Nervous system development related gene expression regulation in the zebrafish embryo after exposure to valproic acid and retinoic acid: A genome wide approach.

The evaluation of chemical and pharmaceutical safety for humans is moving from animal studies to New Approach Methodologies (NAM), reducing animal use and focusing on mechanism of action, whilst enhancing human relevance. In developmental toxicology, the mechanistic approach is facilitated by the assessment of predictive biomarkers, which allow mechanistic pathways perturbation monitoring at the basis of human hazard assessment. In our search for biomarkers of maldevelopment, we focused on chemically-induced perturbation of the retinoic acid signaling pathway (RA-SP), a major pathway implicated in a plethora of developmental processes. A genome-wide expression screening was performed on zebrafish embryos treated with two teratogens, all-trans retinoic acid (ATRA) and valproic acid (VPA), and a non-teratogen reference compound, folic acid (FA). Each compound was found to have a specific mRNA expression profile with 248 genes commonly dysregulated by both teratogenic compounds but not by FA. These genes were implicated in several developmental processes (e.g., the circulatory and nervous system). Given the prominent response of neurodevelopmental gene sets, and the crucial need to better understand developmental neurotoxicity, our study then focused on nervous system development. We found 62 genes that are potential early neurodevelopmental toxicity biomarker candidates. These results advance NAM-based safety assessment evaluation by highlighting the usefulness of the RA-SP in providing early toxicity biomarker candidates.

Retinoic acid signaling pathway perturbation impacts mesodermal-tissue development in the zebrafish embryo: Biomarker candidate identification using transcriptomics.

The zebrafish embryo (ZE) model provides a developmental model well conserved throughout vertebrate embryogenesis, with relevance for early human embryo development. It was employed to search for gene expression biomarkers of compound-induced disruption of mesodermal development. We were particularly interested in the expression of genes related to the retinoic acid signaling pathway (RA-SP), as a major morphogenetic regulating mechanism. We exposed ZE to teratogenic concentrations of valproic acid (VPA) and all-trans retinoic acid (ATRA), using folic acid (FA) as a non-teratogenic control compound shortly after fertilization for 4 h, and performed gene expression analysis by RNA sequencing. We identified 248 genes specifically regulated by both teratogens but not by FA. Further analysis of this gene set revealed 54 GO-terms related to the development of mesodermal tissues, distributed along the paraxial, intermediate, and lateral plate sections of the mesoderm. Gene expression regulation was specific to tissues and was observed for somites, striated muscle, bone, kidney, circulatory system, and blood. Stitch analysis revealed 47 regulated genes related to the RA-SP, which were differentially expressed in the various mesodermal tissues. These genes provide potential molecular biomarkers of mesodermal tissue and organ (mal)formation in the early vertebrate embryo.

Dynamic regulation of gene expression and morphogenesis in the zebrafish embryo test after exposure to all-trans retinoic acid.

The zebrafish embryotoxicity test (ZET) is widely used in developmental toxicology. The analysis of gene expression regulation in ZET after chemical exposure provides mechanistic information about the effects of chemicals on morphogenesis in the test. The gene expression response magnitude has been shown to change with exposure duration. The objective of this work is to study the effect of the exposure duration on the magnitude of gene expression changes in the all-trans retinoic acid (ATRA) signaling pathway in the ZET. Retinoic acid regulation is a key driver of morphogenesis and is therefore employed here as an indicator for the regulation of developmental genes. A teratogenic concentration of 7.5 nM of ATRA was given at 3 hrs post fertilization (hpf) for a range of exposure durations until 120 hrs of development. The expression of a selection of genes related to ATRA signaling and downstream developmental genes was determined. The highest magnitudes of gene expression regulation were observed after 2-24 hrs exposure with an optimal response after 4 hrs. Longer exposures showed a decrease in the gene expression response, although continued exposure to 120 hpf caused malformations and lethality. This study shows that assessment of gene expression regulation at early time points after the onset of exposure in the ZET may be optimal for the prediction of developmental toxicity. We believe these results could help optimize sensitivity in future studies with ZET.