Next-Generation Transgenesis in Zebrafish.

Zebrafish (Danio rerio) is a small tropical fish that has rapidly become an excellent model for biomedical studies. Zebrafish are inexpensive to maintain and breed readily. The young fish are small and relatively transparent which enables researchers to make microscopic observations in the living animal. This research project aims to develop an exciting new technology that will allow us to watch individual cells in the intact animal. To do this we will fluorescently label individual cells which will allow us to follow them using high-resolution microscopy. This new technology will also allow us to target specific molecules within these labelled cells to disrupt their activity. The molecules that we will target play important roles in regeneration, stem cells and cancer. After we disrupt the function of a molecule, we will be able to watch the cell to see whether its movement, division or differentiation potential are affected. For example we can test whether disruption of one molecule in a cell causes it to divide rapidly and form a tumour. This method will even allow us to follow what happens to an individual cell over a period of months or years. These exciting new tools will enable many researchers around the world to answer unresolved questions and will open up many new areas for biomedical research.

We propose to make a large set of creERT2 and lox based lines which will be useful to many Zebrafish researchers including those in the fields of stem cell biology, regeneration and cancer. We will make more than 15 tissue-specific creERT2 transgenes to drive recombination in a range of precursor and differentiated cell types. We will make a set of 3 lineage tracing lines which will enable researchers to permanently label individual cells and their descendants and follow their behaviour (migration, division and differentiation) throughout the life of the animal. In addition, we will make a set of lox based transgenes that will enable researchers to up- or downregulate the activity of more than 7 pathways permanently in a given cell (PathM lines). The following pathways will be targeted in the first instance: FGF, WNT, BMP, Notch, Hedgehog, Hippo, JAK/STAT. Four more PathM lines will be generated at the request of the Zebrafish community. All lines will be freely available as soon as they are established and with the appropriate material transfer agreement(s) in place. We will establish a website to publicise the resource, aid in distribution and to provide a discussion group for trouble-shooting. Our overall aim is to galvanize the use of the Cre/Lox system in Zebrafish research and in doing so provide many new tools for biomedical research.

CONTRIBUTION TO WELLBEING IN THE UK AND GLOBALLY
This project will greatly enhance productivity in the cancer, stem cell and regeneration fields, and thus in the long-term, many patient groups in related fields. Given the breadth of these fields, it would be difficult to underestimate this project's potential impact:
Cancer
Many cancers are of clonal origin and involve the signalling pathways targeted in this application. By generating new methods to induce clonal tumour formation we will be generating new models to enhance our understanding of tumour biology. These models may also present suitable platforms for screens to identify anti-tumour drugs.
Stem cells
Although most ongoing research uses cell culture, it is very important to study stem cells in living animals to fully understand their needs and capabilities and the signals controlling these; the tools developed here will allow this. This knowledge will facilitate our ability to manipulate endogenous stem cells and to predict how transplanted cells should behave when reintroduced during regenerative therapy; it may even allow creation of precursor cells tailored to specific needs.
Regeneration
Zebrafish have a tremendous capacity for regeneration, with mature cell types (e.g. osteoblasts, myocytes) dedifferentiating, proliferating and then redifferentiating. Little is known about which signalling pathways regulate these steps and whether dedifferentiated cells are lineage restricted in vivo; tools made in this project will allow these questions to be answered. If, for example, we could identify which signals trigger dedifferentiation of myocytes, this knowledge might be used to stimulate repair in humans after myocardial infarction.

EXPLOITATION AND APPLICATION
This research will benefit multiple user groups, outside the immediate academic partnership. Most immediately (2-5 years) other academic, charity and industrial parties will benefit from the availability of these tools to use in their own studies and applications. In the medium term (5-15 years), cancer/degenerative disease patients and the NHS would benefit from the therapeutic application of the data obtained with these tools.

Our work has clear potential impacts, within a 5-15 year timescale, on the nation's wealth, through contributing to the efficient development of patentable therapies for cancer, injuries and ageing diseases; and to the nation's health, through providing the basic science allowing the provision of more, and more effective, treatments.

Identification of potential patent filings will be a key agenda item at the internal laboratory meetings. The Universities are agreed in principle that Sheffield will take the lead and that all parties will co-operate to ensure proper protection and exploitation of IP arising from the research. The Department of Biomedical Science (Sheffield) employs a Research Translator (Dr. Susan Smith(SS)). SS is hosted within BMS to facilitate commercialisation of academic research and interaction with industry as appropriate. SS is a biochemist by training and has worked in biotech companies in the US and Germany in several research and development roles, as well as business development. She is a PRINCE2 registered practitioner with experience in project management and, through her work at the University of Sheffield, in knowledge transfer. Her knowledge transfer and industry skills will facilitate building new links to, and ways to engage, commercial beneficiaries, for example through Knowledge Transfer Partnerships. At the University of Bath, the Research Development and Collaborations (RDC) team and technology transfer office (Bath Ventures) will provide a comprehensive framework of support to ensure that outputs are identified and protected and appropriate routes and partnerships are developed to maximise commercial and societal value. Both Sheffield and Bath have successful track records in promoting and exploiting their research outputs.