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2003 31: 62Toxicol PatholJan M. Spitsbergen and Michael L. Kent

Advantages and Current Limitations−−The State of the Art of the Zebrafish Model for Toxicology and Toxicologic Pathology Research

  

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TOXICOLOGIC PATHOLOGY, vol 31(Suppl.), pp 62–87, 2003Copyright C© 2003 by the Society of Toxicologic PathologyDOI: 10.1080/01926230390174959

The State of the Art of the Zebrafish Model for Toxicology and ToxicologicPathology Research—Advantages and Current Limitations

JAN M. SPITSBERGEN1,2 AND MICHAEL L. KENT2,3

1Department of Environmental and Molecular Toxicology and Marine/Freshwater Biomedical Sciences Center,Oregon State University, Corvallis, Oregon 97333

2Zebrafish International Resource Center, University of Oregon, Eugene, Oregon 97403-5274, and3Center for Fish Disease Research, Department of Microbiology, Oregon State University, Corvallis, Oregon 97331-3804

ABSTRACT

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

Keywords. Zebrafish;Danio rerio; genomics; molecular genetics; development; toxicologic pathology; carcinogenesis; toxicology.

INTRODUCTION

As the most numerous and phylogenetically diverse groupof vertebrates, fish teach us important principles about fun-damental processes in vertebrate evolution, development anddisease processes. For over 100 years, fish from primitivehagfish to advanced reef fish have yielded unique insightsinto cell biology, physiology, development, and immunology(258, 418, 545). A high level of conservation of genetic pro-grams controlling development and fundamental physiologicprocesses is present among all vertebrates, as well as betweeninvertebrates and vertebrates. Endocrine systems are highlyconserved between fish and other vertebrates. Fish possessmost of the tissue types of mammals except breast, prostateand lung. Fish have served as useful sentinels to detect envi-ronmental hazards, and as efficient, cost-effective model sys-tems for mechanistic toxicology and risk assessment for manydecades (206, 208). In choosing a model system for conduct-ing particular research, it is essential to realize that no singlemodel is best for addressing all biomedical questions. Eachmodel species has unique strengths and weaknesses (576).

STRENGTHS OF THEZEBRAFISHMODEL SYSTEM

Zebrafish and other aquarium fish species have distinct ad-vantages as models for biomedical research including muchlower husbandry costs than mammals. Oviparous species in-cluding the zebrafish have external fertilization and develop-ment, facilitating access for observation and manipulation ofdeveloping embryos. Oviparous fish species can be cloned

Address correspondence to: Jan Spitsbergen, Deparment of Environ-mental and Molecular Toxicology, 1007 Agricultural and Life SciencesBuilding, Oregon State University, Corvallis, Oregon 97333; e-mail:spitsbej@onid.orst.edu

quite easily, allowing genetic manipulation for study of hap-loids, triploids, or tetraploids with androgenesis or gynogen-esis (107, 489). Zebrafish are easily housed in compact recir-culating systems, breed continuously year-round, and haveshort generation times of approximately 3–5 months (125).The small size of adult zebafish allows efficient, low-costevaluation of all major organs on a limited number of slides(168). The small size of embryos and fry minimizes the costand waste volume for drug and toxicant studies. Thus minuteamounts of expensive metabolites or new targeted drugs canbe rapidly evaluated.

Unique Advantages of the Zebrafish Model for Studyof Development

Among vertebrates, the zebrafish embryo has unrivaledoptical clarity, allowing visual tracing of individual cell fatesthroughout organogenesis. Fluorescent dyes or other mark-ers aid in visualization of cell lineages (104, 105, 273, 279,474). If inhibitors of pigment formation are included in therearing medium, cell lineages can be traced throughout thefirst week of zebrafish development, and immunohistochem-ical or immunofluorescence studies will reveal specific celltypes in whole mount preparations (269). Histological sec-tions of larvae over 1 week of age are required to localizespecific cell types. Alternatively, confocal microscopy canoptically section these thicker larvae (323). A wide array ofhistochemical markers for protein and gene expression allowsidentification of essentially all cell types, and often revealsfunctional capabilities such as synthesis of nitric oxide orspecific neurotransmitters, during development of the majortissues (75, 77, 82, 103, 111, 171, 240, 261, 520, 533, 536,579, 583).

Immunohistochemical studies have quantified cell prolif-eration and cell death in specific tissues during development

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(100, 284, 578). The optical clarity of the embryo coupledwith detailed understanding of basic developmental pro-cesses and a well-established timetable for specific develop-mental milestones allows elegant embryonic manipulationsto distinguish the relative influences of the genetic compo-sition of a specific cell (cell autonomous effects) versus theinfluences of the genetic suite of its surrounding environ-ment (non–cell-autonomous effects). For example, at a pre-cise stage of development, specific neurons can be removedfrom the spinal cord using a micropipette, and can be replacedby those from fish of a different genotype. Then the impact onneuronal fate and innervation of skeletal muscle can be deter-mined (142). Or during various time points in development,single cells, or cell clusters can be removed from specificanatomic fields in the embryo and relocated to other sites toclarify the processes controlling cell fate determination, andrevealing when the fate of certain cell types is irreversiblyspecified (224, 356).

DNA or RNA constructs can be readily microinjected intoembryos at the 1-cell or 2-cell stages to study effects oftransient gene expression. More uniform tissue expression isachieved with RNA injection (228). With injection of RNAat the 2-cell stage, typically half of the embryo expresses theexogenous construct, with the other half acting as an elegantinternal control. Using constructs with a promoter such asthat from a heat shock gene, laser probes can elicit transientexpression of injected constructs in precise cell types at exactstages of development (203, 238, 288).

Because of these advantages of zebrafish for basic devel-opmental biology and molecular development studies, thezebrafish has emerged as the premier vertebrate model forclarification of the roles of specific genes and signaling path-ways in development. The past decade has seen intense world-wide research into molecular genetic mechanisms in cell fatedetermination, pattern formation, morphogenesis and func-tional maturation of heart, blood vessels, brain, eye, ear, nose,neural crest, muscle, cartilage, bone, lymphomyeloid system,skin, kidney, and gonad.

Cell Culture Techniques Using Zebrafish TissuesCell culture methods are established to create primary and

immortal cell lines from adult tissues as well as from embryos(101, 182, 183, 210, 292). Also explants of embryos and adulttissues, such as whole brain, can be cultured to study cell-cellinteractions and metabolism (509).

Zebrafish Genomics InitiativeSeveral transinstitute funding initiatives by the National

Institutes of Health (NIH) in the past 5 years have spawned aflurry of activity using the zebrafish as an animal model forunderstanding human development and disease. The NIH hasfunded research in zebrafish genomics for the past decade.Recently, Britain’s Wellcome Trust has put the zebrafish onthe fast track for complete genome sequencing, with a roughdraft to be available by the summer of 2003. Among fishspecies, the most complete database on genomics, moleculargenetics and embryology is available for the zebrafish. Thesedata are accessible through the Zebrafish Information Net-work <http://zfin.org/ZFIN> (559) coordinated in conjunc-tion with the NIH-funded Zebrafish International Resource

Center (ZIRC) at the University of Oregon. Updated sequenc-ing information is posted on the Sanger Institute/WellcomeTrust web site <http://trace.ensemble.org>.

Zebrafish Comparative GenomicsAlthough a rough draft of the human genome is now avail-

able, the function of most mammalian genes remains un-known. Because of the rapid embryonic development, op-tical clarity of zebrafish embryos, and efficiency of mutantscreens in zebrafish, we are likely to understand the role ofevery gene in development in the zebrafish before we havesuch knowledge for any other vertebrate. It will take muchlonger to clarify the role of these genes in diseases occurringlater in life.

The Zebrafish GenomeZebrafish have 25 pairs of chromosomes compared to

23 pairs in humans. Evolutionary genetic data indicate that awhole-genome duplication event occurred early in the teleostlineage, after separation of the fish lineage from the tetrapodlineage. So for many genes of mammalian species, duplicategenes (paralogs) occur on separate chromosomes in bony fish.Not all of these gene duplicates have been preserved duringevolution (40, 161, 242, 346, 413, 414). On average about20% of the pairs of duplicated genes have been preserveddue to neofunctionalization or subfunctionalization. In neo-functionalization, an entirely new function for a gene evolves.In subfunctionalization, for example, if the original gene ofmammals was expressed in 4 tissues during development,each of the duplicate genes of a bony fish might be expressedin 2 of the 4 tissues (166). Although many scientists contendthat fish are simpler model systems than mammals, in termsof the molecular genetics, fish are actually more complex. Anadditional whole-genome duplication likely occurred in troutand salmon following the initial teleost genome duplication,so the salmonids will often have 4 co-orthologs for each geneof mammals (68).

Mutations in one of a pair of duplicated genes in zebrafishmay give rise to a simpler phenotype than mutation of theortholog in mammals. Also, in cases in which mutation ofthe mammalian ortholog is embryonic lethal, mutation ofone of the zebrafish paralogs may give rise to viable ani-mals, allowing better definiton of gene function and clearerunderstanding of the roles of the gene in signaling pathways.

Investigators worldwide collaborated to create anoligonucleotide-normalized and expressed sequence tag-characterized zebrafish cDNA library which has been used formapping zebrafish genes, for in situ hybridization studies ofgene expression, and now is being used to develop microarraytechnology for comprehensive evaluation of stage-specificgene expression in zebrafish (97, 289). ZFIN contains a vir-tual web-based map of the zebrafish genome, integrating datafrom the various gene mapping projects. A physical map ofthe zebrafish genome is available, constructed using fluores-cence in situ hybridization (FISH) (407). At least 28 groupsof genes are syntenic (together on a single chromosome) inzebrafish and humans (28). This shared synteny has aidedidentification of and positional cloning of candidate diseasegenes (498, 499).

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64 SPITSBERGEN AND KENT TOXICOLOGIC PATHOLOGY

Zebrafish Mutant ScreensOver a decade ago, geneticist Christiane Nüsslein-Volhard

chose zebrafish as the best vertebrate model system for clar-ifying the roles of specific genes in development. Patterningthese studies in zebrafish upon her highly successful Nobel-Prize-winning saturation mutagenesis studies that elucidatedthe role of every gene and every signal pathway in fruit flydevelopment, Nüsslein-Volhard collaborated with WolfgangDriever, then at Massachusetts General Hospital, to conductthe first large-scale mutagenesis studies in zebrafish (72, 112,199, 241, 303, 380). These studies were spectacularly suc-cessful, generating thousands of mutant lines of zebrafishwith defects in essentially every major organ system, aswell as mutant lines with defects in basic embryo pattern-ing. A special issue of the journalDevelopmentin 1996 wasdevoted to describing this cornucopia of mutant lines. Thesuccess of these initial mutant screens has stimulated manyresearch centers around the world to continue conductingmutant screens, working toward a complete understandingof molecular, cellular, and functional development of all ze-brafish tissues and organ systems. Initial screens focusedon several parameters including morphology of embryos us-ing stereoscopic and Nomarsky optics, neuronal pathway ar-rangements, and markers for expression of panels of genes.Now the focus of mutant screens using zebrafish is broaden-ing to consider functional parameters such as lipid digestion(64, 110, 153), behaviors such as mating, the startle responseand feeding, and diseases such as neoplasia or skeletal mal-formations occurring later in life (5, 160, 397). The ingenu-ity of future mutant screens will be limited mainly by man-power and funding—these screens are very labor-intensiveundertakings.

Creating Mutant FishTo date, totipotent embryonic stem cells are not avail-

able for any fish species. Therefore the homologous recom-bination methods used for creating knockout mice cannotbe used in fish. Chemical mutagens includingN-nitroso-N-ethylurea (ENU; 282), psoralens (11), radiation (543), in-sertional mutagenesis using pseudotyped retroviruses (173,174, 186), or dominant negative transgenes can be usedto create stable mutant lines of fish with “knocked down”gene function. For homozygous lethal or recessive muta-tions, screening of haploid embryos or early pressure-derivedembryos (35) containing the genetic material of just oneparent greatly speeds up identification of mutant pheno-types (87). Unlike mammalian species in which haploidembryos do not survive, haploid zebrafish embryos survivefor 4 days. These haploid embryos exhibit a well-definedsuite of brain abnormalities, however, most organs can bescreened for abnormalities associated with mutant genes(543). If transient gene “knockdown” is desired, morpholinoantisense oligonucleotides efficiently achieve null pheno-types for most genes evaluated early in development (106,144, 209). An alternative strategy for gene “knockdown,” al-beit a more technically challenging approach, is the use ofribozymes (544). Recently, the Cre-loxP system has beenused in zebrafish embryos to achieve precise control of geneexpression in a spatially and temporally restricted manner(587).

Creating Transgenic ZebrafishTransgenic zebrafish are useful for study of the pheno-

types resulting from overexpression of selected genes glob-ally, or in a tissue-targeted fashion (256, 313, 398). Trans-genic technology also allows “knockdown” of gene functionusing dominant negative transgenes. Recently, growing num-bers of tissue-specific promoters are becoming available toallow very precise targeting of transgene expression (188,221, 222, 238, 255, 286). Transgenic zebrafish with the greenfluorescent protein marker under control of a specific pro-moter can highlight stage-specific, tissue-specific and cell–type-specific expression of selected genes in developing andadult zebrafish (364, 402).

Transgenes are most commonly introduced by microin-jection of constructs into newly fertilized eggs. Electropora-tion of eggs or sperm has also been used. The most efficientmethod for introducing transgenes into zebrafish eggs uti-lizes pseudotyped retroviruses (173, 174, 186); however, useof retrotransposons and integrase is continually being opti-mized to achieve higher efficiency of stable integration ofmicroinjected transgenes in developing zebrafish (96, 262).

Role of Zebrafish Mutants in Toxicology ResearchMutant lines of zebrafish will help clarify the roles of spe-

cific genes and their associated signaling pathways and net-works in the pathogenesis of toxicant-induced lesions. Dou-ble and triple mutants can clarify the interactions of suitesof genes, and these multiple mutants can be produced moreefficiently and cheaply in zebrafish than in rodents.

Zebrafish Mutant Models for Understanding HumanDevelopment and Disease

Numerous recent review articles have trumpeted praisesof the zebrafish model for understanding human develop-ment and disease (5, 30, 64, 128, 131, 133, 162, 282, 304,505, 530, 550, 552, 537, 539, 574, 592). Active research pro-grams are now present worldwide focusing on normal andabnormal development of essentially all organ systems andtissues shared by zebrafish and man, as well as on a variety ofpathologic lesions, physiologic processes, and disease con-ditions including aging, alcoholism, and drug addiction, forwhich zebrafish can provide mechanistic insight into humandisease pathogenesis.

Normal and Perturbed Hemopoiesis in ZebrafishSeveral hemato-oncology research groups at Harvard

Medical School including those of Leonard Zon and ThomasLook, Graham Lieschke’s group from the Ludwig Institutefor Cancer Research, Victoria, Australia, and Shu Lin’sgroup at the University of Georgia, together with othercollaborators have clarified the role of specific genes innormal and deranged hemopoietic development of zebrafish(6, 42, 53, 121, 310). Several mutant lines of zebrafish haveclinical syndromes resembling diseases of humans, suchas congenital sideroblastic anemia (67), X-linked siderob-lastic anemia (581), hepatoerythropoietic porphyria (547),erythroid myeloproliferative disorders (319). A transgeniczebrafish expressing aRUNX1-CBF2T1translocation showsa preleukemic syndrome (257). The genetic program forsequential specification of pluripotential precursors followed

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by divergence of distinct lineages of lymphoid, myeloidand erythroid cells is highly conserved from zebrafish tohumans, indicating that zebrafish will continue to providevaluable insights into additional lymphomyeloid disordersof humans (6).

The Zebrafish Immune SystemAlthough the immune system of zebrafish has been less

well studied than that of several fish species important incommercial aquaculture such as rainbow trout and channelcatfish, this field will grow in importance as we begin to un-derstand more about infectious diseases of zebrafish. Likeother bony fish, zebrafish have distinct lymphoid subsets,T and B cells. Willett et al (569–572) describe expressionof the recombination activating genes,rag 1 and rag 2 inzebrafish thymus and anterior kidney, the major histolologicsites for production of T cells and B cells, respectively, dur-ing zebrafish development. The Zon laboratory at Children’sHospital in Boston, MA has developed mutant lines withdefective T cell development. These mutant lines fall into5 distinct complementation groups (505, 515, 516). Zebrafishpossess several novel immune-type receptor genes in compar-ison to mammals. These genes may shed light on evolutionof the innate and adaptive immune systems of vertebrates(314). Recent data indicate that zebrafish novel immune-type receptor genes may represent evolutionary intermedi-ates in the establishment of the leukocyte receptor clusterfor cytokine responses in mammals (584). Class I and IImajor histocompatability proteins are mapped and charac-terized in zebrafish (492, 493, 497). The promoter regionof the Class II A genes is highly conserved between mam-mals and zebrafish, indicating conservation of gene regula-tory mechanisms (492). Independent duplications of Bf andC3 components of the complement system occurred duringevolution of the zebrafish (189). Haire et al (201) studiedimmunoglobulin isotypes and the T cell antigen receptor inzebrafish. Herbomel et al (216, 217) show that macrophagesappear in embryos concurrent with embryonic erythrocytes,but at a distinct location compared to other hemopoietic cells.Macrophages develop initially in mesoderm anterior to heart,while primary hemopoiesis occurs ventral to the tail, near theyolk extension. These embryonic macrophages constitute adistinct lineage from the monocyte lineage which gives rise tomacrophages in adult vertebrates including zebrafish (465).Embryonic macrophages require an intact M-CSF signalingsystem to disseminate properly to embryonic tissues, indi-cated by the failure of macrophage dispersal in the panthermutant lacking functional M-CSF receptors. Antigen recep-tors on nonspecific cytotoxic cells are similar in zebrafish andchannel catfish (251).

Blood Coagulation in ZebrafishSpecialized methods are required to analyze coagulation

parameters in zebrafish due to the small size of both early lifestages and adults, however, clotting times can be evaluatedin blood samples from larvae by 30 hours postfertilization(246, 249). Zebrafish possess both intrinsic and extrinsic clot-ting pathways containing the same clotting factors present inmammals. Anticoagulants such as warfarin and hirudin actsimilarly in zebrafish and mammals (244, 245, 247–250).

Although nucleated, zebrafish thrombocytes are functionallysimilar to those of mammals and circulate in the blood by36 hours postfertilization (247). Like mammals, thrombo-cyte activation in zebrafish is blocked by drugs inhibitingcyclooxygenase 1, such as aspirin, but not by inhibitorsof cyclooxygenase 2 (195, 247). Screens to detect mutantswith defective blood coagulation are underway and lines ofzebrafish with specific abnormalities in the coagulation sys-tem are evident (244).

Cardiovascular Development and Cardiovascular MutantsThe clarity of zebrafish embryos and fry, and the ability of

the fish to survive for several days without circulation due tothe diffusion of gases through the skin make this an excellentmodel for study of cardiovascular development (86, 163–165,309, 318, 321, 405, 456, 462, 479, 552, 553, 556, 582, 583).Mutant lines with defects in contractility (458, 580), rhyth-micity (551), heart size, heart tube patterning, valve mor-phogenesis, and cardiac looping are available. Thegridlock(gdl) mutant with abnormal circulation to the tail resemblescoarctation of the aorta in man. Babin et al (25) and Durliatet al (138) cloned the zebrafish orthologs for theapolipopro-tein E (apoE) and A-1 (apoA-1) genes of humans. Thesegenes regulate lipid uptake and distribution in mammals andApoEplays a role in Alzheimer’s disease in man.ApoEandA-1 are highly expressed in the yolk syncytial layer in ze-brafish embryos, a tissue which controls yolk assimilation,and apoE is expressed in brain and eye of developing ze-brafish. The roles of these genes in cardiovascular and braindevelopment and disease in zebrafish remain to be explored.Brant Weinstein of the National Institutes of Health coordi-nates a Web site featuring images of zebrafish cardiovasculardevelopment <http://mgchd1.nichd.nih.gov:8000/zfatlas> or<http://dir.nichd.nih.gov/lmg/uvo/WEINSLAB.html>. Thesite features current information on comparative vasculardevelopment of various organs.

Neural System and Neural CrestSome of the most intense research in the zebafish model

has focused on genetic mechanisms of cell specification andmorphogenesis of the nervous system (52, 142, 357, 358).Mutant lines with extremely specific defects in most compo-nents of the nervous system are available. Also mutant lineswith defects in neural connections are established (237). Theontogeny of specific behaviors is well defined (156, 172, 443),and neurologic functions such as sleep are being investigated(211). Calcium fluxes can be visualized in individual neuronsin the central nervous system of live fry during behaviors suchas the escape response (155, 496, 590). Neuronal metabolicprofiles are described (534).

Migration pathways of neural crest cells and sequentialspecification of particular cell types are documented in ze-brafish (143, 176, 275, 377, 513). Mutant lines with specificdefects in certain aspects of neural crest development areavailable, with lesions including abnormal pigment patterns,abnormal jaw development, and abnormal enteric neural tis-sue (20, 140, 213, 265, 343, 394).

Sensory Systems Including Eye, Ear, Taste Bud, and NoseAt least 50 mutant lines with lesions affecting specific cells

or layers of the eye are available (46, 65, 327, 328, 375, 541).

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66 SPITSBERGEN AND KENT TOXICOLOGIC PATHOLOGY

Also, more sophisticated tests of visual function and ocu-lar motion are being developed (26, 146, 185, 306, 307).All zebrafish mutants with lamination defects in the retinahave anomalies of the retinal pigment epithelium (RPE). Thissuggests that the RPE controls retinal lamination during mor-phogenesis of eye. Themosaic eyes(moe) mutant shows lossof normal retinal lamination, with loss of the normal local-ization of dividing retinal cells to the surface of the RPE.Cell–type-specific antibodies and riboprobes show that allretinal cell types differentiate normally, but are misplaced inthemoemutant (252). Normal and abnormal morpohologicand functional development of the olfactory system is under-stood in detail (287, 426, 561–563). Recent work by Jessenet al (253) shows that the recombination activation genes,rag1 and rag2, which function to generate diversity in theimmune system, may perform a similar function in gener-ating a large repertoire of olfactory receptors. Cellular andmolecular mechanisms controlling development and func-tion of ear (21, 44, 99, 361, 406, 557) and tastebuds (204) areless well understood than other sensory systems in zebrafish,but are currently being investigated.

Normal and Abnormal Kidney DevelopmentEmbryo and fry stages of zebrafish possess a pronephros

with a single glomerulus, whereas juvenile and adult stageshave a mesonephric kidney. Recent investigations have clar-ified molecular and cellular aspects of renal developmentin zebrafish (132, 134, 135, 324–326, 460, 462). Althoughmany aspects of renal development are conserved from ze-brafish to mammals, in contrast to mammals, glial-derivedneurotrophic factor (GDNF) is not required for kidney mor-phogenesis in zebrafish (466). Several zebrafish mutants in-cludingdouble bubbleresemble human autosomal dominantpolycystic kidney disorders.Fleer andelipsadisplay renal-retinal dysplasia similar to that seen in the human Senior-Loken syndrome (135, 271). Zebrafish with inactivating mu-tations in the homeobox genetcf2(vhnf1) show kidney cysts,underdevelopment of pancreas and liver, and reduction in sizeof the otic vesicles (494). This zebrafish mutant line is a goodmodel for study of the MODY5 syndrome (maturity-onset di-abetes of the young, type V) in humans and familial GCKD(glomerulocystic kidney disease).

Musculoskeletal SystemMuch is understood regarding the timing and genetic

mechanisms controlling development of the axial and ap-pendicular skeleton of zebrafish (158, 362). Shannon Fisherof Johns Hopkins University School of Medicine is screen-ing mutant lines using radiography to detect lesions in theskeletal system (160). The zebrafishchihuahua(chi) mutantshows skeletal dysplasia resembling osteogenesis imperfectain man (159).

Molecular and cellular development of skeletal muscle,and neuromuscular function in normal and abnormal ze-brafish are well studied, although not yet fully understood(39, 73, 74, 200, 224, 229, 236, 363, 376). Mutant screenshave identified over 50 genes essential for normal somite de-velopment (136, 137, 486). Mutant lines with abnormalitiessimilar to human Duchenne muscular dystrophy are available(54, 55, 83, 396). Mutant lines with altered motility due to

abnormal neurotransmitter receptors on nerve and muscle aredescribed (385, 459).

Enteric Tract and LiverStudies of normal and abnormal development of the enteric

tract and liver have continued since the initial mutant screenswere conducted in 1996 (98, 193, 285, 390, 424, 425). Mu-tants with abnormally large or small livers, or with abnormalpositioning of the gut and liver are documented (157). Guoet al (198) are using microarray analysis of stage-specificgene expression combined with forward and reverse geneticanalyses to clarify the roles of specific genes in liver develop-ment. Cheng et al (88) describe dysplastic histologic featuresin gut of early life stages of certain mutant lines of zebrafish.

Endocrine Organ SystemsGrowing international interest is focused on development

and function of endocrine systems of vertebrates due to theconcern regarding endocrine-disrupting agents in the envi-ronment. Whereas most endocrine organs are similar struc-turally and functionally in fish and mammals, fish have someunique features. Fish interrenal and chromaffin tissue, the ho-mologs of adrenal medulla and cortex, respectively, are notencapsulated and are located surrounding veins in the ante-rior kidney. Zebrafish thyroid is also not encapsulated andis located surrounding the ventral aorta as it emerges fromthe bulbus arteriosus. Fish, like birds, have ultimobranchialbodies, homologs of mammalian medullary thyroid tissue,which are located in the transverse septum between heart andesophagus. Surprisingly, although the zebrafish does not havea parathyroid gland, it has functional parathyroid hormonesand receptors (438, 439, 524).

Cellular and molecular mechanisms controlling pituitarydevelopment are active topics of current research (139, 293,440, 448), however, knowledge about development of this or-gan is currently less than that regarding most organ systemsof zebrafish. More information is available regarding devel-opment of endocrine pancreas (19, 45, 193, 349, 437), thyroid(66, 149, 150, 316, 317, 417, 425, 431, 558), and pineal gland(167, 192, 263, 589). Whitlock et al (564) show a dual originof cells secreting gonadotropin-releasing hormone (GnRH),with these cells arising in both neural crest and in pituitaryplacode.

Germ Cell Specification, Gonadal Patterningand Sex Determination

Confirmation of expression of the evolutionarily conservedvasagene as a marker of germ cells in zebrafish has facil-itated study of migration, cell proliferation and cell deathpatterns of this cell type (57). Weidinger et al (554, 555) andStars-Gaiano and Lehmann (483) describe the normal migra-tion patterns of zebrafish germ cells and document abnormalhoming of germ cells to presumptive gonad in certain mu-tant lines. Uchida et al (523) document histologic changesoccurring during early differentiation of gonad into patternscharacteristic of male or female. Chiang et al (92, 93) revealthe role ofsox9genes in gonadal differentiation and in latergonadal function in zebrafish. Bauer and Goetz (32) report avariety of mutant lines of zebrafish with abnormal develop-ment of male and female gonads, including lines with defects

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in various stages of spermatogenesis or oocyte maturation.Some information is available on the structure of zebrafishGnRH (415, 517) and gonadotropin receptors (293). Wu et al(577) and Pang and Ge (392, 393) outline signaling pathwayscontrolling ooctye maturation in zebrafish. Li et al (308) suc-cessfully achievedin vitro maturation of zebrafish oocytes,documenting a culture system that may prove useful in fu-ture toxicant studies. Likewise, an in vitro culture system isoptimized to produce functional sperm from zebrafish germcells (444). Zebrafish will provide a model system for studyof gender-related differences in toxicant responses (113).

Basic Embryo Patterning and Craniofacial DevelopmentMuch of the initial embryology studies as well as studies of

mutant lines focused on cellular and molecular mechanismsfor determination of basic body plans and cell fates (273,276–279, 347, 351, 365, 366, 451, 452, 473, 474, 511). Theinitial Tuebingen mutant screen identified a wide variety ofmutants with defective epiboly, axis pattterning, gastrulation,dorso-ventral patterning, or anterior-posterior patterning. Anexample of defective patterning clarifying mechanisms ofhead formation is theheadless(hdl) mutant. This phenotypeoccurs due to defectivetcf3(T-cell factor 3). Appropriatetcf3expression is necessary to repress specificwnt target genes,allowing head formation (270). The1-eyed pinhead(oep)mutant shows defects in L-R asymmetry mimicking somesitus inversussyndromes of man such as dextrocardia, cardiacseptal defects, or gut malrotation associated with mutationsin theCFC gene (47, 49, 194, 450).

Normal and abnormal craniofacial development in ze-brafish has been studied intensely (350, 408, 409, 451). Avariety of mutant lines show phenotypes resembling congen-ital malformations occurring in humans.

Study of Aging Using the Zebrafish ModelKarlovich of Stanford University and colleagues (259)

cloned the Huntington’s disease gene homolog from ze-brafish, and will develop a zebrafish model for this progres-sive neurodegenerative disease of man. Leimer et al (302)cloned thepresenilingene of zebrafish to begin developing amodel of Alzheimer’s disease. Musa et al (367) studied earlylife stage expression of 2 zebrafish orthologs of human amy-loid precursor protein, and found distinct expression patternsfor each of these paralogs in zebrafish. Rubenstein et al (440)are developing a zebrafish model for the study of Parkinson’sdisease, and have demonstrated destruction of dopamin-ergic neurons in zebrafish embryos following treatmentwith 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP),an agent associated with drug-induced Parkinsonism in man.

DATA REGARDING TOXICANT EXPOSURE INZEBRAFISH

Metabolism of Endogenous and Exogenous Agentsand Pharmacokinetics

The database on metabolism in zebrafish is much less com-plete than that available for certain other highly studied fishspecies such as rainbow trout. More data are available re-garding Phase I than Phase II metabolism in zebrafish. Sev-eral cytochrome P450 enzymes from zebrafish have beenmapped, but full cDNA sequences are in the public do-main only forcyp19a, cyp19b, andcyp26. Cyp1a1 (P4501a1)

activity is induced in adults (69, 518) as well as in earlylife stages of zebrafish (13), and in liver cell cultures (102,214, 353) by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).The aryl hydrocarbon receptor (AHR) signaling pathway hasbeen studied extensively in zebrafish (1, 12, 416, 500, 501).Stage-specific expression ofahr, theahr nuclear transloca-tor (arnt), andcyp1a1are documented in early life stages ofzebrafish (13, 335, 336).

Paralogs of aromatase are present on two separate chro-mosomes in zebrafish, due to an ancient chromosomal du-plication event. These enzymes play key roles in sex steroidmetabolism, facilitating androgen conversion to estrogens.Zebrafishcyp19ais expressed in ovary, whereascyp19bisexpressed in brain (93). Appropriate tissue-specific expres-sion of these enzymes is required for normal sexual devel-opment (76, 280, 510, 514). Steroid glucuronides, producedin zebrafish testis by metabolism of androgens, are potentpheromones stimulating mating behavior in females (529).Hu et al (235) and Lai et al (295) studied the regulation ofsteroidogenesis in zebrafish. Zebrafishcyp26plays an essen-tial role in retinoic acid catabolism in zebrafish and mam-mals, allowing precise local regulation of retinoid acitivity(320, 422).

Keizer et al (264) studied species differences in acetyl-choline esterase inhibition by diazinon in fish. They foundthat zebrafish are relatively resistant to diazinon compared toother fish species, because their acetylcholine esterase is rel-atively resistant to inhibition by this pesticide. Donnarummaet al (130) investigated glutathioneS-transferase activity inliver of several fish species. They found no activity of thisenzyme toward 1,2-epoxy-3-(p-nitrophenoxy) propane in ze-brafish liver.

Relatively scant data are available regarding pharmacoki-netics of drugs or environmental agents in zebrafish. Hertland Nagel (220) and Zok et al (591) studied bioconcentrationand metabolism of various substituted anilines in zebrafish.Petersen and Kristensen (403) evaluated uptake and elimina-tion of polycyclic aromatic hydrocarbons by early life stagesof zebrafish. Gorge and Nagel (190) studied toxicokineticsand metabolism of lindane and atrazine in eggs, larvae and ju-venile zebrafish. Andersson et al (9) compared PCB uptake inmature zebrafish from the diet, from intraperitoneal injection,and from implanted intraperitoneal silicone capsules. Neilsonet al (374) investigated bioconcentration, metabolism, andtoxicity of components of bleached pulp effluent to variouslife stages of zebrafish. Wiegand et al (568) evaluated uptake,metabolism and toxicity of the algal toxin, microcystin, fol-lowing bath treatment of various early life stages. Labrot et al(294) studied toxicokinetics of lead and uranium in zebrafish.Wicklund et al (565) investigated effects of zinc on uptake,distribution and elimination of cadmium in zebrafish. Mostpublished zebrafish studies report exposure concentrations,but not tissue concentrations of toxicants, so that quantita-tive comparison of sensitivity between zebrafish and otherspecies is difficult.

Toxic Endpoints in Risk Assessment—Noncancer EndpointsFor decades worldwide, the zebrafish has been used exten-

sively for environmental toxicity testing, in acute and chronicbioassays to generate mandated water quality criteria. Be-cause of the rapid development and optical clarity of early

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life stages, the bulk of the toxicology data for zebrafish re-gards early life stages. To date, the great power of our currentknowledge in molecular genetics, genomics, and mechanisticdevelopmental biology have not been fully utilized in toxi-cology studies. We now have the tools to answer very specificquestions in many areas of toxicology and toxicologic pathol-ogy. Unfortunately, the strain and source of zebrafish used inmany toxicology studies is not reported in the publications.Generalizations regarding the zebrafish based on data froma single genetic line will not likely predict responses for themany wild-type lines and thousands of mutant lines. Much ofthe basic toxicity data for zebrafish is in unpublished reports.

Acute, Subacute, and Chronic Toxicity StudiesAnsari and Kumar (17) studied effects of the insecticide

diazinon on liver protein and nucleic acid metabolism in adultzebrafish. Kumar and Ansari (290) investigated liver toxic-ity of malathion. Roex et al (429) assessed acute toxicity ofparathion and chlorobenzene. Zok et al (591) studied acutetoxicity of substituted anilines. Lanzky and Halling-Sorensen(297) found metronidazole to have low acute toxicity to ze-brafish. Meinelt et al (341) investigated the influences ofcalcium concentration and humic acid on toxicity of acri-flavine to juvenile zebrafish. Roche et al (428) studied acuteand chronic toxicity of colchicine in zebrafish. Labrot et al(294) investigated acute toxicity of lead and uranium in ze-brafish, finding lead relatively nontoxic and uranium highlytoxic. Van den Belt et al (527) evaluated acute toxicity ofcadmium-contaminated clay to zebrafish. Ultraviolet-B lightinduces oxidant stress in adult zebrafish (85).

Early Life Stage ToxicityNearly every class of environmental contaminants has been

evaluated for early life stage toxicity in zebrafish. However,only a few recent studies have investigated molecular mech-anisms of toxicity. Early life stage toxicity of most metalsand several organometallics have been investigated in ze-brafish (91, 117–120, 184, 340, 378, 389, 391, 427, 436,447, 470, 490, 495). Toxicity of a variety of pesticides,organochlorines, and halogenated aromatic hydrocarbons isreported (43, 62, 116, 145, 190, 191, 196, 197, 215, 354,355, 383, 502, 566, 567). Willey and Krone (573) utilizedthevasagene as a marker of primordial germ cells to trackalterations in their homing to gonad caused by endosulfanor nonylphenol. Dong et al (129) appliedin situ terminaltransferase-mediated nick-end-labeling staining (TUNEL) todemonstrate increased cell death in the dorsal midbrain ofTCDD-treated embryos. These investigators could mimicthe TCDD-induced cell death with the AHR agonist beta-naphthoflavone, and prevent the increased cell death with theAHR antagonist alpha-naphthoflavone. Another recent studywhich exploited current molecular genetic tools available forzebrafish demonstrates that TCDD does not inhibit primitiveembryonic erythropoiesis, indicated bygata-1, gata-2mark-ers (41). However, TCDD prevents formation of later defini-tive erythropoiesis revealed by thesclmarker. TCDD causescraniofacial malformations in fish and mammals. TCDD re-duces normal expression of the signaling moleculesonichedgehog(shh) in zebrafish jaw (503). Toxicity of variousdrugs, endogenous signaling molecules and hormones have

been evaluated in zebrafish (33, 202, 218, 219, 224, 383,384, 538, 588). Akimenko and Ekker (2) show that fin mal-formations induced by exogenous all-trans retinoic acid areassociated with anterior duplication of expression domainsof shh. Various industrial chemicals and waste show adverseeffects in developing zebrafish (71, 79, 95, 147, 148, 177,296, 368, 370, 373, 388, 531, 532). Physical stresses suchas magnetic fields may perturb zebafish development (85,179, 488). Skauli et al (469) show an additive interaction ofmagnetic field stress with the hormone progesterone in caus-ing delayed hatching. The algal toxin microcystin suppressesgrowth following early life stage exposure (568).

Reproductive Toxicity and Endocrine DisruptionMost structural classes of toxicants including metals,

organochlorines, and pesticides (15, 18, 58, 148, 152, 429),halogenated aromatic hydrocarbons (387) substituted ani-lines (63), synthetic and natural estrogens (8, 526, 528), andother industrial chemicals (79, 230) have been evaluated forreproductive toxicity in zebrafish (61, 272, 293). Potential fordisruption of endocrine systems in the zebrafish model hasbeen assessed with a growing list of agents (301, 522).

Neurobehavioral ToxicityNeurobehavioral effects of a variety of toxicants, drugs,

and alcohol have been evaluated in developing or adult ze-brafish (491). Samson et al (447) found that impairment ofswimming and predator/prey behavior were much more sen-sitive indicators of toxicity in zebrafish exposed as earlylife stages to methylmercury than were mortality or mor-phologic lesions. Both early and recent studies have investi-gated pathologic lesions and functional impairment of devel-oping zebrafish exposed to ethanol (51, 115, 291). Gerlai et al(181) are using zebrafish to assess genetic factors predispos-ing lines of fish to alcohol preference. Darland and Dowling(114) are screening mutant lines of zebrafish to identify thosewith increased preference for exposure to cocaine and for al-tered responses to cocaine. Thomas (506) studied effects oftetrahydrocannabinol (THC) from marijuana in developingzebrafish.

Disease Resistance and Immune StatusAssays for assessment of disease resistance and immune

competence are developed to a much greater extent in themedaka (34, 78, 586) than in zebrafish. Treatment of zebrafishwith zinc or copper did not consistently increase their sus-ceptibility to intraperitoneally injectedListeria (435). Zincexposure suppresses the humoral immune response againstProteus vulgaris, but not against infectious pancreatic necro-sis virus in adult zebrafish (449).

Biomarkers in ZebrafishSurrogate tests to predict chronic toxicity are gaining favor

for environmental monitoring of human health and other sen-tinel species. Data regarding several biomarkers is availablein zebrafish. Induction of the cytochrome P4501a1 (Cyp1a1)enzyme has been extensively used to indicate exposure of fishto halogenated hydrocarbons and polycyclic aromatic hydro-carbons. Activity of Cyp1a1 is induced in early life stagesas well as adult zebrafish, and in zebrafish cell cultures (13,

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69, 102, 214, 353, 518) following exposure to TCDD. A va-riety of assays of DNA damage may indicate exposure offish to mutagens (126). Transgenic fish containing shuttleplasmid vectors serve as efficient indicators of exposure toenvironmental mutagens (3, 4). Schnurstein and Braunbeck(453) have developed an in vitro system using hepatocytesand gill cells to detect mutagenic agents. Troxel et al (519)measured DNA adducts in adult zebrafish injected with thecarcinogen aflatoxin B1 (AFB1). Zebrafish are quite resistantto AFB1-induced neoplasia compared to the most sensitivevertebrate species, the rainbow trout. Surprisingly, adductlevels in zebrafish were just 4-fold less than those in the highlysensitive rainbow trout, and were comparable to adduct lev-els occurring in relatively sensitive mammalian species. Hsuand Deng (234) quantified adduct levels in various organs ofzebrafish following bath exposure to benzo[a]pyrene. Theyfound highest adduct levels in intestine and liver. Severalinvestigators have measured plasma vitellogenin levels in ze-brafish as an indicator of exposure to environmental estrogens(8, 522).

Use of Zebrafish as Sentinels to DetectEnvironmental Hazards

Carvan et al (80) developed transgenic lines of zebrafishwith response elements to indicate exposure to specific tox-icants. The constructs introduced into the zebrafish includearyl hydrocarbon, electrophile, metal, and estrogen responseelements. Each reponse element is coupled to a reporter suchas luciferase or green fluorescent protein. Zebrafish cell linescontaining such reponse elements coupled to reporter genesare also available (81). Zebrafish embryos are sensitive indi-cators of toxic components in industrial and municipal wasteand landfill leachates (169, 170, 243). Chronic bioassays us-ing whole life-cycle tests with zebrafish can detect mortality,growth suppression, reproductive, and developmental toxic-ity, as well as behavioral toxicity (472).

SPONTANEOUS ANDINDUCED NEOPLASIA IN ZEBRAFISH

Carcinogen-Induced NeoplasiaAlthough the zebrafish was the first fish species in which

experimental carcinogenesis was conducted (by Mearle Stan-ton of the National Cancer Institute in the 1960s; 481, 482),until recently, little additional neoplasia research has usedzebrafish (269, 411, 412).

The most comprehensive carcinogenesis studies to datewith zebrafish were conducted at Oregon State Univer-sity (OSU) with funding from the US Army. This re-search evaluated carcinogenicity of a panel of struc-turally diverse carcinogens at three development stagesin Florida wild-type zebrafish. Carcinogens evaluated inthis work included N-nitrosodiethylamine (DEN), afla-toxin B1 (AFB1), methylazoxy-methanol acetate (MAMA),N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), and 7,12-dimethyl-benz[a]anthracene (DMBA). We administered car-cinogens by bath to late stage embryos or fry at 2–3 weekspostfertilization. In dietary carcinogenesis studies we fed2-month-old juvenile fish a semipurified diet (300) contain-ing carcinogen for 3–9 months. For each carcinogen and the3 developmental stages, we treated fish with at least 3 gradeddoses of carcinogen up to the maximum tolerated dose.

TABLE 1.—Target organs for carcinogen reponses of Florida wild-typezebrafish.

Life stage at Primary SecondaryCarcinogen treatment target tissues target tissue Other targetsa

DMBA Lymphomyeloid;skeletal muscle,thyroid

Embryo Liver NeuralFry Liver, gill, Intestine,

blood vessel neuralJuvenile Intestine Gill

MNNG Gill, ultimo-branchial

Embryo Liver TestisFry Testis Blood vesselJuvenile None None

MAMA Heart, eye, brain,nerve sheath,pancreas, fin

Embryo Liver Blood vesselFry Liver TestisJuveniles Liver Intestine

DEN Notochord,ultimobranchial,intestine, skin

Embryo Liver NoneFry Liver NoneJuvenile None None

AFB1 Intestine, boneEmbryo Liver NoneFry Liver NoneJuvenile Liver Intestine

aTarget tissues affected occasionally following treatment at one or more life stage.

Zebrafish exposed to most of the carcinogens as fry orembryos display a wide variety of neoplasm types derivedfrom epithelial, mesenchymal, neural, and neural crest tis-sues. Liver is a primary target organ for most carcinogens,regardless of developmental stage of fish at exposure. We seethe greatest diversity of histologic types of neoplasia follow-ing early life stage exposure to MAMA, MNNG, or DMBA(212, 476–478, 521). Table 1 indicates the patterns of tar-get organs observed in our carcinogen studies with Floridawild-type zebrafish. Zebrafish of the Florida wt strain areremarkably resistant to carcinogenic effects of AFB1 at alllife stages. In dietary studies, neoplasia was not observedfollowing treatment with MNNG or DEN, and substantialincidences of neoplasia occurred with AFB1 only after di-etary exposure for 9 months to 100 ppm. Typically low ppbconcentrations of AFB1 are used for dietary carcinogenesisstudies in rainbow trout to achieve high incidences of liverneoplasia (27). The relative resistance of juvenile zebrafish tomost dietary carcinogens when compared to juvenile rainbowtrout fed the same carcinogens may occur in part because thegrowth rate of zebrafish slows substantially as they approachmaturity at 3–6 months of age, whereas trout continue to growat a more rapid rate throughout life. The reasons for the rela-tive resistance of Florida wild-type zebrafish to AFB1 at alllife stages are not yet clear. Surprisingly, adduct levels follow-ing adult exposure were just 4-fold less than those observedin rainbow trout, and were comparable to adduct levels oc-curring in relatively sensitive mammalian species (518, 519).Although maximal percent incidences of total neoplasia inthe most sensitive life stage were 67, 66, 62, 37, and 32 forDEN, DMBA, MAMA, AFB1, and MNNG, respectively, atmost a 32% incidence of liver neoplasia or 15% incidence ofgut neoplasia occurred in carcinogen treated fish.

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Beckwith et al (36) of Pennsylvania State University report100% incidence of cutaneous papillomas occurring in 18 ze-brafish of the Florida wild-type line within 1 year following3 adult bath exposures to 2.5–3 mM ENU. We hypothesizedthat early life stage exposure to ENU might speed up the timeto tumor development, and also predicted that the Tuebingenlong fin leopard(TL) mutant line with fin overgrowth wouldlikely develop more papillomas at a faster rate on fins thanwild-type lines. We administered single 1 hr bath exposuresof the maximum tolerated dose of ENU (2.5 mM) to 3-week-old TL fry, and administered 3 doses of 2.5 mM ENU toTuebingen wild-type (TU) fry at 3, 5, and 7 weeks of age.At 1 year posttreatment in the TL line, and at 1 and 2 yearsposttreatment in the TU line, we observed no papillomas. Incarcinogen-treated fish of both lines, we documented a va-riety of neoplasm types including hemangiomas and hepaticand neural neoplasia not observed in vehicle or sham con-trol fish of those lines (Spitsbergen and Kent, unpublisheddata). Several explanations are possible for the differencesin our findings in the TL and TU lines at OSU compared tothe Florida wild-type line at Pennsylvania State University.Perhaps only the Florida wild-type line has skin that respondsto ENU. Perhaps ENU is carcinogenic in zebrafish only afteradult exposure of specific genetic lines. Perhaps the Penn.State University zebrafish colony has a unique oncogenicvirus and/or tumor promoter contributing to papilloma de-velopment. The zebrafish colony at the University of Oregonhouses large numbers of zebrafish of the AB line treated asadults with a similar ENU protocol to that used at Pennsyl-vania State University. Yet cutaneous papillomas are seen atthe University of Oregon only in eggbound female broodstockwith prolapse of the distal intestine and subsequent chronicirritation of the vent, resulting in hyperplasia or papilloma ofthe skin of the vent.

Spontaneous NeoplasiaIn carcinogen studies at OSU, the spontaneous rate of neo-

plasia in the Florida wild-type line at 6–14 months of ageis 1%, based on 3,000 untreated controls. The most com-mon histologic types of spontaneous neoplasia in this lineare seminoma of testis, hepatocellular adenoma, and ade-noma of exocrine pancreas, with intestinal adenocarcinomaless common (476–478).

Michael Kent and Jan Spitsbergen of OSU and MonteWesterfield of Univeristy of Oregon are currently investi-gating spontaneous rates of neoplasia in wild-type and mu-tant lines of zebrafish. We are evaluating the relative rolesof diet, husbandry systems, infectious agents, and genetic in-fluences on rates of neoplasia in various zebrafish lines. Aspart of this research, we are comparing spontaneous tumorincidences at 2 years of age in replicate groups of AB wild-type zebrafish fed 2 different diets (commercial diet or semi-purified diet) and raised at 3 different facitilites at OregonState University and University of Oregon. We have begunto systematically examine large numbers of retired brood-stock histologically over the past year. Since 1999, we havestudied diagnostic cases from moribund fish, fish with grosslesions, or sentinel fish submitted to the diagnostic pathol-ogy service provided by the Zebrafish International ResourceCenter at the University of Oregon. We have examined ap-proximately 500 fish in the diagnostic service. Among these

diagnostic cases, the most common target tissues for neopla-sia are ultimobranchial gland (26/500), testis (22/500), liver(15/500), gut (14/500), peripheral nerve (11/500), and thy-roid (6/500). Less common target tissues in diagnostic casesare eye (5/500), nasal sensory neuroepithelium (3/500), bloodvessel (2/500), fibroblast (1/500), brain (2/500), gill (1/500),lymphomyeloid system (1/500), pancreas (2/500), notochord(1/500), and pigment cell (1/500). In these studies of spon-taneous neoplasia together with our ongoing and historicalcarcinogen studies, benign and malignant neoplasms occur innearly every tissue of zebrafish. Tissues in which we have notyet observed neoplasia are pituitary, glial cells of brain, ovary,and the granulocytic lineage of blood (have seen myelodys-plasia with granulocytes predominating). Sample sizes in ourstudies of retired broodstock are currently too small to drawclear conclusions. Fewer than 100 fish per line have beenexamined from most lines, often of a single cohort raised to-gether in one facility. So far the spectrum of tumor types inmost broodstock lines, wild-type or mutant, is similar to thatin diagnostic cases, with tumors of testis, liver, and gut mostcommon. AB wild-type zebrafish 1.5–2 years of age showincidences of total neoplasia of approximately 10–15%. Upto 50% of males of the AB line develop seminomas of testisby 1.5–2 years of age.

Medical doctor pathologists, veterinary pathologists andfish pathologists are collaborating to complete a review pa-per summarizing current information on neoplasia in ze-brafish. This information was presented in poster form atscientific meetings (37, 38). This manuscript should be sub-mitted within months, and information with images will beavailable on the World Wide Web through ZFIN.

Development of Zebrafish Lines Highly Sensitiveto Neoplasia

Investigations of spontaneous and carcinogen-inducedneoplasia in zebrafish reveal a variety of neoplasms that rarelyoccur even in carcinogen-treated vertebrates of other species(chordoma, pineocytoma, hepatoblastoma, ocular medul-loepithelioma, olfactory esthesioneuroepithelioma). Devel-opment of zebrafish lines that show high incidences ofspecific neoplasms early in life will offer an efficient, cost-effective system for investigating molecular alterations oc-curring during various phases of the evolution of tumors andfor developing novel anticancer therapy.

Jan Spitsbergen, Michael Kent, and Donald Buhler ofOregon State University are investigating carcinogen-induced neoplasia in various wild-type and mutant lines ofzebrafish in order to identify lines that develop high inci-dences of specific histologic types of neoplasia early in life.We have identified several mutant lines that develop 70–90%incidences of liver neoplasia within 6–12 months posttreat-ment. Certain of these lines rapidly develop highly anaplasticneoplasia, including hepatoblastoma. Some of these mutantlines are susceptible to relatively high incidences of multipletumor types including hemangioma/hemangiosarcoma, es-thesioneuroepithelioma/esthesioneuroblastoma of nose, andmyelodysplastic syndrome of hemopoietic tissue. Our find-ing of myelodysplasia at a relatively high incidence (50%incidence or greater) in mutant lines following carcinogenexposure is particularly exciting because, for unexplained

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reasons, myeloid hemopoietic neoplasia has not yet been re-ported in untreated or carcinogen-treated wild-type zebrafishunder the conditions studied to date (475).

Leonard Zon and colleagues at Children’s Hospital inBoston, Massachusetts are investigating carcinogen respon-siveness of mutant lines of zebrafish selected for altered ex-pression of cell cycle genes in embryos and young fry. Severalmutant lines show increased cumulative incidences in neo-plasia following fry bath treatment with MNNG or DMBA,compared to wild-type siblings when sampled at 3, 6, and12 months posttreatment (464). Keith Cheng and colleaguesat Pennsylvania State University have developed lines of ze-brafish with genomic instability and are investigating neo-plasm prevalences in these lines (359, 360). Cheng’s groupis also investigating lines of zebrafish showing dysplasia ofvarious tissues at 7 days of development, predicting that suchlesions will be associated with elevated neoplasm risk laterin life (88). Thomas Look and colleagues at Harvard MedicalSchool are developing a model for neuroblastoma by engi-neering overexpression of zebrafishmycn(420).

Oncogenes and Tumor Suppressor Genes in ZebrafishFunctional motifs in oncogenes and tumor suppressor

genes tend to be highly conserved among vertebrate species,so that study of oncogenes and tumor suppressor genes inzebrafish will likely provide valuable insights into molecularmechanisms of tumorigenesis and genetic susceptibility totumors in humans (124, 525). Because interest in neoplasiaand carcinogenesis in the zebrafish model has only recentlybegun to dramatically expand, unfortunately relatively few ofthe zebrafish orthologs of human tumor suppressor genes andoncogenes have been cloned, fully sequenced and/or mapped.Single orthologs of the human tumor suppressor genesWT1(266, 471),P53(90),MEN 1(268),MADH2,MADH5(127),PTC1, andPTC2(305) are fully sequenced and mapped inzebrafish. Full cDNA sequence and mapping data are avail-able for single orthologs of human oncogenesNRAS(89),RET(48), CMYC(455),CKIT (395, 421),PIM1 (239) andMDM2 (504). In contrast to findings inXenopus, overexpres-sion of mdm2in developing zebrafish does not cause earlyincreased tumor incidence (504).

COMPLICATING FACTORS INZEBRAFISHTOXICOLOGICPATHOLOGY RESEARCH

Shortage of Basic Data on Zebrafish PathologyThere are scant baseline data on zebrafish pathologic le-

sions in infectious and noninfectious diseases. Shortly afterthe widespread use of mutant mice began, industry, academia,and government agencies realized that molecular biology andgenetic approaches alone could not adequately predict andsystematically characterize the complex phenotypes of micewith single or multiple gene mutations. For example, manylines of knockout mice for which the inactive genes are notrelated directly to the immune system still are immunode-ficient and at high risk for opportunistic infection (60, 433,575). Thus rigorously trained comparative pathologists areplaying a growing role in research with mutant mice. To uti-lize the potential of zebrafish fully as an animal model forunderstanding human development, disease, and toxicology,we must greatly advance our knowledge of zebrafish dis-

eases and pathology. Much of the information pertinent tozebrafish infectious diseases is in general reference texts re-garding the topic of aquarium fish diseases (379, 487). Since1999, Michael Kent of Oregon State University, JenniferMatthews of the University of Oregon, and collaborators haveinvestigated epidemiology, prevention and control of impor-tant infectious and noninfectious diseases affecting zebrafishcolonies (50, 334).

Infectious Diseases of ZebrafishTwo infectious diseases that commonly occur in well-

managed zebrafish colonies are microsporidiosis and my-cobacteriosis.Pseudoloma neurophila, a microsporidian in-fects the central nervous system, cranial and spinal nerves,and skeletal muscle of zebrafish. Severely affected fish maybe emaciated, ataxic, or have spinal malformations (123,333). Michael Kent’s research group has developed a poly-merase chain reaction test to screen broodstock and derivespecific pathogen-free lines. We are uncertain whether ver-tical transmission of microsporidia can occur in zebrafish.Unfortunately, these parasites are very difficult to inactivateand remove from recirculating husbandry systems. Zebrafishof various lines derived from eggs obtained in the main colonyor new Zebrafish International Resource Center Colony at theUniversity of Oregon do not develop microsporidiosis whenreared in a flow-through husbandry system at Oregon StateUniversity, but do develop microsporidiosis when raised inthe recirculating systems at University of Oregon. Thus hor-izontal transmission of this parasite seems to be important,however, the role of vertical vs horizontal transmission is stillunclear.Renibacterium salmoninarum, an important bacte-rial disease of salmonids, is spread vertically within eggs,but only a few eggs are usually infected. Yet these few in-fected eggs serve as a source for horizontal transmissionin early life stages held in incubation trays (151).Pseu-doloma presents particular problems for neurobehavioraltoxicity studies. Although Kent’s group is investigating pos-sible drug therapy, no recommended therapy methods are yetavailable.

Piscine mycobacteriosis remains a tenacious problem inaquarium fish colonies. It most often occurs as an oppor-tunistic infection in fish over 1 year of age, especially thosestressed by toxicant exposure or adverse environmental con-ditions. Therefore, mycobacteriosis is a problem in long-term tumor or aging studies. Highly pathogenic strains maycause devastating mortality in otherwise healthy colonies.The sources of infection and epizootiology are not well de-fined. Mycobacteria may enter fish from food or water, andmay possibly be vertically transmitted. Currently no effec-tive chemopreventative or therapeutic regimens are definedfor fish (23). Because mycobacterial antigens are potent im-mune adjuvants, these agents can seriously confound researchin disease resistance or immune responses.

Pseudocapillaria tomentosa, a nematode parasite, infectsthe gut of zebrafish. This parasite occurs commonly incolonies of Florida wild-type lines. Infection causes moder-ate to severe multifocal to diffuse hyperplasia and dysplasiaof the intestine, as well as elevated incidences of intestinalneoplasia. Enteric adenomas and carcinomas occur in closeproximity to profiles of nematodes in the gut. The parasite actsas a tumor promoter in carcinogen experiments. In dietary

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carcinogen studies, zebrafish fed DMBA developed more in-testinal neoplasia if infected with the nematode (267).

To date, a pathogenic viral agent has not yet been isolatedfrom or visualized in zebrafish tissues. We are looking forviral agents with ultrastructural studies of fish tissue, viralisolation procedures in fish cell lines, and disease transmis-sion trials in which young zebrafish are injected with tumortissue or tissue from diseased fish. All vertebrate species thathave been studied intensely are host to multiple pathogenicviruses.

Zebrafish are certainly affected by pathogenic and onco-genic viruses that will eventually be identified. Salmonidviruses infectious hematopoietic necrosis virus and infectiouspancreatic necrosis virus infect zebrafish and zebrafish celllines (315), but do not cause clinical disease (298, 457).

A small amount of published information is available re-garding zebrafish disease diagnostic procedures and infec-tious diseases. Astrofsy et al (22) outline diagnostic tech-niques for clinical investigation of laboratory zebrafish.Menudier et al (345) compared pathogenicity of variousstrains ofListeria in zebrafish and mice, showing that strainsnonpathogenic in mice were sometimes highly pathogenicin zebrafish. Pullium et al (419) document epizootic motileaeromonad septicemia in a zebrafish colony associated withstress due to poor water quality.Edwardsiella ictaluri, a com-mon pathogen of channel catfish, was isolated from moribunddanio during a disease outbreak (546). Mills (352) reports onecological factors influencing digenean trematode infections.

Influence of Diet and Husbandry Systemson Spontaneous Neoplasia

Zebrafish colonies fed commercial diets and maintainedin standard recirculating systems have different patterns ofneoplasia and spontaneous pathologic lesions than those ob-served in the Core Fish Facility of the Marine/FreshwaterCenter at Oregon State University. We have a flow-throughsystem and feed fish a semipurified diet used for over 30 yearsin carcinogenesis studies in fish. We have not yet seen sponta-neous mesenchymal neoplasia, spontaneous neoplasia of thecentral or peripheral nervous system, or spontaneous thyroidneoplasia in any lines of zebrafish in the Oregon State Uni-versity center. In contrast, such neoplasms occur quite com-monly in diagnostic cases from many labs around the world,even in fish just 6 months of age. These findings suggest thatnaturally occurring carcinogens may be present in the diet orenvironment in many zebrafish colonies. Our studies com-paring neoplasm incidences in zebrafish of the AB line fedcommercial or semipurified diets at 3 separate fish facilitieswill help shed light on the influences of diet and husbandryon neoplasm patterns.

The Mystery of Hepatic Megalocytosis in ZebrafishIn diagnostic cases from around the world and in about

50% of groups of retired broodstock from standard hus-bandry systems feeding commercial diets, we see mild tomoderate hepatocyte megalocytosis with karyomegaly. From10 to 100% of the fish in affected lots of broodstock showhepatic megalocytosis. Hepatocyte cytoplasmic volumes andnuclear volumes may be 5–50X normal. We have never seenthis megalocytosis in untreated control fish of any line from

the Core Fish Facility of the Marine/Freshwater Center atOregon State University. Data from many vertebrates in-cluding zebrafish indicate that hepatocyte megalocytosis iscaused by toxicant damage to DNA or the mitotic apparatus(207). The algal toxin microcystin causes hepatic megalocy-tosis in Atlantic salmon (10). We often see high incidences ofhepatocyte megalocytosis in zebrafish following carcinogenexposure. In a given environment, some lines appear moreprone to hepatic megalocytosis. In the flow-through quaran-tine facility at the University of Oregon, the TL line showsmegalocytosis, but the TU, Cologne,another long fin, andknorrig lines do not. The toxicant sources causing megalocy-tosis are uncertain. Dietary components (commercial flake,paramecium cultures), algae or biofilm in hoses/lines, andmetabolites of microbes in biofilters may contribute to theproblem. In addition to increasing the spontaneous incidencesof neoplasia, the toxicant(s) causing megalocytosis may alsocause other health problems, reducing early life stage sur-vival, longevity, reproductive potential, immune competence,and disease resistance.

Bile Duct Hyperplasia in the TL LineIn our studies of retired broodstock at OSU and U. of O.

mild to severe multifocal to diffuse hyperplasia of bile ductsis present in essentially 100% of broodstock of the TL lineby 1.5 year of age. The incidences and severity of these le-sions are similar at a given age in fish raised at Oregon StateUniversity or University of Oregon. The lesions are not asso-ciated with significant inflammation, parasites or other infec-tious agents, however, the severity is greater in males than infemales. Approximately 10% of TL line fish raised in flow-through conditions at the University of Oregon show biliaryneoplasia spontaneously by 1.5 years of age. Bile duct hyper-plasia is not linked to thelong finmutant gene, as broodstockare not homozygous for this dominant gene, and wild-typesiblings have similar incidences and severity of bile duct hy-perplasia. Although it is a double mutant, the TL line has beenused as a background genetic line for perpetuating other mu-tant lines in many zebrafish colonies because it is hardy andfecund, so its background genetics are unique. Although theproliferated bile ducts may occupy 50% of the volume ofliver of TL line fish by 1.5 year of age, except for elevatedincidences of neoplasia, the fish appear clinically healthy andusually breed well. The bile duct hyperplasia seen in TL linezebrafish is not seen in TU wild-type line or in theanotherlong fin(alf ) mutant line on the TU× AB background. TheTL line will be a useful model for study of primary biliary cir-rhosis and bile duct neoplasia, spontaneous and carcinogen-induced, but would be a poor line to use in toxicant studiesin which subtle alterations in bile ducts must be discerned.

The Problem of Skewed Sex Ratios in Cohorts of ZebrafishAt many zebrafish facilities around the world, problems

with skewed sex ratios in cohorts of zebrafish can interferewith natural breeding, and can complicate studies such ascarcinogen or other toxicant bioassays where balanced sexratios in control groups are desired. In contrast to mam-malian species, sex determination in fish is more flexible,often reversible, and less dictated by genetic factors (29,122, 442). The relative influences of and interactions between

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environment, genetics, and toxicants in sex determination inzebrafish are not yet clearly defined. However, genetic aswell as environmental factors appear to influence sex deter-mination. The TU line raised in any of the recirculating orflow-through systems at the University of Oregon tends tobe predominately female, whereas the AB line reared in thesame systems often has an excess of males. We do not seeskewed sex ratios in control groups of Florida wt zebrafishin the Core Fish Facility of the Marine/Freshwater Center atOregon State University, but we often see very skewed sexratios with the German lines TU and TL, and the AB line.We do not see unbalanced sex ratios in rainbow trout in thisfacility.

Histopathology and Toxicologic Pathology LiteratureRegarding Zebrafish

The literature on nonneoplastic pathologic lesions in ze-brafish is sparse compared to that for mammals. A few ref-erences on nutritional pathology are available (141, 342,408). Vithelic and Hyde (540) document light-induced retinaldamage in albino zebrafish. Ferretti and Geraudi (154) andGeraudi et al (180) show retinoic acid-induced cell deathin healing wounds on regenerating fins. Few of the toxicantstudies using zebrafish have investigated light microscopic orultrastructural morphologic alterations. Hisaoka (225, 226)describe histochemical changes including depletion of hep-atic glycogen, and light microscopic morphologic lesions in-cluding necrosis of neuroepithelium of the central nervoussystem occurring in zebrafish following embryo exposureto 2-acetylaminofluorene. Burkhardt-Holm et al (71) reportultrastructural alterations in liver, gill and erythrocytes fol-lowing chronic exposure of embryonic or larval zebrafish to4-chloroaniline. Oulmi and Braunbeck (388) describe ultra-structural alterations in liver and kidney in zebrafish follow-ing embryo microinjection of 4-chloroaniline.

Braunbeck et al (59) document ultrastructural alterations inliver of zebrafish including reduced numbers of peroxisomesfollowing chronic exposure to 4-chloroaniline. Bresch et al(63) evaluated spinal integrity using radiography during a 3-generation life cycle study with 4-chloroaniline. Braunbecket al (58) show hepatic steatosis occurring in zebrafish chron-ically exposed to lindane in a full life cycle test. Strmacand Braunbeck (490) indicate that the cardiovascular sys-tem is a primary target following sublethal exposure of earlylife stages of zebrafish to triphenyl tin acetate. They reportultrastructural alterations in liver including depleted glyco-gen, mitochondrial swelling, and cytoplasmic myelin figures.Dong et al (129) and Henry et al (215) describe light micro-scopic lesions in zebrafish during the early life stage toxicitysyndrome caused by TCDD. As in early life stages of otherspecies of fish exposed to TCDD, the cardiovascular system isthe primary target organ system in zebrafish. Ansari et al (18)and Ansari and Kumar (15) show reduced numbers of matureovarian follicles, hepatomegaly, and alterations in hepatic nu-cleic acid and protein (16, 17) in zebrafish following chronicsublethal exposure to malathion. Olsson et al (383) studiedhistologic lesions in offspring following maternal exposureto PCBs or 17-beta-estradiol. Maternal exposure to PCB-104or 190 causes craniofacial malformations and scoliosis in off-spring. Maternal exposure to PCB-104 or 17-beta-estradiol

causes tubular nephrosis, and 17-beta-estradiol, PCB-104,or PCB-60 increases ooctye atresia in ovaries, and arrestsspermatogenesis in testes in early life stages of progeny. His-tologic alterations occur in gonads of both female and malezebrafish given acute bath exposures as adults to the syntheticestrogen 17-alpha-ethynylestradiol (526). Increased atreticfollicles and arrested follicular development occur in the go-nads of females. In males, spermatogenesis ceases at the sper-matogonial stage.

FUTURE NEEDS ANDFUTURE RESEARCHDIRECTIONS

The utility of early life stages of zebrafish in high through-put screening systems for drug development is already beingexploited (369, 371, 404, 484). The small size of zebrafishembryos and fry, and their ability to be cultured during thefirst week of life in 96 well microtiter plates make this systemideal for drug discovery and safety testing. Most hydrophilicas well as lipophilic agents are readily absorbed from theculture medium of eggs or fry, facilitating efficient testing ofnew agents.

To advance the field of zebrafish toxicologic pathology to-ward the state of the art in mammalian toxicologic pathology,much more data regarding pathologic lesions following acute,subchronic and chronic toxicant exposure will be required.We need to develop a comprehensive database regardingspontaneous and toxicant-induced neoplasia and nonneoplas-tic lesions in the common wt and mutant lines of zebrafish.Spontaneous aging lesions in various strains of zebrafish needto be investigated. Comprehensive data on metabolism andpharmacokinetics of toxicants in various wt and mutant lineswill be essential to support sophisticated toxicologic pathol-ogy research. Very little information is available on DNArepair enzymes in various lines of zebrafish. Once the fullgenomic sequence is available for zebrafish during the com-ing year, this information will speed the identification of andstudy of DNA repair enzymes. The cornucopia of markers forprotein and gene expression generated for studies of zebrafishdevelopment need to be exploited in toxicant studies. To bestutilize the zebrafish as a model system for understandingmechanisms in oncogenesis relevant to man, we must clarifythe members of families of oncogenes and tumor suppres-sor genes to sort out their relative roles in tumorigenesis inzebrafish compared to mammals. Duplicate genes for somemembers of these families are likely to complicate the clar-ification of signaling pathways in the zebrafish. Microarraytechnology will likely help in unraveling the complex signal-ing networks in zebrafish.

In recent years, the Society of Toxicologic Pathology hascoordinated working groups of pathologists focused on spe-cific organ systems in rodents. The poster sessions at STPmeetings and STP-sponsored monographs on proliferativeand nonproliferative lesions for each organ system have es-tablished consensus in the field of toxicologic pathology re-garding terminology for morphologic diagnosis of pathologiclesions in rodents. We need to establish similar pathologyworking groups to define diagnostic criteria for prolifera-tive and nonproliferative lesions in the major organ systemsof zebrafish. An inadequate number of scientists have suf-ficient training in anatomy, histology, and pathology of fishincluding zebrafish to support the growing need for pathology

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74 SPITSBERGEN AND KENT TOXICOLOGIC PATHOLOGY

expertise in the study of zebrafish models for human disease.Some short courses in fish diseases and pathology such as theAquavet program taught each spring at the Marine Biolog-ical Laboratory in Woods Hole, Massachusetts can providean introduction to fish pathology for scientists who alreadyhave basic histology and pathology training.

However, more extended residency and/or graduate train-ing for D.V.M. and M.D. pathologists as well as fish biolo-gists will be required in order to establish adequate diagnosticskills to correctly interpret histologic lesions in fish tissues.

ACKNOWLEDGMENTS

Research at Oregon State University and University ofOregon was funded by US Public Health Service grantsES011587-01 and P30ESO3850 from the National Institutesof Environmental Health Sciences, by grant 3P40RR12546and its supplement 03S1 from the National Center for Re-search Resources, and by US Army contract DAMD 17-91Z1043. We thank Tom Miller, Keri St. Clair, JanelleBishop-Stewart, Sheila Cleveland, Chance MacDonald,Karen Larison, April Mazanac, Bill Trevarrow, and DanArbogast for technical support in our ongoing and pastzebrafish studies.

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