Cell-ECM interactions in the development of the aortic arch arteries

 描述(申请人提供)：胚胎形态发生需要不同细胞类型之间的协调沟通。这些相互作用的失调会导致出生缺陷。体内细胞之间的信号传递发生在细胞外基质(ECM)的背景下，ECM的化学和机械性质都调节细胞信号和祖细胞的命运。细胞外基质成分调节胚胎形态发生的机制尚不清楚。在我们的第一个R01支持的研究过程中，我们有了一个有趣的发现，即ECM糖蛋白Fn1的合成在小鼠胚胎发育过程中受到动态调节，并且Fn1在不同的胚胎前体细胞中的表达高度丰富，这对心血管的形态发生至关重要。Fn1是一种高度保守的脊椎动物糖蛋白，在小鼠和人类中由单一基因编码，先前的研究表明Fn1在心血管发育中是必需的。因此，我们假设Fn1的不同细胞来源在心血管系统的发育过程中发挥着不同的生物学意义。这项研究提供的初步数据表明，由神经脊(NC)合成的Fn1对主动脉弓动脉(AAAs)的发育至关重要，AAAs是一种将含氧血液从心脏输送到身体其他部位的血管系统。当咽弓动脉周围的NC细胞成熟为AAA时，Fn1的mRNA和蛋白在这些血管周围的NC细胞中被高度诱导，我们发现由咽弓动脉周围的NC细胞合成的Fn1调节NC细胞向血管平滑肌细胞(VSMCs)的分化。我们的证据表明，由NC合成的Fn1通过整合素α5β1信号转导和调节肌动蛋白聚合，以一种细胞自主的方式调节NC到VSMC的分化，进而调节Myocardin相关转录因子B从细胞质到细胞核的转位。在细胞核中，MRTFB可以直接激活平滑肌基因的转录，我们发现一个结构性地定位于细胞核的MRTFB缺失突变体拯救了Fn1缺陷的NC细胞的平滑肌分化。在这一新的应用中，我们建议通过以下具体目的来确定Fn1调节NC向VSMC分化的机制以及组织生物力学在这一过程中的作用：1)检验Fn1通过整合素α5β1信号调节NC细胞向VSMC分化的假说；2)检验Fn1和组织生物力学通过调节肌动蛋白细胞骨架动力学和MRTFB的核定位来调节NC向VSMC分化的假说；3)验证以下假设：EIIIA和EIIIB形式的Fn1调节NC向VSMC的分化，以及Fn1在咽弓动脉内皮细胞周围的NC细胞中的诱导是由Smad2依赖的信号介导的。

 DESCRIPTION (provided by applicant): Embryonic morphogenesis requires coordinated communications among diverse cell types. Dysregulation of these interactions results in birth defects. Signaling among cells in vivo occurs in the context of the extracellular matrix (ECM), and both the chemical and mechanical properties of the ECM regulate cell signaling and progenitor cell fates. Mechanisms whereby components of the ECM regulate embryonic morphogenesis are not well understood. In the course of the studies supported by our first R01, we made the intriguing discovery that synthesis of the ECM glycoprotein fibronectin (Fn1) is dynamically regulated during mouse embryonic development, and that the expression of Fn1 is highly enriched in distinct embryonic progenitors important for cardiovascular morphogenesis. Fn1 is a highly conserved vertebrate glycoprotein encoded by a single gene in mice and humans, and previous studies showed that Fn1 is required for cardiovascular development. Therefore, we hypothesized that different cellular sources of Fn1 played distinct, biologically significant roles in the development of the cardiovascular system. Preliminary data presented in this grant demonstrate that Fn1 synthesized by the neural crest (NC) is essential for the development of the aortic arch arteries (AAAs), a system of blood vessels that routs oxygenated blood from the heart to the rest of the body. Fn1 mRNA and protein become highly induced in NC cells surrounding the pharyngeal arch arteries as these blood vessels mature into the AAAs, and we show that Fn1 synthesized by NC cells around the pharyngeal arch arteries regulates the differentiation of NC cells into vascular smooth muscle cells (VSMCs). We present evidence suggesting that Fn1 synthesized by the NC regulates NC-to-VSMC differentiation in a cell- autonomous manner by signaling through integrin α5β1 and regulating actin polymerization, which in turn, regulates the translocation of the myocardin-related transcription factor B (MRTFB) from the cytoplasm into the nucleus. In the nucleus, MRTFB can directly activate the transcription of smooth muscle genes, and we show that a deletion mutant of MRTFB that constitutively localizes to the nucleus rescues smooth muscle differentiation in Fn1-deficient NC cells. In this renewal application we propose to determine the mechanisms by which Fn1 regulates NC-to-VSMC differentiation and the role of tissue biomechanics in this process by addressing the following Specific Aims: 1) To test the hypothesis that Fn1 regulates the differentiation of NC cells into VSMCs by signaling through integrin α5β1; 2) To test the hypothesis that Fn1 and tissue biomechanics regulate NC-to-VSMC differentiation by modulating actin cytoskeletal dynamics and nuclear localization of MRTFB; and 3) To test the hypotheses that EIIIA+ and EIIIB+ forms of Fn1 regulate NC-to-VSMC differentiation and that the induction of Fn1 in NC cells surrounding the endothelium of the pharyngeal arch arteries is mediated by SMAD2-dependent signaling.