ABSTRACT: The whole embryo culture (WEC) model serves as a potential replacement for classical in vivo developmental toxicity testing. In this alternative test, cultured rat embryos are exposed during neurulation and early organogenesis and evaluated for morphological adverse outcomes. Toxicogenomic-based approaches may improve the predictive ability of WEC by providing molecular-based markers associated with chemical exposure which can be compared across multiple parameters (e.g. time, dose, model). Additionally, comparisons between in vitro and in vivo models may identify objective, relevant molecular responses linked with developmental toxicity endpoints in vivo. In this study, using a transcriptomic approach, we compared all-trans-retinoic acid (RA)-exposed and non-exposed Wistar rat embryos derived using WEC (RA, 0.5µg/mL) or in vivo (RA, 50mg/kg, oral gavage) to identify overlapping and non-overlapping effects of RA on RNA expression in parallel with morphological changes. Across six time points (GD 10 + 2-48h), we observed strong similarities in RA-response at the gene (directionality, significance) and functional (e.g. embryonic development, cell differentiation) level which associated with RA-induced adverse morphological effects, including growth reduction as well as alterations of neural tube, limb, branchial, and mandible development. Differences between models in the timing of RA-induced impacts on the expression of select genes related to embryonic development and RA-metabolism associated with specific differential morphological outcomes. This study supports the use of WEC to examine compound-induced molecular responses relative to in vivo, and furthermore, assists in defining the applicability domain of the WEC in determining complementary windows of sensitivity for developmental toxicological investigations. Cultured embryos mimic the morphological developmental progression of embryos (in vivo) undergoing neurulation and early organogenesis. Using available genomics technologies, comparative molecular-based assessments between cultured embryos and in vivo models may further clarify commonalities and dissimilarities which contribute to differences between systems. Therefore, in this study, using a transcriptomic approach, we compared cultured whole rat embryos and embryos in vivo at comparable time points in development (gestational day (GD) 10 + 2-48h, GD 0 = copulatory plug) to assess for commonalities and differences in gene expression in relation to morphology. We reveal strong parallels in time-dependent expression of genes in terms of magnitude, directionality and functionality between whole embryo culture (WEC) and in vivo (rat). Genes changing in expression over time resemble previously hypothesized mechanisms underlying early development in mammalian systems. Furthermore, at the gene and functional level, we identify genes which differ in expression between models, including genes related to development, oxygen transport and metabolism. In summary, our results support the use of WEC for toxicological studies aimed at representing in vivo development during this time-window at the molecular level. Additionally, we indicate genes which differ in expression between models, providing possible insights for improvement of culture conditions.
