Defining Novel Functions Of Centrosomes And Primary Cilia In Neocortical Development

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The development and evolutionary expansion of mammalian cerebral cortex depends on spatiotemporal control of neural progenitor proliferation. Radial glial progenitors (RGPs) are the principal neural stem cell population responsible for producing virtually all cortical neurons. Centrosome is an important organelle in mammalian cell proliferation and differentiation. It serves as a microtubule-organizing center (MTOC) as well as the basal body for ciliogenesis. Unlike typical mammalian cells, RGPs position centrosomes far away from nuclei in the apical endfeet and project primary cilia into the brain ventricles. Interestingly, genetic mutations in genes encoding centrosomal and ciliary proteins underlie many congenital brain malformation disorders. However, the lack of in vivo functional analysis of these cellular organelles in this unique biological context has limited our understanding of the mechanisms underlying normal and diseased development. My dissertation research aimed to address this question by using genetically modified mouse models to define the functions of centrosomes and primary cilia in cortical development. In order to genetically interrogate the function of primary ciliogenesis, I compared conditional knockout (cKO) model of Cep83, together with a second mouse line of Ift88 cKO. These mouse lines were chosen to strategically disrupt the pathway at two distinct steps— ¬¬ basal body docking and axoneme extension. This stepwise genetic approach demonstrated that centrosome positioning controls the physical property of RGPs and consequently their proliferative and neurogenic behaviors. In particular, selective removal of CEP83, a protein required for mother centriolar distal appendage (DAP) assembly, in mouse cortical RGPs eliminates DAPs and disrupts apical membrane docking of centrosome, resulting in apical surface expansion and increased stiffness. Interestingly, this physical change activates mechanically sensitive YAP signaling that promotes RGP proliferation and cortical neurogenesis, eventually leading to an enlarged cortex with abnormal folding. In stark contrast, only subtle, if any, brain defects were observed in Ift88 cKO. Together, these results demonstrate a previously unknown role of centrosome positioning in regulating mechanical features of neural progenitors and therefore the size and formation of the mammalian cortex. Building upon our previous work on Sas4 cKO to characterize cortical neurogenesis in the absence of centrioles, I further analyzed functions of other centrosomal structures in cortical development, including pericentriolar material (PCM), subdistal appendages (sDAPs), and intercentriolar linker. While the loss of sDAPs and/or intercentriolar linker did not yield any significant brain phenotype, the removal of a representative PCM protein PERICENTRIN (PCNT) in a mouse gene-trap line (PcntGt) substantially phenocopied Sas4 cKO. In particular, PcntGt brain exhibited microcephaly due to P53-dependent cell death of neural progenitors. Despite a partial rescue of microcephaly by p53 loss, RGPs of PcntGt p53-/- suffered from progenitor delamination, mitotic delay, and spindle misorientation, albeit to a lesser extent compared to Sas4 p53 cDKO. Together, these results demonstrate that MTOC activity mediated by PCMs is the major function of centrosome in cortical development. In summary, this dissertation defined a novel biophysical function of centrosome positioning in cortical development and determined MTOC as the primary function of centrosome in neural progenitor proliferation.
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Centrosome; Cortical neurogenesis; Megalencephaly; Microcephaly; Primary Cilia; Radial glial progenitor
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Cell & Developmental Biology
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Doctor of Philosophy
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Government Document
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Attribution-NonCommercial-NoDerivatives 4.0 International
dissertation or thesis
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