Highly Sensitive Quantitative Determination of Retinoic Acid Levels, Retinoic Acid Synthesis, and Catabolism in Embryonic Tissue Using a Reporter Cell-Based Method.

The effect of all-trans retinoic acid (RA) on embryogenesis is tissue specific and highly concentration dependent. Using a liquid chromatography/mass spectrometry-based method to quantify trace amounts of RA in embryonic tissue requires expensive specialist facilities. Here, we describe the use of a RA response element (RARE)-lacZ reporter cell-based method, which is simple and cost effective, to measure RA levels in small pieces of tissue from the embryo. We further apply this method to quantitatively assay activities of RA-synthesizing and RA-catabolizing enzymes, the key regulators of RA bioavailability in tissues and developing organs of the embryo.

Perturbation of Retinoid Homeostasis Increases Malformation Risk in Embryos Exposed to Pregestational Diabetes.

Pregestational diabetes is highly associated with an increased risk of birth defects. However, factors that can increase or reduce the expressivity and penetrance of malformations in pregnancies in women with diabetes remain poorly identified. All-trans retinoic acid (RA) plays crucial roles in embryogenesis. Here, we find that Cyp26a1, which encodes a key enzyme for catabolic inactivation of RA required for tight control of local RA concentrations, is significantly downregulated in embryos of diabetic mice. Embryonic tissues expressing Cyp26a1 show reduced efficiency of RA clearance. Embryos exposed to diabetes are thus sensitized to RA and more vulnerable to the deleterious effects of increased RA signaling. Susceptibility to RA teratogenesis is further potentiated in embryos with a preexisting genetic defect of RA metabolism. Increasing RA clearance efficiency using a preconditioning approach can counteract the increased susceptibility to RA teratogenesis in embryos of diabetic mice. Our findings provide new insight into gene-environment interactions that influence individual risk in the manifestation of diabetes-related birth defects and shed light on environmental risk factors and genetic variants for a stratified medicine approach to screening women with diabetes who are of childbearing age and assessing the risk of birth defects during pregnancy.

A paradoxical teratogenic mechanism for retinoic acid.

Retinoic acid, an active metabolite of vitamin A, plays essential signaling roles in mammalian embryogenesis. Nevertheless, it has long been recognized that overexposure to vitamin A or retinoic acid causes widespread teratogenesis in rodents as well as humans. Although it has a short half-life, exposure to high levels of retinoic acid can disrupt development of yet-to-be formed organs, including the metanephros, the embryonic organ which normally differentiates into the mature kidney. Paradoxically, it is known that either an excess or a deficiency of retinoic acid results in similar malformations in some organs, including the mammalian kidney. Accordingly, we hypothesized that excess retinoic acid is teratogenic by inducing a longer lasting, local retinoic acid deficiency. This idea was tested in an established in vivo mouse model in which exposure to excess retinoic acid well before metanephric rudiments exist leads to failure of kidney formation several days later. Results showed that teratogen exposure was followed by decreased levels of Raldh transcripts encoding retinoic acid-synthesizing enzymes and increased levels of Cyp26a1 and Cyp26b1 mRNAs encoding enzymes that catabolize retinoic acid. Concomitantly, there was significant reduction in retinoic acid levels in whole embryos and kidney rudiments. Restoration of retinoic acid levels by maternal supplementation with low doses of retinoic acid following the teratogenic insult rescued metanephric kidney development and abrogated several extrarenal developmental defects. This previously undescribed and unsuspected mechanism provides insight into the molecular pathway of retinoic acid-induced teratogenesis.