Laboratory of Developmental Genetics, The Rockefeller University, 1230 York Avenue, New York, New York 10065, USA.
Nat Commun. 2017 Jan 18;8:14100. doi: 10.1038/ncomms14100.
Visualizing neural-circuit assembly in vivo requires tracking growth of optically resolvable neurites. The Caenorhabditis elegans embryonic nervous system, comprising 222 neurons and 56 glia, is attractive for comprehensive studies of development; however, embryonic reporters are broadly expressed, making single-neurite tracking/manipulation challenging. We present a method, using an infrared laser, for reproducible heat-dependent gene expression in small sublineages (one to four cells) without radiation damage. We go beyond proof-of-principle, and use our system to label and track single neurons during early nervous-system assembly. We uncover a retrograde extension mechanism for axon growth, and reveal the aetiology of axon-guidance defects in sax-3/Robo and vab-1/EphR mutants. We also perform cell-specific rescues, determining DAF-6/patched-related site of action during sensory-organ development. Simultaneous ablation and labelling of cells using our system reveals roles for glia in dendrite extension. Our method can be applied to other optically/IR-transparent organisms, and opens the door to high-resolution systematic analyses of C. elegans morphogenesis.
在体内可视化神经回路组装需要跟踪可分辨的神经突的生长。秀丽隐杆线虫的胚胎神经系统由 222 个神经元和 56 个神经胶质细胞组成，非常适合于发育的综合研究;然而，胚胎报告基因广泛表达，使得单神经突跟踪/操作具有挑战性。我们提出了一种使用红外激光在小亚谱系(一个到四个细胞)中进行可重复的热依赖性基因表达的方法，而不会造成辐射损伤。我们超越了原理验证，并在早期神经系统组装过程中使用我们的系统对单个神经元进行标记和跟踪。我们揭示了轴突生长的逆行延伸机制，并揭示了 sax-3/Robo 和 vab-1/EphR 突变体中轴突导向缺陷的病因。我们还进行了细胞特异性挽救实验，确定了 DAF-6/patched 相关作用位点在感觉器官发育过程中的作用。我们的系统同时进行细胞的消融和标记，揭示了神经胶质细胞在树突延伸中的作用。我们的方法可以应用于其他光/IR 透明的生物体，并为秀丽隐杆线虫形态发生的高分辨率系统分析开辟了道路。
