Tie Tek Modulation of Cardiac Development

描述(申请人提供)：Tie1和Tek(也称为Tie2)是内皮蛋白受体酪氨酸激酶(RTK)。与血管内皮生长因子(VEGF)受体一起，它们是唯一已知的内皮细胞特异性RTK。由于Tie1或Tie2基因缺失对胚胎的致死性，Tie1或Tie2基因在心脏发育调控中的确切作用和机制尚不清楚。此外，Tie1的配体(S)或Tie1激活的靶标尚未确定。我们推测，在心血管系统中，Tie1不仅是Tie2激活的抑制共受体，而且能够自主地传递Tie2的信号。因此，我们建议：1)确定Tie1和Tie2信号在体内心脏发育早期和晚期的独特功能：最近产生的携带Tie1或Tie2等位基因的小鼠将与新的早期心内膜特异性Cre(NFATC1Cre)或后来的瓣膜心内膜Cre系(NFATC1enCre)结合使用，在子宫心脏发育的整个过程中产生阶段特异性的Tie1和Tie2缺失。形态和功能分析将被用来表征心脏特异性RTK减弱所导致的独特表型。2)确定在体内心脏形成的早期和晚期正常的心脏形态发生所需的特定的Tie1-Tie2相互作用。重组酶介导的盒交换(RMCE)将被用来评估潜在的Tie1-Tie2相互作用的关键域 心内膜。我们将产生一系列缺乏Tie1胞外区(ECD)、胞内区(ICD)以及Tie1胞浆区关键酪氨酸激酶结构域(酪氨酸到苯丙氨酸/Y1113F和赖氨酸到丙氨酸/K866A)的cDNA点突变小鼠。这些动物将被杂交到心内膜特定的Cre系，并评估心脏发育中的特定缺陷。3)勾画心血管系统Tie1激活的下游靶点。在缺乏Tie1激活的已知配体的情况下，Tie1信号的血流动力学激活将用于评估Tie1明确缺失/突变的效果。从条件性Tie1突变体获得的永生化心内膜细胞系将暴露在改变的剪应力、湍流和拉伸下，并评估先前描述的归因于Tie激活的信号转导通路的变化。信息量最大的Tie1突变动物将用于RNA-Seq和生物信息学分析，以构建信号网络，定义Tie1的独特单一签名，并帮助描绘心脏内皮细胞自主和非自主的信号通路。

DESCRIPTION (provided by applicant): Tie1 and Tek (also known as Tie2) are endothelial protein receptor tyrosine kinases (RTKs). Along with the vascular endothelial growth factor (VEGF) receptor, these are the only known endothelial cell-specific RTKs. Due to embryonic lethality of panendotheilal deletion of either Tie 1 or Tie2 the exact roles and mechanisms of Tie1 or Tie2 in regulating cardiac development has not been clearly delineated. Furthermore the ligand(s) for Tie1 or the targets of Tie1 activation have not been identified. We hypothesize that in the cardiovascular system Tie1 is not only an inhibitory co-receptor for Tie2 activation, but also capable of signaling autonomously of Tie2. We therefore propose to: 1) Determine the unique functions of Tie1 and Tie2 signaling during early and late stages of cardiac development in vivo: Recently generated mice harboring a floxed allele of Tie1 or Tie2 will be used in conjunction with a novel early endocardial specific (Nfatc1Cre) or a later valvular endocardial Cre line (Nfatc1enCre) to produce stage specific cardiac deletion of Tie1 and Tie2 throughout the continuum of heart development in utero. Morphological and functional analysis will be used to characterize the unique phenotype that results from cardiac specific RTK attenuation. 2) Define the specific Tie1-Tie2 interactions that are required for normal cardiac morphogenesis at both early and late stages of heart formation in vivo. Recombinase mediated cassette exchange (RMCE) will be used to evaluate potential critical domains of Tie1-Tie2 interaction specifically in the endocardium. We will generate a series of cDNA "knock-in" mice lacking the extracellular domain (ECD), intracellular domain (ICD) as well as point mutations in critical tyrosine kinase domains in the cytoplasmic region of Tie1 (tyrosine to phenylalanine/Y1113F and lysine to alanine/K866A). These animals will be crossed to endocardial specific Cre lines and evaluated for specific defects in cardiac development. 3) Delineate down-stream targets of Tie1 activation in the cardiovascular system. In the absence of a known ligand for Tie1 activation, hemodynamic activation of Tie1 signaling will be used to evaluate the effect of defined deletion / mutations of Tie1. Immortalized endocardial cell lines derived from the conditional Tie1 mutants will be exposed to altered shear stress, turbulence and stretch and evaluated for alterations in previously described signal transduction pathways attributed to Tie activation. The most informative Tie1 mutant animals will be used for RNA-Seq and bioinformatic analysis for construction of signaling networks that will define a unique singling signature for Tie1 and help delineate endothelial cell autonomous and non-autonomous signaling pathways in the heart.