Visualize Drosophila Leg Motor Neuron Axons Through the Adult Cuticle

Summary

Here we describe a protocol to visualize the axonal targeting with a florescent protein in adult legs of Drosophila by fixation, mounting, imaging, and post-imaging steps.

Abstract

The majority of work on the neuronal specification has been carried out in genetically and physiologically tractable models such as C. elegans, Drosophila larvae, and fish, which all engage in undulatory movements (like crawling or swimming) as their primary mode of locomotion. However, a more sophisticated understanding of the individual motor neuron (MN) specification—at least in terms of informing disease therapies—demands an equally tractable system that better models the complex appendage-based locomotion schemes of vertebrates. The adult Drosophila locomotor system in charge of walking meets all of these criteria with ease, since in this model it is possible to study the specification of a small number of easily distinguished leg MNs (approximately 50 MNs per leg) both using a vast array of powerful genetic tools, and in the physiological context of an appendage-based locomotion scheme. Here we describe a protocol to visualize the leg muscle innervation in an adult fly.

Introduction

Like the vertebrate limb, the Drosophila adult leg is organized into segments. Each fly leg contains 14 muscles, each of which comprises multiple muscle fibers^1,2. The cell bodies of the adult leg MNs are located in the T1 (prothoracic), T2 (mesothoracic), and T3 (metathoracic) ganglia on each side of the ventral nerve cord (VNC), a structural analogous to the vertebrate spinal cord (Figure 1). There are approximately 50 MNs in each ganglia, which target muscles in four segments of the ipsilateral leg (coxa, trochanter, femur, and tibia) (Figure 1

Protocol

1. Leg Dissection and Fixation

1. Take a glass multi-well plate and fill appropriate number of wells with 70% ethanol. Add 15–20 CO₂-anesthetized flies (of either sex and any age) to each well and by using a brush, gently dab the flies into the ethanol solution until flies are fully submerged.
   NOTE: This step is to remove the hydrophobicity of the cuticle. Do not wash for more than 1 min, because this increases auto-fluorescence of the cuticle.
2. Rinse the flies 3 times with 0.3% nonionic surfactant detergent solution in 1x phosphate buffered saline (PBS). Keep the flies in this solution for at least 10 min.
   NOTE: L....

Results

As shown in Figure 4, this procedure allows excellent imaging of GFP-labeled axons in adult Drosophila legs, together with their terminal arbors. Importantly a clean GFP signal is obtained without any contamination from the fluorescence emitted by the leg cuticle. The signal from the cuticle can then be combined with the GFP signal to identify the positioning of axons in the legs (Figure 4E, Figure 1.......

Discussion

The cuticle of adult Drosophila and of other arthropods, which contains many dark pigments, is a major obstacle for viewing structures inside their body. In addition, it is strongly auto fluorescent which is made worse by fixation. These two features are very problematic for observations of fluorescent dyes or molecules inside the body of animals with an exoskeleton.

The procedure that we have described and that we routinely use in the lab yields clean and detailed images of axon trajectories .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ethanol absolute	Fisher	E/6550DF/17	Absolute analytical reagent grade
nonionic surfactant detergent	Sigma-Aldrich	T8787	Triton X-100, for molecular biology
Fine forceps	Sigma-Aldrich	F6521	Jewelers forceps, Dumont No. 5
Glass multi-well plate	Electron Microscopy Sciences	71563-01	9 cavity Pyrex, 100 mm x 85 mm
PFA	Thermofisher	28908	Pierc 16% Formaldehyde (w/v), Methanol-free
Glycerol	Fisher BioReagents	BP 229-1	Glycerol (Molecular Biology)
Spacers	Sun Jin Lab Co	IS006	iSpacer, four wells, around 12 μL working volume per well, 7 mm diameter, 0.18 mm deep
Square 22 mm x 22 mm coverslips	Fisher Scientific	FIS#12-541-B	No.1.5-0.16 to 0.19 mm thick
Mounting Medium	Vector Laboratories	H-1000	Vectashield Antifade Mounting Medium
Confocal microscope	Carl Zeiss		LSM780; objective used LD LCI Plan-Apochromat 25X/0.8 Imm Korr DIC M27 (oil/ silicon/glycerol/water immersion) (420852-9871-000)
imaging software	Carl Zeiss		ZEN 2011
3D-Image software	ThermoFisher Scientific		Amira 6.4
ImageJ	National Institutes of Health	https://imagej.nih.gov/ij/	ImageJ/FIJI