RADAR Research Archive and Digital Asset Repository Copyright © and Moral Rights for this thesis are retained by the author and/or other copyright owners. A copy can be downloaded for personal non-commercial research or study, without prior permission or charge. This thesis cannot be reproduced or quoted extensively from without first obtaining permission in writing from the copyright holder(s). The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the copyright holders. When referring to this work, the full bibliographic details must be given as follows: North, A. (2011) A genetic screen for genes involved in dendrite morphogenesis of central neurons in Drosophila melanogaster. PhD Thesis. Oxford Brookes University. WWW.BROOKES.AC.UK/GO/RADAR A genetic screen for genes involved in dendrite morphogenesis of central neurons in Drosophila melanogaster Annemarie North B.Sc (Hons), M.Sc Awarded by Oxford Brookes University, Oxford in collaboration with University of Cambridge, Cambridge August 2011 A thesis submitted in partial fulfilment of the requirements of the award of Doctor of Philosophy i Declaration I hereby declare that this thesis is my own work and effort and that it has not been submitted anywhere for any award. Where other sources of information have been used, they have been acknowledged. Signature: ………………………………………. Date: ……………………………………………. i For Hazel and Rachel The true delight is in the finding out rather that the knowing Isaac Asimov Even if you fall on your face, you’re still moving forward Victor Kiam ii Abstract In order to produce a functional nervous system, it is essential that neurons project synaptic terminals into particular regions of the developing nervous system, so as to make connections with appropriate pre- and postsynaptic partners. While axon targeting has been studied extensively, much less is known about how the postsynaptic dendrites grow and branch. To study dendrite morphogenesis, a mosaic loss of function screen was developed and carried out in Drosophila melanogaster for chromosomal regions required for motor neuron dendrite development. Specifically, the Mosaic Analysis with a Repressible Cell Marker (MARCM) method was modified so that individual motor neurons can be made homozygous for a defined genomic aberration in an otherwise wild-type (heterozygous) background and visualised during early larval stages. In addition, 85 defined chromosomal deficiencies were recombined individually onto FRT-carrying chromosomes to be screened for genes involved in dendrite morphogenesis. These recombinant FRT-deficiency chromosomes provided a coverage of ~64.3% of chromosome 2 (3864 annotated genes). After analysis of ~35% of chromosome 2 (2092 annotated genes), 814 central nervous systems and 414 neurons, five genomic regions were identified that had a dendritic phenotype when absent. One of these regions, 2R:23D2;23E, uncovered by FRT40A Df(2L)S2590, was studied in more detail. Overlapping deficiencies were screened to define more precisely the region of chromosome 2 where the loss of gene(s) caused a dendritic phenotype in motor neurons. Of the seven candidate genes identified in this region, only one was shown to have expression in the embryonic central nervous system – the as yet uncharacterised gene CG34393. Based on sequence comparisons, CG34393 is predicted to encode a putative Ras guanyl exchange factor. Expression data from putative homologues in other species and Drosophila genes that are expressed in a similar developmental time course suggest that CG34393 may be involved in synapse development. iii Contents Declaration ............................................................................................................................... i Abstract .................................................................................................................................. iii Figures .................................................................................................................................. vii Tables ..................................................................................................................................... ix Chapter 1 Introduction ........................................................................................................... 1 Features of dendrites ............................................................................................................ 4 Dendritic growth .................................................................................................................... 5 The molecular basis of dendrite growth ............................................................................... 9 Parallels with axon guidance ............................................................................................ 9 Dendritic guidance ......................................................................................................... 10 Intrinsic factors required for dendrite morphogenesis .................................................... 11 Partner recognition and synapse formation ................................................................... 13 Dendrites and disease ........................................................................................................ 16 Genetic screens: Drosophila melanogaster as a genetic model system ........................... 17 Drosophila screens for genes involved in nervous system development .......................... 19 A screen for genes involved in dendrite morphogenesis ................................................... 20 Chapter 2 Method development ......................................................................................... 22 Methods for visualising motor neurons in Drosophila ........................................................ 23 The GAL4/UAS system and visualisation of motor neuron dendrites ............................ 23 Development of a FLP-out system to target individual identified motor neurons .......... 24 Mosaic analysis with a repressible cell marker .............................................................. 26 The MARCM system is modified to target a subset of motor neurons .......................... 28 Assessment of different versions of modified MARCM systems ........................................ 28 Experiments using mutations known to affect dendrite development confirm the function of the modified MARCM system ............................................................................................. 40 Variations of the ‘ftz loop’ MARCM stock ........................................................................... 42 Neuroblast GAL4 drivers ................................................................................................ 42 Red fluorescence protein ............................................................................................... 43 iv Alternative GAL80 expression constructs ...................................................................... 46 Concluding remarks ............................................................................................................ 47 Chapter 3 Loss of function screen ..................................................................................... 48 Generation of a fly stock collection for FLP/FRT mediated mitotic recombination with defined chromosome 2 deficiencies ................................................................................... 49 Coverage of chromosome 2 by recombinant FRT-deficiency stocks ................................. 51 Screening chromosome 2 using the ‘ftz loop’ MARCM method and FRT-deficiency recombinants ...................................................................................................................... 63 Effects of lethality in neurons homozygous for recombinant FRT-deficiencies .................. 64 Regions of chromosome 2 that cause a motor neuron dendrite phenotype when homozygous ....................................................................................................................... 66 Df(2L)al ........................................................................................................................... 68 Df(2L)TE35BC-24 .......................................................................................................... 71 Df(2R)Np5 ...................................................................................................................... 75 Df(2R)Px2 ....................................................................................................................... 78 Bloomington stock 4101 ................................................................................................. 81 Df(2L)S2590 ................................................................................................................... 83 Chapter 4 Df(2L)S2590 ......................................................................................................... 84 Analysis of deletions overlapping with Df(2L)S2590 refines the genomic area containing candidate genes for dendritic development ........................................................................ 87 MARCM analysis of dendritic phenotypes deficiencies that overlap with Df(2L)S2590 ..... 89 Df(2L)BSC28 .................................................................................................................. 89 Df(2L)BSC162 ...............................................................................................................