FIBROBLAST DIFFERENTIATION DURING EYE DEVELOPMENT

描述（改编自申请人的摘要）：该项目将 结缔组织特化过程中成纤维细胞分化研究 组织，特别是集中在角膜细胞的分化， 来自胚胎祖细胞神经嵴群的角膜基质 细胞在头部区域，与分化成纤维细胞在其他 眼和非眼组织。 角膜细胞分化的标志物是 合成三种主要硫酸角质素蛋白聚糖（KSPG）的能力 核心蛋白（Lumican、keratocan、KSPG 25或mimican）并将其糖基化 具有长的高度硫酸化的硫酸角质素（KS）链。 这种“KSyylation” 是角膜KSPG和神经嵴/成纤维细胞的独特特征 分化成角膜细胞。 具体目标是：1）确定是否 选择性剪接发生在3个KSPG核心蛋白的生物合成过程中 在角膜角膜细胞和其他成纤维细胞和结缔组织细胞中 在眼睛和头部区域; 2）鉴定基因组DNA中的调控元件 其控制角膜基质细胞中KSPG的生物合成; 3）鉴定 调节三个KSPG核心糖基化和硫酸化的因子 角膜和其他组织中的蛋白质; 4）表征表达 角膜发育过程中KSPG mRNA和核心蛋白的模式， 特别是与角膜神经支配的时间和模式有关; 5） 确定角膜神经的起源-神经嵴与外胚层 基板; 6）确定角膜发育和神经支配的影响， 干扰1种、2种或所有3种角膜KSPG的生物合成;和7） 确定KSPG受体是否存在于角膜细胞类型上， 在巨噬细胞上显示。 分子生物学方法将应用于 成年牛眼组织，以及鹌鹑和鸡眼组织 胚胎 蛋白聚糖、糖和蛋白质的标准生物化学方法 硫酸根离子将被用来集中在KSPG作为敏感的标志物， 角膜细胞分化 虽然移植的角膜通常 移植物的透明神经再支配非常缓慢（数年）且不完全， 剥夺了患者的角膜触觉敏感性。 研究建议可 阐明KSPG分布与通过以下途径选择的途径之间的关系： 主要来自三叉神经节的角膜神经。

DESCRIPTION (adapted from the applicant's abstract): This project will study fibroblast differentiation during specialization of connective tissues, concentrating especially on differentiation of keratocytes of the corneal stroma from the neural crest population of embryonic progenitor cells in the head region, versus differentiation of fibroblasts in other ocular and non-ocular tissues. Markers of keratocyte differentiation are ability to synthesize the three major keratan sulfate proteoglycan (KSPG) core proteins (lumican, keratocan, KSPG 25 or mimican) and glycosylate them with long, highly sulfated chains of keratan sulfate (KS). Such "KSylation" is a unique feature of corneal KSPGs and of neural crest/fibroblast differentiation into keratocytes. Specific Aims are to: 1) Determine if alternative splicing occurs during biosynthesis of the 3 KSPG core proteins in corneal keratocytes and in other fibroblastic and connective tissue cells in the eye and head regions; 2) Identify regulatory elements in genomic DNA that control biosynthesis of KSPGs in corneal keratocytes; 3) Identify factors that regulate glycosylation and sulfation of the three KSPG core proteins in the cornea and other tissues; 4) Characterize the expression patterns of the KSPG mRNAs and core proteins during corneal development, particularly in relation to timing and pattern of corneal innervation; 5) Determine the origin of corneal nerves - neural crest vs. ectodermal placode; 6) Determine the effect on corneal development and innervation of perturbing the biosynthesis of 1, 2, or all 3 corneal KSPGs; and 7) Determine if receptors for KSPGs occur on corneal cell types, as demonstrated on macrophages. Molecular biology methods will be applied to tissues from adult bovine eyes, as well as from those of quail and chicken embryos. Standard biochemical methods for proteoglycans, sugars, and sulfate ions will be used to focus on KSPGs as sensitive markers of keratocyte differentiation. Although transplanted corneas generally remain transparent re-innervation of the graft is very slow (years) and incomplete, depriving patients of corneal touch sensitivity. Research proposed may elucidate a relationship between KSPG distribution and pathways chosen by corneal nerves that mainly arise from the trigeminal ganglion.