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.

Biofilm growth and survival pose a problem in both medical and industrial fields. Bacteria in biofilms are more tolerant to antibiotic treatment due to the inability of antibiotics to permeate to the bottom layers of cells in a biofilm and the creation of altered microenvironments of bacteria deep within the biofilm. Despite the abundance of information we have about E. coli biofilm growth and maturation, we are still learning how manipulating different signaling pathways influences the formation and fitness of biofilm. Understanding the impact of signaling pathways on biofilm formation may narrow the search for novel small molecule inhibitors or activators that affect biofilm production and stability. Here, we study the influence of the minor sigma transcription factor FliA (RpoF, sigma-28), which controls late-stage flagellar assembly and chemotaxis, on biofilm production and composition at various temperatures in the E. coli strain PHL628, which abundantly produces the extracellular structural protein curli. We examined FliA’s influence on external cellular structures like curli and flagella and the biomolecular composition of the biofilm’s extracellular polymeric substance (EPS) using biochemical assays, immunoblotting, and confocal laser scanning microscopy (CLSM). At 37°C, FliA overexpression results in the dramatic growth of biofilm in polystyrene plates and more modest yet significant biofilm growth on silica slides. We observed no significant differences in curli concentration and carbohydrate concentration in the EPS with FliA overexpression. Still, we did see significant changes in the abundance of EPS protein using CLSM at higher growth temperatures. We also noticed increased flagellin concentration, a major structural protein in flagella, occurred with FliA overexpression, specifically in planktonic cultures. These experiments have aided in narrowing our focus to FliA’s role in changing the protein composition of the EPS, which we will examine in future endeavors.

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.

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.

Biofilms form when planktonic, or freely swimming, bacteria adhere to a surface. Once adhered, connections form with other bacteria through extracellular polymeric substances (EPS). Biofilms can form on medical devices, causing persistent infections in patients, and on industrial equipment, which is known as biofouling. Biofilms present a unique challenge, as they can exhibit a higher tolerance for antibiotics than their planktonic counterparts. Curli are extracellular quaternary protein structures associated with robust biofilm formation in E. coli. They have been shown to aid in initial adhesion of the bacterial cells to a surface.Here, we induced the overexpression of rpoF, also known as fliA and σ28, which codes for a flagellar transcription factor important in late‐stage flagellar assembly, in PHL628 E. coli, which overexpress curli at growth temperatures less than 30 °C. Because rpoF increases cell motility, we originally predicted that rpoF overexpression would decrease the amount of biofilm formation. However, we observed that rpoFoverexpression increased the amount of biofilm formed. We now suspect that the nature of rpoF overexpression's effects on biofilm formation may be through changing the composition of the EPS. To test this hypothesis, we grew biofilm under different conditions, and then analyzed the biomolecule composition of the EPS, as well as the overall biofilm volume using confocal laser scanning microscopy (CLSM). We used the dyes Thioflavin T and Calcofluor White to quantify extracellular amyloid proteins and carbohydrates, respectively. We found that rpoF overexpression increases the amount of extracellular carbohydrates. We also observed more curli formation when rpoF was overexpressed at lower temperatures, but not at higher growth temperatures. This suggests that curli does not account for increased biofilm formation at higher temperatures. In future experiments, we will continue similar imaging and analysis methods with Sypro Ruby (extracellular protein concentration), propidium iodide (eDNA), and DiD’ oil (lipid concentration) to obtain a more complete picture of how rpoF changes the composition of the biofilm EPS.

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.

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