Molecular mechanisms regulating neurogenesis in the developing mouse cerebral cortex [Elektronische Ressource] / Luisa Pinto

Luisa Pinto

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2008
• Nombre de lectures: 19
• Langue: English
• Poids de l'ouvrage: 25 Mo

Extrait

Molecular mechanisms regulating neurogenesis in
the developing mouse cerebral cortex









PhD Thesis



Fakultät für Biologie
der Ludwig-Maximilians-Universität München








Prepared at the Helmholtz Center Munich
German Research Center for Environmental Health (GmbH) – Institute of Stem
Cell Research in the group of Prof. Dr. Magdalena Götz









Luisa Pinto
Munich, August 2008



Examiners of the thesis

1. Prof. Dr. Magdalena Götz
2. Prof. Dr. Mark Hübener
3. Prof. Dr. Benedikt Grothe
4. PD. Dr. Angelika Böttger


thThe thesis was submitted in the 14 of August, 2008
thThe day of oral examination was in the 28 of November, 2008







11 Table of content


1 Table of content …………………………………………………………………. 2
2 Abstract ………..................................................................................................... 9
3 Introduction …………………………………………………………………….. 11
3.1 Progenitor cells and neuron production in the CNS .……………………. 11
3.1.1 Population heterogeneity in CNS development .………………. 11
3.1.2 Forebrain development and regionalization .…………………... 12
3.1.3 Radial glia progenitors in the forebrain .……………………….. 13
3.1.4 Radial glial cells as committed progenitors ……………………. 15
3.1.5 Generation of neurons from two different types of progenitors .. 16
3.1.6 Neuronal production timing and specification during brain
development ………………………………………………………........... 18
3.1.7 Transcriptional regulators of cell fate in the developing
forebrain .………………………………………………………………… 19
3.1.8 Isolation of functionally distinct progenitor subsets by FACS ... 20
3.1.9 Adult neurogenesis from astroglia in mammals ………………. 21
3.2 The transcription factor AP2 γ …………………………………………… 23
3.2.1 AP2 family of transcription factors …………………………… 23
3.2.2 Expression and functions of AP2 γ in the developing and adult
mouse …………………………………………………………………….. 24
4 Abbreviations ....................................................................................................... 26
5 Material and Methods …………………………………………………………. 28
5.1 Animals ………………………………………………………………….. 28
5.2 Genotyping of mice ………………………………………………………
5.3 Fixation of mouse brains and cryosections ……………………………… 29
5.4 Immunostaining …………………………………………………………. 30
5.5 In situ hybridization …………………………………………………….. 31
5.6 Cell culture …………………………………………………………….... 31
5.6.1 Cortex dissection and dissociation ……………………………
5.6.2 Clonal analysis and overexpression in vitro ………………….. 32
5.6.3 Neurospheres cultures ……………………………………….... 32
5.6.4 Dual-luciferase reporter assay ………………………………… 33
5.7 Fluorescence activated cell Sorting (FACS) and cell cycle analysis … 33

25.8 RNA isolation and microarray analysis …………………………………. 34
5.9 Molecular biology ………………………………………………………. 36
5.9.1 Plasmids and viral production ……………………………….... 36
5.9.2 Transformation into chemically competent E.coli …………….. 38
5.9.3 DNA-purification ……………………………………………… 38
5.9.4 DNA restriction digest ……………………………………........ 39
5.9.5 Vector dephosphorylation …………………………………….. 39
5.9.6 Ligation ………………………………………………………... 39
5.10 PCR ……………………………………………………………………… 39
5.11 cDNA synthesis and real-time PCR analysis ………………………….... 40
5.12 In vivo injections ………………………………………………………... 40
5.12.1 In utero injections ……………………………………………... 40
5.12.2 Beads injections ………………………………………………. 41
5.13 BrdU labelling in vivo …………………………………………………… 41
5.14 Data analysis ……………………………………………………………. 41
5.14.1 Quantification of fluorescence intensity at the confocal
microscope ……………………………………………………………… 41
5.14.2 Clonal analysis ………………………………………………... 42
5.14.3 Quantification of PH3-positive cells ………………………….. 42
5.14.4 Interkinetic nuclear migration quantifications ………………… 42
5.14.5 Cortical neuronal layers measurements ……………………….. 42
5.14.6 Statistics ……………………………………………………….. 43
5.15 Materials ………………………………………………………………… 43
5.15.1 Kits …………………………………………………………….. 43
5.15.2 Medium ………………………………………………………… 43
5.15.3 Primers …………………………………………………………. 43
5.15.4 Plasmids and vectors …………………………………………… 46
5.15.5 Primary antibodies …………………………………………….. 47
5.15.6 Secondary antibodies ………………………………………….. 47
5.15.7 Solutions ……………………………………………………….. 48
6 Results …………………………………………………………………………… 49
6.1 Lineage analysis at the transcriptome level – new insights into functionally
distinct radial glial subtypes ……………………………………………………… 49
6.1.1 Separation of distinct radial glia-derived lineages …………….. 49

36.1.2 Prospective isolation of distinct radial glia progenitor subtypes . 51
6.1.3 Transcriptome analysis of distinct radial glial lineages ……….. 53
6.1.4 e analysis of distinct radial glial subtypes ………. 55
6.1.5 Clustering analysis of differential gene expression in subtypes of
E14 radial glial cells …………………………………………………….. 58
low 6.1.5.1 GFP /prominin+ enriched mRNAs ……………… 58
high 6.1.5.2 GFP /promiRNAs ……………... 59
6.1.6 Cell surface genes identified for distinct radial glial subtypes ... 59
6.1.7 Summary of the transcriptome and lineage of distinct radial glia
subsets isolated at mid-neurogenesis …………………………………….. 60
6.1.8 Characterization of cortical radial glial cells at the end of
neurogenesis ……………………………………………………………… 61
6.1.9 Comparison of primary radial glial subsets with radial glia derived
from embryonic stem cells ……………………………………………….. 63
6.2 Expression and function of the transcription factor AP2 γ in a subset of cortical
radial glia ………………………………………………………………………… 64
6.2.1 AP2γ expression in the developing and adult mouse cerebral
cortex …………………………………………………………………….. 64
6.2.2 AP2γ expression in the adult mouse brain …………………….. 65
6.2.3 AP2γ expression in the primate and human cortex ……………. 66
6.2.4 AP2γ deletion in the developing cortex causes decrease in the
number of neurons and misspecification of basal, but not apical progenitors at
mid-neurogenesis ……………………………………………………….... 67
6.2.5 Transcriptome analysis of AP2 γ-/- cortices at mid-neurogenesis 70
6.2.6 Cell death of basal progenitors in the AP2 γ-/- cortex …………. 73
6.2.7 Defects in neuron generation and basal progenitor specification
in the AP2 γ-/- cortex at the end of neurogenesis ………………………... 73
6.2.8 AP2γ deletion reduces upper layer neuron generation ………… 74
6.2.9 Defects in layers II/III callosal projection neurons in the adult
AP2 γ-/- occipital cortex …………………………………………………. 76
6.2.10 Functional defects in visual acuity in the AP2 γ-/- mice ………. 77
6.2.11 AP2 γ overexpression increases the generation of basal
progenitors ………………………………………………………………. 78
6.2.12 Molecular mechanisms regulating AP2 γ in the cortex ………… 80

47 Figures and Tables ............................................................................................... 82
Figure 1: Subtypes of progenitors present in the embryonic mammalian cortex during
development ……………………………………………………………………… 83
Figure 2: Distinct subsets of radial glial cells in the dorsal and ventral
telencephalon …………………………………………………………………….. 85
Figure 3: Astrocytic heterogeneity in the adult mammalian brain ………………. 86
Figure 4: Schematic representation of the genomic fragment and protein structure of
the transcription factor AP2 γ …………………………………………………….. 87
Figure 5: FACS isolation and clonal analysis of distinct radial glia-derived
lineages …………………………………………………………………………... 88
Figure 6: Neurospheres forming capacity of distinct radial glia-derived lineages . 90
Figure 7: Isolation and characterization of distinct radial glial subsets ………….. 91
Figure 8: Generation of Tbr2-positive clones by distinct subsets of radial glial
cells ……………………………………………………………………………… 93
Figure 9: RT-PCR analysis of differentially expressed genes between distinct subsets
of radial glia ……………………………………………………………………… 94
Figure 10: Summary of the transcriptome of GFP+(high and low) and GFP+(high and
low)/prominin+ sorted cells ……………………………………………………… 96
Figure 11: In situ hybridization of mRNAs with differential expression levels in the
high lowGFP /GFP and GFP/prominin double positive radial glia subsets …………. 97
Figure 12: Hierarchical clustering of overlapping genes differentially expressed
high lowbetween GFP /GFP fractions and GFP/prominin double positive fractions
isolated from E14 cortex …………………………………………….…………… 98
Figure 13: Prospective isolation of neurogenic radial glia from E14 cortex by staining
of p75, CD83 and Flrt2 cell surface markers…………………………………...... 99
Figure 14: Summary of the lineage analysis of GFP+(high and low) and GFP+(high
and low)/prominin+ sorted cells …………………………………………………. 100
Figure 15: Characterization of cortical radial glial cells at the end of neurogenesis
(E18) …………………………………………………………………………….. 101
Figure 16: Hierachical clustering of genes differentially expressed between E18
GFP+/prominin+ and E14 GFP/prominin radial glia subsets …………………… 102
Figure 17: In