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«Dissertation for the attainment of the academic degree of Doctor rerum naturalium Given by the Fakultät Mathematik und Naturwissenschaften of the ...»

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The evolution of neuronal progenitor cell division in

mammals: The role of the abnormal spindle-like

microcephaly associated (Aspm) protein and epithelial cell


Dissertation for the attainment of the academic degree of Doctor rerum naturalium

Given by the Fakultät Mathematik und Naturwissenschaften of the Technische

Universität Dresden

Jennifer Fish

Born on the 7th of May, 1972 in Mansfield, Ohio (USA)

Table of contents

Table of contents


I Introduction I Brain Size and Evolution I - 1. Development of the Vertebrate Brain I - 1.1. From neural plate to neural tube I - 1.2. From neural tube to neurogenesis I - 1.3. The vertebrate telencephalon I - 2. Development of the Mammalian Isocortex I - 2.1. Origin and migration of cortical neurons I - 2.2. Isocortical germinal layers I - 2.2.1. Isocortical layering I - 3. Evolution of the Mammalian Isocortex I - 3.1. Evolutionary increase in the SVZ and upper layer neurons I - 3.2. Evolutionary increase in cortical interneurons I - 3.3. Evolutionary increase in surface area I - 4. Lateral Expansion: Hypotheses and Processes I - 4.1. The radial unit hypothesis and Smart’s two rules I - 4.2. The intermediate progenitor hypothesis I - 4.3. Cellular processes implicated in lateral expansion I - 5. Regulation of Symmetric Versus Asymmetric Cell Division I - 5.1. Cell polarity in Drosophila neuroblasts I - 5.2. Regulation of spindle orientation in the Drosophila CNS I - 5.3. Cell polarity in mammalian NE cells I - 5.4. Regulation of spindle orientation in the mammalian CNS I - 6. The abnormal spindle-like microcephaly associated (Aspm) Protein I - 6.1. Primary microcephaly I - 6.2. ASPM mutations I - 6.3. Aspm/ASPM expression in development, adult tissues, and cancer I - 6.4. Adaptive evolution of ASPM I - 6.5. Asp, the Drosophila ortholog of ASPM I - 6.6. Aspm function in mammals I - 7. Aims of this Study I - 7.1. Candidate analysis: Aspm I - 7.2. Comparative analysis: Primate and rodent progenitors II Results II - 1. Candidate Analysis: Aspm II - 1.1. Expression and localization of Aspm in the mouse neuroepithelium II - 1.2. Aspm is down-regulated in NE cells undergoing neurogenic divisions II - 1.3. Knock-down of Aspm results in its loss from spindle poles II - 1.4. Knock-down of Aspm perturbs vertical cleavage plane orientation II - 1.5. Loss of Aspm promotes asymmetric cell division II - 1.6. Increased non-NE fate of NE cell progeny after Aspm knock-down II - 1.7. Increased neuron-likefate of NE cell progeny after Aspm knock-down II - 1.8. Loss of Aspm affects mitotic cells in anaphase II - 1.9. Model for Aspm function during symmetric NE cell divisions II - 1.10. Strategy for testing ASPM in an evolutionary context II - 2. Comparative Analysis: Rodent and Primate Progenitors II - 2.1. Pax6 and Tbr2 in rodent neuronal progenitors II - 2.2. Pax6 in primate basal neuronal progenitors II - 2.3. Pax6 and Tbr2 in human neuronal progenitors II - 2.4. The epithelial characteristics of primate basal neuronal progenitors

III Discussion

III - 1. ASPM and the Evolution of NE Cell Division III - 1.1. Aspm has functionally diverged from Asp III - 1.2. The role of Aspm in mammalian development and evolution III - 1.3. Evolution of the regulation of asymmetric division III - 1.4. Evolution of the regulation of spindle orientation III - 1.5. Evolution of spindle precision III - 1.6. ASPM evolution: Selection for other cellular roles?

III - 2. Constraints on NE Cell Proliferation III - 2.1. The apical membrane as a cell fate determinant III - 2.2 Basal mitotic populations and the SVZ III - 2.3. Epithelial versus non-epithelial progenitors III - 2.4. Delayed versus extended differentiation III - 3. Future Perspectives

–  –  –

IV - 1. Materials IV - 1.1. Antibodies IV - 1.2. Mouse strains and tissue samples IV - 1.2.1 Mouse strains IV - 1.2.2 Monkey tissue IV - 1.2.3 Human tissue IV - 1.3. Buffers and culture media IV - 1.4. Chemicals, enzymes, DNA standards and films IV - 1.5. Devices and computer applications IV - 1.6. Commercial Kits IV - 1.7. Oligonucleotides IV - 1.8. Plasmids IV - 2. Methods IV - 2.1. DNA preparation IV - 2.1.1. Miniprep (QUIAprep spin Miniprep Kit) IV - 2.1.2. Maxiprep (Endofree Plasmid Maxi Kit) IV - 2.2. Genomic cloning via the polymerase chain reaction IV - 2.3. Aspm antibody IV - 2.4. Embryo electroporation IV - 2.4.1. In utero electroporation IV - 2.4.2. Ex utero electroporation and whole embryo culture IV - 2.4.3. Aspm knock-down IV - 2.5. Immunoflourescence IV - 2.6. In situ hybridization IV - 2.6.1. Preparation of DIG labeled probe IV - 2.7. Western Blot IV - 2.8. Mouse handling, embryo collection, and fixation IV - 2.9. Confocal microscopy IV - 2.10. Quantitative Data Analysis IV - 2.10.1. Assessment of Aspm immunofluorescence intensity in Tis21-GFP–negative versus Tis21GFP–positive NE cells IV - 2.10.2. Analysis of cleavage plane orientation and apical membrane distribution IV - 2.10.3. Quantification of abventricular centrosomes IV - 2.10.4. Quantification of Tis21-GFP–negative versus Tis21-GFP–positive NE cell progeny

–  –  –

Among mammals, primates are exceptional for their large brain size relative to body size. Relative brain size, or encephalization, is particularly striking among humans and their direct ancestors. Since the human-chimp split 5 to 7 million years ago, brain size has tripled in the human lineage (Wood & Collard 1999). The focus of this doctoral work is to investigate some of the cell biological mechanisms responsible for this increase in relative brain size. In particular, the processes that regulate symmetric cell division (ultimately generating more progenitors), the constraints on progenitor proliferation, and how neural progenitors have overcome these constraints in the process of primate encephalization are the primary questions of interest. Both functionally analyses in the mouse model system and comparative neurobiology of rodents and primates are used here to address these questions.

Using the mouse model system, the cell biological role of the Aspm (abnormal spindle-like microcephaly associated) protein in regulating brain size was investigated. Specifically, Aspm function in symmetric, proliferative divisions of neuroepithelial (NE) cells was analyzed. It was found that Aspm expression in the mouse neuroepithelium correlates in time and space with symmetric, proliferating divisions. The Aspm protein localizes to NE cell spindle poles during all phases of mitosis, and is down-regulated in cells that undergo asymmetric (neurogenic) cell divisions. Aspm RNAi alters the division plane in NE cells, increasing the likelihood of premature asymmetric division resulting in an increase in non-NE progeny. At least some of the non-NE progeny generated by Aspm RNAi migrate to the neuronal layer and express neuronal markers. Importantly, whatever the fate of the non-NE progeny, their generation comes at the expense of the expansion of the proliferative pool of NE progenitor cells.

These data have contributed to the generation of an hypothesis regarding evolutionary changes in the regulation of spindle orientation in vertebrate and mammalian neural progenitors and their impact on brain size. Specifically, in contrast to invertebrates that regulate the switch from symmetric to asymmetric division through a rotation of the spindle (horizontal versus vertical cleavage), asymmetric NE cell division in vertebrates is accomplished by only a slight deviation in the cleavage plane away from the vertical, apical-basal axis. The requirement for the precise alignment of the spindle along the apical-basal axis in symmetric cell divisions may have contributed to selection on spindle “precision” proteins, thus increasing the number of symmetric NE cell division, and contributing to brain size increases during mammalian evolution.

Previous comparative neurobiological analyses have revealed an increase in basally dividing NE cells in the brain regions of highest proliferation and in species with the largest brains (Smart 1972a,b; Martinez-Cerdeno et al. 2006). The cell biological characteristics of these basally dividing cells are still largely unknown. We found that primate basal progenitors, similar to rodent apical progenitors, are Pax6+.

This suggests that primate basal progenitors may share other properties with rodent apical progenitors, such as maintenance of apical contact. Our previous finding that artificial alteration of cleavage plane in NE cells affects their ability to continue proliferating supports the hypothesis that the apical membrane and junctional complexes are cell fate determinants (Huttner & Kosodo 2005). As such, the need to maintain apical membrane contact appears to be a constraint on proliferation (Smart 1972a,b; Smart et al. 2002). Together, these data favor the hypothesis that primate basally dividing cells maintain apical contact and are epithelial in nature.

I. Introduction

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Mammals are a class of vertebrates recognized for their elaborate social behaviors and intelligence (Figure 1A). Correspondingly, large brain size relative to body size, or encephalization, has evolved in multiple mammalian lineages. The evolution of relatively large brains is particularly evident in the primate lineage, especially among humans and their recent ancestors. The human brain is roughly 3 times as large as the chimp brain, which is remarkable given the similar body size of these two species (Figure 1B). The evolutionary mechanisms responsible for this dramatic increase in brain size have been the basis of scientific research for decades.

To fully understand the pattern and process of brain size evolution, it is useful to consider this question in its historical context. The mechanisms involved in the development of the vertebrate central nervous system (CNS) have provided the foundation for subsequent evolutionary change. From this basis, the cell biological processes regulating proliferation, as well as proliferative constraints, can be interpreted in order to understand the evolutionary changes that have generated the exceptional human brain.

Figure 1: Evolution and brain size in mammals. A, Mammalian phylogeny showing the evolutionary relationships of the mammalian orders. Primates (green asterisk) and Rodentia (red asterisk), the primary orders discussed in this thesis, are highlighted. Image modified from Nishihara et al. 2006. B, Lateral views of adult human, chimp and mouse brains showing their relative size.

Green and red asterisks reflect the position of these species on the phylogeny presented in A. Scale bar, 5 cm. Images modified from Hill & Walsh 2005.

–  –  –

I – 1.1. From neural plate to neural tube The first recognizable manifestation of the brain in the developing vertebrate embryo is the neural plate. The neural plate is induced from the underlying mesoderm, and progressively differentiates along a rostral to caudal gradient.

Initially, the neural plate consists of a monolayer of cuboidal neuroepithelial (NE) cells exhibiting both apical-basal polarity and planar cell polarity (Strutt 2003; Wang et al. 2006). Apical-basal polarity in NE cells is manifested by apical and basolateral membrane compartments with distinct lipid and protein content that are separated by tight junctions (Aaku-Saraste et al. 1996).

At the onset of neurulation, the neural plate invaginates, creating the neural groove. The lateral margins of this groove extend outward into neural folds. These lateral neural folds initially grow convexly into a bulge that faces opposite to the direction of neural tube closure. Convexity is reversed, at least in part, by the elongation of NE cells which reduces their apical area (Jacobson & Tan 1982). This process lengthens and narrows NE cells, leading to convergent extension of the neural plate, resulting in neural tube closure and internalization of the central nervous system. Signaling via the Sonic hedgehog and non-canonical Wnt pathways are required for convergent extension and neural tube closure (Copp et al. 2003; Doudney & Stainer 2005; Wang et al. 2006; Ybot-Gonzalez et al. 2007).

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