斑馬魚血管發育的過程中，目前已知主動靜脈受到許多分子機制調控，但是對於區間血管 (intersegmental vessels, ISV) 及尾部靜脈血管叢 (caudal vein plexus, CVP) 的調控機制是不明確的。先前研究中指出，受Notch訊息路徑調控的轉錄因子islet2 (LIM homeobox gene, Isl2) 及coupTFIb (nr2f1b, orphan nuclear receptor Chicken ovalbumin upstream promoter transcription factor II的家族成員) 參與斑馬魚靜脈的特化及ISV的生長，為了研究isl2及coupTFIb調控血管發育的分子機制，我們利用DNA微陣列的技術尋找受isl2及coupTFIb調控的下游基因，在我的研究中，我挑選有可能調控血管發育的基因follistatin 1a (fsta, 卵泡抑制素)，我之所以對fsta有興趣，主要是因fsta為bone morphogenetic protein (BMP) 的結抗劑，BMP訊息傳遞除了會在胚胎發育早期參與體軸的發育，近期研究更指出BMP訊息傳遞參與CVP及靜脈血管生成的主要訊息路徑。然而對於fsta-BMP訊息路徑是否影響血管發育並無任何文獻，此外Notch與BMP兩個訊息路徑對血管的關聯性也是未知的。
首先我利用胺基酸序列分析fsta在各物種之間的保守性之餘還發現BMP結合位在各物種間保守性高，之後利用in-situ hybridization在斑馬魚胚胎18個體節時期中得知fsta mRNA表達於前側板中胚層及斑馬魚胚胎發育後24-48小時表達於脈管及CVP，因此我認為fsta會去參與斑馬魚血管發育。在fli1 promoter驅動下過度表達fsta，我發現過度表達fsta所造成的血管缺陷與抑制isl2及coupTFIb是相似的，可能fsta會受到isl2/coupTFIb的負向調控。為了了解fsta調控斑馬魚血管發育的分子機制，我利用in-situ hybridization檢測Tg(fli:fsta)是否影響動靜脈標誌物 (stabilin, ephrinb2, flk-1, flt4 and mrc1) 的表達，此外Tg(fli:fsta)藉由TUNEL及AO檢測過度表達得知fsta導致ISV及CVP的缺陷不適因內皮細胞凋亡所造成的。我們注射fsta morpholino並未造成血管缺陷但是卻可以回復過度表達fsta的表徵，證實Tg(fli:fsta)造成血管缺陷的專一性。此外我利用in-situ hybridization檢測fsta mRNA表達量在isl2/coupTFIb mophants中確實是下降，對於fsta是否受isl2/coupTFIb的負向調控，我進一步共同注射fsta及isl2 morphlinos在斑馬魚胚胎中，會回復isl2 morphants所造成的血管缺陷，亦可回復 coupTFIb morphants的缺陷，因此我們得到的結論是fsta受isl2/coupTFIb-Notch訊息傳遞的負向調控，並可能透過參與BMP訊息傳遞做為重要調控CVP及靜脈血管生成的途徑。

While many regulators and signaling molecules that controlling the process of vascular development are described in zebrafish, genetic regulating the vein identity, intersegmental vessels (ISV) and caudal vein plexus (CVP) patterning is still not fully understood. We previously identified the transcription factors Islet2 (LIM homeobox gene, Isl2) and CoupTFIb(orphan nuclear receptor Chicken ovalbumin upstream promoter transcription factor II family) required for specification of the vein and ISV growth mediated by Notch pathway in zebrafish. Microarray analysis showed highly overlapping downstream targets controlled by coupTFIb and isl2, suggesting that coupTFIb and isl2 cooperatively regulate vascular development. In this study, we selectively analyze the functional relevance of one potential target from array hits, follistatin 1a (fsta). Fsta is a bone morphogenetic protein (BMP) antagonist related to axis formation during the development and BMP signaling has been shown involved in venous angiogenesis. However, there is no description of fsta functioning in vascular development so far. The interaction of notch and BMP signalings to regulate vasculization is unknown.
We first examine amino acid sequence comparison and phylogenetic analysis and show fsta is highly conserved among the vertebrates. Our in-situ hybridization results showed fsta mRNA is expressed in the lateral plate mesoderm at 18 somite stage and in vessels at 24-48hpf, suggesting its roles in vasculature. By overexpressing fsta driven by fli1 promoter in zebrafish embryos, we observed the vascular defects in the growth of ISV and CVP, which is mimic the phenotype of isl2/coupTFIb morphants. Consistent with the vascular defects in Tg(fli:fsta) embryo, we observe the changed expression of vascular markers (stabilin, ephrinb2, flk-1, flt4 and mrc1). We further performed TUNEL and AO assays to confirm the cell death is not the reasons to cause the decrease of ISV and CVP growth. On the other hand, knockdown of fsta does not cause vascular defects but it can restore the defects caused by fsta overexpression We further address the genetic interaction between fsta and isl2/coupTFIb, we found fsta expression is increased in isl2/coupTFIb morphants by in-situ hybridization. In addition, knockdown of fsta can partial rescue the vascular defects in coupTFIb and isl2 morphants. These data suggest fsta likely regulated by coupTFIb and isl2. Since fsta involved in BMP signals and coupTFIb and isl2 functions downstream of Notch, we conclude that fsta-BMP signaling play an important role to regulate vascular patterning, which interacts with isl2/coupTFIb-Notch signaling.

目錄

論文審定書 i
中文摘要 ii
Abstract iv
誌謝 vi
目錄 vii
圖表目錄 xi
中英文名詞對照表 xii
Abbreviation xiii
壹、前言 1
模式生物：斑馬魚 1
斑馬魚血管發育 2
VEGF-Notch訊號對動靜脈的分化的影響 3
Follistatin & BMP signaling 3
CoupTFIb與islet2調控血管脈絡的生成及下游目標基因 5
研究動機 6
貳、實驗材料與方法 13
斑馬魚的飼養繁殖以及受精卵收集 13
斑馬魚顯微注射法 13
嗎啉基 (Morpholino) 13
探針製作 14
原位組織染色(In situ hybridization) 14
冷凍組織切片 15
Tg(fli:fsta)及fsta mRNA序列的選殖及備製 16
pGL3-itm2bb upstream選殖 17
Cos-1細胞轉染 18
Total RNA- cDNA製作 18
Genome DNA 19
Quantitative PCR (qPCR) 20
Acridine orange (AO) staining 20
TUNEL Assay 20
參、實驗結果 22
1. CoupTFIb/isl2 mophants在DNA microarray中下游基因的表達 22
2. fsta在脊椎動物中具有高度的保守性 22
3. fsta在斑馬魚胚胎發育過程中表現的位置 23
4. Tg(fli:fsta)與isl2/coupTFIb mophants造成血管缺陷的表徵相似 23
5. Tg(fli:fsta)導致血液循環缺陷及心包膜水腫 24
6. Tg(fli:fsta)導致血管缺陷並非由細胞凋亡所造成 25
7. Tg(fli:fsta)影響血管標誌物表達量 25
8. fstaATG MO對斑馬魚血管發育沒有影響但可回復Tg(fli:fsta) 26
9. fstae3i3 MO專一性測試 26
10. fsta受上游基因isl2及coupTFIb調控 27
11. BMP inhibitor與Tg(fli:fsta)造成的血管缺陷相似，可能調控BMP signaling影響血管新生 27
12. fsta與上游基因Vegf與Notch之間的關係 28
13. fsta受coupTFIb/isl2調控下，在斑馬魚中扮演的角色 28
肆、問題與討論 31
1. fstaMO專一性作用在fsta並不作用在fstb，兩者有沒有交互作用 31
2. fsta是否透過BMP signaling影響血管的生成。 31
3. fstaMO是否會使血管過度生長 32
4. fsta序列中包含Follistatin-Like family，與血管生成是否有關 32
5. DNA microarray中的基因，在血管內皮細胞中可能扮演的角色 33
6. DNA microarray中的hdac4與fsta兩者之間與血管新生的關係 33
7. BMP signailing其他拮抗劑會不會影響靜脈血管生成 33
8. Itm2b跟癡呆症有關，itm2bb在斑馬魚中是否有相似表徵 34
伍、圖 38
圖一、CoupTFIb/isl2 mophants在DNA microarray中下游基因的表達 39
圖二、fsta在脊椎動物中具有高度的保守性 41
圖三、fsta在斑馬魚胚胎發育過程中表現的位置 42
圖四、Tg(fli:fsta)與isl2/coupTFIb mophants造成血管缺陷的表徵相似 43
圖五、Tg(fli:fsta)導致血液循環缺陷及心包膜水腫 44
圖六、Tg(fli:fsta)導致血管缺陷並非由細胞凋亡所造成 45
圖七、Tg(fli:fsta)影響血管標誌物表達量 46
圖八、fstaATG MO對斑馬魚血管發育沒有影響但可回復Tg(fli:fsta) 47
圖九、fstae3i3 MO專一性測試 48
圖十、fsta被上游基因isl2/coupTFIb調控 49
圖十一、BMP inhibitor與Tg(fli:fsta)造成的血管缺陷相似，可能調控BMP signaling影響血管新生 50
圖十二、fsta與上游基因Vegf與Notch之間的關係 51
圖十二、fsta受coupTFIb/isl2調控下，在斑馬魚中扮演的角色 52
陸、表格 53
柒、附錄 56
附件一、藥品配製(斑馬魚) 56
附件二、、藥品配製(細胞培養) 57
附件三、藥品配製(protein及電泳緩衝液及配置條件) 58
附件五、itm2bb在血管生成重要時期的表達位置 62
附件六、itm2bbATG MO造成血管缺陷 63
附件七、itm2bbi2e3 MO專一性的測試 64
附件八、itm2bbATG MO導致血管缺陷與細胞凋亡的關係 65
附件九、itm2bbi2e3 MO導致血液循環缺陷及心包膜水腫 66
附件十、itm2bbATG MO影響血管標誌物表達量 67
附件十一、過度表達itm2bb造成些微的血管缺陷 68
附件十二、斑馬魚itm2bb upstream不同長度區的活性測試 69
附件十三、itm2bb與上游基因Vegf與Notch之間的關係 70
附件十四、pCR4-TOPO-fsta 71
附件十五、pCS2plus-fsta 72
附件十六、pTol/fli/fsta/cg2 73
附件十七、pTol/fli/itm2bb/cg2 74
附件十八、pGL3-itm2bb-3.5k 75
附件十九、pGL3-itm2bb-2.5k 76
附件二十、pGL3-itm2bb-1.5k 77
附件二十一、pGL3-itm2bb-1k 78
捌、參考文獻 79

圖表目錄
圖一、CoupTFIb/isl2 mophants在DNA microarray中下游基因的表達 35
圖二、fsta在脊椎動物中具有高度的保守性 36
圖三、fsta在斑馬魚胚胎發育過程中表現的位置 37
圖五、Tg(fli:fsta)導致血液循環缺陷及心包膜水腫 39
圖六、Tg(fli:fsta)導致血管缺陷並非由細胞凋亡所造成 40
圖七、Tg(fli:fsta)影響血管標誌物表達量 41
圖八、fstaATG MO對斑馬魚血管發育沒有影響但可回復Tg(fli:fsta) 42
圖九、fstae3i3 MO專一性測試 43
圖十、fsta被上游基因isl2/coupTFIb調控 44
圖十一、BMP inhibitor與Tg(fli:fsta)造成的血管缺陷相似，可能調控BMP signaling影響血管新生 45
圖十二、fsta與上游基因Vegf與Notch之間的關係 46
圖十二、fsta受coupTFIb/isl2調控下，在斑馬魚中扮演的角色 47
表一、probe construct、Tol2 construct及itm2bb Luceferace assay construct primer 48
表二、QPCR primer、Mopholino efficiency primer 49
表三、Mopholino及Probe 50

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