Flt4 signaling in vascular and lymphatic development

描述(申请人提供)：循环系统由内皮细胞排列的血管组成，作为红血球的运输网络，红血球携带氧气、免疫细胞和内分泌系统的激素。同时，淋巴系统由盲端血管组成，同样由内皮细胞排列，并与循环系统的血管密切相关。淋巴管收集间质液体并将其返回循环系统，是淋巴样细胞发育和转运的场所，也是消化系统吸收脂肪所必需的。在这两个系统中，启动形成和调节功能的主要细胞类型是内皮细胞。有趣的是，淋巴管内皮细胞在胚胎发育过程中来源于静脉内皮细胞，这表明两种血管类型具有共同的起源。不足为奇的是，许多信号分子是共享的，是循环系统和淋巴系统形成所必需的。其中包括Flt4，一种在血管和淋巴管内皮细胞上表达的受体酪氨酸激酶，与配体血管内皮生长因子c(VEGFC)结合。小鼠或斑马鱼胚胎中Flt4功能的丧失会导致静脉、血管新生血管和淋巴管的一系列缺陷。对Flt4的这种多样化的需求在一定程度上是由其在胚胎发育过程中的高度动态表达所支配的。然而，人们对负责动态表达的上游监管机构知之甚少。Flt4动态作用的另一个方面是它能够激活内皮细胞中广泛的下游信号效应器。在原代细胞系和体内的研究表明，不同的下游效应器可能在胚胎血管和淋巴发育过程中发挥上下文相关的作用。鉴于新的血液和淋巴管的生长在人类发育和病理环境中的重要性，更好地理解Flt4信号的方式是高度相关的。在这个应用程序中，我们将依靠我们的专业知识，使用斑马鱼作为模型来解决这些问题。在目标1中，我们将确定是什么控制了Flt4的动态表达。特别是，我们将研究ERK和ETS转录因子在直接激活Flt4基因增强子诱导TIP细胞表达中的作用。这将通过应用转基因和敲除斑马鱼品系来实现。在目标2中，我们将确定哪些Flt4-近端信号输出对于血管和淋巴发育是必不可少的。这将通过应用位点特异性核酸酶来产生斑马鱼品系，该品系在Flt4细胞质区域具有靶向的酪氨酸缺失。对这些线的分析将使我们能够确定不同的下游效应器在调节Flt4的上下文相关角色中的重要性。

DESCRIPTION (provided by applicant): The circulatory system consists of endothelial cell-lined blood vessels that serve as a transport network for red blood cells, which carry oxygen, immune cells, and hormones of the endocrine system. In parallel, the lymphatic system comprises blind-ended vessels, which are similarly lined by endothelial cells and are closely associated with the blood vessels of the circulatory system. Lymphatic vessels collect and return interstitial fluid to the circulatory system, are a site of lymphoid cell development and transit, and are required for lipid absorption in the digestive system. In both systems, the primary cell type that initiates formation and mediates function is the endothelial cell. Interestingly, lymphatic endothelial cells derive from venous endothelial cells during embryonic development indicating a common origin for both vessel types. Not surprisingly, a number of signaling molecules are shared and required for the formation of the circulatory and lymphatic systems. Among these is Flt4, a receptor tyrosine kinase expressed on both vascular and lymphatic endothelial cells that binds the ligand vascular endothelial growth factor c (Vegfc). Loss of Flt4 function in mouse or zebrafish embryos leads to a range of defects in veins, angiogenic blood vessels, and lymphatic vessels. This diverse requirement for Flt4 is governed, in part, by its highly dynamic expression during embryonic development. However, little is known about upstream regulators responsible for dynamic expression. An additional aspect of the dynamic role of Flt4 is its ability to activate a wide range of downstream signaling effectors in endothelial cells. Studies in primary cell lines and in vivo suggest that distinct downstream effectors may play context dependent roles during embryonic vascular and lymphatic development. Given the importance of new blood and lymphatic vessel growth in both human development and pathological settings, a better understanding of how Flt4 signals is highly relevant. In this application we will rely on our expertise using the zebrafish as a model to address these issues. In Aim 1, we will determine what controls dynamic expression of flt4. In particular, we will investigate the role of ERK and Ets transcription factors in directly activatin enhancers at the flt4 locus to induce tip cell expression. This will be achieved through application of transgenic and knockout zebrafish lines. In Aim 2, we will determine which Flt4-proximal signaling outputs are essential for vascular and lymphatic development. This will be done through application of site-specific nucleases to generate zebrafish lines bearing targeted deletion of tyrosines in the Flt4 cytoplasmic domain. Analysis of these lines will allow us to determine the importance of different downstream effectors in mediating context-dependent roles of Flt4.