Functional analysis of a new dominant-negative allele of miranda during CNS development in Drosophila Inaugural-Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultät der Heinrich-Heine-Universität Düsseldorf Vorgelegt von Chieko Takizawa aus Tokyo, Japan Düsseldorf, 2006 Gedruckt mit Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der Heinrich-Heine-Universität Düsseldorf Tag der mündlich Prüfung: 22. Mai 2006 Berichterstatter: Prof. Dr. A. Wodarz Prof. Dr. E. Knust 1 Introduction..................................................................................................... 1 1.1 Generation of cell diversity .............................................................................. 1 1.2 Development of the central nervous system in Drosophila ............................... 2 1.3 Asymmetric cell division of Drosophila neuroblasts......................................... 4 1.4 Proteins involved in the asymmetric cell division of neuroblasts....................... 5 1.4.1 Basally localized proteins .......................................................................... 6 1.4.2 Apically localized proteins......................................................................... 6 1.4.3 Other proteins involved in asymmetric cell division................................... 8 1.5 Control of daughter cell size asymmetry......................................................... 11 1.6 Asymmetric cell division in other systems: common features and differences. 11 1.6.1 Drosophila sensory organ precursor cells................................................. 11 1.6.2 C. elegans zygote..................................................................................... 12 1.7 Evolutionary conservation of the Par/aPKC complex...................................... 14 1.8 Miranda.......................................................................................................... 14 1.8.1 Function of Miranda ................................................................................ 16 1.8.2 Protein structure ...................................................................................... 17 1.8.3 miranda mutant alleles............................................................................. 17 1.9 Aim of my work............................................................................................. 18 2 Materials and methods.................................................................................. 19 2.1 Genetic screens .............................................................................................. 19 2.1.1 Maternal screen ....................................................................................... 19 2.1.2 Fly stocks ................................................................................................ 20 2.1.3 Generation of germline clones ................................................................. 21 2.1.4 Zygotic screen ......................................................................................... 22 2.2 Immunocytochemistry.................................................................................... 23 2.2.1 Embryo collection and fixation................................................................ 23 2.2.2 Antibody staining (immunofluorescence)................................................. 23 2.2.3 Antibody staining (enzyme-conjugated secondary antibody).................... 24 2.2.5 Antibodies ............................................................................................... 25 2.2.6 DNA label with YoYo-1.......................................................................... 26 2.2.7 Cuticle preparation .................................................................................. 26 2.3 Genetics ......................................................................................................... 26 2.3.1 Fly stocks ................................................................................................ 26 2.3.2 Complementation test .............................................................................. 28 2.3.3 Recombination......................................................................................... 28 2.4 Molecular biology.......................................................................................... 29 2.4.1 Sequencing of the miraE326 genomic DNA................................................ 29 2.4.1.1 Genomic DNA isolation...................................................................... 30 2.4.1.2 PCR (polymerase chain reaction) ........................................................ 31 2.4.2 Cloning of the jaguar cDNA.................................................................... 31 2.4.2.1 Cloning of PCR fragments into plasmids.............................................. 33 2.4.2.2 Transformation of electrocompetent cells............................................. 33 2.4.2.3 Isolation of plasmid DNA .................................................................... 34 2.4.3 Constructs................................................................................................ 34 2.5 Yeast two-hybrid assay .................................................................................. 36 2.5.1 Constructs................................................................................................ 36 2.5.2 Yeast transformation................................................................................ 38 2.5.3 β-galactosidase test.................................................................................. 39 2.6 Biochemistry.................................................................................................. 40 2.6.1 GST-fusion protein.................................................................................. 40 2.6.2 in vitro transcription and translation......................................................... 41 2.6.3 GST pull-down assay............................................................................... 42 2.6.4 SDS-PAGE and Western blot analysis ...................................................... 43 2.7 General laboratory equipment ....................................................................... 44 3 Results............................................................................................................ 45 3.1 Maternal screen.............................................................................................. 45 3.1.1 Strategy ................................................................................................... 45 3.1.2 Results of the maternal screen.................................................................. 47 3.2 Zygotic screen................................................................................................ 49 3.2.1 Strategy ................................................................................................... 49 3.2.2 Results of the zygotic screen.................................................................... 51 3.3 Identification of the mutation of E326 ............................................................ 52 3.3.1 Complementation test .............................................................................. 53 3.3.2 Sequencing of mirandaE326 genomic DNA................................................ 53 3.4 Mislocalization of the mutant Miranda protein in mirandaE326 mutant neuroblasts and epithelial cells.................................................................................... 57 3.5 Comparison with other miranda alleles .......................................................... 61 3.5.1 Mislocalization of the mutant Miranda protein in mirandaE326 allele......... 61 3.5.2 Mislocalization of Prospero in mirandaE326 allele ..................................... 61 3.6 Genetic interaction between mirandaE326 and prospero ................................... 65 3.7 Neuronal cell fate determination..................................................................... 66 3.7.1 Cell fate determination in single mutants for miranda or prospero........... 66 3.7.2 Cell fate determination in mirandaE326 and prospero transheterozygotes... 67 3.8 Interactions between Miranda and its binding partners ................................... 70 3.8.1 Yeast two-hybrid assay............................................................................ 70 3.8.1.1 Miranda and Inscuteable ..................................................................... 70 3.8.1.2 Miranda and Myosin VI ...................................................................... 73 3.8.2 GST pull-down assay............................................................................... 74 4 Discussion ...................................................................................................... 76 4.1 Maternal screen.............................................................................................. 76 4.2 Zygotic screen................................................................................................ 76 4.3 E326 is allelic to mutations in miranda........................................................... 77 4.4 Effect of the mirandaE326 mutation on neural development.............................. 78 4.4.1 Subcellular localization of the cell fate determinant Prospero in neuroblasts 78 4.4.1.1 Association with Prospero and Staufen ............................................... 78 4.4.1.2 Dissociation from Prospero................................................................