Mechanisms for regulating mammalian axon guidance by differential sulphation of heparan sulphate.

Our brain is an organ of fascinating complexity. To function effectively precise connections must be made between cells which are often far apart. Cells in the retina of the eye, called retinal ganglion cells, send out fine processes called axons, which travel along predefined pathways (a process we call ?navigation?) and connect to specialised cells (their ?targets?) within the brain which will transmit visual information collected by our retinas for processing into images. So how do retinal axons make the long journey from the eye to their targets in the brain without going astray? A little like us using a map to navigate from one location to another and deciding whether to turn left or right at a junction to reach our destination, retinal axons respond to signals from their environment to tell them which way to go. These signals are chemicals produced by the different areas of the brain which attract or repel axons and so guide them along the correct path. These signals are identified by receptors on the surface of the axon which cause the axon to turn one way or the other. Axons can make wrong decisions if either the chemical cues are wrong or if the receptors on the axons that respond to the chemical cues are abnormal. One example of how this process can go awry is where special sugars or ?glycoprotein sidechains? found on the surface of cells are not constructed properly. This results in retinal axons failing to respond to cues in the brain telling them where to turn, and ending up in the wrong areas of the brain. We do not yet know why problems with the structure of certain glycoprotein sidechains give rise to problems in the axon?s ability to navigate appropriately to their targets; the experiments in this proposal aim to find out. To investigate this question we will disrupt the formation of particular glycoprotein sidechains and see how this affects the ability of axons to find their targets in the brain. Identifying what happens when particular sugar structures are not formed correctly will allow us to begin to understand the role of these sugars during normal development of the visual system. In the future we may be able to use our knowledge of the normal processed involved in guidance of axons from the eye to the brain to repair retinal axons lost either by disease or injury.

During development, retinal ganglion cells (RGCs) in each eye projects axons to the optic chaism where they are sorted into the optic tracts and navigate on to their targets in the brain. Heparan sulphate proteoglycans (HSPGs) are glycoproteins expressed on the cell surface and in the extracellular matrix. The structure of the carbohydrate component of HSPGs is modified by heparan sulphotransferase enzymes (HSTs). Our work on axon guidance defects in embryos lacking the HST enzymes Hs2st or Hs6st1 has shown that the sulphation patterns produced by these two enzymes are critical for RGC axon guidance involving the normal response to Slit proteins at the optic chiasm (Pratt et al., 2006). We are now, therefore, in a strong position to use a combined genetic and biochemical approach to investigate the underlying mechanism in more depth. We will investigate the proposition that Hs2st is required for the response to Slit1 and Hs6st1 is required for the response to Slit2 because this is the strongest hypothesis based on the available evidence. Our study is designed to accommodate additional possibilities including that the response to other axon guidance molecules at the chiasm are also sensitive to the sulphation of HS. We will use axon tract tracing techniques to examine axon guidance in embryos lacking both Hs2st and Hs6st1 in order to establish the extent to which Hs2st and Hs6st1 have unique functions in this system. We will use similar techniques to examine embryos lacking Hs2st and Slit1 or Hs6st1 and Slit2 to establish the extent to which the response to Slit1 requires Hs2st and the response to Slit2 requires Hs6st1. We will use in vitro axon navigation assays to determine the ability of navigating growth cones to respond to Slit1 and Slit2 when HS sulphation is disrupted. In the third part of the proposal we will use affinity chromatography to address the relationship between HS sulphation by Hs2st and Hs6st1 and the ability of Slit1 and Slit2 protein to physically interact with HSPGs and we will use quantitative immuno-fluorescence to assess the extent to which the cytoskeletal changes that normally occur when RGC growth cones encounter Slit proteins are disrupted when HS sulphation is disrupted. This study will address the fundamental biology of how axons navigate in complex environments found in the developing brain and may have applications in the development of therapies to repair axonal connections damaged by disease or wounding.