Abstract：
Myocilin is an enigmatic glaucoma-associated glycoprotein whose biological role remains incompletely understood. To gain novel insight into its normal function, we used transposon-mediated transgenesis to generate the first zebrafish line stably overexpressing myocilin [Tg(actb1:myoc-2A-mCherry)]. qPCR showed an approximately four-fold increased myocilin expression in transgenic zebrafish embryos (144 hpf). Adult (13 months old) transgenic animals displayed variable and age-dependent ocular anterior segment alterations. Almost 60% of two-year-old male, but not female, transgenic zebrafish developed enlarged eyes with severe asymmetrical and variable abnormalities in the anterior segment, characterized by corneal limbus hypertrophy, and thickening of the cornea, iris, annular ligament and lens capsule. The most severe phenotype presented small or absent ocular anterior chamber and pupils, due to iris overgrowth along with dysplastic retinal growth and optic nerve hypertrophy. Immunohistochemistry revealed increased presence of myocilin in most altered ocular tissues of adult transgenic animals, as well as signs of retinal gliosis and expanded ganglion cells and nerve fibers. The preliminary results indicate that these cells contributed to retinal dysplasia. Visual impairment was demonstrated in all old male transgenic zebrafish. Transcriptomic analysis of the abnormal transgenic eyes identified disrupted expression of genes involved in lens, muscular and extracellular matrix activities, among other processes. In summary, the developed transgenic zebrafish provides a new tool to investigate this puzzling protein and provides evidence for the role of zebrafish myocilin in ocular anterior segment and retinal biology, through the influence of extracellular matrix organization and cellular proliferation.
摘要：
Myocilin是一种神秘的青光眼相关糖蛋白，其生物学作用尚不完全清楚。为了获得对其正常功能的新颖见解，我们使用转座子介导的转基因来产生第一个稳定过表达myocilin的斑马鱼细胞系[Tg（actb1：myoc-2A-mCherry）]。qPCR显示转基因斑马鱼胚胎中的肌蛋白表达增加了约四倍(144hpf)。成年（13个月大）转基因动物表现出可变的和年龄依赖性的眼前节改变。几乎60%的两岁男性,但不是女性，转基因斑马鱼在眼前段出现严重的不对称和可变异常，以角膜缘肥大为特征，角膜增厚，虹膜,环状韧带和晶状体囊。最严重的表型表现为前房和瞳孔小或缺失，由于虹膜过度生长以及视网膜发育不良和视神经肥大。免疫组织化学显示，成年转基因动物的大多数改变的眼部组织中肌蛋白的存在增加，以及视网膜胶质增生和神经节细胞和神经纤维扩张的迹象。初步结果表明，这些细胞有助于视网膜发育不良。所有老年雄性转基因斑马鱼均表现出视力障碍。对异常转基因眼的转录组学分析发现晶状体中涉及的基因表达被破坏，肌肉和细胞外基质活性，在其他过程中。总之,开发的转基因斑马鱼为研究这种令人费解的蛋白质提供了新的工具，并为斑马鱼肌蛋白在眼前节和视网膜生物学中的作用提供了证据，通过影响细胞外基质组织和细胞增殖。
