EPITHELIAL/MESENCHYMAL TRANSFORMATION IN EYE DEVELOPMENT

眼睛发育中的上皮/间质转化

• 批准号: 2163434
• 负责人: ELIZABETH D HAY
• 金额: $ 26.75万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-08-01 至 1997-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2163434
• 关键词: antibody; antisense nucleic acid; cadherins; cell differentiation; cell transformation; chick embryo; collagen; corneal epithelium; developmental neurobiology; embryo /fetus tissue /cell culture; eye; eye neoplasms; fibroblasts; gene expression; gene induction /repression; immunocytochemistry; in situ hybridization; laboratory mouse; lens; melanocyte; mesenchyme; metastasis; nerve; neural crest; protooncogene; transfection; vertebrate embryology

The research proposed in this grant is expected to contribute to understanding mechanisms of differentiation of eye tissues, with particular emphasis on cornea and lens. We describe a gene program for epithelial- mesenchymal transformation (EMT) in normal development that we believe is turned on abnormally in certain optic pathologies. This gene program may be turned on in part for motility (as in primary mesenchyme) or in whole to create true fibroblasts (as in secondary mesenchyme). The outline of the work is as follows. I.Epithelial-mesenchymal transformations contributing to normal eye development. The cephalic neural crest arises by epithelial-mesenchymal transformation from the cranial neural folds. We will use beta- galactosidase expressing, replication incompetent retroviruses to label cells to find out if one group of crest cells (more like secondary mesenchyme) unique to the mouse or chicken head gives rise to corneal fibroblasts and another (more like trunk crest) to corneal nerves and conjuntival melanocytes. Also, we will learn the mesenchyme of origin of the corneal endothelium. Using immunohistochemistry and in situ hybridization, we will look for expression of genes for molecules that might cause EMT: CAMs (cell adhesion molecules), SAMs (substrate adhesion molecules), protooncogenes. II.Tissue phenotype transitions by corneal fibroblasts and epithelia. We expect to be able to transform differentiated corneal fibroblasts to epithelioid cells by transfection with cDNAs for E-cadherin, alpha6beta1 integrin, and syndecan, and to transform differentiated corneal epithelial and endothelial cells to fibroblasts by transfection with c-src and c-mos DNAs, and antibodies to cadherins and syndecan or their antisense DNA. We will examine the transformed cells to assay whether parts or all of the tissue phenotype is turned on or off by a given gene. III.Epithelial-mesenchymal transformation from lens and cornea in collagen gels. We will examine by the retrovirus-label method the developmental potentials of the mesenchymal cells that form from lens and corneal epithelia in gels. Since they look like secondary mesenchyme, we expect they will form cartilage. CAMs, SAMs, and protooncogenes will be examined in the lens epithelium before and after it is induced to give rise to mesenchyme in collagen gels. We will use subtractive hybridization to search for the mesenchymal master gene(s) that is turned on during collagen-induced transformation of lens to fibroblasts and compare its sequence(s) with those of CAMs, SAMs, protooncogenes, Homeobox genes, growth factors, and other candidates to be master genes. IV. Epithelial-mesenchymal transformation in eye pathologies. We describe relevant CAMs, protooncogenes, and master genes in preinvasive and invasive conjunctival tumors, and metaplasias of retinal pigmented and nonpigmented epithelia. We investigate the interesting possibility that the gene program for metastasis is an abnormally reactivated embryonic program for epithelial-mesenchymal transformation.

这项拨款中提议的研究预计将有助于 了解眼组织分化的机制，特别是 重点是角膜和晶状体。我们描述了一个针对上皮细胞的基因程序- 间质转化(EMT)在正常发育中，我们认为是 在某些视神经病变中异常开启。这个基因程序可能 部分打开是为了运动(如在初级间充质中)或全部打开以 创造真正的成纤维细胞(如次级间充质细胞)。《纽约时报》的大纲 具体工作如下。 一、上皮-间充质转化对正常眼的影响 发展。由上皮间充质形成的头神经脊 从脑神经皱褶转化而来。我们将使用测试版- 表达半乳糖苷酶、复制能力不强的逆转录病毒标记 细胞，以找出是否有一组脊细胞(更像是次级细胞 间充质)小鼠或鸡头所特有的形成角膜 成纤维细胞和另一种(更像是树冠)到角膜神经和 结膜黑素细胞。此外，我们还将了解 角膜内皮。应用免疫组织化学和原位技术 杂交，我们将寻找分子的基因表达 可能导致EMT：CAM(细胞黏附分子)，SAM(底物黏附) 分子)、原癌基因。 二、角膜成纤维细胞和上皮细胞的表型转变。我们 有望将分化的角膜成纤维细胞转化为 E-钙粘附素、α6beta1基因转染上皮样细胞 整合素和Syndecan，并转化分化的角膜上皮 C-src和c-mos基因转导成纤维细胞 DNA，钙粘附素和Syndecan的抗体或它们的反义DNA。我们 将检查转化的细胞以检测是否部分或全部 组织表型的开启或关闭取决于给定的基因。 III.胶原蛋白中晶状体和角膜上皮-间充质转化 凝胶。我们将用逆转录病毒标记法检测发育 晶状体和角膜间充质细胞的潜能 凝胶中的上皮细胞。由于它们看起来像次生间充质，我们预计 它们会形成软骨。将检查CAM、SAM和原癌基因 在诱导前后晶状体上皮细胞中产生 胶原蛋白凝胶中的间质。我们将使用消减杂交技术来 寻找启动的间充质主控基因(S) 胶原蛋白诱导晶状体成纤维细胞转化及其比较 序列(S)与CAMS、SAM、原癌基因、同源盒基因、 生长因子等候选基因被掌握。 眼部病理上皮-间充质转化。我们描述了 侵袭前和侵袭中的相关CAM、原癌基因和主控基因 结膜肿瘤，视网膜色素变性和非色素变性 上皮细胞。我们调查了一种有趣的可能性，即 转移计划是一种异常重新激活的胚胎计划 上皮-间充质转化。