Angiogenic mechanisms governing the segregation of blood-brain barrier and fenestrated capillaries derived from a multipotent cerebrovascular niche

Abstract

Cerebrovascular endothelial cell (EC) subtypes characterized by blood-brain barrier (BBB) properties or fenestrated pores are essential components of brain-blood interfaces, supporting brain function and homeostasis. To date, the origins and developmental mechanisms underlying this heterogeneous EC network remain largely unclear. Using single-cell-resolution lineage tracing in zebrafish, we discover a multipotent vascular niche at embryonic capillary borders that generates ECs with BBB or fenestrated molecular identity. RNAscope analysis demonstrates restricted expression of flt4 in sprouting ECs contributing to fenestrated choroid plexus (CP) vasculature, identifying an early molecular distinction from adjacent BBB vessels. Mechanistically, flt4 null and cytoplasmic-domain-deletion mutants exhibit CP vascularization defects when combined with vegfr2 zebrafish paralog deletion. Pharmacological results support this co-requirement of Flt4 and Vegfr2 signaling and suggest the PI3K and ERK pathways as downstream effectors. These findings reveal a specialized developmental origin for BBB and fenestrated EC subtypes, and establish Flt4 as a crucial guidance receptor mediating their angiogenic segregation.