Molecular regulation of angioblast migration during cornea development

描述（由申请人提供）：角膜血管化是一种威胁视力的并发症。角膜血管形成的分子机制尚不清楚，对角膜血管形成的发展也知之甚少。我们建议研究迁移血管前体细胞（成血管细胞）在角膜血管形成过程中的分子调控。之前，我们发现眼周成血管细胞表达神经匹林1 (Nrp1)，这是一种血管生成血管内皮生长因子（VEGF）的双重受体和抗血管生成引导分子Semaphorin3A （Sema3A）。我们正在进行的研究表明，在角膜发育过程中，眼周成血管细胞也表达PlexinD1 a抗血管生成Sema3E受体。我们还发现，抗血管生成细胞因子CXCL14在分化后不久在基质角质细胞中强烈表达。基于这些观察，我们假设促血管生成因子和抗血管生成因子之间的严格调节平衡控制着成血管细胞迁移和角膜无血管形成过程中的血管模式。为了验证这一假设，我们将利用小鼠遗传学和操纵鸟类眼睛的便利性来探索角膜发育过程中与血管发生相关的抑制基因的功能。我们将研究迁移成血管细胞在眼睛发育过程中的行为，以确定它们在何时何地与其他迁移细胞分离，从而产生角膜基质和内皮。我们将通过qPCR、原位杂交和免疫组织化学来确定角膜发育过程中引导基因及其受体的时空表达。我们将在带荧光血管的Tie1:H2B-eYFP转基因鹌鹑胚胎中进行功能丧失和功能获得实验，以阐明抑制分子及其受体在眼周成血管细胞迁移过程中的功能。缺乏每种抑制基因或受体功能的敲除小鼠将被详细检查成血管细胞迁移和角膜血管化的缺陷。体内实验将与体外迁移实验相结合，直接测试抑制分子对眼周成血管细胞从其他眼部组织分离后迁移的影响。以下具体目标将检验我们的假设：确定Nrp1及其配体VEGF和Sema3A在角膜血管形成过程中成血管细胞迁移中的作用。2. 确定PlexinD1在角膜血管形成过程中的作用。3. 确定CXCL14表达在角膜发育过程中的功能意义。我们所提出的研究的成功完成将提供对导致无血管性角膜发展的成血管细胞迁移和血管发生的调节机制的见解。类似的机制可能会抑制正常成人角膜的新生血管形成，从而为发现潜在的治疗性抗血管生成药物铺平道路，用于治疗血管化的成人角膜和针对癌症引起的血管生成。

DESCRIPTION (provided by applicant): Vascularization of the cornea is a vision-threatening complication. The molecular mechanisms underlying corneal avascularity are still not well understood and comparatively little is known about the development of corneal avascularity. We propose to study the molecular regulation of migratory blood vessel precursor cells (angioblasts) during development of corneal avascularity. Previously, we showed that periocular angioblasts express Neuropilin1 (Nrp1) a dual receptor for the angiogenic vascular endothelial growth factor (VEGF) and the anti-angiogenic guidance molecule Semaphorin3A (Sema3A). Our ongoing studies show that periocular angioblasts also express PlexinD1 a receptor for anti-angiogenic Sema3E during cornea development. We also show that the anti-angiogenic cytokine CXCL14 is strongly expressed by stromal keratocytes shortly after differentiation. Based on these observations we hypothesize that a tightly regulated balance between pro- and anti-angiogenic factors controls angioblast migration and vascular patterning during development of corneal avascularity. To test this hypothesis, we will take advantage of mouse genetics and the ease of manipulating avian eyes to explore the function of inhibitory genes associated with vasculogenesis during corneal development. We will examine the behavior of migratory angioblasts during eye development to determine where and when they segregate from other migratory cells that give rise to the cornea stroma and endothelium. We will determine the spatiotemporal expression of guidance genes and their receptors during cornea development by qPCR, in situ hybridization, and immunohistochemistry. We will perform loss- and gain-of-function experiments in Tie1:H2B-eYFP transgenic quail embryos with fluorescent blood vessels to elucidate the function of inhibitory molecules and their receptors during periocular angioblast migration. Knockout mice lacking the function of each inhibitory gene or receptor will be examined in detail for defects in angioblast migration and vascularization of the cornea. In vivo experiments will be coupled with in vitro migration assays to directly test the effect of inhibitory molecules on periocular angioblast migration in isolation from other ocular tissues. The following Specific Aims will test our hypothesis: 1. Determine the role of Nrp1 and its ligands VEGF and Sema3A in angioblast migration during development of corneal avascularity. 2. Identify the role of PlexinD1 during development of corneal avascularity. 3. Determine the functional significance of CXCL14 expression during cornea development. Successful completion of our proposed study will provide insight into the mechanisms that regulate angioblast migration and vasculogenesis leading to development of an avascular cornea. Similar mechanisms may inhibit neovascularization of the normal adult cornea and thus pave the way for discovery of potential therapeutic anti-angiogenesis agents for treating vascularized adult corneas and targeting angiogenesis due to cancer.