Investigating microRNA:target gene interactions in myogenesis

Multi-cellular organisms contain many distinct cell types with very specialized functions. For example, we need skeletal muscle to be able to move while our skin prevents dehydration and protects us from injury and infections. Amazingly all these different cells arise from a single cell, the fertilized egg. The development of an embryo begins when the egg starts dividing to give rise to many cells. Different cells are specified during embryonic development - they are essentially told what to do and what to become by molecular signals that act in the early embryo. These signals often cause specific genes to be switched 'on' or 'off'. If a gene is 'on' it is expressed which means that it is actively transcribed from the DNA in the nucleus of the cell. During the process of transcription, DNA is copied into RNA. These RNA transcripts typically encode proteins and RNA is translated into proteins by a complex cellular machinery. Proteins are the 'movers and shakers' in a cell, they define a cell and they have specific jobs to do. For example, the contraction of skeletal muscle is mediated by fast and slow contractile fibres (made up of proteins). Muscle is a very plastic tissue and depending on whether you train to be a 100 m sprinter or a marathon runner different types of muscle proteins will be expressed. Muscle also has the ability to repair itself (to regenerate) for example after wearing a cast muscle is lost, but it builds up again quickly when the muscle is used again. We are interested in the molecules that control the development of muscle in an embryo, it is known that some of these (including some that we previously discovered, called 'Wnt') are also used during muscle regeneration. In particular we study a class of RNA molecules, which are not translated to make proteins. Here the RNA molecule itself has an important functions. These non-coding RNAs were discovered recently and because they are very small, they were called microRNAs (miRs). They have been found in plants and animals, which means, that they are part of the most basic machinery of life with a very important and fundamental job to do in all cells. This turned out to be the case and in fact microRNAs control whether or not other coding RNAs are translated into protein. A lot of research is being done, to help understand how this is happening and to uncover what type of cellular processes are controlled in this fashion. Our research investigates how cells become different from one another in a developing vertebrate embryo. In particular, we study the genes and molecules that control the decision of a cell to differentiate into skeletal muscle from a multi-potent precursor, as opposed to into bone for example. We recently discovered that two of these new microRNAs (and there are currently more than 400 microRNAs known) are only present in those cells in the embryo, that will go on to make skeletal muscle and we want to understand what the role of these microRNAs is. We have already figured out how the production of the microRNA itself is being switched 'on' or 'off', and we have identified some of the genes controlled by the microRNAs (the 'targets'). Ideally we want to identify all the target genes and we also need to understand how they in turn affect skeletal muscle. Overall we will learn how an embryo makes normal, healthy, working muscle and this will in the long-term benefit people who suffer from various muscle degenerative diseases or age related muscle-loss.

We are interested in the molecular signalling networks controlling early events in vertebrate embryogenesis. We investigate a number of different processes which impact upon these including pattern formation and cell specification as well as cell movement. We want to understand how multi-potent embryonic progenitor cells are instructed to differentiate into distinct cell types at the correct time and in the right place. We use the developing somite in chick embryos, which will give rise to a number of distinct mesoderm cell lineages including skeletal muscle, as a model system to investigate this question. This project will investigate the importance of the skeletal muscle specific microRNA-206 for the specification and differentiation of cells within the developing somite. Preliminary work has demonstrated that miR-206 is specifically expressed in the myotome, the part of the somite that contains skeletal muscle progenitor cells. We have identified and validated some novel targets using a candidate approach. Two of these will be investigated in detail using established methods in the chicken and, together with international collaborators, using genetic manipulations in mouse embryos. The approaches in mouse are highly complementary and represent a significant contribution and thus added value. To characterize the 'miR-206 targetome', we will use unbiased experimental approaches, for which proof of concept has been established. These will be performed in C2C12 cells and in embryonic somites and benefit from bioinformatics expertise. Targets will be validated in cultured cells and embryos using biochemical methods. The precise roles for microRNAs in embryonic development are not completely understood and nothing is known about how they orchestrate embryo myogenesis. This work will make a significant contribution to the understanding of the system of muscle biology and yield more general insights into the fundamental mechanisms employed in vertebrate embryogenesis.

The School of Biological Sciences (BIO) is committed to significant growth in engagement and enterprise. There are two main areas in which the research will have broad impact for a range of beneficiaries across the public, private sectors and general public: 1. Novel microRNA targets. We plan to build upon our discovery that inhibition of microRNA function in developing somites leads to impaired skeletal muscle development. This is one of the first reports of microRNA function in avian embryos and thus an exciting scientific development in a rapidly developing field. 2. Bioinformatics. Our datasets will be valuable for technological development of bioinformatics toolkits and prediction algorithms for miR targets. Who will benefit from the research and how? Public sector: In the public sector, we aim to communicate the findings of our basic research of fundamental processes ultimately to health professionals. There is much interest in skeletal muscle and its ability to differentiate further during the life-time of the organism (during training) and to regenerate after injury or illness and/or age induced muscle loss. Our work will be relevant here as it will uncover the molecular players involved in the generation of muscle during embryo development, some of these components are likely to be involved in healthy tissue turnover and maintenance. We have a core group of researchers in Biological Sciences actively investigating the musculo-skeletal system and good links with the School of Medicine, Health Policy and Practice. Private sector: In the private sector we will be using the research and enterprise office staff to develop CASEing for studentships to investigate the links between molecular targets that respond to miR manipulation in developing muscle. We are currently working towards a CASE partnership with Pfizer to investigate microRNAs differentially expressed in mouse models of muscular dystrophies (with Professor Uli Mayer, UEA). Our research has had previous CASE support (from Astra-Zeneca and Cyclacel) indicating the interest in skeletal muscle regenerative ability in health and disease. General public: We are actively involved with the press office in highlighting research papers and grant successes at a local and national level. Here we benefit from the engagement office, which helps to ensure that the results can be cascaded through local schools to provide immediate impact. The PI has given a 'Science Cafe' style talk about Stem Cells, been engaged in a 'Masterclass on Stem Cells' for Secondary School Teachers from the Eastern Region (organized by the Teacher Training Network, Autumn 2008) and been involved in Open days for GCSE Science students (July 2009) on dmbryo development. Collaborations and partnerships: The research team is a new collaboration and each member has distinct expertise, which aids synergy. In relation to impact, we will collaborate fully on all activities listed here. To facilitate complementary approaches in mouse models we initiated international collaborations with Dr Eran Hornstein (Weizman, Israel) and Dr Benoit Bruneau (Gladstone, USA) who are best placed to push these aspects of the projcet forward effectively. Exploitation and Application: We will be seeking to exploit the research. Specific partnership agreements are already in place for related projects. We will have regular meetings with our enterprise and engagement officer to discuss results and work up opportunities. To protect the research we will have IP and MTA agreements with new collaborators, drawn up by our research contracts office. Capability: All personnel, including PDRAs will engage with the impact agenda. Other staff involved will be our web development officer, our media office who will advise and assist with press releases and our School enterprise and engagement staff. All investigators have a track record of such activities and had training from staff development programmes.