臺灣博碩士論文加值系統

six1基因屬於sine oculis/six家族的一員，其在型態發生、器官生成及細胞分化上扮演重要角色。於果蠅的研究中已發現，six家族、eyeless (pax)家族、eyes absent (eya)家族與dachshund (dach)家族會形成一個協同調節網絡以促進複眼生成。而於six1基因剔除鼠中，Six1缺失會影響軀幹部的肌肉發育，這指出Six1對於軀幹部肌肉發育是必須的，然而其對於頭部肌肉所扮演的角色仍是未知。為進一步了解six1於頭部肌肉發育上的功能，我們選定斑馬魚作為模式動物以探討。因此，我首先利用whole mount in situ hybridization的方式，來觀察six1在斑馬魚頭部肌肉的表現情形。結果顯示six1可於所有的頭部肌肉前趨細胞表現，抑制six1轉譯的實驗則顯示，胸骨舌骨肌(sternohyoideus)、中直肌(medial rectus)、下直肌(inferior rectus)及所有咽弧肌之形成需要six1的功能；並且這些肌肉的myod與myogenin表現亦需要six1的功能。此外，外源性myod mRNA的添加，可以回復生成six1基因抑制劑(six1-MO)所造成缺失的肌肉，這顯示six1可會透過myod而調控頭部肌肉發育。而且我也觀察到pax3基因抑制劑所造成的頭部肌肉缺失與six1基因抑制劑相同，但外源性的six1, myod或myf5 mRNA皆無法回復生成pax3基因抑制劑所造成的肌肉缺失，因此推論six1與pax3不是處於同一基因網絡以調控頭部肌肉發育。除此之外，於注射six1或pax3基因抑制劑的胚胎中，皆可觀察到側鳍(fin bud)與後軸下肌(posterior hypaxial muscle)會缺失。故综合以上實驗結果，在斑馬魚，six1於頭部肌肉發育扮演重要的角色。

six1 gene belongs to the sine oculis/six gene family that plays important roles in morphogenesis, organogenesis, and cell differentiation. Studies in Drosophila have revealed that six family, eyeless (pax family), eyes absent (eya family) and dachshund (dach family) form a synergistic regulatory network to trigger compound eye organogenesis. six1 knockout mice showed that knockdown of six1 causes extensive muscle hypoplasia which affects most of the trunk muscles, indicating that six1 is required for trunk myogenesis. However, the function of six1 in craniofacial myogenesis remains unknown. To further understand the role of six1 in the development of head muscles, we used zebrafish as a model organism. In this study, I used whole mount in situ hybridization to observe the expression pattern of six1 in the head of zebrafish. Results showed that six1 was expressed in all head-muscle progenitor cells. Knockdown of six1 proved that Six1 was required for the development of sternohyoideus, medial rectus, inferior rectus, and all pharyngeal arch muscles. Six1 was also required for the expression of myod and myogenin in these muscles. Exogenous myod mRNA can rescue the loss of the cranial muscles caused by six1-morpholino(MO), indicating myod may be a target gene of Six1 to regulate craniofacial myogenesis. Moreover, we found that the defect of craniofacial muscles caused by pax3-MO phenocopied that of six1-MO. But exogenous six1, myod or myf5 mRNA did not rescue the loss of the cranial muscles caused by injecting pax3-MO, suggesting six1 and pax3 could not function in the same genetic network to regulate development of head muscles. In addition, we also observed that fin bud and posterior hypaxial muscles disappeared in six1 and pax3 morphants . Taken together, our results revealed that six1 played an important role in craniofacial myogenesis.

英文摘要 ------------------------------------------ 1
中文摘要 ------------------------------------------ 2
前言 -------------------------------------------- 3
實驗材料 ------------------------------------------ 10
實驗方法 ------------------------------------------ 14
結果 -------------------------------------------- 21
討論 -------------------------------------------- 30
參考資料 ------------------------------------------ 36
圖表 -------------------------------------------- 43
著作附錄 ------------------------------------------ 54
附錄 -------------------------------------------- 55

Bajard, L., Relaix, F., Lagha, M., Rocancourt, D., Daubas, H. and Buckingham, M. 2006. A novel genetic hierarchy functions during hypaxial myogenesis: Pax3 directly activates Myf5 in muscle progenitor cells in the limb. Genes Dev 20, 2450-2464.

Bessarab, D. A., Chong, S. W. and Korzh, V. 2004. Expression of zebrafish six1 during sensory organ development and myogenesis. Dev Dyn 230,781–786.

Boucher C. A., Carey N., Edwards Y. H., Siciliano M. J. and Johnson K.J. 1996 Cloning of the human SIX1 gene and its assignment to chromosome 14. Genomics 33, 140-142.

Borue, X. and Noden, D. M. 2004. Normal and aberrant craniofacial myogenesis by grafted trunk somatic and segmental plate mesoderm. Development 131, 3967-3980.

Bovolenta P., Mallamaci A., Puelles L. and Boncinelli E. 1998 Expression pattern of cSix3, a member of the Six/sine oculis family of transcription factors. Mech Dev 70, 201-0203.

Braun, T. and Arnold, H. H. 1995. Inactivation of Myf-6 and Myf-5 genes in mice leads to alternations in skeletal muscle development. EMBO. J. 14, 1176-1186.

Brill, G., Kahane, N., Carmeli, C., von Schack, D., Barde, Y. A. and Kalcheim, C. 1995. Epithelialmesenchymal conservation of dermatome progenitors requires neural tube-derived signals: characterization of the role of Neurotrophin-3. Development 121, 2583-2594.

Buckingham, M. 2001. Skeletal muscle formation in vertebrates. Curr. Opin. Genet. Dev. 11, 440 -448.

Chen, R., Amoui, M., Zhang, Z. and Mardon, G. 1997. Dachshund and eyes absent proteins form a complex and function synergistically to induce ectopic eye formation in Drosophila. Cell 91, 893-903.

Cheyette, B., Green, P., Martin, K., Garren, H., Hartenstein, V. and Zipursky, S. 1994. The Drosophila sine oculis locus encodes a homeodomain-containing protein required for the development of the entire visual system. Neuron 12, 977 -996.

Couly, G..F., Coltey ,P.M. and Le Douarin, N.M. 1992. The developmental fate of the cephalic mesoderm in quail–chick chimeras, Development 114, 1–15.

David, N. B., Saint-Etienne, L., Tsang, M., Schilling, T. F. and Rosa, F. M. 2002. Requirement for endoderm and FGF3 in ventral head skeleton formation. Development 129,4457 -4468.

Gage, P. J., Suh, H. and Camper, S. A. 1999. Dosage requirement of Pitx2 for development of multiple organs. Development 126, 4643 -4651.

Giordani, J., Bajard, L., Demignon, J., Daubas, P., Buckingham, M. and Marie, P. 2007. Six proteins regulate the activation of Myf5 expression in embryonic mouse limbs. Proc. Natl. Acad. Sci. USA 27, 11310-11315.

Graham A. and Smith, A. 2001. Patterning the pharyngeal arches. Bioessays 23, 54-61.

Grifone, R., Demignon, J., Houbron, C., Souil, E., Niro, C., Seller, M. J., Hamard, G. and Marie, P. 2005. Six1 and Six4 homeoproteins are required for Pax3 and Mrf expression during myogenesis in the mouse embryo. Develoment 132, 2235-2249.

Hacker ,A. and Guthrie, S. 1998. A distinct developmental programme for the cranial paraxial mesoderm in the chick embryo, Development 125, 3461–3472.

Hasty, P., Bradley, A., Morris, J. H., Edmondson, D. G., Venuti, J. M., Olson, E. N. and Klein, W. H. 1993. Muscle deficiency and neonatal death in mice with a targeted mutation in the myogenin gene. Nature 364, 501-506.

Heath, S. K., Carne, S., Hoyle, C., Johnson, K. J. and Wells, D. J. 1997. Characterisation of expression of mDMAHP, a homeodomain-encoding gene at the murine DM locus. Hum Mol Genet 6, 651-657.

Jacob, M., Jacob, H. J., Wachtler, F. and Christ, B. 1984. Ontogeny of avian extrinsic ocular muscles. I. A light- and electron-microscopic study. Cell Tissue Res. 237, 549 -557.

Kablar, B., Krastel, K., Tajbakhsh , S. and Rudnicki, M. A. 2003. Myf5 and MyoD activation define independent myogenic compartments during embryonic development, Dev. Biol. 258, 307–318.

Kaul, A., Koster, M., Neuhaus , H. and Braun, T. 2000. Myf-5 revisited: loss of early myotome formation does not lead to a rib phenotype in homozygous Myf-5 mutant mice. Cell 102, 17–19.

Kaufman , M. H. and Bard , J. B. L. 1999. The Anatomical Basis of Mouse Development, Academic Press, San Diego, 60–76.

Kawakami, K., Ohto, H., Takizawa, T. and Saito, T. 1996. Identification and expression of six family genes in mouse retina. FEBS Lett 393, 259-263.

Kelly, R. G., Jerome-Majewska, L. A. and Papaioannou, V. E. 2004. The del22q11.2 candidate gene Tbx1 regulates branchiomeric myogenesis. Hum. Mol. Genet. 13, 2829-2840.

Kimmel, C. B., Schilling, T. F. and Hatta, K. 1991. Patterning of body segments of zebrafish embryos. Curr. Top. Dev. Biol. 25, 77-110.

Kimmel, C. B., Miller, C. T. and Keynes, R.J. 2001. Neural crest patterning and the evolution of the jaw. J. Anat. 199, 105-120.
Kitamura, K., Miura, H., Miyagawa-Tomita, S., Yanazawa, M., Katoh-Fukui, Y., Suzuki, R., Ohuchi, H., Suehiro, A., Motegi, Y., Nakahara, Y. et al. 1999. Mouse Pitx2 deficiency leads to anomalies of the ventral body wall, heart, extra- and periocular mesoderm and right pulmonary isomerism. Development 126, 5749 -5758.
Laclef, C., Hamard, G., Demignon, J., Souil, E., Houbron, C. and Maire, P. 2003. Altered myogenesis in Six1-deficient mice. Development 130, 2239-2252.
Lee, H.C., Huang, H.Y., Lin, C.Y., Chen, Y.H., Tsai, H.J., 2006. Foxd3 mediates zebrafish myf5 expression during early somitogenesis. Dev. Biol. 290, 359–372.
Lin, C. Y., Yung, R. F., Lee, H.C., Chen, W.T., Chen, Y.H. and Tsai, H.J. 2006. Myogenic regulatory factors Myf5 and Myod function distinctly during craniofacial myogenesis of zebrafish. Dev. Biol. 299, 594-608.
Lu, J. R., Bassel-Duby, R., Hawkins, A., Chang, P., Valdez, R., Wu, H., Gan, L., Shelton, J. M., Richardson, J. A. and Olson, E. N. 2002. Control of facial muscle development by MyoR and capsulin. Science 298,2378 -2381.
Miller C. T., Yelon, D., Stainier, D. Y. and Kimmel, C. B. 2003. Two endothelin 1 effectors, hand2 and bapx1, pattern ventral pharyngeal cartilage and the jaw joint. Development 130, 1353-1365.
Mootoosamy, R. C. and Dietrich, S. 2002. Distinct regulatory cascades for head and trunk myogenesis. Development 129, 573-583.

Nica, G., Herzoh, W., Sonntag, C., Nowak, M., Schward, H., Zapata, A. G. and Hammerschmid, M. 2006. Eya1 is required for lineage-specific differentiation, but not for cell survival in the zebrafish adenohypophysis. Dev. Biol. 292, 189-204.
Noden, D.M. 1983. The embryonic origins of avian cephalic and cervical muscles and associated connective tissues. Am. J. Anat. 168, 257–276.
Noden, D.M., Marcucio, R., Borycki , A.G. and Emerson Jr.,C.P. 1999. Differentiation of avian craniofacial muscles: I. Patterns of early regulatory gene expression and myosin heavy chain synthesis, Dev. Dyn. 216, 92–116.
Noden, D. M. and Trainor, P. A. 2005. Relations and interactions between cranial mesoderm and neural crest populations. J.Anat. 207, 575-601.
Nowicki, J. L. and Burke, A. C. 2000. Hox genes and morphological identity: axial versus lateral patterning in the vertebrate mesoderm. Development 127, 4265-4275.
Ohto, H., Kamada, S., Tago, K., Tominaga, S. I., Ozaki, H., Sato, S. and Kawakami, K. 1999. Cooperation of Six and Eya in Activation of Their Target Genes through Nuclear Translocation of Eya. Mol. Cell. Biol. 19, 6815–6824
Ohto, H., Takizawa, T., Saito, T., Kobayashi, M., Ikeda, K., Kawakami, K. 1998. Tissue and developmental distribution of Six family gene products. Int J Dev Biol 42, 141-148.
Oliver, G., Mailhos, A., Wehr, R., Copeland, N. G., Jenkins, N.A. and Gruss, P. 1995 Six3, a murine homologue of the sine oculis gene, demarcates the most anterior border of the developing neural plate and is expressed during eye development. Development 121,4045-4055.
Ordahl, C. P. and le Douarin, N. M. 1992. Two myogenic lineages within the developing somite. Development 114,339 -353.
Ozaki, H., Watanabe, Y., Takahashi, K., Kitamura, K., Tanaka, A., Urase, K., Momoi, T., Sudo, K., Sakagami, J., Asano, M. et al. 2001. Six4, a putative myogenin gene regulator, is not essential for mouse embryonal development. Mol. Cell Biol. 21,3343 -3350.
Perry, R. L. and Rudnick, M. A. 2000. Molecular mechanisms regulating myogenic determination and differentiation. Front. Biosci. 5, 750-767.
Pignoni, F., Hu, B., Zavitz, K. H., Xiao, J., Garrity, P. A. and Zipursky, S. L. 1997. The eye-specification proteins So and Eya form a complex and regulate multiple steps in Drosophila eye development. Cell 91, 881 -891.

Puri, P. L. and Sartorelli, V. 2000. Regulation of muscle regulatory factors by DNA-binding, interacting proteins, and posttranscriptional modifications. J. Cell. Physiol. 185, 155-173

Ridgeway, A. G. and Skerjanc, I. S. 2001. Pax3 is essential for skeletal myogenesis and the expression of Six1 and Eya2. J. Biol. Chem. 273, 19033-19039.

Rudnicki, M. A., Schnegelsberg, P. N., Stead, R.H., Braun, T., Arnold , H.H. and Jaenisch, R. 1993. MyoD or Myf-5 is required for the formation of skeletal muscle. Cell 75, 1351–1359.

Schilling , T.F. and Kimmel, C.B. 1994. Segment and cell type lineage restrictions during pharyngeal arch development in the zebrafish embryo. Development 120, 483-494.

Schilling , T.F. and Kimmel, C.B. 1997. Musculoskeletal patterning in the pharyngeal segments of the zebrafish embryo. Development 124, 2945–2960.


Seo, H.C., Drivenes, Ø., Ellingsen, S. and Fjose, A. 1998a. Expression of two zebrafish homologues of the murine Six3 gene demarcates the initial eye primordia. Mech. Dev. 73, 45–57.

Seo, H.C., Drivenes, Ø., Ellingsen, S. and Fjose, A. 1998b. Transient expression of a novel Six3-related zebrafish gene during gastrulation and eye formation. Gene 216, 39–46.

Seo, H.C., Drivenes, Ø. and Fjose, A. 1998c. A zebrafish Six4 homologue with early expression in head mesoderm. Biochim. Biophys. Acta 1442, 427–431.

Seo, H.C., Curtiss, J., Mlodzik, M. and Fjose, A. 1999. Six class homeobox genes in drosophila belong to three distinct families and are involved in head development. Mech Dev 83, 127-139.

Serikaku, M. A. and O''Tousa, J. E. 1994. Sine oculis is a homeobox gene required for Drosophila visual system development. Genetics 138, 1137-1150.

Shih, H. P., Gross, M. K. and Kioussi, C. 2007 Cranial muscle defects of Pitx2 mutants result from specification defects in the first branchial arch. Proc. Natl. Acad. Sci. USA104, 5907-5912

Spitz, F., Demignon, J., Porteu, A., Kahn, A., Concordet, J. P., Daegelen, D. and Maire, P. 1998. Expression of myogenin during embryogenesis is controlled by Six/sine oculis homeoproteins through a conserved MEF3 binding site. Proc. Natl. Acad. Sci. USA 95,14220 -14225.

Tajbakhsh, S. and Buckingham, M. 2000. The birth of muscle progenitor cells in the mouse: spatiotemporal considerations. Curr. Top. Dev. Biol. 48, 225-268.

Wahl, C. M., Noden, D. M. and Baker, R. 1994. Developmental relations between sixth nerve motor neurons and their targets in the chick embryo. Dev. Dyn. 201, 191 -202.

Winterbottom, R. 1974. A descriptive synonomy of the striated muscles of the Teleostei. Proc. Acad. Nat. Sci. Philad. 125, 225-317.

Xu P-X, Zheng W, Huang L, Maire P, Laclef C, Silvius D. 2003. Six1 is required for the early organogenesis of mammalian kidney. Development 130, 3085-3094.

Zhang, W., Behringer, R. R. and Olson, E. N. 1995. Inactivation of the myogenic bHLH gene MRF4 results in up-regulation of myogenin and rib anomalies. Genes Dev. 9, 1388-1399.

Zuber M. E., Perron M., Philpott A., Bang A. and Harris W. A. 1999. Giant eyes in Xenopus laevis by overexpression of XOptx2. Cell 98, 341-352.

Zuber,M. E., Gestri, G., Viczian, A. S., Barsacchi , G. and Harris, W.A. 2003. Specification of the vertebrate eye by a network of eye field transcription factors, Development 130, 5155–5167.