Spatial Transcriptomics Reveals Human Cortical Layer and Area Specification

The human cerebral cortex, pivotal for advanced cognitive functions, is composed of six distinct layers and dozens of functionally specialized areas. These layers and areas are distinguished both molecularly, by diverse neuronal and glial cell subtypes, and structurally, through intricate spatial organization. While single-cell transcriptomics studies have advanced molecular characterization of human cortical development, a critical gap exists due to the loss of spatial context during cell dissociation. Here, we utilized multiplexed error-robust fluorescence in situ hybridization (MERFISH), augmented with deep-learning-based nucleus segmentation, to examine the molecular, cellular, and cytoarchitectural development of human fetal cortex with spatially resolved single-cell resolution. Our extensive spatial atlas, encompassing over 18 million single cells, spans eight cortical areas across four time points in the second and third trimesters. We uncovered an early establishment of the six-layer structure, identifiable by the laminar distribution of excitatory neuron subtypes, three months before the emergence of cytoarchitectural layers. Notably, we discovered two distinct modes of cortical areal specification in the mid-gestation: (1) a continuous, gradual transition observed across most cortical areas along the anterior-posterior axis, and (2) a discrete, abrupt boundary uniquely 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 continuous morphogen gradients dictate mid-gestation cortical arealization. Furthermore, integrating single-nucleus RNA-sequencing with MERFISH revealed an early upregulation of synaptogenesis in V1-specific Layer 4 neurons, implicating a role of synaptogenesis in this discrete border formation. Collectively, our findings underscore the crucial role of spatial relationships in determining the molecular specification of cortical layers and areas. This study provides a valuable resource for the field and establishes a spatially resolved single-cell analysis paradigm, paving the way for a comprehensive developmental atlas of the human brain.