Dataset Information

The Wilms Tumor Gene wt1a Contributes to Blood-Cerebrospinal Fluid Barrier Function in Zebrafish.

ABSTRACT: The Wilms tumor suppressor gene Wt1 encodes a zinc finger transcription factor, which is highly conserved among vertebrates. It is a key regulator of urogenital development and homeostasis but also plays a role in other organs including the spleen and the heart. More recently additional functions for Wt1 in the mammalian central nervous system have been described. In contrast to mammals, bony fish possess two paralogous Wt1 genes, namely wt1a and wt1b. By performing detailed in situ hybridization analyses during zebrafish development, we discovered new expression domains for wt1a in the dorsal hindbrain, the caudal medulla and the spinal cord. Marker analysis identified wt1a expressing cells of the dorsal hindbrain as ependymal cells of the choroid plexus in the myelencephalic ventricle. The choroid plexus acts as a blood-cerebrospinal fluid barrier and thus is crucial for brain homeostasis. By employing wt1a mutant larvae and a dye accumulation assay with fluorescent tracers we demonstrate that Wt1a is required for proper choroid plexus formation and function. Thus, Wt1a contributes to the barrier properties of the choroid plexus in zebrafish, revealing an unexpected role for Wt1 in the zebrafish brain.

SUBMITTER: Hopfenmuller VL

PROVIDER: S-EPMC8786916 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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The Wilms Tumor Gene wt1a Contributes to Blood-Cerebrospinal Fluid Barrier Function in Zebrafish.

Hopfenmüller Vera L VL Perner Birgit B Reuter Hanna H Bates Thomas J D TJD Große Andreas A Englert Christoph C

Frontiers in cell and developmental biology 20220111

The Wilms tumor suppressor gene Wt1 encodes a zinc finger transcription factor, which is highly conserved among vertebrates. It is a key regulator of urogenital development and homeostasis but also plays a role in other organs including the spleen and the heart. More recently additional functions for Wt1 in the mammalian central nervous system have been described. In contrast to mammals, bony fish possess two paralogous Wt1 genes, namely wt1a and wt1b. By performing d ...[more]

PMID: 35087838

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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.

Project description:BackgroundHypertension is an important risk factor for cerebrovascular disease, including stroke and dementia. Both in humans and animal models of hypertension, neuropathological features such as brain atrophy and oedema have been reported. We hypothesised that cerebrovascular damage resulting from chronic hypertension would manifest itself in a more permeable blood-brain barrier and blood-cerebrospinal fluid barrier. In addition, more leaky barriers could potentially contribute to an enhanced interstitial fluid and cerebrospinal fluid formation, which could, in turn, lead to an elevated intracranial pressure.MethodsTo study this, we monitored intracranial pressure and estimated the cerebrospinal fluid production rate in spontaneously hypertensive (SHR) and normotensive rats (Wistar Kyoto, WKY) at 10 months of age. Blood-brain barrier and blood-cerebrospinal fluid barrier integrity was determined by measuring the leakage of fluorescein from the circulation into the brain and cerebrospinal fluid compartment. Prior to sacrifice, a fluorescently labelled lectin was injected into the bloodstream to visualise the vasculature and subsequently study a number of specific vascular characteristics in six different brain regions.ResultsBlood and brain fluorescein levels were not different between the two strains. However, cerebrospinal fluid fluorescein levels were significantly lower in SHR. This could not be explained by a difference in cerebrospinal fluid turnover, as cerebrospinal fluid production rates were similar in SHR and WKY, but may relate to a larger ventricular volume in the hypertensive strain. Also, intracranial pressure was not different between SHR and WKY. Morphometric analysis of capillary volume fraction, number of branches, capillary diameter, and total length did not reveal differences between SHR and WKY.ConclusionIn conclusion, we found no evidence for blood-brain barrier or blood-cerebrospinal fluid barrier leakage to a small solute, fluorescein, in rats with established hypertension.

Dataset Information

The Wilms Tumor Gene wt1a Contributes to Blood-Cerebrospinal Fluid Barrier Function in Zebrafish.

Publications

The Wilms Tumor Gene <i>wt1a</i> Contributes to Blood-Cerebrospinal Fluid Barrier Function in Zebrafish.

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