Single-cell-resolution fate mapping reveals embryonic venous origins of fenestrated hindbrain choroid plexus vasculature

In the brain, endothelial cell (EC) subtypes characterized by blood-brain barrier (BBB) properties or fenestrated pores form essential brain-blood interfaces and exhibit markedly distinct permeability. The choroid plexus (CP) establishes fenestrated vasculature lacking the BBB to efficiently regulate cerebrospinal fluid balance, yet its developmental origins and mechanisms remain poorly defined. Using single-cell-resolution fate mapping in zebrafish, we identify here two venous sources that give rise to the hindbrain myelencephalic CP (mCP) vasculature. RNAscope and BAC transgenic analyses reveal highly abundant and persistent expression of the venous marker <i>flt4</i> in these EC lineages, supporting their identities. Unexpectedly, we find that these venous origins of the mCP vasculature also contribute ECs to diverse cranial vessels, including those that maintain low <i>flt4</i> expression and later acquire BBB characteristics. Functionally, <i>flt4</i> null and cytoplasmic-domain-deletion mutants exacerbate mCP vascularization defects when combined with <i>vegfr2</i> signaling deficiency, without disrupting neighboring BBB-type vessels. Pharmacological data support this co-requirement of Flt4 and Vegfr2 signaling in mCP vascularization and further suggest that the PI3K and ERK pathways are necessary for this process. Together, these findings reveal embryonic venous lineages and molecular pathways required for hindbrain CP vascularization and imply that Flt4 signaling contributes to the angiogenic separation of CP- and BBB-associated capillaries originating from shared embryonic domains.<b>Significance Statement</b> The choroid plexus forms permeable vasculature devoid of the blood-brain barrier. This vasculature is vital for maintaining brain homeostasis by facilitating molecular exchange between blood and cerebrospinal fluid, yet its developmental mechanisms remain poorly understood. In this study, we investigated embryonic endothelial origins and molecular pathways underlying hindbrain choroid plexus vasculature. Our fate mapping results, together with genetic and pharmacological data, show that choroid plexus vasculature arises exclusively from embryonic veins expressing the venous marker <i>flt4</i>, in part dependent on its receptor function. Further expression analyses suggest that differential cerebrovascular <i>flt4</i> expression guides the developmental separation of choroid plexus vasculature and barrier-associated vessels. These findings advance our understanding of choroid plexus vascularization and provide new insights into heterogeneous cerebrovascular development.