level was detected by ELISA and we used mouse muscle grip instrument to detect the strength of mouse muscles. Results In this study, we demonstrated that daidzein could dose-dependently alleviate lovastatin-induced mice skeletal muscle atrophy, reduce serum creatine kinase, and improve muscle grip strength in mice. Mechanistically, daidzein inhibited lovastatin-induced FOXO3a phosphorylation caused by AMPK activation, thereby inhibiting FOXO3a nuclear translocation to restrain the expression of muscle-related proteins Atrogin-1 and MuRF-1. In C2C12 myotube, administration of AMPK-selective inhibitor Compound C recapitulated the therapeutic effects of daidzein against lovastatin-induced myotubes atrophy, while the anti-atrophy effects of daidzein were lost in the presence of AMPK-selective agonist MK-3903. In lovastatin-induced mice muscle atrophy models, Compound C elicited similar anti-atrophy effects as daidzein, but this effect was not potentiated when it was applied in combination with daidzein, suggesting that daidzein exerted therapeutic efficacy dependent on blockage of AMPK activity. Conclusions Our study identified daidzein as an effective component that ameliorated lovastatin-induced skeletal muscle atrophy through blockage of abnormal activation of AMPK/FOXO3a and transcriptional activation of genes encoding downstream muscle-related proteins. Our results also highlighted the therapeutic potential by regulating the AMPK/FOXO3a axis in management of statin-induced myotoxicity.

Objective·To establish a transgenic zebrafish line Tg(amh:mCherry) for studying gonadal development and related diseases, and to trace and analyze the developmental process of zebrafish gonads.Methods·A recombinant transgenic expression vector pTol2-amh-mCherry was constructed by using the upstream promoter sequence of the zebrafish anti-Müllerian hormone (amh) gene coding region and the gene coding sequence of the red fluorescent protein, and was validated by using Sanger sequencing. The recombinant transgenic expression vector and mRNA of Tol2 transposase were co-microinjected into zebrafish embryos, and fluorescent zebrafish were selected by using fluorescence microscope to screen for a stable inherited transgenic zebrafish line Tg(amh:mCherry). In situ hybridization was used to detect whether the location of the fluorescence signals was consistent with the location of endogenous expression of amh. The red fluorescence of transgenic zebrafish from generation F3 was observed by fluorescence microscopy, and the process of gonadal development was tracked and analyzed.Results·The recombinant transgenic expression vector pTol2-amh-mCherry was successfully constructed, and a stable inherited F3 generation transgenic zebrafish line Tg(amh:mCherry) was successfully established. The results of in situ hybridization showed that the location of the red fluorescence signals in the transgenic zebrafish Tg(amh:mCherry) was consistent with the location of endogenous expression of amh. Through observation and analysis of the fluorescence of transgenic zebrafish from F3 generation, five developmental patterns of zebrafish gonads were found.Conclusion·The successful construction of Tg(amh: mCherry) transgenic zebrafish line facilitates the tracer analysis of zebrafish gonadal development, which provides a better experimental model for the study of gonad-related diseases.

BackgroundTributyl phosphate (TBP) is widely used as an organophosphate flame retardant. However, there are limited studies on the toxicity of TBP to aquatic organisms at low levels of exposure. ObjectiveTo investigate the effects of TBP on early development of zebrafish (Danio rerio). MethodsZebrafish embryos were randomly divided into four groups at 2 h post-fertilisation (2 hpf), namely, the 0.01% dimethyl sulfoxide (DMSO) control group and TBP exposure groups (0.02, 0.2 and 2 μg·L−1). The exposure time was from 2 hpf to 120 hpf and the hatching rate, malformation rate, heart rate and body length of zebrafish embryos at 72 hpf, the frequency of tail curling at 24-29 hpf, the locomotor ability at 96 hpf and the survival rate at 120 hpf were evaluated, respectively. The whole-body triiodothyronine (T3) and tetraiodothyronine (T4) levels of juvenile fish were measured by enzyme immunoassay at the end of the infection, and the expression levels of hypothalamic-pituitary-thyroid axis (HPT) and neurodevelopmental-related genes were detected by quantitative real-time PCR (q-PCR). ResultsThe heart rates of zebrafish embryos were significantly decreased in all TBP-treated groups (P

BackgroundIn recent years, a growing number of studies have indicated that perfluorooctanoic acid (PFOA) exposure can impact heart development, though the specific mechanisms remain elusive. The aryl hydrocarbon receptor (AHR) is a critical environmental sensor capable of inducing oxidative stress and cell apoptosis. ObjectiveTo explore the role of AHR in the cardiac developmental toxicity of PFOA by using zebrafish embryo as an in vivo model. MethodsZebrafish embryos at 2 h post-fertilization (2 hpf) were exposed to dimethyl sulfoxide (DMSO) control, 1, 10, 100, and 1000 μg·L−1 of PFOA. The AHR inhibitor CH223191 (CH) was added to the high-concentration group (1000 μg·L−1) to form an intervention group. Under a dissecting microscope, the survival rate, mortality rate, heart rate, and heart malformation rate of 72 hpf zebrafish larvae were assessed. The activity of AHR was measured using 7-ethoxyresorufin-O-dealkylation (EROD) staining. The levels of intracellular and mitochondrial ROS in the heart of zebrafish larvae were assessed using dichloro-dihydro-fluorescein diacetate and MitoSOX™ Red, respectively. Apoptosis was examined using acridine orange (AO) staining and Immunofluorescence method. Total RNA was extracted from dissected hearts, and mRNA expression levels of oxidative stress-related genes (sod2, cat) and apoptosis-related gene (p53) were analyzed using quantitative PCR. ResultsCompared to the DMSO control group, PFOA at even the lowest concentration (1 μg·L−1) increased heart malformation rate (P＜0.05) and reduced heart rate (P＜0.01) in the zebrafish larvae at 72 hpf, and the results showed a clear concentration-response relationship. Adding the AHR inhibitor CH significantly decreased heart malformation rate and restored heart rate to the control group level. The EROD results showed that PFOA at 1000 μg·L−1 increased AHR activity (P＜0.001). Further studies revealed that PFOA caused a dose-dependent increase in intracellular ROS (P

Objective: Yiwei Decoction (YWD) is an ancient TCM formula with historical use in treating chronic atrophic gastritis (CAG). However, its pharmacodynamic mechanisms remain poorly understood. This study aims to study the effect of YWD on CAG through network pharmacology and experimental verification. Methods: UPLC/MS and the SwissADME database were employed to identify active compounds in YWD. YWD targets were screened by SwissTargetPrediction databases, CAG targets were screened by databases, and the two were intersected for PPI network analysis. The target prediction was performed by GO and KEGG analysis. H2O2-induced injury models were established in zebrafish and GES-1 ​cells, which were subsequently treated with varying doses of YWD. Oxidative stress indicators and apoptosis and inflammation levels in zebrafish and GES-1 ​cells were assessed using fluorescence microscopy, flow cytometry, and microplate reader assays. The binding capabilities of the core components and core targets were examined using molecular docking. Q-PCR and Western blot were employed to analyze the expression levels of Nrf2, Keap1, and HO-1, respectively. Results: Forty-nine compounds were identified from YWD. Network pharmacological analysis suggested that YWD may treat CAG by modulating redox-related signaling pathways, inhibiting apoptosis, and reducing inflammation. Subsequent in vitro and in vivo experiments validated these predictions. YWD effectively mitigated H2O2-induced oxidative stress in zebrafish and GES-1 ​cells, suppressing ROS and decreasing apoptosis. YWD reduced inflammation in gastric epithelial cells. Molecular docking results suggested that methylophiopogonanone A and imperatorin may play a key role in the treatment of YWD. Mechanistically, YWD activated the Nrf2 signaling pathway, downregulated Keap1 expression, and upregulated HO-1 and Bcl2 expression. Conclusion: YWD could improve oxidative stress indicators, inhibiting cell apoptosis, reducing inflammation, and its molecular mechanism of the CAG treatment may be through the Keap1/Nrf2/HO-1 pathways, and to promote the body's antioxidant system.

Objective: To investigate the physicochemical properties and functional changes of the polysaccharides prepared from Sargassum fusiforme by different combinations of enzymes. Methods: Six enzyme combinations of cellulase-alcalase (C-A), pectinase-alcalase (P-A), viscozyme-alcalase (V-A), cellulase-neutrase (C-N), pectinase-neutrase (P-N), and viscozyme-neutrase (V-N) were used to assist the extraction of polysaccharides from Sargassum fusiforme. The physicochemical properties, structural composition, and antioxidant activity in vitro and in vivo of Sargassum fusiforme polysaccharides prepared by six combined enzymes were analyzed. Results: The contents of total sugar, uronic acid and sulfate in the polysaccharides prepared by the six combined enzymes changed greatly, and the monosaccharide composition and chemical structure were similar. The total sugar content and polysaccharide yield of the C-N group were the highest. The reducing power of Fe2+ and DPPH radical scavenging capacity of polysaccharides were the highest in the V-N group, amounting to 0.103±0.003 (Abs) and 0.540±0.022 (mg/mL) (IC50), respectively, and in the P-N group, the hydroxyl radical scavenging capacity of polysaccharides was the highest at 1.341±0.265 (mg/mL) (IC50). The polysaccharides prepared by different combined enzymes had different protective effects on AAPH-induced oxidative stress. Among them, compared with the NO group, the polysaccharides in the P-A, C-A, V-A and P-N groups had a stronger ability to reduce reactive oxygen species. The polysaccharides in the P-A, P-N and V-N groups had significant lipid peroxidation inhibition. Compared with the induction group, the polysaccharides in the C-N and V-N groups significantly increased CAT enzyme activity (P

Aortic aneurysm/dissection (AAD) poses a life-threatening cardiovascular emergency with complex mechanisms and a notably high mortality rate. Zebrafish (Danio rerio) serve as valuable models for AAD due to the conservation of their three-layered arterial structure and genome with that of humans. However, the existing studies have predominantly focused on larval zebrafish, leaving a gap in our understanding of adult zebrafish. In this study, we utilized β-Aminopropionic Nitrile (BAPN) impregnation to induce AAD in both larval and adult zebrafish. Following induction, larval zebrafish exhibited a 28% widening of the dorsal aortic diameter (p

Krabbe disease is a sphingolipidosis characterized by the genetic deficiency of the acid hydrolase β-galactosylceramidase (GALC). Most of the studies concerning the biological role of GALC performed on Krabbe patients and Galc-deficient twitcher mice (an authentic animal model of the disease) indicate that the pathogenesis of this disorder is the consequence of the accumulation of the neurotoxic GALC substrate β-galactosylsphingosine (psychosine), ignoring the possibility that this enzyme may exert a wider biological impact. Indeed, limited information is available about the effect of GALC downregulation on the cell lipidome in adult and developing organisms. The teleost zebrafish (Danio rerio) has emerged as a useful platform to model human genetic diseases, including sphingolipidoses, and two GALC co-orthologs have been identified in zebrafish (galca and galcb). Here, we investigated the effect of the competitive and irreversible GALC inhibitor β-galactose-cyclophellitol (GCP) on the lipid profile of zebrafish embryos. Molecular modelling indicates that GCP can be sequestered in the catalytic site of the enzyme and covalently binds human GALC, and the zebrafish Galca and Galcb proteins in a similar manner. Accordingly, GCP inhibits the β-galactosylceramide hydrolase activity of zebrafish in vitro and in vivo, leading to significant alterations of the lipidome of zebrafish embryos. These results indicate that the lack of GALC activity deeply affects the lipidome during the early stages of embryonic development, and thereby provide insights into the pathogenesis of Krabbe disease.

The zebrafish (Danio rerio) has emerged as a model organism for investigating lncRNAs-driven fundamental biological processes, such as circadian rhythms, physiology, metabolism, and various diseases. While state-of-the-art sequencing technologies have identified an increasing number of lncRNAs in zebrafish, their annotations are far from complete. In this study, we collect 28,925 lncRNAs from both the published studies and our own RNA-seq analyses and establish a novel webserver-based database called SUDAZFLNC (https://sudarna.website/). The database, containing 28,925 lncRNAs, 25,432 mRNAs, and 368 miRNAs, provides several crucial features and annotations for the zebrafish RNAs, such as sequence identifiers (IDs), sequence length, hexamer score, coding probabilities, GO and KEGG annotations, and micropeptides. SUDAZFLNC also includes time-course expression profiles of 3288 lncRNAs, 25,432 mRNAs, and 342 miRNAs generated from our RNA-seq experiments, and 149, 4407, and 43 rhythmically expressed lncRNAs, mRNAs, and miRNAs, respectively. Based on the peak expression patterns, we classified these RNAs into morning RNAs, evening RNAs, and night RNAs. Users of the database can access the RNA sequences and their expression profiles by searching the corresponding IDs from the Graphical User Interface (GUI) of the database. The database supports several features to investigate RNA sequences and expression profiles, including BLAST, search of sequence and data, ID conversion, and RNA-RNA interaction prediction. This is the largest curated database of zebrafish RNAs and their expression profiles to date.

Alzheimer’s disease (AD) is an age-related neurodegenerative disorder. Transgenic and pharmacological AD models are extensively studied to understand AD mechanisms and drug discovery. However, they are time-consuming and relatively costly, which hinders the discovery of potential anti-AD therapeutics. Here, we established a new model of AD in larval zebrafish by co-treatment with aluminum chloride (AlCl3) and D-galactose (D-gal) for 72 h. In particular, exposure to 150 μM AlCl3 + 40 mg/mL D-gal, 200 μM AlCl3 + 30 mg/mL D-gal, or 200 μM AlCl3 + 40 mg/mL D-gal successfully induced AD-like symptoms and aging features. Co-treatment with AlCl3 and D-gal caused significant learning and memory deficits, as well as impaired response ability and locomotor capacity in the plus-maze and light/dark test. Moreover, increased acetylcholinesterase and β-galactosidase activities, β-amyloid 1–42 deposition, reduced telomerase activity, elevated interleukin 1 beta mRNA expression, and enhanced reactive oxygen species production were also observed. In conclusion, our zebrafish model is simple, rapid, effective and affordable, incorporating key features of AD and aging, thus may become a unique and powerful tool for high-throughput screening of anti-AD compounds in vivo.

Objectives. Our study aimed to explore the role of the potassium channel KCNK1 in head and neck squamous cell carcinoma, focusing on its impact on tumor growth, invasion, and metastasis. We also investigated the therapeutic potential of quinidine, a known KCNK1 inhibitor, in both in vitro cell lines and a zebrafish patient-derived xenograft (PDX) model. Methods. We established primary cell cultures from head and neck cancer tissues and employed the FaDu cell line for in vitro studies, modulating KCNK1 expression through overexpression and knockdown techniques. We evaluated cell migration, invasion, and proliferation. Additionally, we developed a zebrafish PDX model to assess the impact of quinidine on tumor growth and metastasis in vivo. RNA sequencing and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were conducted to elucidate the molecular mechanisms underlying the role of KCNK1 in cancer progression. Results. Overexpression of KCNK1 in FaDu cells resulted in enhanced cell migration and invasion, whereas its knockdown diminished these processes. In the zebrafish PDX model, quinidine markedly inhibited tumor growth and metastasis, demonstrating a significant reduction in tumor volume and micrometastasis rates compared to the control groups. The molecular analyses indicated that KCNK1 plays a role in critical signaling pathways associated with tumor growth, such as the Ras and MAPK pathways. Conclusion. Our findings highlight the critical role of KCNK1 in promoting tumor growth and metastasis in head and neck cancer. The inhibitory effect of quinidine on tumor progression in the zebrafish PDX model highlights the therapeutic potential of targeting KCNK1. These results suggest that KCNK1 could serve as a valuable therapeutic target for head and neck cancer, warranting further investigation into treatments that target KCNK1.

Abstract Background Evidence has accumulated to demonstrate that intestinal microbiome can inhibit viral infection. However, our knowledge of the signaling pathways and identity of specific commensal microbes that mediate the antiviral response is limited. Zebrafish have emerged as a powerful animal model for study of vertebrate-microbiota interactions. Here, a rhabdoviral infection model in zebrafish allows us to investigate the modes of action of microbiome-mediated antiviral effect. Results We observed that oral antibiotics-treated and germ-free zebrafish exhibited greater spring viremia of carp virus (SVCV) infection. Mechanistically, depletion of the intestinal microbiome alters TLR2-Myd88 signaling and blunts neutrophil response and type I interferon (IFN) antiviral innate immunity. Through 16S rRNA sequencing of the intestinal contents from control and antibiotic(s)-treated fish, we identified a single commensal bacterial species, Cetobacterium somerae, that can restore the TLR2- and neutrophil-dependent type I IFN response to restrict SVCV infection in gnotobiotic zebrafish. Furthermore, we found that C. somerae exopolysaccharides (CsEPS) was the effector molecule that engaged TLR2 to mediate the type I IFN-dependent antiviral function. Conclusions Together, our results suggest a conserved role of intestinal microbiome in regulating type I IFN antiviral response among vertebrates and reveal that the intestinal microbiome inhibits viral infection through a CsEPS-TLR2-type I IFN signaling axis in zebrafish. Video Abstract

The enzymolysis peptide of peony flower was studied herein to promote the development and utilization of this flower. The molecular weight distribution and amino acid composition of the peptide were analyzed, and the beneficial effects of the peptide were studied by assessing their ability to inhibit tyrosinase activity, advanced glycation end products (AGEs) formation, and hyaluronidase in vitro. The in vitro results were further verified in a zebrafish melanin accumulation model, results indicated that the active peptide was a small molecule comprising 15 amino acids, with an average molecular weight of 644 Da. At 10 mg/mL, the respective in vitro inhibition rates for tyrosinase activity, AGEs formation, and hyaluronidase were 88.96%, 90.50%, and 60.43%, respectively. The respective DPPH (2,2-diphenyl-1-picrylhydrazyl) and hydroxyl free radical scavenging rates were 74.77% and 94.74%, respectively. The zebrafish model studies revealed a melanin inhibition rate of 40.59% when the sample concentration was 1000 μg/mL. In conclusion, the results of these in vivo and in vitro studies preliminarily indicate that the peony flower enzymolysis peptide can potentially improve skin health.

Abstract Background Tryptophan is an essential amino acid involved in critical cellular processes in vertebrates, serving as a precursor for serotonin and kynurenine, which are key neuromodulators to influence neural and immune functions. Systematic and quantitative measurement of tryptophan is vital to understanding these processes. Results Here, we utilized a robust and highly responsive green ratiometric indicator for tryptophan (GRIT) to quantitatively measure tryptophan dynamics in bacteria, mitochondria of mammalian cell cultures, human serum, and intact zebrafish. At the cellular scale, these quantitative analyses uncovered differences in tryptophan dynamics across cell types and organelles. At the whole-organism scale, we revealed that inflammation-induced tryptophan concentration increases in zebrafish brain led to elevated serotonin and kynurenine levels, prolonged sleep duration, suggesting a novel metabolic connection between immune response and behavior. Moreover, GRIT’s application in detecting reduced serum tryptophan levels in patients with inflammation symptoms suggests its potential as a high-throughput diagnostic tool. Conclusions In summary, this study introduces GRIT as a powerful method for studying tryptophan metabolism and its broader physiological implications, paving the way for new insights into the metabolic regulation of health and disease across multiple biological scales.

The use and manufacture of nanomaterials are expanding, which is concerning for human health. Graphene oxide (GO), magnesium oxide (MgO), and quercetin (QU) have typically had significant growth because of their diverse range of applications in nanomedicine. They are utilised in the treatment of cancer, medication delivery, tissue engineering, healing of wounds, and biosensor. In this work, MgO-QU/GO nanocomposite was synthesized and its antioxidant was tested in a zebrafish model. Further obtained MgO-QU/GO nanocomposite was characterized through UV, XRD, FTIR, and SEM with EDAX analysis. Zebrafish adult seven test groups were used in the experiment with each group containing 10 fish follows fish fed the control, and exposed to MgO-QU/GO nanocomposite concentration 0+0.0+20.0+40.0+80.0+20+0.0+40+0.0+80+0 mg/L. The outcome shows that the toxicity of the utilised nanocomposite was comparatively low after zebrafish were exposed to MgO-QU/GO and recovered. Exploratory MgO-QU/GO treatment to zebra fish significant by decreased the Lipid Peroxidation (LPO) and elevated the antioxidant. These findings imply that nanocomposite at the concentration considered here might be a workable substitute for water recovery as they have no negative effects on the long-term life and welfare of humans and animals.

Abstract CHD2‐related epilepsy is characterized by early‐onset photosensitive myoclonic epilepsy with developmental delay and a high rate of pharmacoresistance. We sought to evaluate the efficacy of acetazolamide (ACZ) in CHD2‐related epilepsy, due to ACZ's unexpected efficacy in our first patient harboring a pathogenic CHD2 variant. We collected patients from different Eastern European countries with drug‐resistant CHD2‐related epilepsy who were then treated with ACZ. Patients underwent video EEG before and during ACZ treatment. In a zebrafish model of CHD2‐related epilepsy, ictal‐like events were recorded 5 days post‐fertilization after overnight ACZ exposure. Developmental delay preceded the onset of seizures in 10 of the 12 patients. Four had ataxia, and 6 exhibited autistic features. Seizures, primarily myoclonic, began at an average age of 3.4 years and were photosensitive in all 12 patients. Add‐on ACZ treatment controlled photosensitive seizures in all patients: 6 became seizure‐free, and in the remaining 6, seizure frequency decreased by over 75%. Four patients transitioned to ACZ monotherapy. The median follow‐up was 13 months. In the zebrafish model, ACZ exposure reduced ictal‐like events by 72%. ACZ, a well‐tolerated and cost‐effective medication, could be a good option for CHD2‐related epilepsy, predominantly manifesting with myoclonic seizures and photosensitivity. Plain Language Summary Epilepsy associated with CHD2 mutations is often pharmacoresistant and associated with developmental delay and eventually ataxia. There are several generalized seizure types, including generalized tonic–clonic seizures, but the most characteristic are jerks triggered by light stimulation. We collected 12 patients who received acetazolamide, a drug usually given as a diuretic and registered as a mild antiseizure medication. All jerks triggered by light disappeared while the frequency of spontaneous seizures decreased by over 75%. Further studies are needed to confirm this promising finding and identify the mechanism by which an old compound seems to have such a specific antiseizure effect.

Abstract Isl1 has been described as an embryonic master control gene expressed in the pericloacal mesenchyme. Deletion of Isl1 from the genital mesenchyme in mice leads to an ectopic urethral opening and epispadias-like phenotype. Using genome wide association methods, we identified ISL1 as the key susceptibility gene for classic bladder exstrophy (CBE), comprising epispadias and exstrophy of the urinary bladder. The most significant marker (rs6874700) identified in our recent GWAS meta-analysis achieved a p value of 1.48 × 10− 24 within the ISL1 region. In silico analysis of rs6874700 and all other genome-wide significant markers in Linkage Disequilibrium (LD) with rs6874700 (D’ = 1.0; R2 > 0.90) revealed marker rs2303751 (p value 8.12 × 10− 20) as the marker with the highest regulatory effect predicted. Here, we describe a novel 1.2 kb intragenic promoter residing between 6.2 and 7.4 kb downstream of the ISL1 transcription starting site, which is located in the reverse DNA strand and harbors a binding site for EZH2 at the exact region of marker rs2303751. We show, that EZH2 silencing in HEK cells reduces ISL1 expression. We show that ezh2 −/− knockout (KO) zebrafish larvae display tissues specificity of ISL1 regulation with reduced expression of Isl1 in the pronephric region of zebrafish larvae. In addition, a shorter and malformed nephric duct is observed in ezh2 −/− ko zebrafish Tg(wt1ß:eGFP) reporter lines. Our study shows, that Ezh2 is a key regulator of Isl1 during urinary tract formation and suggests tissue specific ISL1 dysregulation as an underlying mechanism for CBE formation.

Abstract The paralogs 9–13 Hox genes in mouse HoxA and HoxD clusters are critical for limb development. When both HoxA and HoxD clusters are deleted in mice, significant limb truncation is observed compared to the phenotypes of single and compound mutants of Hox9-13 genes in these clusters. In zebrafish, mutations in hox13 genes in HoxA- and HoxD-related clusters result in abnormal morphology of pectoral fins, homologous to forelimbs. However, the effect of the simultaneous deletions of entire HoxA- and HoxD-related clusters on pectoral fin development remains unknown. Here, we generated mutants with several combinations of hoxaa, hoxab, and hoxda cluster deletions and analyzed the pectoral fin development. In hoxaa −/− ;hoxab −/− ;hoxda −/− larvae, the endoskeletal disc and the fin-fold are significantly shortened in developing pectoral fins. In addition, we show that this anomaly is due to defects in the pectoral fin growth after the fin bud formation. Furthermore, in the surviving adult mutants, micro-CT scanning reveals defects in the posterior portion of the pectoral fin which is thought to represent latent regions of the limb. Our results further support that the functional role of HoxA and HoxD clusters is conserved in the paired appendage formation in bony fishes.

Abstract Bisphenol A (BPA) is an environmental estrogen which perturbs hormone signaling pathways adversely affecting aquatic organisms. To evaluate the impact of developmental exposure to long term yet environmentally relevant low doses of BPA, wild-type juvenile zebrafish of 35 days post fertilization were treated with BPA (1 and 10 µg/L), treatment control (0.5% v/v methanol) and control for 60 days. Both BPA treatments led to significantly increased morality overtime. Length increment and specific growth rates became significantly high in BPA exposed zebrafish overtime. Obesogenic property of BPA was not evident with longexposure to low BPA doses. A significantly high and BPA dose-dependent female-biased sex ratios were observed following the juvenile exposure. Significantly low swimming speed was recorded in the fish of both BPA-treated tanks than that of control. Condition factor was significantly low in BPA exposed fish indicating the poor-wellness. There were numerous histopathological alterations of gonads, liver and kidney indicating impacts of juvenile exposure in zebrafish. Altered growth, swimming, mortality, feminization and histopathological changes in zebrafish induced by BPA indicate the risks associated with developmental exposures. The findings call for more comprehensive studies to comprehend the ecological risks imposed by low concentrations of environmental estrogens in urban aquatic ecosystems.

Abstract Paqr5b is a gene encoding membrane progesterone receptor γ (mPRγ), which is one of five mPR subtypes. Paqr5b belongs to the progestin and adipoQ receptor (PAQR) family, which consists of 11 genes. To elucidate the physiological functions of the mPR subtypes, we established gene knockout (KO) zebrafish strains by genetically editing seven paqr genes and analyzed their phenotypes. The null-mutant strain of paqr5b (paqr5b −/− ) that we established in this study showed low fecundity, reduced chorion elevation and a high percentage of abnormal embryos. Embryos showed curvature of the spine and an abnormal head morphology. Individuals with abnormal head morphology continued to develop a phenotype of markedly abnormal palatine bone. The length of the brain of paqr5b −/− zebrafish was short, and the position of the cerebellum moved to the front and overlapped with that of the midbrain. Micro-CT scans revealed that the olfactory rosettes (ORs) were so shrunken that they were difficult to identify and connected with the olfactory bulbs (OBs) by thread-like structures. Immunohistochemical staining of OR with an anti-Paqr5b antibody revealed that Paqr5b was extensively expressed in neurons in the OR in wild-type zebrafish, whereas signals were not detected in paqr5b −/− zebrafish. In histological sections, the neurons disappeared, and the lamellar layer of the OR became thinner. These results indicate that Paqr5b is required for the formation of neurons in the OR. This is the first report demonstrating a distinct role for the mPR gene.

Abstract Inherited and acquired muscle diseases are an important cause of morbidity and mortality in human medical and veterinary patients. Researchers use models to study skeletal muscle development and pathology, improve our understanding of disease pathogenesis and explore new treatment options. Experiments on laboratory animals, including murine and canine models, have led to huge advances in congenital myopathy and muscular dystrophy research that have translated into clinical treatment trials in human patients with these debilitating and often fatal conditions. Whilst animal experimentation has enabled many significant and impactful discoveries that otherwise may not have been possible, we have an ethical and moral, and in many countries also a legal, obligation to consider alternatives. This review discusses the models available as alternatives to mammals for muscle development, biology and disease research with a focus on inherited myopathies. Cell culture models can be used to replace animals for some applications: traditional monolayer cultures (for example, using the immortalised C2C12 cell line) are accessible, tractable and inexpensive but developmentally limited to immature myotube stages; more recently, developments in tissue engineering have led to three-dimensional cultures with improved differentiation capabilities. Advances in computer modelling and an improved understanding of pathogenetic mechanisms are likely to herald new models and opportunities for replacement. Where this is not possible, a 3Rs approach advocates partial replacement with the use of less sentient animals (including invertebrates (such as worms Caenorhabditis elegans and fruit flies Drosophila melanogaster) and embryonic stages of small vertebrates such as the zebrafish Danio rerio) alongside refinement of experimental design and improved research practices to reduce the numbers of animals used and the severity of their experience. An understanding of the advantages and disadvantages of potential models is essential for researchers to determine which can best facilitate answering a specific scientific question. Applying 3Rs principles to research not only improves animal welfare but generates high-quality, reproducible and reliable data with translational relevance to human and animal patients.

Abstract Background The development of the zebrafish visual system is significantly influenced by exposure to monochromatic light, yet investigations into its effects during juvenile stages are lacking. This study evaluated the impacts of varying intensities and durations of red and blue monochromatic light on the visual system and dopamine pathways in juvenile zebrafish. Methods Juvenile zebrafish were exposed to red (650 nm) and blue (440 nm, 460 nm) monochromatic lights over four days at intensities ranging from 500 to 10,000 lx, for durations of 6, 10, and 14 h daily. A control group was maintained under standard laboratory conditions. Post-exposure assessments included the optokinetic response (OKR), retinal structural analysis, ocular dopamine levels, and the expression of genes related to dopamine pathways (Th, Dat, and Mao). Results (1) OKR enhancement was observed with increased 440 nm light intensity, while 460 nm and 650 nm light exposures showed initial improvements followed by declines at higher intensities. (2) Retinal thinning in the outer nuclear layer was observed under the most intense (10,000 lx for 14 h) light conditions in the 440 nm and 650 nm groups, while the 460 nm group remained unaffected. (3) Dopamine levels increased with higher intensities in the 440 nm group, whereas the 460 nm group exhibited initial increases followed by decreases. The 650 nm group displayed similar trends but were statistically insignificant compared to the control group. (4) Th expression increased with light intensity in the 440 nm group. Dat showed a rising and then declining pattern, and Mao expression significantly decreased. The 460 nm group exhibited similar patterns for Th and Dat to the behavioral observations, but an inverse pattern for Mao. The 650 nm group presented significant fluctuations in Th and Dat expressions, with pronounced variations in Mao. Conclusions Specific red and blue monochromatic light conditions promote visual system development in juvenile zebrafish. However, exceeding these optimal conditions may impair visual function, highlighting the critical role of dopamine pathway in modulating light-induced effects on the visual system.

Abstract Receptors for activated C kinases (RACKs) have been shown to coordinate PKC-mediated hypertrophic signalling in mice. However, little information is available on its participation in embryonic gene expression. This study investigated the involvement of RACK1 in the expression of embryonic genes in a zebrafish (ZF) ex vivo heart culture model by using phenylephrine (PE) or a growth factors cocktail (GFs) as a prohypertrophic/regeneration stimulus. Blebbistatin (BL) inhibition has also been studied for its ability to block the signal transduction actions of some PEs. qRT‒PCR and immunoblot analyses confirmed the upregulation of RACK1 in the PE- and GFs-treated groups. BL administration counteracted PE-induced hypertrophy and downregulated RACK1 expression. Immunohistochemical analyses of the heart revealed the colocalization of RACK1 and embryonic genes, namely, Gata4, Wt1, and Nfat2, under stimulation, whereas these genes were expressed at lower levels in the BL treatment group. Culturing ZF heart cells activated via GFs treatment increased the expression of RACK1. The overexpression of RACK1 induced by the transfection of recombinant RACK1 cDNA in ZF heart cells increased the expression of embryonic genes, especially after one week of GFs treatment. In summary, these results support the involvement of RACK1 in the induction of embryonic genes during cardiac hypertrophy/GFs stimulation in a fish heart model, which can be used as an alternative study model for mammals.

Chunjiao Lu,* Xuewei Wu,* Xin Meng,* Yi Liu, Ting Yang, Yan Zeng, Yang Chen, Yishan Huang, Zhou Fang, Xiaojun Yang, Juanjuan Luo Engineering Research Center of Key Technique for Biotherapy of Guangdong Province, Shantou University Medical College, Shantou, 515041, People’s Republic of China*These authors contributed equally to this workCorrespondence: Xiaojun Yang; Juanjuan Luo, Shantou University Medical College, 22 Xinling Road, Shantou, 515041, People’s Republic of China, Email yangx@stu.edu.cn; 15jjluo1@stu.edu.cnIntroduction: The potential toxic effects of wastewater discharges containing silver nanoparticles (AgNPs) and their release into aquatic ecosystems on aquatic organisms are becoming a major concern for environmental and human health. However, the potential risks of AgNPs to aquatic organisms, especially for cardiac development by Focal adhesion pathway, are still poorly understood.Methods: The cardiac development of various concentrations of AgNPs in zebrafish were examined using stereoscopic microscope. The expression levels of cardiac development-related genes were analyzed by qRT-PCR and Whole-mount in situ hybridization (WISH). In addition, Illumina high-throughput global transcriptome analysis was performed to explore the potential signaling pathway involved in the treatment of zebrafish embryos by AgNPs after 72 h.Results: We systematically investigated the cardiac developing toxicity of AgNPs on the embryos of zebrafish. The results demonstrated that 2 or 4 mg/L AgNPs exposure induces cardiac developmental malformations, such as the appearance of pericardial edema phenotype. In addition, after 72 h of exposure, the mRNA levels of cardiac development-related genes, such as myh7, myh6, tpm1, nppa, tbx5, tbx20, myl7 and cmlc1, were significantly lower in AgNPs-treated zebrafish embryos than in control zebrafish embryos. Moreover, RNA sequencing, KEGG (Kyoto Encyclopedia of Genes) and Genomes and GSEA (gene set enrichment analysis) of the DEGs (differentially expressed genes) between the AgNPs-exposed and control groups indicated that the downregulated DEGs were mainly enriched in focal adhesion pathways. Further investigations demonstrated that the mRNA levels of focal adhesion pathway-related genes, such as igf1ra, shc3, grb2b, ptk2aa, akt1, itga4, parvaa, akt3b and vcla, were significantly decreased after AgNPs treatment in zebrafish.Conclusion: Thus, our findings illustrated that AgNPs could impair cardiac development by regulating the focal adhesion pathway in zebrafish.Keywords: AgNPs, focal adhesion, cardiac development, zebrafish

Abstract Background The quest for candidate probiotics and prebiotics to develop novel synbiotics for sustainable and profitable fish farming remains a major focus for various stakeholders. In this study, we examined the effects of combining two fungal probiotics, Saccharomyces cerevisiae and Aspergillus niger with extracts of Jerusalem artichoke and white button mushroom to develop a synbiotic formulation to improve the growth and health status of zebrafish (Danio rerio). An initial in vitro study determined the most effective synbiotic combination, which was then tested in a 60-day in vivo nutritional trial using zebrafish (80 ± 1.0 mg) as a model animal. Four experimental diets were prepared: a control diet (basal diet), a prebiotic diet with 100% selected mushroom extract, a probiotic diet with 107 CFU of S. cerevisiae/g of diet, and a synbiotic diet with 107 CFU of S. cerevisiae/g of diet and 100% mushroom extract. As readouts, growth performance, survival, digestive enzyme activity and innate immune responses were evaluated. Results In vitro results showed that the S. cerevisiae cultured in a medium containing 100% mushroom extract exhibited the maximum specific growth rate and shortest doubling time. In the in vivo test with zebrafish, feeding them with a synbiotic diet, developed with S. cerevisiae and mushroom extract, led to a significant improvement in the growth performance of zebrafish (P

Abstract Fish domestication progresses through five levels: from the initial acclimatization to captivity (Level 1), to the life cycle completion in captivity (Level 4), and even to the implementation of selective breeding programs (Level 5). Domestication leads to phenotypic changes over generations, sometimes from the very first generation. Behavioral traits are among the first to change. However, in fish, potential behavioral changes during early domestication have been little studied. Therefore, we studied potential behavioral changes among early and advanced levels of domestication in a model species, the zebrafish (Danio rerio), using a mirror test experiment, commonly used to assess traits involved in activity, aggressiveness, and stress in this species. We compared these traits between wild zebrafish in captivity (F0; Level 1), the first generation of their captive-born offspring (F1; Level 4), and three laboratory strains (AB, TU, and WIK; Level 5). Each fish was individually filmed and tracked using an automated procedure for 5 min. Nine behavioral traits and one activity-related trait were characterized for each individual based on the movements and positioning of the fish. We applied a principal component analysis (PCA) and tested the significance of potential differences between groups using an analysis of similarities (ANOSIM). We applied an indicator value analysis (IndVal) to determine which traits were most expressed by each group. We detected differences between groups and across domestication levels. More specifically, we highlighted differentiations between different levels of domestication (e.g. between F1, AB, TU, and WIK) as early as the beginning of the domestication process (i.e. F0 vs. F1), but also within the same level of domestication (i.e. AB vs. TU). Based on PCA and IndVal, (i) F0 and F1 tended to show stronger expression of stress-related traits than the other groups, (ii) F0 was more active than others, and (iii) TU was more aggressive than AB. Our results confirmed that domestication can change fish behavior, even in the first generation born in captivity, although these modifications remain limited. In contrast, we did not observe any general trends correlated with domestication levels, given that AB and TU diverged in their aggressiveness levels, and WIK differed only from F1. This result needs to be generalized to other species but also considered for domestication for aquaculture. If future studies confirm that the changes observed at the beginning of the domestication process remain limited and that there is no consistent evolutionary trend across generations in fish, this would highlight the crucial importance of selecting the right species from the outset of domestication. It would also emphasize the need to design selective breeding programs that shape fish stocks with the most desirable characteristics. To our knowledge, this study is one of the few to examine the behavior of wild zebrafish alongside laboratory strains, offering a unique insight into the early stages of domestication.

ABSTRACTVirtually every major medical advance of the last century and at still has depended upon research with animals. Zebrafish's journey from the ocean to the laboratory leads to major scientific breakthroughs. Transparency structure of zebrafish helps in monitoring their internal structures and are permitting scientist to see effectes of nano particles in fish. Their organs share the same main features as humans and so can be used to study human developmental processes. Zebrafish congruence 70% of their genes with humans, and 84% of ailment-depended genes have zebrafish congruence. The zebrafish embryos can also genetically modified. Certain fishes like zebrafish are able to regenerate damaged retinal nerve cells. Müller galia cells in retina of zebrafish can transform in response to injury and act like stem cells to regrow the retina and replace all damaged neurons. Though humans have the same exact Müller galia cell, they don’t respond to damaged in the same way. Zebrafish are also very responsive to having their genomes edited. Zebrafish regenerate some tissue such as heart in during larval stage. In additionaly zebrafish are used as an animal model to study pharmocology – how drugs work and what they do to an organism’s body. Aim of this review, here, we review current knowledge of how these specialized structures and model organism by focusing on cellular behaviors and molecular mechanisms, highlighting findings from in vivo models and briefly discussing the recent advances in tissue cell culture and organoids. Review discusses the applications of human organoids models of disease on model organism and outlines the ailment treatments.

Abstract Background Zebrafish are widely used in various research fields and to fulfil the diverse research needs, numerous zebrafish lines are available, each with a unique domestication background, potentially resulting in intraspecies differences in specific biological functions. Few studies have compared multiple zebrafish lines under identical conditions to investigate both inter- and intra-line variability related to different functions. However, such variability could pose a challenge for the reproducibility of results in studies utilising zebrafish, particularly when the line used is not clearly specified. This study assessed growth, stress status (cortisol, serotonin) and reproductive capabilities (maturity, fecundity, fertilisation rate, sperm quality) of four commonly used wild-type zebrafish lines (AB, SJD, TU, WIK) using standardized protocols. Results The stress markers levels were found to be similar across the lines, indicating that the endocrine stress status is robust to diverse domestication histories. Variations were observed in the growth and reproductive parameters. The lines exhibited differences in the timing of puberty (86 dpf for AB and SJD lines vs. 107 dpf for the WIK line) despite achieving similar sizes, suggesting that there are line-specific variations in the induction of maturation. Additionally, the AB line demonstrated higher sperm quality than did the other lines and higher fecundity and fertilization rates than did the SJD line. The AB line also exhibiting a smaller adult size but a heavier brain relative to its body weight. Conclusion These findings emphasize the importance of line selection for zebrafish research, indicating that researchers should consider line-specific traits to ensure the biological relevance and reproducibility of the results.

Abstract To elucidate the lipidomic and metabolomic alterations associated with hypertrophic cardiomyopathy (HCM) pathogenesis, we utilized cmybpc3-/- zebrafish model. Fatty acid profiling revealed variability of 10 fatty acids profiles, with heterozygous (HT) and homozygous (HM) groups exhibiting distinct patterns. Hierarchical cluster analysis and multivariate analyses demonstrated a clear separation of HM from HT and control (CO) groups related to cardiac remodeling. Lipidomic profiling identified 257 annotated lipids, with two significantly dysregulated between CO and HT, and 59 between HM and CO. Acylcarnitines and phosphatidylcholines were identified as key contributors to group differentiation, suggesting a shift in energy source. Untargeted metabolomics revealed 110 and 53 significantly dysregulated metabolites. Pathway enrichment analysis highlighted perturbations in multiple metabolic pathways in the HM group, including nicotinate, nicotinamide, purine, glyoxylate, dicarboxylate, glycerophospholipid, pyrimidine, and amino acid metabolism. Our study provides comprehensive insights into the lipidomic and metabolomic unique signatures associated with cmybpc3-/- induced HCM in zebrafish. The identified biomarkers and dysregulated pathways shed light on the metabolic perturbations underlying HCM pathology, offering potential targets for further investigation and potential new therapeutic interventions.

Abstract DHPS deficiency syndrome is an ultra-rare neurodevelopmental disorder (NDD) which results from biallelic mutations in the gene encoding the enzyme deoxyhypusine synthase (DHPS). DHPS is essential to synthesize hypusine, a rare amino acid formed by post-translational modification of a conserved lysine in eukaryotic initiation factor 5 A (eIF5A). DHPS deficiency syndrome causes epilepsy, cognitive and motor impairments, and mild facial dysmorphology. In mice, a brain-specific genetic deletion of Dhps at birth impairs eIF5AHYP-dependent mRNA translation. This alters expression of proteins required for neuronal development and function, and phenotypically models features of human DHPS deficiency. We studied the role of DHPS in early brain development using a zebrafish loss-of-function model generated by knockdown of dhps expression with an antisense morpholino oligomer (MO) targeting the exon 2/intron 2 (E2I2) splice site of the dhps pre-mRNA. dhps knockdown embryos exhibited dose-dependent developmental delay and dysmorphology, including microcephaly, axis truncation, and body curvature. In dhps knockdown larvae, electrophysiological analysis showed increased epileptiform activity, and confocal microscopy analysis revealed reduced arborisation of GABAergic neurons. Our findings confirm that hypusination of eIF5A by DHPS is needed for early brain development, and zebrafish with an antisense knockdown of dhps model features of DHPS deficiency syndrome.

Metal oxide nanoparticles (MONPs) are widely used in medicine and environmental remediation because of their unique properties. However, their size, surface area, and reactivity can cause toxicity, potentially leading to oxidative stress, inflammation, and cellular or DNA damage. In this study, a nano-quantitative structure–toxicity relationship (nano-QSTR) model was initially developed to assess zebrafish toxicity for 24 MONPs. Previously established 23 first- and second-generation periodic table descriptors, along with five newly proposed third-generation descriptors derived from the periodic table, were employed. Subsequently, to enhance the quality and predictive capability of the nano-QSTR model, a nano-quantitative read across structure–toxicity relationship (nano-qRASTR) model was created. This model integrated read-across descriptors with modeled descriptors from the nano-QSTR approach. The nano-qRASTR model, featuring three attributes, outperformed the previously reported simple QSTR model, despite having one less MONP. This study highlights the effective utilization of the nano-qRASTR algorithm in situations with limited data for modeling, demonstrating superior goodness-of-fit, robustness, and predictability (R2 = 0.81, Q2LOO = 0.70, Q2F1/R2PRED = 0.76) compared to simple QSTR models. Finally, the developed nano-qRASTR model was applied to predict toxicity data for an external dataset comprising 35 MONPs, addressing gaps in zebrafish toxicity assessment.

Although the concept of endocrine disruptors first appeared almost 30 years ago, the relatively recent involvement of these substances in the etiology of metabolic pathologies (obesity, diabetes, hepatic steatosis, etc.) has given rise to the concept of Metabolic Disrupting Chemicals (MDCs). Organs such as the liver and adipose tissue have been well studied in the context of metabolic disruption by these substances. The intestine, however, has been relatively unexplored despite its close link with these organs. In vivo models are useful for the study of the effects of MDCs in the intestine and, in addition, allow investigations into interactions with the rest of the organism. In the latter respect, the zebrafish is an animal model which is used increasingly for the characterization of endocrine disruptors and its use as a model for assessing effects on the intestine will, no doubt, expand. This review aims to highlight the importance of the intestine in metabolism and present the zebrafish as a relevant alternative model for investigating the effect of pollutants in the intestine by focusing, in particular, on cytochrome P450 3A (CYP3A), one of the major molecular players in endogenous and MDCs metabolism in the gut.

Objective: There are some problems in the evaluation of drug cardiotoxicity in zebrafish, such as unsystematic indicators and weak sensitivity. This study aims to explore the advantages and disadvantages of various evaluation methods in rapid cardiotoxicity assessment, and to identify and establish representative and highly sensitive evaluation indicators. Methods: Four typical cardiotoxic drugs (Doxorubicin, 5-Fluorouracil, Ibuprofen and Lidocaine) with different concentrations were selected to act on zebrafish embryos. The death, cardiac malformation, heart rate, blood flow and sinus venosus-bulbus arteriosus (SV-BA) distance of zebrafish in each group were observed and recorded by stereo microscopy. The behavioral changes of zebrafish after drug treatment were counted and analyzed using a zebrafish behavior recorder. Adult zebrafish were used to screen the cardiotoxic expressed genes, and the spatiotemporal expression stability and concentration-dose dependence of the specifically highly expressed genes were studied. Results: All the 4 drugs can reduce the heart rate and blood flow velocity of zebrafish embryos to varying degrees, increase the SV-BA distance of zebrafish embryos, reduce the activity behavior of zebrafish embryos, and have different degrees of inhibitory effects on the cardiac function of zebrafish. Among the selected 12 genes expressed only in the heart, one and only CMLC1 showed high specific expression in the heart of zebrafish. However, doxorubicin did not affect the expression of CMLC1 gene in a concentration-dose dependent manner, and the other three drugs could significantly induce the up-regulation of CMLC1 gene expression. Conclusion: Ethology is a faster and more sensitive method than the traditional cardiotoxicity evaluation indicators (heart rate, blood flow velocity, and SV-BA distance). Zebrafish have a small number of highly heart-specific expressed genes and are not suitable for assessing cardiotoxicity based solely on heart-specific expression of circulating mRNA.

Ischemic stroke (IS) is a pathological condition characterized by the cessation of blood flow due to factors such as thrombosis, inflicting severe damage to the cranial nervous system and resulting in numerous disabilities including memory impairments and hemiplegia. Despite the critical nature of this condition, therapeutic options remain limited, with a pressing challenge being the development of treatments aimed at restoring neurological function. In this study, we leveraged zebrafish, renowned for their exceptional regenerative capabilities, to analyze the pathology of IS and the subsequent recovery process. We induced photothrombosis in the telencephalon utilizing rose bengal and conducted a temporal investigation of changes in cerebral vascular function and learning ability. Our findings revealed that blood flow in young zebrafish was restored approximately 7 days post-IS induction (dpi), with brain function recuperating by 14 dpi. Furthermore, we observed an escalation in the expression of the neural stem marker gene at 3dpi, followed by an upregulation of the differentiated neuron marker at 7 and 14dpi. In the aged IS model, symptoms were exacerbated. While cerebral blood flow was restored in 7 days, similar to young zebrafish, the recovery of learning ability was protracted in aged fish. Moreover, an upregulation of the differentiated neuron marker seen in young fish was not observed in the aged model. Collectively, our analysis of the zebrafish IS model and its comparison with existing rodent models may lay the groundwork for novel IS treatment strategies. Furthermore, the zebrafish IS model may prove beneficial for analyzing the impact of aging on the pathology of IS and the recovery process.