Jagged1 regulates adult neural stem cell maintenance and multipotency through activation of Notch1 [Elektronische Ressource] / vorgelegt von Yves Nyfeler

Yves Nyfeler

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• Publié par: albert-ludwigs-universitat_freiburg
• Publié le: 01 janvier 2005
• Nombre de lectures: 56
• Langue: English
• Poids de l'ouvrage: 2 Mo

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Aus dem Max-Planck-Institut für Immunbiologie,

Freiburg im Breisgau





Jagged1 Regulates Adult Neural Stem Cell Maintenance
and Multipotency through Activation of Notch1









Inaugural-Dissertation
zur
Erlangung der Doktorwürde
der Fakultät für Biologie
der Albert-Ludwigs-Universität
Freiburg i. Br.



vorgelegt von
Yves Nyfeler
aus Bern

Freiburg 2004



1 Die vorliegende Arbeit wurde im Zeitraum von Dezember 2001 bis Dezember 2004 am
Max-Planck-Institut für Immunbiologie in Freiburg i. Br. unter der Anleitung von
Dr. Verdon Taylor und Dr. habil. Rolf Kemler angefertigt.



















Tag der Mündlichen Prüfung: 4. Februar 2005

Referent: Prof. Rolf Kemler

Koreferent: Prof. Wolfgang Driever

3. Prüfer: Prof. Ralf Baumeister
2 SUMMARY 5
ZUSAMMENFASSUNG 7
1 INTRODUCTION 9
1.1 DEVELOPMENT OF THE CNS 9
1.1.1 THE SVZ: THE LARGEST SITE OF NEUROGENESIS IN THE ADULT BRAIN 9
1.1.2 REBUILDING THE DAMAGED CNS 11
1.2 NOTCH SIGNALLING: AN INTRODUCTION 13
1.2.1 FUNCTION OF NOTCH IN LOWER ORGANISMS 13
1.2.2 DISCOVERY AND STRUCTURE OF NOTCH IN HUMANS AND MAMMALS 15
1.2.3 NOTCH SIGNAL TRANSDUCTION IN MAMMALS 17
1.3 NOTCH FUNCTION IN VERTEBRATE CNS DEVELOPMENT 29
1.3.1 NOTCH SIGNALLING IN THE CNS: THE PATHWAY IN SHORT 29
1.3.2 NOTCH1 EXPRESSION IN THE MAMMALIAN CNS 30
1.3.3 NOTCH1 FUNCTION IN THE MAMMALIAN CNS 31
1.4 THE ROLE OF JAGGED1 IN MAMMALIAN DEVELOPMENT 32
1.4.1 JAGGED1 IS A SINGLE SPAN TRANSMEMBRANE PROTEIN 32
1.4.2 JAGGED1 EXPRESSION IN MAMMALIAN DEVELOPMENT 33
1.4.3 BIOCHEMICAL ASPECTS OF LIGAND INTERACTION WITH NOTCH RECEPTORS 34
1.4.4 ALAGILLE SYNDROME IS CAUSED BY A MUTATION IN HUMAN JAGGED1 35
1.4.5 JAGGED1-DEFICIENT MICE 36
2 JAGGED1 REGULATES ADULT NEURAL STEM CELL MAINTENANCE AND
MULTIPOTENCY THROUGH ACTIVATION OF NOTCH1 37
2.1 ABSTRACT & INTRODUCTION 37
2.2 MATERIALS AND METHODS 38
2.2.1 GENERATION OF MICE & BREEDING 38
2.2.2 ISOLATION AND CULTURE OF NEUROSPHERES 38
2.2.3 QUANTIFICATION, IMMUNOFLUORESCENCE AND CELL FATE ANALYSIS 39
2.2.4 WESTERN BLOT ANALYSIS 40
2.2.5 RNA ISOLATION AND RT-PCR ANALYSIS 40
2.2.6 SOLUBLE JAGGED1-FC TREATMENT OF NEURAL STEM CELLS 41
2.3 RESULTS 42
2.3.1 NOTCH1 AND JAGGED1 ARE EXPRESSED IN THE SVZ AND FORM THE NSC NICHE 42
2.3.2 JAGGED1 AND NOTCH1 ARE EXPRESSED BY NEURAL PROGENITORS 44
2.3.3 RE REQUIRED FOR NSC SELF-RENEWAL 46
2.3.4 JAGGED1 AND NOTCH1 REGULATE SELF-RENEWAL BUT NOT CELL LINEAGE
COMMITMENT 49
2.3.5 JAGGED1 CAN MAINTAIN NSC SELF-RENEWAL IN THE ABSENCE OF
TROPHIC FACTORS 51
2.3.6 NSCS MAINTAINED IN SOLUBLE JAGGED1 SHOW INCREASED NEUROGENIC
POTENTIAL 53
2.3.7 RT-PCR ANALYSIS OF POTENTIAL NOTCH1 SIGNALLING TARGETS FIVE DAYS
AFTER CONDITIONAL INACTIVATION OF JAGGED1 AND NOTCH1 55


3 2.4 DISCUSSION 56
2.5 OUTLOOK 57
2.5.1 ADDRESSING THE ROLE OF JAGGED1 IN ADULT NEURAL STEM CELLS IN VIVO 57
2.5.2 PROLIFERATION 59
2.5.3 FUNCTIONAL CONSERVATION OF NOTCH1 IN ADULT AND EMBRYONIC NEURAL
STEM CELLS 60
3 REDUCING NOTCH SIGNALLING IN VIVO : AN ANALYSIS OF PHENOTYPES
IN THE CORTEX AND THE ROSTRAL MIGRATORY STREAM 62
3.1 INTRODUCTION 62
3.2 MATERIALS AND METHODS 65
3.2.1 GENERATION OF MICE, BREEDING AND GENOTYPING 65
3.2.2 IMMUNOHISTOCHEMISTRY 66
3.3 RESULTS AND DISCUSSION 66
3.3.1 ADULT CALBINDIN-CRE FLOXED NOTCH1 MUTANTS SHOW AN INCREASE OF CELL
NUMBER IN THE CEREBRAL CORTEX 66
3.3.2 PERINATAL JAG1/-; NOTCH1/- MICE SHOW REDUCED PROLIFERATION IN THE RMS
AND AN INCREASE IN CELL NUMBER 69
4 GENERAL DISCUSSIONS AND OUTLOOK 74
REFERENCES 78
CURRICULUM VITAE 96
4 SUMMARY

A dogma in neurobiology that has persisted over a long period of time, namely the
inability of the adult mammalian brain to produce new neurons, was finally proven to be
wrong about a decade ago. Ongoing neurogenesis in the adult mammalian brain is now widely
accepted, but the mechanisms controlling development and differentiation of adult neural
stem cells are still largely unknown. The importance of understanding the basic mechanisms
of neurogenesis in the adult brain becomes obvious in the view of possible treatments of
neurological diseases and injuries, which damage CNS neurons. Putative neural stem cells
(NSCs) have been isolated from various regions of the postnatal central nervous system but
Epidermal Growth Factor(EGF)-dependent (adult type) stem cells of the sub-ventricular zone
(SVZ) are among the best described to date. Notch-Delta signalling molecules were shown to
be involved in developmental decisions in many tissues in organisms ranging from
Drosophila to human. Both Notch receptors and their ligands Delta and Jagged are
transmembrane proteins, which require direct cell-cell contact for an active signal to be
induced. Jagged1 belongs to the Delta-Serrate-Lag2 (DSL) family of Notch ligands and is the
only DSL molecule that is expressed in the SVZ of adult mice.
We here present a study that is demonstrating that adult type NSCs isolated from the
SVZ of early postnatal mice are dependent on Notch signalling in vitro. We support the in
vitro findings by illustrating for the first time the stem cell niche in the adult mouse brain in
vivo. Using conditional gene inactivation, we could show that Jagged1, by its action through
Notch1, is pivotal in the maintenance and differentiation of adult-type neural stem cells. As a
result of Jagged1 inactivation, multi-potent neural stem cells fail to maintain their stem cell-
like potential and ability to self-replicate. Self replication of a neural stem cell results in the
formation of so called neurospheres, ball-like structures made of up to 1000 cells after having
been allowed to grow in suspension for 5 to 6 days. We show that Jagged1 deficient
neurospheres grow, but contain 5 fold less NSCs per sphere than control spheres (1.7 +/- 0.53
to 7.6 +/- 0.16) and that they completely loose their stem cell potential over time. However,
Jagged1-deficient neural stem cells are able to generate neurons, glia and oligodendrocytes
and are comparable to wild-type cells regarding their differentiation potential.
Semiquantitative RT-PCR revealed that the direct targets of the Notch-pathway, the
transcripts of the bHLH transcription factors Hes1 and Hes5, show a reduced expression level
upon ablation of Jagged1 . These results could be pheno-copied upon conditional ablation of
Notch1 from neural stem cells in vitro . Ablation of the receptor Notch1 lead to a drastic
5 reduction in the number of NSCs per sphere after infection compared to controls (0.04 +/-
0.01 to 8.1 +/- 1.1). We used soluble Jagged1-Fc (sJ1) to examine whether the number of
NSCs per sphere could be increased. When cultured in the absence of EGF, we found sJ1 to
stimulate sphere formation in a concentration dependent manner, indicating that sJ1 can
replace EGF as a spherogenic factor for NSCs in vitro . In vivo we characterized the stem cell
niche to be comprised of cells lining the SVZ which express Jagged1 and GFAP (Glial Fibric
Acidic Protein, a glial marker) and which are adjacent to Notch1 expressing cells. We
conclude that a Jagged1-mediated Notch1 signal is required by adult-type neural stem cells to
maintain multi-potency and self- renewal capacity.
Further experiments need to address the function of Notch signalling in neural stem
cells in vivo. As both, Notch1 and Jagged1 null mice die early during embryonic
development, a temporally and spatially restricted ablation of these genes is required. Nestin
is an intermediate filament protein widely used as a neural stem cell marker. We are currently
generating mouse lines expressing CreERT2 (Cre-recombinase fused to estrogen receptor,
active only after application of the ERT2-ligand Tamoxifen) under control of the Nestin
promoter. This inducible Cre-line will allow us to examine the effects of targeted gene
ablation in the CNS by administering Tamoxifen to the animal at any time point of interest as
well as to perform lineage tracing of proliferative cells in the CNS. Another approach we used
to examine the effects of reduced Notch signalling was to generate double hemizygous
(Jagged1 /-, Notch1 /-) mice. Mice double heterozygous for Jagged1 and Notch2 have been
used as a model for human Alagille syndrome, a pleiotropic developmental disorder that is
one of the major forms of chronic liver disease in childhood. Preliminary results show that our
double hemizygous Jagged1 /-, Notch1 /- mice die at late embryonic or early postnatal stages
and show a severe phenotype in the rostral migratory stream and the cortex.







6 ZUSAMMENFASSUNG

Ein Dogma der Neurobiologie, das über einen langen Zeitraum vorherrschte, nämlich
dass Gehirne