Imprinted genes defining a novel growth regulatory axis

Correct growth during life in the womb is important for infant survival and is also known to impact health throughout life, including the risk of developing common metabolic disorders such as obesity, diabetes and heart disease. Growth is a highly regulated process but basic questions remain unanswered such as: How are appropriate body size and proportions achieved? Which genes are important and how do they link fetal growth with lifelong health? Through genetic studies in mice we have identified two genes involved in regulating fetal growth that also influence the balance of lean and adipose tissue in later life, along with other aspects of metabolic health. One of these genes, Dlk1, promotes fetal growth and limits adipose accumulation, whereas Grb10 restricts growth and promotes adipose deposition. We have strong evidence that the two genes influence these processes in opposite directions by acting antagonistically in the same regulatory circuit (or pathway). Such regulation, involving positive and negative factors to achieve fine control, is typical of biological systems.
Our next goal is to identify other key components of the Dlk1/Grb10 pathway. Towards this aim, we have constructed a model of how the pathway might work, based on our own studies and those of others in the field. For instance, we know that while Grb10 directly inhibits growth, Dlk1 promotes growth indirectly by inhibiting Grb10. Also, quite a lot is known about the proteins encoded by the Dlk1 and Grb10 genes, from which we can infer the types of molecule they must interact with. In fact each interacts with different cell surface receptor molecules, akin to antennae projecting out from the cell membrane. Dlk1 encodes a signal protein released from one cell that interacts with receptors projecting from another. This type of cell to cell communication is important for coordinating the complex processes of growth and development. Grb10 encodes a molecule that interacts with receptors from within the cell, where it acts to modify how the cell responds to signals coming from the outside. In the case of Grb10 we have identified a strong candidate 'growth' receptor and one of our key goals is to validate and investigate this candidate further. Similarly, we have a candidate protein predicted to inhibit Grb10 as a consequence of Dlk1 signalling, and we will carry out experiments to test the interaction between the inhibitor and Grb10. These candidate molecules represent key components of the pathway, their validation would confirm important mechanisms predicted by our model and would provide the evidence needed to firmly establish the Dlk1/Grb10 growth axis. Finally, we have designed experiments that will enable us to discover additional pathway components, or alternatives should either of the candidates prove false.
Our studies indicate that the pathway involving Dlk1 and Grb10 is novel and important for our understanding of fetal growth regulation. Further, knowledge of this pathway would impact the future development of advice and treatments to prevent growth and metabolic disorders.

Fetal growth is highly regulated in order to achieve appropriate size and proportions at birth. Inappropriate growth can compromise survival and has been linked with increased life-long risk of adverse metabolic health, including some of the most prevalent life-threatening disorders such as obesity, diabetes and cardiovascular disease. In order to better understand these processes we are focussed on two imprinted genes with roles in fetal growth regulation, as well as lean to adipose body proportions and energy metabolism in later life. Dlk1 encodes the delta-like 1 ligand, is expressed from the paternally-inherited allele, promotes fetal growth and inhibits adipose deposition. Conversely, Grb10 encoding a signalling adaptor protein for tyrosine kinase receptors, is expressed from the maternally-inherited allele, inhibits fetal growth and is permissive for adipose deposition. Using mouse genetics we have shown that these oppositely imprinted genes, with antagonistic functions, most likely act within a common pathway. Further, while Grb10 acts to directly inhibit fetal growth, Dlk1 promotes growth by acting as an inhibitor upstream of Grb10. We propose that Dlk1 and Grb10 are defining members of a novel mammalian growth axis. Preliminary biochemical data both supports our genetic studies and has allowed us to develop a model of the Dlk1/Grb10 pathway. Here we propose testing specific aspects of this pathway by identifying additional components and key regulatory mechanisms predicted by our model. We will test candidates we have identified for the physiological 'growth' receptor targeted by Grb10 and for an inhibitor of Grb10 that must exist downstream of Dlk1. We expect to gain insights into fetal growth regulation that will impact the future development of advice and treatments to prevent growth and metabolic disorders.

The proposed work aligns with the MRC mission to support research aiming to improve human health. It is directly relevant to several objectives of the MRC Strategic Plan 2014-2019, within Research Priority Theme 2, Living a Long and Healthy Life. In particular the project is pertinent to the following objectives: 1. Molecular datasets and disease; in terms of "using genetics to understand predispositions to disease, and target treatments to disease subtypes". Also, the text of this objective recognises "the UK's strengths in epigenetics to better understand what triggers epigenetic changes and how they influence disease". 2. Life course perspective. The pathway that is the subject of our proposal forms a link between fetal growth and life-long health status and although we will not engage directly in "population-based research" our work will contribute knowledge of "health and wellbeing from childhood to older age". 3. Lifestyles affecting health. Determine the most effective
strategies for tackling lifestyles that are detrimental to health. Environment and health. Including how the environment and genes interact, particularly in the context of how the maternal environment (e.g. diet during pregnancy pre-weaning) can affect early growth and also life-long health.

Through basic science carried out in a model organism we aim to understand fundamental mechanisms regulating fetal growth, lean to adipose body proportions and metabolic health. This is relevant to some of the most important global health problems including obesity, type 2 diabetes and cardiovascular disease. Progress in human populations is made difficult by the long time-scales involved in studying a process that involves effects on life-long health that result from genetic, epigenetic and environmental factors experienced during early life. There are already major screens for genetic markers underlying human variation in body composition, for example, and our data will complement such population studies, enabling genetic variations to be related to biological function and mechanism. Ultimately this will help us to understand metabolic disease. Further, findings made in the model system we have established could be rapidly assimilated by biomedical researchers and applied to human studies. For instance, genes and proteins that we identify as components of the Dlk1/Grb10 growth regulatory pathway could be investigated as potential markers of health risk in longitudinal studies of people and as potential therapeutic targets for growth and metabolic disorders. Understanding of the links between early life events and long term health could lead to new ways of influencing health through the adoption of simple practices such as changes in maternal and/or infant diet and dietary supplementation. Ultimately therefore, the research could lead to improvements in health and healthcare that would benefit the general public in and beyond the UK.

As the data emerge we will seek to extend our collaborations with researchers involved in the appropriate types of population study. This could lead to exploration of possible health benefits within the lifespan of the project or soon after its completion. Thus, translation of findings from our model system to the development of early markers of health risks could commence within the next few years. The development of new interventions, including dietary advice and supplementation, or the development of drugs targeting effector molecules or pathways would be a longer-term impact.