ORIGIN AND MAINTENANCE OF THE OCULAR SURFACE EPITHELIA

描述（由申请人提供）：眼睛表面的组织和源自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的成分。任何这些腺体功能不足导致干眼病。角膜和角膜缘上皮负责维持眼睛的屈光表面。角膜上皮细胞的分化缺陷或角膜缘sem细胞产生角膜上皮细胞的不足导致严重的眼刺激、炎症、角膜基质的新血管形成和失明。本提案的目的是：1）鉴定在发育过程中负责眼表上皮及其衍生物的正确形成和功能的信号传导系统; 2）鉴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传导途径。该提案中描述的研究表明，Pax 6和BMP信号传导是所有眼表上皮细胞形成和分化所需的关键因素。激光显微切割和微阵列分析鉴定或确认了四种转录因子，它们是不同眼表上皮细胞的早期标志物，并且可能在其分化中发挥重要作用。其中三个因子的表达依赖于Pax 6。该建议还描述了一种新的方法来遗传标记角膜缘干细胞。同时，这些细胞内的基因可以被选择性地删除。这种方法将被用于验证已知在其他成体干细胞中起作用的关键途径。其中三种途径，BMP和SDF-1信号传导以及N-cadherin介导的niche和干细胞之间的粘附，已经被我们在这个项目上的合作者Scheffer Tseng博士证明参与了角膜缘干细胞和niche细胞之间的信号传导。这些通路的功能将在体内通过基因消融和共聚焦成像进行测试。在每种情况下，我们的体内分析将通过在曾博士实验室进行的体外遗传研究进行补充。这种合作是可能的，因为曾博士分离角膜缘干细胞和贴壁壁龛细胞过夜孵育后。我们将把转基因小鼠的眼睛连夜快递给曾博士。他将分离小生境和干细胞，并用他莫昔芬治疗它们以删除靶基因。这项合作将提供迄今为止最广泛的分析，创造和维持角膜缘干细胞生态位的途径。来自这两个目标的信息将是有价值的，用于替换受伤或有缺陷的角膜和结膜上皮细胞使用诱导多能细胞或通过“重编程”的其他上皮细胞类型。

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.