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.

Lack of motor neuron differentiation is an intrinsic property of the mouse secondary neural tube.

The cranial part of the amniote neural tube is formed by folding and fusion of the ectoderm-derived neural plate (primary neurulation). After posterior neuropore closure, however, the caudal neural tube is formed by cavitation of tail bud mesenchyme (secondary neurulation). In mouse embryos, the secondary neural tube expresses several genes important in early patterning and induction, in restricted domains similar to the primary neural tube, yet it does not undergo neuronal differentiation, but subsequently degenerates. Although the secondary neural tube, isolated from surrounding tissues, is responsive to exogenous Sonic Hedgehog proteins in vitro, motor neuron differentiation is never observed. This cannot be attributed to the properties of the secondary notochord, since it is able to induce motor neuron differentiation in naive chick neural plate explants. Taken together, these results support that the lack of motor neuron differentiation is an intrinsic property of the mouse secondary neural tube.

Tea epigallocatechin-3-gallate increases 8-isoprostane level and induces caudal regression in developing rat embryos.

Tea is the most common beverage after water. Concerns have been raised about the safety of tea during pregnancy, especially for embryo development. We aimed at studying the effects of active tea components on developing embryos by in vitro rat embryo culture. Rat embryos during early organogenesis were cultivated in serum supplemented with one of the tea catechins. Developmental hallmarks and malformations (Mal) in the developing embryos were compared and evaluated by a standard morphological scoring system. The embryotoxicity of each tea catechin was classified according to the European Center for the Validation of Alternative Methods. Cell viability was assessed by supervital dye staining, apoptosis by TUNEL assay, and peroxidation by the 8-isoprostane EIA method. We found that (+)-catechin had the least effect on developing embryos (Mal(50)=715.1 mg/L; IC50(Mal)=435 mg/L), whereas (-)-epigallocatechin gallate had the most adverse effect (Mal(50)=54.2 mg/L; IC50(Mal)=45.8 mg/L). The major malformation in affected embryos included caudal retardation with abnormal axial flexion and delayed hind-limb formation. All catechins were classified as nonembryotoxic except (-)-epigallocatechin gallate, which was classified as weakly embryotoxic. With (-)-epigallocatechin gallate, increased numbers of nonviable and apoptotic cells in the malformed embryos were associated with increased embryo 8-isoprostane.

Hypermethylation of RASSF1A in human and rhesus placentas.

The pseudomalignant nature of the placenta prompted us to search for tumor suppressor gene hypermethylation, a phenomenon widely reported in cancer, in the human placenta. Nine tumor suppressor genes were studied. Hypermethylation of the Ras association domain family 1 A (RASSF1A) gene was found in human placentas from all three trimesters of pregnancy but was absent in other fetal tissues. Hypermethylation of Rassf1 was similarly observed in placentas from the rhesus monkey but not the mouse. An inverse relationship between RASSF1A promoter methylation and gene expression was demonstrated by bisulfite sequencing of microdissected placental cells and immunohistochemical staining of placental tissue sections using an anti-RASSF1A antibody. Treatment of choriocarcinoma cell lines, JAR and JEG3, by 5-aza-2'-deoxycytidine and trichostatin A led to reduction in RASSF1A methylation but increased expression. These observations extend the analogy between the primate placenta and malignant tumors to the epigenetic level.

Implication of Wt1 in the pathogenesis of nephrogenic failure in a mouse model of retinoic acid-induced caudal regression syndrome.

Renal malformations are common human birth defects that sometimes occur in the context of the caudal regression syndrome. Here, we found that exposure of pregnant mice to all-trans retinoic acid, at a time when the metanephros has yet to form, causes a failure of kidney development along with caudal regression. Maternal treatment with Am580 (retinoic acid receptor alpha agonist) also induced similar patterns of kidney maldevelopment in the fetus. In metanephroi from retinoic acid-treated pregnancies, renal mesenchyme condensed around the ureteric bud but then failed to differentiate into nephrons, instead undergoing involution by fulminant apoptosis to produce a renal agenesis phenotype. Results of whole organ cultures in serum-free medium, and also tissue recombination experiments, showed that the nephrogenic defect was intrinsic to the kidney and that it resided in the metanephric mesenchyme and not the ureteric bud. Renal mesenchyme from control embryos expressed Wilms' tumor 1 (Wt1), but this transcription factor, which is indispensable for kidney development, failed to express in metanephroi of retinoic acid-exposed embryos. Wt1 expression and organogenesis were both restored, however, when metanephroi from retinoic acid-treated pregnancies were grown in serum-containing media. Our data illuminate the pathobiology of a severe, teratogen-induced kidney malformation.

Maternal diabetes increases the risk of caudal regression caused by retinoic acid.

Maternal diabetes increases the risk of congenital malformations in the offspring of affected pregnancies. This increase arises from the teratogenic effect of the maternal diabetic milieu on the developing embryo, although the mechanism of this action is poorly understood. In the present study, we examined whether the vitamin A metabolite retinoic acid (RA), a common drug with well-known teratogenic properties, may interact with maternal diabetes to alter the incidence of congenital malformations in mice. Our results show that when treated with RA, embryos of diabetic mice are significantly more prone than embryos of nondiabetic mice to develop caudal regression, a defect that is highly associated with diabetic pregnancy in humans. By studying the vestigial tail (Wnt-3a(vt)) mutant, we provide evidence that Wnt-3a, a gene that controls the development of the caudal region, is directly involved in the pathogenic pathway of RA-induced caudal regression. We further show that the molecular basis of the increased susceptibility of embryos of diabetic mice to RA involves enhanced downregulation of Wnt-3a expression. This positive interaction between RA and maternal diabetes may have implications for humans in suggesting increased susceptibility to environmental teratogens during diabetic pregnancy.