在胚胎發育過程中，細胞遷移是一個重要的課題。為了研究這個複雜的調控過程，我們利用一群會在果蠅卵子生成(oogenesis)移動的上皮細胞，稱之為邊境細胞(border cells)。透過篩選新的基因使我們能更進一步研究細胞遷移的基本機制。透過我們的篩選發現，當過度表現dysfusion (dys)這個基因在邊境細胞時，有90%的邊境細胞會產生移動的缺陷。而Dys是一個含有basic helix-loop-helix (bHLH)-PAS結構的轉錄因子，內生性的Dys蛋白廣泛分佈在每個細胞的細胞核膜(nuclear membrane)上。其中也包括邊境細胞，隨著邊境細胞的移動Dys的表現量會逐漸下降，當邊境細胞抵達到卵子(oocyte)時，完全偵測不到Dys的訊號。此外，當dys突變發生在極細胞(polar cells)時會有64%的邊境細胞會產生移動的缺陷。而dys在邊境細胞發生突變時會導致所有的邊境細胞移動產生缺陷。因此，不管是在邊境細胞或是極細胞中dys是不可或缺的。有趣的是，當我們過度表現dys時不只會使邊境細胞產生移動的缺陷，其中接近50%的細胞不會形成細胞叢(cell cluster)。為了要使卵室前端的濾泡細胞(follicle cells)聚集成為邊境細胞的細胞叢，極細胞會分泌配體(ligand)-Unpaired(Upd)來刺激周圍的濾泡細胞的JAK/STAT訊息傳遞鏈，使得濾泡細胞轉變成為會移動的細胞叢。而Upd的分布範圍決定有多少個濾泡細胞形成邊境細胞，在正常卵室中約有4-8顆濾泡細胞會轉變成邊境細胞的細胞叢而開始進行移動，而在過度表現dys的卵室中大約只有3顆的邊境細胞形成。進而我們利用STAT-GFP來分析STAT的活性，發現當過度表現dys會使得STAT-GFP的表現量下降約30%。根據這些結果，Dys影響邊境細胞的移動可能與JAK/STAT訊息的調控有關。

Cell migration is a critical process for animal development. In order to study the complicated process, we focus on a cluster of epithelial cells which are migratory in Drosophila oogenesis called border cells to screen for novel genes for further exploring basic mechanism in cell migration. Of these, dysfusion (dys), which encodes a basic helix-loop-helix (bHLH)-PAS domain transcription factor, was identified to cause nearly 90% of migration defects in border cells when dys was overexpressed. To analyze the function of dys, we generated mutant clones in polar cells and found nearly 64% of border cell cluster fail to migrate. In addition, the mutant clone in border cells also showed 100% defects in motility. The expression of Dys was shown at the nuclear membrane of germline and all follicle cells including border cells but the expression of Dys was reduced once border cells start to migrate, and it was undetectable when border cells reached the oocyte. Therefore Dys is required both in border cells and polar cells during migration. Interestingly, overexpression of UAS-dys in border cells not only severely impeded migration but also made 50% of border cells fail to form a cluster. To recruit neighboring follicle cells into the migratory cluster, polar cells secrete Upd to activate JAK/STAT signaling in adjacent follicle cells. Therefore graded morphogen, Upd, determines how many follicle cells are recruited, which is 4-8 in wild type border cell cluster. Overexpression of dys, the number of recruited follicle cells was reduced to 3.2. To further validate whether the reduced number of border cells in overexpression of dys is through suppression of JAK/STAT signaling, we analyzed the STAT activity by STAT-GFP. In wild type border cells, the intensity of GFP gradually increased during migration. Consistent our hypothesis, we observed nearly 30% reduction of the expression level in STAT-GFP while dys was overexpressed. Taken together, my thesis work demonstrates that the function of Dys in border cell migration might be involved in regulation of JAK/STAT signaling.

Bach, E.A., Ekas, L.A., Ayala-Camargo, A., Flaherty, M.S., Lee, H., Perrimon, N., and Baeg, G.H. (2007). GFP reporters detect the activation of the Drosophila JAK/STAT pathway in vivo. Gene Expression Patterns : GEP 7, 323-331.

Baeg, G.H., Zhou, R., and Perrimon, N. (2005). Genome-wide RNAi analysis of JAK/STAT signaling components in Drosophila. Genes & Development 19, 1861-1870.

Bai, J., Uehara, Y., and Montell, D.J. (2000). Regulation of invasive cell behavior by taiman, a Drosophila protein related to AIB1, a steroid receptor coactivator amplified in breast cancer. Cell 103, 1047-1058.

Beccari, S., Teixeira, L., and Rorth, P. (2002). The JAK/STAT pathway is required for border cell migration during Drosophila oogenesis. Mechanisms of Development 111, 115-123.

Becker, S., Groner, B., and Muller, C.W. (1998). Three-dimensional structure of the Stat3beta homodimer bound to DNA. Nature 394, 145-151.

Binari, R., and Perrimon, N. (1994). Stripe-specific regulation of pair-rule genes by hopscotch, a putative Jak family tyrosine kinase in Drosophila. Genes & Development 8, 300-312.

Brown, S., Hu, N., and Hombria, J.C. (2001). Identification of the first invertebrate interleukin JAK/STAT receptor, the Drosophila gene domeless. Current Biology : CB 11, 1700-1705.

Brown, S., and Zeidler, M.P. (2008). Unphosphorylated STATs go nuclear. Current Opinion in Genetics & Development 18, 455-460.

Chen, H.J., Wang, C.M., Wang, T.W., Liaw, G.J., Hsu, T.H., Lin, T.H., and Yu, J.Y. (2011). The Hippo pathway controls polar cell fate through Notch signaling during Drosophila oogenesis. Developmental Biology 357, 370-379.

Chen, X., Oh, S.W., Zheng, Z., Chen, H.W., Shin, H.H., and Hou, S.X. (2003). Cyclin D-Cdk4 and cyclin E-Cdk2 regulate the Jak/STAT signal transduction pathway in Drosophila. Developmental Cell 4, 179-190.

Chen, X., Vinkemeier, U., Zhao, Y., Jeruzalmi, D., Darnell, J.E., Jr., and Kuriyan, J. (1998). Crystal structure of a tyrosine phosphorylated STAT-1 dimer bound to DNA. Cell 93, 827-839.

Darnell, J.E., Jr. (1997). STATs and gene regulation. Science 277, 1630-1635.
Dearolf, C.R. (1999). JAKs and STATs in invertebrate model organisms. Cellular and Molecular Life Sciences : CMLS 55, 1578-1584.

Forbes, A., and Lehmann, R. (1999). Cell migration in Drosophila. Current Opinion in Genetics & Development 9, 473-478.

Golic, K.G., and Lindquist, S. (1989). The FLP recombinase of yeast catalyzes site-specific recombination in the Drosophila genome. Cell 59, 499-509.

Gonzalez-Reyes, A., and St Johnston, D. (1998). Patterning of the follicle cell epithelium along the anterior-posterior axis during Drosophila oogenesis. Development 125, 2837-2846.

Hafen, E. (1997). Fly Pushing: The theory and practice of Drosophila genetics - Greenspan,RJ. Nature 389, 559-560.

Harrison, D.A. (2012). The Jak/STAT pathway. Cold Spring Harbor Perspectives in Biology 4.

Hedgecock, E.M., Culotti, J.G., Hall, D.H., and Stern, B.D. (1987). Genetics of cell and axon migrations in Caenorhabditis elegans. Development 100, 365-382.

Hou, S.X., Zheng, Z., Chen, X., and Perrimon, N. (2002). The Jak/STAT pathway in model organisms: emerging roles in cell movement. Developmental Cell 3, 765-778.

Hou, X.S., Melnick, M.B., and Perrimon, N. (1996). marelle acts downstream of the Drosophila HOP/JAK kinase and encodes a protein similar to the mammalian STATs. Cell 84, 411-419.

Hou, X.S., and Perrimon, N. (1997). The JAK-STAT pathway in Drosophila. Trends in Genetics : TIG 13, 105-110.

Ihle, J.N. (2001). The Stat family in cytokine signaling. Current Opinion in Cell Biology 13, 211-217.

Jang, A.C., Chang, Y.C., Bai, J., and Montell, D. (2009). Border-cell migration requires integration of spatial and temporal signals by the BTB protein Abrupt. Nature Cell Biology 11, 569-579.

Jiang, L., and Crews, S.T. (2003). The Drosophila dysfusion basic helix-loop-helix (bHLH)-PAS gene controls tracheal fusion and levels of the trachealess bHLH-PAS protein. Molecular and Cellular Biology 23, 5625-5637.

Jiang, L., and Crews, S.T. (2006). Dysfusion transcriptional control of Drosophila tracheal migration, adhesion, and fusion. Molecular and Cellular Biology 26, 6547-6556.

Jiang, L., and Crews, S.T. (2007). Transcriptional specificity of Drosophila dysfusion and the control of tracheal fusion cell gene expression. The Journal of Biological Chemistry 282, 28659-28668.

Kambysellis, M.P., Starmer, T., Smathers, G., and Heed, W.B. (1980). Studies of Oogenesis in Natural-Populations of Drosophilidae .2. Significance of Microclimatic Changes on Oogenesis of Drosophila-Mimica. American Naturalist 115, 67-91.

Kewley, R.J., Whitelaw, M.L., and Chapman-Smith, A. (2004). The mammalian basic helix-loop-helix/PAS family of transcriptional regulators. The International Journal of Biochemistry & Cell Biology 36, 189-204.

Kimble, J.E., and White, J.G. (1981). On the control of germ cell development in Caenorhabditis elegans. Developmental Bology 81, 208-219.

Kisseleva, T., Bhattacharya, S., Braunstein, J., and Schindler, C.W. (2002). Signaling through the JAK/STAT pathway, recent advances and future challenges. Gene 285, 1-24.

Kumar, M.B., Ramadoss, P., Reen, R.K., Vanden Heuvel, J.P., and Perdew, G.H. (2001). The Q-rich subdomain of the human Ah receptor transactivation domain is required for dioxin-mediated transcriptional activity. The Journal of Biological Chemistry 276, 42302-42310.

Lecaudey, V., and Gilmour, D. (2006). Organizing moving groups during morphogenesis. Current Opinion in Cell Biology 18, 102-107.

Leonard, W.J., and O'Shea, J.J. (1998). Jaks and STATs: biological implications. Annual Review of Immunology 16, 293-322.

Levy, D.E., and Darnell, J.E., Jr. (2002). Stats: transcriptional control and biological impact. Nature Reviews Molecular Cell Biology 3, 651-662.

Li, J., Xia, F., and Li, W.X. (2003). Coactivation of STAT and Ras is required for germ cell proliferation and invasive migration in Drosophila. Developmental Cell 5, 787-798.

Litterst, C.M., and Pfitzner, E. (2001). Transcriptional activation by STAT6 requires the direct interaction with NCoA-1. The Journal of Biological Chemistry 276, 45713-45721.

Litterst, C.M., and Pfitzner, E. (2002). An LXXLL motif in the transactivation domain of STAT6 mediates recruitment of NCoA-1/SRC-1. The Journal of Biological Chemistry 277, 36052-36060.

Manseau, L., Baradaran, A., Brower, D., Budhu, A., Elefant, F., Phan, H., Philp, A.V., Yang, M., Glover, D., Kaiser, K., et al. (1997). GAL4 enhancer traps expressed in the embryo, larval brain, imaginal discs, and ovary of Drosophila. Developmental Dynamics : an Official Publication of the American Association of Anatomists 209, 310-322.

Mattout, A., Goldberg, M., Tzur, Y., Margalit, A., and Gruenbaum, Y. (2007). Specific and conserved sequences in D. melanogaster and C. elegans lamins and histone H2A mediate the attachment of lamins to chromosomes. Journal of Cell Science 120, 77-85.

McIntosh, B.E., Hogenesch, J.B., and Bradfield, C.A. (2010). Mammalian Per-Arnt-Sim proteins in environmental adaptation. Annual Review of Physiology 72, 625-645.

Melcer, S., Gruenbaum, Y., and Krohne, G. (2007). Invertebrate lamins. Experimental Cell Research 313, 2157-2166.

Muller, P., Kuttenkeuler, D., Gesellchen, V., Zeidler, M.P., and Boutros, M. (2005). Identification of JAK/STAT signalling components by genome-wide RNA interference. Nature 436, 871-875.

Ooe, N., Saito, K., Mikami, N., Nakatuka, I., and Kaneko, H. (2004). Identification of a novel basic helix-loop-helix-PAS factor, NXF, reveals a Sim2 competitive, positive regulatory role in dendritic-cytoskeleton modulator drebrin gene expression. Molecular and Cellular Biology 24, 608-616.

Orphanides, G., Wu, W.H., Lane, W.S., Hampsey, M., and Reinberg, D. (1999). The chromatin-specific transcription elongation factor FACT comprises human SPT16 and SSRP1 proteins. Nature 400, 284-288.

Parks, S., and Spradling, A. (1987). Spatially Regulated Expression of Chorion Genes during Drosophila Oogenesis. Genes & Development 1, 497-509.

Partch, C.L., and Gardner, K.H. (2010). Coactivator recruitment: a new role for PAS domains in transcriptional regulation by the bHLH-PAS family. Journal of Cellular Physiology 223, 553-557.

Prasad, M., Wang, X., He, L., and Montell, D.J. (2011). Border cell migration: a model system for live imaging and genetic analysis of collective cell movement. Methods in Molecular Biology 769, 277-286.

Rawlings, J.S., Rosler, K.M., and Harrison, D.A. (2004). The JAK/STAT signaling pathway. Journal of Cell Science 117, 1281-1283.

Reich, N.C., and Liu, L. (2006). Tracking STAT nuclear traffic. Nature Reviews Immunology 6, 602-612.

Ridley, A.J., Schwartz, M.A., Burridge, K., Firtel, R.A., Ginsberg, M.H., Borisy, G., Parsons, J.T., and Horwitz, A.R. (2003). Cell migration: integrating signals from front to back. Science 302, 1704-1709.

Rorth, P. (2002). Initiating and guiding migration: lessons from border cells. Trends in Cell Biology 12, 325-331.

Rorth, P., Szabo, K., Bailey, A., Laverty, T., Rehm, J., Rubin, G.M., Weigmann, K., Milan, M., Benes, V., Ansorge, W., et al. (1998). Systematic gain-of-function genetics in Drosophila. Development 125, 1049-1057.

Rubin, G.M., Yandell, M.D., Wortman, J.R., Gabor Miklos, G.L., Nelson, C.R., Hariharan, I.K., Fortini, M.E., Li, P.W., Apweiler, R., Fleischmann, W., et al. (2000). Comparative genomics of the eukaryotes. Science 287, 2204-2215.

Shi, S., Larson, K., Guo, D., Lim, S.J., Dutta, P., Yan, S.J., and Li, W.X. (2008). Drosophila STAT is required for directly maintaining HP1 localization and heterochromatin stability. Nature Cell Biology 10, 489-496.

Silver, D.L., and Montell, D.J. (2001). Paracrine signaling through the JAK/STAT pathway activates invasive behavior of ovarian epithelial cells in Drosophila. Cell 107, 831-841.
Southall, T.D., Elliott, D.A., and Brand, A.H. (2008). The GAL4 System: A Versatile Toolkit for Gene Expression in Drosophila. Cold Spring Harbor Protocols 2008, pdb top49.

Stark, G.R., Kerr, I.M., Williams, B.R., Silverman, R.H., and Schreiber, R.D. (1998). How cells respond to interferons. Annual Review of Biochemistry 67, 227-264.

Teddy, J.M., and Kulesa, P.M. (2004). In vivo evidence for short- and long-range cell communication in cranial neural crest cells. Development 131, 6141-6151.

Torres, I.L., Lopez-Schier, H., and St Johnston, D. (2003). A Notch/Delta-dependent relay mechanism establishes anterior-posterior polarity in Drosophila. Developmental Cell 5, 547-558.

Wegenka, U.M., Buschmann, J., Lutticken, C., Heinrich, P.C., and Horn, F. (1993). Acute-phase response factor, a nuclear factor binding to acute-phase response elements, is rapidly activated by interleukin-6 at the posttranslational level. Molecular and Cellular Biology 13, 276-288.

Xi, R., McGregor, J.R., and Harrison, D.A. (2003). A gradient of JAK pathway activity patterns the anterior-posterior axis of the follicular epithelium. Developmental Cell 4, 167-177.

Yan, R.Q., Small, S., Desplan, C., Dearolf, C.R., and Darnell, J.E. (1996). Identification of a Stat gene that functions in Drosophila development. Cell 84, 421-430.

Yan, S.J., Lim, S.J., Shi, S., Dutta, P., and Li, W.X. (2011). Unphosphorylated STAT and heterochromatin protect genome stability. The FASEB Journal 25, 232-241.

Zeidler, M.P., Bach, E.A., and Perrimon, N. (2000). The roles of the Drosophila JAK/STAT pathway. Oncogene 19, 2598-2606.

Zhao, K., Kas, E., Gonzalez, E., and Laemmli, U.K. (1993). SAR-dependent mobilization of histone H1 by HMG-I/Y in vitro: HMG-I/Y is enriched in H1-depleted chromatin. The EMBO Journal 12, 3237-3247.