李士傑臺灣大學：漁業科學研究所吳玠錞Wu, Chieh-TuiChieh-TuiWu2007-11-282018-07-062007-11-282018-07-062005http://ntur.lib.ntu.edu.tw//handle/246246/59357細胞骨架(cytoskeleton)之調節對細胞正常功能維持及胚發育扮演著極重要之角色，而肌動蛋白絲(actin filament)乃細胞骨架主要成員之一，在細胞內，肌動蛋白絲通常是處於聚合(polymerization)以及去聚合(depolymerization)之間的動態平衡狀態，而這種平衡狀態則為其結合蛋白所調控。Profilin乃大小約15 kDa之肌動蛋白結合蛋白(actin-binding protein)，其主要藉由促進聚合作用來調控肌動蛋白絲的動態平衡。哺乳動物中有不同型的profilin，且其表現位置亦有所不同。在生理功能上，Profilin已知為動物胚分裂及存活所必需的，然而在硬骨魚類中吾等對profilin之了解卻極少！因此，我們選擇了斑馬魚來研究profilin在發育過程中所扮演的角色。 首先，我們成功的選殖到斑馬魚的profilin等型蛋白之一 profilin II (zpfnII)基因，而由此基因轉譯出的蛋白質有140個胺基酸。根據不同物種間的分析，zPfnII和其他物種的Profilin II有超過70%的相似度。在基因表現方面，我們利用RT-PCR發現zpfnII在胚發育早期表現相當高，因此推測它是一個母源性基因，此外zpfnII在成體的各個組織中均有表現。在zpfnII的原位雜合反應結果顯示zpfnII早期亦均勻分布在每個細胞中，而到了24 hpf之後則主要表現在腦以及神經性的組織中。之後，我們發現利用顯微注射異位表現zpfnII則造成體節及脊索之缺陷。然而以morpholino oligonucleotides 降低zpfnII表現量則只在發育晚期造成小魚的體型較短且尾部會有輕微彎曲。綜合以上結果顯示profilin II之正常表現乃為斑馬魚發育早期體節、脊索及體軸正常形成所必需。To study the roles of profilins in embryogenesis, we have identified a profilin II gene in zebrafish. By RT-PCR, a 423 b.p. profilin II transcript was highly expressed during cleavage and blastula periods as maternal transcripts, disappearing and a zygotic expression was not observed until 24 hours post fertilization (hpf). Profilin II expression was observed in all adult tissues examined. By whole-mount in situ hybridization, profilin II was ubiquitously expressed in all blastomeres of early embryos, but localized mainly in brain and neuronal tissues after 24 hpf. Overexpression of profilin II-EGFP caused the partial inhibition of somitogenesis and the distortion of notochord, which resulted in the malformation and truncation of trunk and tail. Furthermore, morpholino knock-down of profilin II induced a shorten body axis and a contorted tail. These results suggest that a normal expression of profilin II is required for the proper development of neuron, somite and possibly body axis formation in zebrafish.Content Content……………………………………………………………………………I 中文摘要…………………………………………………………………………1 Abstract…………………………………………………………………………2 Introduction………………………………………………………………………3 Experimental Procedures………………………………………………………7 Fish Husbandry and Embryo Collection………………………………………7 RT-PCR Analysis ……………………………………………………………7 Profilin II-EGFP Expression Vector Construction…………………………8 Microinjection Procedures……………………………………………………8 In Situ Hybridization on Whole Mount Embryos……………………………9 Embryo Observations and Photography ………………………………………9 Statistical Analysis……………………………………………………………10 Results…………………………………………………………………………11 Sequence and Phylogenetic Analysis of Profilins……………………………11 Expression of Profilin II in the Early Embryos and the Adult Tissues………12 Expression of Profilin II in situ during Development…………………………13 Transient Expression of profilin II-EGFP Results in Defects in Somitogenesis and Notochord Formation …………………………………………………14 Knockdown of profilin II Using Morpholino Oligonucleotides………………17 Discussion………………………………………………………………………18 References………………………………………………………………………23 Figures…………………………………………………………………………282784514 bytesapplication/pdfen-US胚發育肌動蛋白斑馬魚developmentnotochordsomiteactinprofilinzebrafishotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/59357/1/ntu-94-R91243013-1.pdf