Dynamic brain activity requires timely communications between the brain parenchyma and circulating blood. Brain–blood communication is facilitated by intricate networks of brain vasculature, which display striking heterogeneity in structure and function. This vascular cell heterogeneity in the brain is fundamental to mediating diverse brain functions and has long been recognized. However, the molecular basis of this biological phenomenon has only recently begun to be elucidated. Over the past century, various animal species and in vitro systems have contributed to the accumulation of our fundamental and phylogenetic knowledge about brain vasculature, collectively advancing this research field. Historically, dye tracer and microscopic observations have provided valuable insights into the anatomical and functional properties of vasculature across the brain, and these techniques remain an important approach. Additionally, recent advances in molecular genetics and omics technologies have revealed significant molecular heterogeneity within brain endothelial and perivascular cell types. The combination of these conventional and modern approaches has enabled us to identify phenotypic differences between healthy and abnormal conditions at the single-cell level. Accordingly, our understanding of brain vascular cell states during physiological, pathological, and aging processes has rapidly expanded. In this review, we summarize major historical advances and current knowledge on blood endothelial cell heterogeneity in the brain, and discuss important unsolved questions in the field.

Fenestrated and blood-brain barrier (BBB)-forming endothelial cells constitute major brain capillaries, and this vascular heterogeneity is crucial for region-specific neural function and brain homeostasis. How these capillary types emerge in a brain region-specific manner and subsequently establish intra-brain vascular heterogeneity remains unclear. Here, we performed a comparative analysis of vascularization across the zebrafish choroid plexuses (CPs), circumventricular organs (CVOs), and retinal choroid, and show common angiogenic mechanisms critical for fenestrated brain capillary formation. We found that zebrafish deficient for Gpr124, Reck, or Wnt7aa exhibit severely-impaired BBB angiogenesis without any apparent defect in fenestrated capillary formation in the CPs, CVOs, and retinal choroid. Conversely, genetic loss of various Vegf combinations caused significant disruptions in Wnt7/Gpr124/Reck signaling-independent vascularization of these organs. The phenotypic variation and specificity revealed heterogeneous endothelial requirements for Vegfs-dependent angiogenesis during CP and CVO vascularization, identifying unexpected interplay of Vegfc/d and Vegfa in this process. Mechanistically, expression analysis and paracrine activity-deficient vegfc mutant characterization suggest that endothelial cells and non-neuronal specialized cell types present in the CPs and CVOs are major sources of Vegfs responsible for regionally-restricted angiogenic interplay. Thus, brain region-specific presentations and interplay of Vegfc/d and Vegfa control emergence of fenestrated capillaries, providing insight into the mechanisms driving intra-brain vascular heterogeneity and fenestrated vessel formation in other organs.

Phenotypic diversity of brain vasculature is important for brain region-specific neural function, but how this diversity emerges remains unclear. Here we show a core angiogenic mechanism critical for fenestrated brain vessel development via a comparative analysis of the choroid plexuses (CPs) and circumventricular organs (CVOs) in zebrafish, demonstrating vessel-type-selective vascularization mechanisms. We find that zebrafish deficient for Gpr124, Reck, or Wnt7aa exhibit severely-impaired brain angiogenesis without any apparent defect in fenestrated capillary formation in the CPs and CVOs. Conversely, various combinations of Vegfs direct heterogeneous regulation of angiogenesis within and across these organs. Expression analysis suggests that endothelial cells and non-neuronal cell types uniquely present in the CPs and CVOs are major sources of Vegfs. These comparative results reveal an unexpected, vital role for Vegfc in fenestrated vessel development across the brain and suggest that local and inter-tissue heterogeneity of angiogenic regulation promotes phenotypic diversity of brain vasculature.