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.

授權書
口試委員審議通過簽名單
謝誌	Ⅰ
中文摘要	Ⅱ
英文摘要	Ⅲ
目錄	Ⅴ
圖表目錄  VIII
第一章 前言
1.1 Nuclear transcription factor Y (NF-Y) 簡介	1
1.2眼睛的發育	3
1.3眼睛發育的相關因子	5
1.4班馬魚的優勢	6
1.5本論文的研究目的與動機	7
第二章 材料與方法
2.1斑馬魚之飼養	8
2.2胚胎收集 (Embryo fixation)	8
2.3胚胎固定及脫水保存	8
2.4斑馬魚 RNA 的萃取及合成 cDNA	9
2.5聚合酶鏈鎖反應 (Polymerase Chain Reaction, PCR)	10
2.6核酸的接合反應 (DNA ligation)	10
2.7勝任細胞 (Competent cell) 的製備與細胞的轉形作用 
(Transformation)	11
2.8小量質體萃取 (Isolation of Plasmid DNA)	11
2.9合成探針 (Riboprobe)	12
2.10原位雜交法 (Whole-mount in situ hybridization) 	13
2.11抗體染色 (Immunostaining)	15
2.12 TUNEL	16
2.13顯微注射實驗	17
2.14樣品製備及切片 (Embedding and Cryosectioning)	18
2.15蘇木紫與伊紅染色 (H & E staining)	18
2.16顯微照相系統	18
2.17序列分析軟體	19
第三章 結果
3.1斑馬魚 nfyc 與其他脊椎動物的親緣關係比對	20
3.2觀察 nfyc-tv1 及 nfyc-tv2 在胚胎早期發育時期的表現	21
3.3觀察 nfyc 在蛋白質層面的表現	23
3.4注射 nfyc morpholino antisense oligouncleotide knockdown nfyc 造成的影響	23
3.5注射不同濃度 nfyc morpholino antisense oligouncleotide 的存活率及眼睛缺失的比例	24
3.6注射 nfyc morpholino 至 Nav1.6 的斑馬魚及抗體染色	25
3.7 Knockdown nfyc 後造成眼睛大量細胞凋亡及降低細胞增生	26
3.8眼睛發育相關因子 (rx1 及 crx) 調控斑馬魚眼睛發育	27
3.9注射 nfyc morpholino 至 Tg(fli1:EGFP)×Tg(gata1:RFP) 的斑馬魚	29
第四章 討論
4.1 NF-YC 在演化上的保守性	30
4.2 NF-YC可能與眼睛及血管的發育有關	30
4.3 NF-YC影響眼睛發育的可能原因	31
4.4 NF-YC影響影響血管發育的可能原因	33
4.5總結	34
第五章 參考文獻	35
圖表	39
附錄	57

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