Unraveling the developmental logic of cortical long-range projections using in situ sequencing-based neuroanatomy

项目摘要 神经元的连通性允许复杂的功能从由不同神经元组成的电路中出现 类型。在哺乳动物神经系统中，单细胞转录组学的最新进展使其具有吸引力 通过基因表达模式来定义神经元类型(即转录类型)。在高层次上，班级 由转录切除法定义的神经元在其他神经元特性上也是不同的，包括它们的长程 投影图案。然而，更精细的转录类型并不对应于投影模式： 不同转录类型的神经元可以共享相似的投射，并且相同的神经元 转录型可以有不同的投射。这种缺乏良好水平的通信提出了一个问题，即如何 神经元类型被连接到复杂的电路中，此外，细胞类型如何由这两个基因来定义 表达和连通性。了解基因表达和发育之间的关系 投射可能有助于理解这种复杂的关系，因为成年神经元的投射模式是 这是许多发展过程的累积结果。然而，质疑发展关系 基因表达和投影之间的关系是具有挑战性的，因为传统的单细胞解剖学 方法只能映射少数神经元的投影模式，难以关联 在同一细胞中测量的基因表达的投影。在这里，我建议通过以下方式克服这一挑战 大大提高了基于原位测序的神经解剖学方法的分辨率和规模。就地 基于测序的神经解剖学在绘制投影图中实现了高通量和细胞分辨率 用唯一的RNA条形码标记每个神经元。这些RNA条形码和内源mRNA都可以是 进行原位测序，以将许多神经元的投影模式与基因表达平行地联系起来。通过 为了提高原位条码测序的分辨率和吞吐量，我将生成一个 基因表达与全脑投影关系的史无前例的观点 初级和高级视皮层神经元在出生后发育过程中的模式。这就做 用扰动实验来补充这种系统但相关的方法，以确定因果关系 关键基因和投影之间的关系。通过解开基因之间的复杂关系 表达和投射是在循序渐进的发展中建立起来的，这种结合的方法将提供 对大脑皮层神经元类型连接规则的洞察。生成的数据集将为 神经发育疾病模型中远程连接缺陷的未来研究。最后， 改进基于原位测序的神经解剖学将产生广泛的影响，超越发育 这项提案的重点是使类似的系统方法能够理解长期预测 在衰老过程中，跨个体动物和跨物种。

Project Summary The connectivity of neurons allows complex functions to emerge from a circuit composed of diverse neuronal types. In the mammalian nervous system, recent advances in single-cell transcriptomics make it appealing to define neuronal types by their gene expression patterns (i.e. transcriptomic types). At a high level, classes of neurons defined by transcriptomics are also distinct in other neuronal properties, including their long-range projection patterns. Finer-level transcriptomic types, however, do not correspond to projection patterns: neurons of different transcriptomic types may share similar projections, and neurons of the same transcriptomic type can project diversely. This lack of correspondence at a fine level raises the question of how neuronal types are wired into complex circuits and, furthermore, how cell types can be defined by both gene expression and connectivity. Knowing the developmental relationship between gene expression and projections may help understand this complex relationship, because the projection pattern of an adult neuron is the cumulative result of many developmental processes. However, interrogating the developmental relationship between gene expression and projections is challenging, because conventional single-cell anatomical approaches can only map the projection patterns of a small number of neurons and are difficult to associate projections with gene expression measured in the same cells. Here I propose to overcome this challenge by massively improving the resolution and scale of in situ sequencing-based neuroanatomical approaches. In situ sequencing-based neuroanatomy achieves high throughput and cellular resolution in mapping projections by labeling each neuron with a unique RNA barcode. These RNA barcodes and endogenous mRNAs can both be sequenced in situ to associate projection patterns with gene expression for many neurons in parallel. By improving both the resolution and the throughput of in situ barcode sequencing, I will generate an unprecedented view of the relationship between gene expression and the complete brain-wide projection patterns of neurons in the primary and higher visual cortex over the course of post-natal development. I will complement this systematic but correlational approach with perturbation experiments to establish causal relationship between key genes and projections. By unraveling how the complex relationship between gene expression and projections is established step-by-step in development, this combined approach will provide insights into the wiring rules of cortical neuronal types. The dataset generated will provide a reference for future research into long-range connectivity defects in neurodevelopmental disease models. Finally, the improvement in in situ sequencing-based neuroanatomy will achieve broad impact beyond the developmental focus of this proposal by enabling similar systematic approaches in understanding long-range projections during aging, across individual animals, and across species.