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

项目摘要 研究哺乳动物大脑的一个主要挑战是表征大脑的整合特性。 单个神经元，如分子概况，完整形态（树突，轴突，突触）， 连接和活动;此外，这必须在与以下目标相称的规模上进行： 了解大脑中的所有神经元及其电路。虽然目前的单细胞转录组学和 表观基因组分析技术是高度定量的，可扩展的和信息丰富的， 其他神经元细胞类型定义特性（例如单神经元全脑形态和突触连接） 低吞吐量、劳动密集型、可扩展性差并且经常产生部分数据。新生神经元细胞类型 在无脊椎动物（如果蝇）和啮齿动物中的分类研究表明， 数据如轴突投影模式是相关的，但也可以独立于所定义的细胞类别 通过单细胞基因表达。因此，一个完整的和公正的调查哺乳动物神经元细胞普查 应包括正交数据类型，包括分子概况和单个脑组织的全脑形态 神经元最后，对于由独特的转录组学特征定义的新出现的细胞类型， 确定细胞类型的“神经元身份”基因和其它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，并且不能以可扩展的方式容易地表征。 在本提案中（响应RFA MH-21-140），我们将通过以下方式应对这些挑战： 一种新的神经技术叫做重复移码镶嵌术，简称MORF。MORF小鼠可以赋予细胞- 类型特异性，稀疏和明亮的标记神经元和神经胶质细胞，以阐明其完整的形态， 老鼠的大脑MORF小鼠的创新之处在于使用了一种框架外的单核苷酸肽 重复作为一个随机的翻译开关;和它的随机移码导致一个极端的表达 明亮的膜结合免疫报告蛋白在1-5%的遗传定义的神经元。在本提案中，我们 将产生四个下一代MORF小鼠模型，将允许：（1）。精确和稀疏标记 基于两种遗传驱动因子（即，定义神经元细胞类型的两种分子标记）的神经元细胞类型; （二）、依赖Cre的稀疏标记GABA能和皮质突触前蛋白的标记 谷氨酸能神经元;（3）。选择性表达基因组编辑工具在遗传和稀疏标记 神经元支持扰动和多重亚细胞标记;和（4）.发展一个创新的， 集成的多尺度成像和配准管道，以提供分析全脑的概念验证数据 形态和遗传定义的单个神经元的连接。我们的资助可以帮助 可推广的，可扩展的和民主化的工具，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普查的建设 并在单个神经元的分辨率上推进大脑发育、功能和疾病的研究。

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.