Ontogenesis of Tet family of methylcytosine dioxygenase enzyme genes during Japanese rice fish (Oryzias latipes) embryogenesis: Effects of ethanol and 5-azacytidine ABSTRACT The mRNA expression pattern of tet family of methyl cytosine dioxygenase enzymes (ten-eleven translocation, tet1, tet2, and tet3), which catalyze the conversion of 5-methylcytosine (5mC) to 5- hydroxymethylcytosine (5hmC) and initiate 5mC remodeling through active demethylation, was investigated in brain, ovary, and liver of Japanese rice fish (Oryzias latipes) adults, and in whole embryos after fertilization to hatching. Moreover, fertilized eggs were exposed to ethanol or 5- azacytidine (5-azaC), 0-2 day post fertilization (dpf), and tet mRNAs on 2 and 6 dpf were analyzed. Our data indicated that all three tet mRNAs were expressed in brain, liver, and ovary of adult fish with substantial variations. In embryos, all three tet mRNAs showed rhythmic expression, high copies in 1-3 dpf followed by down regulation until hatching. Embryonic exposure either to ethanol or 5-azaC was unable to alter the expression of any of these tet mRNAs in 2 dpf; in 6 dpf, there was an increase in tet expression in response to high levels of ethanol (400-500 mM) and 5-azaC (2 mM). Although we have previously documented that both ethanol and 5-azaC was able to induce fetal alcohol spectrum disorder (FASD)-like phenotypic features in Japanese rice fish, the present study indicates that the DNA demethylation by tet may differ between these two compounds. KEYWORDS: RT-qPCR, epigenetics, Tet, ethanol, 5-azacytidine, Japanese rice fish INTRODUCTION In vertebrates, methylation of DNA at the fifth position of cytosine residues (5-methylcytosine, 5mC) in CpG dinucleotides is associated with gene silencing which is essential for normal development [1]. The “fifth base” (5mC), is maintained by DNA methyl transferase 1 (DNMT1) during DNA replication [2, 3]. However, for activation of genes during fertilization and early stages of embryonic development, remodeling of 5mC (demethylation) is necessary [4-7]. Until now, two types of DNA demethylation processes, active and passive, have been established [8]. In passive demethylation, rapid proliferation by DNA replication occurs in cells and due to downregulation of DNMT1 enzyme 5mC is diluted [9]. In active demethylation, 5mC is enzymatically processed and removed by the ten- eleven translocation proteins (Tet1, Tet2, and Tet3) that convert 5mC to 5-hydroxymethylcytosine (5hmC) and also to its oxidative derivatives 5- formylcytosine (5fC) and 5-carboxylcytosine (5caC) [10, 11]. Mammalian embryos, after fertilization, employ a combination of both active and passive mechanisms of DNA remodeling; however, in zebrafish passive mechanism of 5mC dilution is predominant [12, 13]. It was suggested that the conversion of 5mC to 5hmC and its derivatives by the TET enzymes is the initial step of DNA base- a National Center for Natural Product Research, School of Pharmacy; b Department of BioMolecular Sciences, Pharmacology Division; c Department of BioMolecular Sciences, Pharmacognosy Division, University of Mississippi, University, MS 38677, USA. Asok K. Dasmahapatra a,b, * and Ikhlas A. Khan a,c *Corresponding author: asok@olemiss.edu Trends in Developmental Biology Vol. 10, 2017