Generating zebrafish models of human eye movement disorders to replace mouse models

生成人类眼球运动障碍的斑马鱼模型以替代小鼠模型

• 批准号: NC/L002264/1
• 负责人: Ivana Poparic
• 金额: $ 24.85万
• 依托单位: King's College London
• 依托单位国家: 英国
• 项目类别: Fellowship
• 财政年份: 2014
• 资助国家: 英国
• 起止时间: 2014 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=NC%2FL002264%2F1
• 关键词: Generating; zebrafish; models; human; eye

This research project aims to replace the mouse as an experimental animal with an ethically more desirable animal model, the zebrafish. It also aims to reduce the numbers of animals used by improving experimental approaches using sophisticated imaging techniques. Overall the project will add to our efforts as a society to reduce the use of animals in research, especially the use of mammals. Our project seeks to understand a particular disorder of the human nervous system, and will show that the zebrafish presents a fantastic opportunity which can ultimately be used to produce clinical benefits. Our research focuses on the causes of squint in humans, which affects about 1% of the population. In severe cases, squint can lead to weakness of vision and partial blindness, and is socially debilitating for sufferers. There is currently no effective therapy, apart from surgery which may be also ineffective in the long term. Squint is an eye movement disorder which arises due to faulty wiring of the ocular motor system - the nerves that control the eye muscles. We and our clinical colleagues have found that a genetic form of squint - Duane Retraction syndrome (DRS) - can be caused by mutations in the gene chimaerin1. This gene produces the alpha2-chimaerin protein, which is present in growing nerves and allows them to wire up with the correct muscles during development. We have previously modelled ocular motor development in the chick and the zebrafish embryo, and showed that perturbing the function of alpha2-chimaerin led to wiring defects akin to DRS in humans.Up to now, our research has involved short term experiments; in order to progress our understanding of DRS, we will make permanent 'transgenic' zebrafish, which produce alpha2-chimaerin at different levels. In particular we will create zebrafish which carry known human mutations of alpha2-chimaerin in their genome, accurately recreating the biology of human patients. In these fish we will study the patterns of nerve growth and connections, as well as tracking the fishes' eye movements. As the zebrafish ocular motor system and control of eye movements is closely similar to humans, this will allow us to mimic the human situation. Fluorescent proteins will be introduced into the growing nerves, allowing us to watch and film nerve growth as it happens, and visualise changes that occur due to the faulty alpha2-chimaerin in comparison to normal development. The effects of changing alpha2-chimaerin levels in the transgenic fish will be tested functionally by filming fish eye movements in response to a rotating striped drum. This will allow us to measure the degree of squint manifest by the fish. The measurements of nerve wiring and of eye movements will be compared directly with information from human patients with DRS.Overall, the aim of the project will be to create zebrafish models which can replace the mouse as an experimental system to study DRS. The transgenic lines we generate, as well as our data, will be made available to other researchers shortly after publication. Overall, the numbers of animals we use will be around tenfold less in number than typical for a mouse study. The adult fish used in our study will mostly be maintained for the duration of the project. The types of live imaging experiment we propose also allow us to make numerous detailed measurements, reducing the numbers of animals used overall. In future we intend to use our model system as a test bed to find possible drugs to treat DRS. For example, fish with faulty wiring of the ocular motor system and abnormal eye movements could be tested with candidate drugs to see if they restore normal development and function, leading to the development of therapies for human eye movement disorders. The zebrafish lines we create will exist as a resource for basic and clinical researchers studying disorders of the human nervous system and will in the long term contribute to improvements in human health.

这一研究项目旨在用一种更符合伦理道德的动物模型--斑马鱼--来取代小鼠作为实验动物。它还旨在通过使用复杂的成像技术改进实验方法来减少使用的动物数量。总体而言，该项目将促进我们作为一个社会减少在研究中使用动物，特别是使用哺乳动物的努力。我们的项目试图了解人类神经系统的一种特殊疾病，并将表明斑马鱼提供了一个绝佳的机会，最终可以用来产生临床益处。我们的研究重点是人类斜视的原因，这影响了大约1%的人口。在严重的情况下，斜视会导致视力减弱和部分失明，并会削弱患者的社交能力。目前还没有有效的治疗方法，除了手术，从长远来看也可能无效。斜视是一种眼球运动障碍，由控制眼部肌肉的神经--眼球运动系统--的错误连接引起。我们和我们的临床同事发现，斜视的一种遗传形式-杜安回缩综合征(DRS)-可能是由嵌合蛋白基因突变引起的。这种基因产生α2嵌合体蛋白，这种蛋白存在于生长的神经中，使它们能够在发育过程中与正确的肌肉连接。我们之前已经在雏鸟和斑马鱼胚胎中模拟了眼睛运动的发育，并表明干扰α2-嵌合体的功能会导致人类出现类似于DRS的连接缺陷。到目前为止，我们的研究包括短期实验；为了提高我们对DRS的理解，我们将永久地将产生α2-嵌合体的斑马鱼培育成不同水平的转基因斑马鱼。特别是，我们将创造斑马鱼，这种斑马鱼在其基因组中携带已知的人类α2嵌合体突变，准确地重建人类患者的生物学。在这些鱼中，我们将研究神经生长和连接的模式，以及跟踪鱼的眼睛运动。由于斑马鱼的眼球运动系统和眼球运动的控制与人类非常相似，这将使我们能够模仿人类的情况。荧光蛋白将被引入正在生长的神经中，使我们能够在神经生长发生时观察和拍摄它，并与正常发育相比，可视化由于有缺陷的α2嵌合体而发生的变化。在转基因鱼中，改变α2-嵌合体水平的效果将通过拍摄鱼的眼睛运动来进行功能测试，以响应旋转的条纹滚筒。这将使我们能够测量鱼的斜视程度。神经连接和眼球运动的测量将直接与人类DRS患者的信息进行比较。总体而言，该项目的目标是创建斑马鱼模型，可以取代老鼠作为研究DRS的实验系统。我们产生的转基因株系以及我们的数据将在发表后不久提供给其他研究人员。总体而言，我们使用的动物数量将比典型的小鼠研究少10倍左右。我们研究中使用的成鱼将在项目期间大部分时间内保持不变。我们提出的活体成像实验的类型还允许我们进行大量详细的测量，从而减少了整体使用的动物数量。在未来，我们打算使用我们的模型系统作为试验台，寻找治疗DRS的可能药物。例如，可以用候选药物测试眼睛运动系统有缺陷和眼睛运动异常的鱼类，看看它们是否能恢复正常的发育和功能，从而开发出治疗人类眼球运动障碍的方法。我们创建的斑马鱼品系将作为基础和临床研究人员研究人类神经系统紊乱的资源而存在，并将从长远来看为改善人类健康做出贡献。