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

从受精卵到出生，我们大脑的发育有点像一个建筑项目，需要向脑细胞发出指令，以确保正确的结构在正确的时间出现在正确的位置。这一过程的核心不仅是在正确的时间和地点发出指令，而且是正确地发送和接收指令。考虑到大脑的巨大复杂性，显然脑细胞必须使用相应的细微差别的语言来相互交流。相应地，我们知道，如果犯了错误，如果编码信号的基因不能正常工作，或者在某些情况下，由于环境因素扰乱信号，就可能发生错误，那么大脑可能会发育异常，这可能会对后来的生活产生重大影响。因此，理解细胞交流的语言对于理解正常和异常的大脑发育至关重要。在过去的几年里，科学家们在识别蛋白质信号方面取得了长足的进步，即在细胞之间传递指令的“信号蛋白质”。信号蛋白由源产生并移动到它们的目标，在那里它们被目标细胞表面的受体感知。然后，目标单元响应于该信号改变其行为。一个令科学家困惑的难题是，尽管进行了大量的努力，但与产生大脑所需的复杂指令相比，只发现了相对较少的信号蛋白。我们将注意力集中在一类完全不同的分子--碳水化合物--在细胞信号传递中所扮演的角色。我们特别感兴趣的是碳水化合物和蛋白质之间的相互作用扩大了指令的多样性。这是一个非常大的问题，我们在这里提出的工作将详细研究一种名为“成纤维细胞生长因子8”(简称“FGF8”)的信号分子和一种名为“硫酸乙酰肝素”(简称“HS”)的碳水化合物之间的特殊相互作用。FGF8对大脑发育特别重要，因为细胞对它们遇到的量非常敏感，即使是FGF8水平的微小变化也会对大脑发育产生戏剧性的影响，包括发育缺陷与FGF8剂量变化有关的情况。我们发现，HS的结构调节着Fgf8在发育中大脑的整体有效性，因为HS结构异常的小鼠突变体具有缺陷的Fgf8信号，导致脑畸形。在这项提案中，我们计划在这项工作的基础上，建立HS如何控制Fgf8蛋白在细胞之间的移动以及细胞对Fgf8蛋白做出反应的能力的详细图景。为了帮助我们做到这一点，我们开发了一种培养系统，允许我们应用Fgf8蛋白的来源，然后跟踪其随时间的移动。使用特殊的显微镜结合荧光标记的FGF8，我们实际上可以观察蛋白质在组织中移动的方式，并测量细胞对FGF8的反应。通过比较Fgf8在正常和异常HS下的行为，我们可以建立一幅HS如何正常调节Fgf8以及该过程如何出错的图景。在未来，我们可能能够利用我们对信号的正常过程的知识来帮助再生方法来替换或修复因疾病或损伤而丢失的脑组织。更广泛地说，鉴于信号蛋白在生活的几乎所有方面都扮演着关键角色，对我们系统中碳水化合物和信号蛋白之间相互作用的了解的增加可能会为其他系统提供洞察。

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.