Towards an understanding of the role of apical polarity molecules in neural stem cells and neurogenesis [Elektronische Ressource] / Franziska Weinandy

Franziska.Weinandy

Sujets

• Naturwissenschaften

Informations

• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2010
• Nombre de lectures: 33
• Langue: Deutsch
• Poids de l'ouvrage: 38 Mo

Extrait

TECHNISCHE UNIVERSITÄT MÜNCHEN

Lehrstuhl für Entwicklungsgenetik







Towards an Understanding of the Role of Apical
Polarity Molecules in Neural Stem Cells and
Neurogenesis



Franziska Weinandy







Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für Ernährung,
Landnutzung und Umwelt der Technischen Universität München zur Erlangung des akademischen Grades
eines
Doktors der Naturwissenschaften

genehmigten Dissertation.


Vorsitzender: Univ.-Prof. Dr. A. Gierl
Prüfer der Dissertation:
1. Univ.-Prof. Dr. W. Wurst
2. Univ.-Prof. Dr. M. Götz
(Ludwig-Maximilans-Universität, München)



Die Dissertation wurde am 07.06.2010 bei der Technischen Universität München eingereicht und durch die
Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt am 09.12.2010
angenommen.


Table of Contents
Table of Contents

Table of Contents....................................................................................................................I
Abbreviation .......................................................................................................................... V
Abstract................................................................................................................................. IX
Zusammenfassung............................................................................................................... XI
1 Introduction..1
1.1 ADHERENS JUNCTIONAL MOLECULES AND THEIR INTERACTING PARTNERS................................... 2
1.2 β-CATENIN DEPENDENT WNT SIGNALLING .................................................................................. 7
1.3 FIRST STEPS OF BRAIN DEVELOPMENT: FROM NEURULATION TO DISTINCT BRAIN STRUCTURES ..... 8
1.4 EMBRYONIC AND ADULT PROLIFERATIVE ZONES IN THE MOUSE CEREBRUM................................ 11
1.4.1 Neural stem and progenitor cells in development ...................................................... 12
1.4.2 The adult subependymal zone: origin and cellular composition ................................. 16
1.5 FUNCTION OF ADHERENS JUNCTIONAL AND APICAL PROTEINS IN NEUROGENESIS....................... 19
1.5.1 Adherens junctions in embryonic neurogenesis.......................................................... 19
1.5.2 Apical polarity complexes and embryonic neurogenesis ............................................ 21
1.5.3 β-Catenin dependent Wnt signalling in neurogenesis................................................. 22
1.6 REGIONAL HETEROGENEITY OF NEURAL STEM CELLS AND REGULATORS OF NEURONAL SUBTYPE
SPECIFICATION............................................................................................................................ 25
1.6.1 Neuronal fate determinants in dorsal and ventral forebrain development .................. 25
1.6.2 Generation of diverse olfactory bulb neurons ............................................................. 29
1.6.3 Regional specification of postnatal and adult olfactory interneurons.......................... 30
1.7 AIM OF THE THESIS ................................................................................................................. 33
2 Materials........................................................................................................................35
2.1 COMMONLY USED BUFFERS..................................................................................................... 35
2.2 BULK CHEMICALS ................................................................................................................... 35
2.3 KITS....................................................................................................................................... 37
2.4 PROTEASE INHIBITORS............................................................................................................ 37
2.5 TISSUE CULTURE REAGENTS ................................................................................................... 38
3 Methods.........................................................................................................................39
3.1 ANIMALS AND GENOTYPING 39
3.1.1 Animals 39
3.1.2 Mouse tail DNA extraction........................................................................................... 40
3.1.3 Poly chain reactions .................................................................................................... 40
3.1.4 Agarose gel electrophoresis of DNA ........................................................................... 41
3.2 ANIMAL EXPERIMENTS............................................................................................................. 41
3.2.1 Anaesthesia................................................................................................................. 41
3.2.2 BrdU administration..................................................................................................... 41

- I -
Table of Contents
3.2.3 Tamoxifen treatment ................................................................................................... 42
3.2.4 Perfusion and tissue preparation................................................................................. 42
3.3 TISSUE CULTURE .................................................................................................................... 42
3.3.1 Preparation of coated cover slips................................................................................ 42
3.3.2 Preparation of primary embryonic cerebral cortical cell culture .................................. 42
3.3.3 Floating neurosphere cultures of the adult subependyma zone ................................. 43
3.3.4 Viral infection of floating neurosphere cultures ........................................................... 44
3.3.5 Colony forming neurosphere assay in collagen .......................................................... 44
3.4 VIRUSES................................................................................................................................. 44
3.4.1 Viral expression plasmids............................................................................................ 44
3.4.2 DNA preparation of viral plasmids............................................................................... 46
3.4.3 Retroviral production ................................................................................................... 46
3.4.4 Lentiviral production .................................................................................................... 47
3.4.5 Determination of viral titres.......................................................................................... 48
3.5 IMMUNOCHEMISTRY ................................................................................................................ 48
3.5.1 General method........................................................................................................... 48
3.5.2 Antigen retrieval 49
3.5.3 Tyramide signal amplification...................................................................................... 50
3.6 IN-SITU HYBRIDISATION 52
3.6.1 In vitro transcription: generation of digoxigenin labelled probes ................................. 52
3.6.2 In-situ hybridisation...................................................................................................... 52
3.7 WESTERN BLOT ANALYSIS....................................................................................................... 53
3.7.1 Tissue preparation 53
3.7.2 SDS-polyacrylamide gel electrophoresis (SDS-PAGE) .............................................. 53
3.7.3 Transfer ....................................................................................................................... 54
3.7.4 Blocking, detection and scanning................................................................................ 54
3.7.5 Semi-quantitative western blot analysis ...................................................................... 56
3.8 SUBCELLULAR FRACTIONATION............................................................................................... 56
3.9 β- GALACTOSIDASE REPORTER GENE ASSAY ........................................................................... 56
4 Results...........................................................................................................................58
4.1 INFLUENCE OF CORTICAL α-E-CATENIN DEFICIENCY ON CELLULAR SIGNALLING ......................... 58
Cre/ α-catΔex2fl/fl4.1.1 Protein levels of α-E- and α-N-catenin in the Emx1 cortices .................... 58
4.1.2 Influence of α-E-catenin loss on apical membrane proteins of the Par complex and
Prominin-1.................................................................................................................... 59
4.1.3 Loss of α-E-catenin does not change canonical Wnt signa