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。 与公共健康相关：眼睛表面覆盖着几种类型的上皮细胞，每一种细胞对视力都是必不可少的。在发育早期，眼表是一层未分化的外胚层。随着发育的进行，外胚层特化成角膜、角膜缘、结膜和眼皮上皮。这些上皮细胞产生泪腺、粘液和眉板腺，这些腺体对润滑眼睛表面和形成稳定的泪膜是必不可少的。关于指定这些不同的信号，我们知之甚少 眼表面区域或基因调控需要它们的专门化。识别这些细胞的特殊属性的机制对于了解它们的正常功能和故障以及为受伤的眼睛生成替代组织非常重要。遗传性疾病和物理、化学或免疫损伤会损害眼表上皮细胞的功能。一些实验室正在探索从诱导的多能干细胞或通过对其他类型的成人上皮组织进行重新编程来生成替代组织。通过了解胚胎中指定这些组织的信号通路，将极大地帮助眼表替代组织的生成。这项研究的一个主要目的是确定指定眼表组织和调节因子的信号 以确保它们的正常功能。角膜上皮细胞不断地被细胞增殖所取代。这一过程的祖细胞是位于 角膜缘，角膜周边的区域。我们开发了一种新的方法来标记这些干细胞及其在活动物中的后代，同时删除编码调节成年干细胞功能的关键信号分子的基因。这些分析将得到我们的合作者、世界公认的角膜干细胞生物学专家Scheffer Tseng博士进行的体外研究的补充。总之，我们希望确定建立和维持角膜缘干细胞的因素，以保护角膜上皮的功能，防止失明。就像形成眼表组织所需的发育信号一样，了解指定和维持角膜缘干细胞的因素将极大地帮助恢复或替换损伤后该区域的功能。

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. PUBLIC HEALTH RELEVANCE: The surface of the eye is covered by several types of epithelial cells, each one of which is essential for vision. Early in development, the ocular surface is an undifferentiated layer of ectoderm. As development proceeds, the ectoderm becomes specialized into the corneal, limbal, conjunctival and eyelid epithelia. These epithelia give rise to the lacrimal, mucus and Meibomian glands that are essential to lubricate the ocular surface and form a stable tear film. Little is known about the signals that specify these different regions of the ocular surface or gene regulation needed for their specialization. Identifying the mechanisms that specify the special properties of these cells is important for understanding their normal function and malfunction and for generating replacement tissues for injured eyes. Genetic diseases and physical, chemical or immunological injuries can impair the function of the ocular surface epithelia. Several laboratories are exploring the generation of replacement tissues from induced pluripotent stem cells or by "reprogramming" other types of adult epithelial tissues. Generation of replacement tissues for the ocular surface would be greatly assisted by knowing the signaling pathways that specify these tissues in the embryo. A major aim of this study is to identify the signals that specify the ocular surface tissues and the regulatory factors that assure their proper function. The cells of the corneal epithelium are constantly being replaced by cell proliferation. The progenitor cells for this process are stem cells located in the limbus, a region around the periphery of the cornea. We developed a novel method to label these stem cells and their progeny in the living animal and, at the same time, delete the genes encoding key signaling molecules that regulate the function of adult stem cells. These analyses will be complemented by in vitro studies performed by our collaborator, Dr. Scheffer Tseng, a world- recognized expert in corneal stem cell biology. Together, we expect to define the factors that establish and maintain the limbal stem cells to preserve the function of the corneal epithelium and prevent blindness. As with the developmental signals needed to form the tissues of the ocular surface, understanding the factors that specify and maintain the limbal stem cells will greatly aid in restoring or replacing the function of this region after injury.