Examining the role of Notch signalling in adult hippocampal stem cell maintenance [Elektronische Ressource] / Oliver Karl Heinz Ehm

Oliver Optic

Sujets

• Biologie

Informations

• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2010
• Nombre de lectures: 24
• Langue: Deutsch
• Poids de l'ouvrage: 3 Mo

Extrait

TECHNISCHE UNIVERSITÄT MÜNCHEN
Lehrstuhl für Entwicklungsgenetik





Examining the role of Notch signalling in
adult hippocampal stem cell maintenance




Dissertation von
Oliver Ehm












Helmholtz Zentrum München
Institut für Entwicklungsgenetik




TECHNISCHE UNIVERSITÄT MÜNCHEN

Lehrstuhl für Entwicklungsgenetik



Examining the role of Notch signalling in adult hippocampal stem cell maintenance


Oliver Karl Heinz Ehm




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. S. Scherer
Prüfer der Dissertation:
1. Univ.-Prof. Dr. W. Wurst
2. Univ.-Prof. Dr. A. Gierl




Die Dissertation wurde am 15.12.2009 bei der Technischen Universität München eingereicht und
durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt
am 06.05.2010 angenommen.

























































Table of contents
1 Zusammenfassung............................................................................................................ 3
2 Abstract ............................................................................................................................. 5
3 Introduction ...................................................................................................................... 6
3.1 Adult neurogenesis and the hippocampal neurogenic niche ................................. 6
3.2 Sox gene family ..................................................................................................... 8
3.2.1 Expression of Sox2 in the CNS ............................................................ 10
3.2.2 Sox2 mutants and function of SOX2 in neural stem cells .................... 13
3.3 Notch signalling .................................................................................................. 15
3.4 The objective of this study .................................................................................. 21
4 Materials and Methods .................................................................................................. 22
4.1 Materials.............................................................................................................. 22
4.1.1 Organisms............................................................................................. 22
4.1.2 Software ............................................................................................... 23
4.1.3 Chemicals ............................................................................................. 23
4.1.4 Commercial kits ................................................................................... 24
4.1.5 Buffers and solutions............................................................................ 24
4.1.6 Antibodies ............................................................................................ 29
4.1.7 Plasmids and oligonucleotides ............................................................. 32
4.2 Methods............................................................................................................... 35
4.2.1 Animals ................................................................................................ 35
4.2.2 Tissue processing ................................................................................. 36
4.2.3 Mouse embryos .................................................................................... 36
4.2.4 Cell cultures.......................................................................................... 36
4.2.5 Fluorescent immunohistochemistry ..................................................... 37
4.2.6 Fluorescent immunocytochemistry ...................................................... 38
4.2.7 Electroporation ..................................................................................... 39
4.2.8 Calciumphosphate-mediated transfection ............................................ 39
4.2.9 Transformation of bacteria ................................................................... 39
4.2.10 Luciferase assay ................................................................................... 40
4.2.11 Isolation of DNA.................................................................................. 40
4.2.12 Isolation of RNA .................................................................................. 40
4.2.13 cDNA synthesis.................................................................................... 41
4.2.14 Extraction of DNA fragments from agarose gels................................. 41
4.2.15 Cloning procedures .............................................................................. 41
1 Table of contents
4.2.16 PCR ...................................................................................................... 42
4.2.17 Preparation of nuclear protein fractions and Western Blotting............ 43
4.2.18 Preparation of nuclear protein cell extracts for EMSA ........................ 44
4.2.19 Electrophoretic mobility shift assay (EMSA) ...................................... 45
4.2.20 Co-immunoprecipitations..................................................................... 47
4.2.21 Chromatin immunoprecipitations......................................................... 48
4.2.22 Prediction of transcription factor binding sites (Genomatix)............... 50
4.2.23 Statistical analysis ................................................................................ 50
5 Results ............................................................................................................................. 51
5.1 Notch signalling is differentially active in SOX2 positive stem cells and
neuronally committed cells ................................................................................. 51
5.2 Notch signalling positively regulates Sox2 expression in adult hippocampal
neural stem cells .................................................................................................. 55
5.3 Loss of RBPJ in neural stem cells perturbs hippocampal neurogenesis ........... 61
5.4 RBPJ is essential for long-term neural stem cell maintenance in the adult
hippocampus........................................................................................................ 66
5.5 Wnt/β-catenin signalling is active in adult hippocampal neural stem cells ........ 68
6 Discussion........................................................................................................................ 73
6.1 Notch signalling and Sox2 expression in neural stem and progenitor cells ........ 73
6.2 Notch signalling and stem cell maintenance ....................................................... 75
6.3 Notch signalling and migration ........................................................................... 78
6.4 Integration of Notch signalling with other signalling pathways ......................... 80
6.5 Notch signalling, Hes genes and the control of quiescence and astrocytic
properties in stem cells........................................................................................ 87
6.6 Role of Notch signalling in mature granule neurons........................................... 88
6.7 Notch signalling, hippocampal function and aging............................................. 89
7 Abbreviations.................................................................................................................. 92
8 Literature index.............................................................................................................. 96
9 Acknowledgements....................................................................................................... 114
10 Erklärung...................................................................................................................... 116
11 Lebenslauf ..................................................................................................................... 117


2
kk Zusammenfassung
1 Zusammenfassung

Im Erwachsenenalter werden im Gyrus Dentatus des Hippocampus fortlaufend neue
Nervenzellen aus neuralen Stammzellen gebildet. Die Erhaltung und Differenzierung
neuraler Stammzellen muss in einem ausgewogenen Verhältnis stehen, um die
hippocampale Neurogenese während der gesamten Lebenszeit aufrechtzuerhalten.
Frühere Studien haben gezeigt, dass der Transkriptionsfaktor Sox2 bei der Erhaltung
hippoca