The cerebral cortex is composed of neuronal types with diverse gene expression that are organized into specialized cortical areas. These areas, each with characteristic cytoarchitecture^1,2, connectivity^3,4 and neuronal activity^5,6, are wired into modular networks^3,4,7. However, it remains unclear whether these spatial organizations are reflected in neuronal transcriptomic signatures and how such signatures are established in development. Here we used BARseq, a high-throughput in situ sequencing technique, to interrogate the expression of 104 cell-type marker genes in 10.3 million cells, including 4,194,658 cortical neurons over nine mouse forebrain hemispheres, at cellular resolution. De novo clustering of gene expression in single neurons revealed transcriptomic types consistent with previous single-cell RNA sequencing studies^8,9. The composition of transcriptomic types is highly predictive of cortical area identity. Moreover, areas with similar compositions of transcriptomic types, which we defined as cortical modules, overlap with areas that are highly connected, suggesting that the same modular organization is reflected in both transcriptomic signatures and connectivity. To explore how the transcriptomic profiles of cortical neurons depend on development, we assessed cell-type distributions after neonatal binocular enucleation. Notably, binocular enucleation caused the shifting of the cell-type compositional profiles of visual areas towards neighbouring cortical areas within the same module, suggesting that peripheral inputs sharpen the distinct transcriptomic identities of areas within cortical modules. Enabled by the high throughput, low cost and reproducibility of BARseq, our study provides a proof of principle for the use of large-scale in situ sequencing to both reveal brain-wide molecular architecture and understand its development.

大脑皮层由具有不同基因表达的神经元类型组成，这些神经元被组织成专门的皮层区域。这些区域各自具有特有的细胞结构^1,2、连接性^3,4和神经元活动^5,6，并连接到模块化网络^3,4,7中。然而，目前尚不清楚这些空间组织是否反映在神经元转录组特征中以及这些特征在发育过程中是如何建立的。在这里，我们使用 BARseq（一种高通量原位测序技术）以细胞分辨率询问 1030 万个细胞中 104 个细胞类型标记基因的表达，其中包括 9 个小鼠前脑半球的 4,194,658 个皮质神经元。单个神经元中基因表达的从头聚类揭示了与之前的单细胞 RNA 测序研究一致的转录组类型^8,9。转录组类型的组成可以高度预测皮质区域的身份。此外，具有相似转录组类型组成的区域（我们将其定义为皮质模块）与高度连接的区域重叠，这表明相同的模块组织反映在转录组特征和连接性中。为了探索皮质神经元的转录组学特征如何依赖于发育，我们评估了新生儿双眼剜除后的细胞类型分布。值得注意的是，双眼剜除导致视觉区域的细胞类型组成谱向同一模块内的邻近皮质区域转移，这表明外周输入使皮质模块内区域的不同转录组特性变得更加清晰。凭借 BARseq 的高通量、低成本和可重复性，我们的研究为使用大规​​模原位测序揭示全脑分子结构并了解其发展提供了原理证明。