During embryonic nervous system assembly, mRNA localisation is precisely regulated in growing axons, affording subcellular autonomy by allowing controlled protein expression in space and time. Different sets of mRNAs exhibit different localisation patterns across the axon. Little is known, however, about how mRNAs move in axons, or how these patterns are generated.

Here, I develop a method for live single molecule imaging of identified endogenous mRNA within Xenopus laevis retinal ganglion cell axons using molecular beacon technology coupled with highly inclined and laminated optical sheet (HILO) microscopy. By combining quantitative single molecule imaging with biophysical motion models, I show that β-actin mRNA travels mainly as single copies, and exhibits different motion type frequencies in different axonal sub-compartments. I find that β-actin mRNA density is four-fold enriched in the growth cone central domain compared to the axon shaft and that a modicum of directed transport is vital for delivery of mRNA to the axon tip. Through mathematical modelling it is further demonstrated that directional differences in motor-driven mRNA transport speeds are sufficient to generate β-actin mRNA enrichment at the growth cone.

Distinctive features within the mRNA have been shown to drive localisation and subsequently translation at specified subcellular regions through recognition by different RNA binding proteins. I employed fluorescent recovery after photobleaching (FRAP) in a screen to investigate whether short sequence motifs that were common to axonally translated mRNAs are sufficient to promote axonal translation. I further use this technique to show that the 5’ untranslated regions (UTRs) of two alternative isoforms of the Acot7 gene generate stark differences in local translation within growing axons. Through in situ hybridization, I demonstrate that these translational profiles result from differences in axonal mRNA localisation. Finally, I probe the relative structural and sequence requirements necessary for 5’UTR mediated axonal Acot7 mRNA localisation. Together, these results provide insight into the intrinsic mRNA features necessary for axonal localisation and the subsequent trafficking mechanisms that drive mRNA enrichment to the axon tip of neurons during development.