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.
Keywords: Axon pathfinding; Lymnaea; ROCK; Retinoic acid; Retinoid; miR-124; microRNA.
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