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    題名: 斑馬魚細胞核因子C次單元在胚胎早期的功能分析
    其他題名: Functional analysis of zebrafish NF-YC (nuclear factor Y subunit C) during early embryonic development
    作者: 傅慧娟;Fu, Hui-Chuan
    貢獻者: 淡江大學生命科學研究所碩士班
    陳曜鴻
    關鍵詞: 斑馬魚;細胞核因子C次單元;眼睛發育;Zebrafish;nfyc;morpholino;eye development
    日期: 2011
    上傳時間: 2011-06-16 21:57:16 (UTC+8)
    摘要: NF-Y 是 CCAAT 主要的結合轉錄因子,由 NF-YA、NF-YB、NF-YC 三種次單元所組成。本研究利用斑馬魚作為模式物種,來探討 nfyc 是否會影響眼睛的發育。首先選殖出斑馬魚的 nfyc 基因,發現 nfyc 在斑馬魚存在有兩種型態:nfyc-tv1 (336 個胺基酸) 及 nfyc-tv2 (360 個胺基酸);斑馬魚的 nfyc-tv1 與人類、大鼠、小鼠、雞、牛、爪蟾序列相似度分別為 84%-87%;斑馬魚的 nfyc-tv2 序列相似度則分別為 79%-81%。利用原位雜合反應觀察 nfyc 在斑馬魚胚胎的表現,發現受精後 24 小時 nfyc 表現集中在腦部、眼睛及神經管上。以三天大的斑馬魚進行抗體染色實驗,發現 NF-YC 表現在初級頭竇 (primary head sinus)、心臟及體間血管上。接著顯微注射反股寡核苷酸 (morpholino) 抑制內生性 nfyc 的蛋白轉譯後,測量斑馬魚的單眼大小,發現抑制 nfyc 會造成斑馬魚眼睛 (301±6 μm) 縮小約為野生型斑馬魚眼睛大小 (139±45 μm) 的 46%。再利用基因轉殖魚 Tg(Nav1.6:GFP) 進行 Zn8 和 Zn12 抗體染色,發現抑制 nfyc 會影響神經節細胞層、內叢層、外叢層的表現。而抑制 nfyc 後進行phospho -histone H3 (PH3) 抗體染色及 TUNEL assay,結果顯示抑制 nfyc 會導致眼睛及心臟的細胞增生數量減少,且在腦部及眼睛有細胞凋亡的現象。進一步以 retinal homeobox 1 (rx1) 和 cone-rod homeobox (crx) 的探針進行原位雜合反應,發現抑制 nfyc 後的 crx 表現量降低,而 rx1 表現量則上升,可能是分化無法完全所導致。另外利用血管標記綠螢光及紅血球標記紅螢光之基因轉殖魚 Tg(fli1:EGFP)×Tg(gata1:RFP),發現抑制 nfyc 後的體間血管有受損及血球流速變慢。綜合以上實驗結果,推測 nfyc 可能透過影響斑馬魚視神經及血管的發育,進而對視網膜的分化造成影響。
    Nuclear factor-Y (NF-Y) is a CCAAT-box-binding transcription factor which is composed of three subunits (NF-YA, NF-YB, and NF-YC). In this study, we used zebrafish as an animal model to study their roles during early developmental stage. First, we cloned two types of zebrafish NF-YC genes (nfyc-tv1 and nfyc-tv2) which revealed high sequence similarity with homologs from other species (zebrafish nfyc-tv1 (nfyc-tv2) polypeptide shares sequence similarities of 86%(81%), 87%(81%), 87%(81%), 87%(81%), 87%(81%) and 84%(79%) with the reported nfyc-tv1 (nfyc-tv2) of human, rat, mouse, chicken, bovine and Xenopus, respectively). We observed the expression of nfyc in zebrafish embryos using whole-mount in situ hybridization. In 24-hpf embryos, nfyc expression was found in brain, eyes and neural tube. Immunostaining with NF-YC antibody revealed the expression of NF-YC in primary head sinus, heart and intersegmental vessel. While endogenous nfyc was knocked down by antisense morpholino of nfyc (nfyc-MO), a reduction in eye size was observed in nfyc-MO-injected embryos (0.25 ± 0.02 mm, n=30) compared with wild-type embryos (0.60 ± 0.01 mm, n=30). Immunostaining with neuron-specific antibodies (Zn8 and Zn12) revealed that nfyc-MO affected nfyc expression in ganglion cell layer (GCL), inner plexiform layer (IPL) and outer nuclear layer (OPL), suggesting that nfyc is associated with the development of retinal neurons. Based on immunostaining with phosphor-histone H3 (PH3) antibody, a decrease in proliferating cells was found in eyes and heart of nfyc-MO-injected embryos. TUNEL assay results revealed the apoptosis in head and eyes of nfyc-MO-injected embryos. Our in situ hybridization data showed an increase in retinal homeobox 1 (rx1) accompanied by a decrease in cone-rod homeobox (crx), suggesting an impaired cell differentiation in nfyc-MO-injected embryos. Furthermore, we used transgenic zebrafish Tg(fli1:EGFP)×Tg(gata1:RFP) as a model to observe the intersegmental vessel and blood cells, and found knockdown of nfyc led to damaged blood vessels and slowed blood flow. Taken together, our results suggested that zebrafish nfyc may affect the development of retinal neurons and blood vessels, and further affect zebrafish eye development.
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