Developmental toxicity, antioxidant, and marker enzyme assessment of swertiamarin in zebrafish (Danio rerio).

A secoiridoid glycoside called swertiamarin has been widely used as a herbal medicine for many decades. In particular, swertiamarin from the Enicostema axillare herb has been used as a multipurpose drug to treat innumerable health problems. As this medicine is consumed orally, its toxicity level should be determined. To examine the safety of this compound, toxicology work was done in zebrafish, and this is the first report to describe swertiamarin toxicity in zebrafish. Zebrafish embryos were used in this swertiamarin toxicity study, and morphological changes were observed. Further, the compound was also studied in adult zebrafish to determine the impact of the compound on the fish liver. Enzyme profiling with superoxide dismutase, glutathione peroxidase, catalase, reduced glutathione levels, glutathione S-transferase, lactate dehydrogenase, glutamic oxaloacetic transaminases, lipid peroxidation, Na+ /K+ -ATPase, and glutamic pyruvic transaminases) was evaluated (p ≤ 0.05). Results suggest that swertiamarin is a safe drug only at a low concentration (40 µM). This study also shows that even herbal medicinal compounds may be toxic to humans at higher dosages. Hence, irrespective of whether a drug is synthetic or natural, it needs to be tested for its toxicity before use in humans.

Quercetin mitigates ethanol-induced hepatic steatosis in zebrafish via P2X7R-mediated PI3K/ Keap1/Nrf2 signaling pathway.

Ethnopharmacological relevanceQuercetin is the active component of the higher content in PCP, which exerts various biological activities such as anti-obesity effect, anti-inflammatory and anti-oxidant activities in alcoholic liver disease (ALD). AIM OF THE STUDY: P2X7 receptor (P2X7R) plays an important role in health and disease, which can be activated by extracellular ATP to induce a variety of downstream events, including lipid metabolism, inflammatory molecule release, oxidative stress. However, whether the mechanism of quercetin on ethanol-induced hepatic steatosis via P2X7R-mediated haven't been elucidated. MATERIAL AND METHODS: Zebrafish transgenic (fabp10: EGFP) larvae were treated with 100 µM, 50 µM, 25 µM quercetin for 48 h at 3 days post fertilization (dpf), then soaked in 350 mmol/L ethanol for 32 h, treated with 1 mM ATP (P2X7R activator) for 30min. Serum lipids, liver steatosis, oxidative stress factors were respectively detected. The mRNA levels in the related pathways were measured by quantitative Real-Time PCR (RT-qPCR) to investigate the mechanisms. RESULTS: Quercetin improved the liver function via decreasing ALT, AST and γ-GT level of zebrafish with acute ethanol-induced hepatic steatosis and attenuated hepatic TG, TC accumulation. Additionally, quercetin significantly reduced the MDA content and suppressed the ethanol-induced reduction of hepatic oxidative stress biomarkers GSH, CAT and SOD and significantly down-regulated the expression of P2X7R, and up-regulated the expression of phosphatidylinositol 3-kinase (PI3K), Kelch like ECH associated protein1 (Keap1), Nuclear Factor E2 related factor 2 (Nrf2). Moreover, ATP stimulation activated P2X7R, which further mediated the mRNA expressions of PI3K, Keap1 and Nrf2. CONCLUSION: Quercetin exhibited hepatoprotective capacity in zebrafish model, via regulating P2X7R-mediated PI3K/Keap1/Nrf2 oxidative stress signaling pathway.

Folic acid supplement rescues ethanol-induced developmental defects in the zebrafish embryos.

Fetal alcohol syndrome (FASD) describes a range of birth defects. Mechanisms of FASD-associated defects are not well understood. It has great significance to investigate whether nutrient supplements like folic acid (FA) can effectively rescue ethanol-induced defects. Moreover, it is very important to determine the optimal time for FA supplementation when it can most effectively antagonize the teratogenic effects of ethanol during embryonic development. Our results indicated that ethanol exposure interrupted the development of zebrafish embryos and induced multiple defects in cardiac function, pharyngeal arch arteries, vessel, craniofacial cartilage, pharyngeal arches, brain, somite and hemoglobin formation. The expressions of critical genes that play important roles in above organs such as tbx1, flk-1, hand2, ngn1, huc, titin, gata-1 and c-myb were reduced, and the apoptosis was increased in ethanol-treated group. FA supplementation could reverse ethanol-induced defects, improve the decreased expressions of above genes and reduce the apoptosis. We also found that giving FA at 6-12 h post-fertilization (hpf), which is at the gastrula period (5.25-10 hpf), can obviously prevent the teratogenicity of ethanol. This research provides clues for elucidating the mechanism of fetal abnormalities caused by alcohol intake and for preventing FASD.

Dynamics of actinotrichia regeneration in the adult zebrafish fin.

The skeleton of adult zebrafish fins comprises lepidotrichia, which are dermal bones of the rays, and actinotrichia, which are non-mineralized spicules at the distal margin of the appendage. Little is known about the regenerative dynamics of the actinotrichia-specific structural proteins called Actinodins. Here, we used immunofluorescence analysis to determine the contribution of two paralogous Actinodin proteins, And1/2, in regenerating fins. Both proteins were detected in the secretory organelles in the mesenchymal cells of the blastema, but only And1 was detected in the epithelial cells of the wound epithelium. The analysis of whole mount fins throughout the entire regenerative process and longitudinal sections revealed that And1-positive fibers are complementary to the lepidotrichia. The analysis of another longfin fish, a gain-of-function mutation in the potassium channel kcnk5b, revealed that the long-fin phenotype is associated with an extended size of actinotrichia during homeostasis and regeneration. Finally, we investigated the role of several signaling pathways in actinotrichia formation and maintenance. This revealed that the pulse-inhibition of either TGFß/Activin-ßA or FGF are sufficient to impair deposition of Actinodin during regeneration. Thus, the dynamic turnover of Actinodin during fin regeneration is regulated by multiple factors, including the osteoblasts, growth rate in a potassium channel mutant, and instructive signaling networks between the epithelium and the blastema of the regenerating fin.

Ferulic Acid Promotes Hypertrophic Growth of Fast Skeletal Muscle in Zebrafish Model.

As a widely distributed and natural existing antioxidant, ferulic acid and its functions have been extensively studied in recent decades. In the present study, hypertrophic growth of fast skeletal myofibers was observed in adult zebrafish after ferulic acid administration for 30 days, being reflected in increased body weight, body mass index (BMI), and muscle mass, along with an enlarged cross-sectional area of myofibers. qRT-PCR analyses demonstrated the up-regulation of relative mRNA expression levels of myogenic transcriptional factors (MyoD, myogenin and serum response factor (SRF)) and their target genes encoding sarcomeric unit proteins involved in muscular hypertrophy (skeletal alpha-actin, myosin heavy chain, tropomyosin, and troponin I). Western blot analyses detected a higher phosphorylated level of zTOR (zebrafish target of rapamycin), p70S6K, and 4E-BP1, which suggests an enhanced translation efficiency and protein synthesis capacity of fast skeletal muscle myofibers. These changes in transcription and translation finally converge and lead to higher protein contents in myofibers, as confirmed by elevated levels of myosin heavy chain (MyHC), and an increased muscle mass. To the best of our knowledge, these findings have been reported for the first time in vivo and suggest potential applications of ferulic acid as functional food additives and dietary supplements owing to its ability to promote muscle growth.

Krüpple-like factors 7 and 6a mRNA expression in adult zebrafish central nervous system.

Krüpple-like factors (KLFs) are transcription factors with zinc finger DNA binding domains known to play important roles in brain development and central nervous system (CNS) regeneration. There is little information on KLFs expression in adult vertebrate CNS. In this study, we used in situ hybridization to examine Klf7 mRNA (klf7) and Klf6a mRNA (klf6a) expression in adult zebrafish CNS. Both klfs exhibit wide and similar expression in the zebrafish CNS. Brain areas containing strongly labeled cells include the ventricular regions of the dorsomedial telencephalon, the ventromedial telencephalon, periventricular regions of the thalamus and hypothalamus, torus longitudinalis, stratum periventriculare of the optic tectum, granular regions of the cerebellar body and valvula, and superficial layers of the facial and vagal lobes. In the spinal cord, klf7- and klf6a-expressing cells are found in both the dorsal and ventral horns. Numerous sensory structures (e.g. auditory, lateral line, olfactory and visual) and several motor nuclei (e.g. oculomotor, trigeminal, and vagal motor nuclei) contain klf7- and/or klf6a-expressing cells. Our results may provide useful information for determining these Klfs in maintenance and/or function in adult CNS.

LRRK2 knockdown in zebrafish causes developmental defects, neuronal loss, and synuclein aggregation.

Although mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common cause of genetic Parkinson's disease, their function is largely unknown. LRRK2 is pleiotropic in nature, shown to be involved in neurodegeneration and in more peripheral processes, including kidney functions, in rats and mice. Recent studies in zebrafish have shown conflicting evidence that removal of the LRRK2 WD40 domain may or may not affect dopaminergic neurons and/or locomotion. This study shows that ∼50% LRRK2 knockdown in zebrafish causes not only neuronal loss but also developmental perturbations such as axis curvature defects, ocular abnormalities, and edema in the eyes, lens, and otic vesicles. We further show that LRRK2 knockdown results in significant neuronal loss, including a reduction of dopaminergic neurons. Immunofluorescence demonstrates that endogenous LRRK2 is expressed in the lens, brain, heart, spinal cord, and kidney (pronephros), which mirror the LRRK2 morphant phenotypes observed. LRRK2 knockdown results further in the concomitant upregulation of ß-synuclein, PARK13, and SOD1 and causes ß-synuclein aggregation in the diencephalon, midbrain, hindbrain, and postoptic commissure. LRRK2 knockdown causes mislocalization of the Na(+) /K(+) ATPase protein in the pronephric ducts, suggesting that the edema might be linked to renal malfunction and that LRRK2 might be associated with pronephric duct epithelial cell differentiation. Combined, our study shows that LRRK2 has multifaceted roles in zebrafish and that zebrafish represent a complementary model to further our understanding of this central protein. © 2016 Wiley Periodicals, Inc.

The Effects of Disturbance on Hypothalamus-Pituitary-Thyroid (HPT) Axis in Zebrafish Larvae after Exposure to DEHP.

Di-(2-ethylhexyl) phthalate (DEHP) has the potential to disrupt the thyroid endocrine system, but the underlying mechanism is unknown. In this study, zebrafish (Danio rerio) embryos were exposed to different concentrations of DEHP (0, 40, 100, 200, 400 µg/L) from 2 to 168 hours post fertilization (hpf). Thyroid hormones (THs) levels and transcriptional profiling of key genes related to hypothalamus-pituitary-thyroid (HPT) axis were examined. The result of whole-body thyroxine (T4) and triiodothyronine (T3) indicated that the thyroid hormone homeostasis was disrupted by DEHP in the zebrafish larvae. After exposure to DEHP, the mRNA expressions of thyroid stimulating hormone (tshß) and corticotrophin releasing hormone (crh) genes were increased in a concentration dependent manner, respectively. The expression level of genes involved in thyroid development (nkx2.1 and pax8) and thyroid synthesis (sodium/iodide symporter, nis, thyroglobulin, tg) were also measured. The transcripts of nkx2.1 and tg were significantly increased after DEHP exposure, while those of nis and pax8 had no significant change. Down-regulation of uridinediphosphate-glucuronosyl-transferase (ugt1ab) and up-regulation of thyronine deiodinase (dio2) might change the THs levels. In addition, the transcript of transthyretin (ttr) was up-regulated, while the mRNA levels of thyroid hormone receptors (trα and trß) remained unchanged. All the results demonstrated that exposure to DEHP altered the whole-body thyroid hormones in the zebrafish larvae and changed the expression profiling of key genes related to HPT axis, proving that DEHP induced the thyroid endocrine toxicity and potentially affected the synthesis, regulation and action of thyroid hormones.

Knockdown of zebrafish YY1a can downregulate the phosphatidylserine (PS) receptor expression, leading to induce the abnormal brain and heart development.

BACKGROUND: Yin Yang 1 (YY1) is a ubiquitously expressed GLI-Kruppel zinc finger-containing transcriptional regulator. YY1 plays a fundamental role in normal biologic processes such as embryogenesis, differentiation, and cellular proliferation. YY1 effects on the genes involved in these processes are mediated via initiation, activation, or repression of transcription depending upon the context in which it binds. The role of the multifunctional transcription factor Yin Yang 1 (YY1) in tissue development is poorly understood. In the present, we investigated YY1a role in developing zebrafish on PSR-mediated apoptotic cell engulfment during organic morphogenesis. RESULTS: YY1a is first expressed 0.5 h post-fertilization (hpf), in the whole embryo 12 hpf, and in brain, eyes, and heart 72 hpf by in situ hybridization assay. The nucleotide sequence of zebrafish YY1a transcription factor (clone zfYY1a; HQ 166834) was found to be similar to that of zebrafish YY1a (99 % sequence identity; NM 212617). With the loss-of-function assay, YY1a knockdown by a morpholino oligonucleotide led to downregulation of the phosphatidylserine engulfing receptor zfPSR during embryonic segmentation and to the accumulation of a large number of dead apoptotic cells throughout the entire early embryo, especially in the posterior area. Up to 24 hpf, these cells interfered with embryonic cell migration and cell-cell interactions that normally occur in the brain, heart, eye, and notochord. Finally, with gain-of-function assay, defective morphants could be rescued by injecting both YY1a mRNA and PSR mRNA and trigger resumption of normal development. CONCLUSIONS: Taken together, our results suggest that YY1a regulates PS receptor expression that linked to function of PSR-phagocyte mediated apoptotic cell engulfment during development, especially the development of organs such as the brain and heart. YY1a/PSR-mediated engulfing system may involve in diseases.

Zebrafish embryos as a screen for DNA methylation modifications after compound exposure.

Modified epigenetic programming early in life is proposed to underlie the development of an adverse adult phenotype, known as the Developmental Origins of Health and Disease (DOHaD) concept. Several environmental contaminants have been implicated as modifying factors of the developing epigenome. This underlines the need to investigate this newly recognized toxicological risk and systematically screen for the epigenome modifying potential of compounds. In this study, we examined the applicability of the zebrafish embryo as a screening model for DNA methylation modifications. Embryos were exposed from 0 to 72 h post fertilization (hpf) to bisphenol-A (BPA), diethylstilbestrol, 17α-ethynylestradiol, nickel, cadmium, tributyltin, arsenite, perfluoroctanoic acid, valproic acid, flusilazole, 5-azacytidine (5AC) in subtoxic concentrations. Both global and site-specific methylation was examined. Global methylation was only affected by 5AC. Genome wide locus-specific analysis was performed for BPA exposed embryos using Digital Restriction Enzyme Analysis of Methylation (DREAM), which showed minimal wide scale effects on the genome, whereas potential informative markers were not confirmed by pyrosequencing. Site-specific methylation was examined in the promoter regions of three selected genes vasa, vtg1 and cyp19a2, of which vasa (ddx4) was the most responsive. This analysis distinguished estrogenic compounds from metals by direction and sensitivity of the effect compared to embryotoxicity. In conclusion, the zebrafish embryo is a potential screening tool to examine DNA methylation modifications after xenobiotic exposure. The next step is to examine the adult phenotype of exposed embryos and to analyze molecular mechanisms that potentially link epigenetic effects and altered phenotypes, to support the DOHaD hypothesis.

Tankyrases regulate glucoregulatory mechanisms and somatic growth via the central melanocortin system in zebrafish larvae.

The central melanocortin system is a key regulator of energy homeostasis. Recent studies indicate that tankyrases (TNKSs), which poly(ADP-ribosyl)ate target proteins and direct them toward proteasomal degradation, affect overall metabolism, but the exact molecular mechanisms remain unclear. We used zebrafish larvae as a model to study the mechanisms by which TNKS1b, the zebrafish ortholog of mammalian TNKS1, regulates glucose homeostasis and somatic growth. In situ hybridization revealed that TNKS1b mRNA is prominently expressed in the hypothalamus and pituitary of the embryonic and larval brain. In the pituitary, TNKS1b is coexpressed with pro-opiomelanocortin a (pomca) gene in corticotropes and melanotropes. Knockdown of TNKS1b reduced the linear growth of the larvae, stimulated insulin gene and glucose transporter 4 protein, and suppressed gluconeogenic phosphoenolpyruvate carboxykinase 1 gene. This result indicates rapid glucose utilization and reduction of gluconeogenesis in TNKS1b-deficient larvae. Knockdown of TNKS1b down-regulated pomca expression and diminished α-melanocyte-stimulating hormone in the pars intermedia. Furthermore, down-regulation of TNKS1b suppressed the expression of melanocortin receptor 3 and increased the expression of melanocortin receptor 4. The collective data suggest that TNKS1b modulates glucoregulatory mechanisms and the somatic growth of zebrafish larvae via the central melanocortin system.

A novel zebrafish model of hyperthermia-induced seizures reveals a role for TRPV4 channels and NMDA-type glutamate receptors.

Febrile seizures are the most common seizure type in children under the age of five, but mechanisms underlying seizure generation in vivo remain unclear. Animal models to address this issue primarily focus on immature rodents heated indirectly using a controlled water bath or air blower. Here we describe an in vivo model of hyperthermia-induced seizures in larval zebrafish aged 3 to 7 days post-fertilization (dpf). Bath controlled changes in temperature are rapid and reversible in this model. Acute electrographic seizures following transient hyperthermia showed age-dependence, strain independence, and absence of mortality. Electrographic seizures recorded in the larval zebrafish forebrain were blocked by adding antagonists to the transient receptor potential vanilloid (TRPV4) channel or N-methyl-d-aspartate (NMDA) glutamate receptor to the bathing medium. Application of GABA, GABA re-uptake inhibitors, or TRPV1 antagonist had no effect on hyperthermic seizures. Expression of vanilloid channel and glutamate receptor mRNA was confirmed by quantitative PCR analysis at each developmental stage in larval zebrafish. Taken together, our findings suggest a role of heat-activation of TRPV4 channels and enhanced NMDA receptor-mediated glutamatergic transmission in hyperthermia-induced seizures.

MicroRNA-24 regulates vascularity after myocardial infarction.

BACKGROUND: Myocardial infarction leads to cardiac remodeling and development of heart failure. Insufficient myocardial capillary density after myocardial infarction has been identified as a critical event in this process, although the underlying mechanisms of cardiac angiogenesis are mechanistically not well understood. METHODS AND RESULTS: Here, we show that the small noncoding RNA microRNA-24 (miR-24) is enriched in cardiac endothelial cells and considerably upregulated after cardiac ischemia. MiR-24 induces endothelial cell apoptosis, abolishes endothelial capillary network formation on Matrigel, and inhibits cell sprouting from endothelial spheroids. These effects are mediated through targeting of the endothelium-enriched transcription factor GATA2 and the p21-activated kinase PAK4, which were identified by bioinformatic predictions and validated by luciferase gene reporter assays. Respective downstream signaling cascades involving phosphorylated BAD (Bcl-XL/Bcl-2-associated death promoter) and Sirtuin1 were identified by transcriptome, protein arrays, and chromatin immunoprecipitation analyses. Overexpression of miR-24 or silencing of its targets significantly impaired angiogenesis in zebrafish embryos. Blocking of endothelial miR-24 limited myocardial infarct size of mice via prevention of endothelial apoptosis and enhancement of vascularity, which led to preserved cardiac function and survival. CONCLUSIONS: Our findings indicate that miR-24 acts as a critical regulator of endothelial cell apoptosis and angiogenesis and is suitable for therapeutic intervention in the setting of ischemic heart disease.

Expression of hairy/enhancer of split genes in neural progenitors and neurogenesis domains of the adult zebrafish brain.

All subdivisions of the adult zebrafish brain maintain niches of constitutive neurogenesis, sustained by quiescent and multipotent progenitor populations. In the telencephalon, the latter potential neural stem cells take the shape of radial glia aligned along the ventricle and are controlled by Notch signalling. With the aim of identifying new markers of this cell type and of comparing the effectors of embryonic and adult neurogenesis, we focused on the family of hairy/enhancer of split [E(spl)] genes. We report the expression of seven hairy/E(spl) (her) genes and the new helt gene in three neurogenic areas of the adult zebrafish brain (telencephalon, hypothalamus, and midbrain) in relation to radial glia, proliferation, and neurogenesis. We show that the expression of most her genes in the adult brain characterizes quiescent radial glia, whereas only few are expressed in progenitor domains engaged in active proliferation or neurogenesis. The low proliferation status of most her-positive progenitors contrasts with the embryonic nervous system, in which her genes are expressed in actively dividing progenitors. Likewise, we demonstrate largely overlapping expression domains of a set of her genes in the adult brain, which is in striking contrast to their distinct embryonic expression profiles. Overall, our data provide a consolidated map of her expression, quiescent glia, proliferation, and neurogenesis in these various subdivisions of the adult brain and suggest distinct regulation and function of Her factors in the embryonic and adult contexts.

Sublethal exposure to crude oil during embryonic development alters cardiac morphology and reduces aerobic capacity in adult fish.

Exposure to high concentrations of crude oil produces a lethal syndrome of heart failure in fish embryos. Mortality is caused by cardiotoxic polycyclic aromatic hydrocarbons (PAHs), ubiquitous components of petroleum. Here, we show that transient embryonic exposure to very low concentrations of oil causes toxicity that is sublethal, delayed, and not counteracted by the protective effects of cytochrome P450 induction. Nearly a year after embryonic oil exposure, adult zebrafish showed subtle changes in heart shape and a significant reduction in swimming performance, indicative of reduced cardiac output. These delayed physiological impacts on cardiovascular performance at later life stages provide a potential mechanism linking reduced individual survival to population-level ecosystem responses of fish species to chronic, low-level oil pollution.

Cloning and developmental expression of zebrafish pdzrn3.

Pdzrn3, a member of the PDZRN/SEMCAP/LNX protein family containing a RING finger and two PDZ domains, has been implicated in myoblast and osteoblast differentiation. However, its spatio-temporal expression pattern during embryonic development has not been defined. Here, we describe the cloning and expression pattern of pdzrn3 during zebrafish development. We found that in addition to being expressed in several mesodermal structures, this gene displays specific expression in the central nervous system including rhombomere 1, ventral retina, thalamus and motor neurons, indicating a novel function during neural development. In particular, the absence of expression of pdzrn3 in the ventral retina of noi mutant fish suggests a possible role for this gene in regulating fasciculation and/or navigation of retinal ganglion cell axons.

Sodium benzoate exposure downregulates the expression of tyrosine hydroxylase and dopamine transporter in dopaminergic neurons in developing zebrafish.

BACKGROUND: Recent data have demonstrated that treatment with sodium benzoate (SB) leads to significant developmental defects in motor neuron axons and neuromuscular junctions in zebrafish larvae, thereby implying that SB can be neurotoxic. This study examined whether SB affects the development of dopaminergic neurons in the zebrafish brain. METHODS: Zebrafish embryos were exposed to different concentrations of SB for various durations, during which the survival rates were recorded, the expression of tyrosine hydroxylase (TH) and dopamine transporter (DAT) in the neurons in the ventral diencephalon were detected by in situ hybridization and immunofluorescence, and the locomotor activity of larval zebrafish was measured. RESULTS: The survival rates were significantly decreased with the increase of duration and dose of SB-treatment. Compared to untreated clutch mates (untreated controls), treatment with SB significantly downregulated expression of TH and DAT in neurons in the ventral diencephalon of 3-day post-fertilization (dpf) zebrafish embryos in a dose-dependent manner. Furthermore, there was a marked decrease in locomotor activity in zebrafish larvae at 6dpf in response to SB treatment. CONCLUSIONS: The results suggest that SB exposure can cause significantly decreased survival rates of zebrafish embryos in a time- and dose-dependent manner and downregulated expression of TH and DAT in dopaminergic neurons in the zebrafish ventral diencephalon, which results in decreased locomotor activity of zebrafish larvae. This study may provide some important information for further elucidating the mechanism underlying SB-induced developmental neurotoxicity.

Translational bypass of nonsense mutations in zebrafish rep1, pax2.1 and lamb1 highlights a viable therapeutic option for untreatable genetic eye disease.

The extensive molecular genetic heterogeneity seen with inherited eye disease is a major barrier to the development of gene-based therapeutics. The underlying molecular pathology in a considerable proportion of these diseases however are nonsense mutations leading to premature termination codons. A therapeutic intervention targeted at this abnormality would therefore potentially be relevant to a wide range of inherited eye diseases. We have taken advantage of the ability of aminoglycoside drugs to suppress such nonsense mutations and partially restore full-length, functional protein in a zebrafish model of choroideraemia (chm(ru848); juvenile chorio-retinal degeneration) and in two models of ocular coloboma (noi(tu29a) and gup(m189); congenital optic fissure closure defects). In vitro cell-based assays showed significant readthrough with two drugs, gentamicin and paromomycin, which was confirmed by western blot and in vitro prenylation assays. The presence of either aminoglycoside during zebrafish development in vivo showed remarkable prevention of mutant ocular phenotypes in each model and a reduction in multisystemic defects leading to a 1.5-1.7-fold increase in survival. We also identified a significant reduction in abnormal cell death shown by TUNEL assay. To test the hypothesis that optic fissure closure was apoptosis-dependent, the anti-apoptotic agents, curcumin and zVAD-fmk, were tested in gup(m189) embryos. Both drugs were found to reduce the size of the coloboma, providing molecular evidence that cell death is required for optic fissure remodelling. These findings draw attention to the value of zebrafish models of eye disease as useful preclinical drug screening tools in studies to identify molecular mechanisms amenable to therapeutic intervention.

Expression of zebrafish ROR alpha gene in cerebellar-like structures.

Mouse genetic studies have identified several genes involved in cerebellar development. The mouse mutants staggerer and lurcher are functionally deficient for the retinoid-related orphan receptor alpha (ROR alpha) and glutamate receptor delta2 (Grid2) genes, respectively, and they show similar functional and developmental abnormalities in the cerebellum. Here, we report the cloning and expression pattern of zebrafish ROR alpha orthologues rora1 and rora2, and compare their expression pattern with that of grid2. Expression of rora1 and rora2 is initiated at late gastrula and pharyngula stages, respectively. Both rora1 and rora2 are spatially expressed in the retina and tectum. Expression of rora2 was further observed in the cerebellum, as reported for mammalian ROR alpha. In the adult brain, rora2 and grid2 are coexpressed in brain regions, designated as cerebellar-like structures. These observations suggest an evolutionarily conserved function of ROR alpha orthologues in the vertebrate brain.

The effects of copper and benzo[a]pyrene on retinoids and reproduction in zebrafish.

This study examines whether a link exists between toxicant exposure, retinoids and reproduction in fish. Zebrafish were fed a control diet (8.1 microg Cu/g diet, 0 microg benzo[a]pyrene/g diet) or diets containing elevated copper (100 microg, 500 microg and 1000 microg Cu/g diet) or benzo[a]pyrene (B[a]P; 30 and 150 microg B[a]P/g diet) for 260 days. Toxicant-supplemented diets did not affect growth or mortality rates. While whole body retinoid levels in control zebrafish decreased during the experiment, females exposed to Cu or B[a]P for 200 days or more experienced additional losses of retinyl esters (45-100% depleted) and retinal (45% depleted in B[a]P-fed fish). Despite the reduced retinoids, Cu and B[a]P did not effect reproduction with respect to the number of eggs spawned, fertilization rates or egg retinal content (retinal was instead increased 55-65% in eggs from B[a]P-fed fish). There were no apparent deformities observed in 36 h post fertilization embryos from any treatment. It appears that although internal retinoid stores were depleted in adults, dietary retinoids were sufficient to meet the daily requirement for retinal deposition in the eggs and retinoic acid synthesis. This study has shown that retinoid levels in female zebrafish are sensitive to Cu and B[a]P, and are a good indicator of long-term exposure. It also brings to light the resiliency of the retinoid system in fish and the importance of the diet on the toxicological response. Specifically that dietary retinoids appear to support normal reproduction in the absence of internal retinoid stores.