Mechanisms regulating the formation of the pharyngeal arch arteries

调节咽弓动脉形成的机制

基本信息

批准号：
9540070
负责人：
Sophie Astrof
金额：
$ 39万
依托单位：
THOMAS JEFFERSON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-08-15 至 2018-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9540070
关键词：
Address Animals Aorta Applications Grants Arteries Biological Assay Blood Vessels Branchial arch structure Caliber Capillary Electrophoresis Cell Lineage Cell physiology Cells Chromosome abnormality Confocal Microscopy Data Defect Development DiGeorge Syndrome Disease Dorsal Embryo Endothelial Cells Endothelium Epithelium Extracellular Matrix Fibronectins Genes Glycoproteins Goals Grant Growth Factor Heart Human Image In Situ Hybridization Injections Invaded KDR gene Label Lead Mediating Mesenchyme Methodology Microscope Microscopy Modeling Molecular Morphogenesis Mus Mutagenesis Mutant Strains Mice Mutate Pathology Patients Phenotype Physiological Process Property Regulation Reverse Transcriptase Polymerase Chain Reaction Role Signal Transduction Stains Stem cells Tamoxifen Testing Three-Dimensional Imaging Time Transgenic Mice Vascular Endothelial Growth Factors Western Blotting Work angiogenesis aortic arch congenital heart disorder density human disease innovation insight microscopic imaging migration mouse model multi-photon mutant progenitor reconstruction spatiotemporal stem temporal measurement two-photon vasculogenesis

项目摘要

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.

项目摘要咽弓动脉（PAAS）的形成在Digeorge综合征患者中有缺陷，最多是人类常见的染色体异常。但是，调节的细胞和分子机制 PAA的形成尚不清楚。多年来，人们认为Paas是由过程开发的血管生成，认为Paas形成是由侵入背主动脉造成的分支血管咽弓。但是，最近在突变小鼠中形成了PAA的突变小鼠的研究提出突变表型和咽内原始血管丛的存在拱形与通过血管生成形成的PAA形成的想法不兼容。相反，这些研究建议 PAAS是通过血管生成过程而发展的，咽是咽血管中的paas 内皮祖细胞。我们采用了瞬态标签和细胞谱系跟踪来解决此问题，证明PAA的内皮细胞来自第二个心脏场。此外，我们的时间分辨， 3D共聚焦成像表明，通过将原始血管丛重组为PAA形成PAA。在我们的研究过程，我们发现需要细胞外基质糖蛋白纤连蛋白（FN1）为了形成第四对PAA，以及在ISL1谱系中的条件缺失引起严重先天性主动脉弓缺陷，类似于Digeorge综合征中的缺陷。我们发现数字和 FN1FLOX/ - ; ISL1CRE动物中内皮细胞的密度远低于对照组，并假设FN1 通过调节SHF衍生的内皮祖细胞的分化来介导PAA形成内皮细胞或通过调节内皮祖细胞从SHF迁移到咽部拱门。在此赠款应用中，我们建议测试这些假设并完善时空调节PAA祖细胞发展的机制。然后，我们建议将我们的方法应用于系统地阐明了对Digeorge建模的TBX1 +/-突变体中PAA形成缺陷的基础综合征。我们计划通过解决以下特定目的来实现这些目标：1）确定 FN1调节PAA形态发生的机制； 2）确定VEGFR2阳性细胞的作用在PAA开发中的SHF内； 3）确定由TBX1调节的PAA形成期。我们将使用条件诱变，时间分辨共聚焦成像和3D重建来测试我们的假设并研究FN1在PAA形成过程中生长因子信号传导中的作用。此外，我们将采用创新的实时多光子显微镜来研究内皮细胞的动力学 PAA在生物胚胎中形成，以及FN1和TBX1在此过程中的作用。这些研究的完成将提供对正常PAA形成机制的重要见解，并成为导致的变化先天性心脏病患者的PAA发育不良。