Role of retinoic acid and DNA methylation in differentiation of mammalian embryo in vitro

Background and Purpose: Modified organ culture of rodent postimplantation embryo developed in our laboratory provides favorable conditions for the terminal differentiaion of the main tissues of primitive streak stage rat embryo. To explore a possible relationship between specific DNA methylation and induction differentiation by retinoic acid (RA) during embryonic development, we examined in vitro differentiation of gastrulatlngrat embryos. Material and Methods: Cultivation of gastrulating rat embryos without extraembryonic membranes was performed at the air-liquid interface for two weeks. Two types of media were used: with homologous serum, and serum and protein-free. RA was added to the media in die second week of cultivation period for 48 hours, and for 9 day, and demethylation agents 5-azacytidine (5azaC) was added for 24 hours and 24 hours before RA treatment. The resulting teratoma-like explants were fixed and histologically examined for the presence of various tissues. Results and Conclusions: RA inhibited differentiation of cartilage and stimulated differentiation of gut epithelium more obviouslyin serum-free medium. Optimal epidermal morphogenesis (orthoceratosis) was achieved only in the presence of a critical physiological RA concentration, which resembled our results obtained in serum supplemented medium without RA addition. The dose of 10(-5)M RA prevented keratinization of epidermis in serum-free medium and, stimulated its replacement by mucus secreting epithelia. The same ation in serum supplemented mewas true for epithelial differentiation in serum when we applied RA during the second week of clture after24 hourpretreatment with demethylating agents. Squamous epithelium waslessfrequentwithlittle ifanykeratinizationandmucussecreting epithelium and gland epithelium were more frequent. Embryos should be affected byRA atleast the whole second week of culture to induce differentiation of glands. 5azaC acted differently in the two media used: in serum supplemented medium it stimulated differentiation, whereas in serum-free medium it impeded differentiation of main tissues, except epidermis. Using DNA demethylating and morpfogen retinoic acid in mammalian embryos grown in organ culture we discovered that gene expression was altered, which was shown by complete change in differentiation pattern of various tissues.