Towards an Understanding of the Role of Apical Polarity Molecules

154 Pages·2011·38.2 MB·English

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

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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
4.1.1 Protein levels of α-E- and α-N-catenin in the Emx1Cre/α-catΔex2fl/fl 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 signalling..................................64
4.1.4 Loss of α-E-catenin up-regulates the levels of GSK3β...............................................66
4.1.5 Semi-quantitative analysis of α-E-catenin dependent candidate proteins..................68
4.2 ADHERENS JUNCTIONS AND PAR COMPLEX MOLECULES IN THE ADULT SUBEPENDYMAL ZONE.....70
4.2.1 Apical surface of the adult subependymal zone..........................................................70
4.2.2 Canonical Wnt signalling activity in the adult subependymal zone.............................71
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4.3 FUNCTIONAL ANALYSIS OF ADHERENS JUNCTIONAL AND PAR COMPLEX MOLECULES IN NEUROSPHERE
CULTURES..................................................................................................................................74
4.3.1 Comparative analyses of the function of α- and β-catenin in neurosphere cultures...74
4.3.2 Functional analysis of Par complex molecules in neurosphere cultures.....................80
4.4 THE ROLE OF β-CATENIN IN ADULT NEUROGENESIS..................................................................83
4.4.2 Maintenance of the pinwheel architecture of the adult SEZ despite loss of β-catenin87
4.4.3 β-Catenin deletion does not change number of astrocytes in the lateral subependymal
zone.............................................................................................................................87
4.4.4 Deletion of β-Catenin decreases the number of neuroblasts in the lateral ventricular
wall...............................................................................................................................91
4.4.5 Loss of neuroblasts after β-Catenin deletion is caused by a decreasing stem cell pool
in the lateral ventricular wall........................................................................................92
4.4.6 β-Catenin is required for Tbr2 expression in the RMS................................................92
4.4.7 The role of β-Catenin in neuronal subtype specification in the olfactory bulb.............94
5 Discussion..................................................................................................................104
5.1 SUMMARY............................................................................................................................104
5.2 α-E-CATENIN DEPENDENT SIGNALLING DURING CORTICAL NEUROGENESIS..............................105
5.2.1 The loss of α-E-catenin causes only minor changes in polarity cues.......................105
5.2.2 α-E-Catenin does not control β-catenin signalling during early neurogenesis..........106
5.2.3 Compensatory response of GSK3β to hyper-proliferation caused by loss of α-E-
catenin from the cerebral cortex................................................................................108
5.3 THE APICAL DOMAIN OF ADULT SEZ NEURAL STEM CELLS SHOWS CHARACTERISTIC FEATURES OF
EMBRYONIC VENTRICULAR ZONE CELLS.................................................................................110
5.4 THE PINWHEEL ORGANISATION OF THE ADULT SEZ DOES NOT DEPEND ON THE ADHESIVE FUNCTION
OF β-CATENIN......................................................................................................................112
5.5 THE ROLE OF ADHERENS JUNCTIONAL MOLECULES IN NEUROSPHERE CULTURES.....................113
5.6 THE ROLE OF THE PAR COMPLEX IN NEUROSPHERE CULTURES................................................115
5.7 THE DUAL ROLES OF β-CATENIN IN ADULT NEUROGENESIS IN-VIVO: MAINTENANCE OF THE NEURAL
STEM CELL POOL AND CONTROL OF DORSAL PROGENITOR SUBTYPES.....................................116
5.7.1 The function of β-catenin in maintaining general adult neurogenesis.......................116
5.7.2 The prospective function of β-catenin in neural subtype specification......................118
6 References..................................................................................................................120
7 Acknowledgements....................................................................................................138
Curriculum Vitae …………………………………………………………………………………………………... 139

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Abbreviation
Abbreviation

% Percent
°C Degree Celsius
µg Microgram(s)
µL Microlitre(s)
AJ Adherens Junction(s)
APC Adenomatous Polyposis Coli
aPKC Atypical Protein Kinase C
APS Ammonium Peroxide Disulphate
bHLH Basic helix-loop-helix
BLBP Brain Lipid Binding Protein
BMP Bone Morphogenetic Protein
BrdU 5’-Bromo-2’-Deoxyuridine
BSA Bovine Serum Albumin
CaIB Calbindin
CaIR Calretinin
cDNA complementary DeoxyriboNucleic Acid
CFP Cyan Fluorescent Protein
cm Centimeter(s)
CNS Central Nervous System
CO Carbon Dioxide
2
CP Cortical Plate
Cre Cre Recombinase
Crib Cdc42/Rac Interaction Binding
CSF Cerebrospinal Fluid
Cux1/2 Cut-Like homeoboX1/2
DAPI 4,6-diamidine-2-pheylidoledihydrochloride
dATP DeoxyAdenosine TriPhosphate
dCTP DeoxyCytidine TriPhosphate
DCX Doublecortin
dGTP DeoxyGuanosine TriPhosphate
Dkk Dickkopf
Dlx Distal-Less homeoboX
DMEM Dulbecco's Modified Eagel Medium
DNA DeoxyriboNucleic Acid
dNTP DeoxyNucleoside TriPhosphate
dTTP DeoxyThymidine TriPhosphate
e.g. For example (Latin ‘exempli gratia’)
eCFP enhanced Cyan Fluorescent Protein
EDTA Ethylene-Diamine-tetra-Acetic-Acid
EGF Epidermal Growth Factor
eGFP enhanced Green Fluorescent Protein
Emx1 Empty Spiracles Homeobox 1
EPL External Plexiform Layer
ERT2 estrogen ligand-binding domain
FCS Fetal Calf Serum
FGF Fibroblast growth factor
Fig. Figure
Fzd Frizzeled
g Gram(s)

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Abbreviation
G Gauge
G418 Geneticin
GABA γ-Amino Butyric Acid
GAD Glutamic Acid Decarboxylase
GAPDH GlycerAldehyde 3-Phosphate DeHydrogenase
GC Granule Cells
GCL Granule Cell Layer
GE Ganglionic Eminence
GFAP Glial Fibrillary Acidic Protein
GFP Green Fluorescent Protein
GL Glomerular Layer
GLAST astrocyte-specific glutamate transporter
Gsh1/2 GS HomeoboX1/2
GSK3β Glycogen synthase kinase 3β
h Hour(s)
HBSS Hank's balanced salt solution
HCl Hydrochloric acid
HEPES 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid
HERP Hes Related Protein(s)
Hes Hairy and Enhancer of Split
HESR Hes Related
hGFAP human Glial Fibrillary Acidic Protein
HIV1 Human Immunodeficiency Virus Type 1
i.e. That is (Latin 'id est')
INM Interkinetic Nuclear Migration
IPL Internal Plexiform Layer
IRES Internal Ribosomal Entry Site
ISH In-Situ Hybridisation
IZ Intermediate Zone
JGC JuxtaGlomerular Cells
kDa Kilodalton (molecular mass)
kg Kilogram(s)
LDL Low Density Lipoprotein
LEF Lymphoid Enhancer binding Factor
LGE Lateral Ganglionic Eminences
LGL Lethal Giant Larvae
LRP Receptor Related Protein
LTR Long Terminal Repeat
LTR/SIN Self-Inactivating Long Terminal Repeat
LV Lateral Ventricle
M Molar (mol/litre)
mA Milliampere
MAPK Mitogen Activated Protein Kinase
Mash1 Mammalian Achaete Scute Homolog1
Math1 Mammalian Atonal Homolog
MCL Mitral Cell Layer
mg Milligram(s)
MGE Medial Ganglionic Eminences
min Minute(s)
mL Millilitre(s)
mM Millimolar
mm Millimetre(s)
MMLV Moloney Murine Leukemia Virus
mpi Month Post Induction
mRNA Messenger Ribonucleic Acid
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Abbreviation
mTOR Mammalian Target Of Rapamycin
NaCl Sodium chloride
NCFC Neural Colony Forming Cell
NeuroD1 Neurogenic Differentiation1/2
ng Nanogram(s)
Ngn1/2 Neurogenin1/2
nm Nanometre(s)
nM Nanomolar
N-P40 Nonidet-P40
NSCL1/2 Neuronal Stem Cell Leukemia1/2
OB Olfactory Bulb
Olig2 Oligodendrocyte Transcription Factor 2
ONL Olfactory Nerve Layer
OSN Olfactory sensory neurons
PAGE Polyacrylamide Gel Electrophoresis
Par3/6 Partitioning defective 3/6 homolog
Pax6 Paired BoX gene 6
PB1 Phox/Bem1
PBS Phosphate Buffered Saline
PCNA Proliferating Cell Nuclear Antigen
PCP planer cell polarity
PCR Poly chain reaction
PDL Poly-D-Lysine
PDZ PSD-95/Disclarge/ZO-1
PFA ParaFormAldehyde
PGC PeriGlomerular Cells
PSA-
NCAM Polysialic Acid Neural Cell Adhesion Molecule
PVDF PolyVinyliDene Fluoride
RMS rostral migratory stream
RNA Ribonucleic Acid
RT Room Temperature
SDS Sodium dodecyl sulfate
sec Second(s)
SEM Standard Error of the Mean
Ser9 Serine9
SEZ Subependymal Zone
sFRP small Frizzeled Related Protein(s)
SGZ SubGranular Zone
Shh Sonic Hedgehog
shRNA Small Hairpin Ribonucleic Acid
SV40 Simian vacuolating Virus 40 promotor
SVZ SubVentricular Zone
TAP Transit Amplifying Precursor
Tris/Borate/EDTA, buffer solution containing mixture of Tris base, boric acid
TBE and EDTA
Tbr1/2 T-box brain gene1/2
TBS Tris-buffered saline
TCF/LEF T Cell Factor
TE Tris/EDTA, buffer solution containing mixture of Tris base and EDTA
TEMED TEtraMethylEthylenDiamine
TH Tyrosine Hydroxlyase
TJ Tight Junction(s)
UV Ultraviolet

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Abbreviation
V Volt
vGluT vesicular Glutamate Transporter
VSVG Vesicular Stomatitis Virus G
VZ Ventricular Zone
WIF-1 Wnt-inhibitory factor-1
wpi Weeks Post Induction
WPRE Woodchuck Hepatitis Post-transcriptional Regulatory Element
WT Wild-type(s)
x g Times the acceleration of gravity (9.807 ms2)
β-Gal β-Galactosidase

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