果蠅眼睛發育過程中，眼睛形成基因eyeless、twin of eyeless、 optix、eye gone、 sine oculis、eyes absent 及 dachshund 決定眼睛原質的命運，給予組織形成眼圓盤的潛能。眼圓盤形成之後，原神經基因atonal (ato) 及 daughterless (da)產生basic helix-loop-helix蛋白，並形成 heterdimers而促使細胞分化為感光細胞。眼圓盤的形態溝中，細胞會開始分化，同時表達Ato及較高量的Da。有趣的是：eyeless、twin of eyeless及optix 會在形態溝中、後被抑制到完全不表現。sine oculis 及eyes absent則在形態溝的前、後大量表現，但在形態溝則不表現。dachshund (dac) 則在形態溝前大量表現，而在後面只維持幾排細胞的低量表現。當異位表達eyeless (ey) 或同時異位表達eyes absent與 sine oculis在形態溝中時，造成Ato 表現被抑制及接下來的感光細胞分化失敗。由這些結果推論得知，眼睛形成基因在形態溝中的負調控，對於果蠅複眼的細胞分化是非常重要的。我發現在形態溝中 ato或 da 的突變組織中， Dac 與Ey的表現量會增加。同時異位表現Ato與Da在形態溝前，會造成Dac和Ey 在形態溝前的表現被抑制。當單獨表達Da時，也會造成類似，但較不有效的抑制效果。由原位雜合實驗得知， dac及 ey的負調控至少部份發生在轉譯階段，而對於其他眼睛形成基因，Ato與Da也可在轉譯階段抑制其表現。Da與VP16 的活化區域所建構的融合蛋白，能持續的促進果蠅背甲上感覺剛毛的發育。因此，DaVP16確實是一個活化形式的Da蛋白。而此融合蛋白在眼圓盤中，對Da的表現仍造成類似的抑制性效果。因此，Ato及Da功能上應為一個活化子活化一個抑制子進而抑制眼睛形成基因的表現。其他實驗結果也將在論文中一一討論。

In the Drosophila eye development, eye formation genes eyeless, twin of eyeless, optix, eye gone, sine oculis, eyes absent, and dachshund specify the eye primordium, giving it the potential to form an eye disc. After eye disc formation, the proneural genes atonal (ato) and daughterless (da) encode subunits of heterodimeric basic helix-loop-helix proteins that promote the differentiation of photoreceptors. In the morphogenetic furrow (MF), where photoreceptor differentiation initiates, cells express Ato and higher levels of Da. Interestingly, eyeless, twin of eyeless and optix are downregulated in the MF. sine oculis and eyes absent are highly expressed both anterior and posterior to, but not in, the MF. Expression of dachshund (dac) is at a high level anterior to the MF and repressed to a basal level in the MF. I shown that misexpression of eyeless, or combination of eyes absent and sine oculis across the MF causes Ato repression in the MF, and consequently failure of photoreceptor differentiation in the eye disc. These results suggest that negative regulation of eye formation genes in the MF is necessary for cell differentiation in the compound eye. I observed that expression of Dac and Eyeless is upregulated in da or ato mutant clones in the MF. Co-misexpression of Da and Ato causes Dac and Eyeless being downregulated anterior to the MF, and misexpression of Da alone also causes a similar, but less effective, downregulation. The negative regulation of dac and ey is at least partly at the transcriptional level, which is also observed for other eye formation genes. The fusion protein of Da and VP16 activation domain that functions constitutively to promote sensory bristle development, also causes similar repressive effect on Dac. Therefore, Ato and Da may function as an activator to activate a repressor of the eye formation genes. Several other results will also be presented in this thesis.

Abstract 1
中文摘要 2 Introduction 3 Materials and Methods 6 Results 13 Discussion 23 References 28 Figures 32 Appendix 41

Baker, N. E., and Yu, S-Y. (1997) Proneural function of neurogenic genes in the developing Drosophila eye. Curr. Bio. 7:122-132
Baker, N. E., (2000) Notch signaling in the nervous system. Pieces still missing from the puzzle. BioEssays 22:264-273
Bonini, N. M., Leiserson, W. M., and Benzer, S. (1993) The Eyes Absent Gene: Genetic Control of Cell Survival and Differentiation In the Developing Drosophila Eye. Cell 72:379-395
Brown, N. L., Paddock, S. W., Sattler, C. A., Cronmiller, C., Thoma, B. J., and Carroll, S. B. (1996) daughterless is required for Drosophila photoreceptor cell determination, eye morphogenesis, and cell cycle progression. Developmental Biology 179:65-78
Chen, C. K., and Chien, C. T. (1999) Negative Regulation of atonal in the proneural cluster formation of Drosophila R8 photoreceptors. Proc. Natl. Acad. Sci. UAS 96:5055-5060
Chanut, F., Luk, A., and Heberlein, U. (2000) A screen for dominant modifiers of ro Dom , a mutation that disrupts morphogenetic furrow progression in Drosophila, identifies Groucho and Hairless as regulators of atonal expression. Genetics 156:1203—1217
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 development in Drosophila. Cell 91:893—903
Chen, R., Halder, G., Zhang, Z., and Mardon, G. (1999) Signaling by the TGF-b homolog decapentaplegic functions reiteratively within the network of genes controlling retinal cell fate determination in Drosophila. Development 126:935-943
Collins, R.T., Furukawa, T., Tanese, N., and Triesman, J. E. (1999) Osa associates with the Brahma chromatin remodeling complex and promotes the activation of some target genes. The EMBO Journal 18(24): 7029-7040
Czerny, T., Halder, G., Kloter, U., Souabni, A., Gehring, W. G., and Busslinger, M. (1999) twin of eyeless, a Second Pax-6 Gene of Drosophila, Acts Upstream of eyeless in the Control of Eye Development. Molecular Cell 3: 297—307
Desplan, C. (1997) Eye development: governed by a direcator or a junta? Cell 91: 861-864
de Celis, J. F., Barrio, R., del Arco, A., and Garcia-Bellido, A. (1993) Genetic and molecular characterization of a Notch mutation in its Delta- and Serrate-binding domain in Drosophila. Proc. Natl. Acad. Sci. USA 90:4037-4041.
Dubnicoff, T., Valentine, S. A., Chen, G., Shi, T., Lengyel, J. A., Paroush, Z., and Courey, A. J. (1997) Conversion of dorsal from an activator to a repressor by global corepressor groucho. Genes Dev. 11: 2952-2957.
Fisher, A. L., Ohsako, S., and Caudy, N. (1996) The WRPW motif of the hairy-related basic helix-loop-helix repressor proteins acts as 4-amono-acid transcription repression and protein-protein interaction domain. Mol. Cell. Biol. 16:2670-2677
Giebel, B., Stuttem, I., Hinz, U., and Campos-Ortega, J. A. (1997) Lethal of scute requires overexpression of daughterless to elicit ectopic neuronal development during embryogenesis in Drosophila. Mech. Dev. 63:75-87
Halder, G., Callaerts, P., and Gehring, W. J. (1995) Induction of ectopic eyes by targeted expression of the eyeless gene in Drosophila. Science 267:1788-1792.
Halder, G., Callaerts, P., Flister, S., Walldorf, U., Kloter, U., and Gehring, W. J. (1998) Eyeless initiates the expression of both sine oculis and eyes absent during Drosophila compound eye development Development 125:2181-2191
Heberlein, U., and Moses, K. (1995) Mechanisms of Drosophila retinal morphogenesis: the virtues of being progressive. Cell 81:987-990
Jarman, A. P., Grau, Y., Jan, L. Y., and Jan, Y. N. (1993) atonal is a proneural gene that directs chordotonal organ formation in the Drosophila peripheral nerve system.
Cell (Cambridge, Mass.) 73:1307-1321
Jarman, A. P., Grell, E. H., Ackerman, L., Jan, L. Y., and Jan, Y. N. (1994) atonal is the proneural gene for Drosophila photoreceptors. Nature 369: 398-400
Jimenez, G., and Ish-Horowicz, D. (1997) A chimeric enhancer-of—split transcriptional activator drives neural development and acute-scute expression. Mol. Cell. Biol. 17:4355-4362.
Jimenez, G., Paroush, Z., and Ish-Horowicz, D. (1997) Groucho acts as a corepressor for a subset of negative regulators, including hairy and engrailed. Genes Dev. 11:3072-3082
Kramer, S. G., Jinks, T. M., Schedl, P., and Gergen, J. P. (1999) Direct activation of Sex-lethal transcription by Drosophila runt protein. Development 126:191-200.
Kumar, J. P. and Moses, K. (2001) EGF Receptor and Notch signaling act upstream of Eyeless/Pax6 to control eye specification. Cell 104: 687—697
Li, Y., and Baker, N. E. (2001) Proneural enhancement by Notch overcomes Suppressor-of-Hairless repressor function in the developing Drosophila eye. Curr. Bio. 11:330—338
Ligoxygakis, P., Yu, S-Y., Delidakis, C., and Baker, N. E. (1998) A subset of Notch functions during Drosophila eye development require Su(H) and the E(spl) gene complex. Development 125:2893-2900
Mardon, G., Solomon, N. M., and Rubin, G. M. (1994) dachshund encodes a nuclear protein required for normal eye and leg development in Drosophila. Development 120: 3473-3486
Masucci, J. D., Milten berger, R. J., and Hoffmann, F. M. (1990) Pattern-specific expression of the Drosophila decapentaplegic gene in imaginal disc is regulated by 3’ cis regulatory elements. Genes Dev. 4:2011-2023
Niimi, T., Semiya, M., Kloter, U., Flister, S., and Gehring, W. G. (1999) Directly regulatory interaction of the eyeless protein with eye-specific enhancer in the sine oculis gene during eye induction in Drosophila. Development 126:2253-2260
Paroush, Z., Finley, R. L., Kidd, Jr. T., Wainwright, S.M., Ingham, P. W., Brent, R., and Ish-Horowicz, D. (1994) Groucho is required for Drosophila neurogensis, segmentation, and sex dertermination and directly with hairy- related bHLH proteins. Cell 79:805-815
Paroush, Z., Wainwright, S.M., and Ish-Horowicz, D. (1997) Torso signaling regulates terminal patterning in Drosophila by antagonizing Groucho-mediated repression. Development 124:3827-3834
Pignoni, F., Hu, B., Zavitzr, 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
Seimiya, M., and Gehring, W. J. (2000) The Drosophila homeobox gene optix is capable of inducing ectopic eyes by an eyeless-independent mechanism. Development 127:1879-1886
Spradling, A. C. (1986) P element-mediated transformation in Drosophila: a practical approach by D. B. Robert, IRL, Oxford, I ed., p.175-197
Staehling-Hampton, K., Jackson, P. D., Clark, M. J., Brand, A. H., and Hoffmann, F. M. (1994) Specificity of bone morphogenetic protein-related factors: cell fate and gene expression changes in Drosophila embrys induced by decapentaplegic gene but not A60. Cell. Growth. Differ. 5:585-593
Sun, Y., Jan, L. Y., and Jan, Y. N. (1998) Transcriptional regulation of atonal during development of the Drosophila peripheral nervous system. Development 125: 3731-3740
Toba, G., Ohsako, T., Miyata, N., Ohtsuka, T., Seong, K-H., and Aigaki, T. (1999) The gene search system: a method for efficient detection and rapid molecular identification of genes in Drosophila melanogaster. Genetics 151:725—737
Treisman, J. E., and Heberlein, U. (1998) Eye development in Drosophila: formation of the eye field and control of differentiation. Cur. Top. Dev. Bio. 39:119-158
Triezenberg, S. J., Kingsbury, R. C., and Mcknight, S. L. (1988) Functional disscetion of VP16, the trans-activator of herpes simplex virus immediate early gene expression. Genes Dev.2:718-729
Wolff, T., and Ready, D. F. (1993) Pattern formation in the Drosophila retina. In “The Development of Drosophila melanogaster”(M. Bate and A.M. Arias, eds.), pp. 1277-1325. Cold Spring Harbor Lab. Press