Methoxyacetic acid inhibits histone deacetylase and impairs axial elongation morphogenesis of mouse gastruloids in a retinoic acid signaling-dependent manner.

BACKGROUND: Teratogenic potential has been linked to various industrial compounds. Methoxyacetic acid (MAA) is a primary metabolite of the widely used organic solvent and plasticizer, methoxyethanol and dimethoxyethyl phthalate, respectively. Studies using model animals have shown that MAA acts as the proximate teratogen that causes various malformations in developing embryos. Nonetheless, the molecular mechanisms by which MAA exerts its teratogenic effects are not fully understood. METHODS: Gastruloids of mouse P19C5 pluripotent stem cells, which recapitulate axial elongation morphogenesis of gastrulation-stage embryos, were explored as an in vitro model to investigate the teratogenic action of MAA. Morphometric parameters of gastruloids were measured to evaluate the morphogenetic effect, and transcript levels of various developmental regulator genes were examined to assess the impact on gene expression patterns. The effects of MAA on the level of retinoic acid (RA) signaling and histone deacetylase activity were also measured. RESULTS: MAA reduced axial elongation of gastruloids at concentrations comparable to the teratogenic plasma level (5 mM) in vivo. MAA at 4 mM significantly altered the expression profiles of developmental regulator genes. In particular, it upregulated the RA signaling target genes. The concomitant suppression of RA signaling using a pharmacological agent alleviated the morphogenetic effect of MAA. MAA at 4 mM also significantly reduced the activity of purified histone deacetylase protein. CONCLUSIONS: MAA impaired axial elongation morphogenesis in a RA signaling-dependent manner in mouse gastruloids, possibly through the inhibition of histone deacetylase.

Adverse effect of valproic acid on an in vitro gastrulation model entails activation of retinoic acid signaling.

Valproic acid (VPA), an antiepileptic drug, is a teratogen that causes neural tube and axial skeletal defects, although the mechanisms are not fully understood. We previously established a gastrulation model using mouse P19C5 stem cell embryoid bodies (EBs), which exhibits axial patterning and elongation morphogenesis in vitro. Here, we investigated the effects of VPA on the EB axial morphogenesis to gain insights into its teratogenic mechanisms. Axial elongation and patterning of EBs were inhibited by VPA at therapeutic concentrations. VPA elevated expression levels of various developmental regulators, including Cdx1 and Hoxa1, known transcriptional targets of retinoic acid (RA) signaling. Co-treatment of EBs with VPA and BMS493, an RA receptor antagonist, partially rescued axial elongation as well as gene expression profiles. These results suggest that VPA requires active RA signaling to interfere with EB morphogenesis.

An in vitro gastrulation model recapitulates the morphogenetic impact of pharmacological inhibitors of developmental signaling pathways.

Certain chemical agents act as teratogens, causing birth defects and fetal deaths when pregnant women are exposed to them. The establishment of in vitro models that recapitulate crucial embryonic events is therefore vital to facilitate screening of potential teratogens. Previously, we created a three-dimensional culture method for mouse P19C5 embryonal carcinoma stem cells that, when cultured as embryoid bodies, display elongation morphogenesis resembling gastrulation, which is the critical event resulting in the germ layers and major body axes. Determination of how well this in vitro morphogenesis represents in vivo gastrulation is essential to assess its applicability as well as to identify limitations of the model for detecting teratogenic agents. Here, we investigated the morphological and molecular characteristics of P19C5 morphogenesis using pharmacological agents that are known to cause abnormal patterning in the embryo in vivo by inhibiting major developmental signaling--e.g., involving Wnt, Nodal, Bone morphogenic protein (Bmp), Fibroblast growth factor (Fgf), Retinoic acid, Notch, and Hedgehog pathways. Inhibitors of Wnt, Nodal, Bmp, Fgf, and Retinoic acid signaling caused distinct changes in P19C5 morphogenesis that were quantifiable using morphometric parameters. These five inhibitors, plus the Notch inhibitor, also altered temporal expression profiles of developmental regulator genes in a manner consistent with the in vivo roles of the corresponding signaling pathways. In contrast, the Hedgehog inhibitor did not have any impact on the process, suggesting an absence of active Hedgehog signaling in these embryoid bodies. These results indicate that the P19C5 in vitro gastrulation model is a promising tool to screen for teratogenic agents that interfere with many of the key developmental signals.