Investigating the mechanisms directing oligodendrocyte precursor cell (OPC) development in the zebrafish hindbrain Thesis submitted to the University of Sheffield for the degree of Doctor of Philosophy by Penelope Jane Boyd Department of Neuroscience, University of Sheffield Institute of Molecular and Cell Biology, A*STAR, Singapore October 2014 i ii Acknowledgements I would firstly like to thank my supervisors for the opportunity to work on this project and for their support these last four years. Thanks to Dr Jon Wood for his encouragement when the project wasn’t going well and for his guidance during writing this thesis. Thanks to Prof Sudipto Roy for allowing me to join his group at the IMCB in Singapore, for teaching me about the realities of science and for the freedom to explore the project. Thanks to Dr Vincent Cunliffe for his advisory support during my project. I would like to show my gratitude to the fantastic members of the SR group for making settling into a new group and country very easy, for helping me to learn techniques and for being patient with me. A particular thanks to Dr Semil Choksi, not only for his friendship but for his endless science wisdom. He helped me so much during my time in SR lab and I cannot thank him enough. To my wonderful friends in Singapore, we were more like a family and I’m sure we will remain friends for a long time. To the lovely Harriet, you made my first year in Singapore so much fun. Thanks for looking after me, I couldn’t have done it without you! Charlie, thank you for the much needed coffee breaks and for being there during my last few weeks to help. To Pervin, Dave, Mark, AD and Ajay you guys were fantastic fun to be around. I will forever think fondly of my time there. To the BMS babes in Sheffield, thanks for girls nights and the laughs. To Aimee, my beautiful friend, thank you for the brilliant times we had when we lived together. For the emergency G ’n ’T sessions and your words of encouragement during those final few months and for the email updates whilst I was in Singapore. Thank you to Tarang, for our trips across South East Asia and for your love and reassurance. You put up with my moaning, irrational panics and always managed to make me feel better. To my family who I cherish and couldn’t imagine doing this without them. To my sister, for her friendship. To my beloved spaniels for their slobber therapy sessions. Finally to my Mum and Dad, two of the kindest people with the most wonderful hearts. Their unconditional love and support helped me to have faith in myself especially during my PhD. iii Statement of Contribution I state that all the work presented in this thesis is my own. The in vivo drug screen (Chapter 6: A preliminary in vivo drug screen to identify compounds modulating OPC development) was performed with the Screening laboratory in the Department of Biomedical Science, University of Sheffield. Training of the drug screening protocol was provided by Dr Sarah Baxendale and Miss Celia (CJ) Holdsworth assisted with drug preparation, embryo preparation and automated in situ hybridisation. iv Abstract Oligodendrocytes are the myelinating cells of the central nervous system (CNS) and are implicated in the pathobiology of many CNS diseases including multiple sclerosis and schizophrenia. Oligodendrocyte precursor cells (OPCs) are capable of migrating long distances, but the mechanisms governing the migration of OPCs to their axonal destinations are not fully understood. We sought to understand the extrinsic and intrinsic factors that contribute to the migration of a subset of OPCs in the zebrafish hindbrain. We utilized the zebrafish transgenic tg(olig2:gfp) line to mark OPCs and characterise their normal migration patterns. These cells normally first migrate in a ventral direction, after which they migrate away from the midline dorsolaterally to populate the hindbrain. However, how these cells initiate migration in a ventral direction is not known. We have found that removing Hedgehog signalling at the onset of their migratory phase results in a failure of OPCs to migrate in their normal ventral fashion, while increasing Hedgehog signalling results in hypermigration of these cells. This suggests that Hedgehog might not just be important for OPC fate specification, via induction of transcription factors such as olig2, but may also be actively involved in the development of these cells post-specification. We propose that Sonic Hedgehog acts as a chemoattractant to drive the initial ventral migration of hindbrain OPCs and furthermore provide evidence that it may function at least in part by inducing disc1, a schizophrenia risk factor gene, which has known roles in neural crest and neuronal migration. With several studies supporting a role of DISC1 in neuronal migration it has been hypothesised that DISC1 could also play a role in the process of myelination. White matter abnormalities are consistently reported in schizophrenia patients, and it is hypothesised that OPC migratory defects may cause these documented white matter abnormalities due to early genetic dysfunction. This thesis has provided evidence for a link between a well characterised schizophrenia risk factor gene, early OPC migration events and a fundamental signalling pathway. v List of abbreviations AMP Ampicillin bHLH basic helix loop helix BMP Bone morphogenic protein CNC Cranial Neural Crest CNS central nervous system DISC1 Disrupted in Schizophrenia-1 DPF Days post fertilisation DSB Double strand break DTI diffusor tensor imaging ENU N-ethyl-N-nitrosourea EtOH Ethanol FGF Fibroblast growth factor FEZ1 Fasciculation And Elongation Protein Zeta 1 GFP Green fluorescent protein GWAS Genome wide association studies HPF hours post fertilisation HR Homologous recombination INDELs Insertions and deletions KAN Kanamycin LIS1 Lissencephaly-1 Protein MO Morpholino MS Multiple sclerosis vi MRI magnetic resonance imaging MeOH Methanol MBP Myelin basic protein NDEL1 NudE Neurodevelopment protein 1 like 1 NRG1 Neuregulin-1 NHEJ Non-homologous end joining OLIG2 Oligodendrocyte lineage transcription factor OPC Oligodendrocyte precursor cell OL Oligodendrocyte PCR Polymerase chain reaction PNS Peripheral Nervous System pMN motor neuron RT room temperature SHH Sonic Hedgehog SZ schizophrenia SEM standard error of the mean TALEN transcription activator like endonucleases UTR Untranslated region Y2H Yeast two hybrid vii Table of Contents Acknowledgements…....………………………………………………………………………………….iii Statement of Contribution …….………………………………………………………………………iv Abstract…….…………………………………………………………………………………………………….v List of Abbreviations……………………………………………………………………………………….vi Introduction ............................................................................................ 1 1.1 Oligodendrocytes .......................................................................................... 1 Function in the adult brain ..................................................................... 1 White matter abnormalities in brain disorders ..................................... 1 1.2 Schizophrenia ................................................................................................ 4 The symptoms of schizophrenia ............................................................ 4 High heritability of Schizophrenia .......................................................... 4 The neurodevelopmental hypothesis of schizophrenia ........................ 5 Risk factor genes and genetic overlap between psychiatric disorders .. 6 Risk factor genes and white matter abnormalities ................................ 7 1.3 Disrupted in schizophrenia-1 (DISC1) ............................................................ 7 Discovery of DISC1 ................................................................................. 7 DISC1 in the brain ................................................................................... 8 Interacting partners implicated in neuronal migration ....................... 11 Primary cilia and DISC1 ........................................................................ 13 Evidence for a role of DISC1 in OL development ................................. 14 1.4 Oligodendrocyte development ................................................................... 16 Specification of oligodendrocyte precursor cells ................................. 16 Early gene expression in OPC specification ......................................... 16 viii Proliferation of OPCs ............................................................................ 17 Migration of OPCs to their axonal targets ........................................... 17 Transition from OPC state to differentiated oligodendrocytes ........... 18 Myelination .......................................................................................... 20 1.5 The zebrafish (Danio rerio) as a model system of myelination ................... 20 Advantages of the zebrafish................................................................. 20 Myelination studies in the zebrafish .................................................... 21 Using the zebrafish as a model system for schizophrenia ................... 23 Zebrafish disc1...................................................................................... 23 Zebrafish disc1 in brain development .................................................. 24 OPC development in the zebrafish hindbrain ...................................... 30 1.6 Hypothesis ..................................................................................................