5hmC (48hpf WT dissected heart) 5hMe-Bead-Integrated Click-seq (5hMe-BIC-seq). 5hmC (48hpf WT dissected heart) 5hMe-Bead-Integrated Click-seq (5hMe-BIC-seq)

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. Overall design: Genomic DNA was isolated from wildtype (n=2) dissected hearts using DNeasy Blood & Tissue Kits (Qiagen). At least 40 hearts were collected for each biological group.

十十一易位（Ten-eleven translocation, Tet）酶（Tet1/2/3）可介导5-甲基胞嘧啶（5-methylcytosine, 5mC）的羟化修饰，该过程能够促进DNA去甲基化，进而调控基因表达。
过往针对Tet酶的研究多聚焦于其突变亚型对癌症的影响，而Tet酶在器官发生过程中的正常生理功能仍知之甚少。
本研究通过对复合突变斑马鱼（compound mutant zebrafish）开展分析，发现胚胎心脏内的Tet2/3活性对于心外膜祖细胞（epicardial progenitors）的招募具有必需作用，该过程与房室管（atrial-ventricular canal, AVC）的发育缺陷密切相关。
通过联合甲基化、羟甲基化与转录组分析（transcript profiling），研究人员鉴定出编码激活素A亚基Inhbaa（表达于心内膜（endocardium））与Sox9b（表达于心肌层（myocardium））的基因，可作为Tet2/3的去甲基化靶基因，且这两个基因对于房室管定位的细胞外基质（extracellular matrix, ECM）的组装至关重要，能够促进心外膜祖细胞向发育中的心管（heart tube）迁移。
本研究阐明了在心脏发育进程中，以显著的时空与谱系特异性调控基因表达变化的核心DNA表观遗传修饰机制，揭示了多细胞群在脊椎动物心脏（vertebrate heart）发育过程中的复杂互作关系。
实验设计概述（Overall design）：采用Qiagen公司的DNeasy血液与组织试剂盒（DNeasy Blood & Tissue Kits），从野生型（n=2）解剖获取的心脏中提取基因组DNA。每个生物学重复组（biological group）至少收集40个心脏样本。