Modulation of DNA methylation machineries in Japanese rice fish (Oryzias latipes) embryogenesis by ethanol and 5-azacytidine

Abstract

As a sequel of our investigations on the impact of epigenome in inducing fetal alcohol spectrum disorder (FASD) phenotypes in Japanese rice fish, we have investigated on several DNA methylation machinery genes including DNA methyl transferase 3ba (dnmt3ba) and methyl binding proteins (MBPs), namely, mbd1b, mbd3a, mbd3b, and mecp2 at the transcription level. Studies were made during normal development, from 0day post fertilization (dpf) to hatching, and also exposing the fertilized eggs to ethanol or a DNMT inhibitor, 5-azacytidine (5-azaC). We observed that during development, all these genes followed distinct expression patterns, generally high mRNA copies in early phases (0–1dpf) and significantly low mRNA copies prior to or after hatching. Ethanol (100–500mM, 0–2dpf) was unable to alter any of these mRNAs in 2dpf; additional four day (2–6dpf) maintenance of these embryos in ethanol-free environment, on 6dpf, was also unable to establish any significant difference in these mRNA levels in comparison with the corresponding controls. However, continuous exposure of fertilized eggs in 300mM ethanol, 0–6dpf, showed significantly high mRNA copies only in MBPs (mbd1b, mbd3a, mbd3b, mecp2). 5-azaC (2mM) on 2dpf was able to enhance only mbd3b mRNA. Removal of 5-azaC and maintenance of these embryos in clean medium, 2–6dpf, showed significantly enhanced mbd3b and mecp2 mRNAs compared to corresponding controls on 6dpf. Our studies showed that in Japanese rice fish embryogenesis both ethanol and 5-azaC have the potential to specifically modulate the developmental rhythm of DNA methylation machineries.