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

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.

The major aim of our proposal is to generate zebrafish models of a human eye movement disorder, to replace mouse models. Eye movement disorders affect 1% of the human population. In severe cases, such disorders can result in amblyopia and partial blindness, and there is currently no effective treatment. Duane Retraction Syndrome (DRS) is a congenital form of squint in which horizontal eye movements are defective. Patients with DRS have abnormal wiring of the ocular motor system - the nerves and muscles that control eye movements. We previously showed that DRS can result from mutations in the cytoplasmic signalling molecule alpha2-chimaerin, and demonstrated that transient manipulations of alpha2-chimaerin signalling in the chick and zebrafish model systems leads to defects in ocular motor wiring, akin to DRS in humans. In this proposal, we will make more permanent models of DRS, to act as a resource for the research community. We will generate stable transgenic zebrafish lines with loss- or gain-of-function of alpha2-chimaerin, in order to determine the effects of chimaerin signalling levels and to model DRS. Gain-of-function models will either over-express the wild-type alpha2-chimaerin protein, or will harbour human alpha2-chimaerin mutations. We will map the neuroanatomy of the ocular motor system in these lines, and functionally test the optokinetic reflex to determine the impairment of eye movements. These data will be directly compared with neuroimaging and functional data in humans, to gain insight into the causation of DRS. Our zebrafish models will be disseminated to the research community, as a replacement and a desirable alternative to the mouse. All experimental analysis will be carried out on larvae at 1-5 days post-fertilisation, and therefore not 'protected' species. Neuroanatomical studies will involve live and time-lapse imaging allowing quantitation of multiple parameters from a single animal, thus allowing a reduction of animal usage.

We will impact the 3Rs in several important ways. The first and main impact of this project will be to generate zebrafish models of a human neural disorder, to replace the mouse. By generating zebrafish models of a specific eye movement disorder (Duane Retraction Syndrome), we will show a direct relationship between zebrafish and a human syndrome, and lead to way to others in encouraging them to take up this promising experimental system. Specific patient mutations in a particular gene (alpha2-chimaerin) will be modelled in the zebrafish to accurately recreate anatomical and functional features of the syndrome, in this case miswiring of the ocular motor system and eye movement defects. Our transgenic models represent only a 'mild' severity banding. 360 fish will be maintained over a 3 year period, and natural breeding will be used to generate embryos which will be analysed at 1-4 days post-fertilisation (not therefore 'protected' species). Currently at least 7 other labs use the mouse to model alpha2-chimaerin in knockouts, entailing more severe procedures to generate the lines, and using an estimated 12,000 mice over three years. Of these mice, at least 50% are sacrificed to generate embryos, whereas with the zebrafish the parents are not harmed during breeding to obtain embryos. The second impact of the project is that we will reduce usage of zebrafish by use of live imaging, especially making time-lapse movies of growing nerves and single axons, from which we can extract many different metrics, reaching significance from much smaller numbers of animals (5-10) than for typical studies of neuroanatomy (20 - 50 animals). A key priority of our project will also be animal welfare in our newly designed fish facility, with Dr Poparic personally supervising the upkeep and welfare of the fish lines used.

Dr Poparic will achieve impact by acting as an ambassador for the 3Rs. She will champion this approach initially in our extensive zebrafish community in King's, branching out to colleagues at UCL, then nationally and internationally, and interacting with researchers working in rodent models also. She will give seminars internally, in London networks, at the 3Rs research conference and at international conferences. She will also publicise our approaches to the public, including school students and the general public at MRC- and 3Rs-sponsored events. We will also develop our own lab website to incorporate 3Rs aspects into all of our work, and publish our zebrafish work and models in journals, at conference presentations and in the online resource zebrafishbrain.org.
We will achieve specific clinical impact through our pre-existing collaborations with clinicians working on eye movement disorders, the clinical neurologist Dr Nick Gutowski and the clinical geneticist Dr Sian Ellard in Exeter. In addition we regularly interact with clinicians based at Moorfields eye hospital and Institute of Child Health. We will give presentations there to explain our zebrafish models, how they may be used and to encourage the possibility of building a future network of interested research groups to establish models of other cranial nerve disorders. Here Dr Poparic will encourage other researchers to use the zebrafish rather than the mouse.

We will also interact with advisors in the Technology Transfer Team at King's Business to consider future commercialisation possibilities, and how we might use our models to develop therapies for eye diseases. We will also interact with the third sector via the charity Fight for Sight.