Claudin-5a is essential for the functional formation of both zebrafish blood-brain barrier and blood-cerebrospinal fluid barrier

Abstract

Background: Mammalian Claudin-5 is the main endothelial tight junction component maintaining blood-brain barrier (BBB) permeability, while Claudin-1 and -3 seal the paracellular space of choroid plexus (CP) epithelial cells contributing to the blood-cerebrospinal fluid barrier (BCSFB). In zebrafish, two paralogs of claudin-5a and -5b are expressed while their roles in the formation of BBB and BCSFB are unclear.

Methods: The expression patterns of Claudin-5a and -5b in zebrafish brains were systematically analyzed by immunofluorescence (IF) assay. The developmental functions of Claudin-5a and -5b were characterized by generating of claudin-5a and -5b mutants respectively. Meanwhile, the cerebral inflammation and cell apoptosis in claudin-5a^-/- were assessed by live imaging of transgenic zebrafish, RT-qPCR, IF, and TUNEL assay. The integrity of BBB and BCSFB was evaluated by in vivo angiographic and dye permeation assay. Finally, RT-qPCR, whole-mount RNA in situ hybridization (WISH), and transmission electron microscopy (TEM) analyses were performed to investigate the development of cerebral vessels and choroid plexus.

Results: We showed that Claudin-5a and -5b are both expressed in zebrafish cerebrovascular endothelial cells (ECs). In addition, Claudin-5a was strongly expressed in CP epithelial cells. Loss of Claudin-5b showed no effect on zebrafish vasculogenesis or BBB function. In contrast, the knockout of claudin-5a caused a lethal phenotype of severe whole-brain oedema, ventricular dilatation, and cerebral hernia in zebrafish larvae, although the cerebral vasculogenesis and the development of CP were not altered. In claudin-5a^-/- , although ultrastructural analysis of CP and cerebral capillary showed intact integrity of epithelial and endothelial tight junctions, permeability assay indicated a disruption of both BBB and BCSFB functions. On the molecular level, it was found that ZO-1 was upregulated in the CP epithelium of claudin-5a^-/-, while the notch and shh pathway responsible for CP development was not affected due to loss of Claudin-5a.

Conclusions: Our findings verified a non-functional role of zebrafish Claudin-5b in the BBB and identified Claudin-5a as the ortholog of mammalian Claudin-5, contributing to the development and the functional maintenance of both BBB and BCSFB.

Keywords: Blood-brain barrier; Blood-cerebrospinal fluid barrier; Claudin-5; Tight junction; Zebrafish.