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Identification and Functional Characterization of Unc5a During Neocortical Regionalization

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Identification and Functional Characterization of Unc5a During Neocortical Regionalization Identification and Functional Characterization of unc5A during Neocortical Regionalization Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultäten der Georg-August-Universität zu Göttingen vorgelegt von Ahmet Ucar aus Konya Göttingen, 2007 D 7 Referent : Professor Dr. P. Gruss Korreferent : Privatdozent Dr. S. Hoyer-Fender Tag der mündlichen Prüfung : 4 Mai 2007 Diese Arbeit wurde am Max-Planck-Institut für biophysikalische Chemie, Karl-Friedrich-Bonhoefer-Institut Abteilung: Molekulare Zellbiologie Direktor: Prof. Dr. Peter Gruss durchgeführt. Table of Contents Table of Contents Table of Contents .................................................................................................................. 1 ABSTRACT.......................................................................................................................... 4 ZUSAMMENFASSUNG ...................................................................................................... 6 INTRODUCTION................................................................................................................. 9 Evolutionary view on brain and cerebral cortex ................................................................. 9 Functional and morphological structures of cerebral neocortex ........................................ 13 Cellular composition of cerebral neocortex .................................................................. 13 Functional and morphological features of neocortical regions ...................................... 15 Olfactory cortices..................................................................................................... 15 Motor cortices.......................................................................................................... 16 Somatosensory cortices............................................................................................ 17 Auditory cortices...................................................................................................... 18 Visual cortices ......................................................................................................... 19 Developmental aspects on functionality ....................................................................... 20 Development of cerebral neocortex.................................................................................. 20 Regionalization of cerebral neocortex .......................................................................... 20 Lamination of cortical plate in cerebral neocortex........................................................ 22 Axon guidance systems in cerebral neocortex development ............................................. 24 Molecular mechanisms of ‘Axon Guidance’................................................................. 25 Different strategies of ‘Axon Guidance’ in neural systems ........................................... 26 ‘Axon Guidance’ ligands and receptors........................................................................ 27 SLIT-ROBO system................................................................................................. 27 SEMAPHORIN-NEUROPHILIN / PLEXIN system................................................ 28 EPHRIN-EPH system .............................................................................................. 29 NETRIN system....................................................................................................... 29 Morphogens............................................................................................................. 29 Hormones ................................................................................................................ 30 ‘Axon Guidance’ mechanisms in the development of neocortex................................... 30 Callosal axons.......................................................................................................... 31 Thalamocortical axons ............................................................................................. 31 Cortico-cortical axons .............................................................................................. 33 Netrin-dependent axon guidance systems......................................................................... 34 Netrin ligands in development of the CNS ................................................................... 34 DCC family of receptors in development of CNS......................................................... 35 UNC5 family of receptors in development of CNS....................................................... 37 Aim of this study ............................................................................................................. 38 1 Table of Contents RESULTS ........................................................................................................................... 41 Determination of predominantly expressed genes in different cortical regions of E14.5 mouse embryos................................................................................................................ 41 In situ hybridization analysis of selected genes from the microarray data......................... 44 In silico identification of gene and protein structures ....................................................... 48 Identification of gene-structure of unc5A by RACE and RT-PCR Analysis ..................... 51 Generation of knockout constructs and ES-cell screens.................................................... 54 Generation of chimaeras and screening for germline transmissions .................................. 61 General phenotypic investigations of unc5A knockout mice ............................................ 62 Generation of ‘Conditional Overexpression’ construct and screening for founder animals 68 Creation of double transgenics with Cre-lines .................................................................. 71 Overexpression of UNC5A-Δexon6 in developing neuroretina via ECG-Cre ................... 71 Overexpression of unc5A-Δ exon6 in Neocortex and Hippocampus via Nex-Cre............. 78 DISCUSSION ..................................................................................................................... 85 Microarray analysis of E14.5 mouse cerebral cortices determined the sets of genes showing region-specific expressions among different cortical regions............................................ 87 Expression analyses and in silico identification of candidate genes for functional studies. 89 Identification of unc5A gene structure and its spliced variants ......................................... 92 Generation of unc5A loss-of-function mutants................................................................. 93 Analyses of unc5A loss-of-function phenotypes............................................................... 97 Analysis of unc5A gain-of-function mutants.................................................................. 100 Ectopic overexpression of unc5A-Δexon6 in the embryonic distal neuroretina causes blindness in a background-dependent manner ............................................................ 102 Ectopic overexpression of unc5A-Δexon6 in neocortex and hippocampus.................. 104 MATERIALS AND METHODS ....................................................................................... 106 Organisms ..................................................................................................................... 106 Mouse..................................................................................................................... 106 Cell-Line ................................................................................................................ 106 Bacteria .................................................................................................................. 107 Materials........................................................................................................................ 107 General laboratory equipments ............................................................................... 107 PCR primers ........................................................................................................... 107 Chemicals and enzymes.......................................................................................... 107 Standard solutions .................................................................................................. 108 Basic molecular biology techniques ............................................................................... 108 In silico gene-prediction studies and Bioinformatics ...................................................... 109 In situ hybridization (ISH) analyses ............................................................................... 109 Synthesis and purification of dig-labeled RNA probes ............................................ 109 Preparation of embryonic mouse brain sections ...................................................... 111 2 Table of Contents Non-radioactive RNA-in situ hybridization............................................................. 111 Solutions used in ISH ............................................................................................. 112 RACE analyses for AU-18 (unc5A)............................................................................... 113 Total-RNA isolation ..............................................................................................
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