High Throughput Clonal Analyses of Gliogenesis in Neocortical and Paleocortical areas of the Mouse Brain

项目摘要 大脑在胚胎和出生后早期发育过程中严重依赖于神经元和神经胶质的平衡产生。 最近发展的克隆谱系分析揭示了神经干细胞（NSC）在神经元分化中的行为。 大脑皮层前所未有的单细胞分辨率。然而，克隆原则的形成， 神经干细胞的神经胶质细胞仍然不清楚，尚未使用这些新技术进行系统研究。胶质细胞生成是 它对正常的神经元功能至关重要，当它被破坏时，可能导致各种神经系统疾病。重建 神经胶质细胞是如何从单个神经干细胞中产生并在发育过程中在皮层中组织起来的， 结构-功能关系以及它们如何被克隆特异性因子调节。我们建立了 基于遗传的单细胞谱系追踪技术，利用MADM（双标记镶嵌分析）小鼠， 标记发育中的皮层神经干细胞，并开始解决这一知识缺口。通过这种方法，我们发现了两个 不同的神经胶质细胞群占据了大脑皮层及其相关结构的不同区域 阵这项研究的目标是重建、量化和数学建模 单独标记的NSC在新皮层和古皮层区域的体内。这项工作需要发展 优化的成像和分析工具，以确保定量数据的可靠和可重复的解释。为此我们 正在开发光片显微镜和基于人工智能的自动量化方法，以促进公正和精确的 脑中克隆数据的成像和定量。成功完成我们的研究将导致全面的 单个神经干细胞及其神经胶质后代在不同皮质区域的图谱。我们的方法还将建立一个平台， 详细的定量和计算分析胶质细胞生成，胶质细胞的多样性，以及它们的修复潜力， 在各种神经系统疾病和脑损伤的背景下，在皮层中的再生方法。 更广泛影响的潜力 我们了解大脑重要组成部分神经胶质细胞如何发育的方法具有广泛的意义。 神经胶质发育的中断是脑中一系列病理状况的根源。因此了解 控制胶质细胞生成的基本原理和细胞机制不仅对理解健康的 发育可能受神经胶质细胞系统性生成的控制，但神经胶质细胞生成的异常也可能 导致毁灭性的神经发育障碍和脑瘤。

Project Summary The brain critically relies on balanced production of neurons and glia during embryonic and early postnatal development. Recently developed clonal lineage analysis has revealed the behavior of neural stem cells (NSCs) giving rise to neurons in the cerebral cortex with unprecedented single-cell resolution. However, the clonal principles underlying the formation of glia by NSCs remains unclear and has yet to be systematically investigated using these new technologies. Gliogenesis is critical for proper neuronal functions and when disrupted, it can result in various neurological diseases. Reconstructing how glia are generated from individual NSCs and organized in the cortex during development is essential to understand the structure-function relationships and how they can be modulated by clone-specific factors. We have established a genetically-based single-cell lineage tracing technique utilizing MADM (Mosaic Analysis with Double Markers) mice to label NSCs in the developing cortex and begin to address this knowledge gap. Using this method we have found two distinct populations of glia that occupy different territories of the cortex and its related structure the hippocampal formation. The goal of the proposed research is to reconstruct, quantify, and mathematically model the behavior of individually labeled NSCs in vivo in neocortical and paleocortical areas. This effort requires the development of optimized imaging and analytical tools to ensure reliable and repeatable interpretation of quantitative data. To this end we are developing light sheet microscopy and AI-based automated quantification methods to facilitate unbiased and precise imaging and quantification of clonal data in the brain. Successful completion of our study will result in a comprehensive map of single NSCs and their glial progeny in various cortical regions. Our approach will also establish a platform for detailed quantitative and computational analysis of gliogenesis, glial diversity, and their potential for repair and regenerative approaches in the cortex in the context of various neurological disorders and brain injury. Potential for Broader Impact Our approaches to understand how important constituents of the brain, the glial cells, develop have wide implications. Disruption of glial development is the root of a range of pathological conditions in the brain. Therefore, understanding the basic principles and cellular mechanisms that control gliogenesis is critical to appreciate not only how healthy development may be controlled by systematic production of glial cells, but also how abnormalities in gliogenesis may lead to devastating neurodevelopmental disorders and brain tumors.