Drosophila hemocytes disperse over the entirety of the developing embryo. This protocol demonstrates how to mount and image these migrations using embryos with fluorescently labelled hemocytes.
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Method Article
Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations
In This Article
Summary
Abstract
Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we present a protocol for the mounting of Drosophila melanogaster embryos and subsequent live imaging of fluorescently labeled hemocytes, the embryonic macrophages of this organism. Using the Gal4-uas system1 we drive the expression of a variety of genetically encoded, fluorescently tagged markers in hemocytes to follow their developmental dispersal throughout the embryo. Following collection of embryos at the desired stage of development, the outer chorion is removed and the embryos are then mounted in halocarbon oil between a hydrophobic, gas-permeable membrane and a glass coverslip for live imaging. In addition to gross migratory parameters such as speed and directionality, higher resolution imaging coupled with the use of fluorescent reporters of F-actin and microtubules can provide more detailed information concerning the dynamics of these cytoskeletal components.
Protocol
Preparation
- Obtain appropriate Drosophila lines containing a hemocyte-specific Gal4 driver (e.g. srp-Gal42) and a genetically encoded fluorescent reporter under UAS control (e.g. uas-GFP). Flies homozygous for srp-Gal4,uas-GMA3 or crq-Gal4,uas-GFP4, 5 are particularly useful for imaging purposes (n.b. GMA is GFP fused to the actin-binding domain of moesin); see below for a discussion of the range of Gal4 drivers and uas constructs available (the Bloomington Stock Centre contains a wide variety).
- Typically genetic crosses are carried out such that mutant alle....
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Discussion
The most important elements of this procedure are the selection of healthy embryos with clearly labeled hemocytes and to mount them carefully without damaging them. Once the embryos are in the halocarbon oil they are resistant to dehydration and once mounted can be imaged for several hours. In our hands we can image hemocytes for three hours, with negligible dehydration of the embryo or obvious photo-damage, taking a z-stack of images every three minutes on our Zeiss LSM510 confocal microscope with a 40X objective. Since.......
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Acknowledgements
This protocol has been developed through our work within and in collaboration with the laboratories of Paul Martin and Antonio Jacinto. We thank the Bloomington Stock Centre for its excellent service and the Drosophila community for continuing to share fly lines. BS is currently funded by a BBSRC project grant. WW is funded by a Wellcome Trust Career Development Fellowship.
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Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| Cell strainer | BD Biosciences | 352350 | 70μm pores |
| Halcarbon oil 700 | Sigma-Aldrich | H8898 | |
| Lumox/Petriperm dish | Sarstedt Ltd | 96077305 |
References
- Brand, A. H., Perrimon, N. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development. 118, 401-415 (1993).
- Bruckner, K. The PDGF/VEGF receptor controls blood cell survival in Drosophila. Dev Cell.
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