血管的生長和形成網狀脈絡需要許多基因及訊息傳遞路徑來調控，然而，較少研究闡述關於轉錄因子 (transcription factor) 如何調控靜脈生成及體節間血管(intersegmental vessels, ISV) 脈絡生成的分子機轉。研究指出CoupTFII (COUP transcription factor 2) 在小鼠中對靜脈分化有正向調節作用。而此論文中，我們發現CoupTF1b 對靜脈細胞和尖端細胞的分化生長有重要的影響。
比較不同物種中的CoupTF 家族的胺基酸序列發現CoupTF1b 在不同脊椎動物中的表現有高度的保守性，因此，我推測CoupTF1b 在斑馬魚中可能扮演於相似於小鼠CoupTFII 在血管發育的功能。
利用原位組織染色，我發現CoupTF1b 的mRNA 表現早期15s 時期在腹部側中胚層處，24 hpf 時表現在動靜脈，符合血管發育的時間及脈絡。利用反義核甘酸morpholino 抑制CoupTF1b 的結果中，不論靜脈螢光信號和靜脈專一性標的基因flt4 和mrc1 的表達都有下降的趨勢，證實CoupTF1b 促進靜脈的分化與生成。此外，我們也發現抑制CoupTF1b，會阻礙ISV 生長。為了證明morpholino抑制的專一性，利用CoupTF1b splicing 及CoupTF1b ATG morpholino 得到相似
的血管缺陷型態。為了瞭解抑制CoupTF1b 造成的血管缺陷，是否是因為細胞凋亡所引起，我使用吖啶橙染色 (acridine orange staining) 與TUNEL 分析，我們發現，注射CoupTF1b morpholino 後非特異性細胞凋亡異常增加，有可能是血管表型缺陷原因。
我們進一步測試CoupTF1b 與其他血管生長調控因子， VEGF、Notch 訊息傳遞路徑的相互關係。我們的結果顯示CoupTF1b 對於血管發育斑馬魚扮演不可缺少的角色。

Genetic programs and signaling pathways are required for proper growth and patterning of blood vessels, however, little known about the transcription factors functioning in vein identity and intersegmental vessels (ISV) patterning in zebrafish.
The orphan nuclear receptor Chicken ovalbumin upstream promoter transcription factor II (CoupTFII) positively regulates vein identity in mice. Here we show that the CoupTF1b is important for vein and tip cell identity. Comparison of amino acids sequence and phylogenetic analysis of CoupTF1b orthologs in different species suggesting the CoupTF1b is conserved among vertebrates. CoupTF1b mRNA has a spatiotemporal expression pattern in ventral lateral mesoderm consistent with a role in early vascular specification. Morpholino
knockdown of coupTFIb results in a decrease in both venous fluorescent signals and expression of the vein specific marker flt4 and mrc1. These data suggest that CoupTF1b has novel role in promoting vein identity. In addition, we show loss of CoupTF1b impairs ISV growth, suggesting that CoupTF1b also has an important role in controlling ISV growth. We have confirmed those vascular defects are CoupTF1b specific by using two different types of morpholinos knockdown strategies (ATG and splicing morpholinos). To address whether the decrease in vein signals and the growth defect in ISV result from cell death, we performed TUNEL assay. We showed that an increase in non-specific cell death after morpholino injection is possible cause of the observed vascular phenotype. We further test CoupTF1b likely interact with the Notch signaling pathway to control vascular development. Together, we show CoupTF1b plays an indispensable role for vascular development in zebrafish.

目 錄
論文審定書.....................................................................................................i
致謝 ..............................................................................................................ii
摘要 .............................................................................................................iii
Abstract........................................................................................................iv
一、緒 論......................................................................................................1
1.1脈管生成作用及血管新生成......................................................................1
1.2血管的發育...............................................................................................2
1.3 Notch訊息傳遞........................................................................................4
1.4 VEGF訊息傳遞........................................................................................6
1.5 CoupTF1b轉錄因子................................................................................7
1.6斑馬魚胚胎的時期與發育.........................................................................8
二、實驗材料與方法...................................................................................10
2.1 實驗藥品...............................................................................................10
2.2斑馬魚飼育及胚胎培養...........................................................................10
2.3 野生株及基因轉殖螢光之斑馬魚魚種.....................................................11
2.4聚合酶連鎖反應......................................................................................11
2.5原位組織染色探針合成...........................................................................11
2.6 原位組織雜交........................................................................................13
2.7 Morpholino...........................................................................................14
2.8 Morpholino效能....................................................................................15
2.9 細胞凋亡分析........................................................................................16
2.10 ISV定量方式、成像及統計分析...........................................................17
2.11 冷凍切片.............................................................................................18
2.12 Tol2轉座子系統...................................................................................18
2.13 西方點墨轉漬......................................................................................19
三、實驗結果..............................................................................................20
3.1. CoupTF1b在脊椎動物中具有高度保守性.............................................20
3.2 CoupTF1b表現位置與斑馬魚脈管生成位置相符...................................20
3.3 不同時期CoupTF1b在斑馬魚胚胎的表現脈..........................................21
3.4 確認Coup TF1b morpholino抑制其基因表現的程.................................21
3.5抑制Coup TF1b表現導致斑馬魚血管缺.................................................22
3.6抑制CoupTF1b導致斑馬魚出現心膜腫大、及循環缺陷.........................22
3.7抑造成的血管缺失可藉過度表現CoupTF1b回復，且不同設計的
CoupTF1b morpholino皆可抑制體節間血管生成....................................23
3.8 注射morpholino導致細胞凋亡..............................................................24
3.9 缺少Coup TF1b導致靜脈標的基因表現量下降,但對動脈標的表現量無
明顯影響.................................................................................................25
3.10 過度表現CoupTF1b促進血管新生成..................................................25
3.11 Notch 信號傳遞及 VEGF會調控 CoupTF1b的表現............................26
3.12 Isl2與Coup TF1b的交互作用.............................................................27
3.13 CoupTF1b訊息傳遞調控示意圖.........................................................27
四、總結...................................................................................................28
五、討論...................................................................................................31
5.1人類CoupTF1與斑馬魚CoupTF1b有相異功能性..................................31
5.2 抑制CoupTF1b導致新血管生成缺失的途徑.........................................31
5.3 CoupTF1b 與血管生成的關係.............................................................32
5.3.1 CoupTF1b下游的調控基因對血管生成的影響..................................32
5.3.2. CoupTF1b訊息傳遞調控機制..........................................................33
5.3.3 CoupTFII與CoupTF1b是否同樣在癌症生成的病理過程扮演重要的
角色......................................................................................................34
5.4. CoupTF1b與isl2直接交互作用的關係................................................35
5.5表皮細胞凋亡引起血管新生成缺失.......................................................35
六、參考文獻............................................................................................37
七、口試委員提問.....................................................................................59

圖目錄

圖 1-1 斑馬魚血管脈絡………………………………………………………..…… 2
圖 1-2 (A)不同時期的血管發育過程 (B) Notch調控血管血球生長的分化過程.....4
圖 1-3 Notch 訊息傳遞………………………………………………………...…....5
圖 1-4血管內皮生長因子家族成員及其受體…………………………………...….7
圖 1-5斑馬魚胚胎發育階段………………………………………………………....9
圖3-1 Sequence alignment CoupTF1b family in zebrafish and its ortholog...44
圖3-2 CoupTF1b is expressed in the zebrafish developing vessels。 45
圖3-3 Expression pattern in wt zebrafish。 46
圖3-4 Knockdown efficiency of CoupTF1b Morpholino. 47
圖3-5 Knockdown CoupTF1b results in vascular defects. 48
圖3-6 Loss of CoupTF1b results in pericardial edema, absent parachordal vessels,
subintestinal vessels (SIV) mispattern and circulation defects 50
圖3-7 Knockdown of CoupTF1b causes defects in zebrafish vascular development. 51
圖3-8 An increase in cell death after morpholino injection is the cause of the observed
vascular phenotype. 52
圖3-9 Loss of Coup TF1b causes the decreased expression of vein marker flt4、mrc1
and dab2, but not artery marker ephrin B2 、notch3 and arteriovenous marker
stabilin2 . 53
圖3-10 Coup TF1b is modulated by Notch signal and VEGF . 54
圖3-11 Overexpression of CoupTF1b under the fli promoter increases the number of
venous cells and endothelial cells per ISV. 55
圖3-12 CoupTF1b is modulated by isl2. 57
圖3-13 Schematic representation of the coupTF1b signaling pathway. 58
圖 7-1動靜脈特化發展的過……………………………………………………….60
圖 7-2 Notch配體對脈管生成的分化功能………………………………………..66
圖 7-3 Notch的調控尖端細胞及柄細胞數量的平衡……………………….…….67

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