Developing cell type-specific enhancers and connectivity mapping pipelines for marmosets

项目摘要 尽管遗传工具极大地促进了我们对大脑功能的理解， 主要局限于小鼠。虽然小鼠是神经科学许多领域的基本模型，但也有一些研究。 高级大脑功能的许多方面无法在啮齿动物中充分建模。同样，许多大脑 这些疾病影响高级认知功能，在啮齿类动物中没有明显的相似之处。此外，近期大... 大规模的单细胞转录组学分析揭示了许多神经元类型、连接和基因表达 灵长类动物特有的模式因此，迫切需要新的遗传模型， 结构和功能更接近人类。非人类灵长类动物（NHP）与人类的关系更为密切 比啮齿类动物更好，这反映在它们的大脑发育，结构和生理上。因此， 越来越多的人认识到，它们为研究高级脑功能和脑疾病提供了一个有吸引力的模型。 一个有希望的新兴NHP模型是普通的绒猴，一种新世界的小型灵长类动物， 神经科学和遗传学研究的优势。然而，缺乏具有细胞类型特异性的工具一直是一个难题。 这是推进NHP结构和功能研究的主要障碍。使用组合的单细胞RNA-seq 和单细胞ATAC-seq，现在可以指定短细胞类型特异性增强子序列。如果 经验证，这些增强子将提供一种有效的工具来映射连接和询问功能， 病毒介导的表达。困难在于从数百种或更多种功能增强剂中鉴定功能增强剂。 NHP中数千个提名的潜在增强子序列。在这里，我们建议（1）使用一种新的高 通过体内方法鉴定功能增强子，以及（2）建立全脑回路映射 管道使用纹状体电路，以验证我们的方法，细胞类型特异性连接映射， 绒猴这些研究完成后，将提供急需的基本工具、方法和 用于绒猴脑细胞类型特异性映射和功能询问的计算管道， 健康和疾病模型。

PROJECT SUMMARY Although genetic tools have dramatically advanced our understanding of brain function, they have largely been confined to mice. While mice are essential models for many areas of neuroscience, there are also many aspects of higher brain function that cannot be adequately modeled in rodents. Similarly, many brain disorders affect higher cognitive functions that have no clear parallels in rodents. Furthermore, recent large- scale single cell transcriptomic analyses have revealed many neuron types, connections and gene expression patterns that are unique to primates. Thus, there is an urgent need for new genetic models that have brain structure and function closer to humans. Non-human primates (NHP) are much more closely related to humans than are rodents, and this is reflected in their brain development, structure and physiology. Hence, it is increasingly recognized that they provide an attractive model to study higher brain function and brain disorders. A promising emerging NHP model is the common marmoset, a small new world primate that has many advantages for neuroscience and genetic research. However, lack of tools with cell type specificity has been a major obstacle in advancing structural and functional studies in NHP. With the combined single cell RNA-seq and single cell ATAC-seq, it is now possible to nominate short cell type-specific enhancer sequences. If validated, these enhancers will provide an effective tool to map connectivity and interrogate function using virus mediated expression. The difficulty lies in the identification of functional enhancers from the hundreds or thousands of nominated potential enhancer sequences in NHP. Here we propose (1) to use a novel high throughput in vivo approach to identify functional enhancers, and (2) to establish a whole-brain circuit mapping pipeline for use striatal circuitry to validate our approach for cell type-specific connectivity mapping in marmosets. When completed, these studies will provide much needed essential tools, methods and computational pipelines for cell type-specific mapping and functional interrogation of the marmoset brain in healthy and disease models.