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Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

Published: March 4th, 2013



1Institute of Genetics, University of Mainz, 2Department of Anatomy and Neuroscience, University of Melbourne

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

In this article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the lipophilic fluorescent membrane marker DiI. This method allows the visualization of neuronal cell morphology in great detail. It is possible to label any cell in the CNS: cell bodies of target neurons are visualized under DIC optics or by expression of a fluorescent genetic marker such as GFP. After labeling, the DiI can be transformed into a permanent brown stain by photoconversion to allow visualization of cell morphology with transmitted light and DIC optics. Alternatively, the DiI-labeled cells can be observed directly with confocal microscopy, enabling genetically introduced fluorescent reporter proteins to be colocalised. The technique can be used in any animal, irrespective of genotype, making it possible to analyze mutant phenotypes at single cell resolution.

Knowledge of neuronal morphology at the level of individual cells is a key prerequisite for understanding neuronal connectivity and CNS function. Thus, from the earliest days of neuroscience, researchers have sought to develop single cell labeling techniques (see 7 for a historical treatment of this issue). Classical methods such as Golgi staining provide excellent resolution of neuronal morphology but are not suitable if one seeks to label a particular type of neuron in a directed way, as staining occurs in a random fashion. The development of methods for staining single cells by intracellular or juxtacellular injection of dyes from a microelectrode addres....

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We have used two slightly different variants of the single neuron labeling method in our laboratories. The differences relate to the embryo collection, dechorionisation, devitellinisation and embryo filleting steps. Figure 1 gives an overview of the common and diverging steps of the variants.

1. Preparation of Micro-needles for Embryo Dissection and Micropipettes for Dye Injection

  1. Glass needles for embryo dissection are drawn from glass capillaries (1 mm diameter a.......

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Figure 4 illustrates typical results of the technique, we describe here. Figure 4A shows an example of a DiI filled single interneuron that was cleanly photoconverted. It nicely demonstrates the amount of detail these preparations offer. When viewed under DIC optics the spatial context of the labeled cell within the non-labeled surrounding tissue becomes visible, e.g. the position of the cell body within the cortex and of the fiber projection within the neuropile.

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One major advantage of Drosophila as a model system is that it allows analysis of development and function on the level of single cells. This is especially helpful regarding the nervous system, where the diversity of cell types is exceptionally high and the function and morphology of neighboring cells can be totally different.

The method we present here allows the labeling of individual neurons with a dye that can either be transformed into a permanent stain or examined directl.......

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This work was supported by a grant from the DFG to G.M.T.


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Name Company Catalog Number Comments
Name of Reagent/Material Company Catalogue Number Comments
DAB Sigma-Aldrich D-5905 2-3 mg/ml in 100 mM TRIS-Hcl, pH 7.4
DiI Invitrogen/Molecular Probes D-282 1 mg/ml in ethanol
Formaldehyde Merck Millipore   7,4 % in PBS
Glycerol Roth 3783 70% in PBS
Heptane Glue Beiersdorf AG Cello 31-39-30 * dilute ca. 1 to 1 with n-Heptane
PBS     1x
TRIS Roth 4855  
Vectashield Vector Laboratories H1000  
Confocal Microscope Leica TCS SP2 to view and document labelings in fluorescence
DC - amplifier Dragan Corporation Cornerstone ION-100 to perform the labelings
Coverslips Menzel BB018018A1 18 x 18 mm
Coverslips Menzel BB024060A1 24 x 60 mm
Dissecting Microscope Leica MZ8 to prepare and disect embryos
Flat Capillaries Hilgenberg   outer diameter 1 mm; glas thickness 0.1 mm
Injection Capillaries Science Products GB 100 TF 8P  
Micromanipulator R Leica   to perform the labelings
Model P-97 Sutter Instruments   to pull capillaries
Object Slides Marienfeld Superior 1000000  
Scientific Microscope Zeiss Axioskop 2 mot to view labelings after photoconversion
Scientific Microscope Olympus BX50 to perform the labelings
Sony MC3255 Video Camera Sony/AVT Horn Sony MC3255 to record labelings after photoconversion

Table 1.

  1. Brand, A. H., Perrimon, N. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development. 118, 401-415 (1993).
  2. Broadie, K., Sink, H., Van Vactor, D., Fambrough, D., Whitington, P. M., Bate, M., Goodman, C. S. From growth cone to synapse: the life history of the RP3 motor neuron. Dev. Suppl. , 227-238 (1993).
  3. Campos-Ortega, J., Hartenstein, V. . The Embryonic Development of Drosophila melanogaster. , (1985).
  4. Featherstone, D. E., Chen, K., Broadie, K. Harvesting and Preparing Drosophila Embryos for Electrophysiological Recording and Other Procedures. J. Vis. Exp. (27), e1347 (2009).
  5. Grenningloh, G., Rehm, E. J., Goodman, C. S. Genetic analysis of growth cone guidance in Drosophila: fasciclin II functions as a neuronal recognition molecule. Cell. 67, 45-57 (1991).
  6. Kunz, T., Kraft, K. F., Technau, G. M., Urbach, R. Origin of Drosophila mushroom body neuroblasts and generation of divergent embryonic lineages. Development. 139, 2510-2522 (2012).
  7. Lanciego, J. L., Wouterlood, F. G. A half century of experimental neuroanatomical tracing. J. Chem. Neuroanat. 42, 157-183 (2011).
  8. Lee, T., Luo, L. Mosaic analysis with a repressible cell marker for studies of gene function in neuronal morphogenesis. Neuron. 22, 451-461 (1999).
  9. Levick, W. R. Another tungsten microelectrode. Med. Biol. Eng. 10, 510-515 (1972).
  10. Matthes, D. J., Sink, H., Kolodkin, A. L., Goodman, C. S. Semaphorin II can function as a selective inhibitor of specific synaptic arborizations. Cell. 81, 631-639 (1995).
  11. Murray, M. J., Whitington, P. M. Effects of roundabout on growth cone dynamics, filopodial length, and growth cone morphology at the midline and throughout the neuropile. Journal of Neuroscience. 19, 7901-7912 (1999).
  12. Murray, M. J., Merritt, D. J., Brand, A. H., Whitington, P. M. In vivo dynamics of axon pathfinding in the Drosophilia CNS: a time-lapse study of an identified motorneuron. J. Neurobiol. 37, 607-621 (1998).
  13. Patel, N. H. Imaging neuronal subsets and other cell types in whole-mount Drosophila embryos and larvae using antibody probes. Methods Cell Biol. 44, 445-487 (1994).
  14. Rickert, C., Kunz, T., Harris, K. -. L., Whitington, P. M., Technau, G. M. Morphological characterization of the entire interneuron population reveals principles of neuromere organization in the ventral nerve cord of Drosophila. Journal of Neuroscience. 31, 15870-15883 (2011).
  15. Thomas, J. B., Bastiani, M. J., Bate, M., Goodman, C. S. From grasshopper to Drosophila: a common plan for neuronal development. Nature. 310, 203-207 (1984).
  16. Whitington, P., Harris, K. Early axonogenesis in the embryo of a primitive insect, the silverfish Ctenolepisma longicaudata. Development Genes and Evolution. 205 (5-6), 272-281 (1996).
  17. Whitington, P., Meier, T. Segmentation, neurogenesis and formation of early axonal pathways in the centipede, Ethmostigmus rubripes (Brandt). Development Genes and Evolution. 199, 349-363 (1991).
  18. Whitington, P. M., Seifert, E. Axon growth from limb motorneurons in the locust embryo: the effect of target limb removal on the pattern of axon branching in the periphery. Developmental Biology. 106, 438-449 (1984).
  19. Whitington, P. M., Leach, D., Sandeman, R. Evolutionary change in neural development within the arthropods: axonogenesis in the embryos of two crustaceans. Development. 118, 449-461 (1993).
  20. Williams, D. W., Tyrer, M., Shepherd, D. Tau and tau reporters disrupt central projections of sensory neurons in Drosophila. J. Comp. Neurol. 428, 630-640 (2000).

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