Downregulation of WT1 transcription factor gene expression is required to promote myocardial fate [ATAC-seq]. Downregulation of WT1 transcription factor gene expression is required to promote myocardial fate [ATAC-seq]

During cardiac development, cells from the precardiac mesoderm fuse to form the primordial heart tube, which then grows by addition of further progenitors to the venous and arterial poles. In the zebrafish, wilms tumor 1 transcription factor a (wt1a) and b (wt1b) are expressed in the pericardial mesoderm at the venous pole of the forming heart tube. The pericardial mesoderm forms a single layered mesothelial sheet that contributes to further the growth of the myocardium, and forms the proepicardium. Proepicardial cells are subsequently transferred to the myocardial surface and give rise to the epicardium, the outer layer covering the myocardium in the adult heart. wt1a/b expression is downregulated during the transition from pericardium to myocardium, but remains high in proepicardial cells. Here we show that sustained wt1 expression impaired cardiomyocyte maturation including sarcomere assembly, ultimately affecting heart morphology and cardiac function. ATAC-seq data analysis of cardiomyocytes overexpressing wt1 revealed that chromatin regions associated with myocardial differentiation genes remain closed upon wt1b overexpression in cardiomyocytes, suggesting that wt1 represses a myocardial differentiation program. Indeed, a subset of wt1a/b-expressing cardiomyocytes changed their cell adhesion properties, delaminated from the myocardial epithelium, and upregulated the expression of epicardial genes, as confirmed by in vivo imaging. Thus, we conclude that wt1 acts as a break for cardiomyocyte differentiation by repressing chromatin opening at specific genomic loci and that sustained ectopic expression of wt1 in cardiomyocytes can lead to their transformation into epicardial cells. Overall design: In this study we compared genome accessibility between two zebrafish transgenic lines: Tg(eGFP:5xUAS:RFP; gcryst:cerulean)cn15/Tg(myl7:Gal4)cbg2Tg, which served as the control group and Tg(eGFP:5xUAS:wt1b; gcryst:cerulean)cn15/Tg(myl7:Gal4)cbg2Tg, representing the experimental group. For each group (control and experimental) 3 replicates were analyzed

在心脏发育进程中，心前中胚层的细胞会相互融合形成原始心管，后续通过向心管的静脉极与动脉极补充更多祖细胞，实现心管的生长。在斑马鱼中，肾母细胞瘤1转录因子a（wt1a）与b（wt1b）在发育中心管静脉极的心包中胚层内表达。
心包中胚层可形成单层间皮层，该结构不仅能促进心肌层的进一步生长，还可分化为心外膜原基。心外膜原基细胞随后会被转移至心肌层表面，进而分化为心外膜——成体心脏中覆盖心肌层的外层组织。wt1a/b的表达在从心包向心肌层转化的过程中被下调，但在心外膜原基细胞中仍维持高表达水平。
本研究证实，持续的wt1表达会损害心肌细胞的成熟过程，包括肌节组装，最终对心脏形态与心脏功能造成不良影响。对过表达wt1的心肌细胞进行ATAC-seq数据分析后发现，与心肌分化相关的染色质区域在心肌细胞过表达wt1b时仍处于闭合状态，这提示wt1可抑制心肌分化程序。
进一步研究表明，部分表达wt1a/b的心肌细胞会改变自身细胞黏附特性，从心肌上皮层中脱离，并上调心外膜相关基因的表达，该结果已通过活体成像得到验证。综上，我们得出结论：wt1通过在特定基因组位点抑制染色质开放，充当心肌细胞分化的制动因子；而在心肌细胞中异位持续表达wt1，可促使其转化为心外膜细胞。
实验设计：本研究比较了两种斑马鱼转基因品系的基因组可及性：以Tg(eGFP:5xUAS:RFP; gcryst:cerulean)cn15/Tg(myl7:Gal4)cbg2Tg作为对照组，以Tg(eGFP:5xUAS:wt1b; gcryst:cerulean)cn15/Tg(myl7:Gal4)cbg2Tg作为实验组。每组（对照组与实验组）均设置3个生物学重复进行分析。