Investigating mechanisms which control blood vessel formation and function: How does GPCR signalling control vascular permeability within the Blood-Br

为了确保组织的稳态，必须保护中枢神经系统免受血液中循环的激素，神经递质或病原体的影响，同时仍然允许重要的营养物质到达大脑。为了实现这一点，使中枢神经系统（CNS）血管化的血管显示出独特的特性，称为血脑屏障（BBB）。血脑屏障严重限制血管通透性，从而保护大脑免受损伤和疾病。在包括中风、糖尿病和血管性痴呆在内的疾病期间屏障性质的丧失有助于潜在的病理学和疾病进展。相反，BBB的限制性渗透性对药物递送至CNS提出了挑战。调节血脑屏障通透性的遗传机制知之甚少，但在血管屏障功能异常导致疾病的情况下是潜在的治疗靶点。我们已经确定了一个G蛋白偶联受体（GPCR）的正常血脑屏障通透性所必需的，但它如何实现这一功能仍然是未知的。我们使用斑马鱼来研究血管通透性是如何控制的，因为斑马鱼胚胎是光学半透明的，并且在母体之外发育。这使我们能够用荧光标记血管，并使用显微镜直接观察斑马鱼胚胎中的渗漏血管。在斑马鱼中，血脑屏障在受精后2 - 3天迅速建立，重要的是，调节斑马鱼血管形成和功能的机制与人类高度保守。使用CRISPR/Cas9基因组编辑，我们在GPCR复合物中产生了新的斑马鱼突变体。我们的斑马鱼突变体有一个渗漏的血脑屏障。通过使用具有荧光标记血管的转基因和突变斑马鱼胚胎，该项目将研究通过这种GPCR在调节血管通透性和BBB功能中的信号传导作用。斑马鱼GPCR突变体在发育中的脑血管系统中显示出高渗透性，在这些血管正常完整的阶段，并且BBB是正常功能的。有趣的是，在我们的突变体中大量微泡的外渗表明血管通透性过高是一个跨细胞过程。我们假设我们的GPCR突变体显示跨细胞渗透性途径的激活，导致BBB的渗透性增加。为了验证这一假设，本项目将利用RNA测序来鉴定GPCR介导的血管通透性的转录机制。为了测试候选基因，我们将采用我们团队开发的尖端CRISPR/Cas9和CRISPR干扰技术。为了确定GPCR复合物如何控制血脑屏障通透性，我们将使用共聚焦和光片荧光显微镜对斑马鱼胚胎内的血管形成和功能进行实时成像。该项目将确定控制血管通透性的新分子机制，并可能成为疾病治疗操作的候选者。

To ensure tissue homeostasis, the central nervous system must be protected from hormones, neurotransmitters or pathogens circulating in the blood, while still allowing vital nutrients to reach the brain. To achieve this, blood vessels which vascularize the central nervous system (CNS) display unique properties, termed the blood-brain barrier (BBB). The BBB heavily restricts vessel permeability and thus protects the brain from injury and disease. Loss of barrier properties during diseases including stroke, diabetes and vascular dementia contribute to underlying pathology and disease progression. Conversely, the restrictive permeability of the BBB poses challenges for drug delivery to the CNS. The genetic mechanisms which regulate permeability of the BBB are poorly understood but are potential therapeutic targets where abnormal vascular barrier function contributes to disease. We have identified a G-Protein Coupled Receptor (GPCR) essential for normal BBB permeability, but how it achieves this function remains unknown. We use zebrafish to study how vascular permeability is controlled because zebrafish embryos are optically translucent and develop outside of the parent. This allows us to label blood vessels fluorescently and directly observe leaky blood vessels in zebrafish embryos using a microscope. In zebrafish, the BBB is quickly established between 2 and 3 days post fertilisation and importantly, mechanisms which regulate blood vessel formation and function in zebrafish are highly conserved with humans.Using CRISPR/Cas9 genome editing, we have generated novel zebrafish mutants in a GPCR complex. Our zebrafish mutants possess a leaky BBB. By employing transgenic and mutant zebrafish embryos with fluorescently labelled blood vessels, this project will examine the role of signalling via this GPCR in regulating vessel permeability and BBB function. Zebrafish GPCR mutants display hyperpermeability within the developing brain vasculature at stages where these vessels are normally intact, and the BBB is normally functional. Interestingly, extravasation of large numbers of microvesicles in our mutants suggests vascular hyperpermeability is a transcellular process. We hypothesise that our GPCR mutants display an activation of transcellular permeability pathways leading to increased permeability of the BBB. To test this hypothesis, this project will utilise RNA sequencing to identify the transcriptional mechanisms underlying GPCR -mediated vascular permeability. To test candidate genes, we will employ cutting edge CRISPR/Cas9 and CRISPR interference technologies developed within our group. To determine how the GPCR complex controls BBB permeability, we will perform live imaging of blood vessel formation and function within zebrafish embryos using confocal and lightsheet fluorescence microscopy. This project will identify novel molecular mechanisms which control vessel permeability and which may be candidates for therapeutic manipulation during disease.