FIBROBLAST DIFFERENTIATION DURING EYE DEVELOPMENT

描述(改编自申请者的摘要)：该项目将 结缔组织特化过程中成纤维细胞分化的研究 组织，尤其专注于角质形成细胞的分化 胚胎祖细胞神经脊群的角膜基质 头部区域的细胞与其他区域成纤维细胞的分化 眼组织和非眼组织。角质形成细胞分化的标志物有 三种主要硫酸角蛋白多糖(KSPG)的合成能力 核心蛋白(鲁米肯、角质形成蛋白、KSPG 25或模拟蛋白)并糖基化它们 具有长的、高度硫酸盐化的角蛋白硫酸酯(KS)链。这样的“KSylation” 是角膜KSPG和神经脊/成纤维细胞的独特特征 分化为角质形成细胞。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 角膜基质细胞以及其他成纤维细胞和结缔组织细胞 在眼睛和头部区域；2)确定基因组DNA中的调控元件 控制角膜基质细胞中KSPGs的生物合成；3)鉴定 调节三个KSPG核心的糖基化和硫酸化的因素 角膜和其他组织中的蛋白质；4)表达的特征 角膜发育过程中KSPG mRNAs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的；5) 确定角膜神经的来源--神经脊与外胚层 6)确定对角膜发育和神经支配的影响 扰乱1个、2个或全部3个角膜KSPG的生物合成；以及7) 确定KSPG受体是否存在于角膜细胞类型上，如 在巨噬细胞上显示。分子生物学方法将应用于 成年牛眼睛的组织，以及鹌鹑和鸡的组织 胚胎。蛋白质多糖、糖和蛋白质的标准生物化学方法 硫酸盐离子将被用来作为KSPG的敏感标记 角质形成细胞分化。尽管移植的角膜一般会保留下来 移植物的透明神经再支配非常缓慢(数年)且不完整， 剥夺患者的角膜触觉敏感度。建议的研究可能 阐明KSPG分布与所选择的途径之间的关系 角膜神经主要来自三叉神经节。

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.