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Quantitative mRNA imaging throughout the entire Drosophila brain

Nature Methods volume 14, pages 703–706 (2017)Cite this article

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Abstract

We describe a fluorescence in situ hybridization method that permits detection of the localization and abundance of single mRNAs (smFISH) in cleared whole-mount adult Drosophila brains. The approach is rapid and multiplexable and does not require molecular amplification; it allows facile quantification of mRNA expression with subcellular resolution on a standard confocal microscope. We further demonstrate single-mRNA detection across the entire brain using a custom Bessel beam structured illumination microscope (BB-SIM).

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Figure 1: mRNA quantification in whole-mount Drosophila brains using a confocal microscope.
Figure 2: Single-mRNA detection in whole-mount Drosophila brains.
Figure 3: Spatiotemporal regulation of mRNA expression in whole-mount Drosophila brains.

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Acknowledgements

We thank members of the former Transcription Imaging Consortium, M. Rosbash, M. Diaz, K. Abruzzi, L. Lavis, T. Brown, U. Heberlein, T. Harris and Y. Wu, for valuable suggestions. We are grateful to A. Nern for providing the Mi4, Mi1 and Mi9 transgenic flies and to G. Henry and F. Davis for providing transcriptome profiling information. We thank K. Close for assistance with Drosophila dissections and A. Lemire, L. Wang, R. Ray, K. Aswath and X. Zhang for assistance with molecular biology and characterizing transgenic flies. We also thank E. Betzig J. Jordan, and D. Milkie for consultation on the design of the BB-SIM microscope and the control software for the system; L. Shao for structured illumination analysis code; and A. Taylor for assistance with confocal imaging. Funding for this work was provided by the Howard Hughes Medical Institute.

Author information

Authors and Affiliations

  1. Transcription Imaging Consortium, Howard Hughes Medical Institute Janelia Research Campus, Ashburn, Virginia, USA

    Xi Long, Robert H Singer & Timothée Lionnet

  2. Applied Physics and Instrumentation Group, Howard Hughes Medical Institute Janelia Research Campus, Ashburn, Virginia, USA

    Jennifer Colonell

  3. Fly Functional Connectome, Howard Hughes Medical Institute Janelia Research Campus, Ashburn, Virginia, USA

    Allan M Wong

  4. Department of Anatomy and Structural Biology, Dominick P. Purpura Department of Neuroscience, Gruss Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, New York, USA

    Robert H Singer

  5. Department of Cell Biology, Institute for Systems Genetics, New York University Langone Medical Center, New York, New York, USA

    Timothée Lionnet

Authors
  1. Xi Long
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  2. Jennifer Colonell
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  3. Allan M Wong
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  4. Robert H Singer
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  5. Timothée Lionnet
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Contributions

X.L. and T.L. designed the experiments and wrote the manuscript. X.L. performed the experiments and did confocal measurements. J.C. designed and built the Bessel beam SPIM with SIM microscope and performed measurements using it. X.L., J.C. and T.L. analyzed the data. A.M.W. created the transgenic flies for detecting two GFP transcription sites and contributed to the neurotransmitter experiments. R.H.S. consulted on the research and helped to write the manuscript. All authors edited the manuscript.

Corresponding authors

Correspondence to Xi Long or Timothée Lionnet.

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T.L. has filed patent applications (for example, PCT/US2015/023953) whose value may be affected by this publication.

Integrated supplementary information

Supplementary Figure 1 Improvements in signal-to-noise ratio upon NaBH4 treatment and optimized imaging medium.

a) Confocal images of PDF mRNA labeled with FISH probes (left panel) with and without NaBH4 treatment prior to hybridization. Plot of normalized intensity (I-I0/Imax-I0, where Imax is the maximum intensity observed along the line profile and I0 the dark count level, estimated in parts of the image outside of the brain) as a function of the line length (right panel) indicates that NaBH4 treatment improves signal-to-noise ratio by suppressing the background fluorescence. b) Confocal images of Pdf mRNA labeled with FISH probes using VECTASHIELD mounting media (left panel) and DPX mounting media imaged under identical conditions, rendered with identical brightness and contrast settings.

Supplementary Figure 2 Simultaneous detection of mRNA by FISH and protein by immunostaining.

a) Simultaneous imaging of Pdf mRNA (green) and GFP protein (purple) in whole-mount tissue. Maximum intensity projection of a confocal stack of a Pdf-Gal4; 5xUAS-IVS-mCD8::GFP fly brain. Pdf mRNA (labeled with FISH probes, green) accumulates mainly in the cell bodies of PDF neurons identified by the GFP protein signal (immunofluorescence; magenta). b) Simultaneous imaging of Pdf mRNA (FISH, green) and PDF protein (immunofluorescence, magenta) in whole-mount tissue. Maximum intensity projection of confocal stack of a wild-type fly brain. Pdf mRNA overlaps with the cell bodies of PDF neurons identified by the PDF protein signal. The PDF protein image also shows the known arborizations in the accessory medulla, the l-LNvs connecting the PDF neurons of both hemispheres via fibers in the posterior optic tract, the projections into distal layers of the medullae of both hemispheres, and the s-LNv projections toward the ipsilateral dorsal protocerebrum. c) Simultaneous imaging of Pdf mRNA (FISH, green) and Bruchpilot protein (immunofluorescence, magenta) that labels presynaptic active zones and provides a map of neuropils across the entire brain. Maximum intensity projection of confocal stack of a wild-type fly brain.

Supplementary Figure 3 Quantitative analysis of tim expression in PDF neurons (l-LNvs and s-LNvs) at ZT2 and ZT14.

Cell Intensities were calculated from confocal stacks of wild type flies labeled with FISH probes against tim and Pdf mRNAs. tim expression is reduced at ZT2, while Pdf expression displays only modest changes over the daily cycle. Each circle corresponds to one cell; Mean +/- s.d. are represented next to each bee swarm plot; 3 brains were imaged for ZT2, 4 brains for ZT14. Data for l-LNVs is presented in Figure 1d.

Supplementary Figure 4 RNA FISH against various neurotransmitters in select neuron types demonstrates labeling selectivity.

RNA FISH against various neurotransmitters in 3 cell types chosen for their known expression of 3 distinct neurotransmitters (Mi1, Mi4, Mi9 expressing respectively ChAT, Gad1, VGlut, F.P. Davis, S.R. Eddy, and G.L. Henry, personal communication; Takemura et al., eLife 2017). Driver lines used to label Mi1,Mi4 and Mi9 are described in Strother et al., Neuron 2017). (a) Representative confocal sections of the optic lobe from each driver line; all reporter lines express a HaloTag reporter under the control of Gal4 (HaloTag fluorescent ligand, magenta). FISH probes targeting the following genes: Chat, Gad1, vGlut (green) are overlaid with the HaloTag stain marking each neuron type. The RNA FISH overlap with the HaloTag signal follows the predicted expression pattern. Scale bar, 10 μm. (b) close up of representative regions from panel (a). Scale bar, 2 μm. (c) Intensity quantification of entire optic lobe datasets. Each distribution represents the intensity of the FISH signal for a specific gene in the pixels positive for the HaloTag fluorescence marking a given neuron type.

Supplementary Figure 5 Close up from the image in Figure 1e.

This representative region demonstrate the mutual exclusion of the different neurotransmitters.

Supplementary Figure 6 Simplified optical layout of the SPIM/SIM microscope.

Beams from three lasers (488 nm, 561 nm, 640 nm) are expanded to a diameter of 3 mm, and vertical polarization set with a half wave plate. This combined beam is sent through an Acousto-optic Tunable Filter (AOTF, AA Optoelectronic) and beam expander. From the beam expander, the beam passes through an annular apodization mask (made in house using a laser mill to ablate the desired pattern on a Thorlabs Neutral Density filter) and a pair of galvo mirrors (Cambridge Technology) that allow lateral and axial positioning of the beam. The combination of a liquid crystal variable waveplate (Edmund Optics) and polarizing beamsplitter allows the beam to be directed to either excitation objective; this diagram shows the beam for just one path. The annular mask, X galvo, Z galvo, and back aperture of the excitation objectives are all at conjugate planes, so beam position is constant at those points. When imaging, the illumination plane and detection objective are fixed, and the sample is moved to image through Z to create a volume.

Supplementary Figure 7 Comparison of the number of Pdf (pink) and tim (blue) transcription sites at ZT2 and ZT14 in s-LNvs in wild type flies imaged with the BB-SIM microscope.

The variation of tim and Pdf nuclear foci is in good agreement with previous findings that tim transcription is reduced at ZT2, while Pdf transcription remains constant over the daily cycle. Mean +/- s.d.; 5 brains were imaged for tim ZT2, 4 brains for tim ZT14; 4 brains were imaged for Pdf ZT2 and ZT14;

Supplementary Figure 8 Single-molecule detection of tim mRNA.

a) Low-resolution frontal view of tim mRNA detected with the BB-SIM microscope in a whole-mount wild type fly (ZT14). Red box indicates the location of the right hemisphere PDF neurons. b) Sagittal view of the sample in a. c) Maximum Intensity Projection of a high resolution BB-SIM scan of the region of the PDF neurons (red box in b). High levels of expression are visible in the 8 PDF neurons; lower tim expression is also present in surrounding cells. Bright foci are observed at the sites of active transcription in the nuclei. d) Magnification of panel c displaying individual mRNA spots as well as one transcription site. Individual mRNAs appear as oblong oblique spots because the light sheet observation angle is inclined at a ~45° angle relative to the vertical axis of the image. e) 3D visualization of the region surrounding the PDF neurons (red box in b panel or entire c panel). The color encodes the local density of spots. f) Top: Histogram of the intensity distribution for the spots detected in panel c using a low detection threshold (gray bars). Background detections appear as a low intensity spots while mRNAs accumulate in a distinct high-intensity distribution. The fit to a 2-component lognormal distribution (purple) provides the respective contributions of background (red) and mRNAs (blue) to the overall histogram. Bottom: Jaccard Coefficient quantifying the similarity between the detected spots and the expected mRNA counts, as a function of an arbitrary intensity threshold (the Jaccard calculation is based on the 2-component lognormal fit, see Supplementary Note). The Jaccard Coefficient provides a metric of detection accuracy taking into account both sensitivity and selectivity; it reaches a maximum for the intensity threshold that separates optimally the background from mRNA signal. The maximum value indicates that the spots detected at the optimum are ~90% similar to the original mRNAs. g) Intensity and sizes of tim mRNA spots as a function of depth. The fluorescence intensities and spot sizes of tim mRNAs detected in panel c do not change as a function of depth. Each circle represents one detected spot; the line is a sliding average.

Supplementary Figure 9 FISH Fluorescence intensity from neurons expressing a reporter gene under control of promoters of increasing intensity.

Top: comparison of Gfp mRNA fluorescence intensity per cell in fly lines using confocal microscopy. The GFP reporter is driven by 3, 5, or 10 UAS repeats, resulting in a gradual increase in expression of the Gfp mRNA. In contrast, the endogenous Pdf mRNA levels do not increase with increasing UAS number (bottom). The colors indicate the two different PDF neuron types. Each circle corresponds to one cell; Mean +/− s.d. are represented next to each bee swarm plot; 5 brains were imaged for each condition.

Supplementary Figure 10 Spatial profile of mRNAs expressed by a reporter gene in PDF neurons.

Quantification of the density of HaloTag mRNA molecules at zeitgeber time 2 in PDF neuron processes as a function of distance from the cell body (4 brains totaling 34 l-LNvs, 23 s-lLNvs with respectively 14 and 21 traced processes; mean +/− s.e.m. assuming Poisson counting error).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–10 and Supplementary Notes 1–3

Supplementary Table 1

List of the imaging parameters used in this study.

Supplementary Table 2

List and Sequences of the probe libraries used in this study.

Supplementary Protocol

Whole-mount RNA FISH of Drosophila adult brain

Spatial Distribution of Neurotransmitter Expression in the Adult Drosophila Brain.

3 dimensional rendition of a whole mount brain sample labeled for with probes targeting the following neurotransmitters: Gad1 (magenta), vGlut (yellow), Chat (green); same sample as in Figure 1e.

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Long, X., Colonell, J., Wong, A. et al. Quantitative mRNA imaging throughout the entire Drosophila brain. Nat Methods 14, 703–706 (2017). https://doi.org/10.1038/nmeth.4309

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