Inhibition of cephalic neural tube closure by 5-azacytidine in neurulating rat embryos in vitro

Abstract

Head-fold stage rat embryos (9.5 days of gestation) were cultured for 48 h in rat serum with or without 0.8 microM 5-azacytidine. Incomplete closure of the cephalic neural tube was observed in 5-azacytidine-treated embryos cultured for 48 h (25-somite stage). Control embryos showed complete fusion of cephalic neural folds at 33 h (16-somite stage) in culture. Drug administration or removal experiments revealed that embryos were sensitive to 5-azacytidine during 6-12 h of culture (three to five somite stages). Electron microscopical studies indicated that the arrangement and fine structure of cephalic neuroepithelial cells were almost the same in control and treated embryos. There was no significant difference in DNA and protein contents between control and treated embryos cultured for 36 h. Immunocytochemical observations using 5-methylcytosine-specific antibody revealed that the staining of neuroepithelial cells in the median part of the transversely sectioned cephalic neural plate, and of mesenchymal cells near the apices of the plate, was suppressed by 5-azacytidine. These results suggest that DNA methylation of these cells plays an important role in closure of the cephalic neural tube.