The role of bone morphogenetic protein mediated signalling in the neurogenic niche of adult mouse brain [Elektronische Ressource] / Dilek Colak

Colak Dilek

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2008
• Nombre de lectures: 28
• Langue: Deutsch
• Poids de l'ouvrage: 10 Mo

Extrait

The Role of Bone Morphogenetic Protein Mediated

Signalling in the Neurogenic Niche

of the Adult Mouse Brain




PhD Thesis


Submitted to the Faculty of Biology

Ludwig-Maximillian-University Munich


Prepared in the group Prof. Dr. Magdalena Götz

at the Helmholtz Zentrum München/Institute for
Stem Cell Research






Dilek Colak



1


1. Gutachter: Prof. Benedikt Grothe



2. Gutachter: Prof. Rudiger Klein

Tag der mündlichen Prüfung: 28/07/2008













2
Ehrenwörtliche Versicherung

Ich versichere hiermit ehrenwörtliche, dass die Dissertation von mir selbständing, ohne
unerlaubte Beihilfe angefertigt ist.



München, den......................



............................................................
(Unterschrift)












Erklärung

Hiermit erkläre ich, dass ich mich anderweitig einer Doktorprüfung ohne Erfolg nicht
unterzogen habe.


München, den.......................


............................................................
(Unterschrift)








31 TABLE OF CONTENTS

1. TABLE OF CONTENTS…………………………………………………………...1

2. ABSTRACT……………………………………………………………………………....5

3. INTRODUCTION………………………………………………………………….6

2.1 Neurogenesis persists in the adult mammalian brain……………………………..6

2.2 Neural stem cells and their progeny in adult SEZ………………………………...7

2.3 cells (NSCs) as promising cellular source for the treatment of diseases
in nervous systems……………………………………………………………….10

2.4 Regulators of adult neurogenesis in SEZ………………………………………...13

2.5 TGFβ superfamily and BMP signalling……………………………………….....18

3.5.1 Receptors and intracellular cascade of TGF β superfamily…………………….18

3.5.2 Signalling by three types of Smad proteins…………………………………....19

3.5.3 Versatile roles of BMP signalling in nervous system………………………….20

3.5.3.1 BMPs in early neural development…………………………………………..21

3.5.3.2 BMPs in the developing spinal cord………………………………………....22

3.5.3.3 BMPs in the developing brain………………………………………………..24

3.5.3.4 BMPs in the adult brain……………………………………………………....26

4. ABBREVIATIONS………………………………………………………………....28

5. MATERIALS AND METHODS………………………………………………......31

5.1 Animals………………………………………………………………………….31

5.1.1 Strains………………………………………………………………………….31

5.1.2 Genotyping……………………………………………………………………..31

5.2 Tamoxifen Administration……………………………………………………....34

5.3 Tissue Culture……………………………………………………………….......34

5.3.1 Neurosphere assay……………………………………………………………..34

5.3.2 Differentiation of neurospheres………………………………………………..36

5.4 Histological Procedures and Immunohistochemistry…………………………..36
5.4.1 Cryosections……………………………………………………………….......36

5.4.2 Free floating sections…………………………………………………………..37

5.4.3 BrdU labeling…………………………………………………………………..37
5.4.4 Immunostaining…………………………………………………………….....38

4 5.4.4.1 Tyramid signal amplification………………………………………………39
5.4.4.2 Tunel staining………………………………………………………………39

5.5 In Situ Hybridization………………………………………………………......43

5.5.1 Plasmid preparation and in vitro transcription…………………………….....43

5.5.2 Non-radioactive in situ hybridization……………………………………......45

5.6 In vivo Injections and Cortex Injury…………………………………………..45

5.6.1 Anesthesia…………………………………………………………………....45

5.6.2 Stereotaxic injections…………………………………………………….......45

5.6.3 Viral vectors………………………………………………………………….46

5.6.3.1 Retroviral vectors and retrovirus production……………………………....46

5.6.3.2 Lentivirus vectors and lentivirus production……………………………....47

5.6.4 Noggin Infusion……………………………………………………………...48

5.6.5 Stab wound injury………………………………………………………........49

5.6.6 Transplantation…………………………………………………………........49

5.7 RNA Extraction and Microarray…………………………………………........49

5.7.1 RNA extraction……………………………………………………………...49

5.7.2 Microarray…………………………………………………………………...50

5.8 cDNA Preparation and Real Time (RT) PCR………………………………....51

5.8.1 cDNA synthesis……………………………………………………………...51

5.8.2 Real time (RT) PCR……………………………………………………….....52

5.9 Quntitative Analysis and Statistics……………………………………….........53


6. RESULTS…………………………………………………………………………54

6.1 BMP Signalling in adult subependymal zone…………………………….........54

6.1.1 BMP pathway components are present in the adult subependymal zone……54

6.1.2 Activity of BMP-mediated signalling in adult neural stem cells of the SEZ, but
not SGZ………………………………………………………………………...54

6.1.3 BMP reporter mouse line…………………………………………………….56

6.2 BMP Signalling in non-neurogenic areas……………………………………...57

6.3 Expression pattern of BMP signalling components in the adult cortex after stab
wound…………………………………………………………………………….57

6.4 Conditional deletion of Smad4 in the adult SEZ………………………………58

6.5 Analysis of adult SEZ stem cells and their progeny after Smad4 deletion…….61

6.5.1 Smad4 deletion does not affect neural stem cell number and properties……61
5
6.5.2 Number of transit amplifying precursors is not altered 10 days after Smad4
deletion…………………………………………………………………………..62

6.5.3 Neurogenesis is impaired 10 days after Smad4 deletion……………………..63

6.5.4 Fate mapping of Smad4-/- cells……………………………………………....64

6.5.5 Deletion of Smad4 alters TAP identity by aberrant expression of Olig2…….64
6.5.6 Proliferation of TAPs is impaired 21 days after Smad4 deletion…………….66

6.5.7 BrdU birthdating analysis shows an increase in the number of immature
oligodendrocytes in corpus callosum……………………………………………67

6.5.8 Deletion of Smad4 results in migration of cells to the corpus callosum and
increased oligodendrogenesis……………………………………………………68

6.5.9 Wild type cells transplanted into the Smad4-/- SEZ are not impaired in
neurogenesis……………………………………………………………………..70

6.5.10 Smad4 is required at early stages in the stem cell-derived lineage…………..71

6.6 Discrimination of BMP and TGF β signalling in regard to the contribution to
Smad4 phenotype…………………………………………………………………..72

6.6.1 Extracellular inhibition of BMP signalling via Noggin increases Olig2
expression and decreases neurogenesis………………………………………….72

6.6.2 Inhibition of TGF β signalling does not cause Olig2 up-regulation and thereby
alteration in neurogenesis………………………………………………………..73

6.7 Overexpression of BMP ligands in vivo and in vitro……………………………74

6.7.1 BMP7 reduces proliferation in the adult SEZ………………………………….74

6.7.2 BMP decreases the proliferation of neurosphere forming cells in vitro……….74

6.7.3 BMP regulates proliferation via Sma