Müller B K, Jay D G, Bonhoeffer F
Max-Planck-Institut für Entwicklungsbiologie, Tübingen, Germany.
Curr Biol. 1996 Nov 1;6(11):1497-502. doi: 10.1016/s0960-9822(96)00754-3.
The axons of retinal ganglion neurons from a precise topographic map in the optic tectum in the midbrain, and the guidance of retinal axons by directional cues in the tectum is crucial in this process. Several in vitro systems have been developed in order to identify the molecular basis of these directional cues. Temporal, but not nasal, retinal axons avoid posterior tectal membranes and grow on anterior membranes as a result of repellent guidance activities that are linked by glycosylphosphatidylinositol (GPI) anchors to the posterior membranes. A putative GPI-anchored repulsive guidance molecule with a molecular weight of 33 kDa has previously been characterized. Indirect results from experiments in vitro support the hypothesis that this 33 kDa molecule guides temporal retinal axons.
To assess whether the 33 kDa protein is involved in axon guidance in vitro, we raised monoclonal antibodies against molecules that had been removed from tectal membranes by treatment with phosphatidylinositol-specific phospholipase C, which cleaves GPI anchors. A monoclonal immunoglobulin M, F3D4, recognized the 33 kDa molecule. In combination with chromophore-assisted laser inactivation, F3D4 caused a loss of the repellent activity from posterior tectal membranes in vitro. As a result, temporal retinal fibers were no longer repelled by posterior tectal membranes. This demonstrates that the F3D4 antigen, which we name RGM (repulsive guidance molecule) is involved in the guidance of retinal axons in an assay in vitro. In vivo, the expression of RGM increases from the anterior to the posterior pole of the optic tectum.
These findings not only support the hypothesis that retinal axons are guided by gradients of repulsive guidance molecules but, in combination with earlier studies of receptor kinases and their ligands that act during guidance, argue for the presence of several repellent guidance molecules with similar functions in vitro and expression patterns in vivo.
视网膜神经节神经元的轴突在中脑视顶盖形成精确的拓扑图谱，在此过程中，视顶盖中的定向线索对视网膜轴突的引导至关重要。为了确定这些定向线索的分子基础，已开发了多种体外系统。由于通过糖基磷脂酰肌醇（GPI）锚定连接到后膜的排斥性引导活性，颞侧而非鼻侧的视网膜轴突避开后顶盖膜并在前膜上生长。先前已鉴定出一种分子量为33 kDa的推定GPI锚定排斥性引导分子。体外实验的间接结果支持了这种33 kDa分子引导颞侧视网膜轴突的假说。
为了评估33 kDa蛋白是否参与体外轴突引导，我们制备了针对用磷脂酰肌醇特异性磷脂酶C处理从顶盖膜中去除的分子的单克隆抗体，该酶可切割GPI锚。一种单克隆免疫球蛋白M，F3D4，识别出33 kDa分子。与发色团辅助激光失活相结合，F3D4导致体外后顶盖膜失去排斥活性。结果，颞侧视网膜纤维不再被后顶盖膜排斥。这表明我们命名为RGM（排斥性引导分子）的F3D4抗原参与了体外实验中视网膜轴突的引导。在体内，RGM的表达从视顶盖的前极向后极增加。
这些发现不仅支持视网膜轴突由排斥性引导分子梯度引导的假说，而且与早期关于在引导过程中起作用的受体激酶及其配体的研究相结合，表明在体外存在几种具有相似功能和体内表达模式的排斥性引导分子。
