A small molecule screen identifies in vivo modulators of peripheral nerve regeneration in zebrafish

Adult vertebrates have retained the ability to regenerate peripheral nerves after injury, although regeneration is frequently incomplete, often leading to functional impairments. Small molecule screens using whole organisms have high potential to identify biologically relevant targets, yet currently available assays for in vivo peripheral nerve regeneration are either very laborious and/or require complex technology. Here we take advantage of the optical transparency of larval zebrafish to develop a simple and fast pectoral fin removal assay that measures peripheral nerve regeneration in vivo. Twenty-four hours after fin amputation we observe robust and stereotyped nerve regrowth at the fin base. Similar to laser mediated nerve transection, nerve regrowth after fin amputation requires Schwann cells and FGF signaling, confirming that the fin amputation assay identifies pathways relevant for peripheral nerve regeneration. From a library of small molecules with known targets, we identified 21 compounds that impair peripheral nerve regeneration. Several of these compounds target known regulators of nerve regeneration, further validating the fin removal assay. Twelve of the identified compounds affect targets not previously known to control peripheral nerve regeneration. Using a laser-mediated nerve transection assay we tested ten of those compounds and confirmed six of these compounds to impair peripheral nerve regeneration: an EGFR inhibitor, a glucocorticoid, prostaglandin D2, a retinoic acid agonist, an inhibitor of calcium channels and a topoisomerase I inhibitor. Thus, we established a technically simple assay to rapidly identify valuable entry points into pathways critical for vertebrate peripheral nerve regeneration.成年脊椎动物在损伤后仍保留外周神经再生的能力，尽管再生过程往往不完全，常导致功能障碍。以完整生物为模型开展的小分子筛选（small molecule screens）具备极高潜力，可识别具有生物学意义的靶点，但目前已有的体内（in vivo）外周神经再生检测方法，要么操作极为繁琐费力，要么需要依赖复杂的实验技术。本研究借助斑马鱼幼体（larval zebrafish）的光学透明特性，开发了一种简便快捷的胸鳍切除检测法（pectoral fin removal assay），用于体内监测外周神经再生过程。在鳍切除24小时后，我们可在鳍基部观察到显著且具有典型特征的神经再生现象。与激光介导的神经切断实验（laser-mediated nerve transection）一致，鳍切除后的神经再生依赖于施万细胞（Schwann cells）和成纤维细胞生长因子信号通路（FGF signaling），这证实该胸鳍切除检测法可精准识别与外周神经再生相关的信号通路。我们从已知靶点的小分子化合物库中，筛选出21种可损害外周神经再生的化合物。其中部分化合物靶向已被报道的神经再生调控因子，进一步验证了该检测法的有效性。另有12种化合物所靶向的靶点，此前未被证实可调控外周神经再生。我们采用激光介导的神经切断实验对其中10种化合物进行验证，最终确认6种化合物可显著损害外周神经再生：分别为表皮生长因子受体抑制剂（EGFR inhibitor）、糖皮质激素（glucocorticoid）、前列腺素D2（prostaglandin D2）、维甲酸激动剂（retinoic acid agonist）、钙离子通道抑制剂（inhibitor of calcium channels）以及拓扑异构酶I抑制剂（topoisomerase I inhibitor）。综上，我们建立了一种操作简便的检测方法，可快速识别脊椎动物外周神经再生关键通路的有效研究切入点。