Deciphering the temporal transcriptional landscape of human fetal leptomeninges

Abstract The leptomeninges play a pivotal role in the central nervous system (CNS), serving both as a barrier and as a conduit for fluid and cellular transport. Despite their critical functions, our understanding of leptomeningeal development and maturation during human embryogenesis remains limited. This study seeks to bridge this gap. We conducted single-nucleus RNA sequencing on leptomeningeal tissues from eight human embryos, capturing developmental stages from early fetal to late mid-fetal phases. Our bioinformatic analyses encompassed cell type classification, identification of layer-specific markers, mapping of the arachnoid barrier maturation trajectory, and joint analyses with mouse and aged human leptomeningeal scRNA datasets. Key bioinformatic findings were validated through immunostaining in selected samples. Our study revealed a complex cellular heterogeneity within the developing leptomeninges, identifying distinct subpopulations of fibroblasts, immune cells, and vascular cells. We mapped the transcriptomic dynamics of fibroblast cell types throughout fetal brain development, highlighting a clear maturation process from early fetal to late mid-fetal stages. Comparative analysis with mouse data allowed us to distinguish human-specific layer markers while confirming several conserved markers shared between humans and mice. Joint analysis with aged human datasets identified two unique arachnoid clusters specific to aging leptomeninges. Moreover, we traced the developmental trajectory of the arachnoid barrier, detailing the transcriptomic shifts associated with its gradual formation. Notably, immune cells in early fetal stages were predominantly M2-type macrophages, underscoring a distinctive immune environment. Finally, we explored the molecular interactions between fibroblasts and other cell types, highlighting their coordinated roles in orchestrating leptomeningeal development. Together, our findings provide a comprehensive overview of the cellular and molecular landscape of the developing human leptomeninges.