Mechanisms regulating the formation of the pharyngeal arch arteries

调节咽弓动脉形成的机制

• 批准号: 9702895
• 负责人: Sophie Astrof
• 金额: $ 39.75万
• 依托单位: RBHS-NEW JERSEY MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-05 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9702895
• 关键词: 3-Dimensional; 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; Epithelial; 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; Tamoxifen; Testing; Three-Dimensional Imaging; Time; Transgenic Mice; Vascular Endothelial Growth Factors; Vascular Endothelium; Western Blotting; Work; angiogenesis; aortic arch; confocal imaging; congenital heart disorder; density; endothelial stem cell; human disease; innovation; insight; microscopic imaging; migration; mouse model; multi-photon; mutant; progenitor; reconstruction; spatiotemporal; stem; temporal measurement; two photon microscopy; two-photon; vasculogenesis

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 DiGeorge综合征患者咽弓动脉（PAA）的形成有缺陷， 人类常见的染色体异常。然而，细胞和分子机制调节 PAA的形成还不清楚。多年来，人们一直认为， 血管生成，其中PAA被认为是从背主动脉产生的分支， 无血管的咽弓然而，最近在PAA形成缺陷的突变小鼠中的研究表明， 这表明，突变的表型和存在的原始血管丛内咽 弓形与PAA通过血管生成形成的观点不一致。相反，这些研究表明， PAAs通过血管发生过程发展，其中PAAs从咽部血管新生产生， 内皮祖细胞我们采用瞬时标记和细胞谱系追踪来解决这个问题， 表明PAAs的内皮细胞起源于第二心脏区域。此外，我们的时间分辨， 三维共聚焦成像显示PAA是由原始血管丛重组成PAA而形成的。在 在我们的研究过程中，我们发现细胞外基质糖蛋白纤维连接蛋白（Fn 1）是必需的， 对于第4对PAA的形成，以及Isl 1谱系中Fn 1的条件性缺失导致严重的 先天性主动脉弓缺陷，类似于DiGeorge综合征中观察到的缺陷。我们发现， Fn 1flox/-; Isl 1Cre动物的内皮细胞密度远低于对照组，并假设Fn 1 通过调节SHF衍生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，我们建议测试这些假设，并完善时空 调节PAA祖细胞发育的机制。然后，我们建议将我们的方法应用于 系统地阐明了Tbx 1 +/-突变体中PAA形成缺陷的基础， 综合征我们计划通过以下具体目标来实现这些目标：1）确定 探讨Fn 1对PAA形态发生的调控机制; 2）探讨VEGF 2阳性细胞在PAA形成中的作用 3）确定Tbx 1调控PAA形成的阶段。 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，并研究在PAA形成过程中生长因子信号转导Fn 1的作用。而且我们 将采用创新的活多光子显微镜来研究内皮细胞的动态， 活胚胎中PAA的形成，以及Fn 1和Tbx 1在此过程中的作用。完成这些研究 将提供重要的见解，正常的PAA形成的机制和改变，导致 先天性心脏病患者的PAA发育缺陷。