Área de Genética, Facultad de Medicina de Albacete, Universidad de Castilla-La Mancha, 02006 Albacete, Spain.
Instituto de Investigación en Discapacidades Neurológicas (IDINE), Universidad de Castilla-La Mancha, 02006 Albacete, Spain.
Int J Mol Sci. 2022 Sep 1;23(17):9989. doi: 10.3390/ijms23179989.
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.
肌球蛋白是一种神秘的与青光眼相关的糖蛋白，其生物学功能仍不完全清楚。为了深入了解其正常功能，我们使用转座子介导的转基因技术，生成了第一条稳定过表达肌球蛋白的斑马鱼系[Tg(actb1:myoc-2A-mCherry)]。qPCR 显示转基因斑马鱼胚胎（144 hpf）中的肌球蛋白表达增加了约四倍。成年（13 个月大）转基因动物表现出可变的和年龄依赖性的前眼部改变。近 60%的两岁雄性，但不是雌性，转基因斑马鱼出现了眼睛增大，前眼部有严重的不对称和可变异常，特征为角膜缘肥大，以及角膜、虹膜、环状韧带和晶状体囊增厚。最严重的表型表现为小或不存在的眼前房和瞳孔，这是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存在更多的肌球蛋白，以及视网膜胶质增生和扩大的节细胞和神经纤维的迹象。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都表现出视力障碍。异常转基因眼的转录组分析确定了参与晶状体、肌肉和细胞外基质活动等过程的基因表达失调。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一个新工具，并为斑马鱼肌球蛋白在眼部前节和视网膜生物学中的作用提供了证据，通过细胞外基质组织和细胞增殖的影响。
