HOW DOES DIFFERENTIAL HEPARAN SULPHATE SULPHATION INSTRUCT FIBROBLAST GROWTH FACTOR SIGNALING IN THE DEVELOPING BRAIN?

The development of our brains starting from a fertilised egg up to the time of birth is a little like a building project where instructions need to be given to brain cells to make sure that the right structures end up in the right place at the right times. Central to this process is not only the giving of instructions in the right time and place but also their proper transmission and reception. Given the massive complexity of the brain it is seems obvious that a correspondingly nuanced language must be used by brain cells to communicate with one another. Correspondingly we know that if mistakes are made, which can happen if genes encoding the signals do not function properly or in some cases due to environmental factors disrupting signalling, then the brain can develop abnormally which can have a major impact on subsequent life. Understanding the language of cell communication is therefore of central importance to understanding normal and abnormal brain development.
Over the past few years scientists have made considerable strides in identifying the protein signals, or 'signalling proteins', which transmit instructions between cells. Signalling proteins are produced by a source and move to their target where they are sensed by receptors on the surface of the target cell. The target cell then changes its behaviour in response to the signal. A conundrum which has puzzled scientists is that, despite intense efforts, only a relatively small number of signalling proteins have been identified compared to the complexity of instructions needed to produce the brain. We have focused our attention on the role played by a completely different class of molecules, the carbohydrates, in cell signalling. We are particularly interested in the idea that interactions between carbohydrates and proteins expand the diversity of instructions. This is a very large question and the work we are proposing here will look in detail at a particular interaction between a signalling molecule called 'fibroblast growth factor 8' (or 'Fgf8' for short) and a particular type of carbohydrate called 'Heparan sulphate' (or 'HS' for short).
Fgf8 is particularly important for brain development as cells are very sensitive to the amount they encounter and even small changes in the level of Fgf8 can have a dramatic effect on brain development including situations where developmental defects are associated with altered Fgf8 dose. We have discovered that the structure of HS regulates the overall effectiveness of Fgf8 in the developing brain as mouse mutants with abnormal HS structure have defective Fgf8 signalling leading to brain malformations. In this proposal we plan to build on this work and build up a detailed picture of how HS controls the movement of Fgf8 protein between cells and the ability of cells to respond to Fgf8 protein. To help us do this we have developed a culture system which allows us to apply a source of Fgf8 protein and then track its movement with time. Using a special microscope in combination with a fluorescently tagged Fgf8 we can actually watch the way the protein moves through the tissue and measure how the cells react to Fgf8. By comparing the behaviour of Fgf8 when presented with normal and abnormal HS we can build up a picture of how HS normally regulate Fgf8 and how the process can go awry.
In the future we may be able to use our knowledge of the normal processes of signalling to assist regenerative approaches in replacing or repairing brain tissue lost either by disease or injury. More broadly, given the key role played by signalling proteins in almost all aspects of life, increased understanding of the interaction between carbohydrates and signalling proteins in our system is likely to provide insights into other systems.

Brain development is regulated by cell signalling, molecules move between cells and are sensed by cells. Perturbing any of these steps can cause abnormal development and understanding cell signalling provides avenues for therapeutic intervention. The roles of proteins, secreted morphogens and transcription factors, in cell signalling are reasonably well understood but knowledge of carbohydrates, while generally acknowledged as important, has lagged behind. Fibroblast growth factor proteins are important in brain development and one, Fgf8, has a particularly interesting role as a classic morphogen meaning that cells experiencing different levels of Fgf8 signal adopt distinct fates. Understanding the mechanisms which regulate Fgf8 dosage is therefore critical to understand its roles in brain development. Heparan sulphate (HS), the carbohydrate component of heparan sulphate proteoglycans, consists of ~100 Uronate-Glucosamine disaccharide repeats and is subjected to differential sulphation by enzymes including Hs6st1 and Hs2st generating an extraordinarily high degree of structural diversity with the potential to give differentially sulphated HS the ability to instruct Fgf8 and other signalling pathways in different ways and enhance the functionality of cell signalling. We previously showed that Hs6st1 loss of function embryos have highly abnormal Fgf8 signalling causing a major brain phenotype in which the corpus callosum, the largest axon tact in the mammalian brain, does not form. We will use a combination of cell culture, live cell imaging, pharmacological, and biochemical assays to test the hypotheses that Hs6st1 modified HS is required for the movement of Fgf8 protein to target cells or for their sensitivity to Fgf8 protein. Next we will test the hypothesis that the Hs2st modification to HS has a different role. Overall this work will yield important mechanistic information into the mechanism(s) and specificity by which differential HS sulphation regulates Fgf8.

This work is fundamental biological research and therefore societal and economic benefits are most likely to accrue in the longer term. Our societal and economic impact plans cover general dissemination of the work and public engagement. We anticipate that our research will lead to novel insights into the normal processes of brain development in the womb which have the potential to affect the quality of subsequent life. Therefore even though our research is not directly clinical it may well provide comfort to patients and families of patients affected by neurodevelopmental disorders involving processes touched on by our research. Dr Pratt is a member of the Patrick Wild Centre (Director Prof Peter Kind, Edinburgh University) which has links to Autism patients and mutations affecting Heparan Sulphate have been linked to Autism phenotypes. More tangibly, we will actively disseminate information about our research efforts to the general public (press releases, schools visits, and our Wellcome Trust-funded 'mindyerbrain' Facebook page). Potential for exploitation of IP is difficult to judge at this stage, but there may be potential for outputs from this project. It is likely that a deeper understanding of the function of carbohydrate in cell signalling will have applications in wound healing and regenerative medicine approaches using stem cells. We will actively monitor our research output and draft publications for potential IP opportunities, and protect these where relevant by liaising with the Universities' technology transfer offices. The training of a post-doctoral fellow, Dr Clegg, in advanced imaging techniques will be of high value in contributing to his career development.