Principles of Tissue-wide and Cell-Autonomous Gene Function in Neocortex Formation

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Understanding the fundamental processes that make the brain unique at a cellular and molecular level is the core focus of my lab. Even though neural stem cells (NSCs) produce an enormous diversity of neurons and glia, our understanding of what defines a cell type in the brain is lacking. To address this, the long-term objective of my research program is to investigate the molecular regulators of NSC lineage progression and their role in determining cell types and states in the developing central nervous system. I study the cerebral cortex, an evolutionarily expanded region in humans associated with higher-level processing. Most, if not all, cortical excitatory neurons and glia are derived from a shared pool of NSCs known as the radial glial progenitors (RGPs). The genetic and epigenetic factors regulating RGP temporal progression along their lineage are primarily unknown. The Notch pathway is a master regulator of RGP proliferation and is indispensable for maintaining the balance between neurogenesis and gliogenesis during development. By incorporating state-of-the-art technologies, my lab will dissect the contribution of Notch signalling in RGP lineage progression at single-cell resolution. This work will significantly expand our knowledge of how large gene modules contribute to complex tissues in a cell-type-specific way. To address this, highly qualified personnel (HQP) will apply Mosaic Analysis with Double Markers (MADM) technology, a single-cell genetic approach for in vivo lineage tracing. This proposal is the first application of these genetic technologies in Canada, with my lab uniquely having both access and expert firsthand experience needed to implement the proposed research program efficiently. MADM technology will be applied in both short-term objectives. Short-term objective 1 will measure the requirements of Notch1 and Rbpj in single RGPs during embryonic development. Notch1 is a cell-surface receptor expressed by RGPs in the cortex and upon activation it is cleaved releasing an intracellular domain. Rbpj is the downstream mediator of Notch signalling, regulating its transcriptional response. These experiments will provide a definitive readout of Notch1 and Rbpj regulation of RGP output in defined cortical regions. Short-term objective 2 will test the hypothesis that Notch activity levels determine the fate of progenitors and their progeny. MADM will dissect the functional importance of cis- and trans-interactions of Notch1 with the ligand, Jagged1, in producing the correct number of neurons and glia. This proposal will also address the broader concept of how cell-intrinsic and tissue-wide gene function contributes to normal brain development and tissue homeostasis. This research program represents a newly emerging area of single-cell biology and genetic research. It will significantly advance our understanding of the molecular biology underlying RGP lineage progression at single-cell resolution while training at least 9 HQP.