Department of Biology, Ashland University, Ashland, Ohio, United States of America.
Center for Bioinformatics & Functional Genomics, Miami University, Oxford, Ohio, United States of America.
PLoS One. 2019 Mar 12;14(3):e0211399. doi: 10.1371/journal.pone.0211399. eCollection 2019.
The zebrafish has become a valuable model for examining ocular lens development, physiology and disease. The zebrafish cloche mutant, first described for its loss of hematopoiesis, also shows reduced eye and lens size, interruption in lens cell differentiation and a cataract likely caused by abnormal protein aggregation. To facilitate the use of the cloche mutant for studies on cataract development and prevention we characterized variation in the lens phenotype, quantified changes in gene expression by qRT-PCR and RNA-Seq and compared the ability of two promoters to drive expression of introduced proteins into the cloche lens. We found that the severity of cloche embryo lens cataract varied, while the decrease in lens diameter and retention of nuclei in differentiating lens fiber cells was constant. We found very low expression of both αB-crystallin genes (cryaba and cryabb) at 4 days post fertilization (dpf) by both qRT-PCR and RNA-Seq in cloche, cloche sibling and wildtype embryos and no significant difference in αA-crystallin (cryaa) expression. RNA-Seq analysis of 4 dpf embryos identified transcripts from 25,281 genes, with 1,329 showing statistically significantly different expression between cloche and wildtype samples. Downregulation of eight lens β- and γM-crystallin genes and 22 retinal related genes may reflect a general reduction in eye development and growth. Six stress response genes were upregulated. We did not find misregulation of any known components of lens development gene regulatory networks. These results suggest that the cloche lens cataract is not caused by loss of αA-crystallin or changes to lens gene regulatory networks. Instead, we propose that the cataract results from general physiological stress related to loss of hematopoiesis. Our finding that the zebrafish αA-crystallin promoter drove strong GFP expression in the cloche lens demonstrates its use as a tool for examining the effects of introduced proteins on lens crystallin aggregation and cataract prevention.
斑马鱼已成为研究眼晶状体发育、生理学和疾病的一种有价值的模型。斑马鱼 cloche 突变体最初因其造血功能丧失而被描述，也表现出眼睛和晶状体尺寸减小、晶状体细胞分化中断以及白内障，可能是由异常蛋白质聚集引起的。为了促进 cloche 突变体在白内障发育和预防研究中的应用，我们对晶状体表型的变化进行了表征，通过 qRT-PCR 和 RNA-Seq 定量了基因表达的变化，并比较了两种启动子将引入的蛋白质驱动到 cloche 晶状体中的表达能力。我们发现，cloche 胚胎晶状体白内障的严重程度不同，而晶状体直径的减小和分化的晶状体纤维细胞中核的保留是恒定的。我们发现，在 cloche、cloche 同窝仔鱼和野生型胚胎中，通过 qRT-PCR 和 RNA-Seq 在受精后 4 天（dpf）时都非常低表达两种αB-晶状体蛋白（cryaba 和 cryabb），而αA-晶状体蛋白（cryaa）的表达没有显著差异。对 4 dpf 胚胎的 RNA-Seq 分析鉴定了 25281 个基因的转录本，其中 25281 个基因的转录本在 cloche 和野生型样本之间的表达有统计学显著差异。八个晶状体β-和γM-晶状体蛋白基因和 22 个视网膜相关基因的下调可能反映了眼睛发育和生长的普遍减少。六个应激反应基因上调。我们没有发现任何已知的晶状体发育基因调控网络组件的失调。这些结果表明，cloche 晶状体白内障不是由αA-晶状体蛋白的缺失或晶状体基因调控网络的变化引起的。相反，我们认为白内障是由造血功能丧失引起的一般生理应激引起的。我们发现，斑马鱼αA-晶状体蛋白启动子在 cloche 晶状体中驱动 GFP 的强烈表达，证明其可用作研究引入的蛋白质对晶状体结晶蛋白聚集和白内障预防的影响的工具。
