Zebrafish System Research Articles

Exploring the advantages and limitations of sampling methods commonly used in research facilities for zebrafish health inspections

Examining zebrafish populations for the presence of disease is an integral component of managing fish health in research facilities. Currently, many different strategies are used for zebrafish fish health inspections, which is a scenario that may result in subjective and biased diagnostic evaluations. The goal of this study was to compare the success of pathogen detection between a sample size of randomly selected fish (n = 60) that provides 95% confidence in pathogen detection based on a presumed pathogen prevalence level ≥5%, and other subpopulations and sample numbers commonly submitted for diagnostic testing within a 1000 tank, 30,000 fish, recirculating research system. This included fish collected from a sump tank (n = 53), sentinel fish (n = 11), and fish that were found moribund or freshly dead (n = 18). Additionally, five fish from each subpopulation were collected for histopathologic examination. A second study used retrospective data to examine pathogen distribution between systems (n = 2-5) in multi-system facilities (n = 5) using a sample size of 60 fish per system. For the pathogens detected, results supported the use of representative sample numbers rather than smaller numbers of populations considered more at risk. The exception to this is for the moribund/mortality group, which may be a resource for targeted surveillance of select pathogens. Each system within multi-system facilities should be considered separate units in terms of fish health inspections and biosecurity. Development of these evidence-based standards for fish health inspections in zebrafish systems enhances fish welfare, provides identification of potentially zoonotic pathogens, and ensures scientific integrity and reproducibility of research results.

Analysis of AMB-FUBINACA Biotransformation Pathways in Human Liver Microsome and Zebrafish Systems by Liquid Chromatography-High Resolution Mass Spectrometry.

In this study, the metabolic profiles of a new illicit drug AMB-FUBINACA were investigated using both human liver microsome and zebrafish models. Liquid chromatography Q Extractive HF Hybrid Quadrupole-Orbitrap mass spectrometry (LC-QE-HF-MS) was employed to analyze the metabolic sites and pathways. AMB-FUBINACA was added to the in vitro liver microsome incubation model to simulate the metabolic processes in human body. The results showed that a total of 17 metabolites were generated in the human liver microsome model; the main metabolic pathways of the phase I metabolism included ester hydrolysis, methylation, ester hydrolysis combined with decarboxylation, hydroxylation, ester hydrolysis combined with indazole ring hydroxylation, etc. while glucuronidation served as the main metabolic pathway of the phase II metabolism. The zebrafish system produced a similar result with 16 of the same 17 metabolites identified. The phase I metabolites M3.1 (ester hydrolysis), M1.2 (alkyl chain hydrolysis) and the phase II metabolite M3.2 (M3.1 glucuronide) were recommended to be the potential poisoning markers.

Overview of a Disease Outbreak and Introduction of a Step-by-Step Protocol for the Eradication of Mycobacterium haemophilum in a Zebrafish System.

In 2017, the zebrafish unit at University of Glasgow experienced a detrimental outbreak of pathogenic bacterium, Mycobacterium haemophilum. The presence of other bacterial species was also confirmed by bacteriology growth in the same unit. The affected individuals composed of a wild-origin parental population sourced from India and their F1 offspring generation. Bacteria were diagnostically confirmed to be present systemically in fish and within the water and biofilm of the recirculating zebrafish system. In the absence of a publicly accessible step-by-step disinfectant protocol for these difficult-to-eliminate pathogens, we devised a successful procedure to eradicate mycobacteria and Aeromonas species after colony removal using Cleanline Chlorine tablets (active ingredient Sodium dichloroisocyanurate) and Virkon Aquatic®. Postdisinfection diagnostics did not detect pathogens in the system or in the new fish inhabiting the system that were tested. Newly established fish colonies have not shown similar clinical signs or disease-induced mortality in the 1-year period following system disinfection and repopulation. We present a historical background of the bacterial outbreak and a disinfection method which can be replicated in other zebrafish facilities—at small or large scales—for reliable mycobacterium removal. This procedure can be implemented as a disinfection protocol before the introduction of a new fish population to a previously contaminated system.

The value of sea urchin, Lytechinus variegatus, egesta consumed by shrimp, Litopenaeus vannamei

Sea urchins produce high‐energy, membrane‐bound fecal pellets that contain residual nutrients and large quantities of microbiota. These egesta are readily consumed by the shrimp, Litopenaeus vannamei. Egesta of the sea urchin, Lytechinus variegatus, were evaluated as a feed supplement or total replacement for a commercial shrimp diet. Shrimp were stocked at 0.49 g ± 0.06 g initial body weight and housed individually in 2.8‐L tanks in a commercial recirculating zebrafish system. Shrimp were assigned to one of six diets: commercial shrimp feed, reference sea urchin feed, collected dried sea urchin egesta, collected wet sea urchin egesta, half ration of shrimp feed and half collected wet sea urchin egesta, and egesta naturally produced by two sea urchins in polyculture. Equivalent dry matter amounts of each diet were proffered to shrimp in each treatment twice daily, except for those that had complete access to natural egesta excreted by sea urchins in polyculture. Sea urchins were proffered a reference sea urchin feed at 2% body weight daily. After 27 days, shrimp proffered collected dried or wet egesta did not differ significantly in percent weight gain and showed the lowest weight gain. The percent weight gain of shrimp fed the commercial shrimp diet did not differ significantly from that of the shrimp fed half commercial shrimp diet and half egesta. The highest weight gain was recorded for those shrimp that consumed the untouched egesta produced by sea urchins in polyculture. These data suggest that consumed egesta have noteworthy nutritional value and therefore would be beneficial to the culture of extractive species in an integrated multitrophic aquaculture system.

Analyzing Neuronal Mitochondria in vivo Using Fluorescent Reporters in Zebrafish.

Despite their importance for cellular viability, the actual life history and properties of mitochondria in neurons are still unclear. These organelles are distributed throughout the entirety of the neuron and serve many functions, including: energy production (ATP), iron homeostasis and processing, calcium buffering, and metabolite production, as well as many other lesser known activities. Given their importance, understanding how these organelles are positioned and how their health and function is maintained is critical for many aspects of cell biology. This is best illustrated by the diverse disease literature which demonstrates that abnormal mitochondrial movement, localization, size, or function often correlates with neural pathology. In the following methods article, we will describe the techniques and tools we have optimized to directly visualize mitochondria and analyze mitochondrial lifetime, health, and function in neurons in vivo using fluorescent reporters in the zebrafish. The zebrafish system is ideal for in vivo studies of mitochondrial biology as: (1) neuronal circuits develop rapidly, within days; (2) it is genetically accessible; and (3) embryos and larvae are translucent allowing imaging in a completely intact vertebrate nervous system. Using these tools and techniques, the field is poised to answer questions of mitochondrial biology in the context of neuronal health and function in normal and disease states.

Heart and liver regeneration in zebrafish using silver nanoparticle synthesized from Turbinaria conoides – In vivo

Abstract The present study was aimed to identify the heart and liver regeneration in zebrafish using biosynthesised silver nanoparticles from Turbinaria conoides. This work focussed on the bioactivity of AgNP when incorporated into the zebrafish (Danio rerio) system. We began investigating the in vivo assays: Hepatocyte viability staining after H2O2 treatment, cardiomyocyte response to Ca++, cardio vascular heart rate activity by measuring hypertrophy, cardio vascular pathology and cardio vascular regeneration, liver regeneration and liver pathology, molecular pathway target identification and hypothesis on interacting domain (AgNO3) of the vertebrate model organism. The FTIR results of biosynthesized silver nanoparticles from Turbinaria conoides showed prominent peaks (range between 620.967 and 2854.14). Further, the results of XRD analysis showed the 2γ intense values (38.11 and 70.57) within the ranges of Bragg's reflection. In addition, the SEM analysis determined the size of nanoparticle (50–100 nm). It can be concluded from the present findings that, the biosynthesised silver nanoparticles from the seaweed extract of Turbinaria conoides can be used as potential cardioprotective and liver protective agent.

Cxcr1 mediates recruitment of neutrophils and supports proliferation of tumor-initiating astrocytes in vivo

Neutrophils are first-responders to sites of infection and tissue damage including the inflamed tumor microenvironment. Increasing evidence suggests that crosstalk between tumors and neutrophils can affect the progression of established tumors. However, there is a gap in our understanding of the early events that lead to neutrophil recruitment to oncogene-transformed cells and how these pathways alter tumor progression. Here, we use optically transparent zebrafish larvae to probe the early signals that mediate neutrophil recruitment to Kras-transformed astrocytes. We show that zebrafish larvae with impaired neutrophil function exhibit reduced proliferation of transformed astrocytes supporting a critical role for tumor-associated neutrophils in the early progression of tumorigenesis. Moreover, using mutants and pharmacological inhibition, we show that the chemokine receptor Cxcr1 promotes neutrophil recruitment, proliferation of tumor-initiating cells, and neoplastic mass formation. These findings highlight the power of the larval zebrafish system to image and probe early events in the tumor-initiating microenvironment and demonstrate the potential for neutrophil recruitment signaling pathways such as Cxcl8-Cxcr1 as targets for anti-cancer therapies.

Zebrafish Models of Neurodevelopmental Disorders: Past, Present, and Future.

Zebrafish are increasingly being utilized as a model system to investigate the function of the growing list of risk genes associated with neurodevelopmental disorders. This is due in large part to the unique features of zebrafish that make them an optimal system for this purpose, including rapid, external development of transparent embryos, which enable the direct visualization of the developing nervous system during early stages, large progenies, which provide considerable tractability for performing high-throughput pharmacological screens to identify small molecule suppressors of simple behavioral phenotypes, and ease of genetic manipulation, which has been greatly facilitated by the advent of CRISPR/Cas9 gene editing technologies. This review article focuses on studies that have harnessed these advantages of the zebrafish system for the functional analysis of genes that are strongly associated with the following neurodevelopmental disorders: autism spectrum disorders (ASD), epilepsy, intellectual disability (ID) and schizophrenia. We focus primarily on studies describing early morphological and behavioral phenotypes during embryonic and larval stages resulting from loss of risk gene function. We highlight insights into basic mechanisms of risk gene function gained from these studies as well as limitations of studies to date. Finally, we discuss advances in in vivo neural circuit imaging in zebrafish, which promise to transform research using the zebrafish model by illuminating novel circuit-level mechanisms with relevance to neurodevelopmental disorders.

Comprehensive analysis of coding-lncRNA gene co-expression network uncovers conserved functional lncRNAs in zebrafish

BackgroundZebrafish is a full-developed model system for studying development processes and human disease. Recent studies of deep sequencing had discovered a large number of long non-coding RNAs (lncRNAs) in zebrafish. However, only few of them had been functionally characterized. Therefore, how to take advantage of the mature zebrafish system to deeply investigate the lncRNAs’ function and conservation is really intriguing.ResultsWe systematically collected and analyzed a series of zebrafish RNA-seq data, then combined them with resources from known database and literatures. As a result, we obtained by far the most complete dataset of zebrafish lncRNAs, containing 13,604 lncRNA genes (21,128 transcripts) in total. Based on that, a co-expression network upon zebrafish coding and lncRNA genes was constructed and analyzed, and used to predict the Gene Ontology (GO) and the KEGG annotation of lncRNA. Meanwhile, we made a conservation analysis on zebrafish lncRNA, identifying 1828 conserved zebrafish lncRNA genes (1890 transcripts) that have their putative mammalian orthologs. We also found that zebrafish lncRNAs play important roles in regulation of the development and function of nervous system; these conserved lncRNAs present a significant sequential and functional conservation, with their mammalian counterparts.ConclusionsBy integrative data analysis and construction of coding-lncRNA gene co-expression network, we gained the most comprehensive dataset of zebrafish lncRNAs up to present, as well as their systematic annotations and comprehensive analyses on function and conservation. Our study provides a reliable zebrafish-based platform to deeply explore lncRNA function and mechanism, as well as the lncRNA commonality between zebrafish and human.

Tendon developmental plasticity and functional regeneration

Tendons enable movement by transferring force from muscle to bone. They are comprised of a highly organized collagen‐rich extracellular matrix. Disruption to the development and maintenance of tendons can lead to birth defects and result in injury in adults. We currently have an incomplete understanding of how tendons interact with their neighboring tissues to regulate the development and morphogenesis of the musculoskeletal system. In addition, once mature mammalian tendon tissue is formed, it has limited regenerative potential after injury. Central to this challenge is to uncover new mechanisms underlying tendon cell regulation during musculoskeletal development and regeneration. To accomplish this, we have turned to the zebrafish system because it is highly regenerative and amenable to genetic manipulations and live imaging. We have previously demonstrated that zebrafish tendons have molecular, developmental, morphological and biomechanical similarities to mammalian tendons, thereby making them an excellent vertebrate model to study tendon biology. However, no study has investigated their capacity to regenerate their tendons. Using the zebrafish, we developed methods to perform genetic tendon cell ablation, lineage tracing, live imaging, and mechanical testing to determine if developing and adult zebrafish can regenerate their tendon tissues. Following tendon cell ablation in developing zebrafish embryos, we observe defects in tendon gene expression and ECM composition. Cartilage and muscle tissue morphology was also disrupted following tendon cell loss. Tendon cells and the collagen organization returned after 1–2 weeks and 1–2 months, respectively. Interestingly, the pattern of the attachment sites was also restored, indicating the presence of regulatory programs that recruit and/or promote tendon cell proliferation and differentiation at the previous attachment sites. We are currently focused on identifying the developmental origins of the regenerating tendon cells and the pathways restoring the attachment site pattern. In addition to embryonic tendon regeneration, we also found that adult zebrafish appear to regenerate their collagen organization and mechanical properties after an acute physical injury. Together, our work demonstrates robust tendon regeneration at embryonic and adult stages in the zebrafish. These studies also provide a system to examine the morphogenetic and molecular events underlying tendon regeneration and musculoskeletal tissue‐tissue interactions, which has significance for regenerative biology approaches to musculoskeletal injuries.Support or Funding InformationNICHD HD06953, NIDCR DE024771, Charles H. Hood Foundation, HSCI Seed GrantThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

In vitro analysis of the transcriptional regulatory mechanism of zebrafish pou5f3

Zebrafish pou5f3 (previously named pou2), a close homologue of mouse Oct4, encodes a PouV-family transcription factor. pou5f3 has been implicated in diverse aspects of developmental regulation during embryogenesis. In the present study, we addressed the molecular function of Pou5f3 as a transcriptional regulator and the mechanism by which pou5f3 expression is transcriptionally regulated. We examined the influence of effector genes on the expression of the luciferase gene under the control of the upstream 2.1-kb regulatory DNA of pou5f3 (Luc-2.2) in HEK293T and P19 cells. We first confirmed that Pou5f3 functions as a transcriptional activator both in cultured cells and embryos, which confirmed autoregulation of pou5f3 in embryos. It was further shown that Luc-2.2 was activated synergistically by pou5f3 and sox3, which is similar to the co-operative activity of Oct4 and Sox2 in mice, although synergy between pou5f3 and sox2 was less obvious in this zebrafish system. The effects of pou5f3 deletion constructs on the regulation of Luc-2.2 expression revealed different roles for the three subregions of the N-terminal region in Pou5f3 in terms of its regulatory functions and co-operativity with Sox3. Electrophoretic mobility shift assays confirmed that Pou5f3 and Sox3 proteins specifically bind to adjacent sites in the 2.1-kb DNA and that there is an interaction between the two proteins. The synergy with sox3 was unique to pou5f3—the other POU factor genes examined did not show such synergy in Luc-2.2 regulation. Finally, functional interaction was observed between pou5f3 and sox3 in embryos in terms of the regulation of dorsoventral patterning and convergent extension movement. These findings together demonstrate co-operative functions of pou5f3 and sox3, which are frequently coexpressed in early embryos, in the regulation of early development.

Embryonic exposure to valproic acid affects the histaminergic system and the social behaviour of adult zebrafish (Danio rerio).

Histamine modulates several behaviours and physiological functions, and its deficiency is associated with neuropsychiatric disorders. Gestational intake of valproic acid (VPA) is linked to autism spectrum disorder (ASD), characterized by impaired sociability and stereotypies. VPA effects on the neurochemistry and functional morphology of the histaminergic system in ASD are unclear. Zebrafish are highly social, and given the similarities between zebrafish and human neurotransmitter systems, we have studied the effects of VPA on histamine in zebrafish. Histaminergic, dopaminergic and noradrenergic systems of larval and adult zebrafish exposed to VPA from the end of gastrulation until neural tube formation were studied using HPLC, quantitative PCR, immunocytochemistry and in situ hybridization. Sociability, dark-flash response and locomotion were also studied. Zebrafish larvae exposed to VPA showed decreased locomotion and an abnormal dark-flash response. Additionally, a reduced number of histaminergic neurons, low histamine and altered mRNA expression of key genes of the monoaminergic systems were also detected. The reduced mRNA expression of genes of the studied systems persisted until adulthood. Furthermore, adult VPA-exposed animals presented lower brain levels of noradrenaline and 3,4-dihydroxyphenylacetic acid, along with impaired sociability. VPA exposure in early development causes molecular and neurochemical alterations in zebrafish, which persist into adulthood and accompany impaired sociability. These findings will highlight the possible involvement of the histaminergic system in outcomes related to neuropsychiatric disorders. Furthermore, it supports zebrafish as a tool to investigate mechanisms underlying these disorders.

ALKALs are in vivo ligands for ALK family receptor tyrosine kinases in the neural crest and derived cells

Mutations in anaplastic lymphoma kinase (ALK) are implicated in somatic and familial neuroblastoma, a pediatric tumor of neural crest-derived tissues. Recently, biochemical analyses have identified secreted small ALKAL proteins (FAM150, AUG) as potential ligands for human ALK and the related leukocyte tyrosine kinase (LTK). In the zebrafish Danio rerio, DrLtk, which is similar to human ALK in sequence and domain structure, controls the development of iridophores, neural crest-derived pigment cells. Hence, the zebrafish system allows studying Alk/Ltk and Alkals involvement in neural crest regulation in vivo. Using zebrafish pigment pattern formation, Drosophila eye patterning, and cell culture-based assays, we show that zebrafish Alkals potently activate zebrafish Ltk and human ALK driving downstream signaling events. Overexpression of the three DrAlkals cause ectopic iridophore development, whereas loss-of-function alleles lead to spatially distinct patterns of iridophore loss in zebrafish larvae and adults. alkal loss-of-function triple mutants completely lack iridophores and are larval lethal as is the case for ltk null mutants. Our results provide in vivo evidence of (i) activation of ALK/LTK family receptors by ALKALs and (ii) an involvement of these ligand-receptor complexes in neural crest development.

AmotL2 integrates polarity and junctional cues to modulate cell shape

The assembly of individual epithelial or endothelial cells into a tight cellular sheet requires stringent control of cell packing and organization. These processes are dependent on the establishment and further integration of cellular junctions, the cytoskeleton and the formation of apical-basal polarity. However, little is known how these subcellular events are coordinated. The (Angiomotin) Amot protein family consists of scaffold proteins that interact with junctional cadherins, polarity proteins and the cytoskeleton. In this report, we have studied how these protein complexes integrate to control cellular shapes consistent with organ function. Using gene-inactivating studies in zebrafish and cell culture systems in vitro, we show that Par3 to be essential for localization of AmotL2 to cellular junctions to associate with VE/E-cadherin and subsequently the organization of radial actin filaments. Our data provide mechanistic insight in how critical processes such as aortic lumen expansion as well as epithelial packing into hexagonal shapes are controlled.

Harmless effects of argon plasma on caudal fin regeneration and embryogenesis of zebrafish: novel biological approaches for safe medical applications of bioplasma

The argon plasma jet (Ar-PJ) is widely used in medical fields such as dermatology and dentistry, and it is considered a promising tool for cancer therapy. However, the in vivo effects of Ar-PJ for medical uses have not yet been investigated, and there are no biological tools to determine the appropriate clinical dosages of Ar-PJ. In this study, we used the caudal fin and embryo of zebrafish as novel in vivo tools to evaluate the biosafety of Ar-PJ. Typically, Ar-PJ is known to induce cell death in two-dimensional (2D) cell culture systems. By contrast, no detrimental effects of Ar-PJ were shown in our 3D zebrafish systems composed of 2D cells. The Ar-PJ-treated caudal fins grew by an average length of 0.7 mm, similar to the length of the normally regenerating fins. Remarkably, Ar-PJ did not affect the expression patterns of Wnt8a and β-Catenin, which play important roles in fin regeneration. In the embryo system, 85% of the Ar-PJ-treated embryos hatched, and the lateral length of these embryos was ~3.3 mm, which are equivalent to the lengths of normal embryos. In particular, vasculogenesis, which is the main cellular process during tissue regeneration and embryogenesis, occurred normally under the Ar-PJ dose used in this study. Therefore, our biosafety evaluation tools that use living model systems can be used to provide an experimental guideline to determine the clinically safe dosage of Ar-PJ.

Live imaging of primary ocular vasculature formation in zebrafish

Ocular vasculature consists of the central retinal and ciliary vascular systems, which are essential to maintaining visual function. Many researchers have attempted to determine their origins and development; however, the detailed, stepwise process of ocular vasculature formation has not been established. In zebrafish, two angioblast clusters, the rostral and midbrain organizing centers, form almost all of the cranial vasculature, including the ocular vasculature, and these are from where the cerebral arterial and venous angioblast clusters, respectively, differentiate. In this study, we first determined the anatomical architecture of the primary ocular vasculature and then followed its path from the two cerebral angioblast clusters using a time-lapse analysis of living Tg(flk1:EGFP)k7 zebrafish embryos, in which the endothelial cells specifically expressed enhanced green fluorescent protein. We succeeded in capturing images of the primary ocular vasculature formation and were able to determine the origin of each ocular vessel. In zebrafish, the hyaloid and ciliary arterial systems first organized independently, and then anastomosed via the inner optic circle on the surface of the lens by the lateral transfer of the optic vein. Finally, the choroidal vascular plexus formed around the eyeball to complete the primary ocular vasculature formation. To our knowledge, this study is the first to report successful capture of circular integration of the optic artery and vein, lateral transfer of the optic vein to integrate the hyaloidal and superficial ocular vasculatures, and formation of the choroidal vascular plexus. Furthermore, this new morphological information enables us to assess the entire process of the primary ocular vasculature formation, which will be useful for its precise understanding.

Comparative Developmental Toxicity of Flavonoids Using an Integrative Zebrafish System.

Flavonoids are a large, structurally diverse class of bioactive naturally occurring chemicals commonly detected in breast milk, soy based infant formulas, amniotic fluid, and fetal cord blood. The potential for pervasive early life stage exposures raises concerns for perturbation of embryogenesis, though developmental toxicity and bioactivity information is limited for many flavonoids. Therefore, we evaluated a suite of 24 flavonoid and flavonoid-like chemicals using a zebrafish embryo-larval toxicity bioassay-an alternative model for investigating developmental toxicity of environmentally relevant chemicals. Embryos were exposed to 1-50 µM of each chemical from 6 to 120 h postfertilization (hpf), and assessed for 26 adverse developmental endpoints at 24, 72, and 120 hpf. Behavioral changes were evaluated in morphologically normal animals at 24 and 72 hpf, at 120 hpf using a larval photomotor response (LPR) assay. Gene expression was comparatively evaluated for all compounds for effects on biomarker transcripts indicative of AHR (cyp1a) and ER (cyp19a1b, esr1, lhb, vtg) pathway bioactivity. Overall, 15 of 24 flavonoids elicited adverse effects on one or more of the developmental or behavioral endpoints. Hierarchical clustering and principle component analyses compared toxicity profiles and identified 3 distinct groups of bioactive flavonoids. Despite robust induction of multiple estrogen-responsive biomarkers, co-exposure with ER and GPER antagonists did not ameliorate toxicity, suggesting ER-independence and alternative modes of action. Taken together, these studies demonstrate that development is sensitive to perturbation by bioactive flavonoids in zebrafish that are not related to traditional estrogen receptor mode of action pathways. This integrative zebrafish platform provides a useful framework for evaluating flavonoid developmental toxicity and hazard prioritization.

Expression of miRNA-122 Induced by Liver Toxicants in Zebrafish.

MicroRNA-122 (miRNA-122), also known as liver-specific miRNA, has recently been shown to be a potent biomarker in response to liver injury in mammals. The objective of this study was to examine its expression in response to toxicant treatment and acute liver damage, using the zebrafish system as an alternative model organism. For the hepatotoxicity assay, larval zebrafish were arrayed in 24-well plates. Adult zebrafish were also tested and arrayed in 200 mL cages. Animals were exposed to liver toxicants (tamoxifen or acetaminophen) at various doses, and miRNA-122 expression levels were analyzed using qRT-PCR in dissected liver, brain, heart, and intestine, separately. Our results showed no significant changes in miRNA-122 expression level in tamoxifen-treated larvae; however, miRNA-122 expression was highly induced in tamoxifen-treated adults in a tissue-specific manner. In addition, we observed a histological change in adult liver (0.5 μM) and cell death in larval liver (5 μM) at different doses of tamoxifen. These results indicated that miRNA-122 may be utilized as a liver-specific biomarker for acute liver toxicity in zebrafish.

Zebrafish as a Model for the Study of Solid Malignancies.

Zebrafish cancer models have provided critical insight into understanding the link between aberrant developmental pathways and tumorigenesis. The unique strengths of zebrafish as compared to other vertebrate model systems include the combination of fecundity, readily available and efficient transgenesis techniques, transparency that facilitates in vivo cell lineage tracing, and amenability for high-throughput applications. In addition to early embryo readouts, zebrafish can develop tumors at ages ranging from 2 weeks old to adulthood. Tumorigenesis is driven by genetically introducing oncogenes using selected promoter/tissue-specific expression, with either mosaic expression or with the generation of a stable transgenic line. Here, we detail a research pipeline to facilitate the study of human oncogenes in zebrafish systems. The goals of this approach are to identify conserved developmental pathways that may be critical for tumor development and to create platforms for testing novel therapies.

Generation and analysis of zebrafish melanoma models.

The rapid emergence of the zebrafish as a cancer model has been aided by advances in genetic, chemical, and imaging technologies. Melanoma in particular highlights both the power and challenges associated with cancer modeling inzebrafish. This chapterfocuses on the lessons that have emerged from the melanoma models as paradigmatic of what will apply to nearly all cancer models in the zebrafish system. Wespecifically focus on methodologies related to germline and mosaic transgenic melanoma generation, and how these can be used to deeply interrogate additional cooperating oncogenes or tumor suppressors. These transgenic tumors can in turn be used to generate zebrafish-specific, stable melanoma cell lines which can be fluorescently labeled, modified by cDNA/CRISPR techniques, and used for detailed in vivo imaging of cancer progression in real time. These zebrafish melanoma models are beginning to elucidate both cell intrinsic and microenvironmental factors in melanoma that have broader implications for human disease. We envision that nearly all of the techniques described here can be applied to other zebrafish cancer models, and likely expanded beyond what we describe here.