FIBROBLAST DIFFERENTIATION DURING EYE DEVELOPMENT

长期目标：查明造成性别差异的因素 眼内成纤维细胞的组织特异性群体，重点是 角膜 透明的角膜很特别，因为它含有 硫酸角质素蛋白聚糖（KSPG）的最高浓度 组织和任何身体表面的最高密度的神经末梢 地区 具体目的和方法：1）确定角膜特异性 (and KSPG核心蛋白的共有的）同种型被合成。 他们可能 由选择性RNA剪接或由独立但相关的基因引起。 对角膜polyA+RNA的分析将消除其中一种假设。 2)通过实验确定每种KSPG亚型在角膜移植中的作用。 发展 新鲜分离的鹌鹑颅神经嵴首先将 用携带一种KSPG同种型序列的质粒转染， 反向（反义）方向，然后将用于原位 正时单侧移植到鸡胚中。 嵌合角膜 改变了透明度或神经支配， KSPG。 3)表征调节每种角膜KSPG合成的因素 同种型核心蛋白 将添加生长因子和眼周细胞 体外培养神经嵴和角膜基质细胞，检测KSPG的合成 同种型特异性mRNA和蛋白质。 4)确定之间的关系 KSPG的分子特征和角膜神经的发育。 具体 KS糖胺聚糖链高度或低度的KSPG亚型 硫酸化将用作底物，以测试对神经嵴的影响 分化成纤维细胞或神经，并测试神经突生长 来自三叉神经嵴和/或外胚层基板衍生的细胞 神经节（角膜神经源）。 5)表征推定的受体 KSPG对角膜细胞类型的影响。 角膜细胞表面蛋白质将 允许与KSPG结合（可逆），溶解，表征（天然 和肽N-末端测序），并用于制备抗 全蛋白和/或肽。 使用寡核苷酸探针， 受体氨基酸序列，这些受体蛋白质的cDNA将 并在用反义核酸干扰的嵌合角膜中表达 probes. 健康相关性：刺激角膜细胞的因素 特异性KSPG亚型的合成或使基质基质更多 神经突生长锥允许/穿透可能加速神经再支配 移植的人角膜，目前需要12-18个月的过程。

Long-term objectives: Identify factors causing differentiation of tissue-specific populations of fibroblasts in the eye, with emphasis on the cornea. The transparent cornea is remarkable because it contains the highest concentration of keratan sulfate proteoglycan (KSPG) of any tissue and the highest density of nerve endings of any body surface region. Specific Aims and Methods: 1) Determine how cornea-specific (and shared) isoforms of KSPG core proteins are synthesized. They may arise from alternative RNA splicing or from separate but related genes. Analyses of corneal polyA+RNA will eliminate one of these hypotheses. 2) Experimentally determine roles of each KSPG isoform during corneal development. Freshly isolated quail cranial neural crest first will be transfected with a plasmid carrying sequence for one KSPG isoform in reverse (antisense) orientation and then will be used for orthotopic orthochronic unilateral transplants into chick embryos. Chimeric corneas with altered transparency or innervation will provide data on roles of KSPGs. 3) Characterize factors regulating synthesis of each corneal KSPG isoform core protein. Growth factors and periocular cells will be added to neural crest and keratocytes in vitro to test for synthesis of KSPG isoform-specific mRNAs and proteins. 4) Determine relationships between molecular features of KSPG and development of corneal nerves. Specific KSPG isoforms of high or low degree of KS glycosaminoglycan chain sulfation will be used as substrates to test for effects on neural crest differentiation into fibroblasts or nerves, and to test neurite outgrowth from crest- and/or ectodermal placode-derived cells of the trigeminal ganglion (corneal nerve source). 5) Characterize putative receptors for KSPG on corneal cell types. Corneal cell surface proteins will be allowed to bind (reversibly) to KSPG, solubilized, characterized (native and peptide N-terminal sequencing), and used to make antibody against the whole protein and/or peptides. Using oligonucleotide probes based on receptor amino acid sequence, the cDNAs for these receptor proteins will be isolated, and expression in chimeric corneas perturbed with antisense probes. Health relevance: Factors that would stimulate keratocyte synthesis of specific KSPG isoforms or would make the stromal matrix more permissive/penetrable by neurite growth cones might hasten reinnervation of transplanted human corneas, a process currently requiring 12-18 mos.