Eyes shut homolog is important for the maintenance of photoreceptor morphology and visual function in zebrafish

Mutations in eyes shut homolog (EYS), a gene predominantly expressed in the photoreceptor cells of the retina, are among the most frequent causes of autosomal recessive (ar) retinitis pigmentosa (RP), a progressive retinal disorder. Due to the absence of EYS in several rodent species and its retina-specific expression, still little is known about the exact function of EYS and the pathogenic mechanism underlying EYS-associated RP. We characterized eys in zebrafish, by RT-PCR analysis on zebrafish eye-derived RNA, which led to the identification of a 8,715 nucleotide coding sequence that is divided over 46 exons. The transcript is predicted to encode a 2,905-aa protein that contains 39 EGF-like domains and five laminin A G-like domains, which overall shows 33% identity with human EYS. To study the function of EYS, we generated a stable eysrmc101/rmc101 mutant zebrafish model using CRISPR/Cas9 technology. The introduced lesion is predicted to result in premature termination of protein synthesis and lead to loss of Eys function. Immunohistochemistry on retinal sections revealed that Eys localizes at the region of the connecting cilium and that both rhodopsin and cone transducin are mislocalized in the absence of Eys. Electroretinogram recordings showed diminished b-wave amplitudes in eysrmc101/rmc101 zebrafish (5 dpf) compared to age- and strain-matched wild-type larvae. In addition, decreased locomotor activity in response to light stimuli was observed in eys mutant larvae. Altogether, our study shows that absence of Eys leads to a disorganized retinal architecture and causes visual dysfunction in zebrafish.

眼闭合同源基因（eyes shut homolog, EYS）是一种主要在视网膜感光细胞中表达的基因，该基因的突变是常染色体隐性（autosomal recessive, ar）色素性视网膜炎（retinitis pigmentosa, RP）——一种进行性视网膜疾病——最常见的致病原因之一。由于多种啮齿类动物体内不存在EYS基因，且该基因仅在视网膜中特异性表达，目前学界对EYS的确切功能及其相关RP的致病机制仍知之甚少。本研究通过对斑马鱼眼部来源RNA进行逆转录聚合酶链反应（reverse transcription PCR, RT-PCR）分析，对斑马鱼体内的eys基因进行了功能鉴定，最终获得了一段总长8,715个核苷酸的编码序列，该序列共分为46个外显子。该转录本预计可编码一段含2,905个氨基酸的蛋白质，该蛋白包含39个表皮生长因子样结构域（EGF-like domain）和5个层粘连蛋白A G样结构域，整体与人类EYS的序列同源性达33%。为研究EYS的功能，本研究利用CRISPR/Cas9技术构建了稳定的eysrmc101/rmc101突变斑马鱼模型，该引入的突变位点预计会导致蛋白质合成提前终止，进而使Eys蛋白功能丧失。对视网膜切片进行免疫组织化学检测后发现，Eys蛋白定位于连接纤毛区域；在缺失Eys蛋白的情况下，视紫红质与视锥转导蛋白均出现定位异常。视网膜电图（electroretinogram, ERG）记录结果显示，与年龄和品系匹配的野生型幼鱼相比，eysrmc101/rmc101突变斑马鱼（5日龄，dpf）的b波振幅显著降低。此外，研究还观察到eys突变斑马鱼幼鱼对光刺激的运动活性显著降低。综上，本研究证实，斑马鱼体内Eys蛋白的缺失会导致视网膜结构紊乱，并引发视觉功能障碍。