A quick and versatile protocol for the 3D visualization of transgene expression across the whole body of larval Drosophila

Abstract

Larval Drosophila are used as a genetically accessible study case in many areas of biological research. Here we report a fast, robust and user-friendly procedure for the whole-body multi-fluorescence imaging of Drosophila larvae; the protocol has been optimized specifically for larvae by systematically tackling the pitfalls associated with clearing this small but cuticularized organism. Tests on various fluorescent proteins reveal that the recently introduced monomeric infrared fluorescent protein (mIFP) is particularly suitable for our approach. This approach comprises an effective, low-cost clearing protocol with minimal handling time and reduced toxicity in the reagents employed. It combines a success rate high enough to allow for small-scale screening approaches and a resolution sufficient for cellular-level analyses with light sheet and confocal microscopy. Given that publications and database documentations typically specify expression patterns of transgenic driver lines only within a given organ system of interest, the present procedure should be versatile enough to extend such documentation systematically to the whole body. As examples, the expression patterns of transgenic driver lines covering the majority of neurons, or subsets of chemosensory, central brain or motor neurons, are documented in the context of whole larval body volumes (using nsyb-Gal4, IR76b-Gal4, APL-Gal4 and mushroom body Kenyon cells, or OK371-Gal4, respectively). Notably, the presented protocol allows for triple-color fluorescence imaging with near-infrared, red and yellow fluorescent proteins.

Keywords:

• Tissue clearing
• ethyl cinnamate
• APL neuron
• IR76b
• mushroom body
• near-infrared fluorescent protein

Acknowledgements

Discussions with and comments from Eike Budinger, Nino Mancini, Torsten Stöter, Naoko Toshima, Werner Zuschratter (LIN), Hans Ulrich Dodt and Marco Pende (TU Wien), and expert technical assistance by Bettina Kracht (LIN) are gratefully acknowledged. We thank Rupert D.V. Glasgow (Zaragoza, Spain) for language editing and appreciate the supply of stocks by the Bloomington Drosophila Stock Center (Bloomington, Indiana, USA) (NIH P40OD018537) and the Kyoto Stock Center (Kyoto, Japan), and the supply of plasmid DNA by Thomas Riemensperger (University of Cologne). This piece is dedicated to Marla B. Sokolowski.

Disclosure statement

The authors declare no competing interests.

Additional information

Funding

This study received institutional support by the Otto von Guericke Universität Magdeburg (OVGU), the Wissenschaftsgemeinschaft Gottfried Wilhelm Leibniz (WGL), the Leibniz Institute for Neurobiology (LIN), and benefitted from grant support from the Deutsche Forschungsgemeinschaft (DFG) [GE 1091/4–1 and FOR 2705, to BG].