Mutation of the EPHA2 tyrosine-kinase domain dysregulates cell pattern formation and cytoskeletal gene expression in the lens

Genetic variations in ephrin type-A receptor 2 (EPHA2) have been associated with inherited and age-related forms of cataract in humans. Here we have characterized the eye lens phenotype and transcript profile of germline Epha2 knock-in mutant mice homozygous for either a missense variant associated with age-related cataract in humans (Epha2-Q722) or a novel insertion-deletion mutation (Epha2-indel722) that were both located within the tyrosine-kinase domain of EPHA2. Whole-mount confocal imaging of clear lenses from Epha2-indel722 mice on a fluorescent reporter background revealed misalignment of epithelial-to-fiber cell meridional-rows at the lens equator and severe disturbance of Y-suture formation at the lens poles, whereas, Epha2-Q722 lenses displayed mild disturbance of posterior sutures. Immunofluorescent labeling showed that EPHA2 was mostly localized to lens fiber cell membranes with some sub-membrane localization observed in Epha2-Q722 lenses and diffuse membrane and perinuclear localization in Epha2-indel722 lenses. Immunoprecipitation/blotting studies indicated that EPHA2 formed strong complexes with Src kinase but not with catenin beta 1 or cadherin 2 and was mostly serine phosphorylated in the lens. RNA-sequencing analysis revealed differential expression of several cytoskeleton-associated genes in Epha2-mutant and Epha2-null lenses including strong downregulation of Lgsn and Clic5. Collectively, our data suggest that mutations within the tyrosine-kinase domain of EPHA2 result in lens cell patterning defects and dysregulated expression of several cytoskeletal proteins. mRNA profiles from seven day old lenses of wild type (WT), EPHA2 null, and EPHA2 mutant mice人类促红细胞生成素产生肝细胞受体A类2（ephrin type-A receptor 2, EPHA2）的遗传变异，与人类的遗传性及年龄相关性白内障密切相关。本研究针对两种生殖系Epha2敲入纯合突变小鼠的晶状体表型与转录组特征展开表征：两种突变分别为与人类年龄相关性白内障相关的错义变异（Epha2-Q722），以及一种全新的插入缺失突变（Epha2-indel722），二者均位于EPHA2的酪氨酸激酶结构域内。对携带荧光报告基因背景的Epha2-indel722小鼠透明晶状体进行全标本共聚焦成像，结果显示晶状体赤道部的上皮-纤维细胞子午线排列出现紊乱，晶状体极部的Y型缝形成受到严重干扰；而Epha2-Q722小鼠的晶状体仅表现出后部缝线的轻度紊乱。免疫荧光标记结果显示，EPHA2主要定位于晶状体纤维细胞膜；在Epha2-Q722小鼠晶状体中可见少量膜下定位信号，而在Epha2-indel722小鼠晶状体中则呈现弥散性膜定位与核周定位。免疫沉淀-免疫印迹实验结果表明，EPHA2可与Src激酶（Src kinase）形成稳定复合物，但无法与β-连环蛋白（catenin beta 1）或钙粘蛋白2（cadherin 2）结合，且在晶状体中主要发生丝氨酸磷酸化修饰。RNA测序分析显示，相较于野生型小鼠，Epha2突变型与Epha2敲除型晶状体中多个细胞骨架相关基因的表达存在显著差异，其中Lgsn与Clic5的表达显著下调。综合以上实验数据，本研究表明EPHA2酪氨酸激酶结构域内的突变可导致晶状体细胞排列模式异常，并使多种细胞骨架蛋白的表达失调。本研究采集了7日龄野生型（WT）、EPHA2敲除型及EPHA2突变型小鼠的晶状体mRNA表达谱。