High-Content Screening Assay for Identification of Chemicals Impacting Cardiovascular Function in Zebrafish Embryos

Targeted assays are needed to better
evaluate effects of chemicals
on organogenesis and begin classification of chemicals by toxicologically
relevant modes-of-action. Using transgenic zebrafish (fli1:egfp) that stably express eGFP within vascular endothelial cells, we
have developed and optimized a 384-well-based high-content screening
(HCS) assay that enables us to screen and identify chemicals affecting
cardiovascular function at sublethal, nonteratogenic concentrations.
Following static exposure of one embryo per well from 5 to 72 h postfertilization
(hpf), automated image acquisition procedures and custom image analysis
protocols are used to quantify body length, circulation, heart rate,
pericardial area (a biomarker for cardiac looping defects), and intersegmental
vessel area within freshly hatched live embryos. After optimizing
72 hpf anesthetization procedures, we evaluated each end point across
four independent control plates containing 384 initial embryos per
plate. Survival and imaging success rates across these plates ranged
from 93 to 99% and 42 to 74%, respectively. Criteria were then defined
for assay success and analysis of treatments, and 10 chemicals were
screened for targeted effects on cardiovascular function. Compared
to existing zebrafish-based assays, this method provides a comprehensive
discovery platform with (1) increased sample sizes; (2) broad concentration–response
format; and (3) the ability to identify chemicals that target cardiovascular
function at nonteratogenic concentrations.