FUNCTIONAL ANALYSIS OF DROSOPHILA NEUROTROPHIN AND TOLL RECEPTOR FAMILIES IN THE DEVELOPMENT AND REPAIR OF THE LARVAL CENTRAL NERVOUS SYSTEM by MEI ANN LIM A thesis submitted to the University of Birmingham for the degree of DOCTOR OF PHILOSOPHY School of Biosciences College of Life and Environmental Sciences University of Birmingham January 2015 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. Abstract Drosophila neurotrophins (DNTs) - Spätzle (Spz), DNT1 and DNT2 - and 3 members of the Toll protein family – Toll, Toll-6 and Toll-7, of which Toll is Spz’s receptor – have been shown to promote neuronal survival and motoneuron targeting in embryos. Yet, it remains to be understood (1) whether the DNTs influence cell number and central nervous system (CNS) development after embryonic stages to result in the behaving larva, and in turn (2) whether these events influence larval CNS repair after injury. Here, I investigated the functions of DNTs and Tolls in the formation and repair of the larval CNS, focusing mostly on Spz. I used GAL4 reporters, MiMIC-GFP protein traps and antibodies to the DNTs and Tolls to describe their larval CNS distributions. Interestingly, Spz was restricted to the mechanosensory domain in the ventral nerve cord (VNC). I generated a new loss of function allele for spz and showed that spz mutations affect glial numbers in the larval abdominal VNC. Using stabbing injury in the larval CNS, I showed that loss of spz antagonised wound repair. To conclude, my data show the involvement of Spz in larval CNS development, and that Spz has a prominent role in CNS repair. Dedication To my A-Team member Henry, Sometimes life can be tough. Sometimes the ride can be scary. Sometimes people can be grumps. But I will always love you. Hetty. Acknowledgements Over the past four years I have received support of various kinds from a good number of individuals, thus making my PhD journey a thoughtful and rewarding one. My PhD supervisor, Dr. Alicia Hidalgo, has a knack for being correct. This means that her insight and guidance have been invaluable to my growth as a scientist in the working research environment. To my steadfast supervisor who kept her encouraging ways, thank you for doing your darnedest to keep me on track, and also for providing a conducive writing environment in Room 629. I would like to thank members of the Hidalgo lab, alumni and present. Thanks to Graham, Samaher, Martin, Jill, István, Juliet, Caitríona, Chris, Niki, Maria, Marta, Arin, Neale, Suzana and Gui Yi for discussions of a range of sorts and good company. Thanks Kentaro for straightening me out on matters of the Drosophila larvae and dissections. Simon, for the outreach adventures, ta! Thanks Karthik. Wiesel called Hubel his ‘scientific brother’1. Together, they had carried out experiments every Tuesday and Thursday. Knowing this, I sometimes imagined us as scientific siblings, taking turns harvesting Drosophila virgins as well as acquiring microscopy data on weekends. Our collaboration period was a very fun time. Thanks also to the Ministry of Education of the Brunei Government for my PhD scholarship. Finally, I must acknowledge my father. Dad, you did – and still are – doing your utmost as a father of 6 to support my aspirations and dreams. With love, thank you. 1 Source: SANDRONE, S. 2014. Q&A: Torsten Wiesel. Nature, 514, S11-S12. Table of Contents 1 Introduction .............................................................................................................. 19 1.1 Neurotrophism in Drosophila........................................................................ 20 1.1.1 Definition of Neurotrophic Factors and Neurotrophins ...................... 20 1.1.2 Neurotrophic Factors in Drosophila .................................................. 21 1.1.2.1 Neurotrophins in Drosophila ............................................... 21 1.1.2.2 MANF and CDNF in Drosophila ........................................ 23 1.1.2.3 Netrins in Drosophila .......................................................... 24 1.1.2.4 GDNF Family Signalling in Drosophila .............................. 24 1.1.3 Gliatrophic Factors in Drosophila ..................................................... 25 1.1.4 Review of the Identification of DNTs in the Spätzle Protein Family .. 26 1.1.5 DNT Signalling ................................................................................. 30 1.1.5.1 Molecular Mechanism of Spz Activation ............................. 30 1.1.5.2 Spz in Dorsal-Ventral Patterning and Immunity .................. 31 1.1.5.3 Similarities between Spz, Coagulogen and BDNF ............... 35 1.1.5.4 Mutations in dnt Genes ........................................................ 38 1.1.5.4.1 Point Mutations in spz ........................................... 38 1.1.5.4.2 Mutations in dnt1 and dnt2 Genes ......................... 41 1.1.5.5 Toll Receptors of DNTs ...................................................... 41 1.2 Drosophila as a Model Organism and Technical Approach ........................... 42 1.2.1 Larval CNS Anatomy ........................................................................ 42 1.2.1.1 Mutations Affecting VNC Length ....................................... 44 1.2.1.2 Mutations Affecting Cell Numbers in the Larval VNC ........ 46 1.2.2 Injury in the Larval CNS ................................................................... 47 1.3 Rationale of the Project ................................................................................. 50 1.4 Aims of the Project ....................................................................................... 51 2 Materials and Methods ............................................................................................. 53 2.1 Genetics ........................................................................................................ 53 2.1.1 Housing and Handling Drosophila .................................................... 53 2.1.2 Standard Genetic Mating Protocols.................................................... 60 2.1.3 Genetic Protocol for Cleaning the spz2 Allele of ca1 Mutation ........... 63 2.1.4 Genetic Protocol for Generating dnt2 and spz Loss of Function Alleles . .............................................................................................. 70 2.1.5 Survival Index Assay ......................................................................... 74 2.1.6 Visualising RP2 Neurons ................................................................... 75 2.2 Molecular Biology ........................................................................................ 77 2.2.1 Genomic DNA Extraction ................................................................. 77 2.2.2 Conventional PCR ............................................................................. 77 2.2.3 Inverse PCR ...................................................................................... 79 2.3 Immunohistochemistry.................................................................................. 83 2.4 Microscopy ................................................................................................... 86 2.4.1 Image Acquisition ............................................................................. 86 2.4.2 Time-Lapse Recordings of Stabbed Samples ..................................... 87 2.4.3 Image Processing and Analysis.......................................................... 88 2.5 Phenotypic Analysis...................................................................................... 88 2.5.1 Cuticle Preparations of Unhatched Embryos ...................................... 88 2.5.2 Measurements of CNS Size ............................................................... 89 2.5.3 Sholl Analysis of RP2 Dendrites ....................................................... 90 2.5.4 DeadEasy for Automatic Counting of Cells ....................................... 90 2.5.5 Stabbing Injury to the Larval VNC .................................................... 95 2.5.6 Measurement of Neuropile Wound Size ............................................ 97 2.6 Statistical Methods ........................................................................................ 97 3 Expression and Distribution of DNTs, Toll, Toll6 and Toll7 ................................... 100 3.1 Introduction ................................................................................................ 100 3.1.1 Known Expression Patterns of DNTs, Toll, Toll6 and Toll7 in Larval CNS ............................................................................................ 100 3.1.2 GAL4 Reporters for dnt Genes ........................................................ 101 3.1.3 Mi{MIC}-EGFP Protein Traps for spz and Toll6 .............................. 102 3.1.4 Antibodies for DNTs, Toll and Toll7 ............................................... 102 3.1.5 Specific Aims .................................................................................