Tie Tek Modulation of Cardiac Development

描述（由申请人提供）：Tie 1和Tek（也称为Tie 2）是内皮蛋白受体酪氨酸激酶（RTK）。沿着血管内皮生长因子（VEGF）受体，这些是唯一已知的内皮细胞特异性RTK。由于Tie 1或Tie 2的全内皮缺失的胚胎致死性，Tie 1或Tie 2在调节心脏发育中的确切作用和机制尚未被清楚地描述。此外，尚未鉴定Tie 1的配体或Tie 1活化的靶标。我们推测，在心血管系统中，Tie 1不仅是Tie 2激活的抑制性共受体，而且能够自主地发出Tie 2的信号。因此，我们建议：1）确定Tie 1和Tie 2信号在体内心脏发育早期和晚期阶段的独特功能：最近产生的携带Tie 1或Tie 2的floxed等位基因的小鼠将与新的早期内皮细胞特异性受体结合使用。（Nfatc 1Cre）或后来的瓣膜内支架Cre线（Nfatc 1 enCre）在子宫内心脏发育的整个连续过程中产生Tie 1和Tie 2的阶段特异性心脏缺失。形态和功能分析将用于表征心脏特异性RTK衰减导致的独特表型。2)定义在体内心脏形成的早期和晚期正常心脏形态发生所需的特定Tie 1-Tie 2相互作用。酶介导的盒交换（RMCE）将用于评估Tie 1-Tie 2相互作用的潜在关键结构域，特别是在 内分泌系统我们将产生一系列cDNA“敲入”小鼠，其缺乏胞外结构域（ECD）、胞内结构域（ICD）以及Tie 1胞质区中关键酪氨酸激酶结构域的点突变（酪氨酸至苯丙氨酸/Y1113 F和赖氨酸至丙氨酸/K866 A）。将这些动物与内皮细胞特异性Cre系杂交，并评价心脏发育中的特异性缺陷。3)描绘心血管系统中Tie 1激活的下游靶点。在不存在用于Tie 1活化的已知配体的情况下，Tie 1信号传导的血液动力学活化将用于评价Tie 1的限定缺失/突变的影响。将源自条件Tie 1突变体的永生化内皮细胞系暴露于改变的剪切应力、湍流和拉伸，并评价归因于Tie激活的先前描述的信号转导途径的改变。信息量最大的Tie 1突变动物将用于RNA-Seq和生物信息学分析，以构建信号网络，该信号网络将定义Tie 1的独特单一签名，并帮助描绘心脏中的内皮细胞自主和非自主信号通路。

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.