Fibroblast Differentiation During Eye Development

描述（由申请人提供）：神经嵴衍生的间充质成纤维细胞群在眼睛中分化成不同的细胞类型，包括角膜神经、角膜内皮、基质角膜细胞、非髓鞘形成和髓鞘形成施旺细胞、利姆布斯、结膜、脉络膜、巩膜、眼睑、虹膜、泪腺和其他腺体的结缔组织，在鸟类中分化成巩膜小骨。每个组织形成独特的细胞外基质（ECM），随着发育年龄的变化。免疫细胞化学、原位杂交、质谱法、表面等离子体共振光谱法、敲低和错误表达分析、构建鸡-鹌鹑嵌合胚胎的能力以及激光辅助显微切割（LMD）现在允许检查和操纵眼睛不同部分中的小细胞群中的分化，以更好地评估调节细胞和神经生长锥迁移和分化的信号。假设：每个部位特有的高度带电的ECM结合并释放决定角膜透明度和神经支配以及其他组织的正常功能的生长、分化、亲神经性和神经营养性因子。目的1：确定胚胎眼关键部位的糖结构和结合因子。质谱法将鉴定和定量ECM中存在的特定糖胺聚糖及其结构的细节，包括硫酸基团的结构位置和唾液酸类型的鉴定。目的2：利用三叉神经节神经嵴源性生长锥，探讨角膜感觉神经支配的各个环节的机制。目标3：确定正常谱系来源的非髓鞘形成的雪旺细胞的角膜基质在发展过程中，确定这些细胞表达的标志物蛋白质和干扰ECM合成对他们的分化的影响。重要性：LASEK、LASIK、角膜移植或移植后人角膜愈合和神经再支配的程度由参与ECM的信号决定，特别是在其翻译后修饰中。虽然移植的角膜通常保持透明，但移植物的神经再支配非常缓慢（数年）且通常不完全，剥夺了患者的角膜触觉敏感性和眨眼反射。本文提出的研究将阐明蛋白多糖的形式和分布之间的关系，以及胚胎角膜神经支配过程中角膜神经和无髓鞘雪旺细胞选择的路径。这些数据将有助于设计策略，以更好地再神经支配移植或修改成人角膜。

DESCRIPTION (provided by applicant): Neural crest-derived, mesenchymal, fibroblastic cell populations differentiate into distinct cell-types in the eye, including corneal nerves, corneal endothelium, stromal keratocytes, non-myelinating and myelinating Schwann cells, connective tissues of limbus, conjunctiva, choroid, sclera, eyelids, iris, lacrimal and other glands, and in birds, scleral ossicles. Each tissue forms a unique extracellular matrix (ECM) that changes with developmental age. Immunocytochemistry, in situ hybridization, mass spectrometry, surface plasmon resonance spectrometry, knock-down and mis-expression analysis, ability to construct chick-quail chimeric embryos, and laser- assisted microdissection (LMD) now allow examination and manipulation of differentiation in small groups of cells in different parts of the eye to better assess signals regulating cell and nerve growth cone migrations and differentiation. Hypothesis: highly charged ECMs unique to each site, bind and release growth-, differentiation-, neurotropic-, and neurotrophic-factors that determine corneal transparency and innervation, and normal functions of other tissues. Aim 1: Determine carbohydrate structures and bound factors in key locations of embryonic eyes. Mass spectrometry will identify and quantitate specific glycosaminoglycans present in ECMs and details of their structures, including structural positions of sulfate groups and identification of sialic acid types. Aim 2: Determine the mechanism of each step in corneal sensory innervation by neural crest-derived growth cones of trigeminal ganglion neurons. Aim 3: Determine the normal lineage sources of non-myelinating Schwann cells of corneal stroma during development; identify marker proteins expressed by these cells and effects on their differentiation from perturbing ECM synthesis. Significance: Extent to which human corneas heal and become re-innervated after LASEK, LASIK, PRK or transplantation is determined by signals residing in participating ECMs, particularly in their posttranslational modifications. Although transplanted corneas generally remain transparent, re-innervation of the graft is very slow (years) and often incomplete, depriving patients of corneal touch sensitivity and blink reflex. Research proposed here will elucidate relationships between proteoglycan forms and distributions, and paths chosen by corneal nerves and non-myelinating Schwann cells during innervation of embryonic corneas. These data will facilitate designing strategies to better reinnervate transplanted or modified adult corneas.