Browsing by Subject "cortical development"
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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 Roles for mRNA Regulation in Mammalian Brain Development and Neurodevelopmental Disorders(2018) Lennox, AshleyThe cerebral cortex is an anatomically complex brain structure that controls our higher cognitive functions such as abstract thought and language. The cortex is largely shaped during embryonic development when radial glial progenitors divide and differentiate to produce neurons. Neurons are organized into 6-layers through migration guided by the structural support of radial glial cells. Disruptions in progenitor proliferation or neuronal migration underly diverse neurodevelopmental disorders with life-long impacts on cognitive, psychiatric, and motor functions. Developmental mechanisms that build the brain are regulated by precise gene-expression networks. Here, we uncover two novel layers of post-transcriptional mRNA regulation in the developing cortex. First, we used model models to describe a widespread phenomenon of mRNA localization to the distal structures—the basal process and endfeet—of radial glial progenitors. With live imaging approaches, we detected active mRNA transport to and local translation within radial glial endfeet. Transcriptomic and proteomic analyses revealed that endfeet contain cytoskeletal, signaling, and proteostasis factors that may be locally and dynamically controlled. The second line of investigation focused on the RNA-helicase DDX3X which is frequently mutated in neurodevelopmental disorders. We discovered that Ddx3x is required both in progenitor differentiation and neuronal migration in the developing mouse cortex. DDX3X mutations varied from loss-of-function to missense alleles, and a subset of missense variants caused severe cortical malformations in patients. Biochemical and cell biological assays revealed that dominant missense mutations reduced DDX3X helicase activity and induced formation of RNA-protein aggregates associated with impaired translation, uncovering novel pathologies underlying developmental disorders. Together, these studies extend our understanding of post-transcriptional regulation in brain development and neurodevelopmental disorders.