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

为了确保组织的动态平衡，中枢神经系统必须受到保护，不受血液中循环的激素、神经递质或病原体的影响，同时仍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。在中风、糖尿病和血管性痴呆等疾病中屏障特性的丧失是潜在的病理和疾病进展的原因。相反，血脑屏障的限制性渗透性对向中枢神经系统输送药物构成了挑战。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地方是潜在的治疗靶点。我们已经确定了一个对正常血脑屏障通透性至关重要的G蛋白偶联受体，但它是如何实现这一功能的仍不清楚。我们使用斑马鱼来研究血管通透性是如何控制的，因为斑马鱼的胚胎是光学半透明的，并在亲本之外发育。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泄漏的血管。在斑马鱼中，血脑屏障在受精后2至3天内迅速建立，重要的是，调节斑马鱼血管形成和功能的机制在人类中高度保守。利用CRISPR/Cas9基因组编辑，我们在GPCR复合体中产生了新的斑马鱼突变体。我们的斑马鱼突变体有一个漏血的血脑屏障。通过使用带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发出的信号在调节血管通透性和血脑屏障功能中的作用。斑马鱼GPCR突变体在发育中的脑血管系统中表现出高渗透性，这些血管通常是完整的，血脑屏障正常发挥作用。有趣的是，在我们的突变体中，大量微泡外溢表明血管高通透性是一个跨细胞的过程。我们假设我们的GPCR突变体表现出跨细胞通透性通路的激活，导致血脑屏障通透性增加。为了验证这一假设，该项目将利用RNA测序来确定GPCR介导的血管通透性背后的转录机制。为了测试候选基因，我们将使用我们团队开发的尖端CRISPR/Cas9和CRISPR干扰技术。为了确定GPCR复合体是如何控制血脑屏障通透性的，我们将使用共聚焦和光片荧光显微镜对斑马鱼胚胎中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的分子机制，这些机制可能是疾病期间治疗操作的候选。

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.