CELL ADHESION INDUCED DIFFERENTIATION IN EYE TISSUE

在眼睛发育过程中，特定的细胞类型从祖细胞分化出来， 不同胚胎起源的细胞，以及细胞间的相互作用 形成一个高效的，工作的感觉器官， 不被理解。之后，眼细胞类型必须保持其 分化的表型，以确保眼睛的健康和操作。 导致失明的几种疾病是由分化引起的 例如，视网膜的黄斑变性， 透镜与视网膜色素上皮细胞转分化 （增殖性玻璃体视网膜疾病）。钙粘蛋白是钙依赖性的 细胞-细胞粘附分子是哺乳动物发育所必需的。 我最近发现钙粘蛋白细胞间粘附分子控制 视网膜色素上皮细胞的分化状态;异位 E-cadherin在大鼠视网膜色素上皮细胞系中的表达， RPE-J导致包括重组的表型转化 的组成性蛋白质和诱导合成的特定mRNAS和 产生基底-外侧Na+，K +-ATP酶极性的蛋白质类，锚蛋白亚型 转换、桥粒连接复合体组装和角蛋白丝 组装件.利用这些观察结果，我们现在建议分析机制 用于确定视网膜色素上皮的分化状态 并将这些观察结果扩展到眼睛的其他细胞类型。我们的具体 本研究的目标是：（i）鉴定钙粘蛋白序列 是视网膜色素上皮分化所必需的。 嵌合 内源性视网膜色素上皮细胞之间会产生钙粘蛋白 钙粘蛋白和E-钙粘蛋白，并通过转染RPE-J细胞表达， 定义诱导表型变化所需的序列。（二） 识别钙粘蛋白相关分子， 视网膜色素上皮细胞的诱导分化线索。后 定义在钙粘蛋白诱导的细胞凋亡过程中使用的序列， 差分程序，生化和酵母两种杂交方法将是 用于鉴定与这些序列相互作用的蛋白质。（三） 分析钙粘蛋白诱导透镜上皮细胞分化。像视网膜 色素上皮细胞、透镜上皮细胞不内源性地 表达E-cadherin。我们将测试钙粘蛋白诱导的 这种眼部细胞类型的分化程序。(iv)鉴定新 钙粘蛋白，可以建立和维持分化状态， 透镜上皮细胞和视网膜细胞类型。 我们已经开始搜索 在眼组织中表达的新型钙粘蛋白。 初步 我实验室的证据显示， 在视网膜中表达，这在以前没有描述过。我们 提出用RT-PCR方法广泛筛选新型钙粘蛋白， 克隆编码这些新型钙粘蛋白的全长cDNA。的结果 这些研究将提供钙粘蛋白的详细机制框架， 诱导分化的变化，在眼睛形态发生，并将确定 分化程序中的潜在位点， 眼组织的病理生理过程。

During eye development, specific cell types differentiate from progenitor cells of diverse embryonic origin, and cell-cell interactions orchestrate the formation of a highly efficient, working sensory organ, in ways which are not understood. Later, the ocular cell types must maintain their differentiated phenotype for proper health and operation of the eye. Several diseases leading to loss of sight result from differentiation changes, for example, macular degeneration in the retina, cataracts of the lens and transdifferentiation of the retinal pigment epithelium (proliferative vitreoretinal disorders). Cadherins are calcium dependent cell-cell adhesion molecules that are essential for mammalian development. l have recently shown that cadherin cell-cell adhesion molecules control the differentiation state in retinal pigment epithelial cells; ectopic expression of E-cadherin in a rat retinal pigment epithelium cell line, RPE-J, results in a phenotypic transformation that includes reorganization of constitutive proteins and induced synthesis of specific mRNAS and proteins producing basal-lateral Na+, K+-ATPase polarity, ankyrin isoform switching, desmosome junctional complex assembly and keratin filament assembly. Using these observations, we now propose to analyze mechanisms for determining the differentiated state of the retinal pigment epithelium and extend these observations to other cell types of the eye. Our specific goals in the proposed studies are to: (i) identify cadherin sequences required for retinal pigment epithelium differentiation. Chimeric cadherins will be generated between endogenous retinal pigment epithelium cadherin and E-cadherin and expressed by transfection in RPE-J cells to define the sequences required for inducing phenotypic changes. (ii) Identify cadherin-associated molecules which may transduce cadherin- induced differentiation cues of the retinal pigment epithelium. After defining the sequences which are utilized during the cadherin-induced differentia~on program, biochemical and yeast two hybrid methods will be used to identify proteins which interact with these sequences. (iii) Analyze cadherin-induced differentiation the lens epithelium. Like retinal pigment epithelial cells, lens epithelial cells do not endogenously express E-cadherin. We will test the generality of the cadherin-induced differentiation program in this ocular cell type. (iv) Identify novel cadherins which may establish and maintain the differentiated state of lens epithelial cells and retinal cell types. We have initiated a search for novel cadherins which are expressed in ocular tissues. Preliminary evidence from my laboratory shows that there are cadherin molecules expressed in the retina which have not been previously described there. We propose an extensive screen for novel cadherins using RT-PCR methods, and to clone full length cDNAs encoding these novel cadherins. The results of these studies will provide a detailed mechanistic framework of cadherin- induced differentiation changes during eye morphogenesis and will identify potential sites in the differentiation program which fail during pathophysiological processes in eye tissues.