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.

预计该赠款中提出的研究将有助于 了解眼组织分化的机制，特别是 强调角膜和镜头。 我们描述了上皮的基因程序 我们认为是正常发展中的间充质转化（EMT） 在某些视力病理中异常打开。 这个基因程序可能 部分打开运动性（如主间充质）或从总体上打开 创建真实的成纤维细胞（如次级间质）。 大纲 工作如下。 i.上皮 - 间质转化，导致正常眼 发展。 头皮神经rest由上皮 - 间质产生 从颅神经褶皱转变。 我们将使用beta- 半乳糖苷酶表达，复制无能力的逆转录病毒标记 细胞找出是否一组rest细胞（更像二级 鼠标或鸡头独有的间质）产生角膜 成纤维细胞和另一个（更像是躯干波峰）到角膜神经， 共同细胞。 另外，我们将学习 角膜内皮。 使用免疫组织化学和原位 杂交，我们将寻找分子的基因表达 可能导致EMT：CAM（细胞粘附分子），SAM（底物粘附） 分子），原子基因。 II.角膜成纤维细胞和上皮的细胞表型过渡。 我们 希望能够将分化的角膜成纤维细胞转变为 通过用cDNA转染E-钙粘着蛋白，α6Beta1，上皮细胞通过转染细胞 整联蛋白和syndecan，并改变分化的角膜上皮 和内皮细胞通过C-SRC和C-MOS转染成纤维细胞 DNA，以及对钙粘蛋白和联合can或其反义DNA的抗体。 我们 将检查转化的细胞以测定零件或所有部分 通过给定基因打开或关闭组织表型。 III.上皮 - 间质转化，从胶原蛋白的镜头和角膜 凝胶。 我们将通过逆转录病毒标签法检查发育 由透镜和角膜形成的间充质细胞的电势 凝胶中的上皮。 由于它们看起来像次要间充质，我们希望 它们将形成软骨。 将检查凸轮，SAM和原子基因 在诱导其前后的镜头上皮中产生 胶原蛋白凝胶中的间质。 我们将使用减法杂交 搜索在期间打开的间充质大师基因 胶原蛋白引起的镜头转化为成纤维细胞并比较其 序列与CAM，SAMS，原子基因，同源基因基因的序列 生长因素和其他候选者是主要基因。 iv。眼病理中的上皮间质转化。 我们描述 相关的凸轮，原子基因和侵入性摄入剂中的基因 视网膜色素和未斑点的结膜肿瘤和变质的变质肿瘤 上皮。 我们研究了基因的有趣可能性 转移程序是一个异常重新激活的胚胎程序 上皮间质转化。