In the name of God, the almighty A Study of Neuronal Precursors Using Retrovirus-Mediated Gene Transfer by Mohammad Hajihosseini A thesis submitted to the University of London in part fulfilment for the degree of Doctor of Philosophy (Ph.D) Laboratory of Developmental Neurobiology, National Institute for Medical Research, The Ridgeway, Mill Hill, London. May 1994 ProQuest Number: 10105736 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest 10105736 Published by ProQuest LLC(2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Abstract This study concerns an investigation of factors that may influence the behaviour and development of neuronal precursors derived from embryonic rat cerebral cortex. In this study, single retrovirally-labelled E16 or E14 cortical precursors were cultured amongst unlabelled cells, on monolayers of cortical astrocytes in the presence or absence of basic fibroblast growth factor (bPGF). Several important observation were made when the fate of such cells was analysed after seven days in culture. Most virally-labelled E16 and E14 cortical cells were found to produce clones that were composed of only one of the cell types found in the adult brain; namely neurones, oligodendrocytes, or astrocytes, showing that cortical precursor cells are specified in the phenotypic fate at the time of their isolation from the embryonic cortex. bFGF did not override this specification. However, bFGF was found to act as a survival factor and possibly a mitogen exclusively for neuronal precursor derived from either of the embryonic ages; significantly more and larger neuronal clones were found in the presence of this factor. To test whether bFGF's effects were mediated by cortical astrocytes, on which the embryonic cells were routinely grown, cultures were grown on substrates other than astrocytes in the presence or absence of bFGF. It was discovered that survival of cultures grown in the absence of cortical astrocytes was poor and that bFGF could enhance the survival of neuronal precursors in such cultures, thus arguing for a direct effect by bFGF. However, despite the poor survival of cultures, absence of cortical astrocytes resulted in an increase in the size of neuronal clones even when bFGF was absent This observation suggested that cortical astrocytes inhibit the proliferation of cortical neuronal precursors. In a second line of pursuit, this study investigated whether retroviral DNA can integrate into post-replication DNA of its host. This was addressed by analysing the distribution of viral genes amongst the progeny of single retrovirally-labelled NIH-3T3 cells. This analysis showed that retroviruses integrate almost exclusively into post-replication DNA of host cells as only half the progeny of single infected NIH-3T3 cells were found to inherit the viral genes, a finding that could be used to explain the high frequency of single-cell neuronal clones generated by retrovirally-labelled neural precursors. Results of this study suggested that single-cell neuronal clones arise when only one daughter of an asymmetrically-dividing neuronal precursor inherits the viral genes. 11 Dedication To my dearest parents I l l Acknowledgements First, I would like to thank my supervisor, Dr. Jack Price for his advice, guidance, support, encouragement, and the stimulating discussions throughout the past three and something years, not to forget the unfinished chess match on the train to Cardiff. My thanks also go to my colleagues, both old and new. In particular, I wish to thank Brenda Williams and Joanne Read for introducing me to cell and tissue culture, Linda McNaughton for her unreserved help, Liz Grove for her advice and long discussions about material and immaterial things, and Libert lavachev for carrying out the PCR on single-cell clones relating to data presented in chapter III of this work. It has also been a pleasure to know and work with the newer members of the laboratory, namely, Magdalena Gotz, Norberto Serpente, Beatrice Cousin, Kamala Maruthainer and Rhodri James. Thanks also to Jim Smith and Jeremy Green for their kind gift of Xenopus bFGF, Ivor Mason for providing in vitro translates of FGFs, Roger Morris and Dave Wilkinson for their valued comments on my mid-term report, Johnathan Stoye for his comments on the manuscript of my publication on retroviruses, Nick Goldmann for his help with statistical analysis of data, NIMR biological services for providing timed-pregnant rats, members of NIMR computing laboratory for their generous help with data retrieval and storage, members of NIMR photographic section for their excellent computer drawings and prompt processing of exposed films, members of NIMR Library for their excellent service in obtaining reprints and providing a room for the write up of this work, and the director of studies. Rod King and his secretary Chris Neate for their advice and support. I am also indebted to my external supervisor. Professor Rhona Mirsky, for her generous help and advice. Finally, I gratefully acknowledge the financial support of the Medical Research Council of Great Britain and Northern Ireland in enabling me to accomplish this work. IV Abbreviations AER Apical ectodermal ridge AGF Astroglial growth factor ATP Azidothymidine bFGF basic fibroblast growth factor BDNF Brain derived neurotrophic factor bp base pair CAM Cell adhesion molecule CFSF 5-carboxyfluorescein diacetate, succinidimyl ester cfu colony forming units (measure of viral titre) CNS Central nervous system CNTF Ciliary neurotrophic factor Coumarine 7- amino-4 methylcoumarin - 3 acetic acid DAB Diaminobenzedene DMFM Dulbecco's modified Fagle medium DMF Dimethylformamide DMSO Dimethylsulphoxide DNA Deoxyribonucleic acid DRG Dorsal root ganglia F Embryonic day (e.g. F 16) FDTA Fthylenediaminetetraacetic acid FGF Epidermal growth factor FCS Foetal calf serum GABA Gamma-amino-butyric acid GAP-43 Growth associated protein-43 GFAP Glial fibrillary acidic protein gP glycoprotein HFPFS N- [2-Hydroxyethyllpoperazine-N - [2-ethanesulfonic acid] HIV Human imunodeficiency virus HRP Horse radish peroxidase HSPG Heparan sulphate proteoglycans HSV-1 Herpes Simplex Virus type-1 IGF-I Insulin-like growth factor-I kDa Kilo Daltons KGF Keratinocyte growth factor LIF Leukemia inhibitory factor LRD Lysinated rhodamine dextrans LTR Long term repeat MAP-2 Microtubule associated protein-2 MAP-5 Microtubule associated protein-5 min Minutes MLV Murine leukemia virus MoMLV Moloney Murine leukemia virus NCAM Neural cell adhesion molecule NCS New bom calf serum NGF Nerve growth factor NIH National Institutes of Health NLS Nuclear localisation sequence NP-40 Nonident 40 NR Neural retina NT-3/4 Neurotrophic factors 3 and 4 PBS Phosphate buffered saline Pbs Primer binding site PCR Polymerase chain reaction PDGF Platelet derived growth factor PDL Poly-D-lysine pi Isoelectric point PLC Phospholipase C PNMT Phenylethanolamine-N-methyl transferase PNS Peripheral nervous system R Receptor RNA Ribonucleic acid rpm Rounds per minute RSV Rous Sarcoma Virus sec Seconds SV-40 Simian virus-40 SVZ Subventiicular zone TGF Thyroid growth factor TPA 12-0-tetradecanoyl phorbol-13-acetate VSV-G Vesicular somatitis virus-G VZ Ventricular zone X-gal 5-Bromo-4-chloro-3-indolyl p-D-galactop X-phos 5-Bromo-4-cholo-3-indolyl phosphate ZPA Zone of polarising activity VI Contents Abstract i Dedication ii Acknowledgements iii Abbreviations iv-v Table of contents vi-xi List of Figures and Tables xii Chapter I: General Introduction 1 1.1 Introduction 2 1.2. Appearance of ventricular cells within the neural tube and the ventricular zone 3 1.2.1. Cell cycle times and pattern of VZ cell division 3 1.3. Early hypotheses about generation of different cell types by Ventricular cells 6 1.3.1. Thymidine labelling as means of analysing the period of cytogenesis? 1.3.1.1. Technique and interpretation of findings 7 1.3.1.2. Thymidine labelling; earliest conclusions about generation of cell types by the ventricular zone cells 7 1.3.1.2.1. Period of neurogenesis in the mammalian CNS 8 1.3.1.2.2. Period of gliogenesis in the mammalian CNS 9 1.4. Study of cell lineage 10 1.4.1. Concepts and terminologies 10 1.4.2. Using cell lineage studies to address other developmental questions 11 1.4.3. Possible ways of studying cell lineage/ cell fate 12 1.4.3.1. Direct strategies 12 1.4.3.1.1. Visualisation of cell development under normaski optics 12 1.4.3.1.2 Analysis of the fate of isolated cells 13 1.4.3.2. Indirect strategies 13 1.4.3.2.1. Use of chimeric animals 13 1.4.3.2.2. Use of lineage labels 14 1.4.3.2.3. Use of vital dyes 14 1.4.3.2.4. Use of genetic labels 15 1.4.3.2.4.1. DNA microinjection 15 1.4.3.2.4.2. Retrovirus mediated gene transfer 15 1.4.3.2.4.2.1. Methods of detecting retroviral transgenes or their product 16 1.4.3.2.4.2.2. Disadvantages of the retrovirus method 16 1.4.4. Studies of cell lineage in the vertebrate nervous system 17 v u 1.4.4.1. Neural crest cell lineage studies 17 1.4.4.2. Lineage analysis in the rodent and amphibian retina 18 1.4.4.3. Chick optic tectum and spinal cord 19 1.4.4.3. Rat striatum 22 1.4.4.5.