Retinoic Acid-Mediated Gene Expression in Transgenic Reporter Zebrafish

AbstractRetinoic acid-mediated gene activation is important for normal vertebrate development. The size and nature of retinoic acid make it difficult to identify the precise cellular location of this signaling molecule throughout an embryo. Additionally, retinoic acid (RA) signaling is regulated by a complex combination of receptors, coactivators, and antagonizing proteins. Thus, in order to integrate these signals and identify regions within a whole developing embryo where cells can respond transcriptionally to retinoic acid, we have used a reporter transgenic approach. We have generated several stable lines of transgenic zebrafish which use retinoic acid response elements to drive fluorescent protein expression. In these zebrafish lines, transgene expression is localized to regions of the neural tube, retina, notochord, somites, heart, pronephric ducts, branchial arches, and jaw muscles in embryos and larvae. Transgene expression can be induced in additional regions of the neural tube and retina as well as the immature notochord, hatching gland, enveloping cell layer, and fin by exposing embryos to retinoic acid. Treatment with retinoic acid synthase inhibitors, citral and diethylaminobenzaldehyde (DEAB), during neurulation, greatly reduces transgene expression. DEAB treatment of embryos at gastrulation phenocopies the embryonic effects of vitamin A deprivation or targeted disruption of the RA synthase retinaldehyde dehydrogenase-2 in other vertebrates. Together these data suggest that the reporter expression we see in zebrafish is dependent upon conserved vertebrate pathways of RA synthesis

Brief Embryonic Strychnine Exposure in Zebrafish Causes Long-Term Adult Behavioral Impairment with Indications of Embyronic Synaptic Changes

Zebrafish provide a powerful model of the impacts of embryonic toxicant exposure on neural development that may result in long-term behavioral dysfunction. In this study, zebrafish embryos were treated with 1.5mM strychnine for short embryonic time windows to induce transient changes in inhibitory neural signaling, and were subsequently raised in untreated water until adulthood. PCR analysis showed indications that strychnine exposure altered expression of some genes related to glycinergic, GABAergic and glutamatergic neuronal synapses during embryonic development. In adulthood, treated fish showed significant changes in swimming speed and tank diving behavior compared to controls. Taken together, these data show that a short embryonic exposure to a neurotoxicant can alter development of neural synapses and lead to changes in adult behavior

Laser-scanning velocimetry: A confocal microscopy method for quantitative measurement of cardiovascular performance in zebrafish embryos and larvae

Abstract Background The zebrafish Danio rerio is an important model system for drug discovery and to study cardiovascular development. Using a laser-scanning confocal microscope, we have developed a non-invasive method of measuring cardiac performance in zebrafish embryos and larvae that obtains cardiovascular parameters similar to those obtained using Doppler echocardiography in mammals. A laser scan line placed parallel to the path of blood in the dorsal aorta measures blood cell velocity, from which cardiac output and indices of vascular resistance and contractility are calculated. Results This technique, called laser-scanning velocimetry, was used to quantify the effects of pharmacological, developmental, and genetic modifiers of cardiac function. Laser-scanning velocimetry was applied to analyze the cardiovascular effects of morpholino knockdown of osmosensing scaffold for MEKK3 (OSM), which when mutated causes the human vascular disease cerebral cavernous malformations. OSM-deficient embryos had a constricted aortic arch and markedly increased peak cell velocity, a characteristic indicator of aortic stenosis. Conclusion These data validate laser-scanning velocimetry as a quantitative tool to measure cardiovascular performance for pharmacological and genetic analysis in zebrafish, which requires no specialized equipment other than a laser-scanning confocal microscope

Antioxidant responses and NRF2 in synergistic developmental toxicity of PAHs in zebrafish

Author Posting. © The Authors, 2009. This is the author's version of the work. It is posted here by permission of Oxford University Press for personal use, not for redistribution. The definitive version was published in Toxicological Sciences 109 (2009): 217-227, doi:10.1093/toxsci/kfp038.Early piscine life-stages are sensitive to polycyclic aromatic hydrocarbon (PAH) exposure, which can cause pericardial effusion and craniofacial malformations. We previously reported that certain combinations of PAHs cause synergistic developmental toxicity, as observed with co-exposure to the aryl hydrocarbon receptor (AHR) agonist β-naphthoflavone (BNF) and cytochrome P4501A inhibitor α-naphthoflavone (ANF). Herein, we hypothesized that oxidative stress is a component of this toxicity. We examined induction of antioxidant genes in zebrafish embryos (Danio rerio) exposed to BNF or ANF individually, a BNF+ANF combination, and a pro-oxidant positive control, tert-butylhydroperoxide (tBOOH). We measured total glutathione, and attempted to modulate deformities using the glutathione synthesis inhibitor buthionine sulfoxamine (BSO) and increase glutathione pools with N-acetyl cysteine (NAC). In addition, we used a morpholino to knockdown expression of the antioxidant response element transcription factor NRF2 to determine if this would alter gene expression or increase deformity severity. BNF+ANF co-exposure significantly increased expressions of superoxide dismutase1 and2, glutathione peroxidase 1, pi class glutathione-s-transferase, and glutamate cysteine-ligase to a greater extent than tBOOH, BNF, or ANF alone. BSO pretreatment decreased some glutathione levels, but did not worsen deformities, nor did NAC diminish toxicity. Knockdown of NRF2 increased mortality following tBOOH challenge, prevented significant upregulation of antioxidant genes following both tBOOH and BNF+ANF exposures, and exacerbated BNF+ANF‐related deformities. Collectively, these findings demonstrate that antioxidant responses are a component of PAH synergistic developmental toxicity, and that NRF2 is protective against prooxidant and PAH challenges during development.This work was supported by the National Institute for Environmental Health Sciencessupported Duke University Superfund Basic Research Program (P42 ES10356), National Institute for Environmental Health Sciences‐supported Duke University Integrated Toxicology & Environmental Health Program (TS ES07031), United States Environmental Protection Agency STAR fellowship (to A.T.‐L.), Duke University RJR‐Leon Golberg Memorial Postdoctoral Training Program in Toxicology (to A.T.‐L.), and the Postdoctoral Scholar Program at the Woods Hole Oceanographic Institution, with funding provided by the J. Seward Johnson Fund and The Walter A. and Hope Noyes Smith Chair (to A.T‐L)

Dynamic Zebrafish Interactome Reveals Transcriptional Mechanisms of Dioxin Toxicity

In order to generate hypotheses regarding the mechanisms by which 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin) causes toxicity, we analyzed global gene expression changes in developing zebrafish embryos exposed to this potent toxicant in the context of a dynamic gene network. For this purpose, we also computationally inferred a zebrafish (Danio rerio) interactome based on orthologs and interaction data from other eukaryotes.Using novel computational tools to analyze this interactome, we distinguished between dioxin-dependent and dioxin-independent interactions between proteins, and tracked the temporal propagation of dioxin-dependent transcriptional changes from a few genes that were altered initially, to large groups of biologically coherent genes at later times. The most notable processes altered at later developmental stages were calcium and iron metabolism, embryonic morphogenesis including neuronal and retinal development, a variety of mitochondria-related functions, and generalized stress response (not including induction of antioxidant genes). Within the interactome, many of these responses were connected to cytochrome P4501A (cyp1a) as well as other genes that were dioxin-regulated one day after exposure. This suggests that cyp1a may play a key role initiating the toxic dysregulation of those processes, rather than serving simply as a passive marker of dioxin exposure, as suggested by earlier research.Thus, a powerful microarray experiment coupled with a flexible interactome and multi-pronged interactome tools (which are now made publicly available for microarray analysis and related work) suggest the hypothesis that dioxin, best known in fish as a potent cardioteratogen, has many other targets. Many of these types of toxicity have been observed in mammalian species and are potentially caused by alterations to cyp1a

Zebrafish as a Model for Studying Adult Effects of Challenges to the Embryonic Nervous System

Zebrafish is introduced as a model system to study environmental, chemical, and pharmaceutical challenges to the embryonic nervous system that can affect adult behavior/learning. The characteristics of the zebrafish system that make it possible to examine the developing nervous system in live embryos and larvae are presented. Gene discovery techniques, methodologies to generate fluorescent transgenic indicator embryos, as well as larval and adult zebrafish behavioral assays are described

Transgenic mice expressing a constitutively active retinoic acid receptor in the lens exhibit ocular defects

Retinoic acid receptors (RARs) modulate gene expression following association with retinoic acid (RA). In transient transfection, an RARα-β-galactosidase fusion protein (RAR-LacZ) was able to transactivate expression in the absence of RA. When expressed in the ocular lens of transgenic mice, this constitutively active RAR-LacZ fusion gene resulted in founder and progeny animals that exhibited cataracts and microphthalmia, both being characteristics of retinoid-in-duced teratogenesis. The transgenic phenotypes indicate that retinoid teratogenesis can be mimicked by expression of a constitutively active RAR-LacZ fusion protein in retinoid-sensitive tissues