Transgenic Zebrafish Model Research Articles

An antitumor arabinan from Glehnia littoralis activates immunity and inhibits angiogenesis

Glehnia littoralis is an edible plant with significant medicinal value. To further elucidate the potential functional components for developing antitumor agents or functional foods, the polysaccharides in this plant were investigated, and a homogeneous polysaccharide, GLP90-2, was obtained through extraction and ethanol precipitation. By employing methylation, GC–MS, FT-IR, and NMR analysis, GLP90-2 was identified as an arabinan having a molecular weight of 7.76 × 103 g/mol and consisting of three types of residues: α-l-Araf-(1→, →5)-α-l-Araf-(1→, and →3,5)-α-l-Araf-(1→. The subsequent functional analysis revealed that GLP90-2 suppressed tumor development and metastasis in a zebrafish model. Mechanistic studies have shown that GLP90-2 promoted the maturation of DC2.4 cells and macrophages and enhanced the expression of immune-related cytokines, which may be attributed to the interaction between GLP90-2 and TLR-4. Additionally, GLP90-2 exhibited a strong interaction with PD-1, contributing to the activation of immunity. Furthermore, GLP90-2 suppressed angiogenesis in the transgenic zebrafish model, and this impact may be ascribed to the modulation of the VEGF/VEGFR-2 signaling pathway. All the results indicate that GLP90-2 demonstrates a strong tumor immunotherapy effect in vivo and has high potential for development.

SLAM-ITseq identifies that Nrf2 induces liver regeneration through the pentose phosphate pathway

The liver exhibits a remarkable capacity to regenerate following injury. Despite this unique attribute, toxic injury is a leading cause of liver failure. The temporal processes by which the liver senses injury and initiates regeneration remain unclear. Here, we developed a transgenic zebrafish model wherein hepatocyte-specific expression of uracil phosphoribosyltransferase (UPRT) enabled the implementation of SLAM-ITseq to investigate the nascent transcriptome during initiation of liver injury and regeneration. Using this approach, we identified a rapid metabolic transition from the fed to the fasted state that was followed by induction of the nuclear erythroid 2-related factor (Nrf2) antioxidant program. We find that activation of Nrf2 in hepatocytes is required to induce the pentose phosphate pathway (PPP) and improve survival following liver injury. Mechanistically, we demonstrate that inhibition of the PPP disrupts nucleotide biosynthesis to prevent liver regeneration. Together, these studies provide fundamental insights into the mechanism by which early metabolic adaptation to injury facilitates tissue regeneration.

Cost-effective strategies to knock down genes of interest in the retinas of adult zebrafish.

High throughput sequencing has generated an enormous amount of information about the genes expressed in various cell types and tissues throughout the body, and about how gene expression changes over time and in diseased conditions. This knowledge has made targeted gene knockdowns an important tool in screening and identifying the roles of genes that are differentially expressed among specific cells of interest. While many approaches are available and optimized in mammalian models, there are still several limitations in the zebrafish model. In this article, we describe two approaches to target specific genes in the retina for knockdown: cell-penetrating, translation-blocking Vivo-Morpholino oligonucleotides and commercially available lipid nanoparticle reagents to deliver siRNA. We targeted expression of the PCNA gene in the retina of a P23H rhodopsin transgenic zebrafish model, in which rapidly proliferating progenitor cells replace degenerated rod photoreceptors. Retinas collected 48 h after intravitreal injections in adult zebrafish reveal that both Vivo-Morpholinos and lipid encapsulated siRNAs were able to successfully knock down expression of PCNA. However, only retinas injected with Vivo-Morpholinos showed a significant decrease in the formation of P23H rhodopsin-expressing rods, a downstream effect of PCNA inhibition. Surprisingly, Vivo-Morpholinos were able to exit the injected eye and enter the contralateral non-injected eye to inhibit PCNA expression. In this article we describe the techniques, concentrations, and considerations we found necessary to successfully target and inhibit genes through Vivo-Morpholinos and lipid encapsulated siRNAs.

Modification of a natural diterpene and its antitumor mechanism: Promoting apoptosis, suppressing migration, and inhibiting angiogenesis

The synthesis or derivation of a series of structural analogues based on natural products is a common strategy for discovering antitumor drugs. As a unique natural diterpene derived from Trigonostemon howii, 3,4-seco-sonderianol (1) exhibited moderate cytotoxic effects. To improve the activity, a new diterpene derivative (1b) with a mitochondrial targeting function was synthesized by coupling triphenylphosphine to compound 1. Compared to the parent molecule, the antitumor activity of 1b increased greatly. A series of mechanistic experiments revealed that compound 1b induced cancer cell apoptosis and inhibited cancer cell migration by targeting the mitochondria and regulating the STAT3 and FAK signaling pathways. Meanwhile, 1b was found to inhibit angiogenesis in a transgenic zebrafish model. It is worth noting that 1b also demonstrated excellent antitumor effects in zebrafish tumor xenotransplantation models. All the evidence supports that compound 1b targeting mitochondria has the potential to be a candidate for an antitumor drug.

Calotropis procera: A double edged sword against glioblastoma, inhibiting glioblastoma cell line growth by targeting histone deacetylases (HDAC) and angiogenesis

Despite substantial investments in anti-glioblastoma (GBM) drug discovery over the last decade, progress is limited to preclinical stages, with clinical studies frequently encountering obstacles. Angiogenic and histone deacetylase inhibitors (HDACi) have shown profound results in pre-clinical studies. Investigating a multicomponent anti-cancer remedy that disrupts the tumor angiogenic blood vessels and simultaneously disrupts HDACs, while inducing minimal side effects, is critically needed. The crude extracts derived from medicinal plants serve as a renewable reservoir of anti-tumor drugs, exhibiting reduced toxicity compared to chemically synthesized formulations. Calotropis procera is a traditional medicinal plant, and its anticancer potential against many cancer cell lines has been reported, however its antiangiogenic and HDAC inhibitory action is largely unknown. The anticancer activity of methanol leaf extract of C. procera was tested in three types of human glioblastoma cell lines. Wild-type and transgenic zebrafish embryos were used to evaluate developmental toxicity and angiogenic activity. A human angiogenic antibody array was used to profile angiogenic proteins in the U251 GM cell line. A real-time reverse transcriptase polymerase chain reaction (RT PCR) assay was used to detect the differential expression of eleven HDAC genes in U251 cells treated with C. procera extract. The extract significantly reduced the proliferation of all three types of GBM cell lines and the cytotoxicity was found to be more pronounced in U251 GM cells, with an IC50 value of 2.63 ± 0.23 μg/ml, possibly by arresting the cell cycle at the G2/M transition. The extract did not exhibit toxic effects in zebrafish embryos, even at concentrations as high as 1000 μg/ml. The extract also inhibited angiogenic blood vessel formation in the transgenic zebrafish model in a dose-dependent manner. The results from the angiogenic antibody array have suggested novel angiogenesis targets that can be utilized to treat GBM. Real-time RT PCR analysis has shown that C. procrea extract caused an upregulation of HDAC5, 7, and 10, while the mRNA of HDAC1, 2, 3 and 8 (Class I HDACs), and HDAC4, 6, and 9 (Class II) were downregulated in U251 GM cells. The cytotoxicity of the C. procera extract on GBM cell lines could be due to its dual action by regulation of both tumor angiogenesis and histone deacetylases enzymes. Through this study, the C. procera leaf extract has been suggested as an effective remedy to treat GBM with minimal toxicity. In addition, various novel angiogenic and HDAC targets has been identified which could be helpful in designing better therapeutic strategies to manage glioblastoma multiforme in human patients.

Effects of exposure to 3,6-DBCZ on neurotoxicity and AhR pathway during early life stages of zebrafish (Danio rerio)

Polyhalogenated carbazoles (PHCZs) are emerging environmental pollutants, yet limited information is available on their embryotoxicity and neurotoxicity. Therefore, the current work was performed to investigate the adverse effects of 3,6-dibromocarbazole (3,6-DBCZ), a typical PHCZs homolog, on the early life stages of zebrafish larvae. It revealed that the 96-hour post-fertilization (hpf) median lethal concentration (LC50) value of 3,6-DBCZ in zebrafish larvae was determined to be 0.7988 mg/L. Besides, 3,6-DBCZ reduced survival rates at concentrations ≥ 1 mg/L and decreased hatching rates at ≥ 0.25 mg/L at 48 hpf. In behavior tests, it inhibited locomotor activities and reduced the frequency of recorded acceleration states in response to optesthesia (a sudden bright light stimulus) at concentrations ≥ 160 μg/L. Meanwhile, 3,6-DBCZ exposure decreased the frequency of recorded acceleration states in the startle response (tapping mode) at concentrations ≥ 6.4 μg/L. Pathologically, with the transgenic zebrafish model (hb9-eGFP), we observed a strikingly decreased axon length and number in motor neurons after 3,6-DBCZ treatment, which may be ascribed to the activation of the AhR signaling pathway, as evidenced by the molecular docking analysis and Microscale thermophoresis (MST) assay suggested that 3,6-DBCZ binding to AhR-ARNT2 compound proteins. Through interaction with AhR-ARNT, a striking reduction of the anti-oxidative stress (sod1/2, nqo1, nrf2) and neurodevelopment-related genes (elavl3, gfap, mbp, syn2a) were observed after 3,6-DBCZ challenge, accompanied by a marked increased inflammatory genes (TNFβ, IL1β, IL6). Collectively, our findings reveal a previously unrecognized adverse effect of 3,6-DBCZ on zebrafish neurodevelopment and locomotor behaviors, potentially mediated through the activation of the AhR pathway. Furthermore, it provides direct evidence for the toxic concentrations of 3,6-DBCZ and the potential target signaling in zebrafish larvae, which may be beneficial for the risk assessment of the aquatic ecosystems.

Generation of a Transgenic Zebrafish Model for Pancreatic Beta Cell Regeneration

Background: Diabetes is a major worldwide health problem. It is widely accepted that the beta cell mass decreases in type I diabetes (T1D). Accordingly, beta cell regeneration is a promising approach to increase the beta cell mass in T1D patients. However, the underlying mechanisms of beta cell regeneration have yet to be elucidated. One promising avenue is to create a relevant animal model to explore the underlying molecular and cellular mechanisms of beta cell regeneration. The zebrafish can be considered a model in beta cell regeneration studies because the pancreas structure and gene expression pattern are highly conserved between human and zebrafish. Materials and Methods: In this study, the Tol2 transposase was exploited to generate a Tg(Ins:egfp-nfsB) zebrafish model that expressed a fusion protein composed of enhanced green fluorescent protein (EGFP) and nitroreductase (NTR) under control of the Ins promoter. Results: Metronidazole (MTZ) treatment of Tg(ins:egfp-nfsB) zebrafish larvae led to selective ablation of beta cells. Proof-of-concept evidence for beta cell regeneration in the transgenic larvae was observed two days after withdrawal of MTZ. Conclusion: This study suggests that the Tg(ins:egfp-nfsB) zebrafish can be used as a disease model to study beta cell regeneration and elucidate underlying mechanisms during the regeneration process. [GMJ.2019;8:e1056]

Antitumor Effect of Berberine Analogs in a Canine Mammary Tumor Cell Line and in Zebrafish Reporters via Wnt/β-Catenin and Hippo Pathways.

The heterogeneous nature of human breast cancer (HBC) can still lead to therapy inefficacy and high lethality, and new therapeutics as well as new spontaneous animal models are needed to benefit translational HBC research. Dogs are primarily investigated since they spontaneously develop tumors that share many features with human cancers. In recent years, different natural phytochemicals including berberine, a plant alkaloid, have been reported to have antiproliferative activity in vitro in human cancers and rodent animal models. In this study, we report the antiproliferative activity and mechanism of action of berberine, its active metabolite berberrubine, and eight analogs, on a canine mammary carcinoma cell line and in transgenic zebrafish models. We demonstrate both in vitro and in vivo the significant effects of specific analogs on cell viability via the induction of apoptosis, also identifying their role in inhibiting the Wnt/β-catenin pathway and activating the Hippo signals with a downstream reduction in CTGF expression. In particular, the berberine analogs NAX035 and NAX057 show the highest therapeutic efficacy, deserving further analyses to elucidate their mechanism of action more in detail, and in vivo studies on spontaneous neoplastic diseases are needed, aiming at improving veterinary treatments of cancer as well as translational cancer research.

A novel ERβ high throughput microscopy platform for testing endocrine disrupting chemicals

In this study we present an inducible biosensor model for the Estrogen Receptor Beta (ERβ), GFP-ERβ:PRL-HeLa, a single-cell-based high throughput (HT) in vitro assay that allows direct visualization and measurement of GFP-tagged ERβ binding to ER-specific DNA response elements (EREs), ERβ-induced chromatin remodeling, and monitor transcriptional alterations via mRNA fluorescence in situ hybridization for a prolactin (PRL)-dsRED2 reporter gene. The model was used to accurately (Z’ = 0.58–0.8) differentiate ERβ-selective ligands from ERα ligands when treated with a panel of selective agonists and antagonists. Next, we tested an Environmental Protection Agency (EPA)-provided set of 45 estrogenic reference chemicals with known ERα in vivo activity and identified several that activated ERβ as well, with varying sensitivity, including a subset that is completely novel. We then used an orthogonal ERE-containing transgenic zebrafish (ZF) model to cross validate ERβ and ERα selective activities at the organism level. Using this environmentally relevant ZF assay, some compounds were confirmed to have ERβ activity, validating the GFP-ERβ:PRL-HeLa assay as a screening tool for potential ERβ active endocrine disruptors (EDCs). These data demonstrate the value of sensitive multiplex mechanistic data gathered by the GFP-ERβ:PRL-HeLa assay coupled with an orthogonal zebrafish model to rapidly identify environmentally relevant ERβ EDCs and improve upon currently available screening tools for this understudied nuclear receptor.

Effusanin B Inhibits Lung Cancer by Prompting Apoptosis and Inhibiting Angiogenesis

Cancer is one of the deadliest human diseases, causing high rates of illness and death. Lung cancer has the highest mortality rate among all malignancies worldwide. Effusanin B, a diterpenoid derived from Isodon serra, showed therapeutic potential in treating non-small-cell lung cancer (NSCLC). Further research on the mechanism indicated that effusanin B inhibited the proliferation and migration of A549 cells both in vivo and in vitro. The in vitro activity assay demonstrated that effusanin B exhibited significant anticancer activity. Effusanin B induced apoptosis, promoted cell cycle arrest, increased the production of reactive oxygen species (ROS), and altered the mitochondrial membrane potential (MMP). Based on mechanistic studies, effusanin B was found to inhibit the proliferation and migration of A549 cells by affecting the signal transducer and activator of transcription 3 (STAT3) and focal adhesion kinase (FAK) pathways. Moreover, effusanin B inhibited tumor growth and spread in a zebrafish xenograft model and demonstrated anti-angiogenic effects in a transgenic zebrafish model.

A metagenomic library cloning strategy that promotes high-level expression of captured genes to enable efficient functional screening

Functional screening of environmental DNA (eDNA) libraries is a potentially powerful approach to discover enzymatic "unknown unknowns", but is usually heavily biased toward the tiny subset of genes preferentially transcribed and translated by the screening strain. We have overcome this by preparing an eDNA library via partial digest with restriction enzyme FatI (cuts CATG), causing a substantial proportion of ATG start codons to be precisely aligned with strong plasmid-encoded promoter and ribosome-binding sequences. Whereas we were unable to select nitroreductases from standard metagenome libraries, our FatI strategy yielded 21 nitroreductases spanning eight different enzyme families, each conferring resistance to the nitro-antibiotic niclosamide and sensitivity to the nitro-prodrug metronidazole. We showed expression could be improved by co-expressing rare tRNAs and encoded proteins purified directly using an embedded His6-tag. In a transgenic zebrafish model of metronidazole-mediated targeted cell ablation, our lead MhqN-family nitroreductase proved ∼5-fold more effective than the canonical nitroreductase NfsB.

SAT066 Leukotriene B4 Receptor 2 Mediates Macrophage Migration To Injured β Cells

Abstract Disclosure: E. Ermis: None. T. Nargis: None. A. Kulkarni: None. S. May: None. S.A. Tersey: None. R.G. Mirmira: None. R.M. Anderson: None. Type 1 diabetes (T1D) is an autoimmune disease, characterized in part by islet inflammation and macrophage infiltration leading to β-cell injury and loss. The inflammatory mediator 12/15-lipoxygenase (12/15-LOX) and its lipid product 12-hydroxyeicosatetraenoic acid (12-HETE) have both been implicated in T1D. 12-HETE activates the low affinity receptor leukotriene B4 receptor 2 (LTB4R2, or simply BLT2), which was reported as a low-affinity receptor for 12-HETE, suggesting that BLT2 may play a proinflammatory role. Here, we used both mouse and zebrafish models to probe the roles of BLT2 in macrophage activation and/or recruitment to pancreatic islets during T1D progression. First, we analyzed Blt2 gene expression in a variety of C57BL/6J mouse tissues. We found Blt2 gene expression in bone marrow derived macrophages and peritoneal macrophages, however we found little to no expression in pancreatic islets. Although Blt2-/- mice showed a significantly reduced expression of Blt2, they exhibited overall normal growth and glucose tolerance. To uncover roles for BLT2 in macrophage function, we first interrogated its involvement in the in vitro polarization of proinflammatory macrophages to the M1-like state using LPS and IFN-γ and the M2-like state using IL-4. Under these conditions, we found no differences between Blt2-/- and wildtype macrophages, as assessed by flow cytometry analysis, suggesting that BLT2 does not play a substantial role in macrophage polarization. Next, to query roles for BLT2 in macrophage migration, we employed the transgenic zebrafish line Tg(mpeg:EGFP) in which macrophages are labeled with green fluorescent protein (GFP). Following zebrafish tailfin injury, we quantified macrophage accumulation at the injury at 6 hours post amputation. Inhibitory treatments of zebrafish larvae with either a chemical BLT2 inhibitor (LY255283) or by knockdown of the blt2a gene using an antisense morpholino, both decreased the number of GFP+ macrophages found at the injury site. Moreover, in the latter approach ectopic expression of blt2a mRNA effectively restored the wild-type phenotype. To determine if BLT2 mediates macrophage migration to injured islets, we employed a transgenic zebrafish model of β-cell injury, Tg(ins:NTR), in which metronidazole treatment rapidly induces β-cell injury. We treated the Tg(mpeg: GFP); Tg(ins: NTR) zebrafish with metronidazole and monitored macrophage migration into injured islets. We observed a significant decrease in macrophage migration towards injured β-cells in blt2a-knockdown zebrafish. Collectively, these results show that BLT2 plays a role in macrophage migration associated with tissue injury, including the islet β cell as seen in T1D. Presentation: Saturday, June 17, 2023

Dimethenamid promotes oxidative stress and apoptosis leading to cardiovascular, hepatic, and pancreatic toxicities in zebrafish embryo

Dimethenamid, one of the acetamide herbicides, is widely used on soybeans and corns to inhibit weed growth. Although other acetamide herbicides have been reported to have several toxicities in non-target organisms including developmental toxicity, the toxicity of dimethenamid has not yet been studied. In this research, we utilized the zebrafish animal model to verify the developmental toxicity of dimethenamid. It not only led to morphological abnormalities in zebrafish larvae but also reduced their viability. ROS production and inflammation responses were promoted in zebrafish larvae. Also, uncontrolled apoptosis occurred when the gene expression level related to the cell cycle and apoptosis was altered by dimethenamid. These changes resulted in toxicities in the cardiovascular system, liver, and pancreas are observed in transgenic zebrafish models including fli1a:EGFP and L-fabp:dsRed;elastase:GFP. Dimethenamid triggered morphological defects in the heart and vasculature by altering the mRNA levels related to cardiovascular development. The liver and pancreas were also damaged through not only the changes of their morphology but also through the dysregulation in their function related to metabolic activity. This study shows the developmental defects induced by dimethenamid in zebrafish larvae and the possibility of toxicity in other non-target organisms.

Dual effect of aucubin on promoting VEGFR2 mediated angiogenesis and reducing RANKL-induced bone resorption

BackgroundAngiogenesis is regarded as a critical role in bone repair and regeneration, involving in pathological bone disorders such as osteoporosis. Aucubin, an iridoid glycoside primarily derived from Eucommia ulmoides, is reported to inhibit osteoclast activity, enhance bone formation and promote angiogenesis in osteoporosis models. Our study is to further investigate the anti-osteoporosis effect of aucubin in transgenic medaka, and the pro-angiogenic effect of aucubin and its mechanism of action both in vivo and in vitro.MethodsThe anti-osteoporosis effect of aucubin was confirmed by using RANKL-stimulated bone resorption transgenic medaka. The pro-angiogenic effect of aucubin in vivo was investigated using vascular endothelial growth factor (VEGF) tyrosine kinase inhibitor II (VRI)-induced vascular insufficient transgenic zebrafish model. Furthermore, endothelial cell proliferation, migration, tube formation and the mechanisms were evaluated to identify the pro-angiogenic effect of aucubin in normal and su5416-injured human umbilical vein endothelial cells (HUVECs).ResultsAucubin decreased the resorption of the mineralized bone matrix and centra degradation in heat-shocked transgenic col10α1:nlGFP/rankl:HSE:CFP medaka. Moreover, aucubin reversed VRI-induced vascular insufficiency in zebrafish through regulating flt1, kdr, kdrl, vegfaa, ang-1, ang-2, tie1 and tie2 mRNA expressions in Tg(fli1a:EGFP)y1 or AB wild type zebrafish. Aucubin promoted cell proliferation by upregulating p-mTOR, p-Src, p-MEK, p-Erk1/2, p-Akt and p-FAK in HUVECs. Furthermore, aucubin exhibited a pro-angiogenic effect on su5416-injured HUVECs by promoting their proliferation, migration, and tube formation through regulating the phosphorylation of VEGFR2, MEK, ERK and the ratio of Bcl2-Bax.ConclusionAucubin could reduce bone resorption in RANKL-induced osteoporosis medaka by live imaging. Meanwhile, aucubin exhibited a protective effect in VRI-induced vascular insufficient zebrafish by regulating VEGF-VEGFR and Ang-Tie signaling pathways. Additionally, aucubin promoted the proliferation, migration and tube formation of HUVECs probably by mediating VEGFR2/MEK/ERK, Akt/mTOR and Src/FAK signalling pathways. This study further indicated the dual effect of aucubin on angiogenesis and osteogenesis which may be beneficial to its treatment of osteoporosis.

Abstract P2176: Development Of Integrated Transparent Transgenic Zebrafish Modeling System For Experimental Cardio-oncology Therapeutics

The zebrafish model has provided an excellent platform to study the genetic and molecular approach of basic and translational cardiac research. Zebrafish heart cells are similar to human heart cells at the molecular level and determine the function of genes that control cardiac function and dysfunction. In zebrafish, myl7 is myosin light chain-7 gene and identified as a regulatory gene of heart orthologs to human MYL7. In vertebrates, including zebrafish, murine and human systems, the in-vivo spatial resolution is limited due to the normal opacification of skin and subdermal structures. For in-vivo imaging the skin transparency is primary requirement and to maintain the transparency, blocking the pigmentation needs to maintain. Chemical inhibition treatment is temporary and possible till the organism treated with the chemical inhibitor agent. Zebrafish casper mutant maintain transparency throughout the life and serve as ideal combination of sensitivity and resolution for in-vivo stem cell analyses and in-vivo imaging. To maintain the skin transparency, in this study, we have developed transparent transgenic zebrafish model and established of time lapse in-vivo confocal microscopy to study of cellular phenotype/pathologies of the cardiomyocytes to quantify changes in cardiomyocyte morphology and function overtime by comparing control and cardiac injury by generating casper/ myl7:RFP; annexin-5:YFP transgenic zebrafish. In the field of cardio-oncology, we are developing in-vivo transparent transgenic cellular phenotype model of zebrafish to study of the cell death process (Annexin5) and Inflammatory activity (NFkB) in the microglia, and the resident macrophage of the brain and cardiomyocytes) homozygous casper/ myl7:RFP; annexin-5:YFP/NF-kB:GFP/mpeg1:mcherryFP transgenic strain zebrafish. By using this novel model our lab establishing the role of NFkB and microglia function under normal healthy status and in glioblastomas Our strategist approach to yield crucial new insights into in-vivo cardiomyocyte imaging by confocal microscopy to observe and track the cell death pattern and cardio inflammatory pathways in cardiomyocyte and develop novel therapeutic approaches to treat cardio inflammatory pathology and glioblastoma

Identification of an adverse outcome pathway (AOP) for chemical-induced craniofacial anomalies using the transgenic zebrafish model.

Craniofacial anomalies are one of the most frequent birth defects worldwide and are often caused by genetic and environmental factors such as pharmaceuticals and chemical agents. Although identifying adverse outcome pathways (AOPs) is a central issue for evaluating the teratogenicity, the AOP causing craniofacial anomalies has not been identified. Recently, zebrafish has gained interest as an emerging model for predicting teratogenicity because of high throughput, cost-effectiveness and availability of various tools for examining teratogenic mechanisms. Here, we established zebrafish sox10-EGFP reporter lines to visualize cranial neural crest cells (CNCCs) and have identified the AOPs for craniofacial anomalies. When we exposed the transgenic embryos to teratogens that were reported to cause craniofacial anomalies in mammals, CNCC migration and subsequent morphogenesis of the first pharyngeal arch were impaired at 24 hours post-fertilization. We also found that cell proliferation and apoptosis of the migratory CNCCs were disturbed, which would be key events of the AOP. From these results, we propose that our sox10-EGFP reporter lines serve as a valuable model for detecting craniofacial skeletal abnormalities, from early to late developmental stages. Given that the developmental process of CNCCs around this stage is highly conserved between zebrafish and mammals, our findings can be extrapolated to mammalian craniofacial development and thus help in predicting craniofacial anomalies in human.

Investigation of Kelussia Odoratissima and Angelica Sinensis Similarities in Zebrafish-based In-vivo Bioactivity Assays and Their Chemical Composition.

Kelussia odoratissima and Angelica sinensis are two medicinal plants commonly used in Iran and China, respectively. They have been used in their indigenous traditional medicine, for various diseases including, blood refining, inflammation, cold, flu, stress, cardiovascular diseases, and nervous disorders. This study was conducted to evaluate the volatile oil composition of K. odoratissima leaves (KVL) and A. sinensis root (AVR); we also examined the biological activity of essential oils (EOs) and hydroalcoholic extracts of both plants using two different transgenic zebrafish (Danio rerio) models: angiogenesis and pancreatic beta cell (pBC) regeneration models. Both EOs were isolated by hydrodistillation and analysed by GC and GC/MS. For viability tests, larvae were treated with different concentrations of extracts to determine an appropriate starting concentration. Hydroalcoholic extracts and EOs have been tested in a dose-dependent manner for their biological activity using tissue-specific transgenic zebrafish Tg(fli-1: EGFP) and Tg (ins: GFP-NTR) embryos and larvae. One-way ANOVA was used to compare the mean of pBC area and intersegmental vessels (ISVs) outgrowth between the treatment groups. Eleven compounds were in common to both oils, comprising 51.3% of KVL and 61.7% of AVR, of which 39.3% in KVL and 37.6% in AVR were phthalide structures. Results revealed that both EOs blocked ISVs formation in the Tg (fli-1: EGFP) embryos increased to 10% of the control value, while both hydroalcoholic extracts did not show any anti-angiogenesis effects in these embryos. In addition, AVR has been shown to significantly induce PBC regeneration following ablation in the Tg (ins: GFP-NTR), but its regenerative activity was lower than that of 5'-N-ethylcarboxamidoadenosine (NECA) as a positive control. Taken together, the anti-angiogenesis activity of both EOs could be attributed to the phthalide structures while for the PBC regenerative activity, other compounds including β-Thujaplicinol, exclusively existing in AVR, might be effective. Although the genera, organs, and origin of these plants are different, their similar chemical composition and biological activities make them valuable resources for further investigation in basic medical and pharmaceutical science.

Terbutryn causes developmental toxicity in zebrafish (Danio rerio) via apoptosis and major organ malformation in the early stages of embryogenesis

Terbutryn (2-(ethylamino)-4-(tert-butylamino)-6-(methylthio)-1,3,5-triazine) is a substituted symmetrical triazine herbicide used in agricultural fields to prevent undesired vegetation growth by inhibiting photosynthesis in target weeds. Although terbutryn has various benefits, long-term exposure, misuse, or abuse of terbutryn may cause non-target toxicity and severe ecosystem pollution. To provide a detailed description of the embryonic developmental toxicity of terbutryn, zebrafish (Danio rerio) were exposed to 2, 4, and 6 mg/L of terbutryn and the morphological changes, pathological abnormalities, and developmental endpoints were assessed relative to that of a solvent control. The results showed that terbutryn induces a loss of survivability, reduction in body and eye size, and edema in the yolk sac. Through fluorescence microscopy, blood vessels, motor neurons, and liver development were investigated using transgenic zebrafish models based on fluorescently tagged genes (fllk1:eGFP, olig2:dsRed, and L-fabp:dsRed). Furthermore, cell death by apoptosis in zebrafish caused by terbutryn exposure was evaluated via acridine orange staining, which is a selective fluorescent staining agent. To support the preceding results, gene expression alterations caused by terbutryn exposure in zebrafish larvae were assessed. The overall results indicate that exposure to terbutryn induces apoptosis and disrupts organ development. These embryonic developmental toxicity results suggest that terbutryn should be applied in the right areas at the appropriate rates, concentrations, and quantities.

Fluchloralin induces developmental toxicity in heart, liver, and nervous system during early zebrafish embryogenesis

The zebrafish is a prominent vertebrate model popularly used for toxicity testing because of its rapid development and transparent embryos. Fluchloralin, a dinitroaniline herbicide used to control weeds, inhibits microtubule formation and cell division. The structurally homologous substances ethalfluralin and pendimethalin, which belong to the dinitroaniline family, were found to be genotoxic and to exert developmental toxicity via mitochondrial dysfunction in a zebrafish model. To date, developmental toxicity of fluchloralin in zebrafish has not been reported. In the present study, we identified morphological changes in developing zebrafish, including decreased survival rate and body length, and increased yolk sac edema. In dose-dependent response to fluchloralin exposure, inhibition of neurogenesis in the spinal cord and motor neuron defects were observed in transgenic zebrafish models (olig2:dsRed). Zebrafish exposed to fluchloralin also displayed organ dysfunction in the heart, liver, and pancreas in cmlc2:dsRed and lfabp:dsRed;elastase:GFP transgenic models. Fluchloralin increased cell death in the brain by promoting apoptosis, visualized via acridine orange staining, and by activating apoptosis signaling proteins, including cytochrome c1, zBax, and Bcl-XL. This study provides novel evidence supporting the necessity of controlling pollutants in aquatic environments.

Developmental defects induced by thiabendazole are mediated via apoptosis, oxidative stress and alteration in PI3K/Akt and MAPK pathways in zebrafish

Thiabendazole, a benzimidazole fungicide, is widely used to prevent yield loss in agricultural land by inhibiting plant diseases derived from fungi. As thiabendazole has a stable benzimidazole ring structure, it remains in the environment for an extended period, and its toxic effects on non-target organisms have been reported, indicating the possibility that it could threaten public health. However, little research has been conducted to elucidate the comprehensive mechanisms of its developmental toxicity. Therefore, we used zebrafish, a representative toxicological model that can predict toxicity in aquatic organisms and mammals, to demonstrate the developmental toxicity of thiabendazole. Various morphological malformations were observed, including decreased body length, eye size, and increased heart and yolk sac edema. Apoptosis, reactive oxygen species (ROS) production, and inflammatory response were also triggered by thiabendazole exposure in zebrafish larvae. Furthermore, PI3K/Akt and MAPK signaling pathways important for appropriate organogenesis were significantly changed by thiabendazole. These results led to toxicity in various organs and a reduction in the expression of related genes, including cardiovascular toxicity, neurotoxicity, and hepatic and pancreatic toxicity, which were detected in flk1:eGFP, olig2:dsRED, and L-fabp:dsRed;elastase:GFP transgenic zebrafish models, respectively. Overall, this study partly determined the developmental toxicity of thiabendazole in zebrafish and provided evidence of the environmental hazards of this fungicide.