A Spatiotemporal Atlas of Cerebrovascular Development in Zebrafish: From Vasculatures to Cells and Molecules

The cerebrovascular system plays a pivotal role in brain development and the maintenance of its functions. Although several single-cell transcriptomic studies have delineated the molecular profiles of brain vasculature, integrating these data into a coherent multiscale understanding across molecular, cellular, and vascular network architecture remains a fundamental challenge. Here, we present a comprehensive spatiotemporal atlas of zebrafish cerebrovascular development that bridges these scales by combining in situ sequencing with three-dimensional (3D) vascular network reconstruction. Our quantitative 3D analysis revealed region-specific cerebrovascular growth dynamics at 3, 6, and 11 days post-fertilization. Through single-cell RNA sequencing, we identified endothelial cell (EC) subtypes at these stages and validated their molecular signatures and cross-species conservation during vascular development. Spatial transcriptomics further demonstrated that capillary ECs are the predominant vascular subtype within intracranial vessels, showing progressive enrichment in mesencephalic/metencephalic regions. Importantly, we identified three previously uncharacterized genes (slc16a1a, zgc:158423, and si:ch73-86n18.1) that critically contribute to blood-brain barrier integrity. Our study systematically deciphers the molecular profile of ECs during development, establishing a foundational framework for understanding the dynamic landscape of cerebrovascular development. Overall design: Endothelial-specific Tg(kdrl:eGFP) transgenic zebrafish line was used to isolate the brain endothelial cells by flow cytometry at 3, 6, and 11 dpf, and then performed 10x Genomics scRNA-seq on the sorted cells with 3' labelling strategy.