ORIGIN AND MAINTENANCE OF THE OCULAR SURFACE EPITHELIA

描述(申请人提供)：眼睛表面的组织和由这些组织衍生的分泌腺对视力是必不可少的。泪腺、睑粘液腺和结膜粘液腺分泌泪膜成分。这些腺体的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度。角膜缘上皮细胞分化不足或角膜上皮细胞生成不足会导致严重的眼刺激、炎症、角膜基质新生血管和失明。这项建议的目的是1)确定在发育过程中负责眼表上皮细胞及其衍生物正确形成和功能的信号系统，以及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。这项研究表明，Pax6和BMP信号是所有眼表上皮细胞形成和分化所必需的关键因素。激光显微切割和微阵列分析发现或证实了四个转录因子，它们是不同眼表上皮细胞的早期标志，可能在它们的分化中发挥重要作用。这些因子中有三个依赖于Pax6的表达。这项建议还描述了一种对角膜缘干细胞进行基因标记的新方法。与此同时，这些细胞内的基因可以被选择性地删除。这种方法将被用来灭活已知在其他成人干细胞中起作用的关键途径。其中三条途径，BMP和SDF-1信号通路，以及由N-钙粘蛋白介导的壁龛和干细胞之间的黏附，已经被我们的合作者Scheffer Tseng博士证明参与了角膜缘干细胞和壁龛细胞之间的信号传递。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。在每一种情况下，我们的活体分析都将得到曾博士实验室进行的体外遗传学研究的补充。这种合作是可能的，因为曾博士在隔夜孵育后分离出了角膜缘干细胞和贴壁的壁龛细胞。我们会用隔夜快递把转基因小鼠的眼睛寄给曾博士。他将分离利基细胞和干细胞，并用他莫昔芬治疗它们，以删除目标基因。这项合作将为创建和维持角膜缘干细胞生态位的途径提供迄今为止最广泛的分析。来自这两个目标的信息将对使用诱导多能细胞或通过对其他类型的上皮细胞进行“重新编程”来替换受损或缺陷的角膜和结膜上皮细胞具有价值。

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.