An automated in vivo screening platform for the UK zebrafish research community

Much of what we know about the function of genes and drugs has arisen from so-called screens. Screens are unbiased methods to test gene and chemical compound function. For example, scientists can test how disruption of large numbers of genes affects a biological process of interest to discover which gene controls a given process. Similarly one can test how large numbers of chemical compounds affect specific biological events and discover, for example, how future drugs might affect different cells. Carrying out screens on living animals is very difficult, because one cannot manipulate genes or use chemicals in a sufficiently large number of animals in a time, cost, or ethically reasonable manner. Zebrafish, however, are a laboratory organism in which genetic and chemical screens can be carried out readily. This is because their embryos are available in very large numbers (1000s per day), they develop very rapidly, going from a fertilized egg to a tiny entity with the majority of organs that humans have in under 3 days- before they are technically considered animals. These young organisms are also transparent, such that one can directly observe biological events as they happen over time. New genetic technologies have further refined the ability to visualize in living zebrafish, e.g. by creating fish strains in which cell types of interest fluoresce. Genetic and chemical screens in zebrafish have already taught us a great deal about the fundamental molecular and cellular mechanisms of life and have even led to new clinical trials for the treatment of disease. However, despite being possible, such screens have remained very laborious, manual procedures, that take extended periods of time and that often rely on immediate experimenter's ability to link a gene or chemical treatment with biology. Therefore there is a great need to increase the efficiency of such screens because this will translate directly into new knowledge and insights of importance to health. Our proposal combines two elements, an electronically controlled system that can automatically transfer large numbers of embryonic zebrafish from their dishes via small capillaries onto a microscope stand in a rapid and uniform manner one by one. The second element is the microscope itself, which can carry out high-speed imaging of the individual zebrafish as they pass through the system. One can take overview snapshots of the entire animal to assess general health or organ function, or very detailed 3D images of specific cells of interest. Therefore it is possible to use this system to automatically image and screen hundreds of zebrafish throughout a day in a systematic manner and create a permanent record of the data, all of which represents an enormous increase in the efficiency of current approaches.
This new system will be installed at the University of Edinburgh, where there is a concentration of world-experts in the use of zebrafish as a laboratory model. During the funding period of this award, the applicants will install the system in Edinburgh, and launch it to the wider community. In Edinburgh, the system will support projects focused on understanding the development of motor neurons and glial cells in the nervous system- cell types that are important for normal nervous system function and disrupted in diseases such as motor neuron disease and MS. The system will also be used to gain new insights into pigment cell development and how these cells are transformed in melanoma. Further screening studies will be carried out to better understand how our immune system can sometimes promote tumour growth and to learn how the resident network of immune cells in our brain develops and responds to injury. The wider UK community of zebrafish researchers who have already expressed great interest will use this new technology to elucidate yet further aspects of biology. Thus this new automated screening system for zebrafish will greatly strengthen UK Bioscience for health.

Through the BBSRC's Advanced Life Sciences Research Technology Initiative 2014 we aim to establish an automated screening platform for the UK zebrafish community. Zebrafish are a powerful laboratory organism for the discovery of fundamental molecular and cellular biological mechanisms. The zebrafish has risen to prominence as a model system due to its suitability for genetic and chemical discovery screens, whereby one can assess in a systematic and unbiased manner, how manipulation of genes or treatment with chemicals affect biological events. Following the association of a gene or chemical treatment with a specific phenotype, one can carry out detailed studies using a variety of approaches in both zebrafish and other models. Although such zebrafish screens have been very effective in providing insight into multiple biological processes and seeding drug discovery projects, they are typically time consuming and laborious manual processes that rely on user observation rather than the acquisition of permanent record to identify associations between gene or chemical and phenotype. Here we introduce an integrated system that will automate many aspects of zebrafish screens. A unique microfluidics platform called VAST (Vertebrate Automated Screening Technology) developed by researchers at MIT, can transfer live or fixed zebrafish embryos from dishes or plates to the stage of a microscope and orient them in a uniform pre-defined manner. An associated rapid scanning confocal microscope with brigh-tfield capacity can then automatically acquire images at cellular to whole animal resolution. In-so-doing the system creates a permanent record of data at speeds orders of magnitude beyond current manual approaches. Such an automated system will be the first of its kind in the UK and will be installed at the University of Edinburgh, home to world leading zebrafish researchers, who will coordinate its wider use to boost zebrafish related research and bioscience in the UK.

The present project will establish an automated screening and imaging platform available to the UK zebrafish research community. Projects using this setup will investigate the fundamental mechanisms of nervous system formation, melanocyte development, and immune cell biology in the larval zebrafish, using automated pharmacological and genetic screening, imaging and manipulations that have been previously impossible.

Communications and Engagement
The PIs will attend 6 conferences from October 2014 to September 2015 to advertise the availability of the VAST system. CB and LP will distribute information through EUFishBioMed and the ZDM Society. In late June 2014 we will organise a one-day workshop at the University of Edinburgh to exchange experience gained to that point and train new interested parties (up to 30). Prof Herman Spaink, the only other European VAST screening system user to date, has agreed to speak to support the technical and development angle of the setup. Dr. Asier Unciti-Broceta, an expert in chemical screen development based at the IGMM in Edinburgh, will present the Chemical Biology possibilities using the VAST system. We plan to continue these workshops in subsequent years and will apply for funding through EMBO and/or The Company of Biologists, strengthening UK capacity and the recognition of UK based science excellence in Europe.

The PIs take every opportunity to engage with the public, including patients, their relatives and other interested parties, including potential donors (for example the "Bash at the Brewery", MND Scotland Open Days, and the Edinburgh International Science Festival). CB will attend the Packard Centre for ALS Research in March 2015 and DL will contribute to events organised by the University of Edinburgh Multiple Sclerosis Centre and liaise with the online multiple sclerosis user group shift.ms to spread knowledge about our research and research results to the wider public.
A summary that is suitable for a lay audience will be published on a custom web stie for the new equipment, and the web sites of the CNR, School of Biomedical Sciences and IGMM. We will further ensure dissemination of results through publications in the Fish Societies' Newsletter and by contributions to charity newsletters (e.g. Packard Center, Wellcome Trust, Lister Institute, CRUK). Our research has been featured in the University's "Friends" magazine, targeted to Alumni and donors and we will strive to get another feature in the Friends magazine by liaising with the Press Office. We are in regular contact with the College's press officer, Eleanor Cowie, and will, in coordination with the BBSRC press office, issue a press release when the set up is commissioned and when results are published.

Developmental Biology using zebrafish is a particularly attractive topic to engage pupils in science. CP and LP have run workshops for primary school pupils at local schools in science lessons and workshops and will continue to do so.

Collaboration
The present proposal incorporates an active collaboration between DL and Biogen Idec, who are also contributing financially to project. Success in our award will also directly lead to a collaboration with Union Biometrica on the further development of the VAST system (see letters). The UK wide access will further strengthen collaboration between UK fish groups, one of the aims being the creation of a multi-transgenic fish allowing simultaneous screen of multiple organ systems.

Capability
In the course of this project, > 15 postdoctoral fellows and PhD students will be trained (currently, 16 groups plan to use the system). Furthermore, >20 MSc students from the MSc Integrative Neuroscience will be introduced into the technical and biological potential of automated zebrafish screening. Dr Katy Cole, currently employed as technician, will receive targeted training in Edinburgh and Leiden, to support her career development towards a laboratory manager role.