Selective dendritic localization of mRNA in Drosophila mushroom body output neurons
- Centre for Neural Circuits and Behaviour, University of Oxford, United Kingdom
- Delft Center for Systems and Control, Delft University of Technology, Netherlands
- Department of Biochemistry, University of Oxford, United Kingdom
- Department of Engineering Science, University of Oxford, United Kingdom
Figures
Figure 1
CaMKII and nAChR α1 mRNA visualized in the mushroom body (MB) calyx and γ5β'2a mushroom body output neuron (MBON) dendrites with single-molecule fluorescence in situ hybridization (smFISH).
(A) Schematic of Drosophila MB. smFISH signal was imaged in the calyx, indicated by the dashed box. (B) CaMKII and nAChRα1 mRNAs labeled with smFISH in the MB calyx. Images are maximum intensity …
Figure 2 with 1 supplement
Differential localization of mRNAs in γ5β'2a and γ1pedc>α/β mushroom body output neuron (MBON) dendrites.
(A, A'). CaMKII::YFP mRNA visualized in γ5β'2a MBON dendrites using YFP single-molecule fluorescence in situ hybridization (smFISH) probes. The γ5β'2a MBON is labeled by R66C08-GAL4-driven …
Figure 2—figure supplement 1
Effect of spot detection threshold on false-positive detections.
(A) Signal/background intensity distribution of YFP probe signals in CaMKII::YFP brains relative to control brains with no threshold on signal detection, as in Figure 2D. (B) Proportion of overlap …
Figure 3 with 2 supplements
Learning alters CaMKII mRNA abundance in the γ5β'2a mushroom body output neurons (MBONs).
(A, A'). CaMKII::YFP single-molecule fluorescence in situ hybridization (smFISH) in γ5β'2a MBON dendrites and soma (R66C08-GAL4>UAS-myr::SNAP). Images are maximum intensity projections of ten 0.2 μm …
Figure 3—figure supplement 1
Single-molecule fluorescence in situ hybridization (smFISH) labels different numbers of active CaMKII loci in homozygous and heterozygous flies.
(A–A"). Transcriptionally inactive nucleus visualized with endogenous CaMKII smFISH probes. (B–B"). Monoallelic transcription visualized with endogenous CaMKII smFISH probes. (C–C"). Biallelic …
Figure 3—figure supplement 2
Further unpaired control and quantification of CaMKII::YFP after learning.
(A) Schematic of training protocols. The yellow and red circles represent the two odors. These experiments increased the interval between the two parts of the session from 45 to 180 s to avoid …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Drosophila melanogaster) | CaMKII | NCBI | Gene ID: 43828 | |
| Gene (Drosophila melanogaster) | PKA-R2 | NCBI | Gene ID: 36041 | |
| Gene (Drosophila melanogaster) | Ten-m | NCBI | Gene ID: 40464 | |
| Gene (Drosophila melanogaster) | nAChRα1 | NCBI | Gene ID: 42918 | |
| Gene (Drosophila melanogaster) | nAChRα5 | NCBI | Gene ID: 34826 | |
| Gene (Drosophila melanogaster) | nAChRα6 | NCBI | Gene ID: 34304 | |
| Genetic reagent (Drosophila melanogaster) | R66C08-GAL4 | Bloomington Drosophila Stock Center (Owald et al., 2015) | RRID:BDSC_49412 | |
| Genetic reagent (Drosophila melanogaster) | MB112c-GAL4 | Bloomington Drosophila Stock Center (Perisse et al., 2016) | RRID:BDSC_68263 | |
| Genetic reagent (Drosophila melanogaster) | UAS-myr::SNAPf | Bloomington Drosophila Stock Center | RRID:BDSC_58376 | |
| Genetic reagent (Drosophila melanogaster) | CaMKII::YFP | Kyoto Stock Centre (Lowe et al., 2014) | RRID:DGGR_115127 | |
| Genetic reagent (Drosophila melanogaster) | PKA-R2::YFP | Kyoto Stock Centre (Lowe et al., 2014) | RRID:DGGR_115174 | |
| Genetic reagent (Drosophila melanogaster) | Ten-m::YFP | Kyoto Stock Centre (Lowe et al., 2014) | RRID:DGGR_115131 | |
| Chemical compound | 20% v/v paraformaldehyde | Thermo Fisher Scientific | Cat#15713S | |
| Chemical compound | RNase-free 10× PBS | Thermo Fisher Scientific | Cat#AM9625 | |
| Chemical compound | Triton X-100 | Sigma-Aldrich | Cat#T8787 | |
| Chemical compound | 20× RNase-free SSC | Thermo Fisher Scientific | Cat#AM9763 | |
| Chemical compound | Deionized formamide | Thermo Fisher Scientific | Cat#AM9342 | |
| Chemical compound | 50% dextran sulphate | Millipore | Cat#S4030 | |
| Chemical compound | Vybrant DyeCycle Violet Stain | Thermo Fisher Scientific | Cat#V35003 | |
| Chemical compound | Vectashield anti-fade mounting medium | Vector Laboratories | Cat#H-1000-10 | |
| Chemical compound | JF549-SNAPTag | Grimm et al., 2015 | ||
| Chemical compound | Mineral oil | Sigma-Aldrich | Cat#M5904 | |
| Chemical compound | 4-Methocyclohexanol (98%) | Sigma-Aldrich | Cat#218405 | |
| Chemical compound | 3-Octanol (99%) | Sigma-Aldrich | Cat#153095 | |
| Software, algorithm | FIJI | NIH (Schindelin et al., 2012) | http://fiji.sc/ | |
| Software, algorithm | MATLAB R2019b | The MathWorks, Natick, MA | https://www.mathworks.com/products/matlab.html | |
| Software, algorithm | GraphPad Prism 8 | GraphPad Software, La Jolla, CA | https://www.graphpad.com/scientific-software/prism/ | |
| Software, algorithm | Drosophila brain smFISH analysis | This paper (Mitchell, 2021) | see Data availability section | |
| Software, algorithm | Blender | Blender Foundation, Amsterdam | https://www.blender.org | |
| Software, algorithm | NAVis 0.2.0 | Bates et al., 2020b | https://pypi.org/project/navis/ |
Additional files
-
Supplementary file 1
Oligonucleotide sequences of single-molecule fluorescence in situ hybridization (smFISH) probe sets.
- https://cdn.elifesciences.org/articles/62770/elife-62770-supp1-v3.xlsx
-
Transparent reporting form
- https://cdn.elifesciences.org/articles/62770/elife-62770-transrepform-v3.docx
