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

项目摘要 神经元的连接性使得复杂的功能可以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吸引了 通过它们的基因表达模式（即转录组类型）来定义神经元类型。在高水平上， 由转录组学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 投射模式然而，更精细水平的转录组类型并不对应于投射模式： 不同转录组类型的神经元可以共享相似的投射，并且相同转录组类型的神经元可以共享相似的投射。 转录组型可以投射差异。这种缺乏良好水平的通信引发了一个问题：如何 神经元类型被连接成复杂的电路，此外，细胞类型如何被这两个基因定义， 表达和连通性。了解基因表达与 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 基因表达和投射之间的关系是具有挑战性的，因为传统的单细胞解剖学 这些方法只能映射少量神经元的投影模式， 在相同的细胞中测量基因表达的预测。在此，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。原位 基于测序的神经解剖学在映射投影中实现了高通量和细胞分辨率， 用独特的RNA条形码标记每个神经元。这些RNA条形码和内源性mRNA都可以是 原位测序，将投射模式与许多神经元的基因表达平行关联起来。通过 为了提高原位条形码测序的分辨率和通量，我将生成一个 对基因表达和全脑投射之间关系的前所未有的看法 在出生后的发展过程中，初级和高级视觉皮层的神经元模式。我会 用扰动实验来补充这种系统但相关的方法，以建立因果关系。 关键基因和预测之间的关系。通过揭示基因之间的复杂关系 表达式和投影是在开发过程中逐步建立的，这种组合方法将提供 深入了解皮层神经元类型的布线规则。生成的数据集将为以下方面提供参考： 未来研究神经发育疾病模型中的长距离连接缺陷。最后 基于原位测序的神经解剖学的改进将实现超越发育的广泛影响。 通过在理解长期预测方面采用类似的系统方法， 在不同的动物个体和物种之间。

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.