脊椎動物的血管脈絡在胚胎生長發育的過程中扮演著關鍵性的角色，對於研究基因調控與血管新生具備有非常之重要性。利用斑馬魚做為模式生物之研究已經發現許多基因可以調控血管發育。斑馬魚的前期研究中，我們發現islet2和coup-TF1b(Chicken ovalbumin upstream promoter transcription factor 1b)對於靜脈分化以及Notch訊息路徑會調控的主幹區間血管 (Intersegmental vessel, ISV) 其生成發育扮演著不可或缺的角色。因此，我們探討coup-TF1b下游基因對血管的生長發育之影響，並且評估其生物訊息傳遞路徑。首先我們利用cDNA微陣列(cDNA microarray)的實驗方法，尋找血管的生長發育會有影響的候選基因做為研究方向，我們找到一個新穎的候選基因-麻木蛋白的配體-X1 (ligand of numb-protein X1, lnx1)，搜尋分析此基因的胺基酸序列，因此發現在各種脊椎動物上有高度的相似性，本論文的主要研究目標為探討lnx1對於斑馬魚血管脈絡的生成以及血液循環的影響。研究發現在發育中的血管會增加lnx1 mRNA表現量，利用嗎啉基/反義寡核苷酸(Morpholino，MO)抑制lnx1導致斑馬魚胚胎的血管發育在主幹區間血管與尾部靜脈叢(caudal vein plexus, CVP)無法形成連結蜂巢狀的網狀結構等缺陷，根據這些現象推測lnx1參與靜脈分化以及主幹區間血管的生成。進一步發現缺少lnx1的血管容易導致血液無法正常輸送到全身而聚集於心臟部份，進而影響血液循環，而導致血管生成異常。推測缺少lnx1造成斑馬魚血管內皮細胞死亡進而影響靜脈分化以及主幹區間血管的生成，但當利用吖啶橙染色(Acridine orange staining, AO)與末端脫氧核甘酸轉移酶標定物法(Terminal deoxynucleotidyl transferase dUTP nick end labeling assay, TUNEL assay)等實驗方法發現缺少lnx1並不會造成斑馬魚內皮細胞的死亡，接下來進行測試血管發育缺陷的可能分子機制，實驗研究結果發現缺少lnx1會減少血管動脈與靜脈特異性標誌物的蛋白質表現量(如fl4, flk, ephrin B2, mrc1, stabilin)，最後，實驗結果發現缺少lnx1會影響斑馬魚的血管內皮細胞生長因子(vascular endothelial growth factor, VEGF)家族中的vegfaa和ERK蛋白質表現量，因此推論斑馬魚缺少lnx1是透過減弱或抑制VEGF/ERK 和Notch的訊號傳遞來遲緩血管發育。

Because establishment of blood vessels in vertebrates is highly conserved in evolution, and extremely important in embryogenesis, zebrafish have become a widely accepted model that has been used to identify many genes involved in vascular development. Using zebrafish, we found that transcription factors islet2 and coup-TF1b act via the Notch signaling pathway to control vein and intersegmental vessel (ISV) growth. We further investigated the downstream targets of islet2 and coup-TF1b by microarray, and identified ligand of numb-protein X 1 (lnx1) as a potential downstream target that may mediate vasculogenesis. Amino acid sequence alignment and phylogenetic analysis revealed that lnx1 is highly conserved in vertebrates. To study the effects of lnx1 on blood vessel formation, we first showed that lnx1 mRNA is expressed in developing vessels. Morpholino knockdown of lnx1 led to vascular growth defects wherein a mesh-like pattern was unable to form in the tail vein plexus, suggesting a necessity for lnx1 in promoting ISV and caudal vein plexus (CVP) growth. We further demonstrated that the defects in vessel development were associated with edema and impaired circulation. To address whether cell death contributed to the inhibition of ISV and CVP growth, we performed acridine orange stain (AO stain) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays. The data showed no increase in endothelial cell apoptosis after morpholino injection, suggesting that cell death is not the cause of the observed vascular phenotype. Moreover, we examined the expression of vascular markers (flt4, flk, ephrin B2, mrc1, stabilin) and found the expression was decreased, demonstrating the effect of lnx1 morpholino on blood vessels. Finally, we examined the interaction between various signaling pathways and lnx1. We found inactivation of VEGF and Notch signals reduced the expression of lnx1, and knockdown of lnx1 caused reductions in vegfaa and ERK expression. Overall, we conclude that the loss of lnx1 impairs vascular development, and that this effect is mediated by VEGF-ERK/Notch signaling in zebrafish.

目 錄
論文審定書 i
論文公開授權書 ii
致 謝 iii
中文摘要 iv
Abstract vi
目 錄 viii
圖 次 xiii
表 次 xiv
中英文名詞對照表 xv
Abbreviation xvii
壹、前 言 1
一、 血管新生的發展 1
二、 模式生物-斑馬魚的近況 1
三、 探討斑馬魚血管脈絡的應用 2
四、 動脈與靜脈分化的機制 3
五、 VEGF訊息調控路徑 3
六、 Notch訊息調控在斑馬魚血管生成的機制 4
七、 Islet2與coupTF1b調控班馬魚血管生成與下游基因的表現 4
八、 BMP訊息調控扮演的角色 5
九、 針對lnx1參與斑馬魚胚胎血管發育的研究動機 5
Figure 1. 脈管生成與血管新生 7
Figure 2. 斑馬魚血管脈絡示意圖 8
Figure 3. 血管的發育系統 9
Figure 4. VEGF訊息傳遞路徑示意圖 10
Figure 5. Notch訊息傳遞路徑示意圖 11
Figure 6. BMP訊息傳遞路徑示意圖 12
貳、實驗材料與方法 13
一、 實驗動物:模式生物-斑馬魚 13
1. 斑馬魚魚種分類 13
2. 斑馬魚飼養繁殖 13
3. 斑馬魚受精胚胎收集 13
二、 顯微注射法(microinjection) 14
1. 顯微注射器 14
2. 胚胎的處理與注射過程 14
3. 嗎啉基/反義寡核苷酸(Morpholino，MO) 15
三、 萃取total RNA與cDNA製作 15
1. 萃取total RNA: 15
2. cDNA製作: 16
四、 聚合酶連鎖反應(PCR) 16
五、 即時聚合酶連鎖反應 (real-time PCR) 17
六、 探針製作(mRNA probe) 17
七、 原位組織染色(In situ hybridization) 19
1. 胚胎的前處理: 19
2. 原位組織染色 20
八、 細胞凋亡檢測 21
1. TUNEL Assay 21
2. Acridine orange (AO staining)染色 22
九、 螢光影像攝影 22
十、 冷凍組織切片 22
1. 組織處理 22
2. 切片機使用 23
十一、 自斑馬魚魚體萃取蛋白質 23
1. 萃取蛋白質 23
2. 測蛋白質濃度 24
十二、 西方墨點法 (Western Blot) 24
十三、 影像處理與統計分析軟體 25
叁、研究結果 26
一、 利用DNA microarray 探討islet2/coupTF1b下游基因 26
二、 lnx1在生物資訊的脊椎動物中所具有的演化特性 26
三、 lnx1在zebrafish胚胎發育中其模式的表現 27
四、 Morpholino knockdown zebrafish 在phenotype是否具有專一性 27
五、 證實lnx1 knockdown zebrafish embryos 時導致血管異常 28
六、 使用lnx1ATG morpholino knockdown檢視其血管發育缺陷過程中之影響. 29
七、 觀察lnx1 morpholino knockdown斑馬魚胚胎後，對動脈與靜脈標記物形成缺陷的主因 30
八、 lnx1 morpholino knockdown斑馬魚胚胎後，影響血管生成導致血液循環缺陷與心包膜腫大 31
九、 探討細胞凋亡、增生、分化或遷移對血管生成影響的關聯 31
十、 證實上游基因導致抑制後rescue 對lnx1 morpholino knockdown所形成的缺陷 32
十一、 證實Notch/ Vegf/ BMP訊息調控與lnx1的關聯性 33
十二、 就血管生成的分子機制在lnx1 morpholino knockdown斑馬魚胚胎的表現 35
肆、討 論 36
一、 透過cDNA microarray篩選可能影響血管生成的基因 36
二、 Lnx透過蛋白質結構中的關聯性是否影響血管生成 36
三、 就lnx1與E3泛素連接酶的交互作用 37
四、 Islet2/coupTF1b對於lnx1基因的表現 37
五、 Notch訊息傳遞對調控下游基因Hey/Hes的關聯 38
六、 BMP訊息路徑是否導致lnx1血管缺陷 38
七、 lnx1i1e2 MO與lnx1ATG MO是否導致血管異常缺陷 38
八、 lnx1藉由Numb抑制Notch訊息傳遞調控血管生成 39
九、 Notch訊息傳遞調控抑制islet2/coupTF1b，lnx1為其受負向調控影響血管生成，推論是否影響lnx1過度表現 40
伍、圖 41
圖一、利用DNA microarray 探討islet2/coupTF1b下游基因 42
圖二、lnx1在生物資訊的脊椎動物中所具有的演化特性 44
圖三、lnx1在zebrafish胚胎發育中模式 45
圖四、設計morpholino knockdown zebrafish 在phenotype是否具有專一性 47
圖五、證實lnx1 i1e2 MO knockdown zebrafish embryos 導致血管異常 49
圖六、使用lnx1 ATG MO knockdown檢視其血管發育缺陷過程中之影響 51
圖七、觀察lnx1 i1e2 MO knockdown斑馬魚胚胎後，對動脈與靜脈標記物形成缺陷的主因 53
圖八、lnx1 i1e2 MO knockdown斑馬魚胚胎後，影響血管生成導致血液循環缺陷與心包膜腫大 55
圖九、探討細胞凋亡、增生、分化或遷移對血管生成影響的關聯 57
圖十、證實上游基因coupTF1b抑制lnx1，經由rescue血管缺陷的調控 59
圖十一、證實Notch/ VEGF/ BMP訊息調控與lnx1的關聯性 61
圖十二、血管生成的分子機制在lnx1i1e2 morpholino knockdown斑馬魚胚胎的表現 63
陸、附 錄 64
附件一、觀察lnx1 ATG MO knockdown斑馬魚胚胎後，對動脈與靜脈標記物形成缺陷的主因 65
附件二、lnx1ATG MO knockdown斑馬魚胚胎後，影響血管生成導致血液循環缺陷與心包膜腫大 66
附件三、探討細胞凋亡、增生、分化或遷移對血管生成影響的關聯 68
附件四、溶液與藥品的配製 70
附件五、使用藥品廠牌一覽表 79
附件六、Sequence and Multi-Cloning Site of the pGEM®-T Vector 80
附件七、pGEM®-T Easy Vector Map and Sequence Reference Points 81
柒、表 格 82
捌、參考文獻 84

圖 次
圖一、利用DNA microarray 探討islet2/coupTF1b下游基因 42
圖二、lnx1在生物資訊的脊椎動物中所具有的演化特性 44
圖三、lnx1在zebrafish胚胎發育中模式 45
圖四、設計morpholino knockdown zebrafish 在phenotype是否具有專一性 47
圖五、證實lnx1 i1e2 MO knockdown zebrafish embryos 導致血管異常 49
圖七、觀察lnx1 i1e2 MO knockdown斑馬魚胚胎後，對動脈與靜脈標記物形成缺陷的主因 53
圖八、lnx1 i1e2 MO knockdown斑馬魚胚胎後，影響血管生成導致血液循環缺陷與心包膜腫大 55
圖九、探討細胞凋亡、增生、分化或遷移對血管生成影響的關聯 57
圖十、證實上游基因coupTF1b抑制lnx1，經由rescue血管缺陷的調控 59
圖十一、證實Notch/ VEGF/ BMP訊息調控與lnx1的關聯性 61
圖十二、血管生成的分子機制在lnx1i1e2 morpholino knockdown斑馬魚胚胎的表現 63
附件一、觀察lnx1 ATG MO knockdown斑馬魚胚胎後，對動脈與靜脈標記物形成缺陷的主因 65
附件二、lnx1ATG MO knockdown斑馬魚胚胎後，影響血管生成導致血液循環缺陷與心包膜腫大 66
附件三、探討細胞凋亡、增生、分化或遷移對血管生成影響的關聯 68


表 次
表一、即時聚合酶連鎖反應 (real-time PCR) 82
表二、Mopholino及Probe 82
表三、抗體資料 83
表四、BMP/VEGF/Notch 抑制劑 83

1. Carmeliet P, Jain RK: Angiogenesis in cancer and other diseases. Nature 2000, 407:249-257.
2. Folkman J: Tumor angiogenesis: therapeutic implications. N Engl J Med 1971, 285:1182-1186.
3. Weinstein B: Vascular cell biology in vivo: a new piscine paradigm? Trends Cell Biol 2002, 12:439-445.
4. Amatruda JF, Shepard JL, Stern HM, Zon LI: Zebrafish as a cancer model system. Cancer Cell 2002, 1:229-231.
5. Lieschke GJ, Currie PD: Animal models of human disease: zebrafish swim into view. Nat Rev Genet 2007, 8:353-367.
6. Gore AV, Monzo K, Cha YR, Pan W, Weinstein BM: Vascular development in the zebrafish. Cold Spring Harb Perspect Med 2012, 2:a006684.
7. Adams RH, Alitalo K: Molecular regulation of angiogenesis and lymphangiogenesis. Nat Rev Mol Cell Biol 2007, 8:464-478.
8. Agathon A, Thisse C, Thisse B: The molecular nature of the zebrafish tail organizer. Nature 2003, 424:448-452.
9. Carmeliet P: Angiogenesis in health and disease. Nat Med 2003, 9:653-660.
10. Mudumana SP, Hentschel D, Liu Y, Vasilyev A, Drummond IA: odd skipped related1 reveals a novel role for endoderm in regulating kidney versus vascular cell fate. Development 2008, 135:3355-3367.
11. Pardanaud L, Yassine F, Dieterlen-Lievre F: Relationship between vasculogenesis, angiogenesis and haemopoiesis during avian ontogeny. Development 1989, 105:473-485.
12. Eichmann A, Yuan L, Moyon D, Lenoble F, Pardanaud L, Breant C: Vascular development: from precursor cells to branched arterial and venous networks. Int J Dev Biol 2005, 49:259-267.
13. Blum Y, Belting HG, Ellertsdottir E, Herwig L, Luders F, Affolter M: Complex cell rearrangements during intersegmental vessel sprouting and vessel fusion in the zebrafish embryo. Dev Biol 2008, 316:312-322.
14. Gerhardt H, Golding M, Fruttiger M, Ruhrberg C, Lundkvist A, Abramsson A, Jeltsch M, Mitchell C, Alitalo K, Shima D, Betsholtz C: VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia. J Cell Biol 2003, 161:1163-1177.
15. Jin SW, Beis D, Mitchell T, Chen JN, Stainier DY: Cellular and molecular analyses of vascular tube and lumen formation in zebrafish. Development 2005, 132:5199-5209.
16. Kamei M, Saunders WB, Bayless KJ, Dye L, Davis GE, Weinstein BM: Endothelial tubes assemble from intracellular vacuoles in vivo. Nature 2006, 442:453-456.
17. Schuermann A, Helker CS, Herzog W: Angiogenesis in zebrafish. Semin Cell Dev Biol 2014, 31:106-114.
18. Ridgway J, Zhang G, Wu Y, Stawicki S, Liang WC, Chanthery Y, Kowalski J, Watts RJ, Callahan C, Kasman I, et al: Inhibition of Dll4 signalling inhibits tumour growth by deregulating angiogenesis. Nature 2006, 444:1083-1087.
19. Wiley DM, Kim JD, Hao J, Hong CC, Bautch VL, Jin SW: Distinct signalling pathways regulate sprouting angiogenesis from the dorsal aorta and the axial vein. Nat Cell Biol 2011, 13:686-692.
20. Childs S, Chen JN, Garrity DM, Fishman MC: Patterning of angiogenesis in the zebrafish embryo. Development 2002, 129:973-982.
21. Duarte A, Hirashima M, Benedito R, Trindade A, Diniz P, Bekman E, Costa L, Henrique D, Rossant J: Dosage-sensitive requirement for mouse Dll4 in artery development. Genes Dev 2004, 18:2474-2478.
22. Sorensen I, Adams RH, Gossler A: DLL1-mediated Notch activation regulates endothelial identity in mouse fetal arteries. Blood 2009, 113:5680-5688.
23. Hellstrom M, Phng LK, Gerhardt H: VEGF and Notch signaling: the yin and yang of angiogenic sprouting. Cell Adh Migr 2007, 1:133-136.
24. You LR, Lin FJ, Lee CT, DeMayo FJ, Tsai MJ, Tsai SY: Suppression of Notch signalling by the COUP-TFII transcription factor regulates vein identity. Nature 2005, 435:98-104.
25. Shibuya M: Tyrosine Kinase Receptor Flt/VEGFR Family: Its Characterization Related to Angiogenesis and Cancer. Genes Cancer 2010, 1:1119-1123.
26. Eklund L, Saharinen P: Angiopoietin signaling in the vasculature. Exp Cell Res 2013, 319:1271-1280.
27. Olofsson B, Korpelainen E, Pepper MS, Mandriota SJ, Aase K, Kumar V, Gunji Y, Jeltsch MM, Shibuya M, Alitalo K, Eriksson U: Vascular endothelial growth factor B (VEGF-B) binds to VEGF receptor-1 and regulates plasminogen activator activity in endothelial cells. Proc Natl Acad Sci U S A 1998, 95:11709-11714.
28. Ogawa S, Oku A, Sawano A, Yamaguchi S, Yazaki Y, Shibuya M: A novel type of vascular endothelial growth factor, VEGF-E (NZ-7 VEGF), preferentially utilizes KDR/Flk-1 receptor and carries a potent mitotic activity without heparin-binding domain. J Biol Chem 1998, 273:31273-31282.
29. Dumont DJ, Jussila L, Taipale J, Lymboussaki A, Mustonen T, Pajusola K, Breitman M, Alitalo K: Cardiovascular failure in mouse embryos deficient in VEGF receptor-3. Science 1998, 282:946-949.
30. Kume T: Novel insights into the differential functions of Notch ligands in vascular formation. J Angiogenes Res 2009, 1:8.
31. Dyer LA, Pi X, Patterson C: The role of BMPs in endothelial cell function and dysfunction. Trends Endocrinol Metab 2014, 25:472-480.
32. Norrie JL, Lewandowski JP, Bouldin CM, Amarnath S, Li Q, Vokes MS, Ehrlich LI, Harfe BD, Vokes SA: Dynamics of BMP signaling in limb bud mesenchyme and polydactyly. Dev Biol 2014, 393:270-281.
33. Walsh DW, Godson C, Brazil DP, Martin F: Extracellular BMP-antagonist regulation in development and disease: tied up in knots. Trends Cell Biol 2010, 20:244-256.
34. Kim JD, Kim J: Alk3/Alk3b and Smad5 mediate BMP signaling during lymphatic development in zebrafish. Mol Cells 2014, 37:270-274.
35. van Wijk B, Moorman AF, van den Hoff MJ: Role of bone morphogenetic proteins in cardiac differentiation. Cardiovasc Res 2007, 74:244-255.
36. Wakayama Y, Fukuhara S, Ando K, Matsuda M, Mochizuki N: Cdc42 mediates Bmp-induced sprouting angiogenesis through Fmnl3-driven assembly of endothelial filopodia in zebrafish. Dev Cell 2015, 32:109-122.
37. Kim JD, Kang H, Larrivee B, Lee MY, Mettlen M, Schmid SL, Roman BL, Qyang Y, Eichmann A, Jin SW: Context-dependent proangiogenic function of bone morphogenetic protein signaling is mediated by disabled homolog 2. Dev Cell 2012, 23:441-448.
38. Nie J, McGill MA, Dermer M, Dho SE, Wolting CD, McGlade CJ: LNX functions as a RING type E3 ubiquitin ligase that targets the cell fate determinant Numb for ubiquitin-dependent degradation. EMBO J 2002, 21:93-102.
39. Flynn M, Saha O, Young P: Molecular evolution of the LNX gene family. BMC Evol Biol 2011, 11:235.
40. Even J, Eskander M, Kang J: Bone morphogenetic protein in spine surgery: current and future uses. J Am Acad Orthop Surg 2012, 20:547-552.
41. Oliver G: Lymphatic vasculature development. Nat Rev Immunol 2004, 4:35-45.
42. Covassin LD, Villefranc JA, Kacergis MC, Weinstein BM, Lawson ND: Distinct genetic interactions between multiple Vegf receptors are required for development of different blood vessel types in zebrafish. Proc Natl Acad Sci U S A 2006, 103:6554-6559.
43. Pardali E, Goumans MJ, ten Dijke P: Signaling by members of the TGF-beta family in vascular morphogenesis and disease. Trends Cell Biol 2010, 20:556-567.
44. Holderfield MT, Hughes CC: Crosstalk between vascular endothelial growth factor, notch, and transforming growth factor-beta in vascular morphogenesis. Circ Res 2008, 102:637-652.
45. Fischer A, Gessler M: Delta-Notch--and then? Protein interactions and proposed modes of repression by Hes and Hey bHLH factors. Nucleic Acids Res 2007, 35:4583-4596.
46. Gamez B, Rodriguez-Carballo E, Ventura F: BMP signaling in telencephalic neural cell specification and maturation. Front Cell Neurosci 2013, 7:87.
47. Deshane J, Chen S, Caballero S, Grochot-Przeczek A, Was H, Li Calzi S, Lach R, Hock TD, Chen B, Hill-Kapturczak N, et al: Stromal cell-derived factor 1 promotes angiogenesis via a heme oxygenase 1-dependent mechanism. J Exp Med 2007, 204:605-618.
48. Rual JF, Venkatesan K, Hao T, Hirozane-Kishikawa T, Dricot A, Li N, Berriz GF, Gibbons FD, Dreze M, Ayivi-Guedehoussou N, et al: Towards a proteome-scale map of the human protein-protein interaction network. Nature 2005, 437:1173-1178.
49. Yu Y, Li H, Xue B, Jiang X, Huang K, Ge J, Zhang H, Chen B: SDF-1/CXCR7 axis enhances ovarian cancer cell invasion by MMP-9 expression through p38 MAPK pathway. DNA Cell Biol 2014, 33:543-549.
50. Berezovska O, McLean P, Knowles R, Frosh M, Lu FM, Lux SE, Hyman BT: Notch1 inhibits neurite outgrowth in postmitotic primary neurons. Neuroscience 1999, 93:433-439.
51. Chen J, Xu J, Zhao W, Hu G, Cheng H, Kang Y, Xie Y, Lu Y: Characterization of human LNX, a novel ligand of Numb protein X that is downregulated in human gliomas. Int J Biochem Cell Biol 2005, 37:2273-2283.
52. Heasman J: Morpholino oligos: making sense of antisense? Dev Biol 2002, 243:209-214.
53. Katoh M, Katoh M: NUMB is a break of WNT-Notch signaling cycle. Int J Mol Med 2006, 18:517-521.

圖 次
圖一、利用DNA microarray 探討islet2/coupTF1b下游基因 42
圖二、lnx1在生物資訊的脊椎動物中所具有的演化特性 44
圖三、lnx1在zebrafish胚胎發育中模式 45
圖四、設計morpholino knockdown zebrafish 在phenotype是否具有專一性 47
圖五、證實lnx1 i1e2 MO knockdown zebrafish embryos 導致血管異常 49
圖七、觀察lnx1 i1e2 MO knockdown斑馬魚胚胎後，對動脈與靜脈標記物形成缺陷的主因 53
圖八、lnx1 i1e2 MO knockdown斑馬魚胚胎後，影響血管生成導致血液循環缺陷與心包膜腫大 55
圖九、探討細胞凋亡、增生、分化或遷移對血管生成影響的關聯 57
圖十、證實上游基因coupTF1b抑制lnx1，經由rescue血管缺陷的調控 59
圖十一、證實Notch/ VEGF/ BMP訊息調控與lnx1的關聯性 61
圖十二、血管生成的分子機制在lnx1i1e2 morpholino knockdown斑馬魚胚胎的表現 63
附件一、觀察lnx1 ATG MO knockdown斑馬魚胚胎後，對動脈與靜脈標記物形成缺陷的主因 65
附件二、lnx1ATG MO knockdown斑馬魚胚胎後，影響血管生成導致血液循環缺陷與心包膜腫大 66
附件三、探討細胞凋亡、增生、分化或遷移對血管生成影響的關聯 68


表 次
表一、即時聚合酶連鎖反應 (real-time PCR) 82
表二、Mopholino及Probe 82
表三、抗體資料 83
表四、BMP/VEGF/Notch 抑制劑 83