Early Zebrafish Development Research Articles

Gene profiling of head mesoderm in early zebrafish development: insights into the evolution of cranial mesoderm

BackgroundThe evolution of the head was one of the key events that marked the transition from invertebrates to vertebrates. With the emergence of structures such as eyes and jaws, vertebrates evolved an active and predatory life style and radiated into diversity of large-bodied animals. These organs are moved by cranial muscles that derive embryologically from head mesoderm. Compared with other embryonic components of the head, such as placodes and cranial neural crest cells, our understanding of cranial mesoderm is limited and is restricted to few species.ResultsHere, we report the expression patterns of key genes in zebrafish head mesoderm at very early developmental stages. Apart from a basic anterior–posterior axis marked by a combination of pitx2 and tbx1 expression, we find that most gene expression patterns are poorly conserved between zebrafish and chick, suggesting fewer developmental constraints imposed than in trunk mesoderm. Interestingly, the gene expression patterns clearly show the early establishment of medial–lateral compartmentalisation in zebrafish head mesoderm, comprising a wide medial zone flanked by two narrower strips.ConclusionsIn zebrafish head mesoderm, there is no clear molecular regionalisation along the anteroposterior axis as previously reported in chick embryos. In contrast, the medial–lateral regionalisation is formed at early developmental stages. These patterns correspond to the distinction between paraxial mesoderm and lateral plate mesoderm in the trunk, suggesting a common groundplan for patterning head and trunk mesoderm. By comparison of these expression patterns to that of amphioxus homologues, we argue for an evolutionary link between zebrafish head mesoderm and amphioxus anteriormost somites.

The combined effects of graphene oxide and bisphenol A on oxidative damage in early development of zebrafish

Bisphenol A (BPA) is widely present in natural surface water, and was found to be at the high level of μg/L in the surface water around a BPA manufacturing plant. Mass production and application make BPA ubiquitous in the environment, biota and humans. As a two-dimensional carbon based nanomaterial, graphene oxide (GO) is used in many fields, and there is a great potential for GO to discharge to aquatic environment via various pathways. Thus GO has the potential to interact with organic pollutants, such as BPA, copresent in water system. GO could adsorb organic pollutants in water, and adhere on the surface of organisms, displaying different effects on the behaviors and toxicities of organic pollutants in organisms at different developmental stages. But the intrinsic mechanisms for the different effects remain unclear. To fill this gap, zebrafish ( Danio rerio ) was selected as a vertebrate model to investigate the toxicities of a variety of pollutants and nanomaterials in this study. Embryogenesis is an important growth phase in the zebrafish life cycle. Pollutants can induce excessive reactive oxygen species (ROS) and generate a large number of oxidative intermediate products by destroying the balance between oxidation and antioxidation, thus causing oxidative damage. Superoxide dismutase (SOD) can catalyze the oxidation of superoxide anion (O2−) to O2 and H2O, in an attempt to maintain ROS at a physiological level and protect the body from damage. Catalase (CAT) exists primarily within peroxisomes and removes the H2O2 generated by the long-chain fatty acid β-oxidation pathway. Excessive ROS can attack the polyunsaturated fatty acids in the biomembrane system, resulting in lipid peroxidation. Malondialdehyde (MDA) is the product of irreversible lipid peroxidation, and the change of MDA can reflect the level and damage degree of lipid peroxidation in organisms. Glutathione (GSH), as an important non-enzymatic antioxidant in organism, can be induced and effectively scavenged by external stimulation to regulate oxidative stress. Toxic effects occur in organisms when GSH is gradually reduced. This study focused on the joint effects of GO and BPA on the oxidative responses in zebrafish at early development stage. Zebrafish embryos were individually exposed to BPA (0, 50, 500 µg/L), GO (0, 0.1, 1 mg/L), BPA+GO (50 µg/L+ 0.1 mg/L, 500 µg/L+ 0.1 mg/L, and 50 µg/L +1 mg/L, 500 µg/L +1 mg/L) solutions for 7 d. The levels of SOD, MDA, GSH, CAT, and ROS in the exposed zebrafish larvae were determined. The results indicated that BPA displayed strong oxidative damage effects on zebrafish larvae. However, the presence of GO distinctly alleviated the various indicators of oxidative damages caused by BPA. Based on analyses using the Bliss independence model, GO and BPA exhibited antagonistic effects on the oxidative damage of zebrafish in the early development stage, which indicated that GO could reduce the oxidative damage toxicities of BPA on zebrafish embryos under the experimental conditions. These could be due to the formation of a GO layer on the zebrafish embryos, which depressed the absorption of BPA by zebrafish, leading to reduced BPA accumulation in zebrafish in early developmental stage.

Cross Talk without Cross Tolerance: Effect of Rearing Temperature on the Hypoxia Response of Embryonic Zebrafish.

Environmental stressors, such as warm temperatures and hypoxia, can interact and pose a threat to aquatic species. Cross talk between the hypoxia and heat stress cellular pathways can lead to enhanced cross tolerance between these environmental stressors. In this study, we questioned whether elevated temperatures (from 27° to 32°C) during rearing would enhance the hypoxia-inducible transcription factor-1 (HIF-1)-mediated transcriptional response to hypoxia (5% dissolved O2) in early stages of zebrafish development and whether these differences would be associated with enhanced larval tolerance and survival to hypoxia. We found that embryos reared at 32°C had an enhanced cellular HIF-1 response (elevated hif-1ab and insulin-like growth factor binding-protein mRNA level) and that acute hypoxia (4 h) activated the heat-shock response (elevated hsp70a and hsp90aa mRNA levels). Elevated rearing temperatures and hypoxia exposure also induced precocious hatching, but neither environmental stressor had an effect on the hypoxia tolerance (critical O2 tension) of 4-d-old larvae and did not protect larvae against the lethal effects of a second acute hypoxia exposure. Overall, during early zebrafish development, cross talk between the hypoxia and heat stress cellular pathways at the gene expression level did not confer cross tolerance at the whole-animal level with respect to hypoxia stress.

Vitamin D Receptor Signaling Regulates Craniofacial Cartilage Development in Zebrafish.

Vitamin D plays essential roles in supporting the skeletal system. The active form of vitamin D functions through the vitamin D receptor (VDR). A hereditary vitamin-D-resistant rickets with facial dysmorphism has been reported, but the involvement of VDR signaling during early stages of craniofacial development remains to be elucidated. The present study investigated whether VDR signaling is implicated in zebrafish craniofacial cartilage development using a morpholino-based knockdown approach. Two paralogous VDR genes, vdra and vdrb, have been found in zebrafish embryos. Loss-of-vdra has no discernible effect on cartilage elements, whereas loss-of-vdrb causes reduction and malformation of craniofacial cartilages. Disrupting both vdra and vdrb leads to more severe defects or complete loss of cartilage. Notably, knockdown of vdrb results in elevated expression of follistatin a (fsta), a bone morphogenetic protein (BMP) antagonist, in the adjacent pharyngeal endoderm. Taken together, these findings strongly indicate that VDR signaling is required for early craniofacial cartilage development in zebrafish.

How glyphosate and its associated acidity affect early development in zebrafish (Danio rerio).

BackgroundGlyphosate is among the most extensively used pesticides worldwide. Following the ongoing highly controversial debate on this compound, its potential impact on non-target organisms is a fundamental scientific issue. In its pure compound form, glyphosate is known for its acidic properties.MethodsWe exposed zebrafish (Danio rerio) embryos to concentrations between 10 μM and 10 mM glyphosate in an unbuffered aqueous medium, as well as at pH 7, for 96 hours post fertilization (hpf). Furthermore, we investigated the effects of aqueous media in the range of pH 3 to 8, in comparison with 1 mM glyphosate treatment at the respective pH levels. Additionally, we exposed zebrafish to 7-deoxy-sedoheptulose (7dSh), another substance that interferes with the shikimate pathway by a mechanism analogous to that of glyphosate, at a concentration of one mM. The observed endpoints included mortality, the hatching rate, developmental delays at 24 hpf, the heart rate at 48 hpf and the malformation rate at 96 hpf. LC10/50, EC10 and, if reasonable, EC50 values were determined for unbuffered glyphosate.ResultsThe results revealed high mortalities in all treatments associated with low pH, including high concentrations of unbuffered glyphosate (>500 μM), low pH controls and glyphosate treatments with pH < 3.4. Sublethal endpoints like developmental delays and malformations occurred mainly at higher concentrations of unbuffered glyphosate. In contrast, effects on the hatching rate became particularly prominent in treatments at pH 7, showing that glyphosate significantly accelerates hatching compared with the control and 7dSh, even at the lowest tested concentration. Glyphosate also affected the heart rate, resulting in alterations both at pH 7 and, even more pronounced, in the unbuffered system. In higher concentrations, glyphosate tended to accelerate the heart rate in zebrafish embryos, again, when not masked by the decelerating influence of its low pH. At pH > 4, no mortality occurred, neither in the control nor in glyphosate treatments. At 1 mM, 7dSh did not induce any mortality, developmental delays or malformations; only slightly accelerated hatching and a decelerated heart rate were observed. Our results demonstrate that lethal impacts in zebrafish embryos can be attributed mainly to low pH, but we could also show a pH-independent effect of glyphosate on the development of zebrafish embryos on a sublethal level.

The role of endothelial cilia in postembryonic vascular development.

Cilia are essential for morphogenesis and maintenance of many tissues. Loss-of-function of cilia in early Zebrafish development causes a range of vascular defects, including cerebral hemorrhage and reduced arterial vascular mural cell coverage. In contrast, loss of endothelial cilia in mice has little effect on vascular development. We therefore used a conditional rescue approach to induce endothelial cilia ablation after early embryonic development and examined the effect on vascular development and mural cell development in postembryonic, juvenile, and adult Zebrafish. ift54(elipsa)-mutant Zebrafish are unable to form cilia. We rescued cilia formation and ameliorated the phenotype of ift54 mutants using a novel Tg(ubi:loxP-ift54-loxP-myr-mcherry,myl7:EGFP)sh488 transgene expressing wild-type ift54 flanked by recombinase sites, then used a Tg(kdrl:cre)s898 transgene to induce endothelial-specific inactivation of ift54 at postembryonic ages. Fish without endothelial ift54 function could survive to adulthood and exhibited no vascular defects. Endothelial inactivation of ift54 did not affect development of tagln-positive vascular mural cells around either the aorta or the caudal fin vessels, or formation of vessels after tail fin resection in adult animals. Endothelial cilia are not essential for development and remodeling of the vasculature in juvenile and adult Zebrafish when inactivated after embryogenesis.

Metabolomics profiling of haloperidol and validation of thromboxane-related signaling in the early development of zebrafish

Haloperidol is a common butyrophenone-derivative antipsychotic drug that is used clinically to treat schizophrenia and to control Tourette disorder. Haloperidol has been shown to be an embryonic toxicant and to cause a variety of adverse effects that affect human embryonic development. However, the pathway impaired by haloperidol during the developmental stages remains unclear. To elucidate the innate toxicological pathway of haloperidol, we investigated the lethality of haloperidol during the embryonic development of zebrafish. We observed that haloperidol caused serious morphological changes, with an LD50 of 9.7 x 10−6 ± 2.4 x 10−6 μg/L. Next, we established a systematic approach to perform metabolite profiling in embryonic zebrafish with various concentrations of haloperidol and analyzed the metabolites using ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF MS). A total of 304 metabolites were identified and 86 metabolites were chosen to predict potential pathways. Among the metabolites, we found through prediction that numerous metabolomics-biological pathways are associated with haloperidol, including peroxisome-proliferator-activated receptor (ppar), thromboxane, and mTOR signaling. Quantitative real time-qPCR was then used to validate the gene expression potentially associated with the thromboxane, which is a metabolic product of arachidonic acid and considered to be important for cell proliferation and the inflammatory response. To sum up, analysis of metabolites in the zebrafish model provides a system for mining biomarkers that reflect biological significance and highlight the therapeutic potency in humans. In addition, it may show potential for application to other pharmaceuticals to identify their various activities and clarify functional mechanisms in the future.

Packaging development: how chromatin controls transcription in zebrafish embryogenesis.

How developmental gene expression is activated, co-ordinated and maintained is one of the biggest questions in developmental biology. While transcription factors lead the way in directing developmental gene expression, their accessibility to the correct repertoire of genes can depend on other factors such as DNA methylation, the presence of particular histone variants and post-translational modifications of histones. Collectively, factors that modify DNA or affect its packaging and accessibility contribute to a chromatin landscape that helps to control the timely expression of developmental genes. Zebrafish, perhaps better known for their strength as a model of embryology and organogenesis during development, are coming to the fore as a powerful model for interpreting the role played by chromatin in gene expression. Several recent advances have shown that zebrafish exhibit both similarities and differences to other models (and humans) in the way that they employ chromatin mechanisms of gene regulation. Here, I review how chromatin influences developmental transcriptional programmes during early zebrafish development, patterning and organogenesis. Lastly, I briefly highlight the importance of zebrafish chromatin research towards the understanding of human disease and transgenerational inheritance.

Knock-Down of Specific Thyroid Hormone Receptor Isoforms Impairs Body Plan Development in Zebrafish.

The role of thyroid hormones (THs) in development has been extensively studied, however, the specific molecular mechanisms involved are far from being clear. THs act by binding to TH nuclear receptors (TR) that act as ligand-dependent transcription factors to regulate TH-dependent gene expression. Like vertebrates, zebrafish express different isoforms of functional Tr alpha and beta, some of which can bind alternative ligands like 3,5-T2. In this study, we first analyzed the effects of exogenous T3 and 3,5-T2 exposure during embryogenesis. The percentage of affected embryos was similar to those vehicle-injected, suggesting that the early exposure to low TH levels is not sufficient to elicit effects upon the phenotype of the embryo. We then generated crispants for four isoforms of thr to learn more about the role of these receptors in early development. We found that crispant larvae from thraa and a newly identified l-thrb+, but not thrab and canonical thrb1 showed profound deleterious effects upon symmetry and laterality, suggesting early novel roles for these Tr isoforms in the body plan developmental program. Since critical events that determine cell fate start in the late gastrula, we tested if some genes that are expressed during early developmental stages could indeed be TH targets. We identify early development genes, like sox10 and eve, that were specifically over-expressed in thraa and l-thrb+ crispants, suggesting that these specific thr isoforms function as transcription repressors for these genes, while transcription of zic and ets appear to be thraa and l-thrb+-mediated, respectively. Overall, present results show that TH signaling participates in early zebrafish development and identify Tr isoform-specific mediated regulation of early gene expression.

Cellular responses to ionizing radiation change quickly over time during early development in zebrafish.

Animal cells constantly receive information about and respond to environmental factors, including ionizing radiation. Although it is crucial for a cell to repair radiation‐induced DNA damage to ensure survival, cellular responses to radiation exposure during early embryonic development remain unclear. In this study, we analyzed the effects of ionizing radiation in zebrafish embryos and found that radiation‐induced γH2AX foci formation and cell cycle arrest did not occur until the gastrula stage, despite the presence of major DNA repair‐related gene transcripts, passed on as maternal factors. Interestingly, P21/WAF1 accumulation began ∼6 h post‐fertilization, although p21 mRNA was upregulated by irradiation at 2 or 4 h post‐fertilization. These results suggest that the cellular responses of zebrafish embryos at 2 or 4 h post‐fertilization to radiation failed to overcome P21 protein accumulation and further signaling. Regulation of P21/WAF1 protein stabilization appears to be a key factor in the response to genotoxins during early embryogenesis.

The dynein adaptor Hook2 plays essential roles in mitotic progression and cytokinesis.

Hook proteins are evolutionarily conserved dynein adaptors that promote assembly of highly processive dynein-dynactin motor complexes. Mammals express three Hook paralogs, namely Hook1, Hook2, and Hook3, that have distinct subcellular localizations and expectedly, distinct cellular functions. Here we demonstrate that Hook2 binds to and promotes dynein-dynactin assembly specifically during mitosis. During the late G2 phase, Hook2 mediates dynein-dynactin localization at the nuclear envelope (NE), which is required for centrosome anchoring to the NE. Independent of its binding to dynein, Hook2 regulates microtubule nucleation at the centrosome; accordingly, Hook2-depleted cells have reduced astral microtubules and spindle positioning defects. Besides the centrosome, Hook2 localizes to and recruits dynactin and dynein to the central spindle. Dynactin-dependent targeting of centralspindlin complex to the midzone is abrogated upon Hook2 depletion; accordingly, Hook2 depletion results in cytokinesis failure. We find that the zebrafish Hook2 homologue promotes dynein-dynactin association and was essential for zebrafish early development. Together, these results suggest that Hook2 mediates assembly of the dynein-dynactin complex and regulates mitotic progression and cytokinesis.

The Use of Complementary Luminescent and Fluorescent Techniques for Imaging Ca2+ Signaling Events During the Early Development of Zebrafish (Danio rerio).

We have visualized many of the Ca2+ signaling events that occur during the early stages of zebrafish development using complementary luminescent and fluorescent imaging techniques. We initially microinject embryos with the luminescent Ca2+ reporter, f-holo-aequorin, and using a custom-designed luminescent imaging system, we can obtain pan-embryonic visual information continually for up to the first ~24h postfertilization (hpf). Once we know approximately when and where to look for these Ca2+ signaling events within a complex developing embryo, we then repeat the experiment using a fluorescent Ca2+ reporter such as calcium green-1 dextran and use confocal laser scanning microscopy to provide time-lapse series of higher-resolution images. These protocols allow us to identify the specific cell types and even the particular subcellular domain (e.g., nucleus or cytoplasm) generating the Ca2+ signal. Here, we outline the techniques we use to precisely microinject f-holo-aequorin or calcium green-1 dextran into embryos without affecting their viability or development. We also describe how to inject specific regions of early embryos in order to load localized embryonic domains with a particular Ca2+ reporter. These same techniques can also be used to introduce other membrane-impermeable reagents into embryos, including Ca2+ channel antagonists, Ca2+ chelators, fluorescent dyes, RNA, and DNA.

Zebrafish embryos exposed to deltamethrin exhibit abnormalities despite induced expression of related genes (you, you-too, momo and u-boot)

Evaluation of the toxic effects of a widely used synthetic pyrethroid, deltamethrin (DM), was carried out in this study. This pesticide is preferred for pest control because of its low environmental persistence and toxicity. We investigated the expression pattern of four genes, namely, you ( you), yot ( you-too), momo ( mom) and ubo ( u-boot) during early development of zebrafish, that is, from 12 hpf to 48 hpf stages. These stages are selected as most of the important developmental aspects take place during this period. All four genes are known to play a vital role in development of notochord and somites. To understand the effect of DM on development, embryos of 4 hpf stage were exposed to two concentrations (100 and 200 µg/L) of DM, and observations were made at 12, 24 and 48 hpf stages. Our earlier studies have shown phenotypic abnormalities such as notochord bending, tail deformation, yolk sac and pericardial edema, lightening of body and eye pigmentation and interfered in somite patterning, during these stages of development. Understanding the relationship of phenotypic abnormalities with these four genes has been our primary objective. These four genes were analyzed by Reverse transcription (RT)-polymerase chain reaction and intensity of the bands has shown induction in their expression after exposure to the toxicant. In spite of the expression of genes, it was noticed that DM caused abnormalities. It can be said from the results that translational pathway could have been affected.

Comparative Toxicity Evaluation of Targeted Anticancer Therapeutics in Embryonic Zebrafish and Sea Urchin Models

Cancer drug resistance and poor selectivity towards cancer cells demand the constant search for new therapeutics. PI3K-Akt-mTOR and RAS-MAPK-ERK signaling pathways are key mechanisms involved in cell survival, proliferation, differentiation, and metabolism and their deregulation in cancer can pro-mote development of therapy resistance. We investigated the effects of targeted inhibitors (wortmannin, GSK690693, AZD2014 and tipifarnib) towards these two pathways on early zebrafish and sea urchin development to assess their toxicity in normal, fast proliferating cells. PI3K inhibitor wortmannin and RAS inhibitor tipifarnib displayed highest toxicity while GSK690693, a pan-Akt kinase inhibitor, exhib-ited a less significant impact on embryo survival and development. Moreover, inhibition of the upstream part of the PI3K-Akt-mTOR pathway (wortmannin/GSK690693 co-treatment) produced a synergistic effect and impacted zebrafish embryo survival and development at much lower concentrations. Dual mTORCl/mTORC2 inhibitor AZD2014 showed no considerable effects on embryonic cells of zebrafish in concentrations substantially toxic in cancer cells. AZD2014 also caused the least prominent effects on sea urchin embryo development compared to other inhibitors. Significant toxicity of AZD2014 in human cancer cells, its capacity to sensitize resistant cancers, lower antiproliferative activity against human normal cell lines and fast proliferating embryonic cells could make this agent a promising candidate for anticancer therapy.

Centrosomal protein FOR20 is essential for cilia-dependent development in zebrafish embryos.

Centrosomal proteins play critical roles in ciliogenesis. Mutations in many centrosomal proteins have been documented to contribute to developmental defects and cilium-related diseases. Centrosomal protein fibroblast growth factor receptor 1 oncogene partner-related protein of 20 kDa (FOR20) is crucial for ciliogenesis in mammalian cells and the unicellular eukaryote Paramecium; however, the biologic significance of FOR20 in vertebrate development remains unclear. We cloned the zebrafish homolog of the for20 gene and found that for20 mRNA is enriched in ciliated tissues during early zebrafish development. Knockdown of for20 by morpholino oligonucleotides in zebrafish results in multiple ciliary phenotypes, including curved body, hydrocephaly, pericardial edema, kidney cysts, and left-right asymmetry defects. for20 morphants show reduced number and length of cilia in Kupffer's vesicle and pronephric ducts. High-speed video microscopy reveals that cilia in most for20 morphants are consistently paralyzed or beat arrhythmically. To confirm the ciliary phenotypes of for20 morphants, we used the CRISPR/Cas9 system to disrupt for20 gene in zebrafish. for20 mutants exhibit multiple ciliary phenotypes resembling the defects in for20 morphants. All of these phenotypes in for20 morphants and mutants are significantly reversed by exogenous expression of for20 mRNA. Taken together, these data suggest that FOR20 is required for cilium-mediated processes during zebrafish embryogenesis.-Xie, S., Jin, J., Xu, Z., Huang, Y., Zhang, W., Zhao, L., Lo, L. J., Peng, J., Liu, W., Wang, F., Shu, Q., Zhou, T. Centrosomal protein FOR20 is essential for cilia-dependent development in zebrafish embryos.

Knockdown of calcium-binding calb2a and calb2b genes indicates the key regulator of the early development of the zebrafish, Danio rerio.

The present study initiates our investigation regarding the role of calb2a and calb2b genes that are expressed in the central nervous system, including the multiple tissues during early embryonic development of zebrafish. In this study, we have adopted individual and combined morpholino-mediated inactivation approach to investigate the functions of calb2a and calb2b in early development of the zebrafish. We have found that calb2a and calb2b morpholino alone failed to generate an obvious phenotype; however, morphological inspection in early developmental stages of calb2a and calb2b combined knockdown morphants show abnormal neural plate folding in midbrain-hindbrain region. In addition to this, combinatorial loss of these mRNA leads to severe hydrocephalus, axial curvature defect, and yolk sac edema in later developmental stages. Also, the combined knockdown of calb2a and calb2b are found to be associated with an impaired touchdown and swimming performance in the zebrafish. Co-injection of the calb2a and calb2b morpholino oligonucleotide cocktail with human CALB2 mRNA leads to the rescue of the strong phenotype. This study provided the first comprehensive analyses of the zebrafish Calb2a and Calb2b proteins; we havefound that Calb2a and Calb2b are highly conserved across vertebrate species and originated from the same ancestral gene long back in theevolution. Homology modeling and docking with the similar structure and Ca2+ binding sites for both proteins provide the evidence that both the proteins may have similar function and one can compensate for the loss of other. Collectively, these findings confirm the unique and essential functions of calb2a and calb2b genes in the early development of the zebrafish.

MRNA expression of antioxidant and biotransformation enzymes in zebrafish (Danio rerio) embryos after exposure to the tricyclic antidepressant amitriptyline

Now-a-days, the occurrence of antidepressant residues in surface waters has become a major concern. Amitriptyline (AMI) has been described to treat depression and other disorders for decades. However, little is known about its effect on non-target organisms. The aim of this study was to assess the potential impact of AMI on the mRNA expression of antioxidant and detoxification enzymes during the early embryonic development of zebrafish (Danio rerio). Fertilized D. rerio embryos were exposed to AMI at concentrations of 300 ng/L and 30 μg/L and sampled 24, 48, 96, and 144 h post fertilization (hpf) to assess the mRNA expressions of cytochrome P450 1A1, glutathione-S-transferase, glutathione peroxidase, superoxide dismutase, and catalase.The time courses of the mRNA expressions of antioxidant and detoxification enzymes revealed characteristic changes during embryonic development causing generally transient changes post hatching; however, AMI did not cause any significant impact except in the case of CAT after 144 h, which was significantly upregulated by the AMI concentration of 30 μg/L. The results suggest that the antidepressant AMI causes only moderate to minor impacts on antioxidant and detoxification enzymes during early embryonic development of the non-target organism D. rerio and that CAT is the only biomarker affected by AMI.

Developmental protein kinase C hyper-activation results in microcephaly and behavioral abnormalities in zebrafish

Susceptible genetic polymorphisms and altered expression levels of protein kinase C (PKC)-encoding genes suggest overactivation of PKC in autism spectrum disorder (ASD) development. To delineate the pathological role of PKC, we pharmacologically stimulated its activity during the early development of zebrafish. Results demonstrated that PKC hyper-activation perturbs zebrafish development and induces a long-lasting head size deficit. The anatomical and cellular analysis revealed reduced neural precursor proliferation and newborn neuron formation. β-Catenin that is essential for brain growth is dramatically degraded. Stabilization of β-catenin by gsk3β inhibition partially restores the head size deficit. In addition, the neuropathogenic effect of developmental PKC hyper-activation was further supported by the alterations in the behavioral domain including motor abnormalities, heightened stress reactivity and impaired habituation learning. Taken together, by causally connecting early-life PKC hyper-activation to these neuropathological traits and the impaired neurogenesis, these results suggest that PKC could be a critical pathway in ASD pathogenesis.

Generation of Cas9 transgenic zebrafish and their application in establishing an ERV-deficient animal model

ObjectivesTo investigate the effect of endogenous Cas9 on genome editing efficiency in transgenic zebrafish.ResultsHere we have constructed a transgenic zebrafish strain that can be screened by pigment deficiency. Compared with the traditional CRISPR injection method, the transgenic zebrafish can improve the efficiency of genome editing significantly. At the same time, we first observed that the phenotype of vertebral malformation in early embryonic development of zebrafish after ZFERV knockout.ConclusionsThe transgenic zebrafish with expressed Cas9, is more efficient in genome editing. And the results of ZFERV knockout indicated that ERV may affect the vertebral development by Notch1/Delta D signal pathway.

Genome-wide identification of the interactions between key genes and pathways provide new insights into the toxicity of bisphenol F and S during early development in zebrafish

Bisphenol F (BPF) and bisphenol S (BPS) have been widely used as alternatives to bisphenol A (BPA). With their increasing use, BPF and BPS have also been released into the environment; thus, their potential risks to aquatic organisms and humans are drawing attention. The objective of this study was to identify the interactions between key pathways and hub genes in zebrafish following BPF and BPS exposure, and to evaluate the potential risks to human health. We identified three key pathways using KEGG over-representation test and Gene Set Enrichment Analysis (GSEA): ‘Necroptosis,’ ‘Adipocytokine signaling pathway,’ and ‘C-type lectin receptor signaling pathway.’ Moreover, three hub genes (mst1ra, prkcdb, and pik3cb) and detailed interactions among the pathways were examined by the analyses of PPI network, subcellular location, and shortest-pathway. Surprisingly, all three pathways were strongly associated with a potential risk of cancer, as reported previously. In addition, the results of KOBAS shown in ‘Pathways in Cancer’ and ‘Cancers’ belong to the top 10 terms in pathway enrichment analyses using genes related to BPF or BPS in human, as was found using GenCLiP. Moreover, the Kaplan-Meier survival analysis was performed using homologenes (MST1R, PIK3CB and PRKCD) of hub genes in human to evaluate whether exposure to bisphenols may adversely affect breast cancer. Taken together, these studies demonstrate the potential carcinogenicity of BPF and BPS. To our knowledge, this is the first study on three overlapping key pathways and three hub genes to investigate BPF and BPS exposure-related mechanisms and subsequent interactions in zebrafish.