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

了解使大脑在细胞和分子水平上独一无二的基本过程是我实验室的核心重点。尽管神经干细胞（NSC）产生了大量的神经元和神经胶质细胞，但我们对大脑中细胞类型的定义缺乏了解。为了解决这个问题，我的研究计划的长期目标是研究NSC谱系进展的分子调节因子及其在确定发育中的中枢神经系统的细胞类型和状态中的作用。我研究的是大脑皮层，这是一个在人类进化过程中不断扩大的区域，与更高层次的处理有关。大多数（如果不是全部的话）皮质兴奋性神经元和神经胶质细胞来源于被称为放射状神经胶质祖细胞（RGP）的NSC的共享池。调节RGP时间进程的遗传和表观遗传因素沿着其谱系主要是未知的。Notch通路是RGP增殖的主要调节因子，并且对于维持发育过程中神经发生和胶质发生之间的平衡是不可或缺的。通过结合最先进的技术，我的实验室将在单细胞分辨率下剖析Notch信号在RGP谱系进展中的贡献。这项工作将大大扩展我们对大基因模块如何以细胞类型特异性方式对复杂组织做出贡献的知识。为了解决这一问题，高素质人员（HQP）将应用双标记镶嵌分析（MADM）技术，这是一种用于体内谱系追踪的单细胞遗传方法。这项提案是这些基因技术在加拿大的首次应用，我的实验室拥有有效实施拟议研究计划所需的独特访问和专家第一手经验。MADM技术将应用于这两个短期目标。短期目标1将测量胚胎发育期间单个RGP中Notch1和Rbpj的需求。Notch1是由皮质中的RGP表达的细胞表面受体，并且在激活时其被切割释放细胞内结构域。Rbpj是Notch信号传导的下游介质，调节其转录反应。这些实验将提供一个明确的读出的Notch1和Rbpj的RGP输出的规定皮层区域的调节。短期目标2将检验Notch活性水平决定祖细胞及其后代命运的假设。MADM将剖析Notch1与配体Jagged1的顺式和反式相互作用在产生正确数量的神经元和神经胶质中的功能重要性。这项提案还将解决更广泛的概念，细胞内在和组织范围的基因功能如何有助于正常的大脑发育和组织稳态。该研究项目代表了单细胞生物学和遗传学研究的一个新兴领域。它将显著推进我们对RGP谱系进展的分子生物学基础的理解，同时训练至少9个HQP。

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.