Novel System to Automate Zebrafish Angiogenesis Assays

DESCRIPTION (provided by applicant): The zebrafish, Danio rerio, has become widely accepted as a model for human disease. There is strong interest in developing zebrafish as a model organism for use in high throughput drug discovery assays. Advantages of using zebrafish in drug discovery include the ease with which they absorb candidate drug molecules and their genetic and physiologic similarity to humans. Thus far, high throughput screening in zebrafish has been limited to embryos and very young larvae. This is because the vasculature and many of the organs that are most interesting to researchers are best viewed from the side of the animal. Once the swim bladder inflates the larvae generally float upright making visualization of internal structures difficult. This is especially true in the multiwell microplate formats that are used in most high-throughput drug discovery. Efforts to resolve this important problem have centered on use of confocal imaging techniques or powerful image deconvolution software to assemble 3-D reconstructions of the animal. These efforts have not achieved the desired spatial resolution and typically require 1 to 2 minutes to image a single well. Physical Sciences Inc. (PSI) has invented a novel technology that will be adaptable to standard image analysis systems that are already in use in drug discovery laboratories. PSI's technology will allow researchers to generate clear side-views of older zebrafish larvae in microwell arrays. By providing a side view of the zebrafish, the technology will enable generation of quality images in seconds per well versus minutes per well for current techniques, hi Phase I we will generate fluorescence images of the vasculature of transgenic zebrafish that express Enhanced Green Fluorescent Protein (EGFP) under control of thefli-1 regulatory sequences. We will compare images of larvae with normal vascular development with those of larvae treated with a VEGF inhibitor and which have incompletely developed vasculature. These experiments will serve as a model for Phase II development of a fully-automated, high-throughput, drug-discovery assay in zebrafish for novel angiogenesis inhibitors.