Next-generation MORF Mice for Scalable Brainwide Morphological Mapping and Genetic Perturbation of Single Neurons

下一代 MORF 小鼠，用于可扩展的全脑形态映射和单神经元的遗传扰动

• 批准号: 10370248
• 负责人: Hong-Wei Dong
• 金额: $ 435.37万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-10 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10370248
• 关键词: 3-Dimensional; Address; Atlases; Axon; BRAIN initiative; Biological; Brain; Brain Mapping; Brain region; Cells; Cellular Morphology; Censuses; Cerebral hemisphere; Characteristics; Classification; DNA; Data; Data Analyses; Dendrites; Development; Disease; Drosophila genus; Feedback; Frequencies; Gene Activation; Gene Expression; Genes; Genetic; Genetic Models; Glutamates; Goals; Grant; Image; Individual; Information Technology; Intrinsic factor; Invertebrates; Label; Light; Link; Maps; Mediating; Membrane; Modeling; Molecular; Molecular Profiling; Morphology; Mosaicism; Motor Cortex; Mus; Neuroglia; Neurons; Organelles; Pattern; Physiology; Property; Proteins; Protocols documentation; Reporter; Reporter Genes; Resolution; Rodent; Role; Structure; Subcellular structure; Surveys; Synapses; System; Techniques; Technology; Testing; base; bioinformatics pipeline; brain cell; cell type; density; epigenome; epigenomics; experience; flexibility; genome editing; high resolution imaging; image registration; imaging informatics; informatics tool; innovation; molecular marker; mouse genetics; mouse model; neurotechnology; next generation; novel; presynaptic; programs; rapid growth; recombinase; reconstruction; response; selective expression; tissue processing; tool; transcriptomics

PROJECT SUMMARY A major challenge in studying the mammalian brain is to characterize the integrative properties of individual neurons, such as molecular profiles, complete morphology (dendrites, axons, synapses), connectivity, and activity; furthermore, this must be done at a scale that is commensurate with the goal of understanding all the neurons and their circuitry in the brain. While current single-cell transcriptomic and epigenomic profiling techniques are highly quantitative, scalable and informative, the technologies to study other neuronal cell-type defining properties(e.g. single-neuron brain-wide morphology and synaptic connectivity) are low throughput, labor intensive, poorly scalable and often yield partial data. Emerging neuronal cell type classification studies in invertebrates (e.g. Drosophila) and in rodents suggest that the neuronal morphological data such as axonal projection patterns are correlated, but may also be independent to the cell classes defined by single-cell gene expression. Thus, a complete and unbiased survey of mammalian neuronal cell census should include orthogonal data types consisting of both molecular profiles and brainwide morphology of single neurons. Finally, for emerging new cell types defined by unique transcriptomic profiles, the causal links between the cell-type-defining “neuronal identity” genes and other cell-type-specific features, such as morphology, synaptic connectivity and activity, remain elusive and cannot be readily characterized in a scalable manner. In this proposal (in response to RFA MH-21-140), we will address these challenges by building upon a novel neurotechnology called Mosaicism with Repeat Frameshift, or MORF. MORF mice can confer cell- type specific, sparse and brightly labeling of neurons and glia to illuminate their complete morphologies in the mouse brain. The innovative aspect of the MORF mice is the use of an out-of-frame mononucleotide repeat as a stochastic translational switch; and its random frameshift leads to the expression of an extremely bright membrane-bound immunoreporter protein in 1-5% of genetically-defined neurons. In this proposal, we will generate four next-generation MORF mouse models that will allow: (1). precise and sparse labeling of neuronal cell types based on two genetic drivers (i.e. two molecular markers that define the neuronal cell type); (2). Cre-dependent labeling of endogenous presynaptic proteins in sparsely labeled GABAergic and cortical glutamatergic neurons; (3). selective expression of genome-editing tools in genetically and sparsely labeled neurons to support perturbation and multiplex subcellular labeling; and (4). development of an innovative and integrative multiscale imaging and registration pipeline to provide proof-of-concept data that analyzes brainwide morphology and connectivity of genetically-defined single neurons. Together, our grant may help to develop generalizable, scalable and democratizable tools to advance the study of neuronal morphology, synapses and connectivity, and genetic perturbation. These tools will facilitate the construction of mammalian brain cell census and advance the study of brain development, function and disease at the resolution of single neurons.

项目概要 研究哺乳动物大脑的一个主要挑战是表征大脑的综合特性 单个神经元，例如分子谱、完整形态（树突、轴突、突触）， 此外，这必须以与目标相称的规模进行。 了解大脑中的所有神经元及其电路。 表观基因组分析技术具有高度定量性、可扩展性和信息丰富性，是研究的技术 其他神经元细胞类型定义属性（例如单神经元全脑形态和突触连接） 吞吐量低、劳动强度大、可扩展性差，并且经常产生部分数据。 无脊椎动物（例如果蝇）和啮齿动物的分类研究表明，神经元形态学 轴突投影模式等数据是相关的，但也可能独立于定义的细胞类别 因此，对哺乳动物神经细胞普查进行了完整且公正的调查。 应包括由分子谱和单个脑的全脑形态组成的正交数据类型 最后，对于由独特的转录组图谱定义的新兴细胞类型，它们之间的因果关系。 细胞类型定义的“神经元身份”基因和其他细胞类型特异性特征，例如形态、 突触连接和活动仍然难以捉摸，并且无法以可扩展的方式轻松表征。 在本提案中（回应 RFA MH-21-140），我们将通过建立在 一种称为重复移码镶嵌（MORF）小鼠的新型神经技术可以赋予细胞- 对神经元和神经胶质细胞进行类型特异性、稀疏且明亮的标记，以阐明它们的完整形态 MORF 小鼠的创新之处在于使用了框外单核苷酸。 作为随机平移开关重复；并且其随机移码导致极端的表达 在该提案中，我们在 1-5% 的基因定义的神经元中发现了明亮的膜结合免疫报告蛋白。 将生成四个下一代 MORF 小鼠模型，这些模型将允许：（1）。 基于两种遗传驱动因素的神经元细胞类型（即定义神经元细胞类型的两种分子标记）； (2). 稀疏标记的 GABA 能和皮质中内源性突触前蛋白的 Cre 依赖性标记 基因组编辑工具的选择性表达和稀疏标记；(3)。 神经元支持扰动和多重亚细胞标记；以及（4）。 集成多尺度成像和配准管道，提供全脑双胞胎的概念验证数据 我们的资助可能有助于基因定义的单个神经元的形态学和连接性的发展。 可推广、可扩展和民主化的工具，以推进神经形态、突触和神经元的研究 连接性和遗传扰动这些工具将有助于哺乳动物脑细胞普查的构建。 并以单个神经元的分辨率推进大脑发育、功能和疾病的研究。

项目成果

Epitope-preserving magnified analysis of proteome (eMAP).

蛋白质组的表位保留放大分析 (eMAP)。

• 发表时间: 2021-11-12
• 影响因子: 13.6
• 作者: Park, Joha;Khan, Sarim;Yun, Dae Hee;Ku, Taeyun;Villa, Katherine L;Lee, Jiachen E;Zhang, Qiangge;Park, Juhyuk;Feng, Guoping;Nedivi, Elly;Chung, Kwanghun
• 通讯作者: Chung, Kwanghun