基因調控和訊息傳遞路徑對於血管的生成非常重要。但是，目前對於靜脈特化、區間血管的生成以及尾部靜脈血管叢形成的分子機制仍不清楚。我們實驗室之前發現轉錄因子 isl2 與 nr2f1b 對於靜脈的特化與區間血管的生成非常重要。另外也發現 nr2f1a 對於血管的生成占有相當重要的角色。胺基酸序列比對與親緣關係說明 nr2f1a 在脊椎動物具有高度的保守性。從原位組織染色得知 nr2f1a 在 18 S 時期表現在背部的側中胚層，而在 24-30 小時表現的位置，可推測其在血管的角色。當使用 morpholino knockdown nr2f1a 表現時，會造成區間血管生長的缺陷和尾部靜脈血管叢無法形成網狀的脈絡，利用 tol2 質體來過度表現 nr2f1a 。發現血管缺陷可以被挽救，證實 nr2f1a 在調控區間血管和尾部靜脈血管叢的生長的專一性。脈管的缺陷會進而導致發育後期的心包膜水腫與血液循環的缺陷，證實其功能性的缺失。透過 TUNEL assay 和 AO staining 驗證。結果顯示注射 nr2f1a morpholino 後血管內皮細胞並沒有增加凋亡，因此推測區間血管與尾部靜脈血管叢的缺陷並不是由細胞凋亡造成。透過觀察基因轉殖魚種 Tg(kdrl:mCherry;fli:nEGFP)y7 的表現，我分析區間血管或脈管細胞數，以及觀察區間血管生長，證實血管生長缺陷可能是因為影響內皮細胞增生及移動所造成。為了得知 nr2f1a knockdown 影響血管生成的分子機制與訊息。觀察不同脈管基因的表現。結果發現在 nr2f1a morphant 中，動脈基因 (ephrinb2) 和區間血管/靜脈基因 (flt4 , mrc1, stabilin) 的表現有下降的趨勢，可知 nr2f1a 調節血管生長的角色。利用 rbpsuh morphant 和 DAPT 藥物抑制 notch 訊息，分析 nr2f1a 基因的變化，推測其受 notch 訊息的正向調控。總結以上， nr2f1a 在斑馬魚血管發育中扮演相當重要的角色。

Genetic regulators and signaling pathways are important for the formation of blood vessels. However, less well understood about the transcription factors controlling in vein identify, intersegmental vessels (ISV) growth and caudal vein plexus (CVP) formation in zebrafish. We previously identified the transcription factor Islet2 (Isl2) and nuclear receptor subfamily 2 group F member IB (nr2f1b) are required for vein specification and ISV growth in zebrafish. Here we show the nuclear receptor subfamily member 1A (nr2f1a) is also important for zebrafish vascular development.
Amino acid sequence alignment and phylogenetic analysis confirmed nr2f1a is highly conserved among the vertebrates. Our in situ hybridization results showed nr2f1a mRNA is expressed in lateral posterior mesoderm at 18 somite stage and in vessels at 24-30 hpf, suggesting its roles in vasculature. Consistent with that, morpholino-based knockdown of nr2fla impairs ISV growth and fails to develop fenestrated vascular structure in CVP, meanwhile, we also used tol2 plasmid to overexpress nr2f1a .If the phenotype of vascular be rescued, suggested that nr2f1a has an important role in controlling ISV and CVP growth. In addition, nr2f1a morphant showed pericardial edema and circulation defect. To address whether the growth impairment of ISV and CVP results from cell death, we performed TUNEL assay and AO staining. We showed that no increased cell death in endothelial cells after morpholino injection, indicating the growth defect of ISV and CVP is not due to the cell death. To test molecular mechanisms and signaling that nr2f1a associated with, we first examined the expression of vascular markers. We found loss of nr2f1a results in a decreased expression of aorta specific marker, ephrinb2 and vein/ISV specific markers, flt4, mrc1 and stabilin, suggested the regulatory role of nr2f1a in controlling vascular development. We further showed that nr2f1a likely interact with the Notch signaling by examining nr2f1a expression in rbpsuh morphants and DAPT-treatment embryos. Together, we show nr2f1a plays a critical role for vascular development in zebrafish.

章節目錄 i
圖表目錄 iii
中英文名詞對照/縮寫 iv
Abbreviation v
中文摘要 vi
Abstract vii
前言 1
血管的發育 2
Notch路徑在心血管發育過程中會調控細胞的二分化 3
動靜脈特化的過程 4
血管生成 (vasculogenesis) 6
出芽性血管新生 (angiogenesis) 6
NR2Fs 家族 7
研究nr2f1a的動機 7
材料與方法 13
實驗材料 13
實驗方法 15
斑馬魚培養 15
斑馬魚受精卵收集與培養 15
顯微注射 (Morpholino microinjection) 15
RNA抽取 16
cDNA 製作 17
探針製作 17
原位組織染色(In situ hybridation) 18
Acridine orange (AO) staining 19
TUNEL Assay 19
Quantitative PCR(Q-PCR) 19
Transposase 製作 21
勝任細胞製作 21
Tol2 過度表現 21
實驗結果 23
Nr2f1a 在脊椎動物中具有高度保守性 23
Nr2f1a在不同時期的表現位置 23
斑馬魚 nr2f1a 基因影響血管發育之功能 24
血管發育的缺陷是否專一性由 nr2f1a 造成 25
確認 nr21fa 造成的血管缺陷 26
Nr2f1a knockdown 後的血管缺陷衍生的表型 26
血管生長缺陷的原因 27
nr2f1a knockdown 造成動靜脈基因表現下降而造成血管缺陷 27
使用 tol2 技術挽救 nr2f1a 所造成的缺陷 28
Notch訊息調控 nr2f1a 以促進靜脈分化以及區間血管生長 28
VEGFR2訊息調控nr2f1a表現以促進血管生長 29
問題與討論 31
References 33

1. Patterson C (2009) Torturing a blood vessel. Nat Med 15: 137-138.
2. Revencu N, Vikkula M (2006) Cerebral cavernous malformation: new molecular and clinical insights. J Med Genet 43: 716-721.
3. Childs S, Chen JN, Garrity DM, Fishman MC (2002) Patterning of angiogenesis in the zebrafish embryo. Development 129: 973-982.
4. Christie TL, Carter A, Rollins EL, Childs SJ (2010) Syk and Zap-70 function redundantly to promote angioblast migration. Dev Biol 340: 22-29.
5. Lamont RE, Lamont EJ, Childs SJ (2009) Antagonistic interactions among Plexins regulate the timing of intersegmental vessel formation. Dev Biol 331: 199-209.
6. Liu J, Fraser SD, Faloon PW, Rollins EL, Vom Berg J, et al. (2007) A betaPix Pak2a signaling pathway regulates cerebral vascular stability in zebrafish. Proc Natl Acad Sci U S A 104: 13990-13995.
7. Torres-Vazquez J, Gitler AD, Fraser SD, Berk JD, Van NP, et al. (2004) Semaphorin-plexin signaling guides patterning of the developing vasculature. Dev Cell 7: 117-123.
8. Zhong TP, Childs S, Leu JP, Fishman MC (2001) Gridlock signalling pathway fashions the first embryonic artery. Nature 414: 216-220.
9. Mudumana SP, Hentschel D, Liu Y, Vasilyev A, Drummond IA (2008) odd skipped related1 reveals a novel role for endoderm in regulating kidney versus vascular cell fate. Development 135: 3355-3367.
10. Pardanaud L, Yassine F, Dieterlen-Lievre F (1989) Relationship between vasculogenesis, angiogenesis and haemopoiesis during avian ontogeny. Development 105: 473-485.
11. Choi K, Kennedy M, Kazarov A, Papadimitriou JC, Keller G (1998) A common precursor for hematopoietic and endothelial cells. Development 125: 725-732.
12. Vogeli KM, Jin SW, Martin GR, Stainier DY (2006) A common progenitor for haematopoietic and endothelial lineages in the zebrafish gastrula. Nature 443: 337-339.
13. Blum Y, Belting HG, Ellertsdottir E, Herwig L, Luders F, et al. (2008) Complex cell rearrangements during intersegmental vessel sprouting and vessel fusion in the zebrafish embryo. Dev Biol 316: 312-322.
14. Gerhardt H, Golding M, Fruttiger M, Ruhrberg C, Lundkvist A, et al. (2003) VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia. J Cell Biol 161: 1163-1177.
15. Jin SW, Beis D, Mitchell T, Chen JN, Stainier DY (2005) Cellular and molecular analyses of vascular tube and lumen formation in zebrafish. Development 132: 5199-5209.
16. Kamei M, Saunders WB, Bayless KJ, Dye L, Davis GE, et al. (2006) Endothelial tubes assemble from intracellular vacuoles in vivo. Nature 442: 453-456.
17. Ridgway J, Zhang G, Wu Y, Stawicki S, Liang WC, et al. (2006) Inhibition of Dll4 signalling inhibits tumour growth by deregulating angiogenesis. Nature 444: 1083-1087.
18. Noguera-Troise I, Daly C, Papadopoulos NJ, Coetzee S, Boland P, et al. (2006) Blockade of Dll4 inhibits tumour growth by promoting non-productive angiogenesis. Nature 444: 1032-1037.
19. Fischer A, Schumacher N, Maier M, Sendtner M, Gessler M (2004) The Notch target genes Hey1 and Hey2 are required for embryonic vascular development. Genes Dev 18: 901-911.
20. Leslie JD, Ariza-McNaughton L, Bermange AL, McAdow R, Johnson SL, et al. (2007) Endothelial signalling by the Notch ligand Delta-like 4 restricts angiogenesis. Development 134: 839-844.
21. Siekmann AF, Lawson ND (2007) Notch signalling limits angiogenic cell behaviour in developing zebrafish arteries. Nature 445: 781-784.
22. Hellstrom M, Phng LK, Hofmann JJ, Wallgard E, Coultas L, et al. (2007) Dll4 signalling through Notch1 regulates formation of tip cells during angiogenesis. Nature 445: 776-780.
23. Adams RH, Wilkinson GA, Weiss C, Diella F, Gale NW, et al. (1999) Roles of ephrinB ligands and EphB receptors in cardiovascular development: demarcation of arterial/venous domains, vascular morphogenesis, and sprouting angiogenesis. Genes Dev 13: 295-306.
24. Hong CC, Peterson QP, Hong JY, Peterson RT (2006) Artery/vein specification is governed by opposing phosphatidylinositol-3 kinase and MAP kinase/ERK signaling. Curr Biol 16: 1366-1372.
25. Lawson ND, Scheer N, Pham VN, Kim CH, Chitnis AB, et al. (2001) Notch signaling is required for arterial-venous differentiation during embryonic vascular development. Development 128: 3675-3683.
26. Wang HU, Chen ZF, Anderson DJ (1998) Molecular distinction and angiogenic interaction between embryonic arteries and veins revealed by ephrin-B2 and its receptor Eph-B4. Cell 93: 741-753.
27. Sorensen I, Adams RH, Gossler A (2009) DLL1-mediated Notch activation regulates endothelial identity in mouse fetal arteries. Blood 113: 5680-5688.
28. Duarte A, Hirashima M, Benedito R, Trindade A, Diniz P, et al. (2004) Dosage-sensitive requirement for mouse Dll4 in artery development. Genes Dev 18: 2474-2478.
29. Covassin LD, Villefranc JA, Kacergis MC, Weinstein BM, Lawson ND (2006) 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 103: 6554-6559.
30. Kaipainen A, Korhonen J, Mustonen T, van Hinsbergh VW, Fang GH, et al. (1995) Expression of the fms-like tyrosine kinase 4 gene becomes restricted to lymphatic endothelium during development. Proc Natl Acad Sci U S A 92: 3566-3570.
31. You LR, Lin FJ, Lee CT, DeMayo FJ, Tsai MJ, et al. (2005) Suppression of Notch signalling by the COUP-TFII transcription factor regulates vein identity. Nature 435: 98-104.
32. Lamont RE, Childs S (2006) MAPping out arteries and veins. Sci STKE 2006: pe39.
33. Koch S, Tugues S, Li X, Gualandi L, Claesson-Welsh L (2011) Signal transduction by vascular endothelial growth factor receptors. Biochem J 437: 169-183.
34. Cunningham SA, Stephan CC, Arrate MP, Ayer KG, Brock TA (1997) Identification of the extracellular domains of Flt-1 that mediate ligand interactions. Biochem Biophys Res Commun 231: 596-599.
35. Takahashi T, Yamaguchi S, Chida K, Shibuya M (2001) A single autophosphorylation site on KDR/Flk-1 is essential for VEGF-A-dependent activation of PLC-gamma and DNA synthesis in vascular endothelial cells. EMBO J 20: 2768-2778.
36. Goldie LC, Nix MK, Hirschi KK (2008) Embryonic vasculogenesis and hematopoietic specification. Organogenesis 4: 257-263.
37. Eichmann A, Yuan L, Moyon D, Lenoble F, Pardanaud L, et al. (2005) Vascular development: from precursor cells to branched arterial and venous networks. Int J Dev Biol 49: 259-267.
38. Furuya M, Nishiyama M, Kasuya Y, Kimura S, Ishikura H (2005) Pathophysiology of tumor neovascularization. Vasc Health Risk Manag 1: 277-290.
39. Swift MR, Weinstein BM (2009) Arterial-venous specification during development. Circ Res 104: 576-588.
40. De Smet F, Segura I, De Bock K, Hohensinner PJ, Carmeliet P (2009) Mechanisms of vessel branching: filopodia on endothelial tip cells lead the way. Arterioscler Thromb Vasc Biol 29: 639-649.
41. Lu X, Le Noble F, Yuan L, Jiang Q, De Lafarge B, et al. (2004) The netrin receptor UNC5B mediates guidance events controlling morphogenesis of the vascular system. Nature 432: 179-186.
42. Ruhrberg C, Gerhardt H, Golding M, Watson R, Ioannidou S, et al. (2002) Spatially restricted patterning cues provided by heparin-binding VEGF-A control blood vessel branching morphogenesis. Genes Dev 16: 2684-2698.
43. Love CE, Prince VE (2012) Expression and retinoic acid regulation of the zebrafish nr2f orphan nuclear receptor genes. Dev Dyn 241: 1603-1615.
44. Wang Z, Benoit G, Liu J, Prasad S, Aarnisalo P, et al. (2003) Structure and function of Nurr1 identifies a class of ligand-independent nuclear receptors. Nature 423: 555-560.
45. Kruse SW, Suino-Powell K, Zhou XE, Kretschman JE, Reynolds R, et al. (2008) Identification of COUP-TFII orphan nuclear receptor as a retinoic acid-activated receptor. PLoS Biol 6: e227.
46. Pereira FA, Tsai MJ, Tsai SY (2000) COUP-TF orphan nuclear receptors in development and differentiation. Cell Mol Life Sci 57: 1388-1398.
47. Qiu Y, Krishnan V, Pereira FA, Tsai SY, Tsai MJ (1996) Chicken ovalbumin upstream promoter-transcription factors and their regulation. J Steroid Biochem Mol Biol 56: 81-85.
48. Tsai SY, Tsai MJ (1997) Chick ovalbumin upstream promoter-transcription factors (COUP-TFs): coming of age. Endocr Rev 18: 229-240.
49. Park JI, Tsai SY, Tsai MJ (2003) Molecular mechanism of chicken ovalbumin upstream promoter-transcription factor (COUP-TF) actions. Keio J Med 52: 174-181.
50. Choi J, Dong L, Ahn J, Dao D, Hammerschmidt M, et al. (2007) FoxH1 negatively modulates flk1 gene expression and vascular formation in zebrafish. Dev Biol 304: 735-744.
51. Lawson ND, Weinstein BM (2002) In vivo imaging of embryonic vascular development using transgenic zebrafish. Dev Biol 248: 307-318.
52. Roman BL, Pham VN, Lawson ND, Kulik M, Childs S, et al. (2002) Disruption of acvrl1 increases endothelial cell number in zebrafish cranial vessels. Development 129: 3009-3019.
53. Patel-Hett S, D'Amore PA (2011) Signal transduction in vasculogenesis and developmental angiogenesis. Int J Dev Biol 55: 353-363.
54. Smits BM, Haag JD, Rissman AI, Sharma D, Tran A, et al. (2013) The gene desert mammary carcinoma susceptibility locus Mcs1a regulates Nr2f1 modifying mammary epithelial cell differentiation and proliferation. PLoS Genet 9: e1003549.
55. Montemayor C, Montemayor OA, Ridgeway A, Lin F, Wheeler DA, et al. (2010) Genome-wide analysis of binding sites and direct target genes of the orphan nuclear receptor NR2F1/COUP-TFI. PLoS One 5: e8910.