Browsing by Subject "radial glia"
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Item Embargo Characterization of Basal Endfeet Reveals Roles for Local Gene Regulation in Radial Glia and Cortical Development(2023) D'Arcy, Brooke RRadial glial cells (RGCs) are essential for the generation and organization of neurons in the cerebral cortex. RGCs have an elongated bipolar morphology with basal and apical endfeet that reside in distinct niches. Yet, how this subcellular compartmentalization of RGCs controls cortical development is largely unknown. Here, we employ in vivo proximity labeling, in the mouse, using unfused BirA to generate the first subcellular proteome of RGCs and uncover new principles governing local control of cortical development. We discover a cohort of proteins that are significantly enriched in RGC basal endfeet, with MYH9 and MYH10 among the most abundant. Myh9 and Myh10 transcripts also localize to endfeet with distinct temporal dynamics. Although they each encode isoforms of non-muscle myosin II heavy chain, Myh9 and Myh10 have drastically different requirements for RGC integrity. Myh9 loss from RGCs decreases branching complexity and causes endfoot protrusion through the basement membrane. In contrast, Myh10 controls endfoot adhesion, as mutants have unattached apical and basal endfeet. Finally, we show that Myh9- and Myh10-mediated regulation of RGC complexity and endfoot position non-cell autonomously controls interneuron number and organization in the marginal zone. The first part of this study demonstrates the utility of in vivo proximity labeling for dissecting local control of complex systems, and reveals new mechanisms for dictating RGC integrity and cortical architecture. In the second portion of this work, we have developed a method for purification of endfeet from the embryonic mouse brain and employed it to discover the first global transcriptome of RGC endfeet. Analysis at E15.5 revealed that the network of localized mRNAs is much more extensive than previously appreciated. There are over 3,000 transcripts localized to RGC endfeet and 870 of them are highly enriched in the endfeet compared to the cell body. These data uncovered hundreds of new genes in endfeet and also reinforced our previous findings that cytoskeletal regulators and ECM components are especially important in endfeet. Exploration of the newly discovered localized transcripts will provide valuable insights into additional RGC functions and allow us to assess potential signaling interactions between endfeet and surrounding cells. We also propose a method for subcellular gene knockdown in which we can modulate mRNA levels of a gene of interest in the cell body and endfeet independently in vivo. Through these studies we have discovered vital roles for subcellular gene regulation in RGCs and developed tools to facilitate future studies.
Item Open Access Effects of prolonged mitosis on neural stem cells in vivo during development(2020) Mitchell-Dick, Aaron MMicrocephaly patients are born with a brain size >3 standard deviations below normal and have mild to severe cognitive deficits. 12 microcephaly-linked genes identified in human genetics studies encode microtubule/centrosome-associated proteins and mutations in these genes are strongly tied to disrupted mitotic processes in neural stem cells during cortical development. Yet, how perturbed neural stem cell mitosis kinetics affects cell fate following neural stem cell division is not well understood. Our lab recently discovered prolonging mitosis of mouse neural progenitors, either ex vivo or in vitro, alters fate decisions forcing early increased neurogenic divisions at the expense of maintaining the stem cell pool. Yet, the consequences of prolonged mitosis in vivo, and directly in human stem cells, remain unexplored. Additionally, how prolonged mitosis mechanistically affects cell stress response and cell fate decisions during development is not well-studied.
Through in vivo pharmacological approaches, and in vitro culture of human neural progenitors, I provide evidence that prolonged mitosis in vivo directly alters cell fate, and that this consequence of prolonged mitosis is conserved from mice to humans. I find prolonged mitosis of neural stem cells in vivo results in increased phosphorylation of H2AX in mitosis, and increased pATR in a subset of newborn cells. P53 is then activated in a subset of daughter cells and upregulates downstream target genes. Within approximately the first cell cycle, prolonged mitosis results in an increase in neurogenic fates in the daughter cell population at the expense of progenitor renewal. Conditional loss of P53 rescues these effects on cell fate, while loss of BAX does not. Additionally, I find that time to cell death occurs on a log-normal distribution within the population. These experiments suggest that identifying the factors sensitive to prolonged prometaphase/mitosis arrest that transduce P53 activating signals is critical for our understanding of microcephaly etiologies. Together, data presented in this thesis suggest prolonged mitosis directly alters cell fate in vivo during cortical development and in human neural stem cells, that response to prolonged mitosis is relatively specific, involving P53 signaling, and that prolonged mitosis is a main contributing factor to microcephaly as a result of mitotic gene disruption.