Zebrafish blood vessel epicardial substance (Bves) plays a critical role in regulating epidermal barrier and eye development

• Main Authors: Yu-Ching Wu, 吳侑靜
• Other Authors: 陳瑞芬
• Format: Others
• Language: en_US
• Published: 2014
• Online Access: http://ndltd.ncl.edu.tw/handle/03094541606277545817

博士 === 國立臺灣大學 === 生命科學系 === 102 === Bves is widely observed in the cell junction of epithelium in the skin, epicardium, intestine, and cornea of developmental embryos and mature adults. However, it is not clear how Bves confers its role in intercellular adhesion. Because LacZ-knockin Bves-null mice are not observed with any overt phenotypes and fish has the advantage of aquatic environment to investigate epithelial adhesion, we choose zebrafish as animal model in this thesis. First, we identify the zebrafish bves gene and protein structure, and find it remarkable appears in the heart, eyes, skin, and brain. Furthermore, we use MOs to interfere with Bves protein expression in zebrafish. The epidermal barrier function is disrupted after knockdown of Bves, and these zBves morphants are sensitive to osmotic stress. Therefore, the first part of this thesis is to explore the Bves function by study of zebrafish epidermis. Immunofluorescent studies demonstrate that the Claudin protein expression and the aPKC aggregation around the cell junctions are disintegrated in zBves morphants. The results of rescue experiment not only show tjp-2 and aPKC mRNA could rescue the mortality and defect rates in zBves morphants but imply the regulatory relationship between Bves, aPKC, and tight junction. However, the expression and assembly of zBves are not influenced by aPKC-MO. Therefore, immunoprecipitation experiments are performed to examine their relation and show the association between zBves and aPKC. Taken together, these results in this part of thesis indicate the loss of zBves affects the location of aPKC in epidermis and both of them are indispensable to claudin expression. The second part of this thesis is to examine the role of zBves in eye development, because cell polarity during eye development determines the normal retinal lamination and differentiation of photoreceptor cells in the retina. We generate a transgenic zebrafish line- Tg(zbves:EGFP)- to investigate the expression pattern of Bves in the retina. Immunostaining with different specific antibodies from retinal cells and transmission electron microscopy analyses are used to identify the morphological defects in normal and Bves knockdown zebrafish. These results show that zBves is located at the apical junctions of embryonic retinal neuroepithelia during retinogenesis; later, it is strongly expressed around inner plexiform layer and retinal pigment epithelium. The loss of normal retinal lamination and cellular polarity is found with undifferentiational photoreceptor cells in Bves knockdown zebrafish. In this part of thesis, our results indicate that disruption of Bves will result in a loss of normal retinal lamination.