FIBROBLAST DIFFERENTIATION DURING EYE DEVELOPMENT

神经嵴分化的最独特的产物， 角膜是硫酸角质素蛋白聚糖（KSPG）。 这个细胞外的， 硫酸化的N-连接糖蛋白在角膜中比在 任何其他组织。 虽然KS的良好化学表征 多糖已经进行了，很少有人知道的核心 KSPG蛋白 一些研究表明，多种形式的角膜 KSPG。 我们的初步数据表明， 与KSPG共价或非共价缔合。 合成 KS多糖通过分离的、分化的角膜基质细胞 在体外，角膜细胞（keratocytes）的增殖仅发生很短的时间。 在角膜伤口愈合过程中， 检测到瘢痕中的KS。 在正常的角膜发育过程中， 禽胚胎神经嵴细胞不表达成熟的 KS直到细胞侵入原代角膜基质后。 角膜神经在细胞外KS分泌后不久侵入基质。 检测到 在这种背景下，我们建议测试三个 理论 假设1：有两类KSPG： 存在于包括角膜在内的大多数组织中的“组成型”形式，以及 一种“组织特异性”形式; KSPG结合蛋白与 两种形式。 将分离鸡角膜KSPG核心蛋白，10- 将确定每个N-末端序列的20个残基， 将合成复制这些序列的肽 化学上，将制备针对每一种的多克隆抗体， 肽，并且抗原的表达将定位在 正在发育小鸡的眼睛和其他组织 假设2：角膜- 特异性KSPG合成受细胞外 环境：1）来自其他角膜细胞类型的可扩散因子 2）基质的不溶性成分， 细胞外基质，或3）物理力和/或脱水 基质。 抗体将用于确定是否或何时 KSPG核心蛋白抗原在体外停止表达。 一 用于神经嵴分化的器官培养系统将允许 角膜细胞类型的实验性改变和 细胞外基质 另一个器官培养系统将检测 在改变的基质中角膜细胞分化的稳定性， 测试恒定拉伸应力对KSPG合成的影响。 假设3：神经侵入角膜基质， 胚胎发生对KSPG细胞外沉积的反应 基质。 N-连接糖基化和加工的抑制剂 将用于干扰KSPG的合成，以及对角膜的影响。 将记录神经支配。

The most distinctive product of neural crest differentiation in the cornea is keratan sulfate proteoglycan (KSPG). This extracellular, sulfated, N-linked glycoprotein is more abundant in cornea than in any other tissue. Although good chemical characterization of KS polysaccharide has been performed, little is known about the core proteins of KSPG. Some studies suggest multiple forms of corneal KSPG. Our preliminary data suggest as many as 5 proteins associated covalently or non-covalently with KSPG. Synthesis of KS polysaccharide by isolated, differentiated corneal stroma cells (keratocytes) in vitro occurs for only short periods of time. During corneal wound healing, synthesis of antigenically altered KS in the scar is detected. During normal corneal development in avian embryos, neural crest cells do not express antigens of mature KS until after the cells invade the primary corneal stromal. Corneal nerves invade the stroma shortly after extracellular KS is detected. Given this background, we propose to test three theories. Hypothesis 1: There are two classes of KSPG: a "constitutive" form present in most tissues including cornea, and a "tissue-specific" form; KSPG-binding proteins are associated with both forms. Chick corneal KSPG core proteins will be isolated, 10- 20 residues of each N-terminal sequence will be determined, peptides duplicating these sequences will be synthesized chemically, polyclonal antibodies will be prepared against each peptide, and expression of the antigens will be localized in developing chick eyes and other tissues. Hypothesis 2: Cornea- specific KSPG synthesis is regulated by the extracellular environment: 1) diffusible factors from other corneal cell-types or aqueous humor, 2) insoluble components of the stromal extracellular matrix, or 3) physical forces and/or dehydration of the stroma. Antibodies will be used to determine whether or when KSPG core protein antigens stop being expressed in vitro. One organ culture system for neural crest differentiation will allow experimental alteration of corneal cell-types and perturbation of extracellular matrix. Another organ culture system will assay stability of keratocyte differentiation within altered matrices and test the effect of constant tensile stress on KSPG synthesis. Hypothesis 3: Nerves invade the corneal stroma during embryogenesis in response to extracellular deposition of KSPG in the stroma. Inhibitors of N-linked glycosylation and processing will be used to perturb synthesis of KSPG, and effects on corneal innervation will be recorded.