Congenital diaphragmatic hernia after exposure to a triple retinoic acid antagonist during pregnancy.

AIM: To establish a mouse model for the study of congenital defects, using exposure of pregnant females to the teratogen BMS-189453, a multiple retinoic acid competitive antagonist.We found not less than 60% of fetuses had transposition of the great arteries and l5% had other congenital heart defects such as double outlet right ventricle, tetralogy of Fallot, truncus and right aortic arch. Newborns exposed in utero to BMS-189453 were affected by thymus aplasia or hypoplasia, and severe congenital anomalies of the central nervous system due to neural tube defects. An anterior rotation of the right lung was also frequently present in our model. We also report a case of murine congenital diaphragmatic hernia associated with thymic aplasia and transposition of the great arteries. CONCLUSION: These findings support the hypothesis that the combination of diaphragmatic hernia and congenital heart defects may be related to an alteration of the retinoic acid signaling pathways.

Hif1α down-regulation is associated with transposition of great arteries in mice treated with a retinoic acid antagonist.

BACKGROUND: Congenital heart defect (CHD) account for 25% of all human congenital abnormalities. However, very few CHD-causing genes have been identified so far. A promising approach for the identification of essential cardiac regulators whose mutations may be linked to human CHD, is the molecular and genetic analysis of heart development. With the use of a triple retinoic acid competitive antagonist (BMS189453) we previously developed a mouse model of congenital heart defects (81%), thymic abnormalities (98%) and neural tube defects (20%). D-TGA (D-transposition of great arteries) was the most prevalent cardiac defect observed (61%). Recently we were able to partially rescue this abnormal phenotype (CHD were reduced to 64.8%, p = 0.05), by oral administration of folic acid (FA). Now we have performed a microarray analysis in our mouse models to discover genes/transcripts potentially implicated in the pathogenesis of this CHD. RESULTS: We analysed mouse embryos (8.5 dpc) treated with BMS189453 alone and with BMS189453 plus folic acid (FA) by microarray and qRT-PCR. By selecting a fold change (FC) ≥ ± 1.5, we detected 447 genes that were differentially expressed in BMS-treated embryos vs. untreated control embryos, while 239 genes were differentially expressed in BMS-treated embryos whose mothers had also received FA supplementation vs. BMS-treated embryos. On the basis of microarray and qRT-PCR results, we further analysed the Hif1α gene. In fact Hif1α is down-regulated in BMS-treated embryos vs. untreated controls (FCmicro = -1.79; FCqRT-PCR = -1.76; p = 0.005) and its expression level is increased in BMS+FA-treated embryos compared to BMS-treated embryos (FCmicro = +1.17; FCqRT-PCR = +1.28: p = 0.005). Immunofluorescence experiments confirmed the under-expression of Hif1α protein in BMS-treated embryos compared to untreated and BMS+FA-treated embryos and, moreover, we demonstrated that at 8.5 dpc, Hif1α is mainly expressed in the embryo heart region. CONCLUSIONS: We propose that Hif1α down-regulation in response to blocking retinoic acid binding may contribute to the development of cardiac defects in mouse newborns. In line with our hypothesis, when Hif1α expression level is restored (by supplementation of folic acid), a decrement of CHD is found. To the best of our knowledge, this is the first report that links retinoic acid metabolism to Hif1α regulation and the development of D-TGA.

Folic acid and methionine in the prevention of teratogen-induced congenital defects in mice.

INTRODUCTION: Periconceptional supplementation with multivitamins containing folic acid reduces the risk of congenital malformations. We have previously investigated the effect on the murine development of a multiple retinoic acid competitive antagonist, Bristol-Myers-Squibb 189453, showing that treated fetuses were affected with heart defects, thymus aplasia or hypoplasia, and severe anomalies of the central nervous system. Hereby, we analyzed the effects of nutritive therapy involving folic acid and methionine on teratogen-induced congenital defects in mice. MATERIALS AND METHODS: A total of 132 outbred CD1 litters were studied. Pregnant mice were divided into four experimental groups, and an oral supplementation of H(2)O or folic acid, or methionine, or folic acid+methionine was administered from 0.5 days postcoitum until the end of pregnancy. At 7.5 days postcoitum, mice from all these groups were administered Bristol-Myers-Squibb 189453 to induce the teratogenic effect. At the end of pregnancy, fetuses were dissected and tissues were analyzed by histology and flow cytometric assays. RESULTS: Folic acid reduces congenital heart diseases from 81.3% to 64.8%, neural tube defects from 20.3% to 3.7%, and thymus abnormalities from 98.4% to 27.8%, restoring a normal number of differentiated thymus cells. Methionine is less effective in contrasting congenital heart diseases and neural tube defects, and induces thymus cell proliferation but not differentiation. Folic acid+methionine weakly reduce congenital heart diseases and neural tube defects, but consistently reduce the incidence of fetuses affected with thymus pathologies from 98.4% to 67.7%. CONCLUSIONS: Our results suggest that folic acid and methionine periconceptional supplementations may influence the incidence of congenital defects and may probably induce negative selection of embryos presenting developmental anomalies.

A multiple retinoic acid antagonist induces conotruncal anomalies, including transposition of the great arteries, in mice.

BACKGROUND: The morphogenetic mechanisms that are responsible for the transposition of the great arteries are still largely unknown, mainly because this malformation is very difficult to experimentally reproduce. The aim of the present study was to test the effect of BMS-189453, a retinoic acid antagonist, on murine heart morphogenesis. METHODS: We administered this drug at 5 mg/kg body weight (twice, at a 12-h interval) to pregnant mice on 6.25/6.75 days postcoitum (dpc) (Group A), 6.75/7.25 dpc (Group B), 7.25/7.75 dpc (Group C), 7.75/8.25 dpc (Group D), or 8.25/8.75 dpc (Group E). At birth, the anatomical features of fetuses were evaluated by stereomicroscopic examination. RESULTS: In Group A (18 fetuses), cardiovascular anatomy was normal in 10 (56%) cases, and 8 (44%) fetuses presented with transposition of the great arteries. In Group B, no fetuses were obtained. In Group C (78 fetuses), cardiovascular anatomy was normal in 19 (24%) cases, while 59 (76%) mice presented with various types of cardiac defects, including 48 transpositions of the great arteries (61%). In Group D (80 fetuses), cardiac defects were seen in 22 (27%) mice: 14 of these (17%) were transpositions of the great arteries. In Group E (72 fetuses), cardiovascular anatomy was normal in all cases. Of 248 fetuses analyzed, 87% presented with thymic aplasia or hypoplasia, and 20% presented with meroanencephalia and/or rachischisis. CONCLUSIONS: Transposition of the great arteries can be consistently reproduced in mice by administration of a retinoic acid competitive antagonist on 7.5 dpc.

Expression and role of PDGF-BB and PDGFR-beta during testis morphogenesis in the mouse embryo.

The role played by PDGF in testis morphogenesis is still incompletely understood. The present study investigates the expression and potential role of platelet-derived growth factor-BB (PDGF-BB) and its receptor, PDGF receptor beta (PDGFR-beta), during mouse testis cord formation, and the possibility that the growth factor may be involved in the migration to the gonad of mesenchymal cells of mesonephric origin. Studies from this laboratory have previously shown that mesenchymal cells that migrate from the mesonephros into the gonad, to form peritubular myoid cells and most of the intertubular cells, can be identified by the presence on their surface of the p75 neurotrophin receptor (p75NTR), and can be isolated to near-purity by immunomagnetic selection with anti-p75NTR antibody. We show here that mesonephric p75NTR(+) cells also bear the PDGFR-beta, and are able to migrate and proliferate in vitro in response to PDGF-BB. PDGF-BB is expressed at higher levels in male than female developing gonads, suggesting a role for this factor in testis development. Such a role is further supported by the observation that addition of PDGF-BB to serum-free medium is sufficient to allow organ-cultured male 11.5 days post-coitum urogenital ridges to form testis cords. Finally, we show that mesonephric cell motility and growth induced by exposure to PDGF-BB involve mitogen-activated protein kinases (MAPK) and phosphatidylinositol-3 kinase (PI3-K) pathways, as MAPK inhibitor U0126 and PI3K inhibitor Ly294002 inhibit migration and proliferation in vitro assays. The present findings support the hypothesis that the PDGF/PDGFR system plays a key role in testis morphogenesis in the mouse embryo.