Vascular endothelial cells (vECs) in the brain exhibit structural and functional heterogeneity. Fenestrated, permeable brain vasculature mediates neuroendocrine function, body-fluid regulation, and neural immune responses; however, its vascular formation remains poorly understood. Here, we show that specific combinations of vascular endothelial growth factors (Vegfs) are required to selectively drive fenestrated vessel formation in the zebrafish myelencephalic choroid plexus (mCP). We found that the combined, but not individual, loss of Vegfab, Vegfc, and Vegfd causes severely impaired mCP vascularization with little effect on neighboring non-fenestrated brain vessel formation, demonstrating fenestrated-vEC-specific angiogenic requirements. This Vegfs-mediated vessel-selective patterning also involves Ccbe1. Expression analyses, cell-type-specific ablation, and paracrine activity-deficient vegfc mutant characterization suggest that vEC-autonomous Vegfc and meningeal fibroblast-derived Vegfab and Vegfd are critical for mCP vascularization. These results define molecular cues and cell types critical for directing fenestrated CP vascularization and indicate that vECs’ distinct molecular requirements for angiogenesis underlie brain vessel heterogeneity.

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.

The zebrafish is a valuable vertebrate model to study cardiovascular formation and function due to the facile visualization and rapid development of the circulatory system in its externally growing embryos. Despite having distinct advantages, zebrafish have paralogs of many important genes, making reverse genetics approaches inefficient since generating animals bearing multiple gene mutations requires substantial efforts. Here, we present a simple and robust synthetic CRISPR RNA/Cas9-based mutagenesis approach for generating biallelic F0 zebrafish knockouts. Using a dual-guide synthetic CRISPR RNA/Cas9 ribonucleoprotein (dgRNP) system, we compared the efficiency of biallelic gene disruptions following the injections of one, two, and three dgRNPs per gene into the cytoplasm or yolk. We show that simultaneous cytoplasmic injections of three distinct dgRNPs per gene into one-cell stage embryos resulted in the most efficient and consistent biallelic gene disruptions. Importantly, this triple dgRNP approach enables efficient inactivation of cell autonomous and cell non-autonomous gene function, likely due to the low mosaicism of biallelic disruptions. In support of this finding, we provide evidence that the F0 animals generated by this method fully phenocopied the endothelial and peri-vascular defects observed in corresponding stable mutant homozygotes. Moreover, this approach faithfully recapitulated the trunk vessel phenotypes resulting from the genetic interaction between two vegfr2 zebrafish paralogs. Mechanistically, investigation of genome editing and mRNA decay indicates that the combined mutagenic actions of three dgRNPs per gene lead to an increased probability of frameshift mutations, enabling efficient biallelic gene disruptions. Therefore, our approach offers a highly robust genetic platform to quickly assess novel and redundant gene function in F0 zebrafish.