肌動蛋白結合蛋白profilin在斑馬魚胚發育過程中之研究

Abstract

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.