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.

项目摘要 研究哺乳动物大脑的主要挑战是表征 单个神经元，例如分子特征，完全形态（树突，轴突，突触）， 连通性和活动；此外，这必须以与目标相称的规模完成 了解大脑中所有神经元及其电路。而当前的单细胞转录组和 表观基因组分析技术是高度定量，可扩展性和信息性的，是研究的技术 其他神经元细胞类型定义特性（例如，单神经元的脑部形态和突触连通性） 吞吐量低，劳动力密集，可扩展性差，通常会产生部分数据。新兴的神经元细胞类型 无脊椎动物（例如果蝇）和啮齿动物中的分类研究表明神经元形态学 诸如轴突投影模式之类的数据是相关的，但也可能与定义的单元格独立 通过单细胞基因表达。这是对哺乳动物神经元细胞人口普查的完整而无偏的调查 应包括由单个分子曲线和脑形态组成的正交数据类型 神经元。最后，对于由唯一的转录组轮廓定义的新的细胞类型 细胞型定义的“神经元认同”基因和其他细胞类型特异性特征，例如形态学， 突触连通性和活性仍然难以捉摸，不能以可扩展的方式轻易地表征。 在此提案中（响应RFA MH-21-140），我们将通过建立来解决这些挑战 一种新型的神经技术，称为镶嵌物，并重复移交或Morf。 MORF小鼠可以会议的细胞 - 特定于神经元和神经胶质的特定，稀疏和明亮的标记，以阐明其完整的形态 小鼠大脑。 MORF小鼠的创新方面是使用框外单核苷酸 重复作为随机翻译开关；它的随机移码会导致极端的表达 在1-5％的遗传定义神经元中，明亮的膜结合免疫蛋白蛋白。在这个建议中，我们 将生成四个下一代MORF鼠标模型，该模型将允许：（1）。精确和稀疏标签 基于两个遗传驱动因素的神经元细胞类型（即两个定义神经元细胞类型的分子标记）； （2）。稀疏标记的GABA能和皮质的内源性突触前蛋白的CRE依赖性标记 谷氨酸能神经元； （3）。基因组编辑工具在遗传和稀疏标记中的选择性表达 神经元支持扰动和多个亚细胞标记； （4）。创新和 集成多尺度成像和注册管道，以提供概念验证数据，以大脑分析 遗传定义的单神经元的形态和连通性。我们的赠款共同有助于发展 可推广，可扩展和民主化的工具，以推进神经元形态，突触和 连通性和遗传扰动。这些工具将促进哺乳动物脑细胞人口普查的构建 并在单个神经元的分辨率下推进对大脑发育，功能和疾病的研究。

项目成果

Epitope-preserving magnified analysis of proteome (eMAP).

• DOI: 10.1126/sciadv.abf6589
• 发表时间: 2021-11-12
• 期刊: Science advances
• 影响因子: 13.6
• 作者: Park J;Khan S;Yun DH;Ku T;Villa KL;Lee JE;Zhang Q;Park J;Feng G;Nedivi E;Chung K
• 通讯作者: Chung K