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Developmental Biology

Visualize Drosophila Leg Motor Neuron Axons Through the Adult Cuticle

Published: October 30th, 2018

DOI:

10.3791/58365

1Institut de Génomique Fonctionnelle de Lyon, ENS de Lyon, CNRS, 2Departments of Biochemistry and Molecular Biophysics, and Neuroscience, Mortimer B. Zuckerman Mind Brain Behavior Institute, Columbia University, 3Neuroscience Program, NYU School of Medicine

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.

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.

Like the vertebrate limb, the Drosophila adult leg is organized into segments. Each fly leg contains 14 muscles, each of which comprises multiple muscle fibers1,2. 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

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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% .......

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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.......

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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 .......

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We thank Robert Renard for preparing fly food medium. This work was supported by an NIH grant NS070644 to R.S.M. and funding from the ALS Association (#256), FRM (#AJE20170537445) and ATIP-Avenir Program to J.E.

....

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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, 100x85 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 22x22 mm coverslips Fisher Scientific FIS#12-541-B No.1.5 -0.16 to 0.19mm 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

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