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A novel transgenic zebrafish model for blood-brain and blood-retinal barrier development.

ABSTRACT: Development and maintenance of the blood-brain and blood-retinal barrier is critical for the homeostasis of brain and retinal tissue. Despite decades of research our knowledge of the formation and maintenance of the blood-brain (BBB) and blood-retinal (BRB) barrier is very limited. We have established an in vivo model to study the development and maintenance of these barriers by generating a transgenic zebrafish line that expresses a vitamin D-binding protein fused with enhanced green fluorescent protein (DBP-EGFP) in blood plasma, as an endogenous tracer.The temporal establishment of the BBB and BRB was examined using this transgenic line and the results were compared with that obtained by injection of fluorescent dyes into the sinus venosus of embryos at various stages of development. We also examined the expression of claudin-5, a component of tight junctions during the first 4 days of development. We observed that the BBB of zebrafish starts to develop by 3 dpf, with expression of claudin-5 in the central arteries preceding it at 2 dpf. The hyaloid vasculature in the zebrafish retina develops a barrier function at 3 dpf, which endows the zebrafish with unique advantages for studying the BRB.Zebrafish embryos develop BBB and BRB function simultaneously by 3 dpf, which is regulated by tight junction proteins. The Tg(l-fabp:DBP-EGFP) zebrafish will have great advantages in studying development and maintenance of the blood-neural barrier, which is a new application for the widely used vertebrate model.

SUBMITTER: Xie J

PROVIDER: S-EPMC2914679 | biostudies-literature | 2010 Jul

REPOSITORIES: biostudies-literature

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A novel transgenic zebrafish model for blood-brain and blood-retinal barrier development.

Xie Jing J Farage Eric E Sugimoto Masahiko M Anand-Apte Bela B

BMC developmental biology 20100723

<h4>Background</h4>Development and maintenance of the blood-brain and blood-retinal barrier is critical for the homeostasis of brain and retinal tissue. Despite decades of research our knowledge of the formation and maintenance of the blood-brain (BBB) and blood-retinal (BRB) barrier is very limited. We have established an in vivo model to study the development and maintenance of these barriers by generating a transgenic zebrafish line that expresses a vitamin D-binding protein fused with enhanc ...[more]

PMID: 20653957

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Project description:Alzheimer's disease is widely described since the discovery of histopathological lesions in Mrs. Auguste Deter in 1906. However to date, there is no effective treatment to deal with the many cellular and molecular alterations. The complexity is even higher with the growing evidence of involvement of the peripheral blood mononuclear cells (PBMCs). Indeed, monocytes and T cells are shown in the cerebral parenchyma of AD patients, and these cells grafted to the periphery are able to go through the blood-brain barrier (BBB) in transgenic mouse models. It is known that BBB is disrupted at a late stage of AD. Chemokines represent major regulators of the transmigration of PBMCs, but many data were obtained on AD animal models. No data are available on the role of AD BBB in a healthy brain parenchyma. Therefore, the purpose of this study was to analyze the longitudinal chemokine profile expression in a BBB model from AD transgenic mice versus wild-type (WT) mice.A primary mouse BBB model was used with a luminal compartment either AD or WT and an abluminal compartment WT consisting of astrocytes and microglia. PBMCs were extracted by a ficoll gradient and incubated in the transwell with a direct contact with the luminal side, including the endothelial cells and pericytes. Then, the complete BBB model was incubated during 48 h, before supernatants and cell lysates were collected. Chemokines were quantified by X-MAP® luminex technology.Abluminal CX3CL1 production increased in 12-month-old AD BBB while CX3CL1 levels decreased in luminal lysates. CCL3 in luminal compartment increased with aging and was significantly different compared to AD BBB at 12 months. In addition, abluminal CCL2 in 12-month-old AD BBB greatly decreased compared to levels in WT BBB. On the contrary, no modification was observed for CCL4, CCL5, and CXCL10.These first findings highlighted the impact of AD luminal compartment on chemokine signature in a healthy brain parenchyma, suggesting new therapeutic or diagnostic approaches.

Project description:BackgroundMammalian 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.MethodsThe 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.ResultsWe 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.ConclusionsOur 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.

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A novel transgenic zebrafish model for blood-brain and blood-retinal barrier development.

Publications

A novel transgenic zebrafish model for blood-brain and blood-retinal barrier development.

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