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研究生:林佳穎
研究生(外文):Chia-ying Lin
論文名稱:探討斑馬魚10-甲醛四氫葉酸去氫酶在胚胎發育過程中所扮演的角色
論文名稱(外文):Studies on the Significance of 10-Formyltetrahydrofolate Dehydrogenase in Zebrafish Embryogenesis
指導教授:傅子芳
指導教授(外文):Tzu-Fun Fu
學位類別:碩士
校院名稱:國立成功大學
系所名稱:醫事技術學系
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:英文
論文頁數:75
中文關鍵詞:胚胎發育10-甲醛四氫葉酸去氫酶斑馬魚
外文關鍵詞:Embryogenesis10-Formyltetrahydrofolate DehydrogenaseZebrafish
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10-甲醛四氫葉酸去氫酶參與由葉酸所媒介的單碳循環,主要的催化反應是將10-甲醛四氫葉酸轉變成四氫葉酸及二氧化碳。在哺乳類動物的肝臟細胞中富含10-甲醛四氫葉酸去氫酶,大約佔了所有蛋白百分之ㄧ的比例。然而,10-甲醛四氫葉酸去氫酶真正的生理功能至今仍不清楚,因此我們以斑馬魚為模式動物探討10-甲醛四氫葉酸去氫酶在胚胎發育過程中所扮演的角色。我們利用西方點墨法和細胞免疫染色法偵測10-甲醛四氫葉酸去氫酶表現情形。實驗結果發現,在胚胎發育過程中,10-甲醛四氫葉酸去氫酶在受精後一小時即有少量表現,在三到四天有最大的表現量,主要集中在腦部及肝臟附近。為了探討10-甲醛四氫葉酸去氫酶的弁遄A我們利用antisense morpholino oligonucleotides降低10-甲醛四氫葉酸去氫酶蛋白表現。我們初步發現降低10-甲醛四氫葉酸去氫酶蛋白表現,造成三天大的斑馬魚心臟功能出現異常,微心腔腫大,心搏速率減緩,體軸呈現彎曲,嚴重者甚至沒有尾巴。進一步藉由全覆式原位雜交法,發現斑馬魚的心血管,神經,脊索以及體軸發育出現異常。為了更加確認心臟發育過程,我們也利用顯微注射法將10-甲醛四氫葉酸去氫酶專一性的antisense morpholino oligonucleotides打入Tg(cmlc2::egfp)胚胎中。我們發現降低10-甲醛四氫葉酸去氫酶表現會導致斑馬魚在形成心環過程中出現異常。最後,這些缺陷可以部分被斑馬魚的10-甲醛四氫葉酸去氫酶去氧核醣核�˙藺M10-甲醛四氫葉酸去氫酶下游產物,四氫葉酸,補修回來。綜合以上實驗結果,我們推測10-甲醛四氫葉酸去氫酶可能在斑馬魚的心臟發育過程中扮演一個重要的角色。
10-Formyltetrahydrofolate dehydrogenase (FDH, EC 1.5.1.6), a folate-dependent enzyme in one-carbon metabolism, catalyses the conversion of 10-formyltetrahydrofolate and NADP+ to tetrahydrofolate (THF), CO2 and NADPH. FDH is very abundant in mammalian liver and constitutes 1% of soluble protein in liver. Currently, FDH has only been characterized in tissueculture cells and no functional in vivo or developmental roles have been described. In this study, we used zebrafish as animal model to investigate the role of FDH in embryonic development. We performed Western blot and whole-mount immunocytochemistry hybridization (ICC) to determine the expression pattern of FDH. We found that FDH expressed as early as 1 hour post-fertilization (hpf) and reached its expression peak at 3-dpf. To examine the function of FDH, we used antisense morpholino oligonucleotides (MO) to block its protein translation. Knockdown of FDH caused pericardial edema, dysfunction heart, body curve, no tail and all accompanied by the lower heart rate at 3-dpf. Analysis by whole mount in situ hybridization (WISH) showed that FDH morphants displayed the cardiovascular, nerve, notochord and somite development defects. To further monitor the heart development, we injected the FDH specific MO to transgenic zebrafish, Tg (cmlc2::egfp) embryos. We found that FDH morphants showed the heart looping defects in the early stage. Additionally, these effects could be partial rescued by the zFDH cap mRNA and THF. All these findings suggested that the FDH may play a crucial role in the cardiac development.
Abstract (in Chinese) I

Abstract (in English) II

Acknowledgements III

Abbreviations Ⅳ

Table of Contents Ⅴ

List of Figures Ⅸ

List of Tables Ⅸ

List of Appendixes Ⅸ

Background Introduction 1
1. Folate 1
(1) Introduction 1
(2) Structure of folate 1
(3) Metabolism of folate 2
(4) Folate-mediated one-carbon metabolism 2
(5) Folate deficiency 3

2. 10-formyltetrahydrofolate dehydrogenase (FDH) 3
(1) The structure of FDH 3
(2) The role of FDH 4
(3) Methanol toxicity in different species 5
(4) FDH deficient mice 5

3. Zebrafish (Danio rerio) 6
(1) Zebrafish as an animal model 6
(2) Overview of zebrafish development 7
(3) Zebrafish heart development 8

Specific aims 10

Materials 11
1. Zebrafish strains 11
2. Cell lines 11
3. Complement cell 11
4. Plasmid 11

Methods 12
1. Cell culture 12
2. Transfectioin 12
3. Fish care and maintenance 13
4. Embryonic protein extraction and Western blot 13
5. Cloning of zFDH 3’UTR cDNA 14
6. RNA probe synthesis 15
7. Whole-mount in situ hybridization 15
8. Whole-mount antibody hybridization 17
9. Morpholino injection 18
10. Subcloning of the zFDH full-length, N-terminal, C-terminal sequence 19
11. Subcloning of the EGFP sequence 19
12. In vitro transcription 20
13. Rescue by mRNA, tetrahydrofolate (THF) 20

Results 21
1. The mecleotide sequence of zFDH 3’UTR 21
2. Confirm the expression constructs 21
3. Distribution of zFDH protein in zebrafish embryos and larvae 22
4. Knockdown of FDH by morpholino (MO) 22
5. Defective heart morphogenesis and function
in FDH morphant 24
6. Rescue FDH morphants by zFDH mRNA and tetrahydrofolate 24
7. Knockdown of FDH affects formation of brain, notochord
and blood vascular markers 25
8. Over-expression of FDH does not affect p53 protein level during embryogenesis 25

Discussion 27
1. Early expression pattern analysis 27
2. Specificity and efficiency of the MO1 and MO2 27
3. The role of FDH in heart development 28
4. FDH morphant and folate pool unbalance 28
5. Overexpression and knockdown of FDH 29
6. The function of FDH during the embryosgenesis 29

Future Experiments 31

References 32

Figures 36

Tables 51

Appendixes 52

Author 75
Abraham, T. W. (2001). Preparation of nonradioactive probes for in situ hybridization. Methods 23, 297-302.
Anguera, M. C., Field, M. S., Perry, C., Ghandour, H., Chiang, E. P., Selhub, J., Shane, B. and Stover, P. J. (2006). Regulation of folate-mediated one-carbon metabolism by 10-formyltetrahydrofolate dehydrogenase. J Biol Chem 281, 18335-42.
Blom, H. J., Shaw, G. M., den Heijer, M. and Finnell, R. H. (2006). Neural tube defects and folate: case far from closed. Nat Rev Neurosci 7, 724-31.
Champion, K. M., Cook, R. J., Tollaksen, S. L. and Giometti, C. S. (1994). Identification of a heritable deficiency of the folate-dependent enzyme 10-formyltetrahydrofolate dehydrogenase in mice. Proc Natl Acad Sci U S A 91, 11338-42.
Cook, R. J. (2001). Disruption of histidine catabolism in NEUT2 mice. Arch Biochem Biophys 392, 226-32.
Cook, R. J., Champion, K. M. and Giometti, C. S. (2001). Methanol toxicity and formate oxidation in NEUT2 mice. Arch Biochem Biophys 393, 192-8.
Cook, R. J., Lloyd, R. S. and Wagner, C. (1991). Isolation and characterization of cDNA clones for rat liver 10-formyltetrahydrofolate dehydrogenase. J Biol Chem 266, 4965-73.
Djukic, A. (2007). Folate-responsive neurologic diseases. Pediatr Neurol 37, 387-97.
Donato, H., Krupenko, N. I., Tsybovsky, Y. and Krupenko, S. A. (2007). 10-formyltetrahydrofolate dehydrogenase requires a 4'-phosphopantetheine prosthetic group for catalysis. J Biol Chem 282, 34159-66.
Ekker, S. C., Ungar, A. R., Greenstein, P., von Kessler, D. P., Porter, J. A., Moon, R. T. and Beachy, P. A. (1995). Patterning activities of vertebrate hedgehog proteins in the developing eye and brain. Curr Biol 5, 944-55.
Gisondi, P., Fantuzzi, F., Malerba, M. and Girolomoni, G. (2007). Folic acid in general medicine and dermatology. J Dermatolog Treat 18, 138-46.
Grunwald, D. J. and Eisen, J. S. (2002). Headwaters of the zebrafish -- emergence of a new model vertebrate. Nat Rev Genet 3, 717-24.
Huang, C. J., Tu, C. T., Hsiao, C. D., Hsieh, F. J. and Tsai, H. J. (2003). Germ-line transmission of a myocardium-specific GFP transgene reveals critical regulatory elements in the cardiac myosin light chain 2 promoter of zebrafish. Dev Dyn 228, 30-40.
Johlin, F. C., Fortman, C. S., Nghiem, D. D. and Tephly, T. R. (1987). Studies on the role of folic acid and folate-dependent enzymes in human methanol poisoning. Mol Pharmacol 31, 557-61.
Johlin, F. C., Swain, E., Smith, C. and Tephly, T. R. (1989). Studies on the mechanism of methanol poisoning: purification and comparison of rat and human liver 10-formyltetrahydrofolate dehydrogenase. Mol Pharmacol 35, 745-50.
Jowett, T. (2001). Double in situ hybridization techniques in zebrafish. Methods 23, 345-58.
Kari, G., Rodeck, U. and Dicker, A. P. (2007). Zebrafish: an emerging model system for human disease and drug discovery. Clin Pharmacol Ther 82, 70-80.
Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B. and Schilling, T. F. (1995). Stages of embryonic development of the zebrafish. Dev Dyn 203, 253-310.
Krauss, S., Johansen, T., Korzh, V. and Fjose, A. (1991). Expression of the zebrafish paired box gene pax[zf-b] during early neurogenesis. Development 113, 1193-206.
Krupenko, S. A. and Oleinik, N. V. (2002). 10-formyltetrahydrofolate dehydrogenase, one of the major folate enzymes, is down-regulated in tumor tissues and possesses suppressor effects on cancer cells. Cell Growth Differ 13, 227-36.
Krupenko, S. A., Wagner, C. and Cook, R. J. (1997). Domain structure of rat 10-formyltetrahydrofolate dehydrogenase. Resolution of the amino-terminal domain as 10-formyltetrahydrofolate hydrolase. J Biol Chem 272, 10273-8.
Langheinrich, U. (2003). Zebrafish: a new model on the pharmaceutical catwalk. Bioessays 25, 904-12.
Larson, J. D., Wadman, S. A., Chen, E., Kerley, L., Clark, K. J., Eide, M., Lippert, S., Nasevicius, A., Ekker, S. C., Hackett, P. B. et al. (2004). Expression of VE-cadherin in zebrafish embryos: a new tool to evaluate vascular development. Dev Dyn 231, 204-13.
Li, G. M., Presnell, S. R. and Gu, L. (2003). Folate deficiency, mismatch repair-dependent apoptosis, and human disease. J Nutr Biochem 14, 568-75.
Lieschke, G. J. and Currie, P. D. (2007). Animal models of human disease: zebrafish swim into view. Nat Rev Genet 8, 353-67.
Link, V., Shevchenko, A. and Heisenberg, C. P. (2006). Proteomics of early zebrafish embryos. BMC Dev Biol 6, 1.
Lohr, J. L. and Yost, H. J. (2000). Vertebrate model systems in the study of early heart development: Xenopus and zebrafish. Am J Med Genet 97, 248-57.
Martinasevic, M. K., Rios, G. R., Miller, M. W. and Tephly, T. R. (1999). Folate and folate-dependent enzymes associated with rat CNS development. Dev Neurosci 21, 29-35.
McGrath, P. and Li, C. Q. (2008). Zebrafish: a predictive model for assessing drug-induced toxicity. Drug Discov Today 13, 394-401.
Muskiet, F. A. (2005). The importance of (early) folate status to primary and secondary coronary artery disease prevention. Reprod Toxicol 20, 403-10.
Neymeyer, V., Tephly, T. R. and Miller, M. W. (1997). Folate and 10-formyltetrahydrofolate dehydrogenase (FDH) expression in the central nervous system of the mature rat. Brain Res 766, 195-204.
Novak, A. E. and Ribera, A. B. (2003). Immunocytochemistry as a tool for zebrafish developmental neurobiology. Methods Cell Sci 25, 79-83.
Oleinik, N. V., Krupenko, N. I. and Krupenko, S. A. (2007). Cooperation between JNK1 and JNK2 in activation of p53 apoptotic pathway. Oncogene 26, 7222-30.
Oleinik, N. V., Krupenko, N. I., Priest, D. G. and Krupenko, S. A. (2005). Cancer cells activate p53 in response to 10-formyltetrahydrofolate dehydrogenase expression. Biochem J 391, 503-11.
Oleinik, N. V. and Krupenko, S. A. (2003). Ectopic expression of 10-formyltetrahydrofolate dehydrogenase in A549 cells induces G1 cell cycle arrest and apoptosis. Mol Cancer Res 1, 577-88.
Oxtoby, E. and Jowett, T. (1993). Cloning of the zebrafish krox-20 gene (krx-20) and its expression during hindbrain development. Nucleic Acids Res 21, 1087-95.
Pichler, F. B., Laurenson, S., Williams, L. C., Dodd, A., Copp, B. R. and Love, D. R. (2003). Chemical discovery and global gene expression analysis in zebrafish. Nat Biotechnol 21, 879-83.
Rebeille, F., Ravanel, S., Marquet, A., Mendel, R. R., Webb, M. E., Smith, A. G. and Warren, M. J. (2007). Roles of vitamins B5, B8, B9, B12 and molybdenum cofactor at cellular and organismal levels. Nat Prod Rep 24, 949-62.
Ro, H., Soun, K., Kim, E. J. and Rhee, M. (2004). Novel vector systems optimized for injecting in vitro-synthesized mRNA into zebrafish embryos. Mol Cells 17, 373-6.
Robu, M. E., Larson, J. D., Nasevicius, A., Beiraghi, S., Brenner, C., Farber, S. A. and Ekker, S. C. (2007). p53 activation by knockdown technologies. PLoS Genet 3, e78.
Schirch, D., Villar, E., Maras, B., Barra, D. and Schirch, V. (1994). Domain structure and function of 10-formyltetrahydrofolate dehydrogenase. J Biol Chem 269, 24728-35.
Schulte-Merker, S., van Eeden, F. J., Halpern, M. E., Kimmel, C. B. and Nusslein-Volhard, C. (1994). no tail (ntl) is the zebrafish homologue of the mouse T (Brachyury) gene. Development 120, 1009-15.
Stainier, D. Y. (2001). Zebrafish genetics and vertebrate heart formation. Nat Rev Genet 2, 39-48.
Steegers-Theunissen, R. P. (1995). Folate metabolism and neural tube defects: a review. Eur J Obstet Gynecol Reprod Biol 61, 39-48.
Sun, S. N., Gui, Y. H., Song, H. Y., Zhong, T., Wang, Y. X. and Jiang, Q. (2007a). [Folic acid antagonist methotrexate causes the development malformation of heart and down-regulates the BMP2b and HAS2 expressions in zebrafish]. Zhongguo Dang Dai Er Ke Za Zhi 9, 159-63.
Sun, S. N., Gui, Y. H., Wang, Y. X., Qian, L. X., Jiang, Q., Liu, D. and Song, H. Y. (2007b). Effect of dihydrofolate reductase gene knock-down on the expression of heart and neural crest derivatives expressed transcript 2 in zebrafish cardiac development. Chin Med J (Engl) 120, 1166-71.
Svoboda, K. R., Linares, A. E. and Ribera, A. B. (2001). Activity regulates programmed cell death of zebrafish Rohon-Beard neurons. Development 128, 3511-20.
Thisse, B., Heyer, V., Lux, A., Alunni, V., Degrave, A., Seiliez, I., Kirchner, J., Parkhill, J. P. and Thisse, C. (2004). Spatial and temporal expression of the zebrafish genome by large-scale in situ hybridization screening. Methods Cell Biol 77, 505-19.
Thisse, C. and Thisse, B. (2008). High-resolution in situ hybridization to whole-mount zebrafish embryos. Nat Protoc 3, 59-69.
Trinh, L. A. and Stainier, D. Y. (2004). Cardiac development. Methods Cell Biol 76, 455-73.
Ueland, P. M., Midttun, O., Windelberg, A., Svardal, A., Skalevik, R. and Hustad, S. (2007). Quantitative profiling of folate and one-carbon metabolism in large-scale epidemiological studies by mass spectrometry. Clin Chem Lab Med 45, 1737-45.
Vogel, A. M. and Weinstein, B. M. (2000). Studying vascular development in the zebrafish. Trends Cardiovasc Med 10, 352-60.
Weinberg, E. S., Allende, M. L., Kelly, C. S., Abdelhamid, A., Murakami, T., Andermann, P., Doerre, O. G., Grunwald, D. J. and Riggleman, B. (1996). Developmental regulation of zebrafish MyoD in wild-type, no tail and spadetail embryos. Development 122, 271-80.
Westerfield, M. (2000). The Zebrafish Book: A Guide for the Laboratory
Use of Zebrafish (Danio rerio), Fourth Edition.
Yelon, D., Horne, S. A. and Stainier, D. Y. (1999). Restricted expression of cardiac myosin genes reveals regulated aspects of heart tube assembly in zebrafish. Dev Biol 214, 23-37.
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