Genome-wide chromatin accessibility profile of zebrafish cardiomyocytes throughout heart development. Genome-wide chromatin accessibility profile of zebrafish cardiomyocytes throughout heart development

By using transgenic zebrafish lines Tg(nxk2.5:GFP) (Witzel et al. 2012) and Tg(myl7:EGFP) (D'Amico et al. 2007), we have characterized chromatin accessibility of FACS-isolated CM (GFP+) from developing zebrafish heart at 24, 48 and 72 hpf, corresponding to heart tube formation, chamber formation and differentiation and heart maturation, respectively. GFP- cells were used as a control. We have identified cardiac regulatory networks playing a crucial role in heart morphogenesis. To validate their importance in heart development, we employed zebrafish mutants of cardiac transciption factors Gata5, Hand2 and Tbx5a, the disruption of which were previously linked to impaired migration of the cardiac primordia to the embryonic midline, reduced number of myocardial precursors and failure of heart looping, respectively (Reiter et al. 1999; Yelon et al. 2000; Garrity et al. 2002). ATAC-seq was performed from homozygous gata5tm236a/tm236a, tbx5am21/ m21, hand2s6/s6 mutant 72 hpf embryos in Tg(myl7:EGFP) genetic background. Homozygous mutant embryos for analyses were selected on the basis of their phenotypes of cardia bifida (gata5tm236a/tm236a, hand2s6/s6) or heart-string (tbx5am21/ m21) . Overall design: Examination of chromatin accessibility maps of cardiomycytes at 3 stages of heart development

本研究采用转基因斑马鱼品系Tg(nxk2.5:GFP)（Witzel等，2012）与Tg(myl7:EGFP)（D'Amico等，2007），针对发育中斑马鱼心脏在受精后24、48及72小时（hours post fertilization, hpf，分别对应心脏管形成、心室腔形成与分化、心脏成熟三个阶段）的荧光激活细胞分选（Fluorescence-Activated Cell Sorting, FACS）分离得到的GFP阳性心肌细胞（cardiomyocytes, CM）开展染色质开放性分析，并以GFP阴性细胞作为对照。本研究已鉴定出在心脏形态发生过程中发挥关键作用的心脏调控网络。为验证上述调控网络在心脏发育中的重要性，我们使用了心脏转录因子Gata5、Hand2及Tbx5a的斑马鱼突变体——既往研究显示，这三类转录因子的功能缺失分别会导致心脏原基向胚胎中线迁移受阻、心肌前体细胞数量减少以及心脏环化失败（Reiter等，1999；Yelon等，2000；Garrity等，2002）。我们对处于Tg(myl7:EGFP)遗传背景下的纯合突变体gata5tm236a/tm236a、tbx5am21/m21及hand2s6/s6在受精后72小时的胚胎进行了转座酶可及性测序（Assay for Transposase-Accessible Chromatin using sequencing, ATAC-seq）。用于后续分析的纯合突变胚胎依据其表型进行筛选：gata5tm236a/tm236a与hand2s6/s6突变体表现为心脏二分畸形（cardia bifida），tbx5am21/m21突变体则呈现心索样表型（heart-string）。本实验整体设计为：检测心脏发育三个阶段的心肌细胞染色质开放性图谱。