進階搜尋


   電子論文尚未授權公開,紙本請查館藏目錄
(※如查詢不到或館藏狀況顯示「閉架不公開」,表示該本論文不在書庫,無法取用。)
系統識別號 U0026-1707201417442100
論文名稱(中文) 系統性篩選參與果蠅骨骼肌發育基因之研究
論文名稱(英文) A systematic approach to identify novel genes involved in Drosophila somatic muscle development
校院名稱 成功大學
系所名稱(中) 生命科學系
系所名稱(英) Department of Life Sciences
學年度 102
學期 2
出版年 103
研究生(中文) 巫峻豪
研究生(英文) Chun-Hao Wu
學號 l56011041
學位類別 碩士
語文別 中文
論文頁數 68頁
口試委員 指導教授-劉雅心
召集委員-王浩文
口試委員-張松彬
中文關鍵字 果蠅骨骼肌發育 
英文關鍵字 Drosophila somatic muscle development  twist  Mef2  transcriptional regulation 
學科別分類
中文摘要 果蠅胚胎發育的過程中,骨骼肌由中胚層細胞特化而來的。先前的研究指出,轉錄因子Twist和Mef2 (Myocyte enhancer factor 2)在骨骼肌的發育過程中扮演了相當重要的角色。他們藉由調控其下游相關基因的轉錄作用,進而影響整個肌肉發育分化。近年來有數個研究以Chip-on-chip或是ChIP-Seq的方式有系統地建立了整個果蠅基因體中胚層和其下游發育過程包括骨骼肌發育的基因調控網絡 (gene regulatory network)。但大多數經由功能基因體學方法找出的Twist或Mef2所結合的DNA區域和其所相關聯的下游基因在果蠅骨骼肌發育過程有無功能則尚未被釐清。為了驗證這些下游基因在果蠅骨骼肌發育過程中的角色並篩選出新的基因來增進對果蠅骨骼肌發育過程的了解,我首先分析整理了Twist和Mef2在整個果蠅基因體的結合位點資料,並結合現有的基因表現資料庫 BDGP in situ database選出受Twist和Mef2結合的區域和其所相關聯的可能候選基因,再以原位雜合染色實驗並利用RNAi果蠅株在果蠅胚胎中胚層細胞抑制基因的表現,找到9個表現在中胚層以及骨骼肌細胞中且其在中胚層細胞的表現量下降時會影響果蠅骨骼肌的正常發育的基因,證實了這些受Twist和Mef2結合的DNA區域其所相關聯的下游基因在果蠅骨骼肌發育過程有所作用。
英文摘要 During Drosophila embryogenesis, somatic muscle is derived from the mesoderm. Transcription factors Twist and Mef2 (Myocyte enhancer factor 2) have been shown playing critical roles in regulating Drosophila mesoderm and somatic muscle development. Several studies have established the gene regulatory network of Drosophila mesoderm development by using ChIP-on-chip or ChIP-seq against key mesoderm transcriptional regulators, including Twist and Mef2. However, it is not clear whether most of the identified bound regions of Twist and Mef2 and their associated target genes play roles in Drosophila somatic muscle development. In order to understand functions of these target genes associated with the Twist- and Mef2- bound regions and identify novel genes involved in Drosophila somatic muscle development, I selected several genes for further studies based on the Twist- and Mef2- binding profiles and information of expression patterns from BDGP in situ database to select Twist- and Mef2- bound regions and their associated target genes. I then performed-1) in situ hybridization experiments to examine the expression patterns of the selected genes, 2) tissue-specific RNAi experiments to understand the functions of the selected genes for somatic muscle development, and 3) ectopic expression of transcription factor Mef2 to know the regulatory relationships between Mef2 and the selected genes. In total, I found 9 genes that were expressed in the mesoderm or the somatic muscles, and showed malformation of somatic musculature when the expression levels of the selected genes were reduced in the mesoderm. These preliminary results suggest these Twist- and Mef2- bound regions and their associated genes play roles in Drosophila somatic muscle development.
論文目次 目錄

中文摘要…………………………………………………………………………P1
Extended Abstract ………………………………………………………………P2
致謝………………………………………………………………………………P6
目錄………………………………………………………………………………P7
圖目錄……………………………………………………………………………P10
縮寫表……………………………………………………………………………P11
前言………………………………………………………………………………P13
一、果蠅中胚層及骨骼肌發育………………………………………………P13
二、Twist (twi)和中胚層發育的關係 ………………………………………P14
三、Myocyte enhancer factor 2 (Mef2)和骨骼肌發育的關係……………P15
四、骨骼肌發育的基因調控…………………………………………………P16
實驗目的…………………………………………………………………………P18
材料與方法………………………………………………………………………P19
一、材料………………………………………………………………………P19
(一)、動物實驗材料………………………………………………………P19
(二)、動物實驗藥品………………………………………………………P20
(三)、EST clones來自Berkeley Drosophila Genome Project (BDGP) P20
(四)、寡核苷酸購自波仕特生物科技公司………………………………P21
(五)、抗體…………………………………………………………………P21
(六)、試劑組………………………………………………………………P22
二、方法………………………………………………………………………P22
(一)、果蠅株交配及收集胚胎實驗………………………………………P22
1. 內源性基因表現實驗 (Endogenous gene expression)……………P22
2. RNAi表現型篩選實驗 (RNAi phenotype)…………………………P22
3. 特定組織性表現實驗 (Ectopic expression)………………………P22
(二)、利用EST clones製備探針…………………………………………P23
1. 質體DNA萃取及EST clone確認實驗 …………………………P23
2. 聚合脢連鎖反應 (Polymerase chain reaction) …………………P24
3. 探針製備 (Probe labeling) ………………………………………P25
(三)、果蠅胚胎處理及免疫染色實驗……………………………………P26
1. 胚胎福馬林固定 (Embryo fixation) ………………………………P26
2. 原位雜合反應 (in situ hybridization) ……………………………P26
3. 免疫螢光染色 ………………………………………………………P27
結果………………………………………………………………………………P29
一、 分析Twist和Mef2的全基因體結合位點資料,整理出111個主要由
Twist和Mef2調控的CRMs…………………………………………P29
二、 利用BDGP expression pattern以及Flybase gene ontology (GO) term
在111個CRMs與其相關聯的基因中選出91個候選基因………P30
三、 原位雜合反應實驗顯示一共有15個候選基因有表現在中胚層、骨骼
肌前驅細胞、骨骼肌細胞中 …………………………………………P31
四、 利用RNAi果蠅株在果蠅中胚層細胞中減低候選基因的表現,對骨骼
肌發育造成程度不一的影響 …………………………………………P32
五、 利用特定組織性表現實驗觀察候選基因和Mef2基因間的調控關係
,僅有ppa基因可被異位表現的Mef2誘發表現…………………P32
討論………………………………………………………………………………P34
一、 表現在中胚層及骨骼肌細胞的候選基因對於骨骼肌發育的影響 …P34
二、 在中胚層及骨骼肌細胞減低候選基因表現對於骨骼肌發育的影響 P34
三、 異位表現Mef2對於候選基因表現所產生的影響 …………………P35
結論………………………………………………………………………………P36
參考資料…………………………………………………………………………P37


圖目錄

圖ㄧ、系統性篩選參與骨骼肌發育過程候選基因的流程圖…………………P46
圖二、候選基因在果蠅胚胎中的內源性表現…………………………………P47
圖三、抑制候選基因對骨骼肌型態產生的影響………………………………P50
圖四、外胚層異位表現Mef2對候選基因表現的影響………………………P52
圖五、系統性篩選參與骨骼肌發育過程候選基因之總結……………………P53
表一 、候選基因決定之分類整理 ……………………………………………P54
表二、進行RNAi表現型篩選實驗之果蠅株整理……………………………P66
表三、進行特定組織性表現實驗之候選基因整理……………………………P68
參考文獻 參考資料

Azpiazu, N., Lawrence, P. A., Vincent, J. P. and Frasch, M. (1996). Segmentation and specification of the Drosophila mesoderm. Genes & development 10, 3183-3194.
Bate, M., Rushton, E. and Currie, D. A. (1991). Cells with persistent twist expression are the embryonic precursors of adult muscles in Drosophila. Development 113, 79-89.
Bate, M. and Rushton, E. (1993). Myogenesis and muscle patterning in Drosophila. Comptes rendus de l'Academie des sciences. Serie III, Sciences de la vie 316, 1047-1061.
Baylies, M. K., Martinez Arias, A. and Bate, M. (1995). wingless is required for the formation of a subset of muscle founder cells during Drosophila embryogenesis. Development 121, 3829-3837.
Baylies, M. K. and Bate, M. (1996). twist: a myogenic switch in Drosophila. Science 272, 1481-1484.
Baylies, M. K., Bate, M. and Ruiz Gomez, M. (1998). Myogenesis: a view from Drosophila. Cell 93, 921-927.
Black, B. L. and Olson, E. N. (1998). Transcriptional control of muscle development by myocyte enhancer factor-2 (MEF2) proteins. Annual review of cell and developmental biology 14, 167-196.
Borkowski, O. M., Brown, N. H. and Bate, M. (1995). Anterior-posterior subdivision and the diversification of the mesoderm in Drosophila. Development 121, 4183-4193.
Bour, B. A., O'Brien, M. A., Lockwood, W. L., Goldstein, E. S., Bodmer, R., Taghert, P. H., Abmayr, S. M. and Nguyen, H. T. (1995). Drosophila MEF2, a transcription factor that is essential for myogenesis. Genes & development 9, 730-741.
Brand, A. H. and Perrimon, N. (1993). Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118, 401-415.
Cadigan, K. M., Grossniklaus, U. and Gehring, W. J. (1994a). Functional redundancy: the respective roles of the two sloppy paired genes in Drosophila segmentation. Proceedings of the National Academy of Sciences of the United States of America 91, 6324-6328.
Cadigan, K. M., Grossniklaus, U. and Gehring, W. J. (1994b). Localized expression of sloppy paired protein maintains the polarity of Drosophila parasegments. Genes & development 8, 899-913.
Carmena, A., Bate, M. and Jimenez, F. (1995). Lethal of scute, a proneural gene, participates in the specification of muscle progenitors during Drosophila embryogenesis. Genes & development 9, 2373-2383.
Carmena, A., Murugasu-Oei, B., Menon, D., Jimenez, F. and Chia, W. (1998). Inscuteable and numb mediate asymmetric muscle progenitor cell divisions during Drosophila myogenesis. Genes & development 12, 304-315.
Casal, J. and Leptin, M. (1996). Identification of novel genes in Drosophila reveals the complex regulation of early gene activity in the mesoderm. Proceedings of the National Academy of Sciences of the United States of America 93, 10327-10332.
Castanon, I., Von Stetina, S., Kass, J. and Baylies, M. K. (2001). Dimerization partners determine the activity of the Twist bHLH protein during Drosophila mesoderm development. Development 128, 3145-3159.
Chambers, A. E., Logan, M., Kotecha, S., Towers, N., Sparrow, D. and Mohun, T. J. (1994). The RSRF/MEF2 protein SL1 regulates cardiac muscle-specific transcription of a myosin light-chain gene in Xenopus embryos. Genes & development 8, 1324-1334.
Cripps, R. M., Black, B. L., Zhao, B., Lien, C. L., Schulz, R. A. and Olson, E. N. (1998). The myogenic regulatory gene Mef2 is a direct target for transcriptional activation by Twist during Drosophila myogenesis. Genes & development 12, 422-434.
Cripps, R. M., Lovato, T. L. and Olson, E. N. (2004). Positive autoregulation of the Myocyte enhancer factor-2 myogenic control gene during somatic muscle development in Drosophila. Developmental biology 267, 536-547.
Damm, C., Wolk, A., Buttgereit, D., Loher, K., Wagner, E., Lilly, B., Olson, E. N., Hasenpusch-Theil, K. and Renkawitz-Pohl, R. (1998). Independent regulatory elements in the upstream region of the Drosophila beta 3 tubulin gene (beta Tub60D) guide expression in the dorsal vessel and the somatic muscles. Developmental biology 199, 138-149.
Davidson, E. H., Rast, J. P., Oliveri, P., Ransick, A., Calestani, C., Yuh, C. H., Minokawa, T., Amore, G., Hinman, V., Arenas-Mena, C., et al. (2002). A genomic regulatory network for development. Science 295, 1669-1678.
Dietzl, G., Chen, D., Schnorrer, F., Su, K. C., Barinova, Y., Fellner, M., Gasser, B., Kinsey, K., Oppel, S., Scheiblauer, S., et al. (2007). A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila. Nature 448, 151-156.
Duan, H., Skeath, J. B. and Nguyen, H. T. (2001). Drosophila Lame duck, a novel member of the Gli superfamily, acts as a key regulator of myogenesis by controlling fusion-competent myoblast development. Development 128, 4489-4500.
Frasch, M. (1995). Induction of visceral and cardiac mesoderm by ectodermal Dpp in the early Drosophila embryo. Nature 374, 464-467.
Furlong, E. E., Andersen, E. C., Null, B., White, K. P. and Scott, M. P. (2001). Patterns of gene expression during Drosophila mesoderm development. Science 293, 1629-1633.
Gajewski, K., Kim, Y., Choi, C. Y. and Schulz, R. A. (1998). Combinatorial control of Drosophila mef2 gene expression in cardiac and somatic muscle cell lineages. Development genes and evolution 208, 382-392.
Grossniklaus, U., Pearson, R. K. and Gehring, W. J. (1992). The Drosophila sloppy paired locus encodes two proteins involved in segmentation that show homology to mammalian transcription factors. Genes & development 6, 1030-1051.
Inoue, T., Wang, M., Ririe, T. O., Fernandes, J. S. and Sternberg, P. W. (2005). Transcriptional network underlying Caenorhabditis elegans vulval development. Proceedings of the National Academy of Sciences of the United States of America 102, 4972-4977.
Ip, Y. T., Park, R. E., Kosman, D., Yazdanbakhsh, K. and Levine, M. (1992). dorsal-twist interactions establish snail expression in the presumptive mesoderm of the Drosophila embryo. Genes & development 6, 1518-1530.
Koide, T., Hayata, T. and Cho, K. W. (2005). Xenopus as a model system to study transcriptional regulatory networks. Proceedings of the National Academy of Sciences of the United States of America 102, 4943-4948.
Lawrence, P. A., Bodmer, R. and Vincent, J. P. (1995). Segmental patterning of heart precursors in Drosophila. Development 121, 4303-4308.
Lee, Y. M., Park, T., Schulz, R. A. and Kim, Y. (1997). Twist-mediated activation of the NK-4 homeobox gene in the visceral mesoderm of Drosophila requires two distinct clusters of E-box regulatory elements. The Journal of biological chemistry 272, 17531-17541.
Leptin, M. (1991). twist and snail as positive and negative regulators during Drosophila mesoderm development. Genes & development 5, 1568-1576.
Lilly, B., Zhao, B., Ranganayakulu, G., Paterson, B. M., Schulz, R. A. and Olson, E. N. (1995). Requirement of MADS domain transcription factor D-MEF2 for muscle formation in Drosophila. Science 267, 688-693.
Lin, M. H., Bour, B. A., Abmayr, S. M. and Storti, R. V. (1997a). Ectopic expression of MEF2 in the epidermis induces epidermal expression of muscle genes and abnormal muscle development in Drosophila. Developmental biology 182, 240-255.
Lin, Q., Schwarz, J., Bucana, C. and Olson, E. N. (1997b). Control of mouse cardiac morphogenesis and myogenesis by transcription factor MEF2C. Science 276, 1404-1407.
Liu, Y. H., Jakobsen, J. S., Valentin, G., Amarantos, I., Gilmour, D. T. and Furlong, E. E. (2009). A systematic analysis of Tinman function reveals Eya and JAK-STAT signaling as essential regulators of muscle development. Developmental cell 16, 280-291.
Markstein, M., Zinzen, R., Markstein, P., Yee, K. P., Erives, A., Stathopoulos, A. and Levine, M. (2004). A regulatory code for neurogenic gene expression in the Drosophila embryo. Development 131, 2387-2394.
Navankasattusas, S., Zhu, H., Garcia, A. V., Evans, S. M. and Chien, K. R. (1992). A ubiquitous factor (HF-1a) and a distinct muscle factor (HF-1b/MEF-2) form an E-box-independent pathway for cardiac muscle gene expression. Molecular and cellular biology 12, 1469-1479.
Naya, F. J., Black, B. L., Wu, H., Bassel-Duby, R., Richardson, J. A., Hill, J. A. and Olson, E. N. (2002). Mitochondrial deficiency and cardiac sudden death in mice lacking the MEF2A transcription factor. Nature medicine 8, 1303-1309.
Nguyen, H. T., Bodmer, R., Abmayr, S. M., McDermott, J. C. and Spoerel, N. A. (1994). D-mef2: a Drosophila mesoderm-specific MADS box-containing gene with a biphasic expression profile during embryogenesis. Proceedings of the National Academy of Sciences of the United States of America 91, 7520-7524.
Nguyen, H. T. and Xu, X. (1998). Drosophila mef2 expression during mesoderm development is controlled by a complex array of cis-acting regulatory modules. Developmental biology 204, 550-566.
Ni, J. Q., Liu, L. P., Binari, R., Hardy, R., Shim, H. S., Cavallaro, A., Booker, M., Pfeiffer, B. D., Markstein, M., Wang, H., et al. (2009). A Drosophila resource of transgenic RNAi lines for neurogenetics. Genetics 182, 1089-1100.
Park, M., Wu, X., Golden, K., Axelrod, J. D. and Bodmer, R. (1996). The wingless signaling pathway is directly involved in Drosophila heart development. Developmental biology 177, 104-116.
Ranganayakulu, G., Schulz, R. A. and Olson, E. N. (1996). Wingless signaling induces nautilus expression in the ventral mesoderm of the Drosophila embryo. Developmental biology 176, 143-148.
Riechmann, V., Irion, U., Wilson, R., Grosskortenhaus, R. and Leptin, M. (1997). Control of cell fates and segmentation in the Drosophila mesoderm. Development 124, 2915-2922.
Ruiz Gomez, M. and Bate, M. (1997). Segregation of myogenic lineages in Drosophila requires numb. Development 124, 4857-4866.
Sandmann, T., Jensen, L. J., Jakobsen, J. S., Karzynski, M. M., Eichenlaub, M. P., Bork, P. and Furlong, E. E. (2006). A temporal map of transcription factor activity: mef2 directly regulates target genes at all stages of muscle development. Developmental cell 10, 797-807.
Sandmann, T., Girardot, C., Brehme, M., Tongprasit, W., Stolc, V. and Furlong, E. E. (2007). A core transcriptional network for early mesoderm development in Drosophila melanogaster. Genes & development 21, 436-449.
Schroeder, M. D., Pearce, M., Fak, J., Fan, H., Unnerstall, U., Emberly, E., Rajewsky, N., Siggia, E. D. and Gaul, U. (2004). Transcriptional control in the segmentation gene network of Drosophila. PLoS biology 2, E271.
Shirokawa, J. M. and Courey, A. J. (1997). A direct contact between the dorsal rel homology domain and Twist may mediate transcriptional synergy. Molecular and cellular biology 17, 3345-3355.
Sokol, N. S. and Ambros, V. (2005). Mesodermally expressed Drosophila microRNA-1 is regulated by Twist and is required in muscles during larval growth. Genes & development 19, 2343-2354.
Staehling-Hampton, K., Hoffmann, F. M., Baylies, M. K., Rushton, E. and Bate, M. (1994). dpp induces mesodermal gene expression in Drosophila. Nature 372, 783-786.
Stathopoulos, A. and Levine, M. (2002). Whole-genome expression profiles identify gene batteries in Drosophila. Developmental cell 3, 464-465.
Stathopoulos, A. and Levine, M. (2005). Genomic regulatory networks and animal development. Developmental cell 9, 449-462.
Thisse, B., el Messal, M. and Perrin-Schmitt, F. (1987). The twist gene: isolation of a Drosophila zygotic gene necessary for the establishment of dorsoventral pattern. Nucleic acids research 15, 3439-3453.
Tomancak, P., Beaton, A., Weiszmann, R., Kwan, E., Shu, S., Lewis, S. E., Richards, S., Ashburner, M., Hartenstein, V., Celniker, S. E., et al. (2002). Systematic determination of patterns of gene expression during Drosophila embryogenesis. Genome biology 3, RESEARCH0088.
Wang, L., Fan, C., Topol, S. E., Topol, E. J. and Wang, Q. (2003). Mutation of MEF2A in an inherited disorder with features of coronary artery disease. Science 302, 1578-1581.
Wu, X., Golden, K. and Bodmer, R. (1995). Heart development in Drosophila requires the segment polarity gene wingless. Developmental biology 169, 619-628.
Yin, Z., Xu, X. L. and Frasch, M. (1997). Regulation of the twist target gene tinman by modular cis-regulatory elements during early mesoderm development. Development 124, 4971-4982.
Zaffran, S., Kuchler, A., Lee, H. H. and Frasch, M. (2001). biniou (FoxF), a central component in a regulatory network controlling visceral mesoderm development and midgut morphogenesis in Drosophila. Genes & development 15, 2900-2915.
Zinzen, R. P., Girardot, C., Gagneur, J., Braun, M. and Furlong, E. E. (2009). Combinatorial binding predicts spatio-temporal cis-regulatory activity. Nature 462, 65-70.
論文全文使用權限
  • 同意授權校內瀏覽/列印電子全文服務,於2019-07-30起公開。


  • 如您有疑問,請聯絡圖書館
    聯絡電話:(06)2757575#65773
    聯絡E-mail:etds@email.ncku.edu.tw