The role of cldnh during the early retinal development in zebrafish
The role of cldnh during the early retinal development in zebrafish
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cldnh 在斑马鱼早期视网膜发育中的作用
DOI：
10.1016/j.exer.2020.108207
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发表时间：
2020-11-01
影响因子：
3.4
通讯作者：
Luo, Yan
中科院分区：
文献类型：
--
作者：
Lu, Jing;Liu, Ruyuan;Luo, Yan
紧密连接蛋白-3是紧密连接的一个组成部分，最近已显示在视网膜神经节细胞、视网膜色素细胞和视网膜血管内皮细胞中表达。然而，claudin-3在神经视网膜及其血管发育中的作用仍然不确定。本研究旨在探讨斑马鱼claudin-h（cldnh）在神经视网膜及其血管发育中的作用。利用cldnh吗啉代寡核苷酸（MO）和cldnh mRNA对转绿色荧光蛋白基因斑马鱼的Cldnh水平进行遗传操作，以研究基因功能。用聚合酶链反应和免疫荧光染色法分析cldnh的表达。使用苏木精-伊红染色、荧光染料注射、共聚焦体内成像、BrdU标记、TUNEL测定、RNA测序和Western印迹检测cldnh MO-吗啉代眼中改变的形态学、细胞和分子事件。我们证明了cldnh蛋白在斑马鱼的神经视网膜和视网膜血管的前身玻璃体血管中表达。Cldnh敲低可延迟神经视网膜分层并降低其厚度，这可能与atoh 7、pcdh 17、crx、neurod 1、insm 1a、sox 9 b和cdh 11等视网膜发育相关基因的下调以及tp 53、cdkn 1a和casp 8等细胞周期和凋亡相关基因的上调有关。Cldnh基因敲低还可使玻璃体血管密度降低，管腔化中断，这可能与hlx 1和my 17血管形成相关基因表达下调有关。总之，cldnh是斑马鱼神经视网膜及其血管正常发育所必需的，为阐明其在视网膜血管或炎症性疾病发病机制中的作用提供了基础。
Claudin-3, an integral component of tight junction, has recently been shown to be expressed in retinal ganglion cells, retinal pigment cells, and retinal vascular endothelial cells. However, the role of claudin-3 in the development of the neural retina and its vessels remains undefined. This study aimed to investigate the role of zebrafish claudin-h (cldnh), the closest ortholog of mouse and human claudin-3, in the development of the neural retina and its vessels. Cldnh levels in green fluorescent protein transgenic zebrafish were genetically manipulated by cldnh morpholino oligonucleotide (MO) and cldnh mRNA to investigate gene function. The expression of cldnh was analyzed using polymerase chain reaction and immunofluorescence staining. The altered morphological, cellular and molecular events in the cldnh MO-morphant eyes were detected using hematoxylin-eosin staining, fluorescent dye injection, confocal in vivo imaging, BrdU labeling, TUNEL assay, RNA sequencing, and Western blot. We demonstrated that the cldnh protein was expressed in the neural retina and the hyaloid vessel which is the predecessor of the retinal vessel in zebrafish. Cldnh knockdown delayed lamination of the neural retina and reduced its thickness, which might be associated with the downregulation of the retinal development-related genes of atoh7, pcdh17, crx, neurod1, insm1a, sox9b and cdh11, and the upregulation of the cell cycle and apoptosis-associated genes of tp53, cdkn1a and casp8. Cldnh knockdown also reduced the density and interrupted the lumenization of the hyaloid vessels, which might be owing to the downregulation of the vessel formation-related genes of hlx1 and my17. In conclusion, cldnh was required for the normal development of the neural retina and its vessels in zebrafish, providing a basis for elucidating its role in the pathogenesis of retinal vascular or inflammatory diseases.