Lineage mapping within the mouse outflow tract

描述(申请人提供)：先天性心脏缺陷(CHDS)是美国头号出生缺陷，也是导致儿童死亡的主要非传染性原因。随着心外细胞群的定植，原始心管经历隔膜和瓣膜形成，将其转变为四腔心。这些心外细胞群包括心前区、心脏神经脊和心外膜来源的细胞。尽管存在许多跟踪EPDCs和心脏NC来源的细胞迁移到哺乳动物心脏的标记物，但这些标记物中没有一个只专门标记这些细胞亚群，而且一旦细胞到达心脏，所有这些标记物就被关闭-使得这些细胞到达心脏后的角色和功能变得难以捉摸。考虑到流出道间充质缓冲细胞在流出道形态发生和冠心病发病机制中的最终作用，这是一个特别的缺陷。我们对广泛表达的基因Periostin转录调控的研究揭示了一个3.9kb的转录调控模块驱动周围神经的表达，并发现了一个新的“流出道源细胞”(OFTDC)亚群。此外，我们发现，在间隔和瓣膜形成之前和期间，OFTDC的后代取代了许多最初的经济和房室(AV)缓冲间充质细胞。这些新的数据表明了一种心脏发育的新范式，在这种范式中，心脏的两个肢体可以通过它们的垫子相互作用，并且发育中的小鼠心脏的许多非心肌细胞瞬时表达3.9kb的Periostin模块。因此，我们假设在流出道和房室垫之间存在细胞交换，成熟的流出道和房室瓣最终来自这种迁移的内皮来源的OFTDC缓冲细胞亚群。这些结果展示了一种全新的心脏发生学前景，即大多数成熟心脏的间充质细胞来自胚胎流出道本身内的一小部分细胞，而不是像以前认为的那样来自心脏外。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OFTDC在胎儿、新生和成年小鼠中的最终命运。目标2将确定OFTDC的来源。目的3分离表达OFTDCs的3.9kbperi增强型绿色荧光蛋白(EGFP)并鉴定差异表达基因。目的4将通过基因消融每个瞬时表达3.9kb Periostin启动子/增强子的细胞来确定OFTDC缓冲发育和心脏形态发生的需求。

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.