During development and regeneration, growth cones at the tips of extending axons navigate through a complex environment to establish accurate connections with appropriate targets. Growth cones can respond rapidly to classical and non-classical guidance cues in their environment, often requiring local protein synthesis. In vertebrate growth cones, local protein synthesis in response to classical cues can require regulation by microRNAs (miRNAs), a class of small, conserved, non-coding RNAs that post-transcriptionally regulate gene expression. However, less is known of how miRNAs mediate growth cone responses to non-classical cues (such as retinoic acid (RA)), specifically in invertebrates. Here, we utilized adult regenerating invertebrate motorneurons to study miRNA regulation of growth cone attraction to RA, shown to require local protein synthesis. In situ hybridization revealed the presence of miR-124 in growth cones of regenerating ciliary motorneurons of the mollusc Lymnaea stagnalis. Changes in the spatiotemporal distribution of miR-124 occurred following application of RA, and dysregulation of miR-124 (with mimic injection), disrupted RA-induced growth cone turning in a time-dependent manner. This behavioural regulation by miR-124 was altered when the neurite was transected, and the growth cone completely separated from the soma. miR-124 did not, however, appear to be involved in growth cone attraction to serotonin, a response independent of local protein synthesis. Finally, we provide evidence that a downstream effector of RhoGTPases, ROCK, is a potential target of miR-124 during RA-induced growth cone responses. These data advance our current understanding of how microRNAs might mediate cue- and context-dependent behaviours during axon guidance.

在发育和再生过程中，延伸轴突尖端的生长锥在复杂的环境中导航，以与适当的目标建立准确的连接。生长锥可以快速响应其环境中的经典和非经典指导线索，通常需要局部蛋白质合成。在脊椎动物生长锥中，响应经典线索的局部蛋白质合成可能需要通过 microRNA (miRNA) 进行调节，这是一类小的、保守的、非编码 RNA，可在转录后调节基因表达。然而，对于 miRNAs 如何介导生长锥对非经典线索（如视黄酸 (RA)）的反应知之甚少，特别是在无脊椎动物中。在这里，我们利用成年再生无脊椎动物运动神经元来研究 miRNA 对生长锥对 RA 的吸引力的调节，显示需要局部蛋白质合成。原位杂交揭示了在软体动物再生纤毛运动神经元的生长锥中存在 miR-124茯苓 。应用 RA 后 miR-124 的时空分布发生变化，并且 miR-124 的失调（模拟注射）以时间依赖性方式破坏了 RA 诱导的生长锥转动。当神经突被横切时，miR-124 的这种行为调节发生了改变，生长锥与胞体完全分离。然而，miR-124 似乎并不参与生长锥对血清素的吸引，这是一种独立于局部蛋白质合成的反应。最后，我们提供的证据表明，RhoGTPases 的下游效应物 ROCK 是 RA 诱导的生长锥反应过程中 miR-124 的潜在靶标。这些数据促进了我们目前对 microRNA 在轴突引导过程中如何介导线索和上下文相关行为的理解。