Molecular genetic analysis of a sine oculis enhancer and The leventina gene as a model system to study human macular degeneration in Drosophila Inauguraldissertation zur Erlangung der Würde eines Doktors der Philosophie vorgelegt der Philosophisch-Naturwissenschaftlichen Fakultät der Universität Basel von Tobias Samuel Pauli Wahlern (BE) Ausgeführt unter der Leitung von: Prof. Dr. Walter J. Gehring Abteilung Zellbiologie Biozentrum der Universität Basel CH-4056 Basel 2004 Genehmigt von der Philosophisch-Naturwissenschaftlichen Fakultät auf Antrag der Herren Prof. W. J. Gehring Prof. M. Affolter Prof. H. Reichert Referent Koreferent Vorsitz .............................. Basel, den 14. Dezember 2004 Prof. Hans-Jakob Wirz Dekan Philosophisch-Naturwissenschaftliche Fakultät Universität Basel Acknowledgements Acknowledgements I am grateful to Professor Walter J. Gehring for giving me the opportunity to perform my thesis in his laboratory. I thank him especially for the way he shared his enthusiasm for developmental biology and research in general. For the performance of this thesis many people were very helpful. Not only that I got a lot of help in molecular biology and fly genetics but I really had a good time in this laboratory due to all the friendship and fun I could participate in. Therefore I would like to thank all people that helped me in one way or the other to achieve this work. Namely: Paul Baumgartner, Sacha Glardon, Makiko Seimiya, Jorge Blanco, Nicole Grieder, Greta Backhaus, Lydia Michaut, Christopher Brink, Urs Kloter, Georgios Pyrowolakis, Britta Hartmann, Stefan Wenmalm, Frédéric Prince, Tomoko Nagao, George Gentsch, Markus Affolter, Bernadette Bruno, Roland Kirchhofer, Karin Mauro, Georges Martin, Gina Evora, Claudio Punzo, Daniel Felix, Raphael Fünfschilling, Lukas Keller, Sabine Dettwiler, Salome Röck, Clemens Cabernard, Vera Niederkofler, Ute Nussbaumer, Marc Neumann, Silvia Arber, Verena Widmer, Simon Hippenmeyer, Diana Blank, Myriam Schaub, Ilias Charlafti, Erika Oesch, Liliane Devaja, Andy Ebner, Shiuyang Kuo, Hiroshi Suga and the one and only Arabelle Pfrunder and my family. I would like to thank specially those people who collaborated closely with me during my thesis. Many thanks to Makiko Seimiya who introduced me into the sine oculis project and made injecting a quite comfortable task. Thanks a lot for all the discussions and the shared interest for this project. Many thanks also to Christopher Brink and Sacha Glardon for their help on paper work in any language. Besides this I thank you two for the fun we had in and outside the laboratory. Many thanks also to Paul Baumgartner who introduced me into molecular biology. Thanks for always sharing enthusiasm for sophisticated cloning strategies. Many thanks to Urs Kloter, Frédéric Prince and Nicole Grieder for their help on fly genetics and other puzzling things. Many thanks also to Sabine Dettwiler and Verena Widmer for their generous help with the baculovirus system. Many thanks to Arabelle Pfrunder for all the interest in my work and all the help on plenty of different topics. 3 Acknowledgements I am indebted to the ‘Fondation suisse de recherche sur les maladies musculaire’ for their generous financial support. Special thanks also to my supervisor committee Prof. M. Affolter, Prof. H. Reichert and also Prof. W. J. Gehring. 4 Table of contents 1. ABBREVIATIONS ..............................................................................................................................7 2. THE LEVENTINA GENE AS A MODEL SYSTEM TO STUDY HUMAN MACULAR DEGENERATION IN DROSOPHILA...................................................................................................9 2.1. SUMMARY........................................................................................................................................9 2.2. INTRODUCTION ..............................................................................................................................11 2.2.1. Malattia Leventinese ..............................................................................................................11 2.2.2. EFEMP1.................................................................................................................................12 2.2.3. The Notch receptor.................................................................................................................13 2.2.4. Pax-6......................................................................................................................................15 2.2.5. Eye development in Drosophila and vertebrates....................................................................18 2.2.6. Homologies between human and Drosophila eye development .............................................20 2.2.7. Why using Drosophila as a model system to study a human eye disease...............................24 2.2.8. Model for Malattia Leventinese .............................................................................................27 2.3. MATERIALS AND METHODS ...........................................................................................................28 2.3.1. Fly work .................................................................................................................................28 2.3.2. Constructs ..............................................................................................................................30 2.3.3. Molecular methods.................................................................................................................34 2.3.4. Histology ................................................................................................................................37 2.3.5. Cell Culture Methods .............................................................................................................37 2.4. RESULTS ........................................................................................................................................40 2.4.1. In silico structure comparison between EFEMP1 and known Notch ligands ........................40 2.4.2. Overexpression of EFEMP1 / EFEMP1Arg345Trp in Drosophila..............................................41 2.4.3. Expression of EFEMP1 together with human Notch-1 and a chimeric Notch.......................44 2.4.4. Expression of a mammalian protein in Drosophila ...............................................................45 2.4.5. Notch activity measurement using a cell culture assay..........................................................47 2.4.6. In vivo assay to detect Notch activity .....................................................................................50 2.4.7. Expression of a non-secreted version of EFEMP1.................................................................52 2.4.8. Pull-down assay .....................................................................................................................53 2.4.9. EFEMP1 in aging flies...........................................................................................................54 2.4.10. Exposure of flies to constant light........................................................................................55 2.4.11. Drosophila Notch EGF10-13 as template for Aptamers......................................................55 2.5. DISCUSSION ...................................................................................................................................59 3. MOLECULAR GENETIC ANALYSIS OF A SINE OCULIS ENHANCER...............................65 3.1. SUMMARY......................................................................................................................................65 3.2. INTRODUCTION ..............................................................................................................................66 3.3. RESULTS ........................................................................................................................................69 3.3.1. Defining a minimal eye/ocelli specific enhancer of the so gene.............................................69 3.3.2. sine oculis is able to recognize its own enhancer...................................................................70 3.3.3. so10-soAE-LacZ and so7-LacZ are not expressed in the ocellar region of so2 mutant flies..71 3.3.4. 4xsoAE is not expressed in so3 clones....................................................................................72 3.3.5. 4xsoAE-LacZ is induced in ectopic eyes and in cell culture ..................................................73 3.3.6. Defining a consensus sequence for SO - DNA interaction.....................................................73 3.3.7. Genome-wide search for potential sine oculis target genes...................................................74 3.3.8. eyeless is a direct target of so ................................................................................................74 3.3.9. hh is a direct target of so........................................................................................................75 3.4. DISCUSSION ...................................................................................................................................78 3.4.1. so autoregulation is essential for ocelli development ............................................................78 3.4.2. so directly regulates ey in eye development ...........................................................................79