脊椎動物胚胎血管的發育過程中，已有許多的訊號調控分子已經被發現，但是在靜脈生成以及區間血管 (Intersegmental vessel, ISV) 的生成所受的訊號調控目前還是知道甚少。我們團隊之前發現轉錄因子islet2(Isl2)和coupTFIb藉由Notch訊號的負向調控，影響靜脈以及區間血管的生成，為了了解轉錄因子islet2(Isl2)和coupTFIb的調控下游基因分子機制，我們進行微陣列的實驗，來找尋調控血管發育潛在的下游基因。
我們利用微陣列分析的方式，將isl2 knockdown的胚胎與coupTF1b knockdown胚胎與野生型的胚胎做比較。結果顯示出許多受coupTFIb和isl2所重疊調控的下游目標，推測coupTFIb和isl2可能共同調節血管的生成，或是藉由同一條路徑來調節。
我們進一步選擇一些感興趣基因，如Cpn1，Foxo3b，Fst1，Lnx1，Mmp2和Ftr82，他們的基因表達量在isl2和coupTF1b knockdown的胚胎中有高度的改變。利用原位組織的方式，我們發現這些基因在斑馬魚胚胎早期發育過程中表現在血管上，推測可能與血管生成有關係。在這些基因中，ftr82屬於一個脊索動物大家族TRIM的其中一員，含有一個RING-B-box domain 、Coiled Coil motif和 C-terminal domains。然而，目前沒有任何與血管發育有關的研究。因此，我接下來進一步測試ftr82基因對於血管的功能。
使用Morpholino專一抑制ftr82 pre-mRNA發現在區間血管以及尾部靜脈血管叢的生長缺陷，這代表ftr82有促進區間血管和尾部靜脈血管叢(Caudal vein plexus, CVP)增長中的作用。接下來也利用MO efficiency以及ATG morpholino證實了為專一性抑制ftr82的結果。此外抑制ftr82基因下，斑馬魚胚胎的動脈和靜脈特異性標誌物ephrinb2和flt4的表達都有下降的趨勢，顯示其對血管生成的影響。為了想知道這些缺陷是否因為血管內皮細胞的死亡而導致，我們進行TUNEL分析。結果顯示細胞凋亡並沒有發生在血管細胞上，這表示血管生成的缺陷不是因為內皮細胞死亡的緣故。另外從Tg (fli:nGFP,kdrl:mcherry)觀察到ISV 細胞數減少以及移動停滯，顯示ftr82 morphant抑制了血管內皮細胞的增生以及移動。
總括而論，我們找出許多受islet2及coupTFIb調控血管發育的目標基因。另外我發現ftr82在血管發育的斑馬魚扮演著一個重要的角色。

Many regulators and signaling molecules that control the process of vascular development have been described. However, genetic elements regulate the vein identity and intrasegmental vessels (ISV) patterning are still remained unclear. We previously identified the transcription factors islet2 (Isl2) and coupTFIb required for specification of the vein and ISV growth mediated by notch pathway in zebrafish. In order to understand the molecular mechanisms of how isl2 and coupTFIb promote the vascular development, we performed the microarray experiments to identify the potential downstream targets.
In this study, we first examined the changes in the transcriptome between isl2 knockdown, coupTF1b knockdown and wild-type embryos. Our microarray data showed highly overlapping downstream targets of coupTFIb and isl2, suggesting that coupTFIb and isl2 cooperatively regulate endothelial cell identity, likely in the same pathway.
We further confirmed the increased expression of several genes, such as cpn1, foxo3b, fst1, lnx1, mmp2 and ftr82, in isl2 and coupTFIb double knockdown morphants by in-situ hybridization. In addition, those genes were spatiotemporally expressed in vessels during early development suggesting their functions in vascular development. Among those genes, ftr82 (finTRIM family, member 82) belongs to a large family of tripartite motif proteins (TRIM) containing a RING-B-box-Coiled Coil motif followed by different C-terminal domains. However, no description related to vascular function so far.
Thus, we then examined the vascular function of ftr82. ftr82 mRNA is expressed in developing vessels and loss of ftr82 by morpholino knockdown impairs the growth of ISV and CVP (caudal vein plexus), suggesting the role of ftr82 in promoting ISV and CVP growth. We further showed the ftr82 MO knockdown worked efficiently. We performed TUNEL assay to address whether the cell death contributes to the decrease of ISV and CVP growth, Morpholino- knockdown ftr82 results in the decrease expression of the vein specific marker flt4 and the artery marker ephrnb2. The data showed no increase cell death in the endothelial cells after morpholino injection, suggesting that cell death is not the cause of the observed vascular phenotype.
Taken together, in this study, We identified several potential vascular-specific targets downstream of isl2 and coupTFIb. We also show ftr82 paly an important role for vascular development in zebrafish.

論文審定書 i
致謝 ii
中文摘要 iv
Abstract vi
目錄 viii
圖表目錄 xi
壹、前言 1
1.1血管生成的重要性 1
1.2 斑馬魚作為模式生物的優點 1
1.3 斑馬魚血管脈絡形成 2
1.4 Notch 訊號在斑馬魚血管生成中扮演重要的調控機制 3
1.5動靜脈分化上的不同藉由不同的訊號所調控 3
1.6斑馬魚區間血管 (Intersegmental vessel, ISV) 和尾部靜脈血管叢 ( Caudal vein plexus, CVP) 的特化 4
1.7 Islet及CoupTF基因選擇 5
1.8研究目的 5
貳、實驗材料與方法 12
2.1實驗材料 12
2.1.1 斑馬魚品系 12
2.1.2 Morpholino 12
2.2實驗步驟 13
2.2.1斑馬魚的飼養與受精胚胎的收集 13
2.2.2 RNA全覆式原位雜交(RNA whole-mount in situ hybridization) 13
2.2.2-A斑馬魚胚胎之收集及固定 13
2.2.2-B RNA全覆式原位雜交(RNA whole-mount in situ hybridization) 14
2.2.3 冷凍組織切片(LEICA 3050S) 16
2.2.4斑馬魚total RNA的抽取 ( GeneMark ) 17
2.2.5 cDNA 製備(Roche) 18
2.2.6 DNA clean/extraction KIT (使用Genemark KIT) 18
2.2.7 DIG RNA Labeling Probe製備 (使用Roche KIT) 19
2.2.8 microinjection顯微注射 21
2.2.9 Morpholino efficiency 21
2.2.8定量即時聚合酶鏈鎖反應（Quantitative real time polymerase chain reaction） 22
2.2.9 TUNEL ASSAY 22
2.3影像成相系統 23
參、研究結果 25
3.1 利用Microarray篩選下游基因的結果 25
3.2檢測特定下游基因在斑馬魚胚胎的表現位置? 25
3.3 使用coupTFIb/isl2 MO的斑馬魚胚胎測定六個候選基因是否吻合microarray的結果 26
3.4針對coupTFIb/isl2 轉錄因子所調控DNA序列，針對下游目標基因進行Promoter 序列分析 26
3.5探討ftr82觀察在斑馬魚胚胎的表現 27
3.6進一步使用ftr82 e1i1 morpholino 注射至Tg(kdrl:eGFP)斑馬魚胚胎 ，觀察胚胎血管生成的變化 27
3.7測試ftr82 e1i1 morpholino 是否專一結合目標基因的pre-mRNA 28
3.8檢測ftr82 konckdown所造成的phenotype是否具專一性 29
3.9探討ftr82影響血管新生的過程是細胞凋亡或是影響血管細胞的增生或移動 29
3.10觀察Ftr82是否會影響動靜脈標定基因的表現 30
肆、討論及未來研究方向 32
4.1 isl2/coupTF1b抑制ftr82造成血管缺失 34
4.2確認isl2/coupTF1b直接還是間接影響ftr82基因的表現 35
4.3 isl2&coupTF1b弱化後的血管缺陷是否可用 ftr82MO給挽救(rescue) 36
4.4 過度表現ftr82基因，是否與isl2/coupTF1bMO的表現型結果相似 36
4.5目前已知血管訊號調控與Ftr82基因之間的關係 37
4.5 FTR82在斑馬魚中扮演的腳色 37
伍、參考資料 39
七、實驗結果 45
附錄 60
實驗藥品 60
口試問題Q&A 63

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