Downregulation of WT1 transcription factor gene expression is required to promote myocardial fate [RNA-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. Dorsal pericardium, proepicardium and heart-tube were manually dissected, with tungsten needles, from 60 hpf epi:eGFP;myl7:mRFP zebrafish larvae. A minimum of 10 of each tissue/organ were collected and pooled for each sample. For pericardium, two samples were analysed. For the proepicardium 3 samples were analysed. For heart tube 3 samples were analysed.

在心脏发育过程中，心前中胚层（precardiac mesoderm）的细胞相互融合形成原始心管（primordial heart tube），后续通过向心管的静脉极与动脉极添加更多祖细胞，使其进一步生长。
在斑马鱼中，肾母细胞瘤1转录因子a（wilms tumor 1 transcription factor a，简称wt1a）与b（wt1b）在发育中心脏管静脉极处的心包中胚层（pericardial mesoderm）中表达。
心包中胚层会形成单层间皮片层（mesothelial sheet），既参与心肌层（myocardium）的进一步生长，同时也会分化为心外膜原基（proepicardium）。
随后，心外膜原基细胞会被转移至心肌层表面，并分化为心外膜（epicardium）——成体心脏中覆盖心肌层的外层结构。
wt1a/b的表达在从心包中胚层向心肌层的转化过程中会被下调，但在心外膜原基细胞中仍保持高表达水平。
本研究发现，持续的wt1表达会损害心肌细胞（cardiomyocyte）的成熟过程，包括肌节组装（sarcomere assembly），最终影响心脏形态与心功能。
对过表达wt1的心肌细胞进行转座酶可及性测序（ATAC-seq）数据分析后发现，在心肌细胞中过表达wt1b时，与心肌分化基因相关的染色质区域仍处于闭合状态，这表明wt1会抑制心肌分化程序。
确实，表达wt1a/b的一部分心肌细胞会改变其细胞黏附特性，从心肌上皮层脱离，并上调心外膜相关基因的表达，这一点已通过活体成像得到验证。
综上，我们得出结论：wt1通过抑制特定基因组位点处的染色质开放，对心肌细胞分化起到抑制作用；而在心肌细胞中持续异位表达wt1，则可促使其转化为心外膜细胞。
本研究使用钨针，从60小时受精后（hours post fertilization，简称hpf）的epi:eGFP;myl7:mRFP转基因斑马鱼幼体中手动分离背部心包、心外膜原基与心管。
每个样本需收集至少10个相同组织/器官并混合。
其中，背部心包样本共分析2份。
心外膜原基样本共分析3份。
心管样本共分析3份。