Claudin-5a is essential for the functional formation of both zebrafish blood-brain barrier and blood-cerebrospinal fluid barrier.
Claudin-5a is essential for the functional formation of both zebrafish blood-brain barrier and blood-cerebrospinal fluid barrier.
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DOI：
10.1186/s12987-022-00337-9
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发表时间：
2022-06-03
影响因子：
7.3
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中科院分区：
文献类型：
--
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哺乳动物Claudin-5是维持血脑屏障（BBB）通透性的主要内皮紧密连接成分，而Claudin-1和-3封闭脉络膜丛（CP）上皮细胞的细胞旁间隙，形成血脑脊液屏障（BCSFB）。在斑马鱼中，有两种类似于cludin -5a和-5b的基因表达，但它们在BBB和BCSFB形成中的作用尚不清楚。采用免疫荧光（IF）法系统分析了斑马鱼脑组织中Claudin-5a和-5b的表达模式。Claudin-5a和-5b的发育功能分别通过产生Claudin-5a和-5b突变体来表征。同时，采用转基因斑马鱼活体成像、RT-qPCR、IF、TUNEL等检测方法评估cludin -5a-/-的脑炎症和细胞凋亡情况。通过体内血管造影和染色渗透试验评估血脑屏障和BCSFB的完整性。最后，采用RT-qPCR、全载体RNA原位杂交（WISH）和透射电镜（TEM）观察大鼠脑血管和脉络膜丛的发育情况。我们发现cludin -5a和-5b都在斑马鱼脑血管内皮细胞（ECs）中表达。此外，Claudin-5a在CP上皮细胞中强烈表达。Claudin-5b的缺失对斑马鱼血管生成或血脑屏障功能没有影响。相比之下，敲除claudin-5a会导致斑马鱼幼虫出现严重的全脑水肿、心室扩张和脑疝的致死性表型，尽管脑血管发生和CP的发育没有改变。在claudin-5a-/-中，尽管CP和脑毛细血管的超微结构分析显示上皮和内皮紧密连接的完整性完好，但通透性分析显示血脑屏障和BCSFB功能均受到破坏。在分子水平上，我们发现在claudin-5a-/-的CP上皮中ZO-1表达上调，而负责CP发育的notch和shh通路不受claudin-5a缺失的影响。我们的研究结果证实了斑马鱼的Claudin-5b在血脑屏障中的非功能性作用，并确定了Claudin-5a是哺乳动物Claudin-5的同源物，有助于血脑屏障和BCSFB的发展和功能维持。在线版本包含补充材料，可在10.1186/s12987-022-00337-9获得。
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. 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. 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. 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. The online version contains supplementary material available at 10.1186/s12987-022-00337-9.
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