Mechanisms regulating the formation of the pharyngeal arch arteries

项目总结 DiGeorge综合征患者的咽弓动脉(PaaS)形成缺陷最多 人类常见的染色体异常。然而，细胞和分子机制调节 对PAA组的认识还不是很清楚。多年来，人们一直认为PaaS是通过该过程开发的 血管生成，由此认为PaaS形成为起源于入侵的背主动脉的分支 无血管咽弓。然而，最近对PAA形成缺陷突变小鼠的研究 提示突变的表型和咽部原始血管丛的存在 拱门与PaaS是通过血管生成形成的观点不相容。相反，这些研究表明 PaaS是通过血管生成过程发展起来的，由此PaaS从咽部血管产生 内皮祖细胞。我们使用了瞬时标记和细胞谱系跟踪来解决这个问题，并 证明PaaS的内皮细胞起源于第二心野。此外，我们的时间分辨， 三维共聚焦成像显示，PaaS是由原始血管丛重组为PAA形成的。在……里面 在研究过程中，我们发现细胞外基质糖蛋白纤维连接蛋白(Fn1)是必需的 对于第4对PaaS的形成和Isl1谱系中Fn1的条件性缺失导致了严重的 先天性主动脉弓缺陷，与DiGeorge综合征相似。我们发现这个数字和 Fn1flx/-；Isl1Cre动物的内皮细胞密度明显低于对照组，并推测Fn1 通过调节SHF来源的内皮祖细胞分化为 内皮细胞或通过调节内皮祖细胞从SHF迁移到咽部 拱门。在这项拨款申请中，我们建议检验这些假说，并改进时空 PAA前体细胞发育的调控机制。然后，我们建议将我们的方法应用于 系统阐明模拟DiGeorge的Tbx1+/-突变体中PAA形成缺陷的基础 综合症。我们计划通过解决以下具体目标来实现这些目标：1)确定 Fn1调控PAA形态发生的机制；2)确定VEGFR2阳性细胞的作用 在PAA发育的SHF内；3)确定Tbx1调控的PAa形成的阶段(S)。 我们将使用条件突变、时间分辨共聚焦成像和3D重建来测试我们的 假设，并探讨Fn1在PAA形成过程中生长因子信号转导中的作用。此外，我们 将使用一种创新的实时多光子显微镜来研究在 活体胚胎中PAA的形成以及Fn1和Tbx1在这一过程中的作用。完成这些研究 将为正常PAA的形成机制和引起 先天性心脏病患者的PAA发育缺陷。

PROJECT SUMMARY Formation of the pharyngeal arch arteries (PAAs) is defective in patients with DiGeorge syndrome, the most common chromosomal abnormality in humans. However, the cellular and molecular mechanisms regulating PAA formation are not well understood. For many years, it was believed that PAAs developed by the process of angiogenesis, whereby PAAs were thought to form as branches stemming from the dorsal aorta that invade the avascular pharyngeal arches. However, recent studies in mutant mice with defective PAA formation suggested that the mutant phenotypes and the presence of a primitive vascular plexus within the pharyngeal arches were incompatible with the idea that PAAs formed via angiogenesis. Instead, these studies suggested that PAAs develop by the process of vasculogenesis, whereby PAAs arise de-novo from pharyngeal vascular endothelial progenitors. We employed transient labeling and cell lineage tracking to resolve this issue and demonstrated that endothelial cells of PAAs arise from the second heart field. Furthermore, our time-resolved, 3D confocal imaging showed that PAAs form by reorganization of the primitive vascular plexus into the PAA. In the course of our studies, we discovered that the extracellular matrix glycoprotein fibronectin (Fn1) is required for the formation of the 4th pair of PAAs, and that conditional deletion of Fn1 in the Isl1 lineage caused severe congenital aortic arch defects, similar to those observed in DiGeorge syndrome. We found that the number and density of endothelial cells in Fn1flox/-;Isl1Cre animals is much lower than in controls, and hypothesize that Fn1 mediates PAA formation by regulating the differentiation of SHF-derived endothelial progenitors into endothelial cells or by regulating the migration of endothelial progenitors from the SHF into the pharyngeal arches. In this grant application, we propose to test these hypotheses and to refine the spatio-temporal mechanisms regulating the development of PAA progenitors. We then propose to apply our methodology to systematically elucidate the basis for the PAA formation defects in Tbx1+/- mutants that model the DiGeorge syndrome. We plan to achieve these goals by addressing the following specific aims: 1) To determine the mechanisms, by which Fn1 regulates PAA morphogenesis; 2) To determine the role of VEGFR2positive cells within the SHF in PAA development; and 3) To determine the stage(s) of PAA formation regulated by Tbx1. We will use conditional mutagenesis, time-resolved confocal imaging and 3D reconstruction to test our hypotheses, and investigate the role of Fn1 in growth factor signaling during PAA formation. Furthermore, we will employ an innovative live multi-photon microscopy to investigate the dynamics of endothelial cells during PAA formation in living embryos, and the roles of Fn1 and Tbx1 in this process. Completion of these studies will provide important insights into the mechanisms of normal PAA formation and into alterations that cause defective PAA development in patients with congenital heart disease.