建立ziwi:GFP-UtrCH基因轉殖斑馬魚以監測受精卵肌動蛋白之變化

Abstract

肌動蛋白(Actin)是真核生物含量最高的蛋白質之一，也是細胞骨架的必要元素之一。肌動蛋白絲(F-actin)由肌動蛋白單元(G-actin)構成，參與很多重要的生理功能，包含細胞形狀的形成及維持、細胞移動、細胞內物質運輸和細胞分裂，所以發展監測肌動蛋白絲變化的工具對其功能的研究相當重要。受精作用從精子接觸到卵子開始，過程中會伴隨著一系列劇烈的變化，最後發育成一全新的生命個體。在硬骨魚的卵表面上有一個由肌動蛋白絲構成的漏斗狀結構稱為卵膜孔(micropyle)，精子會從此處進入魚卵中。而在海膽及老鼠的研究中已經證實肌動蛋白絲於受精之後會立刻進行重組，更有研究指出，肌動蛋白絲的調控跟受精後會出現的鈣離子波動有關。目前在斑馬魚研究中缺乏適合監測肌動蛋白絲的工具，為了研究肌動蛋白絲在受精及胚胎發育時期所扮演的角色，我們發展了一個方法來監測斑馬魚卵中的肌動蛋白絲變化。我們在斑馬魚卵中利用ziwi促進子驅動與綠螢光蛋白融合的utrophin (GFP-UtrCH)的表現。在雌性斑馬魚中，ziwi促進子可以在所有分化階段的卵細胞(包含成熟卵)中驅動基因表現，而GFP-UtrCH是一個可以做為肌動蛋白絲探針的重組蛋白。經由與肌動蛋白絲劑rhodamine phalloidin染色結果的比對，我確認在ziwi:GFP-UtrCH 基因轉殖魚卵和胚胎中的螢光訊號可以即時反映肌動蛋白絲的位置及變化。接著我用ziwi:GFP-UtrCH 基因轉殖魚卵實行體外受精並可以同時監測到卵中肌動蛋白絲的變化。此基因轉殖斑馬魚未來將可作為研究肌動蛋白絲在受精及胚胎發育時期變化的有力工具。

Actin is one of the most abundant proteins in eukaryote and is an essential component of cytoskeleton. Filamentous actin (F-actin), assembled by polymerization of globular actin (G-actin) monomers, is involved in many crucial physiological functions, such as the formation and maintenance of cell shape, cell motility, cell division and intracellular trafficking. Consequently, the development of tools for monitoring F-actin dynamic is important to understand the functional roles of actin during development and other physiological processes. Fertilization starts with sperm contacting eggs, and followed by dramatic changes inside egg to form a complex muticellular organism. In teleost, there is a specific sperm entry site called micropyle, a dimple-like structure consisting of circular tuft of microvilli and containing a meshwork of actin filaments. Furthermore, the F-actin network of unfertilized egg rearranges immediately to the egg cortex during/after fertilization in species such as sea urchin and mouse. Some studies also indicate that the fine regulation of actin filament may be related to calcium wave trigger by fertilization. I was interested in studying actin dynamic during fertilization and early embryogenesis. Due to the lack of a suitable F-actin reporting line in zebrafish, I used a ziwi promoter to drive the maternal expression of green fluorescent protein-utrophin (GFP-UtrCH) fusion proteins. The ziwi promoter is a germ cell-specific promoter and GFP-UtrCH is an F-actin probe, which can bind F-actin by its calponin homology domain. In various development stages of Tg(ziwi:GFP-UtrCH) embryos, the fluorescent protein signal faithfully represent the distribution of F-actin that resembles the rhodamine phalloidin staining patterns. I also performed in vitro fertilization using the unfertilized egg from Tg(ziwi:GFP-UtrCH) and observed dramatic reorganization of cortical actin. This transgenic line is thus an effective tool to study the regulation of F-actin dynamics during fertilization and subsequent development.