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 cellularlevel 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.

Rewarding capacity of optogenetically activating a giant GABAergic central-brain interneuron in larvalDrosophila

“…Fluorescence signals that can be observed across wavelengths and in both the experimental genotype (APL>mCherry-CAAX) and the genetic controls (+>mCherry-CAAX and APL>+ as the effector and the driver control, respectively) constitute autofluorescence and allow rich bodily detail to be discerned (Figure 2). We note that signals from fluorescent food particles contribute to individually-variable signals along the alimentary canal (Kobler et al, 2021).…”

“…This took advantage of a combination of autofluorescence signals with fluorescence from UAS-mCherry-CAAX as the effector. These signals can be detected conveniently under a light-sheet microscope upon clearing the sample (Kobler et al, 2021).…”

“…In addition, APL-GAL4 was crossed to UAS-mIFP/MB247>mCherry-CAAX (Kobler et al, 2021) to label APL together with the mushroom body (Movie 2). In both cases our results confirm that the primary neurite of APL splits to send projections separately into the calyx and the lobes of the mushroom bodies (Figure 3B'', Movie 2) (Masuda-Nakagawa et al, 2014;Mayseless et al, 2018;Saumweber et al, 2018).…”

Ankyrin2 is required for neuronal morphogenesis and long-term memory and interacts genetically with HDAC4

“…Similarly, pan-neuronal knockdown of Ank2 with elav-GAL4 impaired one hour olfactory memory (HIGHAM et al 2019) and mushroom body-specific knockdown with nSyb-GAL4 impaired threehour memory when tested in the olfactory conditioning assay (WALKINSHAW et al 2015). However, in these studies the drivers are expressed in larvae at which time the mushroom body is developing (NICOLAI et al 2003;OGIENKO et al 2020;KOBLER et al 2021), and an intact mushroom body is required for normal olfactory STM (HEISENBERG et al 1985) and associative STM lasting longer than 30 mins (MCBRIDE et al 1999). Furthermore, output from γ neurons is required for short-term courtship memory (KELEMAN et al 2012) and knockdown of Ank2 in γ neurons during mushroom body development was recently found to result in a shortened axon initial segment in third instar larval brains (SPURRIER et al 2019).…”

Transcending boundaries: from quantitative genetics to single genes

“…Chun-Fang Wu and Atulya Iyengar describe a fly seizure EEG that measures field potential activity in the Drosophila brain (Iyengar & Wu, 2021). Oliver Kobler, Bertram Gerber, and colleagues report a new protocol for the 3 D visualization of transgene expression across the whole body of Drosophila larva (Kobler et al, 2021). Gene Robinson and colleagues examine neurodevelopmental and transcriptomic effects of CRISPR/Cas9induced somatic orco mutation in honey bees (Chen et al, 2021).…”