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

这个研究项目的目的是用一种道德上更可取的动物模型——斑马鱼来取代老鼠作为实验动物。它还旨在通过使用先进的成像技术改进实验方法来减少使用的动物数量。总的来说，这个项目将增加我们作为一个社会的努力，以减少在研究中使用动物，特别是使用哺乳动物。我们的项目旨在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，最终可以用于产生临床效益。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，斜视会导致视力虚弱和部分失明，并对患者造成社交障碍。目前没有有效的治疗方法，除了手术，从长远来看也可能无效。斜视是一种眼部运动障碍，它是由眼运动系统（控制眼部肌肉的神经）的线路故障引起的。我们和我们的临床同事已经发现一种遗传形式的斜视——杜安内缩综合征（DRS）——可以由基因嵌合蛋白1的突变引起。这种基因产生α 2-嵌合蛋白，这种蛋白存在于生长中的神经中，并使它们在发育过程中与正确的肌肉连接起来。我们之前在鸡和斑马鱼胚胎中建立了眼运动发育模型，结果表明，干扰α 2-嵌合蛋白的功能会导致类似于人类DRS的线路缺陷。到目前为止，我们的研究涉及短期实验；为了加深我们对DRS的理解，我们将制造永久性的“转基因”斑马鱼，它们可以产生不同水平的α - 2嵌合蛋白。特别是，我们将创造出基因组中携带已知人类α 2嵌合蛋白突变的斑马鱼，准确地重现人类患者的生物学特征。在这些鱼身上，我们将研究神经生长和连接的模式，以及追踪鱼的眼球运动。由于斑马鱼的眼运动系统和对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。荧光蛋白将被引入到生长中的神经中，使我们能够观察和拍摄神经生长的过程，并将由于α 2嵌合蛋白缺陷而导致的变化与正常发育进行比较。改变转基因鱼体内α - 2嵌合蛋白水平的影响将通过拍摄鱼眼对旋转条纹鼓的反应来进行功能测试。这将使我们能够测量出鱼的斜视程度。神经线路和眼球运动的测量结果将直接与DRS患者的信息进行比较。总的来说，该项目的目标是创建斑马鱼模型，以取代小鼠作为研究DRS的实验系统。我们培育的转基因品系，以及我们的数据，将在发表后不久提供给其他研究人员。总的来说，我们使用的动物数量将比典型的小鼠研究少十倍左右。我们研究中使用的成鱼大部分将在项目期间维持。我们提出的实时成像实验类型还允许我们进行许多详细的测量，减少了使用的动物总数。在未来，我们打算使用我们的模型系统作为一个试验台，以寻找可能的药物治疗DRS。例如，可以用候选药物测试眼运动系统线路有缺陷和眼球运动异常的鱼，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疗法。我们创造的斑马鱼系将作为基础和临床研究人员研究人类神经系统疾病的资源而存在，并将长期有助于改善人类健康。

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.