Multiplex Detection of Gene Expression in the Intact Drosophila Brain Using Expansion-Assisted Iterative Fluorescence In Situ Hybridization

Resumo

Expansion-Assisted Iterative Fluorescence In Situ Hybridization (EASI-FISH) is a robust technique that combines expansion microscopy with fluorescence in situ hybridization (FISH) to detect the expression of multiple genes in thick tissue. This protocol outlines recent advancements in the method and its adaptation for labeling the intact Drosophila central nervous system..

Resumo

Understanding gene expression is essential for deciphering cellular functions. However, methods for analyzing the expression of numerous genes in situ within a given tissue remain limited. The EASI-FISH protocol described here has been adapted to detect the expression of dozens of genes in the intact adult Drosophila central nervous system (CNS) using commercially available reagents. This protocol includes a new gel formulation that enhances gel robustness, enabling multiple rounds of hybridization and allowing the embedding of multiple brains per gel. This improvement increases throughput, facilitates optimal comparison of experimental conditions, and reduces reagent costs. Additionally, by employing the GAL4-UAS system for co-detection of green fluorescent protein (GFP), gene expression can be visualized in specific neuronal or glial cell types. Notably, the high resolution achieved through expansion microscopy, combined with the sensitivity of the method, allows for the detection of single RNA transcripts. This approach effectively integrates high image quality with high throughput, making it a powerful tool for studying gene expression throughout the intact fly brain.

Introdução

FISH was first utilized in Drosophila over 40 years ago to map genes on polytene chromosomes^1,2. Today, single-molecule FISH is the gold standard for spatially localizing and quantifying mRNA transcripts in intact tissue³. However, due to issues of sample integrity and RNA loss, whole mount single molecule FISH techniques in Drosophila have been limited to only a few genes that can be analyzed in the same sample^4,5,6,7. While the recent development of barcoding (e.g.^8,9,10) increases the number of transcripts that can be detected up to several hundred even in thick tissue^11,12, the establishment of such protocols requires a considerable investment of resources, which could be a hurdle to implementation. Indeed, many inventive spatial transcriptomic techniques never spread beyond their institutions of origin¹³. Therefore, a straightforward and comparatively inexpensive method to detect the expression of dozens of genes in the same intact brain would facilitate various research objectives: validation of RNA seq and cell atlas data, mapping of molecular cell types across the CNS, quantifying developmental, sex or species-specific differences in gene expression, or measuring changes in gene expression induced by the large variety of genetic or environmental perturbations available in Drosophila.

Expansion microscopy is a technique that allows researchers to observe tissues at higher resolution by expanding them in a swellable hydrogel¹⁴. EASI-FISH combines expansion microscopy and RNA FISH¹⁵, which utilizes the hybridization chain reaction (HCR), a non-enzymatic technique to detect oligo probe binding¹⁶. EASI-FISH was first developed as an alternative approach to thin tissue FISH to map expression patterns of dozens of genes in thick (300 µm) tissue slices of mouse lateral hypothalamus¹⁷. This method is ideal for application to adult fly brains, given this tissue is less than 300 µm thick, and a prototype fly EASI-FISH protocol has recently been described¹⁸. Here, dissected and fixed Drosophila brains are embedded in a swellable hydrogel, where mRNA and proteins are covalently anchored to the gel polymer with the alkylating agents Melphalan-X and Acryloyl-X (Ac-X), respectively. After protein digestion, which allows for isotropic expansion of the brain, gene-specific DNA oligo probes are hybridized to mRNA in the tissue to locate transcripts of interest. Fluorescently tagged DNA hairpins are then added in a second hybridization step. Multiple hairpin molecules polymerize in the HCR reaction to form meta-stable DNA polymers. This leads to an amplification of the signal for individual transcripts, which can be detected as bright spots by light sheet or confocal microscopy.

The covalent anchoring of mRNA and protein to the gel matrix and proteolytic digestion of proteins after gel formation creates an environment where mRNA transcripts are relatively stable and potentially more accessible to probe binding. The stability of mRNA and the gel permits multiple rounds of re-probing and imaging, with the stripping of DNA probes and HCR product by incubation with the enzyme DNase1 between rounds^15,17. Furthermore, expansion of the sample reduces autofluorescence and light scattering by molecular de-crowding and effectively increases image resolution¹⁴. Therefore, individual mRNA transcripts can be more easily resolved.

GFP sustains the procedure sufficiently to remain detectable by fluorescence or post-expansion antibody binding. Therefore, this reporter can be used to mark and segment cell types of interest. In conjunction with the many neuron-specific split-GAL4 driver lines available (e.g.,^19,20), this opens up the possibility of gaining further insight into the molecular identity of a neuronal type or identifying molecular subtypes that cannot be easily distinguished by their anatomy.

Described here is an updated version of the prototype fly EASI-FISH protocol¹⁸ that features excellent mRNA retention, removal of technical difficulties, and greater robustness of the gel. Modifications include increasing the PFA concentration, lowering the temperature of fixation, and facilitating sample preparation by mounting the brains before incubation with Melphalan-X and Ac-X. This protocol also incorporates a new gel recipe to increase gel robustness, which effectively reduces gel damage when performing many rounds of FISH and imaging. These modifications make the protocol more straightforward to perform, decreasing both processing time and reagent use, and enhancing confidence in consistent results. Importantly, the capability of multiplexing is increased such that dozens of genes can be investigated in the same brain over a period of months. This is illustrated by the expression patterns of several neuronal genes in the adult Drosophila brain across a range of expression levels.

Protocolo

This protocol was performed on adult (5-6 day old) Drosophila melanogaster females, but any sex or Drosophila species is applicable. For gel anchor recipes, see Supplementary Table 1; for the gel recipe, Supplementary Table 2; and for other EASI-FISH reagents, Supplementary Table 3. For a day-to-day materials and reagents guide, see Supplementary Table 4. For light sheet microscope gel holder, see Supplementary File 1. Figure 1A-C shows a schematic depiction of the embedding of brains in gels before expansion and probe hybridization (steps 2.1 through 3.8). Figure 1D is a photograph of the embedded brains, Figure 1E shows a gel holder for light sheet microscopy, and Figure 1F depicts the timeline of a typical experiment. The commercial details of the reagents and the equipment used are listed in the Table of Materials.

1. Fly brain dissection and fixation

1. Dissect fly brains or CNS in cold S2 medium (for more dissection details, see reference²¹)
2. To fix the brains, add up to 20 brains or 10 CNS to 2 mL of cold 4% PFA/PBS and incubate overnight on a nutator at 4°C.
   CAUTION: PFA is harmful if swallowed or inhaled, irritant to skin and eyes, and may be carcinogenic.
3. Rinse the sample (1x) with 2 mL of cold PBT (0.5%, see Supplementary Table 4), then wash the sample with 2 mL of cold PBT (4x) for 15 min on a nutator at 4 °C.
4. Rinse the sample (1x) with 2 mL of cold 70% EtOH. Store the brains in 2 mL 70% EtOH at 4 °C for up to 6 months.

2. Mounting brains and adding RNA and protein linkers (Day 1)

NOTE: Wear gloves at all times and consistently wipe down equipment and workspace with RNase Away. Gloves are required (1) to keep RNases away from samples and (2) as protection from several reagents that pose a health hazard.

1. Prepare gel chambers by wiping a non-charged slide with an RNase decontamination reagent. Remove the opaque film protecting the adhesive from the silicone gasket, adhere the gasket (with up to 4 chambers) to the slide, and coat each chamber surface with 1 µL poly-lysine using a P20 pipette tip. Air dry and repeat the process.
   NOTE: Step 2.1 can be done the day before.
2. Transfer the brains to a 0.2 mL PCR tube (up to 4 brains per tube) and rehydrate for (2x) 5 min in 150 µL of PBT (0.1%) and (2x) for 5 min in PBS.
3. To mount the brains, add 40 µL of PBS per chamber, then gently stick down the brains in a row in the center of the chamber with fine tweezers, leaving some space between the brains (see Figure 1D). Mounting up to 6 brains or 2 CNS per chamber is possible.
4. Remove PBS and immediately add 50 µL of 20 mM MOPS buffer. Incubate for 30 min at room temperature (RT).
5. While brains are equilibrating in MOPS buffer, thaw the Melphalan-X and Ac-X stock solutions and prepare 75 µL of Melphalan-X/Ac-X solution per chamber (dilute Melphalan-X stock 1:1 with MOPS buffer and add Ac-X at 1:100). Vortex and spin briefly to collect the solution; time the preparation such that the solution is ready at the end of the 30 min incubation time, not earlier.
   CAUTION: Melphalan-X is harmful if swallowed or inhaled, irritant to skin and eyes, and may be carcinogenic.
6. Remove all MOPS buffer and add 50 µL of Melphalan-X/Ac-X solution.
7. Cover the chamber by placing a coverslip on top, but do not use the gasket adhesive to seal. Place the chamber in a humidified box and incubate protected from light overnight at RT.

3. Gelation and Proteinase K digestion (Day 2)

1. Carefully remove the coverslip and Melphalan-X/Ac-X, and wash mounted brains (2x) for 2 min with 100 µL of PBT (0.1%) and (1x) for 2 min with 100 µL of PBS.
2. Thaw TREx-1000 solution, 4HT, TEMED, and APS and keep on ice.
   NOTE: TREx-1000 can precipitate at -20 °C; vortex well to completely dissolve after thawing.
   CAUTION: These reagents are harmful or toxic when swallowed or if inhaled, as well as skin and eye irritants.
3. Prepare the gel solution by mixing TREx-1000 with 4HT, TEMED, and APS at a ratio of 94:2:2:2. Vortex and keep on ice. For each chamber, prepare 150 µL of gel solution.
4. Remove PBS from the chamber with a pipette tip and carefully wick away any remaining PBS with a lint-free wipe. For each chamber, immediately add 40 µL of gel solution on top of the brains. Incubate at 4 °C for 10 min.
5. Remove the gel solution and add 40 µL of fresh gel solution, incubating at 4 °C for another 10 min.
   NOTE: Collect gel waste solution in a microcentrifuge tube and polymerize before discarding.
6. Remove the gel solution and peel the clear protective film off the gasket's surface adhesive. Add a final 45 µL of fresh gel solution, gently place a coverslip over the chamber, and avoid trapping any air bubbles. Press the coverslip to seal the chamber and incubate at 4 °C for 10 min (for a total of 30 min equilibration at 4 °C).
7. Polymerize the gel at 37 °C for 1.5 h.
8. Once the gels have set, allow them to cool on the bench for a few minutes. Carefully take off the coverslip and remove the silicone gasket with a new razor blade. Next, trim the gel into a rectangle and cut the top right-hand corner to track sample orientation.
9. Remove the gels from the slide with a fine paintbrush wetted with a small amount of ProK Buffer and transfer them individually to 2 mL microcentrifuge tubes.
10. Incubate each gel with 500 µL of ProK buffer and 5 µL of ProK enzyme (1:100) at 37 °C overnight.

4. DNA digestion and probe hybridization (Day 3)

1. Wash the gels (3x) for 10 min with 1 mL of PBS at RT.
   NOTE: Use fine-tip transfer pipettes to avoid damaging the gel when replacing the solutions.
2. Incubate gels for 30 min in 1 mL of of DNase1 buffer at 37 °C.
3. Add 50 µL of DNase1 enzyme to 450 µL of DNase buffer. Gently mix, but do not vortex. Incubate each gel in 500 µL of DNase1 for 2 h 37 °C. Wash (4x) 15 min with 1 mL of PBS at RT.
   NOTE: Gels can be stored in 5x SSC at 4 °C before or after the DNase digest/washes for several weeks.
4. Thaw hybridization (hyb) and probe wash buffer. Incubate the gels in 500 µL of hyb buffer with no probes for 30 min at 37 °C.
   CAUTION: The hyb and probe wash buffer contain formamide, which is suspected of causing cancer.
5. Dilute the probes 1:100 in hyb buffer (10 nM), preparing 300 µL per gel. Incubate the gels with hyb buffer and probes overnight at 37 °C. Keep the probe wash buffer and PBS at 37 °C.

5. Probe washes (Day 4)

1. Wash the gels (3x) for 30 min with 750 µL of probe wash buffer at 37 °C.
2. Wash the gels (3x) for 30 min, then (3x) for 1 h with 1 mL of PBS at 37 °C.
3. Wash the gels in 2 mL of PBS at RT overnight or 4 °C over the weekend.

6. Hybridization Chain Reaction (HCR) with fluorescent hairpins (Day 5)

NOTE: For the HCR principle, see reference²².

1. Incubate the gels in 500 µL of amplification (amp) buffer for 30 min at RT.
2. Heat the fluorescent hairpins at 95°C for 90 sec in a PCR machine and rapidly cool to 25°C for 30 min.
3. For each probe, dilute matching hairpin pairs (h1 and h2) 1:50 in amp buffer, preparing 300 µL per gel and vortex.
4. Incubate the gels with the hairpin mixture for 3 h at RT in the dark.
5. Wash the gels (2x) for 15 min with 750 µL of 5x SSCT and (2x) for 30 min with 1 mL of 0.5x SSCT at RT.
6. If brains express GFP, stain with 500 µL of anti-GFP-AF488 antibody (1:500) in PBT (0.1%) containing 5 mg/mL of ultrapure BSA and incubate overnight (or over the weekend) at 4 °C.

7. Mounting of gels and imaging (Day 6)

1. After antibody incubation, rinse the gels once and wash (1x) for 30 min with 1 mL of PBT (0.1%), followed by (3x) for 30 min with 1 mL of PBS.
   NOTE: Washing in PBS will result in 2x expansion; for 3x expansion, wash with 0.05x SSC.
2. While washing, coat the gel attachment surface of the light sheet microscope gel holder twice with 1 µL of poly-lysine, allowing it to dry at 37 °C between coats. Similarly, for confocal imaging, coat the gel attachment area of a glass bottom dish with repeated applications of poly-lysine.
3. Stain the gels for 30 min at RT in 1 mL of 5 µg/mL DAPI/PBS or DAPI/0.05x SSC, or alternatively, stain overnight at 4 °C.
4. To mount the gel on the gel holder (light sheet microscope) or glass bottom dish (confocal microscope), place the gel on a coverslip and carefully dry the edges with a lint-free wipe. Ensure that the gel is orientated so that the tissue will be closest to the objective. Using a paintbrush, gently place the gel onto the poly-lysine surface in one movement. For imaging, bathe gel in PBS (or 0.05x SSC if expanding to 3x).
   NOTE: After imaging, gels can be gently removed from the poly-lysine surface with a wet paintbrush and transferred to a 2 mL microcentrifuge tube.
5. For immediate re-probing, proceed to day 7. For longer-term storage (1 week to 6 months), store gels in 1 mL of 5x SSC at 4 °C. When ready to use, wash gels (3x) for 10 min with 1 mL of PBS before proceeding.
   NOTE: The extent of expansion is generally consistent in the buffers described (~2x in PBS and ~3x in 0.05x SSC). To check the expansion factor, measure the size of a brain before and after expansion in images taken at a fluorescence stereomicroscope. To visualize the brain after expansion, briefly stain it with DAPI. The approximate expansion factor is the post-expansion size divided by the pre-expansion size.

8. Stripping probes and hairpins for multiplexing (Day 7)

1. Incubate the gels for 30 min in 1 mL of DNase1 buffer at 37°C.
2. Add 50 µL of DNase1 to 450 µL of DNase buffer. Gently mix, but do not vortex. Incubate the gels in 500 µL of DNase1 for 2 h at 37 °C.
3. Wash (4x) for 15 min with 1 mL of PBS at RT. Gels are now ready for the next round of hybridization (Day 3, step 6).

Resultados

The performance of this modified EASI-FISH protocol is demonstrated by detecting the expression of several different neurotransmitter-associated and neuropeptide genes across the adult Drosophila brain over multiple rounds. Typically, gelled and expanded brains were hybridized with two or three probes per round, followed by HCR with hairpin pairs conjugated with Alexa Fluor (AF) 488 (unless GFP was expressed), AF546, and AF647 or Janelia Fluor (JF) 669. Imaging was mostly performed on a light sheet microscope, a...

Discussão

The described EASI-FISH protocol for the intact Drosophila nervous system enables the detection of dozens of transcripts in the same brain or CNS. It is robust, straightforward, and sensitive, and it can be combined with GFP detection to identify subsets of neurons or glia. Since this method is easy to implement, it should facilitate research by Drosophila labs with an interest in mapping and quantifying gene expression in the intact fly brain.

EASI-FISH provides several adva...

Materiais

Name	Company	Catalog Number	Comments
0.2 mL PCR tubes	USA Scientific	1402-4700
0.5 mL screw-cap microcentrifuge tubes, Amber	USA Scientific	1405-9707
0.5M EDTA, PH 8, RNase free	Invitrogen	AM9260G
10M NaOH	Sigma	72068
10x PBS	Fisher	BP 3994
20x SSC	ThermoFisher	AM 9763
40% Acrylamide	Bio-Rad	1610140
4-Hydroxy-TEMPO (4HT)	Sigma	176141
5 M NaCl RNase-free	Thermofisher	AM97060G
5 ml Transfer Pipet- Fine tip	Global Scientific	134070-S20
6 Well Glass Bottom Plate	Cellvis	P06-1.5H-N
Acetonitrile, anhydrous	ThermoFisher	042311-K7
Acrylic Acid	TCI	A0141
Acryloyl-X	ThermoFisher	A20770
Ammonium persulphate (APS)	Sigma	A3678
Amplification Buffer	Molecular Instruments	https://store.molecularinstruments.com/new-bundle/rna-fish
Anhydrous DMSO	Invitrogen	D12345
AstA_B2	Molecular Instruments	PRR477
ChAT_B1	Molecular Instruments	PRG115
Crz_B5	Molecular Instruments	PRR480
DAPI	Sigma	D9542
FIJI	Open source	https://imagej.net/software/fiji/
FMRFa_B2	Molecular Instruments	PRR487
GAD1_B5	Molecular Instruments	RTE581
GFP Polyclonal Antibody, Alexa Fluor 488	ThermoFisher	A-21311
Hybridization Buffer	Molecular Instruments	https://store.molecularinstruments.com/new-bundle/rna-fish
Imaris Stitcher	Oxford Instruments	https://imaris.oxinst.com/products/imaris-stitcher
Imaris viewer	Oxford Instruments	https://imaris.oxinst.com/imaris-viewer
JF-669, SE	Tocris	6420
Melphalan	Caymen Chemicals	16665
MOPs Buffer	Fisher	BP308-100
N,N Methylene-Bis-acrylamide	Bio-Rad	1610142
N,N,N’,N’-Tetramethylethylenediamine (TEMED)	Sigma	T22500
Nuclease Free Water	Ambion	AM9932
Oligo Probes	Molecular Instruments	https://store.molecularinstruments.com/new-bundle/rna-fish
Paraformaldehyde (PFA)	EMS	15710
PBS	Fisher	BP24384
Photoflo-200	EMS	74257
Poly-L-lysine hydrobromide (polylysine)	Sigma	P1524
Probe Wash Buffer	Molecular Instruments	https://store.molecularinstruments.com/new-bundle/rna-fish
Proteinase K	NEB	P8107S
QIAquick Nucleotide Removal Kit	Qiagen	28306
RNase Away	ThermoFisher	7003, decontamination reagent
RNase-Free DNase1	Qiagen	79254
S2 Medium	ThermoFisher	21720024
Silicone Gaskets	Invitrogen	P24743
Tdc2_B5	Molecular Instruments	RTE880
Tk_B5	Molecular Instruments	PRR484
UltaPure 10% SDS	Thermofisher	15553027
Ultrapure BSA	ThermoFisher	AM2616
Unconjugated and Alexa Fluor-conjugated Hairpins	Molecular Instruments	https://store.molecularinstruments.com/new-bundle/rna-fish
VAChT_B1	Molecular Instruments	RTA904
vGLUT_B2	Molecular Instruments	RTB212
Zeis Z7 Gel Holder	Janelia	CAD files avaiable on request
Zeiss LSM 980 confocal Inverted microscope	Zeiss	https://www.janelia.org/node/46947/#lsm980
Zeiss Z7 light sheet microscope	Zeiss	https://www.zeiss.com/microscopy/us/products/light-microscopes/light-sheet-microscopes/lightsheet-7.html