Post-transcriptional control of axon growth and guidance in Drosophila – POSAXODRO

Cell-specific neuron morphology dictates the functional connectivity between a neuron and its targets and hence determines the way the information is processed within the nervous system. In vivo, developing neurons extend axonal processes through a complex environment to find their appropriate targets. While much progress has been made in identifying guidance cues and their receptors, relatively little is known about the intracellular mechanisms by which axons convert these signals into directional decisions. Post-transcriptional regulatory mechanisms, however, have recently emerged as a critical component underlying the establishment of precise neuronal connectivity. In particular, active transport of mRNA to axons, coupled to local translation seems to largely contribute to the fast nucleus-independent response of axons to external cues, and thus to play a key role in axon growth and guidance. While local translation of few specific mRNAs has been demonstrated in response to guidance cues ex vivo, the biological relevance of this process still remains to be investigated in vivo. Furthermore, the precise molecular mechanisms controlling axonal mRNA transport and translation largely remain to be explored. Here, we propose to use Drosophila as a new model organism to i) functionally and dynamically study axonal mRNA targeting and translation in the context of a living organism, and ii) identify the trans-acting factors involved in this process and their target mRNAs.

So far, our work has focused on the conserved RNA transport factor Imp/ZBP1. Using Drosophila adult Mushroom Body (MB) neurons as a model system, we have shown that this RNA binding protein is actively transported to growing MB ? axons, and that its function is required cell-autonomously for directed growth and branching of axonal processes. Combining biochemical and genetic assays, we have shown that Imp associates with and regulates the expression of an mRNA encoding a regulator of the actin cytoskeleton (F-actin polymerization factor). Our first main objective is now to dynamically image and analyze growing axons (both wild-type and imp mutant axons) on one hand, and protein-RNA complexes being transported within axons on the other hand. These experiments rely on a live-imaging protocol we have recently established, and will allow us i) to quantitatively describe the axon growth/guidance phenotypes associated with the disruption of axonal mRNA transport, and ii) to characterize the mode of transport of Imp-containing particles. The second main objective of this proposal is to study how Imp regulates the expression of its multiple mRNA targets, and what are the rules underlying the regulation of its post-transcriptional network in vivo. This part of the project relies on a genome-wide RIP-chip experiment we have already performed. Last, we will extend our study by performing a genetic screen to identify new conserved RNA binding proteins specifically involved in axon growth and guidance. With this screen, we hope to identify both partners of Imp and proteins with complementary functions.