Qian Xuyu, Coleman Kyle, Jiang Shunzhou, Kriz Andrea J, Marciano Jack H, Luo Chunyu, Cai Chunhui, Manam Monica Devi, Caglayan Emre, Lai Abbe, Exposito-Alonso David, Otani Aoi, Ghosh Urmi, Shao Diane D, Andersen Rebecca E, Neil Jennifer E, Johnson Robert, LeFevre Alexandra, Hecht Jonathan L, Micali Nicola, Sestan Nenad, Rakic Pasko, Miller Michael B, Sun Liang, Stringer Carsen, Li Mingyao, Walsh Christopher A
Division of Genetics and Genomics, Department of Pediatrics, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA.
Howard Hughes Medical Institute, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA.
Nature. 2025 May 14. doi: 10.1038/s41586-025-09010-1.
The human cerebral cortex is composed of six layers and dozens of areas that are molecularly and structurally distinct. Although single-cell transcriptomic studies have advanced the molecular characterization of human cortical development, a substantial gap exists owing to the loss of spatial context during cell dissociation. Here we used multiplexed error-robust fluorescence in situ hybridization (MERFISH), augmented with deep-learning-based nucleus segmentation, to examine the molecular, cellular and cytoarchitectural development of the human fetal cortex with spatially resolved single-cell resolution. Our extensive spatial atlas, encompassing more than 18 million single cells, spans eight cortical areas across seven developmental time points. We uncovered the early establishment of the six-layer structure, identifiable by the laminar distribution of excitatory neuron subtypes, 3 months before the emergence of cytoarchitectural layers. Notably, we discovered two distinct modes of cortical areal specification during mid-gestation: (1) a continuous, gradual transition observed across most cortical areas along the anterior-posterior axis and (2) a discrete, abrupt boundary specifically identified between the primary (V1) and secondary (V2) visual cortices as early as gestational week 20. This sharp binary transition in V1-V2 neuronal subtypes challenges the notion that mid-gestation cortical arealization involves only gradient-like transitions. Furthermore, integrating single-nucleus RNA sequencing with MERFISH revealed an early upregulation of synaptogenesis in V1-specific layer 4 neurons. Collectively, our findings underscore the crucial role of spatial relationships in determining the molecular specification of cortical layers and areas. This study establishes a spatially resolved single-cell analysis paradigm and paves the way for the construction of a comprehensive developmental atlas of the human brain.
人类大脑皮层由六层和数十个在分子和结构上截然不同的区域组成。尽管单细胞转录组学研究推进了人类皮层发育的分子特征描述，但由于细胞解离过程中空间背景的丢失，仍存在巨大差距。在这里，我们使用了多重抗误差荧光原位杂交（MERFISH），并辅以基于深度学习的细胞核分割技术，以单细胞分辨率在空间上解析人类胎儿皮层的分子、细胞和细胞结构发育。我们广泛的空间图谱涵盖了超过1800万个单细胞，跨越七个发育时间点的八个皮层区域。我们发现六层结构在细胞结构层出现前3个月就已通过兴奋性神经元亚型的分层分布得以确定。值得注意的是，我们发现在妊娠中期皮层区域特化存在两种不同模式：（1）在大多数皮层区域沿前后轴观察到的连续、渐进过渡；（2）早在妊娠第20周就在初级（V1）和次级（V2）视觉皮层之间明确识别出的离散、突然的边界。V1-V2神经元亚型的这种急剧二元转变挑战了妊娠中期皮层区域化仅涉及梯度样过渡的观点。此外，将单核RNA测序与MERFISH相结合揭示了V1特异性第4层神经元中突触发生的早期上调。总体而言，我们的研究结果强调了空间关系在确定皮层层和区域分子特化中的关键作用。这项研究建立了一种空间解析的单细胞分析范式，为构建人类大脑全面发育图谱铺平了道路。
