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RoCes ofReetin and (Disa6(edl in NeuraC<Deve(opment in Submitted to the University of London in 2007 in partial fulfilment o f the requirements for the award of PhD M 6 i UCL University College London UMI Number: U592718 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. Dissertation Publishing UMI U592718 Published by ProQuest LLC 2013. Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Acknowledgments I wouldlike to thanfjandoffer my deepest gratitude to (ProfessorSteve W/ilsonforgiving me this great opportunity to work-in fiis (aS at VCL and fo r his hefp and advice. I am very grateful to P>r Marina Mione fo r her “matemaCsupervision” and a d times she spent discussing results and ideas. I would like also to thanks Dr Marina %aspimah fo r her precious advice on the anatomy o f the zeSrafish 6rainfor thefirst Chapter of results. I am very gratefud to Parham delice and Alessandro Cjujfanti from Ifom, dire Institute o f “Molecular Oncology fo r their help in the 6ioinformatics part o f the Chapter 4. I would particularly like to thankjDr Monica dnsinifor her support and advice, without her help I wouldperhaps not have started a PhD at VCL. I am very grateful to Professor John Scholes, Claire, Pom, Jacque and Sdvia fo r suggestions and proof reading my thesis and dlorencia fo r the transplant technique and Cjaia, P)iz and a d the fantastic people o f the zeSrafish group at VCL for support. I would like to thankjDr Corinne Houart fo r ad the helpful discussion, especially at the Beginning o f my project. I wouldlike also to thanffadthe nice people at the Haematology Department at Queen Mary’s and in particular Professor dinSarr Cotter, P>r Vlnka Johansson, D r Pghecca Auer and David (Badard fo r their advice especiady on my dnglish writing. I must thankjny Beautifulfamily, my parents Hedo and (jrazia, my sisters dgle (poor her! She was in London during my writing), and Marilena, my Srother Paolo, Cinzia and Cd fo r their support and love. I cannot forget to than !^ my 6oyfriend Stefano always close to me with his mind and heart .... 6etween the phone and the shy, tra il telefono e d cielo (“6uonanottefioredino”De Qregori) I am very grateful to dahrizia my flatmate, fo r the scientific discussions (ad the Italians in London are 6iologists!H) Sut overall fo r herfriendship during 6eautifuland hard moments here. I would like to thanfjLuca fo r his help in each situation; I start this journey with him Sut things, most o f the time, do not end as we expect. dinally, I would like to thank, Mary Pauls, my singing teacher, the singing group, Mozart and Handel fo r ad the wonderful music that helped and will help me during my journey. Abstract Abstract Roles of Reelin and Disabledl in Neural Development in Zebrafish Reelin and Disabledl (Dabl) are known to be involved in neuronal migration in vertebrates and in particular in radial migration. Reelin is a secreted protein that can bind several receptors and the signal is intracellular transduced by Dabl. The final targets of the Reelin pathway are molecules that regulate cytoskeletal remodeling. In my project I studied the expression pattern of reelin and dabl in several areas of the zebrafish CNS. The differences found in the telencephalic expression of reelin and dabl between teleosts and tetrapods are likely to arise from the process of eversion, which is specific for the teleost telencephalon. On the contrary, all the other regions of the CNS present a conservative pattern of expression, in comparable structures among vertebrates, with the only exception of the olfactory bulb that does not express reelin in zebrafish. As the dabl gene shows a high degree of complexity in mammals and mice, I studied the genomic organization of the dabl gene in zebrafish and found a similarly complex organization. In order to study the functions of the Reelin pathway in neuronal migration in zebrafish, I performed loss of function experiments with morpholino antisense oligonucleotides. I found defects at level of several 1 Abstract neuronal groups including the facial branchiomotor nucleus, the Mauthner neurons and neural crests. To investigate whether the role of dabl in the migration of these neuronal groups may be that of conveying a Reelin signal, I attempt to rescue these phenotypes by overexpressing full length dabl or truncated forms of the protein that lack the Reelin or the CDK5 responsive domains. It appears that the presence of the tyrosine domain, but not of the CDK5 phosphorylation domain, is necessary for partial rescuing of most of these phenotypes. I also found that some populations of neurons that express dabl show defects in neurites growth in morpholino injected embryos. 2 Contents Contents A b stra ct ..............................................................................................................................1 Roles of Reelin and Disabledl in Neural Development in Zebrafish 1 Contents ..............................................................................................................................3 List of Tables and Figures ..............................................................................................9 Abbreviations ................................................................................................................. 11 CHAPTER 1..................................................................................................................... 14 1.1. General Introduction ........................................................................................... 14 1.2. Factors and signals involved in neuron migration.................................15 1.2.1. Molecules involved in cytoskeletal changes during migration.... 16 1.2.2. Chemoattractant and chemorepellent molecules are involved in neuron migration .................................................................................................. 20 1.2.3. Neurite growth and its relation with neuronal migration ............. 21 1.3. General modes of migrations........................................................................22 1.3.1 Radial m ig ratio n ......................................................................................... 22 1.3.2. M ultipolar m igration .................................................................................24 1.3.2. Tangential migration .................................................................................25 1.3.3. Chain migration ......................................................................................... 27 1.3. Radial and tangential migrations generate complexity of connections and circuitry in the developing brain ......................................28 1.4. The Reelin signalling pathway and neuronal migration..................... 30 1.4.1. R eelin .............................................................................................................30 1.4.2. Reelin receptors .......................................................................................... 34 1.4.2.1 VLDLR and A poE R 2...............................................................................34 1.4.2.2. Integrins receptors ..................................................................................36