ORIGIN AND MAINTENANCE OF THE OCULAR SURFACE EPITHELIA

描述（由申请人提供）：眼睛表面的组织和来源于这些组织的分泌腺对视力至关重要。泪腺、睑板腺和结膜粘液腺分泌泪膜成分。这些腺体功能不足会导致干眼症。角膜和角膜缘上皮负责维持眼睛的屈光表面。角膜上皮细胞分化不足或角膜缘上皮细胞生成不足可导致严重的眼部刺激、炎症、角膜基质新生血管形成和失明。本研究的目的是：1)确定在眼表上皮及其衍生物发育过程中负责正常形成和功能的信号系统；2)确定建立和维持角膜缘干细胞以提供功能性角膜上皮细胞的信号通路。本提案的研究表明Pax6和BMP信号是所有眼表上皮形成和分化所需的关键因素。激光显微解剖和微阵列分析鉴定或证实了四种转录因子，它们是不同眼表上皮的早期标记物，可能在其分化中起重要作用。其中三个因子的表达依赖于Pax6。提出了一种新的角膜缘干细胞基因标记方法。同时，这些细胞内的基因可以被选择性地删除。这种方法将用于灭活已知在其他成体干细胞中起作用的关键通路。其中三种途径，BMP和SDF-1信号通路以及N-cadherin介导的生态位和干细胞之间的粘附，已经被我们在这个项目中的合作者Scheffer Tseng博士证明参与了角膜缘干细胞和生态位细胞之间的信号传导。这些通路的功能将通过基因消融和共聚焦成像在体内进行测试。在每种情况下，我们的体内分析都将辅以曾博士实验室进行的体外基因研究。这一合作之所以成为可能，是因为曾博士在过夜孵育后分离出了角膜缘干细胞和贴壁壁龛细胞。我们会把转基因老鼠的眼睛用快递寄给曾博士。他将分离生态位和干细胞，并用他莫昔芬处理它们，以删除目标基因。这项合作将提供迄今为止最广泛的关于创造和维持角膜缘干细胞生态位的途径的分析。从这两个目标中获得的信息对于使用诱导多能细胞或通过其他上皮细胞类型的“重编程”来替代受损或有缺陷的角膜和结膜上皮具有重要价值。

DESCRIPTION (provided by applicant): The tissues on the surface of the eye and the secretory glands that are derived from these tissues are essential for vision. The lacrimal, Meibomian and conjunctival mucus glands secrete the components of the tear film. Insufficient function of any of these glands leads to dry eye disease. The corneal and limbal epithelia are responsible for maintaining the refractive surface of the eye. Deficiencies in the differentiation f the corneal epithelial cells or insufficient generation of corneal epithelial cells by the limbal sem cells leads to severe ocular irritation, inflammation, neovascularization of the corneal stroma and blindness. The aims of this proposal are 1) to identify the signaling systems that are responsible for the proper formation and function of the ocular surface epithelia and their derivatives during development and 2) to identify the signaling pathways that establish and maintain the limbal stem cells to provide functional corneal epithelial cells. The studies described in this proposal show that Pax6 and BMP signaling are key factors required for the formation and differentiation of all of the ocular surface epithelia. Laser microdissection and microarray analysis identified or confirmed four transcription factors that are early markers for the different ocular surface epithelia and are likely to play important roles in their differentiaton. Three of these factors depend on Pax6 for their expression. This proposal also describes a new method to genetically mark the limbal stem cells. At the same time, genes within these cells can be selectively deleted. This method will be used to inactivate critical pathways known to function in other adult stem cells. Three of these pathways, BMP and SDF-1 signaling and adhesion between niche and stem cells mediated by N-cadherin, have been shown by our collaborator on this project, Dr. Scheffer Tseng, to be involved in signaling between limbal stem and niche cells. The functions of these pathways will be tested in vivo by genetic ablation and confocal imaging. In each case, our vivo analyses will be complemented by in vitro genetic studies performed in Dr. Tseng's laboratory. This collaboration is possible because Dr. Tseng isolates limbal stem and adherent niche cells after overnight incubation. We will send eyes from our genetically-modified mice to Dr. Tseng by overnight courier. He will isolate the niche and stem cells and treat them with tamoxifen to delete the targeted genes. This collaboration will provide the most extensive analysis to date of the pathways that create and maintain the limbal stem cell niche. Information derived from both aims will be valuable for the replacement of injured or defective corneal and conjunctival epithelia using induced pluripotent cells or by "reprogramming" of other epithelial cell types.