Gene Expression and Functional Analysis of Cofilins during Development in Zebrafish, Danio rerio

肌動蛋白在真核生物中對細胞骨架組織重組佔有極重要地位，它可調控細胞活動力，細胞遷移，細胞膜變動和細胞質分裂。在動物胚發育過程中，細胞遷移和細胞質分裂則可影響細胞命運之決定和組織分化之過程。肌動蛋白細絲在生物體內有絲狀和球狀兩種，藉著聚合作用和去聚合作用，絲狀和球狀肌動蛋白細絲維持著一個動態平衡而調節細胞生理功能。 Cofilin 是一個約18 kDa 的肌動蛋白結合蛋白，它主要調控生物體內肌動蛋白去聚合作用，維持肌動蛋白絲動態平衡。在前人的研究中，cofilin 在不同的物種中皆存在，包括哺乳動物和植物，其具有兩種不同類型－非肌肉型和肌肉型，而在斑馬魚基因資料庫(NCBI database)中找到兩型cofilin，但其基因表現及功能卻尚未分析，因此我們利用RT-PCR分析，發現cofilin 在不同時期的斑馬魚胚皆有表現，而不同成魚組織中主要發現在肌肉、卵巢、精巢、腎和腸等組織中；更進一步利用原位雜合反應方法證明 cofilin 1 主要表現在表皮、鰓弓、脊椎神經、體節、胸鰭和原腎管；而cofilin 2 則主要表現在小腦、視網膜、中樞神經系統、胸鰭和原腎管。在功能性分析中，利用overexpression及Morpholino以降低cofilin 1 蛋白質生成，兩者均造成頭部輕微畸形，軀幹及尾部嚴重扭曲和鰓弓部份或全部的缺失。綜合以上的結果顯示，cofilin 1在斑馬魚胚發育與鰓弓，體軸形成及神經發育可能扮演重要的角色。Actin dynamics is of vital importance of cytoskeleton reorganization in eukaryotic organisms. Proper control of actin dynamics is necessary for regulating cell motility, cell migration, membrane ruffling and cytokinesis. Tightly control of cell migration and cytokinesis is a crucial for the determination of cell fate and tissue differentiation during embryonic development. Two types of actin, filamentous and globular actin, coexist in living cells. The equilibrium between filamentous and globular actin rely on the tightly regulated polymerization and depolymerization of filamentous actins. Cofilins are actin binding proteins with a molecular weight around 18 kDa. By regulating actin assembly, cofilins are shown to be critical mediators in actin filament dynamics. Cofilins have been characterized in various organisms, including mammals and plants. In zebrafish, two cofilin isoforms, cofilin 1 and 2 have been registered in NCBI database. However, virtually no expression or functional assay regarding cofilins has been published in zebrafish. Here we show that both cofilin 1 and 2 are expressed in zebrafish embryos at all stages examined and they are ubiquitously transcribed in various adult tissues detected by RT-PCR analysis. Cofilins are highly expressed in muscle, ovary, testis, kidney and intestine. Furthermore, by whole-mount in situ hybridization analysis, it was demonstrated that Cofilin 1 is mainly expressed to the embryonic epidermis, branchial arches, spinal cord, pectoral fin, somite and pronephric duct and cofilin 2 is expressed to the cerebellum, retina, central neuron system, pectoral fin and pronephric duct. Overexpression and knockdown of cofilin 1 caused similar defects of brain, contortion of trunk and tail, and partial or complete missing of branchial arches. These results suggest that cofilin 1 is required for development of brain, branchial arches and body axis formation in zebrafish.ACKNOWLEDGEMENTS………………………………………………………Ⅰ LIST OF FIGURES…………………………………………………………………Ⅱ ABSTRACT IN CHINESE…………………………………………………………Ⅲ ABSTRACT…………………………………………………………………………Ⅳ Introduction…………………………………………………………………………1 Materials and Methods………………………………………………………………6 Fish breeding and embryo collection………………………………………………6 RT-PCR analysis……………………………………………………………………6 Protein expression and purification…………………………………………………7 Plasmid construction for overexpressing GST-fusion protein in E.coli.………7 Expression and purification of the recombinant protein cofilin 1…………7 Gel electrophoresis and the determination of protein concentration……9 Whole-mount in situ hybridization…………………………………………………9 Morpholino antisense oligonucleotides and microinjection procedures…………10 Alcian blue skeletal staining………………………………………………………10 Embryo observations and photography……………………………………………11 Statistical analysis…………………………………………………………………11 Results………………………………………………………………………………12 Sequence and structure analyses of zebrafish cofilin 1 and 2………………12 Sequence and phylogenetic analyses of cofilin 1 and 2…………………………12 Expression of cofilins in the early embryos and the adult tissues…………………13 Expression of cofilins in situ during development………………………………14 Over-expressions of recombinant protein cofilin 1 during embryogenesis………15 Knockdown of cofilin 1 using morpholino antisense oligonucleotides……17 Morphology of branchial arches with cofilin 1 injected by alcian blue……18 Discussion……………………………………………………………………………19 References…………………………………………………………………………28 Figures………………………………………………………………………………4