Function of Nephronectin in the corneal ECM during development, homeostasis, and wound healing

前眼发育不全以及角膜疾病和损伤是导致眼部缺陷的主要原因。 和失明。眼周神经脊细胞(PNC)是一种多潜能的胚胎细胞群， 提供重要的信号并对角膜的细胞和细胞外成分做出贡献，但 这些过程背后的分子机制仍然不是很清楚。我们已经确定了一部小说 一种新近发现的细胞外基质(ECM)蛋白--肾连接素(Npnt)在角膜组织中的表达 发展。尽管Npnt已被证明在各种发育过程中发挥作用，并在 包括癌症转移在内的病理条件，目前还没有在角膜中进行研究。这一项目旨在 通过检验Npnt促进细胞的假说来了解Npnt在角膜中的作用机制 角膜发育、动态平衡和伤口愈合过程中的迁移和附着。我们正在进行的 研究证实，Npnt在推测的角膜细胞外基质中表达，而它的主要受体 ITGα8在迁移前和迁移期间由PNC表达。此外，我们还观察到了 Npnt在鸡上皮和小鼠内皮细胞基底膜上的定位，并且它是 在小鼠的整个成年期都保持着。基于这些观察，我们将利用鼠标 遗传学和鸟眼的易操纵性结合了Npnt表达的时空差异 在这两个模型中，提供了一个全面的了解Npnt在角膜中的功能。我们的 雏鸡Npnt基因敲除和过度表达的初步研究显示，角膜变薄和增厚， 分别进行了分析。此外，我们还发现，Npnt基因的敲除会导致角膜上皮缺陷，并且 NPNT/ITGα8信号通路在体外促进PNC迁移。我们将进一步研究NPNT在 PNC迁移并在角膜上皮基底膜和胚胎角膜创面愈合。 对NpnT基因敲除小鼠的分析将表明NpnT在不同的PNC迁移模式中的功能， 后弹力膜的形成和维持，以及在成人角膜伤口愈合中的作用。我们 还将进行蛋白质组学分析，以确定NPNT相互作用的伙伴，并确定如何缺乏 NPNT影响角膜细胞外基质和基底膜蛋白。所有建议的研究都有补充 鸡胚眼的显微操作、体外培养和分子生物学技术，以及 对基因功能的药理抑制。以下具体目标将检验我们的假设： (1)确定肾凝素在PNC向角膜迁移过程中的作用。 (2)探讨Npnt在角膜上皮和内皮基底膜中的作用。 (3)探讨Npnt在胚胎和成人角膜伤口愈合过程中的作用。 拟议的研究将揭示Npnt在细胞过程中的正常所需的新功能。 角膜的发育、功能和修复。

Dysgenesis of the anterior eye together with corneal diseases and injuries are major causes of ocular defects and loss of vision. The periocular neural crest cells (pNC) are multipotent embryonic cell population that provide crucial signals and contribute to the cellular and extracellular components of the cornea, but the molecular mechanisms underlying these processes are still not well understood. We have identified novel expression of a recently discovered extracellular matrix (ECM) protein, nephronectin (Npnt), during corneal development. Although Npnt has been shown to function in various developmental processes, and identified in pathological conditions including cancer metastasis, it has not been studied in the cornea. This project seeks to understand the mechanisms of Npnt function in the cornea by testing the hypothesis that Npnt promotes cell migration and attachment during corneal development, homeostasis and wound healing. Our ongoing studies have identified that Npnt is expressed in the ECM of the presumptive cornea whereas its major receptor Itgα8 is expressed by the pNC prior to and during migration. In addition, we observed spatial differences in the localization of Npnt to the chick epithelial and mouse endothelial basement membranes, and that it is maintained throughout adulthood in mice. Based on these observations, we will take advantage of mouse genetics and the ease of manipulating avian eyes, combined the spatiotemporal differences in Npnt expression in the two models, to provide a comprehensive understanding of the function of Npnt in the cornea. Our preliminary studies of Npnt knockdown and overexpression in chick show corneal thinning and thickening, respectively. In addition, we show that knockdown of Npnt causes corneal epithelial defects and that Npnt/Itgα8 signaling augments pNC migration in vitro. We will further examine the function of Npnt during pNC migration and in the corneal epithelial basement membrane and embryonic corneal wound healing. Analysis of Npnt knockout mice will indicate the function of Npnt during a different pattern of pNC migration, formation and maintenance of the Descemet's membrane, and function in adult corneal wound healing. We will also perform proteomic analysis to identify Npnt interacting partners and determine how the absence of Npnt affects the corneal ECM and basement membrane proteins. All proposed studies are supplemented with micromanipulation of chick embryonic eyes, in vitro culture and molecular biology techniques, and pharmacological inhibition of gene function. The following Specific Aims will test our hypothesis: (1) Determine the role of nephronectin during migration of pNC into the cornea. (2) Investigate the role of Npnt in the corneal epithelial and endothelial basement membranes. (3) Determine the function of Npnt during embryonic and adult corneal wound healing. The proposed studies will reveal novel functions of Npnt in cellular processes that are required for normal development, function, and repair of the cornea.