The Role of Drosophila Bx42/SKIP in Cell Cycle D i s s e r t a t i o n zur Erlangung des akademischen Grades d o c t o r r e r u m n a t u r a l i u m (Dr. rer. nat.) im Fach Biologie eingereicht an der Lebenswissenschaftlichen Fakultät der Humboldt-Universität zu Berlin von Diplom-Biologin Shaza Dehne Präsidentin der Humboldt-Universität zu Berlin Prof. Dr.-Ing. Dr. Sabine Kunst Dekanin/Dekan der Lebenswissenschaftlichen Fakultät Prof. Dr. Richard Lucius Gutachter/innen: 1. Prof. Dr. Harald Saumweber 2. Prof. Dr. Christian Schmitz-Linneweber 3. Prof. Dr. Achim Leutz Tag der mündlichen Prüfung: 30.11.2016 Contents Abstract Abstrakt 1 Introduction .................................................................. 10 1.1 Bx42: Identification, protein structure and function .................................................. 11 1.2 Bx42/SNW/SKIP is an essential protein family, conserved in evolution and involved in several biological processes ............................................................................................ 13 1.2.1 Involvement of Bx42/SNW/SKIP in signaling pathways ................................ 16 1.2.1.1 Involvement in nuclear receptor pathways ................................................ 16 1.2.1.2 Involvement in the Notch signaling pathway ............................................ 17 1.2.1.3 Involvement in the TGF-ß/Dpp signal pathway ........................................ 19 1.2.2 Involvement of Bx42/SNW/SKIP in RNA splicing ......................................... 20 1.2.3 Bx42/SNW/SKIP protein family and cell cycle regulation .............................. 22 1.2.3.1 Short overview on cell cycle regulation. ................................................... 22 1.2.3.2 Evidence for Bx42/SNW/SKIP contribution to cell cycle regulation ....... 24 1.3 Drosophila eye imaginal disc: a model to study proliferation and cell cycle regulation during development. ........................................................................................... 27 1.3.2. Signal transduction and proliferation in Eye imaginal disc................................ 28 1.3.2.1. Hh and Dpp arrest cells in G1 ................................................................... 28 1.3.2.2. N signaling drives cell cycle reentry in the SMW ..................................... 29 1.3.2.3. EGFR holds R2-R5 cells in G1 phase and promotes G2/M progression of other cells during the SMW ..................................................................................... 30 1.4 Non-proliferative state and cell fate decisions ........................................................... 30 1.5 Aim of my work ......................................................................................................... 33 2 Materials and Methods ................................................ 34 2.1 Materials ..................................................................................................................... 34 2.1.1 Bacterial- strains ............................................................................................... 34 2.1.2 Vectors .............................................................................................................. 34 2.1.3 Primers used in this work ................................................................................. 34 2.1.4 Primary and secondary antibodies used in this work........................................ 35 2.1.5 Fly strains ......................................................................................................... 36 2.2 General molecular biological methods....................................................................... 39 2.2.1 The synthesis of recombinant plasmid: ............................................................ 39 2.2.2 Polymerase chain reaction (PCR) ..................................................................... 39 2.2.3 DNA ligation .................................................................................................... 39 1 2.2.4 Bacterial Transformation .................................................................................. 40 2.2.4.1 Plasmid Transformation and Antibiotic Selection..................................... 40 2.2.5 Preparation of Plasmid DNA by Alkaline Lysis with SDS - minipreparation . 40 2.2.6 Preparation of Plasmid DNA using QIAfilter plasmid Midi kit (QIAGEN) .... 41 2.2.7 RNAi methods .................................................................................................. 41 2.2.7.1 Preparation of templates by PCR to be used for in vitro transcription ...... 41 2.2.7.2 In vitro transcription .................................................................................. 43 2.2.7.3 RNA Precipitation with Ammonium Acetate ............................................ 43 2.2.7.4 RNAi treatment of cells ............................................................................. 43 2.2.8 Semi q RT-PCR ................................................................................................ 43 2.2.8.1 RNA Isolation ............................................................................................ 43 2.2.8.2 DNase Treatment ....................................................................................... 44 2.2.8.3 First Strand cDNA Synthesis ..................................................................... 44 2.2.9 FACS Analysis ................................................................................................. 45 2.2.10 SDS- Polyacrylamide Gel electrophoresis (SDS-PAGE) ................................. 46 2.2.10.1 Coomassie staining .................................................................................... 47 2.2.10.2 Western blot ............................................................................................... 47 2.3 Working with flies...................................................................................................... 47 2.3.1 Acridine orange staining ................................................................................... 47 2.3.2 Immunostaining of Imaginal discs.................................................................... 48 2.3.3 EdU incorporation ............................................................................................ 48 2.3.3.1 Labeling cells with EdU ............................................................................ 48 2.3.3.2 Fixation and permeabilzation .................................................................... 48 2.3.3.3 EdU Detection ........................................................................................... 49 2.3.3.4 DNA Staining ............................................................................................ 49 2.3.4 X Gal staining ................................................................................................... 49 2.3.5 RNA in situ hybridisation on imaginal discs .................................................... 49 2.3.5.1 Preparation of the Dig RNA labeled probe ............................................... 49 2.3.5.2 Preparation of imaginal discs for in situ hybridization.............................. 50 2.3.5.3 Prehybridization and hybridization of the prepared imaginal discs .......... 50 2.3.5.4 Detection of in situ signal .......................................................................... 50 2.4 Culture and manipulation of S2 cells ......................................................................... 51 2.4.1 Culturing S2 cells ............................................................................................. 52 2.4.1.1 Initiating cell culture from frozen stocks ................................................... 52 2.4.1.2 Passaging the S2 Cells ............................................................................... 52 2.4.2 Cell titer determination ..................................................................................... 52 2.4.3 Vital staining with Trypan blue ........................................................................ 53 2.4.4 Immunostaining of S2 cells .............................................................................. 53 2.4.5 EdU incorporation ............................................................................................ 54 3 Results ........................................................................... 55 2 3.1 The role of Bx42/SKIP in cell cycle control during Drosophila eye development ... 55 3.1.1 Expression of the dominant negative Bx42-SNW in late 3rd instar eye discs results in reduction of eye size ..................................................................................... 55 3.1.2 Bx42-SNW induced reduction in eye size corresponds with a block of the cell cycle at the G2/M transition. ........................................................................................ 58 3.1.2.1 Replication labelling of eye discs expressing Bx42/SNW under the control of GMR-GAL4 is indistinguishable from controls ................................................. 60 3.1.2.2 The number of M-phase cells within the second mitotic wave region (SMW) is reduced in GMR-GAL4/UAS-Bx42-SNW animals ............................... 61 3.1.2.3 Cell death in eye discs of GMR-GAL4/UAS-Bx42-SNW animals is increased .................................................................................................................. 62 3.1.3 Identification of genes involved in cell cycle regulation that modify the Bx42- SNW eye size phenotype. ............................................................................................