Table of figures ................................................ 6
Table of tables ................................................. 7
Abbreviations ................................................... 8
Summary ......................................................... 9
Acknowledgements ............................................... 11
1 Introduction ................................................ 12
1.1 Development of the mammalian neocortex ................. 12
1.1.1 The mammalian embryonic neural tube ............. 12
1.1.2 The development and organisation of the
neocortex ....................................... 13
1.2 Neural progenitor cells of the mammalian neocortex ..... 15
1.2.1 Neuroepithelial cells ........................... 15
1.2.2 Radial glial cells .............................. 15
1.2.3 Basal (intermediate) progenitor cells ........... 15
1.2.4 Neural progenitor lineages and modes of
division ........................................ 16
1.2.5 Mitosis of neural progenitor cells .............. 17
1.2.6 Apoptosis of neural progenitor cells ............ 17
1.3 Neural progenitor proliferation and differentiation .... 18
1.3.1 Onset and gradient of neurogenesis .............. 18
1.3.2 Symmetric versus asymmetric progenitor cell
division ........................................ 18
1.4 Regulation of neocortex size and cytoarchitecture ...... 19
1.4.1 Radial unit hypothesis and cortical size ........ 19
1.4.2 Subventricular zone progenitors and cortical
size ............................................ 19
1.4.3 Radial neuronal migration ....................... 20
1.4.4 Tangential neuronal migration ................... 20
1.4.5 Human genetic disorders of cortical
development ..................................... 21
1.4.6 Neural progenitor apoptosis and brain size
regulation ...................................... 22
1.5 Autosomal recessive primary microcephaly ............... 23
1.5.1 Clinical definitions ............................ 23
1.5.2 Mapped loci and identified genes ................ 23
1.5.3 Molecular evolution of microcephaly genes ....... 24
1.6 Aspm ................................................... 26
1.6.1 Primary microcephaly (MCPH5) .................... 26
1.6.2 Aspm protein domains and interaction partners ... 28
1.6.3 Expression pattern and subcellular
localisation .................................... 29
1.6.4 Abnormal spindle - Drosophila melanogaster ...... 29
1.6.5 Role of ASPM in brain development ............... 30
1.6.6 ASPM and brain size evolution ................... 30
1.7 Brca1 .................................................. 31
1.7.1 Tumour suppression and breast cancer
susceptibility .................................. 31
1.7.2 Brca1 protein domains and interaction
partners ........................................ 31
1.7.3 Expression pattern and subcellular
localisation .................................... 32
1.7.4 Brca1 knockout mouse models ..................... 32
1.7.5 Role of Brca1 in brain development .............. 32
1.8 Aims of this study ..................................... 33
1.8.1 Regulation of cortical size ..................... 33
1.8.2 The function of Aspm in cortical development .... 33
1.8.3 The function of Brca1 in cortical development ... 33
2 Results ..................................................... 34
2.1 Role of Aspm in the development of the neocortex ....... 34
2.1.1 Generation of Aspm gene trap mutant mice ........ 34
2.1.2 Generation of ASPM humanised mice ............... 36
2.1.3 Brain size reduction in Aspm mutants, rescued
by HsASPM ....................................... 36
2.1.4 Subcellular localisation of mutant Aspm
proteins ........................................ 39
2.1.5 Mitotic progression and cleavage plane
orientation ..................................... 43
2.2 Role of Brca1 in the development of the neocortex ...... 45
2.2.1 Cerebral cortex-specific ablation of Brca1 ...... 45
2.2.2 Conditional Brca1 ablation results in smaller
neocortex ....................................... 45
2.2.3 Cortical layering is not perturbed upon Brca1
ablation ........................................ 47
2.2.4 Reduction in deep-layer but not upper-layer
neurons upon Brca1 ablation ..................... 50
2.2.5 Ventricular surface and progenitor number
reduction upon Brca1 ablation ................... 51
2.2.6 Conditional Brca1 ablation causes apoptosis in
progenitors and neurons ......................... 51
2.2.7 Conditional Brca1 ablation does not alter the
mitotic index ................................... 55
2.2.8 No alteration in the Tbr2 lineage upon Brca1
ablation ........................................ 57
2.2.9 Increase in nuclear translocation of p53 upon
Brca1 ablation .................................. 58
3 Discussion .................................................. 60
3.1 Role of Aspm in the development of the neocortex ....... 60
3.1.1 Aspm as a regulator of brain size ............... 60
3.1.2 Aspm and regulation of cleavage plane
orientation ..................................... 61
3.1.3 Function of the Aspm C-terminal domain .......... 62
3.2 Role of В real in the development of the neocortex ..... 64
3.2.1 Brca1 and the regulation of neocortex size ...... 64
3.2.2 Specificity and extent of increased apoptosis ... 64
3.2.3 Molecular cause of increased apoptosis upon
Brca1 ablation .................................. 66
3.2.4 Role of Brca1 in mammalian brain evolution ...... 66
4 Materials and Methods ....................................... 67
4.1 Materials .............................................. 67
4.1.1 Equipment ....................................... 67
4.1.2 Chemicals ....................................... 68
4.1.3 Reagents and buffers ............................ 68
4.1.4 Kits ............................................ 70
4.1.5 Antibodies ...................................... 71
4.1.6 Oligonucleotides ................................ 71
4.1.7 Plasmids ........................................ 73
4.1.8 Bacterial strains and cell lines ................ 73
4.1.9 Mouse lines ..................................... 73
4.1.10 Computer software ............................... 74
4.2 Methods ................................................ 75
4.2.1 Tissue dissection, processing, and sectioning ... 75
4.2.2 Cell culture and transfection ................... 75
4.2.3 Immunofluorescence staining ..................... 75
4.2.4 TUNEL staining .................................. 75
4.2.5 BrdU administration and detection ............... 75
4.2.6 In situ hybridisation ........................... 76
4.2.7 Nissl staining .................................. 76
4.2.8 Nucleic acid extraction and purification, PCR,
and cloning ..................................... 76
4.2.9 Genotyping of mouse lines ....................... 77
4.2.10 Brain size measurement on histological
sections ........................................ 78
4.2.11 Microscopy and image processing ................. 78
4.2.12 Statistical data analysis and graph plotting .... 78
5 References .................................................. 79
6 List of publications ........................................ 97
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