Analysis of axonal trafficking via a novel live imaging technique reveals distinct Hedgehog transport kinetics

Joseph R. Daniele,Rehan M. Baqri,Samuel Kunes

doi:10.1242/bio.024075

Abstract

ABSTRACTThe Drosophila melanogaster (Dmel) eye is an ideal model to study development, intracellular signaling, behavior, and neurodegenerative disease. Interestingly, dynamic data are not commonly employed to investigate eye-specific disease models. Using axonal transport of the morphogen Hedgehog (Hh), which is integral to Dmel eye-brain development and implicated in stem cell maintenance and neoplastic disease, we demonstrate the ability to comprehensively quantify and characterize its trafficking in various neuron types and a neurodegeneration model in live early third-instar larval Drosophila. We find that neuronal Hh, whose kinetics have not been reported previously, favors fast anterograde transport and varies in speed and flux with respect to axonal position. This suggests distinct trafficking pathways along the axon. Lastly, we report abnormal transport of Hh in an accepted model of photoreceptor neurodegeneration. As a technical complement to existing eye-specific disease models, we demonstrate the ability to directly visualize transport in real time in intact and live animals and track secreted cargoes from the axon to their release points. Particle dynamics can now be precisely calculated and we posit that this method could be conveniently applied to characterizing disease pathogenesis and genetic screening in other established models of neurodegeneration.

Highlights

Since the discovery of the first eye-specific mutant in 1910, the Drosophila melanogaster (Dmel) visual system has been the focus of innumerable genetic screens, ranging from research in development, intracellular signaling, behavior, and in particular, neurodegenerative disease
In addition to the practical implications, Dmel disease models are popular because many (>75%) genes associated with human disease are conserved between fly and human and the basic fundamentals of cell biology are similar between these two species
This disparity between static and dynamic data is significant in the field of development especially with respect to the morphogen Hedgehog (Hh), which is trafficked down Dmel photoreceptor axons and is integral to eye-brain development

Summary

Introduction

Since the discovery of the first eye-specific mutant (white) in 1910, the Drosophila melanogaster (Dmel) visual system has been the focus of innumerable genetic screens, ranging from research in development, intracellular signaling, behavior, and in particular, neurodegenerative disease. The developing visual system has been a model of choice since the fly eye is amenable to genetic disruption and is dispensable for the organism’s survival (Lenz et al, 2013; Sang and Jackson, 2005). A select few of these models have focused on dynamic data such as the velocity, flux, and positional distribution of pathogenically relevant proteins (e.g. PINK1, polyglutaminecontaining proteins, Lis1) or organelles in these mutated tissues (e.g. mitochondria) (Liu et al, 2000, 1; Stowers et al, 2002; Wang et al, 2011; Wyan-Ching Mimi Lee et al, 2004) This disparity between static and dynamic data is significant in the field of development especially with respect to the morphogen Hedgehog (Hh), which is trafficked down Dmel photoreceptor axons and is integral to eye-brain development. Amongst Hh-expressing tissues (Dmel embryo and wing disc, planarian CNS regeneration, and vertebrate central nervous system (CNS), limb, organs, and foregut) developing photoreceptor axons offer a unique and accessible means to study Hh intracellular trafficking (Jiang and Hui, 2008; Yazawa et al, 2009)

Methods

Results

Discussion

Conclusion