Jan M. Spitsbergen

The state of the art of the zebrafish model for toxicology and toxicologic pathology research--advantages and current limitations.

The zebrafish (Danio rerio ) is now the pre-eminent vertebrate model system for clarification of the roles of specific genes and signaling pathways in development. The zebrafish genome will be completely sequenced within the next 1—2 years. Together with the substantial historical database regarding basic developmental biology, toxicology, and gene transfer, the rich foundation of molecular genetic and genomic data makes zebrafish a powerful model system for clarifying mechanisms in toxicity. In contrast to the highly advanced knowledge base on molecular developmental genetics in zebrafish, our database regarding infectious and noninfectious diseases and pathologic lesions in zebrafish lags far behind the information available on most other domestic mammalian and avian species, particularly rodents. Currently, minimal data are available regarding spontaneous neoplasm rates or spontaneous aging lesions in any of the commonly used wild-type or mutant lines of zebrafish. Therefore, to fully utilize the potential of zebrafish as an animal model for understanding human development, disease, and toxicology we must greatly advance our knowledge on zebrafish diseases and pathology.

Conservation of gene expression signatures between zebrafish and human liver tumors and tumor progression.

The zebrafish (Danio rerio) has been long advocated as a model for cancer research, but little is known about the real molecular similarities between zebrafish and human tumors. Comparative analysis of microarray data from zebrafish liver tumors with those from four human tumor types revealed molecular conservation at various levels between fish and human tumors. This approach provides a useful strategy for identifying an expression signature that is strongly associated with a disease phenotype.

Pathologic alterations in early life stages of lake trout, Salvelinus namaycush, exposed to 2,3,7,8-tetrachlorodibenzo- p-dioxin as

Fertilized lake trout eggs exposed to vehicle or graded concentrations of [3H]-2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) in water for 48 h accumulated 0, < 15, 40, or 400 ppt (parts per trillion) [3H]-TCDD. The exposed eggs were then transferred to flowing [3H]-TCDD free water where they remained throughout early development. Lake trout embryos developed normally in all groups until one week prior to hatch. At this time retrobulbar, meningeal and subcutaneous hemorrhages were evident in many embryos and sac fry that were derived from eggs containing 400 ppt [3H]-TCDD. High mortality prior to or during hatching accompanied these lesions. All sac fry which survived hatching in the 400 ppt TCDD group developed severe subcutaneous edema, with cessation of blood circulation in the yolk sac and body. Necrosis of the retina, brain, and spinal cord occurred in morbid embryos and sac fry. All sac fry in the 400 ppt TCDD group showed arrested development from the time of hatching and all died prior to swim-up. One percent of sac fry in the vehicle control and < 15 ppt TCDD groups, and 2% in the 40 ppt TCDD group, exhibited blue-sac disease, an edematous syndrome identical grossly and nearly identical microscopically to that observed in the 400 ppt TCDD group. Thus, the cardiovascular system appears to be the initial tissue affected in both the TCDD toxicity syndrome and in blue-sac disease of developing lake trout. Lesions in other organs including brain, retina and liver develop as a result of circulatory derangements, anemia and hypoxia.

Zebrafish models for translational neuroscience research: from tank to bedside

The zebrafish (Danio rerio) is emerging as a new important species for studying mechanisms of brain function and dysfunction. Focusing on selected central nervous system (CNS) disorders (brain cancer, epilepsy, and anxiety) and using them as examples, we discuss the value of zebrafish models in translational neuroscience. We further evaluate the contribution of zebrafish to neuroimaging, circuit level, and drug discovery research. Outlining the role of zebrafish in modeling a wide range of human brain disorders, we also summarize recent applications and existing challenges in this field. Finally, we emphasize the potential of zebrafish models in behavioral phenomics and high-throughput genetic/small molecule screening, which is critical for CNS drug discovery and identifying novel candidate genes.

Neoplasia in Zebrafish (Danio rerio) Treated with 7,12-Diniethylbenz[a]anthracene by Two Exposure Routes at Different Developmental Stages

Using zebrafish, Danio rerio, initial pioneering work in the 1960s revealed carcinogen responsiveness of fish, yet very few subsequent tumorigenesis investigations have utilized this species. We exposed embryos (60 hours postfertilization) and fry (3 week posthatch) to 7,12-dimethylbenz[a]anthracene (DMBA) by immersion in aqueous solutions for 24 hours, at concentrations of 0-1 or 0-5 ppm (mg/L), respectively. Juvenile zebrafish 2 months posthatch were fed a diet containing 0-1,000 ppm DMBA for 4 months. Fish were sampled for histologic evaluation at 7-12 months after the onset of carcinogen treatment. Fry were most responsive to DMBA and showed the widest diversity of target tissues and histologic types of neoplasia, having several types of epithelial, mesenchymal, and neural neoplasia. The principal target tissues for carcinogenic response were liver following embryo or fry exposure, with gill and blood vessel the second and third most responsive tissues in fry. Intestine was the primary target and gill a secondary target in fish that received dietary DMBA as juveniles. These studies indicate that young zebrafish are most responsive to DMBA, showing a greater diversity of neoplasm types than rainbow trout. Thus, zebrafish are a valuable model system in which to study mechanistic aspects of the carcinogenesis process.

Naturally Occurring Thiamine Deficiency Causing Reproductive Failure in Finger Lakes Atlantic Salmon and Great Lakes Lake Trout

Abstract A maternally transmitted, noninfectious disease known as the Cayuga syndrome caused 100% mortality in larval offspring of wild-caught landlocked Atlantic salmon Salmo salar from several of New Yorks Finger Lakes. Survival of lake trout Salvelinus namaycush from Lakes Erie and Ontario was also impaired, but not until yolk absorption was nearly complete; moreover, mortality was greatly reduced relative to that of the salmon (range: 5–87%). Tissue concentrations of thiamine hydrochloride were severely reduced in these salmonid fish relative to unaffected control stocks. Afflicted Atlantic salmon treated with thiamine by yolk-sac injection or by bath immersion recovered completely from the Cayuga syndrome, as evidenced by the quantified reversal of abnormal swimming behaviors only 2 d after treatment and by the excellent survival (>95%) of the treated Atlantic salmon through 1.5 months of feeding. These data represent the first evidence of a vitamin deficiency causing the complete reproductive failu...

Eosinophils in the zebrafish: prospective isolation, characterization, and eosinophilia induction by helminth determinants

Eosinophils are granulocytic leukocytes implicated in numerous aspects of immunity and disease. The precise functions of eosinophils, however, remain enigmatic. Alternative models to study eosinophil biology may thus yield novel insights into their function. Eosinophilic cells have been observed in zebrafish but have not been thoroughly characterized. We used a gata2:eGFP transgenic animal to enable prospective isolation and characterization of zebrafish eosinophils, and demonstrate that all gata2(hi) cells in adult hematopoietic tissues are eosinophils. Although eosinophils are rare in most organs, they are readily isolated from whole kidney marrow and abundant within the peritoneal cavity. Molecular analyses demonstrate that zebrafish eosinophils express genes important for the activities of mammalian eosinophils. In addition, gata2(hi) cells degranulate in response to helminth extract. Chronic exposure to helminth- related allergens resulted in profound eosinophilia, demonstrating that eosinophil responses to allergens have been conserved over evolution. Importantly, infection of adult zebrafish with Pseudocapillaria tomentosa, a natural nematode pathogen of teleosts, caused marked increases in eosinophil number within the intestine. Together, these observations support a conserved role for eosinophils in the response to helminth antigens or infection and provide a new model to better understand how parasitic worms activate, co-opt, or evade the vertebrate immune response.

An inducible krasV12 transgenic zebrafish model for liver tumorigenesis and chemical drug screening

SUMMARY Because Ras signaling is frequently activated by major hepatocellular carcinoma etiological factors, a transgenic zebrafish constitutively expressing the krasV12 oncogene in the liver was previously generated by our laboratory. Although this model depicted and uncovered the conservation between zebrafish and human liver tumorigenesis, the low tumor incidence and early mortality limit its use for further studies of tumor progression and inhibition. Here, we employed a mifepristone-inducible transgenic system to achieve inducible krasV12 expression in the liver. The system consisted of two transgenic lines: the liver-driver line had a liver-specific fabp10 promoter to produce the LexPR chimeric transactivator, and the Ras-effector line contained a LexA-binding site to control EGFP-krasV12 expression. In double-transgenic zebrafish (driver-effector) embryos and adults, we demonstrated mifepristone-inducible EGFP-krasV12 expression in the liver. Robust and homogeneous liver tumors developed in 100% of double-transgenic fish after 1 month of induction and the tumors progressed from hyperplasia by 1 week post-treatment (wpt) to carcinoma by 4 wpt. Strikingly, liver tumorigenesis was found to be ‘addicted’ to Ras signaling for tumor maintenance, because mifepristone withdrawal led to tumor regression via cell death in transgenic fish. We further demonstrated the potential use of the transparent EGFP-krasV12 larvae in inhibitor treatments to suppress Ras-driven liver tumorigenesis by targeting its downstream effectors, including the Raf-MEK-ERK and PI3K-AKT-mTOR pathways. Collectively, this mifepristone-inducible and reversible krasV12 transgenic system offers a novel model for understanding hepatocarcinogenesis and a high-throughput screening platform for anti-cancer drugs.

Normal anatomy and histology of the adult zebrafish.

The zebrafish has been shown to be an excellent vertebrate model for studying the roles of specific genes and signaling pathways. The sequencing of its genome and the relative ease with which gene modifications can be performed have led to the creation of numerous human disease models that can be used for testing the potential and the toxicity of new pharmaceutical compounds. Many pharmaceutical companies already use the zebrafish for prescreening purposes. So far, the focus has been on ecotoxicity and the effects on embryonic development, but there is a trend to expand the use of the zebrafish with acute, subchronic, and chronic toxicity studies that are currently still carried out with the more conventional test animals such as rodents. However, before we can fully realize the potential of the zebrafish as an animal model for understanding human development, disease, and toxicology, we must first greatly advance our knowledge of normal zebrafish physiology, anatomy, and histology. To further this knowledge, we describe, in the present article, location and histology of the major zebrafish organ systems with a brief description of their function.

Morphologic lesions and acute toxicity in rainbow trout (Salmo Gairdneri) treated with 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin

To determine effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on growth, mortality, and morphologic lesions in rainbow trout, juvenile Shasta or Wytheville strain fish, obtained from 4 hatcheries, were administered graded single doses of TCDD, 0.1-125 micrograms/kg, ip. TCDD doses of 25 and 125 micrograms/kg caused 85% lethality 2-4 wk after treatment. At these high doses, death occurred before body weight loss could be detected. A lower dose of 5 micrograms/kg caused decreased growth and cumulative mortality of 20% after 11 wk. Stress associated with netting and weighing the fish at weekly intervals significantly shortened the delay period prior to TCDD-induced lethality. Gross and microscopic lesions were evident in rainbow trout treated with 10 micrograms TCDD/kg, but not in fish treated with 1 or 0.1 microgram/kg. Morphologic lesions occurred consistently in epithelial and lymphomyeloid tissues of TCDD-treated fish. Lymphomyeloid lesions included thymic involution, splenic lymphoid depletion, and hypocellularity of hematopoietic tissues in the head kidney and trunk kidney. In association with decreased hematopoiesis, peripheral leukopenia and thrombocytopenia occurred in Shasta strain yearling trout treated with 1 microgram/kg or more TCDD. Regarding epithelial lesions, all 4 hatchery strains treated with 10 micrograms/kg or more TCDD showed multifocal necrosis of gastric cardiac glandular mucosa, 3 of 4 hatchery strains showed vacuolar inclusions in exocrine pancreatic cells, and 2 of 4 hatchery strains showed fin necrosis. The severity and character of lesions in the liver and gastric mucosa varied markedly between hatchery strains of trout. One hatchery strain showed no hepatic lesions, two showed mild hepatocyte lesions, and one exhibited severe diffuse hepatopathy. In this severely affected hatchery strain, hyaline intracytoplasmic inclusions occurred in hepatocytes at 14 and 34 d after TCDD exposure, and bile-duct hyperplasia occurred at 34 d following TCDD exposure. One of 4 hatchery strains showed atrophy of serous gastric glands and 1 of 4 hatchery strains showed hyperplasia of these same glands at 25 and 34 d, respectively, following TCDD treatment. Thus, lymphomyeloid and epithelial tissues are the primary targets for TCDD-induced pathologic lesions in rainbow trout, and the incidence and severity of these lesions is influenced by the strain of trout used and the hatchery from which the trout were obtained.