Role of integrin a5b1 in vascular patterning and the formation of the pharyngeal arch arteries

项目摘要先天性心脏病(CHD)是新生儿死亡和发病率的重要原因 全世界。主动脉弓动脉及其主要分支的缺陷是最严重的。 导致先天性心脏病的畸形。AAA及其分支的形成是由于AAA的不对称重构 三对两侧对称的咽弓动脉(PaaS)，3、4和6.左侧第4个PAA的缺陷 尤其具有破坏性，导致B型主动脉弓(IAA-B)中断。IAA-B扰乱了 体循环和出生后是致命的。IAA-B的遗传原因大多未知，强调了一种 迫切需要了解调节左侧第4 PAA形成的基因和机制。PAA地层是 一个多步骤的过程：PAA内皮细胞(EC)前体细胞在e7.5和e7.5出现在第二心区(SHF) 在接下来的两天内填满咽弓，最终形成一个小的SHF神经丛- 取材于第4弓E10.0之间的血管。在其形成后，EC神经丛逐渐重塑 通过在第四个拱门中间合并成一个大船，第四个PAA。Fn1或整合素a5b1的缺失 在Isl1谱系中，导致右侧和左侧第4 PAA的缺陷形成，并导致IAA-B。我的实验室有 证明了Fn1和整合素a5b1调节的细胞-ECM相互作用在多个 以细胞类型特异性的方式调节第4个PaaS的形成和重塑的阶段。在这笔赠款中，我们向 Fn1和整合素a5b1在维持SHF来源的血管系统同侧模式中的作用 在咽部以9.5秒的速度。我们的数据显示，Isl1谱系中Fn1或整合素a5b1的缺失会破坏 SHF来源的细胞对称地分布在左右咽弓，导致 左侧与右侧的SHF派生单元格的数量。在后期，在E10和E10.75之间，Fn1和整合素 A5b1通过介导咽部EC神经丛向第4 PAA的重塑来调节血管构型。 我们发表的数据显示，Fn1和整合素a5b1介导了EC-非细胞-PAA神经丛到PAA的重塑。 调节负性血管引导线索的表达和激活的自主方式 VEGFR2和ERK1/2位于第4弓。确定Fn1和整合素a5b1调节血管内皮细胞生长的机制 SHF来源的血管系统的模式，我们将使用实时成像，定量免疫荧光显微镜， 遗传上位性分析和单细胞RNAseq检验两个假设1)Fn1和整合素a5b1 在Isl1谱系中的表达通过调节维持SHF来源的细胞在咽部的平衡分配 细胞外基质组装和同侧SHF细胞迁移；2)Fn1和整合素a5b1调节神经丛到PAA 通过调节EC引导信号的表达和ERK1/2的激活来重塑。 建议的研究将为细胞-细胞外基质相互作用在血管生成中的作用提供重要的见解。 SHF来源的血管系统与冠心病的发病机制。

PROJECT SUMMARY Congenital heart disease (CHD) is a significant cause of neonatal mortality and morbidity worldwide. Defects in the aortic arch artery (AAA) and its major branches are among the most severe malformations that cause CHD. The AAA and its branches arise through the asymmetrical remodeling of the three bilaterally symmetrical pairs of the pharyngeal arch arteries (PAAs), 3, 4, and 6. Defects in the left 4th PAA are particularly devastating, leading to the interruption of the aortic arch type B (IAA-B). IAA-B disrupts the systemic circulation and is lethal after birth. The genetic causes of IAA-B are mostly unknown, underscoring a critical need to understand genes and mechanisms regulating the formation of the left 4th PAA. PAA formation is a multi-step process: PAA endothelial cell (EC) progenitors arise in the second heart field (SHF) by E7.5 and populate pharyngeal arches within the next two days, culminating in the formation of a plexus of small SHF- derived blood vessels between by E10.0 in the 4th arch. Following its formation, the EC plexus gradually remodels by merging in the middle of the 4th arch into one large vessel, the 4th PAA. The deletion of Fn1 or integrin a5b1 in the Isl1 lineages results in the defective formation of the right and left 4th PAA and leads to IAA-B. My lab has demonstrated that cell-ECM interactions regulated by Fn1 and integrin a5b1 function reiteratively, at multiple stages to regulate the 4th PAAs’ formation and remodeling in a cell-type-specific manner. In this grant, we present data implicating Fn1 and integrin a5b1 in maintaining the ipsilateral patterning of the SHF-derived vasculature in the pharynx at E9.5. Our data show that the deletion of Fn1 or integrin a5b1 in the Isl1 lineage disrupts symmetrical allocation of SHF-derived cells to the left and right pharyngeal arches, causing an imbalance in the number of SHF-derived cells on the left vs. the right. At a later stage, between E10 and E10.75, Fn1 and integrin a5b1 regulate vascular patterning by mediating the remodeling of the pharyngeal EC plexus into the 4th PAA. Our published data show that Fn1 and integrin a5b1 mediate the plexus-to-PAA remodeling in an EC-non-cell- autonomous manner by regulating the expression of negative vascular guidance cues and the activation of VEGFR2 and Erk1/2 in the 4th arch. To determine the mechanisms by which Fn1 and integrin a5b1 regulate the patterning of SHF-derived vasculature, we will use live imaging, quantitative immunofluorescence microscopy, genetic epistasis analysis, and single-cell RNAseq to test two hypotheses 1) that Fn1 and integrin a5b1 expressed in the Isl1 lineage maintain the balanced allocation of SHF-derived cells in the pharynx by regulating ECM assembly and ipsilateral SHF cell migration and 2) that Fn1 and integrin a5b1 regulate plexus-to-PAA remodeling by modulating the expression of EC guidance cues and the activation of Erk1/2. Completing the proposed studies will provide essential insights into the functions of cell-ECM interactions in the development of SHF-derived vasculature and CHD pathogenesis.