In-vivo Function and Mechanism of the Long non-coding RNA Locus Handsdown

心脏功能障碍是现代社会发病率和死亡率的主要原因之一，几乎每100个新生儿中就有1个表现出心脏发育畸形。此外，心肌梗死等心脏损伤往往导致心脏重构不适应，导致充血性心力衰竭，给社会和经济带来巨大负担。虽然心脏的再生潜力非常有限，但目前正在进行许多努力来增强再生或将具有再生潜力的细胞移植给患者。然而，深入了解心脏在正常情况下是如何发育的，对于开发有效的心脏病再生疗法至关重要。心脏是胚胎发育过程中最先发育的器官，从很早的时候就含有可收缩的心肌细胞形式的功能细胞。出生后不久，小鼠和人类的心脏都具有暂时有限的潜力，可以将受伤的心肌组织再生为功能齐全的收缩组织。虽然许多蛋白质编码rna在心脏发育中的作用已经得到了很好的研究，但一类长链非编码rna （lncRNAs）及其在心脏发育和疾病中的作用直到最近才成为人们关注的焦点。实际上，脊椎动物转录组中最丰富的RNA转录本是长链非编码RNA (lncRNAs)，转录本长度超过200个核苷酸，不编码蛋白质。lncrna与表观遗传变化、发育缺陷和癌症有关，敲除lncrna有时是致命的。我们之前的研究表明，lncRNA fendr与Polycomb抑制复合体2 （PRC2）相互作用，从而调节早期胚胎心脏细胞谱系中的必需基因。我们发现了几个在早期胚胎心脏中具有特异性表达的新型lncrna，并表明其中一个特定位点对胚胎发育至关重要。为此，我们使用CRISPR/Cas9基因组编辑技术删除了小鼠胚胎干细胞（ESCs）中的lncRNA位点，并从这些ESCs中生成小鼠胚胎。由此产生的胚胎是致命的，除了其他缺陷外，心脏发育不良。我们建议使用最先进的基因组工具和直接从基因工程干细胞中产生的胚胎和小鼠来分析这种新的心脏特异性lncRNA位点。我们将进一步根据由此产生的RNA转录物或潜在的基因组背景来定义该lncRNA位点的分子机制。通过了解lncrna在正常心脏发育过程中的功能，我们可能会定义新的lncrna，这些lncrna可能是重新激活成人心脏再生潜能或增强移植细胞再生潜能的有效靶点。此外，通过反义RNA技术（GapmerR）成功下调lncRNA转录本可能允许上调心脏特异性基因，有利于心脏损伤后的修复。

Heart malfunction is one of the major causes of morbidity and lethality in modern society and nearly 1 in 100 newborns exhibit developmental malformations of the heart. In addition, heart injuries such as myocardial infarction impose an enormous societal and economic burden as they often result in maladaptive heart remodeling leading to congestive heart failure. While the heart has very limited regenerative potential, numerous efforts are currently undertaken to enhance regeneration or transplant cells with regenerative potential to patients. However, an in-depth knowledge of how the heart develops under normal circumstances is crucial for the development of effective regenerative therapies for heart disease.The heart is the first organ to develop during embryogenesis, containing functional cells in the form of contractile cardiomyocytes from very early on. Shortly after birth, the heart of both mice and humans has the temporally-restricted potential to regenerate injured heart muscle tissue into fully functional contractile tissue. While the role of many protein-coding RNAs in heart development has been well-studied, the class of long non-coding RNAs (lncRNAs) and their role in heart development and disease has only recently shifted into focus. Actually, the most abundant RNA transcripts in the vertebrate transcriptome are long noncoding RNAs (lncRNAs), transcripts that are longer than 200 nucleotides and do not encode proteins. LncRNAs have been associated with epigenetic changes, developmental defects and cancer, and knockouts are sometimes lethal. We showed previously that the lncRNA Fendrr interacts with the Polycomb repressive complex 2 (PRC2) and thereby regulates essential genes in the early embryonic cardiac cell lineage. We identified several novel lncRNAs with specific expression in the early embryonic heart and show that one particular locus is essential for embryonic development. To that end, we deleted the lncRNA locus in mouse embryonic stem cells (ESCs) using CRISPR/Cas9 genome editing and generated mouse embryos from these ESCs. The resulting embryos are embryonic lethal with, amongst other defects, mis-development of the heart. We propose here to analyze this novel heart specific lncRNA locus in vivo using state-of the art genomic tools and the generation of embryos and mice directly from genetically engineered stem cells. We will furthermore define the molecular mechanism of this lncRNA locus with respect to the RNA transcripts generated from it or the underlying genomic context.By understanding how lncRNAs function during normal heart development, we are likely to define new lncRNAs that may be valid targets to reactivate regenerative potential in adult hearts or enhance the regenerative potential of transplanted cells. Furthermore, successful downregulation of the lncRNA transcript by antisense RNA technology (GapmerR) might allow for the upregulation of heart specific genes, beneficial for cardiac repair after injury.