The role of microRNAs in Drosophila larval locomotion

microRNAs (miRNAs) are small, non-coding RNAs that negatively regulate genetic expression and have been shown to regulate motor control in several animals, including Drosophila melanogaster. Previous work in our laboratory showed that miRNA-based regulation of self-righting behaviour in Drosophila larvae is pervasive and we therefore endeavour to establish the behaviour’s neural substrates. Here, we use neuronal reconstruction to identify neurons connected to the LT-1/2 motor neurons, themselves essential for self-righting. Combining this with connectomics work, we produce a neural wiring diagram for self-righting which we functionally test by thermogenetically inhibiting individual cellular components - finding that the normal activity of most neurons in our wiring diagram is essential for normal self-righting. Building on this, we examine miRNA regulation in general larval locomotion by screening a mutant collection affecting most miRNAs expressed in early-stage Drosophila larvae. Using high-throughput larval tracking, we observe that the vast majority of miRNA mutants show reduced crawling speed and impacted bending and pausing behaviours. Examining how consistent miRNA-dependent effects are through development, we also test a subset of miRNA mutants at the third-instar larval stage. We demonstrate that effects of specific mutations vary over development, suggesting a complex relationship between miRNA function and larval development. Finally, we select miR-133 for detailed mechanistic study, find that its mutation produces significant increases in rearing and identify tryptophan hydroxylase (Trh) as a potential regulatory target. Target expression analysis confirms an increase in Trh expression and serotonin production in miR-133 mutants. Moreover, gene expression analysis shows miR-133 expression within serotonergic neurons and we successfully phenocopy the increased rearing observed in miR-133 mutant larvae by overexpressing Trh. Altogether, our studies demonstrate the general role of miRNA regulation in Drosophila larval locomotion. Constructing a neuronal wiring diagram for self-righting also identifies the behaviour’s neural basis and opens possibilities for future mechanistic study.