紅血球生成素（Erythropoietin, EPO）是一種醣蛋白激素，能控制紅血球前驅細胞的生存、增殖和分化。EPO最初是因為造血過程中起關鍵作用而得名，而後研究發現它在神經發育中也扮演了關鍵作用。EPO受體（EPO receptor, EPO-R）為第一類細胞激素受體中的一種，可藉由各種蛋白激酶和STAT轉錄因子來證明其作用。目前為止，有關EPO如何促進神經細胞生成的詳細機制尚不清楚。而在這項研究中，主要在探討Epo信號傳遞系統影響神經發育的機制。實驗中，利用morpholino敲落epo基因，會減少xiap / birc4和ciap1 / birc2的轉錄作用以及抑制了神經的分化。此外，敲落xiap / birc4和ciap1 / birc2也影響到神經發育，證實了XIAP和cIAP1在神經發育中扮演著相當重要的角色。實驗發現以AG490抑制Jak2或者敲落stat5基因皆可降低xiap / birc4和ciap1 / birc2的轉錄，抑制神經的生成。利用異位表達的xiap / birc4和ciap1 / birc2 mRNA顯微注射入胚胎中，可以復原敲落Epo或以AG490處理的胚胎內神經的分化。總而言之，此項研究表明Epo信號傳遞會藉由激活Jak2 / STAT5並上調xiap / birc4和ciap1 / birc2轉錄來調節胚胎神經的發育。

Erythropoietin (EPO) is a glycoprotein cytokine required for the survival, proliferation and differentiation of the erythrocytic progenitors. Erythropoietin (EPO) was originally named because of their pivotal contribution to hematopoiesis, but it also plays critical roles in neurogenesis. EPO receptor (EPO-R) is a member of class I cytokine receptor family that excerts its effects through various protein kinases and STAT transcription factors. The mechanism how EPO modulates neurogenesis still remains to be clarified. In this study, I would like to investigate how Epo signaling mediate neurogenesis. It shows that morpholino mediated knockdown of epo results in decrease of xiap/birc4 and ciap1/birc2 transcription and inhibition of neuronal differentiation. Furthermore, knockdown of xiap/birc4 and ciap1/birc2 also abrogates neural development, suggesting that XIAP and cIAP1 play important roles in neurogenesis. Inhibiting Jak2 activity with AG490 or knockdown of stat5 also blocks transcription of xiap/birc4 and ciap1/birc2 and inhibits neural development. Ectopic xiap/birc4 and ciap1/birc2 mRNA could rescue the neuronal differentiation in epo morphants and AG490-treated embryos. In summary, this study suggests that Epo signaling modulates embryonic neurogenesis by activating Jak2/Stat5 and upregulating xiap/birc4 and ciap1/birc2 transcriptions.

摘要 i
ABSTRACT ii
TABLE OF CONTENTS iii
FIGURE INDEX v
CHAPTER I INTRODUCTION 1
1.1. Erythropoietin (EPO) 1
1.2. JAK2 / STAT5 1
1.3. Inhibitor of Apoptosis (IAPs Family) 3
1.4. Expression of Neuro-D (nrd) and elav (HuC) during neuronal differentiation 3
CHAPTER II MATERIALS AND METHODS 5
2.1. Materials 5
2.1.1. Model Animal 5
2.1.2. Competent cells and Plasmids 5
2.1.3. Chemicals used for experiments 5
2.1.4. Enzymes and commercial kits 5
2.1.5. Experimental Chemical Preparation 5
2.2. Methods 6
2.2.1. Collect zebrafish embryos and treatment drugs 6
2.2.2. Morpholino injection 7
2.2.3. Ribonucleic Acid Extraction (Total RNA Extraction) 7
2.2.4. cDNA preparation 8
2.2.5. Polymerase Chain Reaction (PCR) 8
2.2.6. DNA Ligation 8
2.2.7. Transformation 8
2.2.8. Plasmid Extraction (Miniprep kit) 9
2.2.9. DNA Amplification by PCR 9
2.2.10. Making RNA Probe 9
2.2.11. Whole mount in situ Hybridization 10
2.2.12. Quantitative RT-PCR (qPCR) 11
2.2.13. mRNA Rescue Essay 12
2.2.14. Polyadenylation 13
CHAPTER III RESULTS 14
3.1. Transcriptions of XIAP and cIAP1 are controlled by Epo-Jak2 signaling 14
3.2. Jak2/Stat5-IAPs signaling pathway protects neural cells against apoptosis 14
3.3. Epo-Jak2/Stat5-IAPs signaling pathway is important for neural differentiation 15
CHAPTER IV DISCUSSION 16
REFERENCES 17
FIGURES SECTION 20
APPENDIX 37

Agnello, D., Bigini, P., Villa, P., Mennini, T., Cerami, A., Brines, M.L., Ghezzi, P., 2002. Erythropoietin exerts an anti-inflammatory effect on the CNS in a model of experimental autoimmune encephalomyelitis. Brain Research 952, 128-134.
Aoto, K., Trainor, P.A., 2014. Co-ordinated brain and craniofacial development depend upon Patched1/XIAP regulation of cell survival. Human molecular genetics 24, 698-713.
Babon, Jeffrey J., Lucet, Isabelle S., Murphy, James M., Nicola, Nicos A., Varghese, Leila N., 2014. The molecular regulation of Janus kinase (JAK) activation. Biochemical Journal 462, 1-13.
Cattaneo, E., Conti, L., De-Fraja, C., 1999. Signalling through the JAK–STAT pathway in the developing brain. Trends in Neurosciences 22, 365-369.
Chen, C., Edelstein, L.C., Gélinas, C., 2000. The Rel/NF-κB family directly activates expression of the apoptosis inhibitor Bcl-xL. Molecular and cellular biology 20, 2687-2695.
Chu, C.-Y., Cheng, C.-H., Chen, G.-D., Chen, Y.-C., Hung, C.-C., Huang, K.-Y., Huang, C.-J., 2007. The zebrafish erythropoietin: Functional identification and biochemical characterization. FEBS Letters 581, 4265-4271.
De Almagro, M., Vucic, D., 2012. The inhibitor of apoptosis (IAP) proteins are critical regulators of signaling pathways and targets for anti-cancer therapy. Experimental oncology.
Digicaylioglu, M., Lipton, S.A., 2001. Erythropoietin-mediated neuroprotection involves cross-talk between Jak2 and NF-κB signalling cascades. Nature 412, 641-647.
Dubrez-Daloz, L., Dupoux, A., Cartier, J., 2014. IAPS : More than just inhibitors of apoptosis proteins. Cell Cycle 7, 1036-1046.
Elliott, S., Pham, E., Macdougall, I.C., 2008. Erythropoietins: a common mechanism of action. Experimental hematology 36, 1573-1584.
Galbán, S., Duckett, C.S., 2010. XIAP as a ubiquitin ligase in cellular signaling. Cell death and differentiation 17, 54.
Grasso, G., Sfacteria, A., Meli, F., Fodale, V., Buemi, M., Iacopino, D.G., 2007. Neuroprotection by erythropoietin administration after experimental traumatic brain injury. Brain Research 1182, 99-105.
Hanson, A.J., Wallace, H.A., Freeman, T.J., Beauchamp, R.D., Lee, L.A., Lee, E., 2012. XIAP monoubiquitylates Groucho/TLE to promote canonical Wnt signaling. Molecular cell 45, 619-628.
Hirata, Y., Takahashi, M., Morishita, T., Noguchi, T., Matsuzawa, A., 2017. Post-translational modifications of the TAK1-TAB complex. International journal of molecular sciences 18, 205.
Hitchcock, P., Kakuk-Atkins, L., 2004. The basic helix-loop-helix transcription factorneuroD is expressed in the rod lineage of the teleost retina. The Journal of Comparative Neurology 477, 108-117.
Infante, P., Lospinoso Severini, L., Bernardi, F., Bufalieri, F., Di Marcotullio, L., 2019. Targeting Hedgehog Signalling through the Ubiquitylation Process: The Multiple Roles of the HECT-E3 Ligase Itch. Cells 8, 98.
Jelkmann, W., Bohlius, J., Hallek, M., Sytkowski, A.J., 2008. The erythropoietin receptor in normal and cancer tissues. Critical Reviews in Oncology/Hematology 67, 39-61.
Jowett, T., Lettice, L., 1994. Whole-mount in situ hybridizationon zebrafish embryos using a mixture of digoxigenin- and fluorescein-labelled probes. Trends in Genetics 10, 73-74.
Junk, A.K., Mammis, A., Savitz, S.I., Singh, M., Roth, S., Malhotra, S., Rosenbaum, P.S., Cerami, A., Brines, M., Rosenbaum, D.M., 2002. Erythropoietin administration protects retinal neurons from acute ischemia-reperfusion injury. Proceedings of the National Academy of Sciences 99, 10659-10664.
Kaneko, N., Kako, E., Sawamoto, K., 2013. Enhancement of ventricular-subventricular zone-derived neurogenesis and oligodendrogenesis by erythropoietin and its derivatives. Frontiers in Cellular Neuroscience 7.
Kim, C.-H., Ueshima, E., Muraoka, O., Tanaka, H., Yeo, S.-Y., Huh, T.-L., Miki, N., 1996. Zebrafish elav/HuC homologue as a very early neuronal marker. Neuroscience Letters 216, 109-112.
Kim, S.I., Choi, M.E., 2012. TGF-β-activated kinase-1: New insights into the mechanism of TGF-β signaling and kidney disease. Kidney research and clinical practice 31, 94-105.
Lawoko-Kerali, G., Rivolta, M.N., Lawlor, P., Cacciabue-Rivolta, D.I., Langton-Hewer, C., Hikke van Doorninck, J., Holley, M.C., 2004. GATA3 and NeuroD distinguish auditory and vestibular neurons during development of the mammalian inner ear. Mechanisms of Development 121, 287-299.
Lee, J.E., 1997. NeuroD and Neurogenesis. Developmental Neuroscience 19, 27-32.
Ma, A.C.H., Ward, A.C., Liang, R., Leung, A.Y.H., 2007. The role of jak2a in zebrafish hematopoiesis. Blood 110, 1824-1830.
Mueller, T., Wullimann, M.F., 2002. Expression domains of neuroD (nrd) in the early postembryonic zebrafish brain. Brain Research Bulletin 57, 377-379.
O'Shea, J.J., Schwartz, D.M., Villarino, A.V., Gadina, M., McInnes, I.B., Laurence, A., 2015. The JAK-STAT Pathway: Impact on Human Disease and Therapeutic Intervention. Annual Review of Medicine 66, 311-328.
Ochocinska, M.J., Hitchcock, P.F., 2007. Dynamic expression of the basic helix-loop-helix transcription factor neuroD in the rod and cone photoreceptor lineages in the retina of the embryonic and larval zebrafish. The Journal of Comparative Neurology 501, 1-12.
Orkin, S.H., Zon, L.I., 2008. Hematopoiesis: An Evolving Paradigm for Stem Cell Biology. Cell 132, 631-644.
Paffett-Lugassy, N., Hsia, N., Fraenkel, P.G., Paw, B., Leshinsky, I., Barut, B., Bahary, N., Caro, J., Handin, R., Zon, L.I., 2007. Functional conservation of erythropoietin signaling in zebrafish. Blood 110, 2718-2726.
Reffey, S.B., Wurthner, J.U., Parks, W.T., Roberts, A.B., Duckett, C.S., 2001. X-linked inhibitor of apoptosis protein functions as a cofactor in transforming growth factor-β signaling. Journal of Biological Chemistry 276, 26542-26549.
Sakai, K., Gofuku, M., Kitagawa, Y., Ogasawara, T., Hirose, G., Yamazaki, M., Koh, C.S., Yanagisawa, N., Steinman, L., 1994. A Hippocampal Protein Associated with Paraneoplastic Neurologic Syndrome and Small Cell Lung Carcinoma. Biochemical and Biophysical Research Communications 199, 1200-1208.
Szabo, A., Dalmau, J., Manley, G., Rosenfeld, M., Wong, E., Henson, J., Posner, J.B., Furneaux, H.M., 1991. HuD, a paraneoplastic encephalomyelitis antigen, contains RNA-binding domains and is homologous to Elav and sex-lethal. Cell 67, 325-333.
Yamaguchi, K., Nagai, S.i., Ninomiya‐Tsuji, J., Nishita, M., Tamai, K., Irie, K., Ueno, N., Nishida, E., Shibuya, H., Matsumoto, K., 1999. XIAP, a cellular member of the inhibitor of apoptosis protein family, links the receptors to TAB1–TAK1 in the BMP signaling pathway. The EMBO journal 18, 179-187.
Zhang, F., Wang, S., Cao, G., Gao, Y., Chen, J., 2007. Signal transducers and activators of transcription 5 contributes to erythropoietin-mediated neuroprotection against hippocampal neuronal death after transient global cerebral ischemia. Neurobiology of Disease 25, 45-53.