常見的神經和血管系統共同指引分子是受到複雜的神經血管交互作用精確調控。正如預期，某些起初被定義為血管生成的因子，在後來也被發現可在生長過程中做為神經指引分子。這些因子即被命名為“angioneurin”。於先前的研究中我們團隊發現一個在脊椎動物中高度保留的蛋白，稱為凝血酶敏感蛋白區域包含蛋白7(THSD7A)。我們首先發現在血管新生的過程中，THSD7A能夠引導內皮細胞的遷移和血管形成。此外我們也發現斑馬魚的Thsd7a是一個神經蛋白，且在發育過程中的節間血管型態的生成扮演重要角色。最近我們更進一步發現，功能性的THSD7A是一個N端修飾的可溶性醣蛋白，其可以透過FAK的機制來促進內皮細胞的遷移。然而，關於thsd7a的確切表現位置和其在神經系統的影響仍未釐清。在本篇研究中，我們發現斑馬魚的thsd7a表現在初級運動神經內，若在Tg(kdr:EGFP/mnx1:TagRFP) 基因轉殖斑馬魚上使用morpholino減低Thsd7a的表現，會造成初級運動神經生長路徑和節間血管生長產生異常。更具體來說，延髓運動神經(RoP)軸突和parachordal chain血管會產生缺失或者扭曲。值得注意的是，這些異常的表型與之前文獻所提及的netrin1a以及dcc表現下降所引起的異常表型是類似的，這意味著，thsd7a可能在神經血管發育中涉及netrin1a訊號傳遞。在RTq-PCR和ISH的實驗結果顯示，當thsd7a的表現被抑制時netrin1a的表現確實也會跟著下降。此外，RoP軸突和parachordal chain血管的缺陷能夠成功被netrin1a mRNA拯救回來。總體來說，我們的研究結果指出斑馬魚的Thsd7a是一個表現在初級運動神經內的神經蛋白，且藉由調控netrin1a的表現來引導RoP軸突和parachordal chain血管的形成。此外，我們的結果也說明Thsd7a是一個非常有潛力的angioneurin並參與在發育和/或重新構築神經以及血管系統中。最終，Thsd7a的神經血管引導作用也許能夠在未來提供獨特的治療潛力。

The common guidance cues share by the neural and vascular systems are under precise control of complex neurovascular interactions. As expected, some factors initially identified as angiogenic factors also serve as neuronal guidance molecules during development. These molecules are thus named as “angioneurin”. Previously, our group has discovered a novel protein called thrombospondin type I domain containing 7A (THSD7A) that is highly conserved among the vertebrates. We first found that THSD7A is able to mediate endothelial cells migration and tube formation during angiogenesis. We later showed that zebrafish thsd7a ortholog is a neural protein required for intersegmental vessels (ISVs) patterning during development. Recently, we further revealed that the functional THDS7A is a soluble N-glycoprotein that promotes endothelial cell migration via a FAK-dependent mechanism. However, the exact origin of zebrafish thsd7a and its effect on neural system remains unclear. In this study, we discovered that zebrafish thsd7a is expressed in the primary motoneurons. Morpholino (MO) knockdown of Thsd7a activity resulted in aberrant primary motoneuron pathfinding and ISV sprouting in the Tg(kdr:EGFP/mnx1:TagRFP) double transgenic zebrafish. Specifically, rostral motoneuron (RoP) axon and the parachordal chain (PAC) were either absent or distorted in the thsd7a morphants. Strikingly, the morphant’s phenotypes are reminiscent of those observed in the netrin1a and deleted in colorectal cancer (dcc) morphants shown in the literatures, implying that thsd7a may involve in the netrin1a signaling during neurovascular development. The Real Time-quantitative PCR analysis (RTq-PCR) and in situ hybridization (ISH) results showed that netrin1a expression level was indeed decreased in the thsd7a morphants. Moreover, RoP axon and PAC defect could be rescued by the addition of netrin1a mRNA. Our findings indicate that zebrafish Thsd7a is a neural protein expressed in primary motoneuron. It is required for PAC and RoP axon formation by regulating netrin1a expression. Taken together, our data supports the notion that Thsd7a is a potent angioneurin involved in the developing and/or remodeling of both neural and vascular systems. Finally, the neurovascular guidance function of Thsd7a may offer a unique therapeutic potential in the future.

1. Herbert, S.P. and D.Y. Stainier, Molecular control of endothelial cell behaviour during blood vessel morphogenesis. Nat Rev Mol Cell Biol, 2011. 12(9): p. 551-64.
2. Lamalice, L., F. Le Boeuf, and J. Huot, Endothelial cell migration during angiogenesis. Circ Res, 2007. 100(6): p. 782-94.
3. De Smet, F., et al., Mechanisms of vessel branching: filopodia on endothelial tip cells lead the way. Arterioscler Thromb Vasc Biol, 2009. 29(5): p. 639-49.
4. Childs, S., et al., Patterning of angiogenesis in the zebrafish embryo. Development, 2002. 129(4): p. 973-82.
5. Covassin, L.D., et al., 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(17): p. 6554-9.
6. Phng, L.K. and H. Gerhardt, Angiogenesis: a team effort coordinated by notch. Dev Cell, 2009. 16(2): p. 196-208.
7. Carmeliet, P. and M. Tessier-Lavigne, Common mechanisms of nerve and blood vessel wiring. Nature, 2005. 436(7048): p. 193-200.
8. Tam, S.J. and R.J. Watts, Connecting vascular and nervous system development: angiogenesis and the blood-brain barrier. Annu Rev Neurosci, 2010. 33: p. 379-408.
9. Wallace, C.S., et al., Evidence that angiogenesis lags behind neuron and astrocyte growth in experience-dependent plasticity. Dev Psychobiol, 2011. 53(5): p. 435-42.
10. Miller, G., Origins. On the origin of the nervous system. Science, 2009. 325(5936): p. 24-6.
11. Eichmann, A., et al., Guidance of vascular and neural network formation. Curr Opin Neurobiol, 2005. 15(1): p. 108-15.
12. Klagsbrun, M. and A. Eichmann, A role for axon guidance receptors and ligands in blood vessel development and tumor angiogenesis. Cytokine Growth Factor Rev, 2005. 16(4-5): p. 535-48.
13. Adams, R.H. and A. Eichmann, Axon guidance molecules in vascular patterning. Cold Spring Harb Perspect Biol, 2010. 2(5): p. a001875.
14. Zacchigna, S., D. Lambrechts, and P. Carmeliet, Neurovascular signalling defects in neurodegeneration. Nat Rev Neurosci, 2008. 9(3): p. 169-81.
15. Lu, X., et al., The netrin receptor UNC5B mediates guidance events controlling morphogenesis of the vascular system. Nature, 2004. 432(7014): p. 179-86.
16. Keino-Masu, K., et al., Deleted in Colorectal Cancer (DCC) encodes a netrin receptor. Cell, 1996. 87(2): p. 175-85.
17. Fazeli, A., et al., Phenotype of mice lacking functional Deleted in colorectal cancer (Dcc) gene. Nature, 1997. 386(6627): p. 796-804.
18. Hong, K., et al., A ligand-gated association between cytoplasmic domains of UNC5 and DCC family receptors converts netrin-induced growth cone attraction to repulsion. Cell, 1999. 97(7): p. 927-41.
19. Srinivasan, K., et al., Netrin-1/neogenin interaction stabilizes multipotent progenitor cap cells during mammary gland morphogenesis. Dev Cell, 2003. 4(3): p. 371-82.
20. Kang, J.S., et al., Netrins and neogenin promote myotube formation. J Cell Biol, 2004. 167(3): p. 493-504.
21. Liu, Y., et al., Novel role for Netrins in regulating epithelial behavior during lung branching morphogenesis. Curr Biol, 2004. 14(10): p. 897-905.
22. Yebra, M., et al., Recognition of the neural chemoattractant Netrin-1 by integrins alpha6beta4 and alpha3beta1 regulates epithelial cell adhesion and migration. Dev Cell, 2003. 5(5): p. 695-707.
23. Eisen, J.S., Motoneuronal development in the embryonic zebrafish. Development, 1991. Suppl 2: p. 141-7.
24. Moreno, R.L. and A.B. Ribera, Developmental regulation of subtype-specific motor neuron excitability. Ann N Y Acad Sci, 2010. 1198: p. 201-7.
25. Myers, P.Z., J.S. Eisen, and M. Westerfield, Development and axonal outgrowth of identified motoneurons in the zebrafish. J Neurosci, 1986. 6(8): p. 2278-89.
26. Hutchinson, S.A. and J.S. Eisen, Islet1 and Islet2 have equivalent abilities to promote motoneuron formation and to specify motoneuron subtype identity. Development, 2006. 133(11): p. 2137-47.
27. Lim, A.H., et al., Motoneurons are essential for vascular pathfinding. Development, 2011. 138(17): p. 3847-57.
28. Kuo, M.W., et al., Soluble THSD7A is an N-glycoprotein that promotes endothelial cell migration and tube formation in angiogenesis. PLoS One, 2011. 6(12): p. e29000.
29. Wang, C.H., et al., Thrombospondin type I domain containing 7A (THSD7A) mediates endothelial cell migration and tube formation. J Cell Physiol, 2010. 222(3): p. 685-94.
30. Wang, C.H., et al., Zebrafish Thsd7a is a neural protein required for angiogenic patterning during development. Dev Dyn, 2011. 240(6): p. 1412-21.
31. Lawson, N.D. and B.M. Weinstein, Arteries and veins: making a difference with zebrafish. Nat Rev Genet, 2002. 3(9): p. 674-82.
32. Kimmel, C.B., et al., Stages of embryonic development of the zebrafish. Dev Dyn, 1995. 203(3): p. 253-310.
33. Kwan, K.M., et al., The Tol2kit: a multisite gateway-based construction kit for Tol2 transposon transgenesis constructs. Dev Dyn, 2007. 236(11): p. 3088-99.
34. Narayanan, K. and Q. Chen, Bacterial artificial chromosome mutagenesis using recombineering. J Biomed Biotechnol, 2011. 2011: p. 971296.
35. Court, D.L., J.A. Sawitzke, and L.C. Thomason, Genetic engineering using homologous recombination. Annu Rev Genet, 2002. 36: p. 361-88.
36. Lauderdale, J.D., N.M. Davis, and J.Y. Kuwada, Axon tracts correlate with netrin-1a expression in the zebrafish embryo. Mol Cell Neurosci, 1997. 9(4): p. 293-313.
37. Park, K.W., et al., Identification of new netrin family members in zebrafish: developmental expression of netrin 2 and netrin 4. Dev Dyn, 2005. 234(3): p. 726-31.
38. Hale, L.A., D.K. Fowler, and J.S. Eisen, Netrin signaling breaks the equivalence between two identified zebrafish motoneurons revealing a new role of intermediate targets. PLoS One, 2011. 6(10): p. e25841.
39. Wilson, B.D., et al., Netrins promote developmental and therapeutic angiogenesis. Science, 2006. 313(5787): p. 640-4.
40. Navankasattusas, S., et al., The netrin receptor UNC5B promotes angiogenesis in specific vascular beds. Development, 2008. 135(4): p. 659-67.
41. Nikolopoulos, S.N. and F.G. Giancotti, Netrin-integrin signaling in epithelial morphogenesis, axon guidance and vascular patterning. Cell Cycle, 2005. 4(3): p. e131-5.
42. Lai Wing Sun, K., J.P. Correia, and T.E. Kennedy, Netrins: versatile extracellular cues with diverse functions. Development, 2011. 138(11): p. 2153-69.
43. Long, H., et al., Conserved roles for Slit and Robo proteins in midline commissural axon guidance. Neuron, 2004. 42(2): p. 213-23.
44. Bedell, V.M., et al., roundabout4 is essential for angiogenesis in vivo. Proc Natl Acad Sci U S A, 2005. 102(18): p. 6373-8.
45. Zhang, B., et al., Repulsive axon guidance molecule Slit3 is a novel angiogenic factor. Blood, 2009. 114(19): p. 4300-9.
46. Luo, Y., D. Raible, and J.A. Raper, Collapsin: a protein in brain that induces the collapse and paralysis of neuronal growth cones. Cell, 1993. 75(2): p. 217-27.
47. Yu, H.H., C. Houart, and C.B. Moens, Cloning and embryonic expression of zebrafish neuropilin genes. Gene Expr Patterns, 2004. 4(4): p. 371-8.
48. Feldner, J., et al., Neuropilin-1a is involved in trunk motor axon outgrowth in embryonic zebrafish. Dev Dyn, 2005. 234(3): p. 535-49.
49. Brennan, C., et al., Two Eph receptor tyrosine kinase ligands control axon growth and may be involved in the creation of the retinotectal map in the zebrafish. Development, 1997. 124(3): p. 655-64.
50. Wang, H.U. and D.J. Anderson, Eph family transmembrane ligands can mediate repulsive guidance of trunk neural crest migration and motor axon outgrowth. Neuron, 1997. 18(3): p. 383-96.
51. Gerety, S.S. and D.J. Anderson, Cardiovascular ephrinB2 function is essential for embryonic angiogenesis. Development, 2002. 129(6): p. 1397-410.
52. Gerety, S.S., et al., Symmetrical mutant phenotypes of the receptor EphB4 and its specific transmembrane ligand ephrin-B2 in cardiovascular development. Mol Cell, 1999. 4(3): p. 403-14.
53. Adams, R.H., et al., Roles of ephrinB ligands and EphB receptors in cardiovascular development: demarcation of arterial/venous domains, vascular morphogenesis, and sprouting angiogenesis. Genes Dev, 1999. 13(3): p. 295-306.
54. Bouvree, K., et al., Netrin-1 inhibits sprouting angiogenesis in developing avian embryos. Dev Biol, 2008. 318(1): p. 172-83.
55. Larrivee, B., et al., Activation of the UNC5B receptor by Netrin-1 inhibits sprouting angiogenesis. Genes Dev, 2007. 21(19): p. 2433-47.
56. Arakawa, H., p53, apoptosis and axon-guidance molecules. Cell Death Differ, 2005. 12(8): p. 1057-65.
57. Tanikawa, C., et al., p53RDL1 regulates p53-dependent apoptosis. Nat Cell Biol, 2003. 5(3): p. 216-23.
58. Arakawa, H., Netrin-1 and its receptors in tumorigenesis. Nat Rev Cancer, 2004. 4(12): p. 978-87.
59. Goldschneider, D. and P. Mehlen, Dependence receptors: a new paradigm in cell signaling and cancer therapy. Oncogene, 2010. 29(13): p. 1865-82.
60. Park, K.W., et al., The axonal attractant Netrin-1 is an angiogenic factor. Proc Natl Acad Sci U S A, 2004. 101(46): p. 16210-5.
61. Alcantara, S., et al., Netrin 1 acts as an attractive or as a repulsive cue for distinct migrating neurons during the development of the cerebellar system. Development, 2000. 127(7): p. 1359-72.
62. Serafini, T., et al., The netrins define a family of axon outgrowth-promoting proteins homologous to C. elegans UNC-6. Cell, 1994. 78(3): p. 409-24.
63. Dent, E.W., et al., Netrin-1 and semaphorin 3A promote or inhibit cortical axon branching, respectively, by reorganization of the cytoskeleton. J Neurosci, 2004. 24(12): p. 3002-12.
64. Xu, B., et al., Critical roles for the netrin receptor deleted in colorectal cancer in dopaminergic neuronal precursor migration, axon guidance, and axon arborization. Neuroscience, 2010. 169(2): p. 932-49.
65. Jarjour, A.A., et al., Netrin-1 is a chemorepellent for oligodendrocyte precursor cells in the embryonic spinal cord. J Neurosci, 2003. 23(9): p. 3735-44.
66. Colamarino, S.A. and M. Tessier-Lavigne, The axonal chemoattractant netrin-1 is also a chemorepellent for trochlear motor axons. Cell, 1995. 81(4): p. 621-9.
67. Clarke, J.D., et al., Sensory physiology, anatomy and immunohistochemistry of Rohon-Beard neurones in embryos of Xenopus laevis. J Physiol, 1984. 348: p. 511-25.
68. Roberts, A., Early functional organization of spinal neurons in developing lower vertebrates. Brain Res Bull, 2000. 53(5): p. 585-93.
69. Lamborghini, J.E., Disappearance of Rohon-Beard neurons from the spinal cord of larval Xenopus laevis. J Comp Neurol, 1987. 264(1): p. 47-55.
70. Castets, M. and P. Mehlen, Netrin-1 role in angiogenesis: to be or not to be a pro-angiogenic factor? Cell Cycle, 2010. 9(8): p. 1466-71.
71. Castets, M., et al., Inhibition of endothelial cell apoptosis by netrin-1 during angiogenesis. Dev Cell, 2009. 16(4): p. 614-20.