by Dr P. M. Eimon
Zebrafish embryo screens enable the rapid, cost effective identification of compounds with desired biological activities. These screens also allow large numbers of compounds to be analysed in vivo early in the drug discovery process to eliminate those with unfavourable acute and organ-specific toxicities. The full power of zebrafish-based assays and disease models is realised when they are coupled with fluorescent reporters or other robust, quantitative readouts that can be rapidly screened and analysed, even by non-zebrafish specialists, using high content imaging systems.
The zebrafish (Danio rerio) is a leading model organism for the study of vertebrate developmental biology. The same traits that make zebrafish embryos attractive to academic researchers also make them ideal for disease modelling, drug discovery and safety testing [1]. Molecular pathways, organ systems and physiology are well conserved between zebrafish and mammals. Embryos develop rapidly outside the mother, are optically transparent and are permeable to a wide range of drugs and small molecules. Within five days of fertilisation, most major organ systems are present in the larvae. Because zebrafish embryos can be arrayed in multi-well plates (up to 384-well) and tolerate prolonged exposure to DMSO (up to 1%), they are easily adapted to high throughput screening needs. The zebrafish genome has been sequenced and numerous mutant lines are available (Zebrafish International Resource Center, Eugene, OR and Znomics, Portland, OR). Transgenic zebrafish lines have been developed in which organs or tissues of interest are labelled with fluorescent proteins and embryos from a number of these lines are commercially available (Zygogen, Atlanta, GA). Individual gene targets can be knocked down in vivo in zebrafish embryos using morpholino antisense oligonucleotides (Gene Tools, Philomath, OR). Finally, zebrafish embryos are an important alternative for researchers looking to meet regulatory mandates that promote reduced laboratory animal testing. This article does not aim to be a comprehensive review of zebrafish disease and toxicity models, but rather highlights ways in which transgenic technology has been used to develop and automate assays in the areas of oncology and cardiac toxicity.
ONCOLOGY
Angiogenesis is essential for the growth and metastasis of solid tumours and plays a causative role in macular degeneration and diabetic retinopathy. Conventional in vivo models used to evaluate anti-angiogenic compounds (e.g. the matrigel plug assay and the corneal model in mice) are expensive, time-consuming and require comparatively large quantities of test compound, making them unsuitable for early-stage screening assays. Zebrafish embryos offer an important alternative to mammalian angiogenesis assays. The zebrafish vascular system is well characterised and molecular pathways underlying angiogenesis are known to be highly conserved. Consequently, numerous angiogenesis inhibitors have been shown to affect the formation of zebrafish blood vessels.
Researchers have recently developed a quantitative zebrafish-based angiogenesis assay that preserves the biological complexity of in vivo models. This angiogenesis assay enables much higher-throughput screening than is feasible in traditional animal models [2]. The assay utilises a transgenic zebrafish line in which expression of Aequorea coerulescens green fluorescent protein (AcGFP) is driven by the vegfr2 promoter. Embryos obtained from these transgenic lines express fluorescent protein throughout their developing vasculature, including the prominent network of angiogenic vessels that form in the trunk of the embryo beginning at one day post fertilisation (dpf). This convenient fluorescent readout eliminates the need for time-consuming detection steps (e.g. alkaline phosphatase staining or microangiography) and provides a straightforward real-time readout of angiogenesis. For compound library screening, embryos are arrayed into 384-well plates and test compounds are applied using a robotic liquid handling instrument. Following overnight incubation, a fluorescent image of each embryo is captured using an automated imaging system and analysed using standard image analysis software [Figure 1].
Transgenic zebrafish with fluorescent blood vessels also play an important role in two recent publications describing zebrafish xenograft models [3, 4]. In both instances, mammalian tumour cell lines were successfully grafted into transgenic zebrafish and vessel fluorescence was used to monitor subsequent tumour neovascularisation in vivo. Stoletov et al. report that when human tumour cells (also fluorescently labelled) are injected into the peritoneal cavity of immunosuppressed zebrafish, they exhibit invasive behaviour, induce angiogenesis and interact with the newly formed vessels. In contrast to traditional animal models, the zebrafish system allows real time non-invasive monitoring of these highly dynamic processes.
A number of valuable transgenic zebrafish cancer models have also been developed. Researchers have generated transgenic fish expressing the oncogenic TEL-AML1 fusion in order to model precursor B cell acute lymphoblastic leukaemia
(B-ALL) [5]. Approximately 3% of transgenic fish develop leukaemia within 8-12 months and leukaemic cells have been successfully transplanted to irradiated wild-type recipients. A transgenic zebrafish model of T cell acute lymphoblastic leukaemia (T-ALL) has also been developed by using the rag2 promoter to drive expression of EGFP-labelled mouse c-Myc [6]. Leukaemia in this transgenic line is highly penetrant and typically renders the animals moribund prior to reaching sexual maturity. To circumvent this difficulty, researchers have devised a conditional transgenic line using the Cre/lox system in which T-ALL can be induced by injection of Cre RNA into one-cell-stage embryos.
CARDIOTOXICITY
Juvenile and adult fish have long played an important role in many areas of safety and toxicity testing, particularly in environmental risk assessment and hazard classification. Because researchers and governmental regulatory bodies have become increasingly sensitive to the need to replace, reduce and refine the use of laboratory animals whenever feasible, there is a growing movement to perform toxicity assays during the early life stage of the fish. The reason for this is most clearly laid out in the Council of the European Communities Directive 86/609/EEC on the protection of experimental animals. In this directive, protected research animals are defined as “non-human vertebrate, including free-living larval and/or reproducing larval forms, but excluding foetal or embryonic forms.” In the case of zebrafish, “embryonic form” is generally regarded as extending from the time of fertilisation until the onset of independent feeding (~4-5 dpf). Even assays performed on later-stage zebrafish larvae may facilitate a significant overall reduction in the total number of mammals used for drug testing.
In the realm of safety and toxicity testing, zebrafish are a particularly attractive model for cardiotoxicity screening [1]. Zebrafish have a two-chambered heart that develops rapidly. Contractile waves are first observed at 1 dpf and the basic architecture of the heart is in place by 2 dpf. Voltage clamp analysis of embryonic cardiocytes reveals a repertoire of ion-channel currents analogous to those present in mammals. Cardiac safety and toxicity assays are significantly aided by the fact that zebrafish embryos and larvae tolerate severe disruptions of cardiac function and can survive for up to a week without blood circulation.
Measurement of the heart rate by simple microscopic examination has been used for years to detect cardiotoxicity in zebrafish embryos, while immunohistochemistry and histology have enabled analysis of cardiac morphology in a wide range of mutant lines. Absence of a heart beat is also one of several key parameters used to determine lethality in regulatory guidelines being developed to test chemicals in fish embryos (OECD Draft Test Guidelines on Fish Embryo Toxicity Test - 31 May 2006). Recently, the development of transgenic lines in which fluorescent reporters are expressed under the control of heart tissue-specific promoters has greatly enhanced the value and utility of zebrafish cardiotoxicity assays. These lines enhance monitoring of embryonic heart rate and aid in the analysis of specific cardiac structures for compound-induced toxicity.
Drug-induced long QT syndrome (LQTS) predisposes individuals to life-threatening arrhythmias and is therefore a major target of preclinical drug safety programmes. Adult zebrafish electrocardiograms show that drugs associated with a prolonged QT interval in humans also cause QT increases in fish [7]. Two studies published in 2003 demonstrated that drugs known to induce LQTS in humans elicit bradycardia and a 2:1 atrioventricular block (ventricle beating exactly half as often as the atrium) in zebrafish embryos [8, 9]. Milan et al. demonstrated that the correlation between zebrafish bradycardia and LQTS is very strong, with 22 out of 23 positive compounds being identified correctly in the assay. Therapeutic compounds that produce LQTS often do so via blockade of the cardiac potassium channel hERG (human ether-a-go-go-related gene). Direct inhibition of the zebrafish homologue of hERG (zerg) using morpholino antisense oliognucleotides also induces bradycardia and a 2:1 atrioventricular block, confirming the effects of potassium channel blockade.
Transgenic zebrafish have been instrumental in developing a high-throughput heart-rate assay suitable for small molecule screening. Researchers have used the cmlc2 (cardiac myosin light chain 2) promoter to develop transgenic lines that express fluorescent reporter proteins exclusively in the myocardium by 1 dpf [10]. Short videos of transgenic embryos are acquired with a high content imaging system and an automated algorithm extracts heart rates based on changes in fluorescent intensity, thereby eliminating the need for laborious visual scoring [Figure 2]. Heart rates can be scored accurately over a large linear dynamic range (0-300+ beats per minute) at an approximate rate of one embryo every 22 seconds. The cmlc2 promoter has also been used to express the Ca2+-sensitive fluorescent protein G-CaMP in the zebrafish heart [11]. These embryos exhibit repetitive fluorescent waves representing systolic Ca2+ release, allowing compounds or mutations that alter Ca2+ transients in all or part of the heart to be analysed.
SUMMARY
Zebrafish embryos are a robust in vivo model system suitable for medium-to-high throughput drug screening. Automated zebrafish assays have been developed in a number of therapeutic areas as well as for toxicity and safety assessment. Transgenic zebrafish embryos with fluorescent organs allow rapid imaging and quantitative analysis on existing platforms designed for cell-based assays. Use of zebrafish embryos also aids researchers in the drive to replace, reduce and refine the use of laboratory animals.
REFERENCES
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2. Tran TC, Sneed B, Haider J, Blavo D, White A, Aiyejorun T, Baranowski TC, Rubinstein AL, Doan TN, Dingledine R et al. Automated, quantitative screening assay for antiangiogenic compounds using transgenic zebrafish. Cancer Res 2007; 67: 11386-92.
3. Nicoli S, Ribatti D, Cotelli F and Presta M. Mammalian tumor xenografts induce neovascularization in zebrafish embryos. Cancer Res 2007; 67: 2927-31.
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5. Sabaawy HE, Azuma M, Embree LJ, Tsai HJ, Starost MF and Hickstein DD. TEL-AML1 transgenic zebrafish model of precursor B cell acute lymphoblastic leukemia. Proc Natl Acad Sci U S A 2006; 103: 15166-71.
6. Langenau DM, Feng H, Berghmans S, Kanki JP, Kutok JL and Look AT. Cre/lox-regulated transgenic zebrafish model with conditional myc-induced T cell acute lymphoblastic leukemia. Proc Natl Acad Sci U S A 2005; 102: 6068-73.
7. Milan DJ, Jones IL, Ellinor PT and MacRae CA. In vivo recording of adult zebrafish electrocardiogram and assessment of drug-induced QT prolongation. Am J Physiol Heart Circ Physiol 2006; 291: H269-73.
8. Langheinrich U, Vacun G and Wagner T. Zebrafish embryos express an orthologue of HERG and are sensitive toward a range of QT-prolonging drugs inducing severe arrhythmia. Toxicol Appl Pharmacol 2003; 193: 370-82.
9. Milan DJ, Peterson TA, Ruskin JN, Peterson RT and MacRae CA. Drugs that induce repolarization abnormalities cause bradycardia in zebrafish. Circulation 2003; 107: 1355-8.
10. Burns CG, Milan DJ, Grande EJ, Rottbauer, W, MacRae CA and Fishman MC. High-throughput assay for small molecules that modulate zebrafish embryonic heart rate. Nat Chem Biol 2005; 1: 263-4.
11. Arnaout R, Ferrer T, Huisken J, Spitzer K, Stainier DY, Tristani-Firouzi M and Chi NC. Zebrafish model for human long QT syndrome. Proc Natl Acad Sci U S A 2007; 104: 11316-21.
THE AUTHOR
Dr Peter Eimon
Director of Research
Zygogen, LLC
Atlanta, GA, USA
Tel +1 404-523-7309
e-mail: peter@zygogen.com
