臺灣博碩士論文加值系統

本論文的主要目的是要研究Agr2基因表現的調節機制，首先利用Ensembl Genome Browser搜尋引擎搜尋斑馬魚Agr2 upstream /promoter DNA序列與轉錄區之DNA、使用promoter scan分析Agr2預測保守的promoter region及TATA Box位置、利用Family Relations II軟體比較不同物種之Agr2 upstream/promoter DNA 序列來預測是否有保守的區域及利用網站Genomatix中的Matinspector軟體來預測了6kbp Agr2 upstream/promoter DNA序列可能有哪些轉錄因子結合區域，上述所獲得之資訊用來設計製備不同長度的Agr2 upstream /promoter DNA sequence接合入TolⅡ-EGFP transposon vector中，分別獲得：[-6038EGFP]、[-5276EGFP]、[-4188EGFP]、[-3284EGFP]與[-2049EGFP]質體DNA與transposase mRNA共同注射入斑馬魚胚胎1~2細胞時期，觀察從24~120 hpf間綠色螢光蛋白表現位置，結果顯示[-6038EGFP]和[-5276EGFP]注射的斑馬魚胚胎觀察到的綠色螢光蛋白表現位置與內生性Agr2 mRNA表現位置相似度很高，觀察結果顯示24~120hpf期間綠色螢光蛋白主要會表現在孵化腺、黏液細胞、耳半規管、咽、食道、腸道杯狀細胞；[-4188EGFP]與[-3284EGFP]注射的斑馬魚胚胎所觀察到的綠色螢光蛋白表現位置只在耳半規管與黏液細胞，而其內胚層衍生器官如咽與腸道杯狀細胞則無綠色螢光蛋白表現；最後[-2049EGFP]注射的斑馬魚胚胎多數胚胎則無綠色螢光蛋白表現，若有綠色螢光蛋白表現則為非專一性的在肌肉或脊索的mosaic pattern。綜而言之，Agr2 upstream /promoter DNA序列在-5276至-4188有調控綠色螢光蛋白表現在不同內胚層衍生器官如咽與腸道杯狀細胞之功能，而在 -3284至-2049間則具有調控綠色螢光蛋白表現在耳半規管與黏液細胞的調節序列。最後將[-6038EGFP]注射的斑馬魚胚胎所觀察綠色螢光蛋白表現與內生性Agr2 mRNA表現位置相仿的斑馬魚胚胎經過三個月的飼養，經交配所生下之F1子代，觀察發現F1子代綠色螢光蛋白表現位置與內生性Agr2 mRNA分佈情形可說是幾乎完全相符。

The main goal of my thesis is to study the regulatory mechanism of anterior gradient 2 (Agr2) gene expression in zebrafish. At the begining, I used Ensembl Genome Browser to search and download zebrafish Agr2 upstream/promoter DNA sequence, used Promoter scan to predict potential promoter and TATA box regions, used Family Relations II to search for conserved regions of Agr2 upstream/promoter DNA among different species, and used Matinspector of Genomatix website to localize possible transcription factor binding motifs in 6kbp Agr2 upstream/ promoter DNA sequence. The obtained information was used to design different lengths of Agr2 upstream/promoter DNA sequences in order to ligate them into Tol II-EGFP transposon vector. Thus, I have obtained respective recombinant plasmids including [-6038EGFP], [-5276EGFP], [-4188EGFP], [-3284EGFP] and [-2049EGFP] plasmids. After Co-injection of both the respective plasmid and transposase mRNA into 1~2 cells of zebrafish embryo, EGFP expression pattern in injected embryos was monitored during 24 to 120 h of development. Results of EGFP expression pattern of respective [-6038EGFP]- and [-5276EGFP]-injected embryos show that they share high similarity with endogenous Agr2 mRNA expression pattern. EGFP is mainly expressed in hatching gland, mucous cells, otic vesicle, pharynx, esophagus, and goblet cells in the intestine. EGFP expression patterns in respective [-4188EGFP]- and [-3284EGFP]-injected embryos are restricted in mucous cells and otic vesicles, while no EGFP expression can be detected in any endoderm derived organs such as pharynx and goblet cells in the intestine. Most of [-2049EGFP]-injected embryos show either no EGFP expression or non-specific muscle or notochord expression. In conclusion, DNA sequence within -5276~ -4188 of Agr2 upstream/promoter may regulate EGFP expression in endoderm derived organs such as pharynx and goblet cells in the intestine, while DNA sequence within -3284~ -2049 of Agr2 upstream/promoter may regulate EGFP expression in otic vesicles and mucous cells. Finally, those [-6038EGFP]-injected embryos showing EGFP expression pattern similar to endogenous Agr2 mRNA localization were raised to adulthood after three months. EGFP expression patterns in F1 embryos from Tg(Agr2 6K:EGFP) transgenic fish is the same as endogenous Agr2 mRNA distribution during 24 to 120
hpf of development.

誌謝 i
中文摘要 iii
英文摘要 v
目錄 vii
實驗方法目錄 viii
圖目錄 x
前言 1
實驗方法 21
實驗結果 43
討論 49
參考文獻 56
圖表 62
附錄 86

Alexander, J., Rothenberg, M., Henry, G. L. and Stainier, D. Y. (1999). casanova plays an early and essential role in endoderm formation in zebrafish. Dev Biol 215, 343-357.
Alexander, J. and Stainier, D. Y. (1999). A molecular pathway leading to endoderm formation in zebrafish. Curr Biol 9, 1147-1157.
Amatruda, J. F., Shepard, J. L., Stern, H. M. and Zon, L. I. (2002). Zebrafish as a cancer model system. Cancer Cell 1, 229-231.
Andreu, P., Colnot, S., Godard, C., Gad, S., Chafey, P., Niwa-Kawakita, M., Laurent-Puig, P., Kahn, A., Robine, S., Perret, C. et al. (2005). Crypt-restricted proliferation and commitment to the Paneth cell lineage following Apc loss in the mouse intestine. Development 132, 1443-1451.
Ao, A., Wang, H., Kamarajugadda, S. and Lu, J. (2008). Involvement of estrogen-related receptors in transcriptional response to hypoxia and growth of solid tumors. Proc Natl Acad Sci U S A 105, 7821-7826.
Artavanis-Tsakonas, S., Rand, M. D. and Lake, R. J. (1999). Notch signaling: cell fate control and signal integration in development. Science 284, 770-776.
Berghmans, S., Jette, C., Langenau, D., Hsu, K., Stewart, R., Look, T. and Kanki, J. P. (2005). Making waves in cancer research: new models in the zebrafish. Biotechniques 39, 227-237.
Bjornson, C. R., Griffin, K. J., Farr, G. H., 3rd, Terashima, A., Himeda, C., Kikuchi, Y. and Kimelman, D. (2005). Eomesodermin is a localized maternal determinant required for endoderm induction in zebrafish. Dev Cell 9, 523-533.
Burns, C. G., Milan, D. J., Grande, E. J., Rottbauer, W., MacRae, C. A. and Fishman, M. C. (2005). High-throughput assay for small molecules that modulate zebrafish embryonic heart rate. Nat Chem Biol 1, 263-264.
Byrd, C. A. and Brunjes, P. C. (1995). Organization of the olfactory system in the adult zebrafish: histological, immunohistochemical, and quantitative analysis. J Comp Neurol 358, 247-259.
Chen, S. and Kimelman, D. (2000). The role of the yolk syncytial layer in germ layer patterning in zebrafish. Development 127, 4681-4689.
Chen, Y. H., Wang, Y. H., Chang, M. Y., Lin, C. Y., Weng, C. W., Westerfield, M. and Tsai, H. J. (2007). Multiple upstream modules regulate zebrafish myf5 expression. BMC Dev Biol 7, 1.
Choo, B. G., Kondrichin, I., Parinov, S., Emelyanov, A., Go, W., Toh, W. C. and Korzh, V. (2006). Zebrafish transgenic Enhancer TRAP line database (ZETRAP). BMC Dev Biol 6, 5.
Crosnier, U., Vargesson, N., Gschmeissner, S., Ariza-McNaughton, L., Morrison, A. and Lewis, J. (2005). Delta-Notch signalling controls commitment to a secretory fate in the zebrafish intestine. Development 132, 1093-1104.
David, N. B. and Rosa, F. M. (2001). Cell autonomous commitment to an endodermal fate and behaviour by activation of Nodal signalling. Development 128, 3937-3947.
de Santa Barbara, P., van den Brink, G. R. and Roberts, D. J. (2003). Development and differentiation of the intestinal epithelium. Cell Mol Life Sci 60, 1322-1332.
De Strooper, B., Annaert, W., Cupers, P., Saftig, P., Craessaerts, K., Mumm, J. S., Schroeter, E. H., Schrijvers, V., Wolfe, M. S., Ray, W. J. et al. (1999). A presenilin-1-dependent gamma-secretase-like protease mediates release of Notch intracellular domain. Nature 398, 518-522.
den Hertog, J. (2005). Chemical genetics: Drug screens in Zebrafish. Biosci Rep 25, 289-297.
Escaffit, F., Pare, F., Gauthier, R., Rivard, N., Boudreau, F. and Beaulieu, J. F. (2006). Cdx2 modulates proliferation in normal human intestinal epithelial crypt cells. Biochem Biophys Res Commun 342, 66-72.
Feldman, B., Dougan, S. T., Schier, A. F. and Talbot, W. S. (2000). Nodal-related signals establish mesendodermal fate and trunk neural identity in zebrafish. Curr Biol 10, 531-534.
Feldman, B., Gates, M. A., Egan, E. S., Dougan, S. T., Rennebeck, G., Sirotkin, H. I., Schier, A. F. and Talbot, W. S. (1998). Zebrafish organizer development and germ-layer formation require nodal-related signals. Nature 395, 181-185.
Griffin, K. J., Amacher, S. L., Kimmel, C. B. and Kimelman, D. (1998). Molecular identification of spadetail: regulation of zebrafish trunk and tail mesoderm formation by T-box genes. Development 125, 3379-3388.
Gritsman, K., Zhang, J., Cheng, S., Heckscher, E., Talbot, W. S. and Schier, A. F. (1999). The EGF-CFC protein one-eyed pinhead is essential for nodal signaling. Cell 97, 121-132.
Haffter, P., Granato, M., Brand, M., Mullins, M. C., Hammerschmidt, M., Kane, D. A., Odenthal, J., van Eeden, F. J., Jiang, Y. J., Heisenberg, C. P. et al. (1996). The identification of genes with unique and essential functions in the development of the zebrafish, Danio rerio. Development 123, 1-36.
Horne-Badovinac, S., Rebagliati, M. and Stainier, D. Y. (2003). A cellular framework for gut-looping morphogenesis in zebrafish. Science 302, 662-665.
Jarriault, S., Brou, C., Logeat, F., Schroeter, E. H., Kopan, R. and Israel, A. (1995). Signalling downstream of activated mammalian Notch. Nature 377, 355-358.
Jenny, M., Uhl, C., Roche, C., Duluc, I., Guillermin, V., Guillemot, F., Jensen, J., Kedinger, M. and Gradwohl, G. (2002). Neurogenin3 is differentially required for endocrine cell fate specification in the intestinal and gastric epithelium. EMBO J 21, 6338-6347.
Jensen, J., Pedersen, E. E., Galante, P., Hald, J., Heller, R. S., Ishibashi, M., Kageyama, R., Guillemot, F., Serup, P. and Madsen, O. D. (2000). Control of endodermal endocrine development by Hes-1. Nat Genet 24, 36-44.
Kawakami, K. and Shima, A. (1999). Identification of the Tol2 transposase of the medaka fish Oryzias latipes that catalyzes excision of a nonautonomous Tol2 element in zebrafish Danio rerio. Gene 240, 239-244.
Kawakami, K., Takeda, H., Kawakami, N., Kobayashi, M., Matsuda, N. and Mishina, M. (2004). A transposon-mediated gene trap approach identifies developmentally regulated genes in zebrafish. Dev Cell 7, 133-144.
Kikuchi, Y., Agathon, A., Alexander, J., Thisse, C., Waldron, S., Yelon, D., Thisse, B. and Stainier, D. Y. (2001). casanova encodes a novel Sox-related protein necessary and sufficient for early endoderm formation in zebrafish. Genes Dev 15, 1493-1505.
Kikuchi, Y., Trinh, L. A., Reiter, J. F., Alexander, J., Yelon, D. and Stainier, D. Y. (2000). The zebrafish bonnie and clyde gene encodes a Mix family homeodomain protein that regulates the generation of endodermal precursors. Genes Dev 14, 1279-1289.
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.
Kimmel, C. B., Warga, R. M. and Schilling, T. F. (1990). Origin and organization of the zebrafish fate map. Development 108, 581-594.
Koga, A., Suzuki, M., Inagaki, H., Bessho, Y. and Hori, H. (1996). Transposable element in fish. Nature 383, 30.
Kotani, T. and Kawakami, K. (2008). Misty somites, a maternal effect gene identified by transposon-mediated insertional mutagenesis in zebrafish that is essential for the somite boundary maintenance. Dev Biol 316, 383-396.
Kunwar, P. S., Zimmerman, S., Bennett, J. T., Chen, Y., Whitman, M. and Schier, A. F. (2003). Mixer/Bon and FoxH1/Sur have overlapping and divergent roles in Nodal signaling and mesendoderm induction. Development 130, 5589-5599.
Langheinrich, U. (2003). Zebrafish: a new model on the pharmaceutical catwalk. Bioessays 25, 904-912.
Liu, D., Rudland, P. S., Sibson, D. R., Platt-Higgins, A. and Barraclough, R. (2005a). Human homologue of cement gland protein, a novel metastasis inducer associated with breast carcinomas. Cancer Research 65, 3796-3805.
Liu, D., Rudland, P. S., Sibson, D. R., Platt-Higgins, A. and Barraclough, R. (2005b). Human homologue of cement gland protein, a novel metastasis inducer associated with breast carcinomas. Cancer Res 65, 3796-3805.
Lunde, K., Belting, H. G. and Driever, W. (2004). Zebrafish pou5f1/pou2, homolog of mammalian Oct4, functions in the endoderm specification cascade. Curr Biol 14, 48-55.
Madison, B. B., Braunstein, K., Kuizon, E., Portman, K., Qiao, X. T. and Gumucio, D. L. (2005). Epithelial hedgehog signals pattern the intestinal crypt-villus axis. Development 132, 279-289.
Mutoh, H., Satoh, K., Kita, H., Sakamoto, H., Hayakawa, H., Yamamoto, H., Isoda, N., Tamada, K., Ido, K. and Sugano, K. (2005). Cdx2 specifies the differentiation of morphological as well as functional absorptive enterocytes of the small intestine. Int J Dev Biol 49, 867-871.
Naya, F. J., Huang, H. P., Qiu, Y., Mutoh, H., DeMayo, F. J., Leiter, A. B. and Tsai, M. J. (1997). Diabetes, defective pancreatic morphogenesis, and abnormal enteroendocrine differentiation in BETA2/neuroD-deficient mice. Genes Dev 11, 2323-2334.
Ng, A. N., de Jong-Curtain, T. A., Mawdsley, D. J., White, S. J., Shin, J., Appel, B., Dong, P. D., Stainier, D. Y. and Heath, J. K. (2005). Formation of the digestive system in zebrafish: III. Intestinal epithelium morphogenesis. Dev Biol 286, 114-135.
North, T. E. and Zon, L. I. (2003). Modeling human hematopoietic and cardiovascular diseases in zebrafish. Dev Dyn 228, 568-583.
Novoselov, V. V., Alexandrova, E. M., Ermakova, G. V. and Zaraisky, A. G. (2003). Expression zones of three novel genes abut the developing anterior neural plate of Xenopus embryo. Gene Expr Patterns 3, 225-230.
O'Kane, C. J. and Gehring, W. J. (1987). Detection in situ of genomic regulatory elements in Drosophila. Proc Natl Acad Sci U S A 84, 9123-9127.
Ober, E. A. and Schulte-Merker, S. (1999). Signals from the yolk cell induce mesoderm, neuroectoderm, the trunk organizer, and the notochord in zebrafish. Dev Biol 215, 167-181.
Parinov, S., Kondrichin, I., Korzh, V. and Emelyanov, A. (2004). Tol2 transposon-mediated enhancer trap to identify developmentally regulated zebrafish genes in vivo. Dev Dyn 231, 449-459.
Pesce, M. and Scholer, H. R. (2000). Oct-4: control of totipotency and germline determination. Mol Reprod Dev 55, 452-457.
Postlethwait, J. H., Woods, I. G., Ngo-Hazelett, P., Yan, Y. L., Kelly, P. D., Chu, F., Huang, H., Hill-Force, A. and Talbot, W. S. (2000). Zebrafish comparative genomics and the origins of vertebrate chromosomes. Genome Res 10, 1890-902.
Poulain, M. and Lepage, T. (2002). Mezzo, a paired-like homeobox protein is an immediate target of Nodal signalling and regulates endoderm specification in zebrafish. Development 129, 4901-4914.
Randall, R. A., Germain, S., Inman, G. J., Bates, P. A. and Hill, C. S. (2002). Different Smad2 partners bind a common hydrophobic pocket in Smad2 via a defined proline-rich motif. EMBO J 21, 145-156.
Rebagliati, M. R., Toyama, R., Haffter, P. and Dawid, I. B. (1998). cyclops encodes a nodal-related factor involved in midline signaling. Proc Natl Acad Sci U S A 95, 9932-9937.
Reiter, J. F., Alexander, J., Rodaway, A., Yelon, D., Patient, R., Holder, N. and Stainier, D. Y. (1999). Gata5 is required for the development of the heart and endoderm in zebrafish. Genes Dev 13, 2983-2995.
Reiter, J. F., Kikuchi, Y. and Stainier, D. Y. (2001). Multiple roles for Gata5 in zebrafish endoderm formation. Development 128, 125-135.
Rinkwitz-Brandt, S., Arnold, H. H. and Bober, E. (1996). Regionalized expression of Nkx5-1, Nkx5-2, Pax2 and sek genes during mouse inner ear development. Hear Res 99, 129-138.
Rodaway, A., Takeda, H., Koshida, S., Broadbent, J., Price, B., Smith, J. C., Patient, R. and Holder, N. (1999). Induction of the mesendoderm in the zebrafish germ ring by yolk cell-derived TGF-beta family signals and discrimination of mesoderm and endoderm by FGF. Development 126, 3067-3078.
Sancho, E., Batlle, E. and Clevers, H. (2003). Live and let die in the intestinal epithelium. Curr Opin Cell Biol 15, 763-770.
Sansom, O. J., Reed, K. R., Hayes, A. J., Ireland, H., Brinkmann, H., Newton, I. P., Batlle, E., Simon-Assmann, P., Clevers, H., Nathke, I. S. et al. (2004). Loss of Apc in vivo immediately perturbs Wnt signaling, differentiation, and migration. Genes Dev 18, 1385-1390.
Sasai, Y., Kageyama, R., Tagawa, Y., Shigemoto, R. and Nakanishi, S. (1992). Two mammalian helix-loop-helix factors structurally related to Drosophila hairy and Enhancer of split. Genes Dev 6, 2620-2634.
Shih, L. J., Lu, Y. F., Chen, Y. H., Lin, C. C., Chen, J. A. and Hwang, S. P. (2007). Characterization of the agr2 gene, a homologue of X. laevis anterior gradient 2, from the zebrafish, Danio rerio. Gene Expr Patterns 7, 452-460.
Shroyer, N. F., Wallis, D., Venken, K. J., Bellen, H. J. and Zoghbi, H. Y. (2005). Gfi1 functions downstream of Math1 to control intestinal secretory cell subtype allocation and differentiation. Genes Dev 19, 2412-2417.
Silberg, D. G., Swain, G. P., Suh, E. R. and Traber, P. G. (2000). Cdx1 and cdx2 expression during intestinal development. Gastroenterology 119, 961-971.
Sirotkin, H. I., Gates, M. A., Kelly, P. D., Schier, A. F. and Talbot, W. S. (2000). Fast1 is required for the development of dorsal axial structures in zebrafish. Curr Biol 10, 1051-1054.
Sive, H. and Bradley, L. (1996). A sticky problem: the Xenopus cement gland as a paradigm for anteroposterior patterning. Dev Dyn 205, 265-280.
Sive, H. L., Hattori, K. and Weintraub, H. (1989). Progressive determination during formation of the anteroposterior axis in Xenopus laevis. Cell 58, 171-180.
Stainier, D. Y. (2002). A glimpse into the molecular entrails of endoderm formation. Genes Dev 16, 893-907.
Tam, P. P., Kanai-Azuma, M. and Kanai, Y. (2003). Early endoderm development in vertebrates: lineage differentiation and morphogenetic function. Curr Opin Genet Dev 13, 393-400.
Thompson, D. A. and Weigel, R. J. (1998). hAG-2, the human homologue of the Xenopus laevis cement gland gene XAG-2, is coexpressed with estrogen receptor in breast cancer cell lines. Biochem Biophys Res Commun 251, 111-116.
van de Wetering, M., Sancho, E., Verweij, C., de Lau, W., Oving, I., Hurlstone, A., van der Horn, K., Batlle, E., Coudreuse, D., Haramis, A. P. et al. (2002). The beta-catenin/TCF-4 complex imposes a crypt progenitor phenotype on colorectal cancer cells. Cell 111, 241-250.
Wallace, K. N., Akhter, S., Smith, E. M., Lorent, K. and Pack, M. (2005). Intestinal growth and differentiation in zebrafish. Mech Dev 122, 157-173.
Warga, R. M. and Nusslein-Volhard, C. (1999). Origin and development of the zebrafish endoderm. Development 126, 827-838.
Yang, Q., Bermingham, N. A., Finegold, M. J. and Zoghbi, H. Y. (2001). Requirement of Math1 for secretory cell lineage commitment in the mouse intestine. Science 294, 2155-2158.
Zon, L. I. (1999). Zebrafish: a new model for human disease. Genome Res 9, 99-100.
Zorn, A. M. and Wells, J. M. (2007). Molecular basis of vertebrate endoderm development. Int Rev Cytol 259, 49-111.
Zweitzig, D. R., Smirnov, D. A., Connelly, M. C., Terstappen, L. W., O'Hara, S. M. and Moran, E. (2007). Physiological stress induces the metastasis marker AGR2 in breast cancer cells. Mol Cell Biochem 306, 255-260.