- 學位論文
斑馬魚Bves在皮膚上皮細胞屏障功能及眼睛發育中扮演的重要角色
Zebrafish blood vessel epicardial substance (Bves) plays a critical role in regulating epidermal barrier and eye development
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摘要
Bves被廣泛地觀察到會表現在上皮組織，例如皮膚，心外膜，小腸，角膜的細胞連合處。但是，目前還不清楚Bves在細胞間黏附扮演什麼樣的角色。由於小鼠剔除Bves基因後，沒有明顯的的生理和病理表現，我們認為Bves的功能在哺乳類由於演化上可能己有其他蛋白質可以替代，所以小鼠剔除Bves基因後不會有特異的表現型，因此我們想尋找其他更原始的動物模式來探討其真正的生理功能，而本篇論文則選擇斑馬魚做為實驗動物模式。首先，我們找出並定義斑馬魚Bves基因跟蛋白，並發現Bves顯著表現在心臟、眼睛、表皮跟腦。進一步，我們利用MOs干擾Bves蛋白的正常表現，發現這些斑馬魚對滲透壓極敏感並且有表皮屏障功能受損的情形，因此，本論文的第一個部份則是利用表皮組織去探討Bves功能。我們利用免疫螢光染色的實驗去觀察細胞連合處蛋白的表現，結果發現破壞Bves表現導致細胞膜上Claudin蛋白消失以及aPKC蛋白無法高密度地聚集在細胞連合處。mRNA rescue實驗發現tjp-2/ZO-2跟aPKC mRNA會顯著地救回zBves缺失後導致的嚴重死亡率及畸形率，這樣的結果更暗示Bves、aPKC跟tight junction間的關連，然而在缺乏aPKC的情況下zBves的表現並不會受到太大影響。我們進一步執行免疫沉澱的實驗去觀察Bves、aPKC跟Claudin之間的關係，實驗結果指出 zBves與aPKC蛋白有交互連結的關係，而在zBves的缺損的狀況下，aPKC跟Claudin蛋白之間的連結及磷酸化動作會大幅降低。綜合這部份的結果，我們推斷zBves的缺損影響aPKC在細胞中的表現位置，進而干擾細胞交界蛋白claudin在交界處的出現及功能。在Bves knockdown的實驗中我們發現除了皮膚屏蔽功能受到影響，魚體內各層的發育包括眼球發育都變亂了，因此論文的另一個部份是探討Bves是如何透過影響細胞黏附功能而對眼睛發育造成影響。眼睛的發育過程中的細胞的極性決定了正常視網膜和感光細胞的分化，而在之前的研究已知Bves的功能與維持上皮細胞極性有密不可分的重要性。因此在這個部份的研究中，我們製造了轉基因斑馬魚Tg(zbves:EGFP)，觀察Bves在眼睛發育中的表現位置，並研究zbves對視網膜分層的影響。我們針對各層的細胞做免疫抗體染色及穿透式電子顯微鏡觀察正常和Bves缺陷的斑馬魚。實驗結果發現在正常的斑馬魚， 早期Bves蛋白會先表現在胚胎視網膜神經上皮，隨著視網膜的發育，強烈地分布在內網狀層(IPL)周圍和視網膜色素上皮(RPE)。與此相反，Bves的缺損造成視網膜的分層混亂甚至於消失，其中還發現未成熟甚至於未分化的感光細胞。因此在眼睛發育的研究中，我們發現Bves會影響視網膜的分層及感光細胞的分化。
並列摘要
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.
並列關鍵字
zBves ； cell permeabilization ； tight junctions ； epidermal barrier ； retinogenesis ； lamination參考文獻
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