Abstract

The vertebrate brain requires precisely patterned vasculature, yet the molecular and cellular dynamics of cerebrovascular development remain incompletely understood. While single-cell studies have profiled brain vasculature, integrating these data across molecular, cellular, and architectural scales remains challenging. We combine in situ sequencing, single-cell transcriptomics, and 3D vascular reconstruction to map zebrafish brain endothelial development (3 - 11 dpf). Quantitative 3D analysis reveals a developmental shift from lateral vascularization to intraparenchymal angiogenesis, coinciding with blood-brain barrier (BBB) maturation. We identify 6 endothelial subtypes with conserved mammalian signatures, showing capillary endothelial cells (CapECs), the predominant intracranial subtype exhibiting stage-specific enrichment of transporter and tight junction modules. Spatial transcriptomics show progressive CapEC enrichment in mesencephalic/metencephalic regions versus arterial EC accumulation in prosencephalic vessels. Functional assays confirm BBB competency by 11 dpf, while genetic perturbation of three uncharacterized CapEC-enriched genes ( slc16a1a, zgc:158423, cldc1 ) disrupts vascular patterning and BBB integrity. This multidimensional atlas provides an evolutionary perspective on cerebrovascular development, bridges molecular profiles with vascular architecture, and offers insights into developmental dynamics across scales. This study maps a spatiotemporal atlas of zebrafish cerebrovascular development, linking genes and cells to 3D vascular architecture. It reveals how vessel networks form and identifies key genes essential for blood-brain barrier integrity.