Lineage mapping within the mouse outflow tract

描述（由申请人提供）：先天性心脏缺陷（CHDs）是美国排名第一的出生缺陷，也是儿童死亡的主要非传染性原因。随着心外细胞群的定植，原始心管经历分离和瓣膜形成，将其转化为四腔心脏。这些心外细胞群包括心前野、心神经嵴和心外膜细胞。尽管存在许多标记物来跟踪EPDCs和心脏nc来源的细胞向哺乳动物心脏的迁移，但这些标记物中没有一个专门标记这些细胞亚群，而且，一旦细胞到达心脏，所有这些标记物都被关闭，这使得这些细胞到达心脏后的作用和功能难以捉摸。当考虑到流出道间充质缓冲细胞在冠心病流出道形态发生和发病中的最终作用时，这是一个特别的缺点。我们对广表达基因periostin的转录调控研究揭示了一个3.9kb的转录调控模块，该模块驱动外周神经和一种新的“流出道衍生细胞”（oftdc）亚群的表达。此外，我们发现oftdc的后代在分离和瓣膜形成之前和期间取代了许多初始的普通和房室（AV）缓冲间充质细胞。这些新数据提示了心脏发育的一种新模式，即心脏的两个肢体可能通过它们的缓冲相互作用，并且发育中的小鼠心脏的许多非心肌细胞短暂表达3.9kb-骨膜蛋白模块。因此，我们假设流出道和房室缓冲细胞之间存在细胞交换，成熟的流出道和房室瓣膜最终来源于这种迁移的内皮衍生的OFTDC缓冲细胞亚群。这些结果对心脏发生提出了一个全新的观点，即大多数成熟心脏的间充质细胞来自胚胎流出道内的一小部分细胞，而不是以前认为的来自心脏外来源。为了验证这一新的假设，我们提出了四个相互关联的目标：目标1将决定胎儿、新生儿和成年小鼠中oftdc的最终命运。目标2将确定oftdc的起源。目的3将分离表达oftdc的3.9kbperi增强绿色荧光蛋白（EGFP），并鉴定差异表达基因。目标4将确定oftdc通过基因消融每个细胞来缓冲发育和心脏形态发生的需求，因为每个细胞短暂地表达3.9kb的骨膜蛋白启动子/增强子。

DESCRIPTION (provided by applicant): Congenital heart defects (CHDs) are the #1 birth defect in the United States and the leading non-infectious cause of death in children. Following the colonization of extracardiac cell populations, the primitive heart tube undergoes septation and valvar formation to convert it into a four-chambered heart. These extracardiac cell populations include the anterior heart field, cardiac neural crest and epicardially-derived cells. Although many markers exist to follow the migration of both the EPDCs and cardiac NC-derived cells into the mammalian heart, none of these markers specifically mark only these cell subpopulations and furthermore, all of these markers are turned off as soon as the cells reach the heart - making the role and function of these cells once they reach the heart elusive. This is a particular drawback when considering the ultimate role of the outflow tract mesenchymal cushion cells within outflow tract morphogenesis and pathogenesis of CHDs. Our study of the transcriptional regulation of the broadly expressed gene, periostin has revealed a 3.9kb transcriptional regulatory module that drives expression in the peripheral nerves and a novel subpopulation of "outflow tract-derived cells" (OFTDCs). Furthermore, we find that descendents of the OFTDCs replace many of the initial conommcal and atrioventricular (AV) cushion mesenchymal cells just prior to and during septation and valvar formation. These novel data suggest a new paradigm for heart development in which the two limbs of the heart may interact through their cushions and that many of the non-cardiomyocytes of the developing mouse heart transiently express the 3.9kb-periostin module. Thus, we hypothesize that there is cell exchange between the outflow track and AV cushions and that the mature outflow and AV valves are ultimately derived from this migratory endothelial-derived OFTDC subpopulation of cushion cells. These results present a radical new outlook on cardiogenesis, where the majority of the mature heart's mesenchymal cells arise from a small subpopulation of cells within the embryonic outflow tract itself, and not from extra-cardiac sources as previously believed. To test this novel hypothesis, we propose four interrelated aims: Aim 1 will determine the ultimate fate of the OFTDCs in the fetus, newborn and adult mouse. Aim 2 will determine the origin of the OFTDCs. Aim 3 will isolate the 3.9kbperi-enhanced green fluorescent protein (EGFP) expressing OFTDCs and identify differentially-expressed genes. Aim 4 will determine the requirement of the OFTDCs to cushion development and heart morphogenesis by genetically ablating each cell as it transiently expresses the 3.9kb periostin promoter/enhancer.