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

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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.