Retinal degeneration in rpgra mutant zebrafish

Frontiers in Cell and Developmental Biology

Cell and Developmental Biology

Jingzhen Li¹  Pan Gao¹  Yuwen Huang¹  Xiang Chen¹  Mugen Liu¹  Zhaohui Tang¹  Danna Jia¹  Shanshan Yu²  Yayun Qin³  Xiliang Liu⁴  Fei Liu⁵  Shanshan Han⁶ 

[1] Key Laboratory of Molecular Biophysics of Ministry of Education, Department of Genetics and Developmental Biology, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, Hubei, China;Key Laboratory of Molecular Biophysics of Ministry of Education, Department of Genetics and Developmental Biology, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, Hubei, China;Institute of Visual Neuroscience and Stem Cell Engineering, College of Life Sciences and Health, Wuhan University of Science and Technology, Wuhan, Hubei, China;Key Laboratory of Molecular Biophysics of Ministry of Education, Department of Genetics and Developmental Biology, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, Hubei, China;Maternal and Child Health Hospital of Hubei Province, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China;Key Laboratory of Molecular Biophysics of Ministry of Education, Department of Genetics and Developmental Biology, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, Hubei, China;Sansure Biotech Inc., Changsha, Hunan, China;Key Laboratory of Molecular Biophysics of Ministry of Education, Department of Genetics and Developmental Biology, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, Hubei, China;State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Innovation Academy for Seed Design, Chinese Academy of Science, Wuhan, Hubei, China;Medical College, China Three Gorges University, Yichang, China;The Institute of Infection and Inflammation, China Three Gorges University, Yichang, Hubei, China;

关键词: rpgra;    zebrafish;    TALEN;    ciliary transport;    RAB8A;    retinal degeneration;   

DOI  :  10.3389/fcell.2023.1169941

 received in 2023-02-20, accepted in 2023-05-24,  发布年份 2023

来源: Frontiers

【 摘 要 】

Introduction: Pathogenic mutations in RPGRORF15, one of two major human RPGR isoforms, were responsible for most X-linked retinitis pigmentosa cases. Previous studies have shown that RPGR plays a critical role in ciliary protein transport. However, the precise mechanisms of disease triggered by RPGRORF15 mutations have yet to be clearly defined. There are two homologous genes in zebrafish, rpgra and rpgrb. Zebrafish rpgra has a single transcript homologous to human RPGRORF15; rpgrb has two major transcripts: rpgrbex1-17 and rpgrbORF15, similar to human RPGRex1-19 and RPGRORF15, respectively. rpgrb knockdown in zebrafish resulted in both abnormal development and increased cell death in the dysplastic retina. However, the impact of knocking down rpgra in zebrafish remains undetermined. Here, we constructed a rpgra mutant zebrafish model to investigate the retina defect and related molecular mechanism.Methods: we utilized transcription activator-like effector nuclease (TALEN) to generate a rpgra mutant zebrafish. Western blot was used to determine protein expression. RT-PCR was used to quantify gene transcription levels. The visual function of embryonic zebrafish was detected by electroretinography. Immunohistochemistry was used to observe the pathological changes in the retina of mutant zebrafish and transmission electron microscope was employed to view subcellular structure of photoreceptor cells.Results: A homozygous rpgra mutant zebrafish with c.1675_1678delins21 mutation was successfully constructed. Despite the normal morphological development of the retina at 5 days post-fertilization, visual dysfunction was observed in the mutant zebrafish. Further histological and immunofluorescence assays indicated that rpgra mutant zebrafish retina photoreceptors progressively began to degenerate at 3-6 months. Additionally, the mislocalization of cone outer segment proteins (Opn1lw and Gnb3) and the accumulation of vacuole-like structures around the connecting cilium below the OSs were observed in mutant zebrafish. Furthermore, Rab8a, a key regulator of opsin-carrier vesicle trafficking, exhibited decreased expression and evident mislocalization in mutant zebrafish.Discussion: This study generated a novel rpgra mutant zebrafish model, which showed retinal degeneration. our data suggested Rpgra is necessary for the ciliary transport of cone-associated proteins, and further investigation is required to determine its function in rods. The rpgra mutant zebrafish constructed in this study may help us gain a better understanding of the molecular mechanism of retinal degeneration caused by RPGRORF15 mutation and find some useful treatment in the future.

【 授权许可】

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Copyright © 2023 Liu, Han, Liu, Yu, Qin, Li, Jia, Gao, Chen, Tang, Liu and Huang.