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論文名稱(外文):Expression profiles and functional analyses of the mouse double homeobox gene : mDux
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譯出具有同源箱區的蛋白質(homeodomain proteins)來調控胚胎發
育過程的細胞分化及決定細胞的命運。之前實驗室根據同源箱區的高度保留性氨基酸序列來設計退化性核酸引子(degenerated oligonucleotide primers),以polymerase chain reaction(PCR)的方式篩選到一個新的同源箱基因,且可同時轉譯出兩個同源箱區,因此將它並名為mDux (murine double homeobox gene), mDux基因可能是人類DUX4基因在小鼠的同源基因,研究指出,DUX4基因可能是造成顏面及肩胛肱肌肉失養症(FSHD:Facioscapulohumeral Muscular Dystrophy)的候選基因。mDux基因具有兩種轉錄物,mDux與mDux-s。以RT-PCR分析mDux基因在成鼠組織之表現,顯示mDux基因主要表現於小鼠性腺、眼球和大腦。以切片原位雜交技術和免疫組織化學染色分析卵巢、睪丸和眼睛組織,顯示mDux表現於卵母細胞、精原細胞和神經節細胞。進一步以全胚胎原位雜交分析胚胎發育時期mDux基因的表現,顯示mDux在早期胚胎主要表現在四肢和尾巴,進一步以免疫組織化學染色分析不同天數胚胎,顯示mDux蛋白表現在四肢的纖維狀已分化肌肉細胞,與MyoD染到的單核肌纖維母細胞明顯的不同,以RT-PCR分析比較mDux與MyoD基因在四肢發育過程中的表現趨勢,發現mDux與MyoD一樣在四肢肌肉發育過程持續的表現,但成熟肌肉組織測不到mDux與MyoD基因的表現,由於mDux基因的表現由胚胎12.5到15.5有明顯的增加,推測mDux與MyoD可能表現在不同分化時期的四肢肌肉細胞,另外我們也證實了mDux蛋白表現於體腔骨骼肌,包括epaxial及hypaxial 中的肌纖維細胞。以RT-PCR分析不同培養分化時期之肌纖維母細胞細胞萃取RNA發現,mDux的表現趨勢和myogenin的表現趨勢相近。同時以細胞免疫螢光染色技術分析顯示,mDux表現在肌管(myotube)肌肉細胞的細胞核中。以RNAi抑制肌纖維母細胞內mDux基因的表現,造成分化中肌纖維母細胞死亡,對於mDux在肌肉發育的影響還需進一步的研究。我們也將建立肌肉專一性表達mDux基因的基因轉殖鼠,分析當轉殖鼠成體肌肉過度表達mDux基因時,是否出現類似FSHD病人的肌肉萎縮現象。

The homeobox gene products act as transcription factors that regulate the cell differentiation and decide the cell fates during animal developmental process. We performed a degenerated oligonucleotide polymerase chain reaction (PCR) to screen and isolate a novel homeobox gene, murine double homeobox gene (mDux), which encodes two homeodomains. mDux gene is the ortholog of human DUX4 gene, which is one of candidate genes of causing facioscapulohumeral muscular dystrophy (FSHD). The mDux has two different transcripts froms: mDux and mDux-s. mDux was predominantly expressed in gonad, eye and brain by reverse transcription (RT)-PCR analysis. Moreover, mDux expressed in oocyte, spermatogonia and ganglion cell was confirmed by in situ hybridization and immunohistochemical staining. Further, whole mount in situ hybridization results show that mDux transcripts expressed in limbs and tail beginning at E9.5 and maintained to E12. MDux signals were localized in fiber-like muscle cells but not in the mononuclear cells that were labeled with MyoD (myoblast marker gene) signals by section in situ hybridization and immuno-histochemistry analyses. During limb myogenesis, mDux expressions, similar to MyoD, were continuously in postnatal limbs (3d) but slightly detectable in adult muscles by RT-PCR analysis. In addition, mDux also expressed in the trunk muscle, including epaxial and hypaxial myofiber. During trunk myogenesis, mDux expression was beginning at E12.5 embryo and increased in E13.5 embryo, myotube formation stage. We also examined the mDux expression during in vitro differentiation of C2C12 (myoblast) cell line. The mDux expression was slightly detected in confluent myocyte, but largely increased in differentiated cells following 2days in differentiation medium by RT-PCR analysis. The expression pattern of mDux is more similar to myogenin and both gene expression increase following myotube formation by RT-PCR analysis. Meanwhile, cellular immunofluorescence analysis also detected the mDux expression in nuclear of myotube. Furthermore, inhibition the expression of mDux in differentiated myoblast by mDux small interference RNA caused the cell death of differentiated myoblast. The influence of mDux in myogenesis must be clarified further. In the future, we will establish the muscle specific overexpression of mDux transgenic mice system to understand whether overexpression of mDux results in the present of FSHD phenotype.


一、 摘要-------------------------------1
二、 前言-------------------------------5
三、 材料與方法------------------------18
四、 結果------------------------------31
五、 討論------------------------------41
六、 圖表------------------------------50
七、 參考文獻--------------------------67
八、 附錄------------------------------71
九、 附圖------------------------------75

蔡子勖96年度大專生國科會計畫:Molecular cloning and characterization of a novel double homeobox gene, mDux。2007。

Apiou, F., Flagiello, D., Cillo, C., Malfoy, B., Poupon, M.F., and Dutrillaux, B. (1996). Fine mapping of human HOX gene clusters. Cytogenet Cell Genet 73, 114-115.

Arnold, H.H., and Winter, B. (1998). Muscle differentiation: more complexity to the network of myogenic regulators. Curr Opin Genet Dev 8, 539-544.

Azuma, N., Nishina, S., Yanagisawa, H., Okuyama, T., and Yamada, M. (1996). PAX6 missense mutation in isolated foveal hypoplasia. Nat Genet 13, 141-142.

Banerjee-Basu, S., and Baxevanis, A.D. (2001). Molecular evolution of the homeodomain family of transcription factors. Nucleic Acids Res 29, 3258-3269.

Bosnakovski, D., Lamb, S., Simsek, T., Xu, Z., Belayew, A., Perlingeiro, R., and Kyba, M. (2008). DUX4c, an FSHD candidate gene, interferes with myogenic regulators and abolishes myoblast differentiation. Exp Neurol.

Buffinger, N., and Stockdale, F.E. (1995). Myogenic specification of somites is mediated by diffusible factors. Dev Biol 169, 96-108.

Cho, S.J., and Park, S.C. (2008). Paired-like subclass homeobox genes from the clitellate annelid Perionyx excavatus. Biochem Genet 46, 737-743.

Clapp, J., Mitchell, L.M., Bolland, D.J., Fantes, J., Corcoran, A.E., Scotting, P.J., Armour, J.A., and Hewitt, J.E. (2007). Evolutionary conservation of a coding function for D4Z4, the tandem DNA repeat mutated in facioscapulohumeral muscular dystrophy. Am J Hum Genet 81, 264-279.

Cossu, G., and Borello, U. (1999). Wnt signaling and the activation of myogenesis in mammals. Embo J 18, 6867-6872.

Dixit, M., Ansseau, E., Tassin, A., Winokur, S., Shi, R., Qian, H., Sauvage, S., Matteotti, C., van Acker, A.M., Leo, O., et al. (2007). DUX4, a candidate gene of facioscapulohumeral muscular dystrophy, encodes a transcriptional activator of PITX1. Proc Natl Acad Sci U S A 104, 18157-18162.

Fisher, J., and Upadhyaya, M. (1997). Molecular genetics of facioscapulohumeral muscular dystrophy (FSHD). Neuromuscul Disord 7, 55-62.

Froeschle, A., Alric, S., Kitzmann, M., Carnac, G., Aurade, F., Rochette-Egly, C., and Bonnieu, A. (1998). Retinoic acid receptors and muscle b-HLH proteins: partners in retinoid-induced myogenesis. Oncogene 16, 3369-3378.

Gabellini, D., D''Antona, G., Moggio, M., Prelle, A., Zecca, C., Adami, R., Angeletti, B.,
Ciscato, P., Pellegrino, M.A., Bottinelli, R., et al. (2006). Facioscapulohumeral muscular dystrophy in mice overexpressing FRG1. Nature 439, 973-977.

Gabellini, D., Green, M.R., and Tupler, R. (2002). Inappropriate gene activation in FSHD: a repressor complex binds a chromosomal repeat deleted in dystrophic muscle. Cell 110, 339-348.

Gehring, W.J. (1987). Homeo boxes in the study of development. Science 236, 1245-1252.

Gehring, W.J., Qian, Y.Q., Billeter, M., Furukubo-Tokunaga, K., Schier, A.F., Resendez-Perez, D., Affolter, M., Otting, G., and Wuthrich, K. (1994). Homeodomain-DNA recognition. Cell 78, 211-223.

Holland, P.W., and Takahashi, T. (2005). The evolution of homeobox genes: Implications for the study of brain development. Brain Res Bull 66, 484-490.

Kablar, B., Krastel, K., Ying, C., Asakura, A., Tapscott, S.J., and Rudnicki, M.A. (1997). MyoD and Myf-5 differentially regulate the development of limb versus trunk skeletal muscle. Development 124, 4729-4738.

Kania, A., Johnson, R.L., and Jessell, T.M. (2000). Coordinate roles for LIM homeobox genes in directing the dorsoventral trajectory of motor axons in the vertebrate limb. Cell 102, 161-173.

Kawazu, M., Yamamoto, G., Yoshimi, M., Yamamoto, K., Asai, T., Ichikawa, M., Seo, S., Nakagawa, M., Chiba, S., Kurokawa, M., et al. (2007). Expression profiling of immature thymocytes revealed a novel homeobox gene that regulates double-negative thymocyte development. J Immunol 179, 5335-5345.

Kowaljow, V., Marcowycz, A., Ansseau, E., Conde, C.B., Sauvage, S., Matteotti, C., Arias, C., Corona, E.D., Nunez, N.G., Leo, O., et al. (2007). The DUX4 gene at the FSHD1A locus encodes a pro-apoptotic protein. Neuromuscul Disord 17, 611-623.

Li, G., Harton, J.A., Zhu, X., and Ting, J.P. (2001). Downregulation of CIITA function by protein kinase a (PKA)-mediated phosphorylation: mechanism of prostaglandin E, cyclic AMP, and PKA inhibition of class II major histocompatibility complex expression in monocytic lines. Mol Cell Biol 21, 4626-4635.

Manak, J.R., and Scott, M.P. (1994). A class act: conservation of homeodomain protein functions. Dev Suppl, 61-77.

Mitashov, V.I., Arsanto, J.P., and Thouveny, Y. (2000). [Expression of glial, neurospecific, and extracellular antigens during retinal regeneration in adult newts]. Izv Akad Nauk Ser Biol, 282-289.

Molkentin, J.D., and Olson, E.N. (1996). Defining the regulatory networks for muscle development. Curr Opin Genet Dev 6, 445-453.

Moshiri, A., Gonzalez, E., Tagawa, K., Maeda, H., Wang, M., Frishman, L.J., and Wang, S.W. (2008). Near complete loss of retinal ganglion cells in the math5/brn3b double knockout elicits severe reductions of other cell types during retinal development. Dev Biol 316, 214-227.

Mu, X. and W. H. Klein (2004). "A gene regulatory hierarchy for retinal ganglion cell specification and differentiation." Semin Cell Dev Biol 15(1): 115-23.

Ordahl, C.P., and Williams, B.A. (1998). Knowing chops from chuck: roasting myoD redundancy. Bioessays 20, 357-362.

Ranganayakulu, G., Elliott, D.A., Harvey, R.P., and Olson, E.N. (1998). Divergent roles for NK-2 class homeobox genes in cardiogenesis in flies and mice. Development 125, 3037-3048.

Rhodes, S.J., DiMattia, G.E., and Rosenfeld, M.G. (1994). Transcriptional mechanisms in anterior pituitary cell differentiation. Curr Opin Genet Dev 4, 709-717.
Rudnicki, M.A., and Jaenisch, R. (1995). The MyoD family of transcription factors and skeletal myogenesis. Bioessays 17, 203-209.

Stern, H.M., Brown, A.M., and Hauschka, S.D. (1995). Myogenesis in paraxial mesoderm: preferential induction by dorsal neural tube and by cells expressing Wnt-1. Development 121, 3675-3686.

Studer, M., Lumsden, A., Ariza-McNaughton, L., Bradley, A., and Krumlauf, R. (1996). Altered segmental identity and abnormal migration of motor neurons in mice lacking Hoxb-1. Nature 384, 630-634.

Tupler, R., and Gabellini, D. (2004). Molecular basis of facioscapulohumeral muscular dystrophy. Cell Mol Life Sci 61, 557-566.

Walther, C., and Gruss, P. (1991). Pax-6, a murine paired box gene, is expressed in the developing CNS. Development 113, 1435-1449.

Yutzey, K.E., Rhodes, S.J., and Konieczny, S.F. (1990). Differential trans activation associated with the muscle regulatory factors MyoD1, myogenin, and MRF4. Mol Cell Biol 10, 3934-3944.

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