DRUG DISCOVERY AND TESTING USING HIGH-THROUGHPUT MORPHOLOGICAL AND BEHAVIORAL SCREENING OF ZEBRAFISH LARVAE

Ravindra Peravali, Eduard Gursky, Daniel Marcato, Johannes Stegmaier, Helmut Breitwieser, Christian Pylatiuk, Robert Geisler, Jos van Wezel, Ralf Mikut and Uwe Straehle

Institute of Toxicology and Genetics, Karlsruhe Institute of Technology, Hermann-von-Helmholtz Platz 1, 76344 Eggenstein-Leopoldshafen, Germany

Abstract

In recent years fish models have been increasingly adopted for identifying novel compounds and in in vivo testing of drugs and in toxicological studies. Specifically, the zebrafish (Danio rerio) embryo has emerged as a robust model for such investigations owing to its transparency, fecundity and genetic capabilities including transgenesis and gene knock-out and genomic similarities to human genes. Furthermore, the importance of this model is reflected by the OECD’s developing and establishing rigorous guidelines for Fish Embryo Toxicity (FET) testing based on the zebrafish. Besides in-depth mechanistic studies, the zebrafish embryo model permits systematic screening of large numbers of compounds in parallel in a fast and unsupervised manner. To this end, the European Zebrafish Resource Center (EZRC) has exploited advances in imaging technologies, data storage and high-performance computing to establish screening platforms and pipelines for identifying and evaluating compounds using the zebrafish.

Recently we have integrated morphological and behavioral phenotypes in a systematic manner to evaluate and test novel and uncharacterized compounds in high-throughput. We identify morphological and behavioral phenotypes of zebrafish embryos/larvae exposed to the chemicals at 3 different developmental stages: from 1 day post fertilization (dpf) to 5 dpf.

For morphological phenotypes, using an automated and intelligent microscopy platform that is developed on an Olympus Scan^R microscope (but can be adapted to any microscope), we detect mortality, developmental disorders and malformations without human intervention. For behavioral phenotypes, we have established a high-throughput Photomotor Response (PMR) platform that is used to study the effect of neuroactive and psychotropic drugs at 30 hours post fertilization (hpf). This automated platform captures the behavior of embryos (treated with chemicals) prior to, during and after presenting a series of light stimuli. At 5 dpf we study the effect of drugs on spontaneous locomotion using a second platform based on a robotic imaging system. A robotic arm equipped with a high-resolution camera is used to measure locomotion in zebrafish larvae. The nature and the kinematic parameters of locomotion are used as markers to evaluate the effects of different drugs on normal spontaneous movement. Importantly, all the platforms presented include specialized data analysis pipelines that automatically extract quantitative phenotypic read-outs in real-time. We discuss the mathematical algorithms that are used in such data analyses. To facilitate such high-throughput and high-content screens we present the LSDF (Large Scale Data Facility) our data storage infrastructure that has on-line storage and archive capacity for several peta-bytes.