Fibroblast Differentiation During Eye Development

描述(申请人提供)：神经脊来源的、间充质的、成纤维细胞的细胞群在眼睛中分化为不同的细胞类型，包括角膜神经、角膜内皮、基质角质细胞、非髓鞘和有髓雪旺细胞、角膜缘结缔组织、结膜、脉络膜、巩膜、眼睑、虹膜、泪腺和其他腺体，鸟类的巩膜听骨。每个组织形成一种独特的细胞外基质(ECM)，随着发育年龄的变化而变化。免疫细胞化学、原位杂交、质谱学、表面等离子共振光谱、击倒和错误表达分析、构建鸡-鹌鹑嵌合胚胎的能力，以及激光辅助显微切割(LMD)现在可以检查和操纵眼睛不同部位的小群细胞的分化，以更好地评估调控细胞和神经生长锥迁移和分化的信号。假设：每个部位特有的高电荷ECM，结合和释放生长、分化、神经营养和神经营养因子，决定角膜的透明度和神经支配，以及其他组织的正常功能。目的1：测定胚胎眼关键部位的碳水化合物结构和结合因子。质谱仪将鉴定和定量存在于ECM中的特定糖胺多聚糖及其结构的细节，包括硫酸盐基团的结构位置和唾液酸类型的鉴定。目的：探讨三叉神经节神经元生长锥在角膜感觉神经支配中的作用机制。目的3：确定发育过程中角膜基质非髓鞘雪旺细胞的正常谱系来源；鉴定这些细胞表达的标志蛋白及其对其分化的影响；干扰细胞外基质的合成。意义：LASEK、LASIK、PRK或移植后人类角膜愈合和重新神经支配的程度由参与的ECM中的信号决定，特别是在它们的翻译后修饰中。虽然移植的角膜通常保持透明，但移植物的再神经支配非常缓慢(数年)，而且往往是不完整的，使患者丧失了角膜触摸敏感性和眨眼反射。这项研究将阐明蛋白多糖的形式和分布，以及在胚胎角膜神经支配过程中，角膜神经和非髓鞘雪旺细胞选择的路径之间的关系。这些数据将有助于设计策略，以更好地重新支配移植或改良的成人角膜。

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.