Embryonic development is orchestrated by the action of morphogens, which spread out from a local source and activate, in a field of target cells, different cellular programs based on their concentration gradient. Fibroblast growth factor 8 (Fgf8) is a morphogen with important functions in embryonic organizing centers. It forms a gradient in the extracellular space by free diffusion, interaction with the extracellular matrix (ECM), and receptor-mediated endocytosis. However, morphogen gradient regulation by ECM is still poorly understood. Here, we show that specific heparan sulfate proteoglycans (HSPGs) bind Fgf8 with different affinities directly in the ECM of living zebrafish embryos, thus affecting its diffusion and signaling. Using single-molecule fluorescence correlation spectroscopy, we quantify this binding in vivo, and find two different modes of interaction. First, reducing or increasing the concentration of specific HSPGs in the extracellular space alters Fgf8 diffusion and, thus, its gradient shape. Second, ternary complex formation of Fgf8 ligand with Fgf receptors and HSPGs at the cell surface requires HSPG attachment to the cell membrane. Together, our results show that graded Fgf8 morphogen distribution is achieved by constraining free Fgf8 diffusion through successive interactions with HSPGs at the cell surface and in ECM space.

中文翻译：

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细胞外基质中硫酸乙酰肝素蛋白聚糖对 Fgf8 形态发生梯度的微调


胚胎发育是由形态发生素的作用精心策划的，形态发生素从局部来源扩散出来，并在靶细胞区域中根据其浓度梯度激活不同的细胞程序。成纤维细胞生长因子 8 （Fgf8） 是一种在胚胎组织中心具有重要功能的形态发生素。它通过自由扩散、与细胞外基质 （ECM） 的相互作用和受体介导的内吞作用在细胞外间隙形成梯度。然而，ECM 对形态发生素梯度的调控仍然知之甚少。在这里，我们表明特异性硫酸乙酰肝素蛋白聚糖 （HSPGs） 直接在活斑马鱼胚胎的 ECM 中以不同的亲和力结合 Fgf8，从而影响其扩散和信号传导。使用单分子荧光相关光谱，我们在体内量化这种结合，并找到两种不同的相互作用模式。首先，减少或增加细胞外间隙中特定 HSPG 的浓度会改变 Fgf8 的扩散，从而改变其梯度形状。其次，Fgf8 配体与细胞表面的 Fgf 受体和 HSPG 形成三元复合物需要 HSPG 附着在细胞膜上。总之，我们的结果表明，分级的 Fgf8 形态发生素分布是通过在细胞表面和 ECM 空间中与 HSPGs 连续相互作用来限制自由 Fgf8 扩散来实现的。