Published: October 30th, 2018
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
1. Leg Dissection and Fixation
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.......
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 .......
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.....
|Absolute analytical reagent grade
|nonionic surfactant detergent
|Triton X-100, for molecular biology
|Jewelers forceps, Dumont No. 5
|Glass multi-well plate
|Electron Microscopy Sciences
|9 cavity Pyrex, 100x85 mm
|Pierc 16% Formaldehyde (w/v), Methanol-free
|Glycerol (Molecular Biology)
|Sun Jin Lab Co
|iSpacer, four wells, around 12 μL working volume per well, 7 mm diameter, 0.18 mm deep
|Square 22x22 mm coverslips
|No.1.5 -0.16 to 0.19mm thick
|Vectashield Antifade Mounting Medium
|LSM780; objective used LD LCI Plan-Apochromat
25x/0,8 Imm Korr DIC M27 (oil/
|National Institutes of Health
Copyright © 2024 MyJoVE Corporation. All rights reserved