臺灣博碩士論文加值系統

pri/tal 為一具有 polycistronic 現象的基因且於上皮細胞的形態發育及 pattern formation 皆扮演重要的角色。由之前 in situ hybridization 的結果，我們認為pri/tal 可能受到 EGFR 訊號的調控。因此，在本實驗中，我想要進一步探討 pri/tal 與 EGFR 之間的調控關係為正向調控或負向調控。EGFR 訊號傳遞途徑參與許多果蠅重要的發育過程，例如：chorodtonal organ recruitment 、翅脈的形成及 follicle cells patterning 等等。所以我們利用遺傳學及 in situ hybridization的方法來證明 pri/tal 於 follicle cells 及chordotonal organ 的表達是受到 EGFR 訊號調控。然而，我們也發現當我大量表達 EGFR transcription repressor Yan 時，pri/tal mRNA 也會異位表達在 eye disc。因此我們推測 EGFR 訊號對於 pri/tal 基因的調控會因組織的不同而有不同的調控。過去已知 EGFR 訊號傳遞途徑下游有兩個 ETS 家族且能辨認ETS binding site的轉錄因子 Pointed 和 Yan。因此我們利用 ETS binding site 尋找 pri/tal 的調控區域，並分析這些調控區是否受到 EGFR訊號的調控。於本實驗結果知， pri/tal 基因上游 5k的調控區內包含leg disc pretarsal region、presumptive joint、antennal segment、follicle cells 及embryo tracheal precursor等不同位置的表達。我們以遺傳學方法證明 pri/tal 2.9kb 調控區於 follicle cells，的確受到 EGFR 訊號的影響。所以 pri/tal 的2.9kb reporter 確實受到 EGFR 訊號的調控。

pri/tal was identified as a polycistronic gene in Drosophila and it has important roles in epithelial morphogenesis and pattern formation in Drosophila. Based on our in situ hybridization data, we suggest that pri/tal may be regulated by the EGFR signaling. Thus, I want to futher study between pri/tal and the EGFR regulative relations for the positive regulation or negative regulation. We knew that EGFR pathway is essential for many development processes in Drosophila, including chordotonal organ recruitment, wing vein formation, and follicle cells patterning, etc. Therefore, we use the genetics and in situ hybridization method proved that EGFR signaling regulated pri/tal in follicle cells and chordotonal organ. But, we also found that when overexpressed EGFR transcription repressor Yan, pri/tal was ectopically expressed in eye disc. Thus, we suggested that pri/tal expression is regulated by EGFR signaling in different tissue. EGFR signaling has two transcription factors, Pointed and Yan and they belong to Ets family and they can recognize the ETS binding site. Therefore I use the ETS binding site to search for pri/tal regulatory region, and analyzes these regulatory region whether to regulate by the EGFR signaling. Here we reported that pri/tal upstream 5k region including leg disc pretarsal region, presumptive joint, antennal segment, follicle cells and embryo tracheal precursor, and so on different expression. We use the genetics method to porve that the 2.9kb pri/tal regulatory region in follicle cells, indeed regulated by EGFR signaling. Therefore, pri/tal 2.9k reporter was regulated by the EGFR signaling in the follicle cells.

目錄
誌 謝 iv
中文摘要 v
Abstract vi
目錄 vii
圖表目錄 ix
背景介紹 1
Epidermal Growth Factor Receptor (EGFR) 訊號傳遞的調控 1
ETS (E twenty-six) 家族成員—Yan 和Pointed 2
果蠅眼睛發育(eye development) 3
果蠅翅膀發育 (wing development) 4
果蠅卵子發育 (oogenesis) 4
pri/tal 基因的介紹及功能 5
實驗材料與方法 8
使用的果蠅株 (Fly strain) 8
Genomic DNA 萃取 8
DNA 質體構築 (Construct) 9
1. 製備RNA 探針之質體 9
2. Promoter constructs 10
3. Polycistronic DNA constructs 10
in situ Hybridization 實驗方法 10
1. RNA探針的製備 10
2. in situ Hybridization of Drosophila imaginal disc 11
3. in situ Hybridization of Drosophila ovary 12
Immunohistochemistry and microscopy 13
1. X-gal staining of imaginal disc 13
2. X-gal staining of ovary 13
3. X-gal staining of embryo 14
4. 影像擷取 14
細胞培養 (Cell culture) 14
1. MDCK cell line 14
2. Margaret S2 cell 17
實驗結果 19
pri/tal mRNA 表達的 pattern 19
1. pri/tal 於 imaginal disc的表達與 EGFR 訊號途徑間關係 20
2. pri/tal於follicle cells的角色與 EGFR 訊號傳遞之間的關係 22
pri/tal promoter analysis 23
1. 以 ETS binding site 作為搜尋調控區的標準 24
2. 分析不同長度的 reporter 片段於果蠅組織中的表達位置 24
3. 分析pri/tal 2.9-LacZ reporter 調控區受到 EGFR 訊號調控 25
分析 pri/tal polycistronic 現象於果蠅及哺乳動物細胞 26
討論 29
pri/tal的表達與 EGFR 訊號傳遞的關係 29
pri/tal 基因的表達受到EGFR 訊號傳遞調控 30
哺乳動物細胞中 polycistronic 現象是否存在 31
參考文獻 33
附 錄 39

圖表目錄

Fig. 1 pri/tal 基因構造特性。 41
Fig. 2 pri/tal mRNA follicle cells 的 pattern 及與 EGFR訊號途徑間的關係。 42
Fig. 3 pri/tal mRNA 於 wild type imaginal disc 表達的 pattern。 44
Fig. 4 以 EQ1-Gal4 大量表達 EGFR 訊號於 all follicle cells。 46
Fig. 5 以 Dpp-Gal4 大量表達 EGFR 訊號於 leg disc chordotonal organ。 47
Fig. 6 GMR-Gal4 大量表達 EGFR 訊號於 eye disc posterior to morphogenic furrow 的區域。 48
Fig. 7 pri/tal reporter 於果蠅 disc 、 follicle cells 及 embryo的表達 pattern。 49
Fig. 8 pri/tal reporter 於果蠅 embryo的表達 pattern。 51
Fig. 9 分析 pri/tal 2.9-LacZ reporter 調控區受到 EGFR 訊號調控。 53
Fig. 10 分析 pri/tal polycistronic 現象於果蠅及哺乳動物細胞 (MDCK cell)。 54
Fig. 11 pri/tal 上游調控區中 ETS binding site 與另一果蠅種 (D. ps : Drosophila pseudoabscura) 之間 Homology比較。 56

Table 1 Gal4 line 測試結果 43
Table 2 Reporter analysis pattern 總覽 52

參考文獻

1. Albagli O, Klaes A, Ferreira E, Leprince D, Klämbt C. (1996). Function of ets genes is conserved between vertebrates and Drosophila. Mech Dev. 59(1), 29-40.
2. Baena-López LA, Pastor-Pareja JC, Resino J. (2003). Wg and Egfr signalling antagonise the development of the peripodial epithelium in Drosophila wing discs. Development. 130(26), 6497-6506.
3. Baker DA, Mille-Baker B, Wainwright SM, Ish-Horowicz D, Dibb NJ. (2001). Mae mediates MAP kinase phosphorylation of Ets transcription factors in Drosophila. Nature. 411(6835), 330-334.
4. Barolo S, Carver LA, Posakony JW. (2000). GFP and beta-galactosidase transformation vectors for promoter/enhancer analysis in Drosophila. Biotechniques. 29(4), 726-732.
5. Behan KJ, Nichols CD, Cheung TL, Farlow A, Hogan BM, Batterham P, Pollock JA. (2002). Yan regulates Lozenge during Drosophila eye development. Dev Genes Evol. 212(6), 267-276.
6. Bellen, H. J., Levis, R. W., Liao, G., He, Y., Carlson, J. W., Tsang, G., Evans-Holm, M., Hiesinger, P. R., Schulze, K. L., Rubin, G. M., Hoskins, R. A., and Spradling, A. C. (2004). The BDGP gene disruption project: single transposon insertions associated with 40% of Drosophila genes. Genetics 167, 761-781.
7. Buskirk CV, Schüpbach T. (1999). Versatility in signaling: multiple resposes to EGF receptor activation during Drosophila oogenesis. Trends in Cell Biol. 9, 1-4.
8. Charroux B, Freeman M, Kerridge S, Baonza A. (2006). Atrophin contributes to the negative regulation of epidermal growth factor receptor signaling in Drosophila. Dev Biol. 291 (2), 278-290.
9. Dammai V, Hsu T. (2003). EGF-dependent and independent activation of MAP kinas during Drosophila oogenesis. Anat. Rec. A Drscov. Mol. Cell Evol. Biol. 272(1), 377-382.
10. Díaz-Benjumea, F. and Hafen, E. (1994). The sevenless signalling cassette mediates Drosophila EGF receptor function during epidermal development. Development 120, 569-578.
11. Freeman, M. (1996). Reitrerative use of the EGF receptor trigger differentiation of all cell type in the Drosophila eye. Cell 87, 651-660.
12. Galindo MI, Pueyo JI, Fouix S, Bishop SA, Couso JP. (2007). Peptides encoded by short ORFs control development and define a new eukaryotic gene family. PLoS Biol. 5(5), 1052-1062.
13. Gabay L, Seger R, Shilo BZ. (1997). In situ Activation pattern of Drosophila EGF receptor pathway during development. Science. 277, 1103-1106.
14. Heberlein, U. and Moses, K. (1995). Mechanisms of Drosophila retinal morphogenesis: the virtues of being progressive. Cell 81, 987-990.
15. Hsu T, Schulz RA. (2000). Sequence and functional properties of Ets genes in the model organism Drosophila. Oncogene. 19 (55), 6409-6416.
16. J.P. Kumar, M. Tio, F. Hsiung, S. Akopyan, L. Gabay, R. Seger et al. (1998). Dissecting the roles of the Drosophila EGF receptor in eye development and MAP kinase activation. Development. 125, 3875-3885.
17. Kondo T, Hashimoto Y, Kato K, Inagaki S, Hayashi S, Kageyama Y. (2007). Small peptide regulators of actin-based cell morphogenesis encoded by a polycistronic mRNA. Nat Cell Biol. 9(6), 660-665.
18. Martín-Blanco E, Roch F, Noll E, Baonza A, Duffy JB, Perrimon N. (1999). A temporal switch in DER signaling controls the specification and differentiation of veins and interveins in the Drosophila wing. Development.126 (24), 5739-5747.
19. Morimoto AM, Jordan KC, Tietze K, Britton JS, O'Neill EM, Ruohola-Baker H. (1996). Pointed, an ETS domain transcription factor, negatively regulates the EGF receptor pathway in Drosophila oogenesis. Development. 122 (12), 3745-3754.
20. Nagaso, H., Murata, T., Day, N. & Yokoyama, K. K. (2001). Simultaneous detection of RNA and protein by in situ hybridization and immunological staining. J. Histochem. Cytochem. 49, 1177-1182
21. Nakamura Y, Kagesawa T, Nishikawa M, Hayashi Y, Kobayashi S, Niimi T, Matsuno K. (2007). Soma-dependent modulations contribute to divergence of rhomboid expression during evolution of Drosophila eggshell morphology. Development. 134 (8), 1529-1537.
22. Nakamura Y, Matsuno K. (2003). Species-specific activation of EGF receptor signaling underlies evolutionary in the dorsal appendage number of the genus Drosophila eggshells. Mech. Dev. 120, 897-907.
23. O’Keefe D, Prober DA, Moyle PS, Rickoll WL, Edgar BA. (2007). Egfr/Ras signaling regulates DE-cadherin.Shotgun localization to control vwin morphogenesis in the Drosophila wing. Dev. Biol. 311, 25-39.
24. Pai LM, Wang PY, Chen SR, Barcelo G, Chang WL, Nilson L, Schüpbach T. (2006). Differential effects of Cbl isoforms on Egfr signaling in Drosophila. Mech. Dev. 123(6), 450-462.
25. Pai LM, Barcelo G, Schüpbach T. (2000). D-cbl, a negative regulator of the Egfr pathway is required for dorsoventral patterning in Drosophila oogenesis. Cell. 103 (1), 51-61.
26. Rabay I, Rubin GM. (1995). Yan functions as a general inhibitor of differentiation and is negatively regulated by actiation of the Ras1/MAPK pathway. Cell. 81, 857-866.
27. Roignant JY, Hamel S, Janody F, Treisman JE. (2006). The novel SAM domain protein Aveugle is required for Raf activation in the Drosophila EGF receptor signaling pathway. Genes Dev. 20(7), 795-806.
28. Rogers EM, Brennan CA, Mortimer NT, Cook S, Morris AR, Moses K. (2005). Pointed regulates an eye-specific transcriptional enhancer in the Drosophila hedgehog gene, which is required for the movement of the morphogenetic furrow. Development. 132 (21), 4833-4843.
29. Sapir A, Schweitzer R, Shilo BZ. (1995). Sequential activation of the EGF receptor pathway during Drosophila oogenesis establishes the dorsoventral axis. Development. 125, 191-200.
30. Savard J, Marques-Souza H, Aranda M, Tautz D. (2006). A segmentation gene in Tribolium produces a polycistronic mRNA that codes for multiple conserved peptides. Cell 126, 559-569.
31. Sharrocks AD, Brown AL, Ling Y, Yates PR. (1997). The ETS-domain transcription factor family. Int J Biochem Cell Biol. 29 (12), 1371-1387.
32. Shilo BZ. (2003). Signaling by the Drosophila epidermal growth factor receptor pathway during development. Exp. Cell Res. 284, 140-149.
33. Simcox A. (1997). Differential requirement for EGF-like ligands in Drosophila wing development. Mech. Dev. 62, 41-50.
34. Sun Y, Jan LY, Jan YN. (1998). Transcritional regulation of atonal during development of the Drosophila peripheral nervous system. Development. 125, 3731-3740.
35. Thio GL, Ray RP, Bacelo G, Schüpbach T. (2000). Localization of gurken RNA in Drosophila oogenesis requires elements in the 5’ and 3’ regions of the transcript. Dev. Biol. 221, 435-446.
36. Vivekanand P, Tootle TL, Rebay I. (2004). MAE, a dual regulator of the EGFR signaling pathway, is a target of the Ets transcription factors PNT and YAN. Mech Dev. 121 (12), 1469-1479.
37. Vivekanand P, Rebay I. (2006). Intersection of signal transduction pathways and development. Annu. Rev. Genet. 40, 139-157.
38. Voas MG, Rebay I. (2004). Signaling interaction during development insights from the Drosophila eye. Dev. Dyn. 299(1), 162-175.
39. Wolff T, Ready DF. (1993). in the Development of Drosophila melanogaster, eds. Bate M, Marthinex A. 1277-1326.
40. zur Lage P, Jarman AP. (1999). Antagonism of EGFR and notch signalling in the reiterative recruitment of Drosophila adult chordotonal sense organ precursors. Development. 126(14), 3149-3157.
41. zur Lage P, Powell LM, Prentice D R.A., Maclaughlin P, P. Jarman A. (2004). EGF receptor signaling trigger recruitment of Drosophila sense organ precursors by stimulating proneural gene autoregulation. Dev.Cell.7, 687-696.