Neuronal cell types are classically defined by their molecular properties, anatomy and functions. Although recent advances in single-cell genomics have led to high-resolution molecular characterization of cell type diversity in the brain¹, neuronal cell types are often studied out of the context of their anatomical properties. To improve our understanding of the relationship between molecular and anatomical features that define cortical neurons, here we combined retrograde labelling with single-nucleus DNA methylation sequencing to link neural epigenomic properties to projections. We examined 11,827 single neocortical neurons from 63 cortico-cortical and cortico-subcortical long-distance projections. Our results showed unique epigenetic signatures of projection neurons that correspond to their laminar and regional location and projection patterns. On the basis of their epigenomes, intra-telencephalic cells that project to different cortical targets could be further distinguished, and some layer 5 neurons that project to extra-telencephalic targets (L5 ET) formed separate clusters that aligned with their axonal projections. Such separation varied between cortical areas, which suggests that there are area-specific differences in L5 ET subtypes, which were further validated by anatomical studies. Notably, a population of cortico-cortical projection neurons clustered with L5 ET rather than intra-telencephalic neurons, which suggests that a population of L5 ET cortical neurons projects to both targets. We verified the existence of these neurons by dual retrograde labelling and anterograde tracing of cortico-cortical projection neurons, which revealed axon terminals in extra-telencephalic targets including the thalamus, superior colliculus and pons. These findings highlight the power of single-cell epigenomic approaches to connect the molecular properties of neurons with their anatomical and projection properties.

神经元细胞类型通常由其分子特性、解剖结构和功能来定义。尽管单细胞基因组学的最新进展已经实现了大脑细胞类型多样性的高分辨率分子表征¹，神经元细胞类型的研究通常脱离其解剖学特性。为了加深我们对定义皮质神经元的分子和解剖特征之间关系的理解，我们将逆行标记与单核 DNA 甲基化测序相结合，将神经表观基因组特性与预测联系起来。我们检查了来自 63 个皮质-皮质和皮质-皮质下长距离投影的 11,827 个单个新皮质神经元。我们的结果显示了投射神经元独特的表观遗传特征，与其层状和区域位置以及投射模式相对应。根据表观基因组，可以进一步区分投射到不同皮质目标的端脑内细胞，一些投射到端脑外目标（L5 ET）的第 5 层神经元形成与其轴突投射对齐的单独簇。这种分离在皮质区域之间存在差异，这表明 L5 ET 亚型存在区域特异性差异，这通过解剖学研究得到了进一步验证。值得注意的是，一群皮质-皮质投射神经元与 L5 ET 而不是端脑内神经元聚集在一起，这表明一群 L5 ET 皮质神经元投射到两个目标。我们通过皮质-皮质投射神经元的双重逆行标记和顺行追踪来验证这些神经元的存在，这揭示了包括丘脑、上丘和脑桥在内的端脑外目标中的轴突末端。