脊椎動物在胚胎生長的過程中，血管發育的扮演重要角色，我們以斑馬魚作為模式生物，研究血管發育的分子機制:基因如何調控動靜脈的分化、區間血管(intersegmental vessel, ISV)與尾部靜脈叢caudal vein plexus (CVP)的生長。其中區間血管與尾部靜脈叢生長的相關訊息傳遞路徑研究仍然不完整。先前實驗室成員利用反義寡核苷酸(morpholino，MO)抑制ligand of numb-protein X1 (lnx1)表現，發現會導致斑馬魚胚胎的區間血管(ISV)發育缺陷與尾部靜脈叢(CVP)無法形成網狀脈絡，進而產生血液循環缺陷及心包膜腫大的現象；藉由AO染色與TUNEL分析發現區間血管內皮細胞數目減少，並非是由細胞凋亡所致，推測是減少血管內皮細胞增生與遷移能力。
本研究將延續先前的結果，探討lnx1 MO造成血管缺陷的專一性以及lnx1如何調控血管發育的分子機制。為了檢測morpholino對lnx1專一性以及作用效率，我們注射splicing morpholino，證實有相似的血管缺陷結果。此外，建構能與lnx1ATG結合且表現綠螢光的質體，將lnx1 ATG -GFP mRNA和lnx1ATGMO一起注射，結果顯示lnx1ATGMO能阻斷lnx1ATG位置，使lnx1 ATG -GFP mRNA無法結合而不表現綠螢光。另外我也建構過度表現lnx1載體，發現過度表現lnx1能回復lnx1 MO造成的血管缺陷，證實lnx1ATGMO的專一性以及效力。
我們進一步探討血管發育的分子機制，當注射lnx1 MO後，原位組織染色及qPCR結果顯示血管相關基因(flt4, flk, ephrin B2, mrc1, stabilin)表現量皆有下降的情形，符合血管缺陷的表徵，我們也發現lnx1與VEGF和BMP訊息路徑之間有交互作用，當抑制VEGF和BMP訊息路徑，會減低lnx1的表現量，而注射lnx1 MO造成VEGF和BMP相關訊息路徑的蛋白質表現降低。
綜合以上結果，我們認為lnx1缺失造成血管發育缺陷，是藉由影響VEGF 和 BMP路徑而造成斑馬魚血管發育的缺陷。

The establishment of blood vessels is important for embryo growth and survival in vertebrate. Using zebrafish as a model organism to study vascular development has been shown many molecules that are important for artery-vein identication, intersegmental vessel (ISV) patterning and caudal vein plexus (CVP) formation. However, the understanding of molecule mechanisms for ISV and CVP formation remains incomplete. In the previous study, we found that morpholino knockdown of lnx1 impairs the growth of ISV and CVP, and we further observed the edema and circulation defects associated with the vessel impariment. We next demonstrated the reduction of ISV endothelial cells and the defect of CVP sprouting in lnx1 morphants by using Tg(Kdrl:mcherry;fli1a:negfp)y7. AO staining and TUNEL assay showed that vascular defects are not results from cell death, but due to decrease of cell proliferation and/or migration.
In this study, I confirmed the specificity of lnx1 MO for vascular defects, and examine lnx1 how to regulate molecular mechanisms that lnx1 control vascular development. To test the specificity of lnx1 morpholino knockdown, we performed the 2nd MO interfere block splicing site. The result showed vascular defects similar to lnx1ATG MO in lnx1i1e2 morphants. In addition, I build EGFP expression construct that can bind to lnx1ATG MO target site. After injecting lnx1 ATG -GFP mRNA with lnx1ATGMO, it will block EGFP expression. And lnx1 overexpression can reduce the vascular defects caused by lnx1ATG MO. According to these results, I confirmed the specificity of lnx1 morpholino knockdown.
Finally, I examined the molecule mechanism of vascular development regulated by lnx1. I analyzed the relationship between lnx1 and vascular marker genes, and found knockdown of lnx1 reduces the expression of vascular markers. flt4, flk, ephrinB2, mrc1 and stabilin. Besides, I also studied the interaction among lnx1, VEGF and BMP signals；I found inactivation of VEGF and BMP signals reduce the expression of lnx1, and knockdown of lnx1 reduces the protein levels related to VEGF and BMP signal pathways.
Together, the loss of lnx1 impairs vascular development, and this effect is mediated by VEGF and BMP signalings in zebrafish.

論文審定書 i
致謝 ii
中文摘要 iii
Abstract iv
圖次 ix
表次 x
Abbreviation xi
壹、前言 1
一、血管生成發育的重要性 1
二、模式生物:斑馬魚 1
三、斑馬魚血管發育 2
四、VEGF與Notch訊息調控動靜脈分化 3
五、BMP訊息調控路徑 4
六、lnx1對斑馬魚胚胎血管發育的影響與研究動機 5
貳、實驗材料方法 7
一、斑馬魚品系及繁養殖 7
二、斑馬魚受精卵收集與培養 7
三、顯微注射法(microinjection) 7
四、嗎啉基/反義寡核苷酸(morpholino， MO) 8
五、mRNA 合成 8
六、Tol2-lnx1 質體建構 8
七、pCSDest-lnx1質體建構 9
八、lnx1 ATG MO質體建構 10
九、探針(probe)製作 10
十、原位組織染色 (In situ hybridization) 10
十一、抑制劑處理胚胎 11
十二、Total RNA 萃取與 cDNA 的製作 12
十三、即時定量聚合酶連鎖反應(Real-time Quantitative Polymerase Chain Reaction， Q-PCR ) 12
十四、免疫螢光染色(immunofluorescent, IF) 13
十五、萃取蛋白質(total protein extraction) 13
十六、西方墨點法(western blot) 14
十七、影像處理 14
十八、統計分析軟體 14
參、實驗結果 15
一、 lnx1ATG morpholin Knockdown與斑馬魚血管缺陷專一性 15
二、 過度表現lnx1不影響血管異常增生或缺陷 17
三、Knockdown lnx1造成血管缺陷原因 17
四、lnx1與血管發育相關路徑 18
五、總結 20
肆、問題與討論 21
一、 過度表現lnx1不導致血管發育缺陷 21
二、 過度表現lnx1無法回復因抑制VEGF/BMP訊息路徑所致的血管缺失 21
三、 注射lnx1 MO後，lnx1蛋白質表現量無下降 22
四、 利用control MO檢測MO專一性 22
五、 lnx1與lnx2蛋白質結構相似是否也會影響血管生成 22
六、 lnx1、numb及Notch訊息傳遞路徑對胚胎斑馬魚血管生成的關係 23
七、lnx1可能與PKCα進行交互作用影響血管新生 23
伍、圖 25
陸、表 37
柒、參考文獻 40
捌、附錄 43
附件一、Knockdown lnx1導致斑馬魚血管發育缺陷 43
附件二、Knockdown lnx1所導致血管缺陷是主因並非是細胞凋亡 44
附件三、過度表達lnx1無法能回復SU5416與DMH1造成的血管缺陷 45
附件四、pCSDest-lnx1基因建構圖 46
附件五、pDestTol2CG-fli-lnx1-RFP 基因建構圖 47
附件六、哺乳類動物LNX家族蛋白質結構域圖 48
附件七、藥品配置 49

1. Lawson, N.D. and B.M. Weinstein, Arteries and veins: making a difference with zebrafish. Nat Rev Genet, 2002. 3(9): p. 674-82.
2. Wilkinson, R.N. and F.J. van Eeden, The zebrafish as a model of vascular development and disease. Prog Mol Biol Transl Sci, 2014. 124: p. 93-122.
3. Brown, D.R., et al., Advances in the Study of Heart Development and Disease Using Zebrafish. J Cardiovasc Dev Dis, 2016. 3(2).
4. Lieschke, G.J. and P.D. Currie, Animal models of human disease: zebrafish swim into view. Nat Rev Genet, 2007. 8(5): p. 353-67.
5. Howe, K., et al., The zebrafish reference genome sequence and its relationship to the human genome. Nature, 2013. 496(7446): p. 498-503.
6. Adams, R.H. and K. Alitalo, Molecular regulation of angiogenesis and lymphangiogenesis. Nat Rev Mol Cell Biol, 2007. 8(6): p. 464-78.
7. Ellertsdottir, E., et al., Vascular morphogenesis in the zebrafish embryo. Dev Biol, 2010. 341(1): p. 56-65.
8. Blanco, R. and H. Gerhardt, VEGF and Notch in tip and stalk cell selection. Cold Spring Harb Perspect Med, 2013. 3(1): p. a006569.
9. Kashiwada, T., et al., beta-Catenin-dependent transcription is central to Bmp-mediated formation of venous vessels. Development, 2015. 142(3): p. 497-509.
10. Wiley, D.M., et al., Distinct signalling pathways regulate sprouting angiogenesis from the dorsal aorta and the axial vein. Nat Cell Biol, 2011. 13(6): p. 686-92.
11. Shibuya, M., Tyrosine Kinase Receptor Flt/VEGFR Family: Its Characterization Related to Angiogenesis and Cancer. Genes Cancer, 2010. 1(11): p. 1119-23.
12. Kuchler, A.M., et al., Development of the zebrafish lymphatic system requires VEGFC signaling. Curr Biol, 2006. 16(12): p. 1244-8.
13. Kume, T., Novel insights into the differential functions of Notch ligands in vascular formation. J Angiogenes Res, 2009. 1: p. 8.
14. Lee, C.Y., et al., Notch signaling functions as a cell-fate switch between the endothelial and hematopoietic lineages. Curr Biol, 2009. 19(19): p. 1616-22.
15. Leslie, J.D., et al., Endothelial signalling by the Notch ligand Delta-like 4 restricts angiogenesis. Development, 2007. 134(5): p. 839-44.
16. Siekmann, A.F. and N.D. Lawson, Notch signalling limits angiogenic cell behaviour in developing zebrafish arteries. Nature, 2007. 445(7129): p. 781-4.
17. Lawson, N.D., et al., Notch signaling is required for arterial-venous differentiation during embryonic vascular development. Development, 2001. 128(19): p. 3675-83.
18. Kume, T., Specification of arterial, venous, and lymphatic endothelial cells during embryonic development. Histol Histopathol, 2010. 25(5): p. 637-46.
19. Wakayama, Y., et al., Cdc42 mediates Bmp-induced sprouting angiogenesis through Fmnl3-driven assembly of endothelial filopodia in zebrafish. Dev Cell, 2015. 32(1): p. 109-22.
20. Wang, R.N., et al., Bone Morphogenetic Protein (BMP) signaling in development and human diseases. Genes Dis, 2014. 1(1): p. 87-105.
21. Cai, J., et al., BMP signaling in vascular diseases. FEBS Lett, 2012. 586(14): p. 1993-2002.
22. Dyer, L.A., X. Pi, and C. Patterson, The role of BMPs in endothelial cell function and dysfunction. Trends Endocrinol Metab, 2014. 25(9): p. 472-80.
23. Flynn, M., O. Saha, and P. Young, Molecular evolution of the LNX gene family. BMC Evol Biol, 2011. 11: p. 235.
24. Kansaku, A., et al., Ligand-of-Numb protein X is an endocytic scaffold for junctional adhesion molecule 4. Oncogene, 2006. 25(37): p. 5071-84.
25. Dho, S.E., et al., The mammalian numb phosphotyrosine-binding domain. Characterization of binding specificity and identification of a novel PDZ domain-containing numb binding protein, LNX. J Biol Chem, 1998. 273(15): p. 9179-87.
26. Nie, J., S.S. Li, and C.J. McGlade, A novel PTB-PDZ domain interaction mediates isoform-specific ubiquitylation of mammalian Numb. J Biol Chem, 2004. 279(20): p. 20807-15.
27. Lai, E.C., Protein degradation: four E3s for the notch pathway. Curr Biol, 2002. 12(2): p. R74-8.
28. Nie, J., et al., LNX functions as a RING type E3 ubiquitin ligase that targets the cell fate determinant Numb for ubiquitin-dependent degradation. Embo j, 2002. 21(1-2): p. 93-102.
29. Guo, Z., et al., Proteomics strategy to identify substrates of LNX, a PDZ domain-containing E3 ubiquitin ligase. J Proteome Res, 2012. 11(10): p. 4847-62.
30. Wolting, C.D., et al., Biochemical and computational analysis of LNX1 interacting proteins. PLoS One, 2011. 6(11): p. e26248.
31. Giles, F.J., The vascular endothelial growth factor (VEGF) signaling pathway: a therapeutic target in patients with hematologic malignancies. Oncologist, 2001. 6 Suppl 5: p. 32-9.
32. Cantley, L.C., The phosphoinositide 3-kinase pathway. Science, 2002. 296(5573): p. 1655-7.
33. Cuadrado, A. and A.R. Nebreda, Mechanisms and functions of p38 MAPK signalling. Biochem J, 2010. 429(3): p. 403-17.
34. Coffman, J.A., et al., Evaluation of developmental phenotypes produced by morpholino antisense targeting of a sea urchin Runx gene. BMC Biol, 2004. 2: p. 6.
35. Rice, D.S., G.M. Northcutt, and C. Kurschner, The Lnx family proteins function as molecular scaffolds for Numb family proteins. Mol Cell Neurosci, 2001. 18(5): p. 525-40.
36. D'Agostino, M., et al., Ligand of Numb proteins LNX1p80 and LNX2 interact with the human glycoprotein CD8alpha and promote its ubiquitylation and endocytosis. J Cell Sci, 2011. 124(Pt 21): p. 3545-56.
37. Won, M., H. Ro, and I.B. Dawid, Lnx2 ubiquitin ligase is essential for exocrine cell differentiation in the early zebrafish pancreas. Proc Natl Acad Sci U S A, 2015. 112(40): p. 12426-31.
38. Hellstrom, M., et al., Dll4 signalling through Notch1 regulates formation of tip cells during angiogenesis. Nature, 2007. 445(7129): p. 776-80.
39. Bresciani, E., et al., Zebrafish numb and numblike are involved in primitive erythrocyte differentiation. PLoS One, 2010. 5(12): p. e14296.
40. Xu, H., et al., Protein kinase C alpha promotes angiogenic activity of human endothelial cells via induction of vascular endothelial growth factor. Cardiovasc Res, 2008. 78(2): p. 349-55.
41. Wong, C. and Z.G. Jin, Protein kinase C-dependent protein kinase D activation modulates ERK signal pathway and endothelial cell proliferation by vascular endothelial growth factor. J Biol Chem, 2005. 280(39): p. 33262-9.