Function of MCPH1 in Neurogenesis Dissertation zur Erlangung des mathematisch-naturwissenschaftlichen Doktorgrades "Doctor rerum naturalium" der Georg-August-Universität Göttingen vorgelegt von Ralph Gruber aus Illertissen Göttingen 2011 2 Mitglieder des Betreuungsausschusses: Referent: PD Dr. Wilfried Kramer Koreferent: Prof. Dr. Zhao-Qi Wang Tag der mündlichen Prüfung: 11. April 2011 Table of Contents 3 Table of Contents Table of Contents ......................................................................................................................3 Abstract ..................................................................................................................................... 8 Zusammenfassung ...................................................................................................................9 1. Introduction .........................................................................................................................10 1.1 Development of the cerebral cortex ................................................................................10 1.1.1 Early events in neural tube development ..................................................................10 1.1.2 Morphology of the developing forebrain ...................................................................12 1.1.3 Neuronal stem and progenitor cells of the dorsal telencephalon ..............................14 1.1.4 Corticogenesis ..........................................................................................................16 1.1.4.1 Cortical neurogenesis ........................................................................................16 1.1.4.2 Gliogenic phase of cortical development ...........................................................17 1.1.5 Mechanisms regulating self-renewal and differentiation of neuronal progenitor cells ........................................................................................................................................... 18 1.1.5.1 Transition from NEs to RGCs .............................................................................18 1.1.5.2 Symmetric and asymmetric cell division of neuronal progenitor cells ................ 19 1.1.5.3 Control of mitotic spindle orientation ..................................................................22 1.2 Regulation of cell division ................................................................................................24 1.2.1 Cell cycle control .......................................................................................................24 1.2.2 Regulation of mitotic entry ........................................................................................25 1.2.3 Mitotic progression ....................................................................................................26 1.2.4 Role of centrosomes in the cell division ....................................................................27 1.2.4.1 Centrosomal organization ..................................................................................27 1.2.4.2 The centrosome cycle ........................................................................................28 1.3 Primary microcephaly ......................................................................................................30 1.3.1 Clinical features of human primary microcephaly .....................................................30 1.3.2 MCPH1/microcephalin/BRIT1 ...................................................................................32 1.3.3 MCPH2/WDR62 ........................................................................................................34 Table of Contents 4 1.3.4 MCPH3/CDK5RAP2 .................................................................................................35 1.3.5 MCPH4/CEP152 .......................................................................................................36 1.3.6 MCPH5/ASPM ..........................................................................................................37 1.3.7 MCPH6/CENPJ ........................................................................................................38 1.3.8 MCPH7/STIL .............................................................................................................39 1.3.9 Common role of MCPH proteins in cortical development .........................................40 1.4 Aim of the study ...............................................................................................................41 2. Material and Methods .........................................................................................................42 2.1 Material ............................................................................................................................42 2.1.1 Cell culture media and solutions ...............................................................................42 2.1.2 Antibodies .................................................................................................................43 2.1.3 Animals .....................................................................................................................44 2.1.4 Microscopy ................................................................................................................45 2.2 Molecular biological methods ..........................................................................................45 2.2.1 DNA extraction ..........................................................................................................45 2.2.2 Polymerase chain reaction (PCR) ............................................................................46 2.2.3 Construction of short hairpin RNA (shRNA)-vectors .................................................47 2.3 Mouse embryonic fibroblast (MEF) culture and methods ................................................48 2.3.1 Isolation and culture conditions of MEFs ..................................................................48 2.3.2 Immortalization of MEFs ...........................................................................................48 2.3.3 Transfection of immortalized MEFs ..........................................................................49 2.3.4 Immunocytochemistry of transfected MEFs ..............................................................49 2.4 Mouse embryonic neuronal progenitor cell culture and methods ....................................50 2.4.1 Isolation and culture conditions of neuronal progenitor cells ....................................50 2.4.2 Primary neurosphere formation ................................................................................51 2.4.3 Determination of self-renewal capacity of neuronal progenitor cells ........................ 52 2.4.4 Immunocytochemistry of neuronal progenitor cells ..................................................52 2.4.5 Metaphase chromosome preparation .......................................................................53 2.4.6 Pair cell assay ...........................................................................................................54 2.5 Protein analysis ...............................................................................................................55 2.5.1 Whole cell protein isolation .......................................................................................55 Table of Contents 5 2.5.2 Electrophoretic protein separation ............................................................................55 2.5.3 Immunoblot analysis .................................................................................................56 2.6 Histological techniques ....................................................................................................57 2.6.1 Isolation and fixation of embryonic and postnatal mouse brains ..............................57 2.6.2 Preparation of paraffin embedded mouse brain sections .........................................57 2.6.3 Preparation of cryosections of mouse brains ............................................................58 2.6.4 Hematoxylin and Eosin (H&E)-staining .....................................................................58 2.6.5 Immunohistochemistry ..............................................................................................60 2.6.6 In vivo bromodeoxyuridine (BrdU)-proliferation assay ..............................................60 2.6.7 Cell cycle exit index ..................................................................................................61 2.6.8 Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) on mouse brain sections .....................................................................................................................62 2.7 Mouse behavioral tests ...................................................................................................63 2.7.1 Barnes maze test ......................................................................................................63 2.7.2 Rotarod test ..............................................................................................................64 2.7.3 Balance bar test ........................................................................................................64 3. Results ................................................................................................................................. 65 3.1 Deletion of Mcph1 in mice leads