The effects of retinoic acid and valproic acid on gene expression in the zebrafishembryotoxicitytest: combining microarray analysis with whole mount in situ hybridization

Abstract

The zebrafish embryo toxicity test (ZET) is a promising alternative for animal testing in developmental toxicity, reducing the great amounts of animals used at present. Before implementation of the ZET, precise characterization of the model, with regard to developmental processes, has to be performed before validation of the test is possible. The classical method of interpretation of the ZET is morphological evaluation, which is consideredrather subjective. Transcriptomics holds great promise as a novel way to interpret the ZET in a much more accurate, sensitive and objective way. Furthermore, these techniques help to increase our understanding of the toxic mechanisms of different teratogens during embryological development. In this study, two known developmentally toxic compounds, retinoic acid (RA) and valproic acid (VPA) were used. Gene expression patterns were analyzed by microarray analysis and in situ hybridization of eleven selected genes, and were compared to the morphological effects, making use of the general morphology scoring system. The current study demonstrated comparable results for ISH and microarray analysis, and showed gene expression to be more sensitive than morphological effects seen in the zebrafish embryo after exposure to both VPA and RA. However, the technique of ISH predominantly showed to be suitable for research purposes, whereas microarray analysis is also applicable as a high-throughput screening method.