Enhanced Reduced Representation Bisulfite Sequencing of 48hpf WT and tet2/3 DM dissected heart

Ten-eleven translocation (Tet) enzymes (Tet1/2/3) mediate 5-methylcytosine (5mC) hydroxylation, which can facilitate DNA demethylation and thereby impact gene expression. Studied mostly for how mutant isoforms impact cancer, the normal roles for Tet enzymes during organogenesis are largely unknown. By analyzing compound mutant zebrafish, we discovered a requirement for Tet2/3 activity in embryonic heart for recruitment of epicardial progenitors, associated with defects in development of the atrial-ventricular canal (AVC). Through a combination of methylation, hydroxymethylation, and transcript profiling, the genes encoding the ActivinA subunit Inhbaa (in endocardium) and Sox9b (in myocardium) were implicated as demethylation targets of Tet2/3 and critical for organization of AVC-localized extracellular matrix (ECM), facilitating migration of epicardial progenitors onto the developing heart tube. This study elucidates essential DNA epigenetic modifications that govern gene expression changes during cardiac development with striking temporal and lineage specificities, highlighting complex interactions in multiple cell populations during development of the vertebrate heart. For reduced representation bisulfite sequencing genomic DNA was isolated from 48hpf WT and tet2/3 DM dissected hearts using the DNeasy Blood & Tissue Kit (Qiagen, 69504) following manufacturer’s guidelines. One replicate was done for each sample.