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Brain endothelial cells (BECs) play an important role in maintaining central nervous system (CNS) homeostasis through blood-brain barrier (BBB) functions. In addition to actively regulating material exchange between the circulation and CNS, BECs express low baseline levels of adhesion receptors, which limits entry of leukocytes. Supporting cells in the neurovascular unit, such as astrocytes and pericytes, are crucial for promoting this immune privilege phenotype in BECs. However, the molecular mediators governing this phenotype are unclear. Here, we explored how BBB immune privilege is influenced by IQGAP2, a scaffold protein associated with elevated barrier function. Using single-cell RNA sequencing and immunohistology, we show that BECs from mice lacking Iqgap2 exhibit a profound inflammatory signature, including extensive upregulation of adhesion receptors and antigen-processing machinery. Further, in zebrafish and mice, loss of Iqgap2 increases infiltration of peripheral leukocytes into the brain. Overall, our results implicate IQGAP2 as an essential regulator of BBB immune privilege.

The blood-brain barrier (BBB) is a unique set of properties of the brain vasculature which severely restricts its permeability to proteins and small molecules. Classic chick-quail chimera studies showed that these properties are not intrinsic to the brain vasculature but rather are induced by surrounding neural tissue. Here we identify Spock1 as a candidate neuronal signal for regulating BBB permeability in zebrafish and mice. Mosaic genetic analysis shows that neuronally-expressed Spock1 is cell non-autonomously required for a functional BBB. Leakage in spock1 mutants is associated with altered extracellular matrix (ECM), increased endothelial transcytosis, and altered pericyte-endothelial interactions. Furthermore, a single dose of recombinant SPOCK1 partially restores BBB function in spock1 mutants by quenching gelatinase activity and restoring vascular expression of BBB genes including mcamb. These analyses support a model in which neuronally secreted Spock1 induces BBB properties by altering the ECM, thereby regulating pericyte-endothelial interactions and downstream vascular gene expression.One-Sentence SummarySpock1 is a signal secreted by neurons that induces barrier properties in the brain vasculature

Background Directed DNA methylation on N6-adenine (6mA), N4-cytosine (4mC), and C5-cytosine (5mC) can potentially increase DNA coding capacity and regulate a variety of biological functions. These modifications are relatively abundant in bacteria, occurring in about a percent of all bases of most bacteria. Until recently, 5mC and its oxidized derivatives were thought to be the only directed DNA methylation events in metazoa. New and more sensitive detection techniques (ultra-high performance liquid chromatography coupled with mass spectrometry (UHPLC-ms/ms) and single molecule real-time sequencing (SMRTseq)) have suggested that 6mA and 4mC modifications could be present in a variety of metazoa. Results Here, we find that both of these techniques are prone to inaccuracies, which overestimate DNA methylation concentrations in metazoan genomic DNA. Artifacts can arise from methylated bacterial DNA contamination of enzyme preparations used to digest DNA and contaminating bacterial DNA in eukaryotic DNA preparations. Moreover, DNA sonication introduces a novel modified base from 5mC that has a retention time near 4mC that can be confused with 4mC. Our analyses also suggest that SMRTseq systematically overestimates 4mC in prokaryotic and eukaryotic DNA and 6mA in DNA samples in which it is rare. Using UHPLC-ms/ms designed to minimize and subtract artifacts, we find low to undetectable levels of 4mC and 6mA in genomes of representative worms, insects, amphibians, birds, rodents and primates under normal growth conditions. We also find that mammalian cells incorporate exogenous methylated nucleosides into their genome, suggesting that a portion of 6mA modifications could derive from incorporation of nucleosides from bacteria in food or microbiota. However, gDNA samples from gnotobiotic mouse tissues found rare (0.9–3.7 ppm) 6mA modifications above background. Conclusions Altogether these data demonstrate that 6mA and 4mC are rarer in metazoa than previously reported, and highlight the importance of careful sample preparation and measurement, and need for more accurate sequencing techniques. Electronic supplementary material The online version of this article (10.1186/s12864-019-5754-6) contains supplementary material, which is available to authorized users.

The blood–brain barrier (BBB) restricts free access of molecules between the blood and the brain and is essential for regulating the neural microenvironment. Here, we describe how the BBB was initially characterized and how the current field evaluates barrier properties. We next detail the cellular nature of the BBB and discuss both the conservation and variation of BBB function across taxa. Finally, we examine our current understanding of mouse and zebrafish model systems, as we expect that comparison of the BBB across organisms will provide insight into the human BBB under normal physiological conditions and in neurological diseases.

As an optically transparent model organism with an endothelial blood-brain barrier (BBB), zebrafish offer a powerful tool to study the vertebrate BBB. However, the precise developmental profile of functional zebrafish BBB acquisition and the subcellular and molecular mechanisms governing the zebrafish BBB remain poorly characterized. Here we find a spatiotemporal gradient of barrier acquisition. Moreover, we capture the dynamics of developmental BBB leakage using live imaging, revealing a combination of steady accumulation in the parenchyma and sporadic bursts of tracer leakage. Electron microscopy studies further reveal that this steady accumulation results from high levels of transcytosis that are eventually suppressed, sealing the BBB. Finally, we demonstrate a key mammalian BBB regulator Mfsd2a, which inhibits transcytosis, plays a conserved role in zebrafish. Mfsd2aa mutants display increased larval and adult BBB permeability due to increased transcytosis. Our findings indicate a conserved developmental program of barrier acquisition between zebrafish and mice.