Rationale: The transcription factor NFATC1 has been implicated in cardiac valve formation in humans and mice, but we know little about the underlying mechanisms. To gain mechanistic understanding of cardiac valve formation at single cell resolution and insights into the role of NFATC1 in this process, we used the zebrafish model as it offers unique attributes for live imaging and facile genetics. Objective: To understand the role of Nfatc1 in cardiac valve formation. Methods and Results: Using the zebrafish atrioventricular (AV) valve, we focus on the valve interstitial cells (VICs) which confer biomechanical strength to the cardiac valve leaflets. We find that initially AV endocardial cells (ECs) migrate collectively into the cardiac jelly to form a bilayered structure; subsequently, the cells that led this migration invade the extracellular matrix (ECM) between the two EC monolayers, undergo endothelial-to-mesenchymal transition as marked by loss of intercellular adhesion, and differentiate into VICs. These cells proliferate and are joined by a few neural crest-derived cells. VIC expansion as well as a switch from a pro-migratory to an elastic ECM drive valve leaflet elongation. Functional analysis of Nfatc1 reveals its requirement during VIC development. Zebrafish nfatc1 mutants form significantly fewer VICs due to reduced proliferation and impaired recruitment of endocardial and neural crest cells during the early stages of VIC development. With high-speed microscopy and echocardiography, we show that reduced VIC formation correlates with valvular dysfunction and severe retrograde blood flow that persist into adulthood. Analysis of downstream effectors reveals that Nfatc1 promotes the expression of twist1b, a well-known regulator of epithelial-to-mesenchymal transition. Conclusions: Our study sheds light on the function of Nfatc1 in zebrafish cardiac valve development and reveals its role in VIC formation. It also further establishes the zebrafish as a powerful model to carry out longitudinal studies of valve formation and function.

中文翻译：

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Nfatc1在斑马鱼的心脏瓣膜发育过程中促进间质细胞形成。

原理：转录因子NFATC1与人类和小鼠的心脏瓣膜形成有关，但我们对其潜在机制了解甚少。为了在单细胞分辨率下获得对心脏瓣膜形成的机械理解，并深入了解NFATC1在此过程中的作用，我们使用了斑马鱼模型，因为它为实时成像和便捷的遗传学提供了独特的属性。目的：了解Nfatc1在心脏瓣膜形成中的作用。方法和结果：我们使用斑马鱼房室瓣（AV），着眼于瓣膜间质细胞（VIC），这些细胞赋予心脏瓣膜小叶生物力学强度。我们发现最初的AV心内膜细胞（ECs）共同迁移到into胶中形成双层结构。后来，导致这种迁移的细胞侵入了两个EC单层之间的细胞外基质（ECM），经历了从内皮到间充质的转变，这一过程以细胞间粘附力的丧失为标志，并分化为VIC。这些细胞增殖，并由一些神经rest来源的细胞连接。VIC扩展以及从迁移前的切换到弹性ECM驱动瓣叶的延长。Nfatc1的功能分析揭示了其在VIC开发过程中的需求。斑马鱼nfatc1突变体形成的VIC明显减少，这是由于在VIC研发的早期阶段增殖减少以及心内膜​​和神经c细胞募集受损。借助高速显微镜和超声心动图，我们显示出减少的VIC形成与瓣膜功能障碍和严重逆行的血流（持续到成年）有关。对下游效应子的分析表明，Nfatc1促进了Twist1b的表达，twist1b是上皮-间充质转化的著名调节剂。结论：我们的研究揭示了Nfatc1在斑马鱼心脏瓣膜发育中的功能，并揭示了其在VIC形成中的作用。它还进一步将斑马鱼确立为进行瓣膜形成和功能的纵向研究的有力模型。