Hybridization Chain Reaction RNA Whole-Mount Fluorescence In situ Hybridization of Chemosensory Genes in Mosquito Olfactory Appendages

Summary

The article describes the methods and reagents necessary to perform hybridization chain reaction RNA whole-mount fluorescence in situ hybridization (HCR RNA WM-FISH) to reveal insights into the spatial and cellular resolution of chemosensory receptor genes in the mosquito antenna and maxillary palp.

Abstract

Mosquitoes are effective vectors of deadly diseases and can navigate their chemical environment using chemosensory receptors expressed in their olfactory appendages. Understanding how chemosensory receptors are spatially organized in the peripheral olfactory appendages can offer insights into how odor is encoded in the mosquito olfactory system and inform new ways to combat the spread of mosquito-borne diseases. The emergence of third-generation hybridization chain reaction RNA whole-mount fluorescence in situ hybridization (HCR RNA WM-FISH) allows for spatial mapping and simultaneous expression profiling of multiple chemosensory genes. Here, we describe a stepwise approach for performing HCR RNA WM-FISH on the Anopheles mosquito antenna and maxillary palp. We investigated the sensitivity of this technique by examining the expression profile of ionotropic olfactory receptors. We asked if the HCR WM-FISH technique described was suitable for multiplexed studies by tethering RNA probes to three spectrally distinct fluorophores. Results provided evidence that HCR RNA WM-FISH is robustly sensitive to simultaneously detect multiple chemosensory genes in the antenna and maxillary palp olfactory appendages. Further investigations attest to the suitability of HCR WM-FISH for co-expression profiling of double and triple RNA targets. This technique, when applied with modifications, could be adaptable to localize genes of interest in the olfactory tissues of other insect species or in other appendages.

Introduction

Mosquito vectors such as Anopheles gambiae rely on a rich repertoire of chemosensory genes expressed in their peripheral olfactory appendages to thrive in a complex chemical world and identify behaviorally relevant odors emanating from human hosts, detect nectar sources, and locate oviposition sites¹. The mosquito antenna and the maxillary palp are enriched with chemosensory genes that drive odor detection in these olfactory appendages. Three main classes of ligand-gated ion channels drive odor detection in mosquitoes' olfactory appendages: the Odorant receptors (ORs), which function with an obligate Odorant receptor co-receptor (Orco); the Ionotropic receptors (IRs), which interact with one or more IR coreceptors (IR8a, IR25a, and IR76b); the chemosensory Gustatory receptors (GRs), which function as a complex of three proteins to detect carbon dioxide (CO₂)^1,2.

RNA fluorescence in situ hybridization is a powerful tool for detecting the expression of endogenous mRNA³. In general, this method utilizes a fluorophore-tagged single stranded nucleic acid probe with sequence complementary to a target mRNA. Binding of the fluorescent RNA probe to the target RNA allows identification of cells expressing a transcript of interest. Recent advancements now enable the detection of transcripts in whole-mount mosquito tissues^4,5. The first generation of hybridization chain reaction (HCR) technology used an RNA-based HCR amplifier; this was improved upon in a second-generation method that instead used engineered DNA for the HCR amplifier^6,7. This upgrade resulted in a 10x increase in signal, a dramatic decrease in production cost, and significant improvement in the durability of reagents^6,7.

In the protocol, we describe the utilization of a third generation HCR whole-mount RNA fluorescence in situ hybridization (HCR RNA WM-FISH) method designed for detecting the spatial localization and expression of any gene^8,9. This two-step method first utilizes nucleic acid probes specific for the mRNA of interest, but which also contain an initiator recognition sequence; the second step utilizes fluorophore-tagged hairpins which bind to the initiator sequence to amplify the fluorescent signal (Figure 1). This method also allows for the multiplexing of two or more RNA probes and amplifying probe signals to facilitate RNA detection and quantification⁸. Visualizing the transcript abundance and RNA localization patterns of chemosensory genes expressed in the olfactory appendages offers the first line of insight into chemosensory gene functions and odor coding.

Protocol

1. Considerations and preparation of materials

1. Decide if whole-mount or cryo-section of tissue will be appropriate. This protocol is optimized for whole-mount in situ imaging of RNA in the Anopheles mosquito antenna and maxillary palp without cryo-sectioning. If samples are thicker than 5 mm, cryo-sectioning is recommended to enable probe penetration.
2. Identify the genes of interest and copy the sequences including introns and exons from a suitable database. Transcribe the gene sequence into RNA for synthesis.
3. Determine whether to purchase probes from commercial vendors or if probes will be synthesized in the laboratory.
   NOTE: In this study, the in situ probes were purchased from a commercial vendor (see Table of Materials). Alternatively, it can be synthesized as previously reported¹⁰. Reagents used in the study are described in Table 1. Materials required to prepare the reagents are listed in the Table of Materials.

2. Tissue pre-fixation

1. Anesthetize 10-15 adult female or male Anopheles coluzzii mosquitoes (strain N'Gousso), aged 5-10 days post emergence, by collecting them with a mouth aspirator into a paper cup or plastic tube and placing them in a bucket containing ice. A successful cold-induced anesthesia can be confirmed when the mosquitoes are immobile.
2. Decapitate them by removing the heads from the neck region with a pair of forceps. Using the non-dominant hand grab the thorax with forceps and separate the neck from the body with forceps held by the dominant hand. Place heads in a 1.5 mL tube containing 500 µL of chitinase-chymotrypsin dimethyl sulfoxide buffer (CCD buffer) on ice.
   NOTE: Freezing animals can deform the antennae; a quick knockdown on ice is recommended. The mosquito's age during testing may vary depending on the project. It is important to confirm and coordinate their physiological status, such as whether they are blood-fed, starved, or mated. In this study, olfactory appendages were sampled from mosquitoes that had been blood-fed and mated.
3. Pre-heat mosquito heads in CCD buffer at 37 ˚ C on a heat block for 5 min and then transfer the tube containing the mosquito heads to a hybridization oven and rotate at 37 ˚ C. Incubation time in CCD buffer depends on tissue type. For female Anopheles antennae, use 20 min, male antennae require 15 min, while longer incubation time (1 h) is needed for the maxillary palps.
4. Transfer the entire content of the tube into a dissecting watch glass. Gently pour sample into the depression of a watch (dissection) glass. If mosquito heads are stuck inside the tube, use a pipette to add CCD buffer into the tube and rinse out the head.
5. Use forceps to carefully transfer the heads or any antennae/palps detached during incubation and fix in 1 mL of the pre-fixative.
   NOTE: Leftover CCD buffer can be preserved at -20 ˚ C. Buffer may be reused up to 3x. If the pre-fixative turns slightly brownish because of the CCD buffer carryover by the tissues, replace it with another 1 mL of fixative.
6. Rotate heads in pre-fixative for 24 h at 4˚ C using a nutator.
   ​NOTE: Rocking speed for the nutator used in this study was non-adjustable; the default speed set (12 rpm) by the manufacturer was used.

3. Tissue dissection

1. Rinse heads 4x (5 min per wash) with 1 mL of 0.1% PBS-Tween on ice. To avoid sample loss, use a pipette attached to gel loading tip to remove the liquid.
   NOTE: Two quick washes followed by a 10 min long wash, and a final quick wash could be performed instead of step 3.1.
2. Transfer the heads in the tube into a dissecting watch glass. Gently pour sample into the depression of a watch glass. Rinse out mosquito heads that are stuck inside the tube with 0.1% PBS-Tween.
3. Under a dissecting microscope, remove tissues of interest (antennae/palps) from the head with sharp forceps. Hold the posterior part of the head with forceps and grab an antenna with another forceps from the base. Clean forceps with paper moistened with RNAse-free solvent. Remove the palps using the same process.
4. Transfer the antennae and palps with forceps into empty DNA/RNase-free tubes placed on ice. Separate the different tissue parts into pre-labeled different tubes.
5. Dehydrate tissue in 400 µL of solvent containing a mixture of methanol (MeOH 80%) and Dimethyl sulfoxide (DMSO 20%) for 1 h at room temperature.
   NOTE: There is no need to place tissue on a nutator; let it sit on a tube rack at room temperature on the lab bench. It is recommended to use fresh solvent mixture. For a 500 µL stock solution, mix 400 µL of MeOH with 100 µL of DMSO.
6. Replace the dehydrating reagent with 400 µL of absolute (100%) methanol and dehydrate tissues overnight at -20 ˚C. Allow tissues to settle by gravity and use a pipette to remove the liquid.
   ​NOTE: Samples are viable for extended period of dehydration, up to 4 nights have been tested without loss of signal.

4. Tissue post-fixation

1. Rehydrate tissues in a four-step series of graded 400 µL MeOH/PBS-Tween for 10 min on ice. Start with 75% MeOH/25% PBS-Tween followed by 50% MeOH/ 50% PBS-Tween, then 25% MeOH/ 75% PBS-Tween and finally 100% PBS-Tween.
   NOTE: A gel loading tip with a tiny opening can be used to remove the buffer from the tube to avoid losing the tissues. Buffer removal can be done under a dissecting microscope.
2. Wash with 400 µL of phosphate-buffered saline containing 0.1% Tween-20 (PBS-T) for 10 min at room temperature. Wash by placing the sample on a nutator.
3. Make a 20 µg/mL Proteinase-K solution and incubate in 400 µL of Proteinase-K solution for 30 min at room temperature.
   NOTE: Dilute 20 mg/mL Proteinase-K stock (1000x stock solution) i.e., 2 µL in 2 mL of 0.1% PBS-Tween. The left over can be stored in a -20 ˚ C freezer.
4. Stop enzymatic digestion of tissue by washing 2x for 10 min with 400 µL of 0.1% PBS-tween.
5. Add 400 µL of the post-fixative and incubate for 20 min at room temperature. Wash 3x, 15 min per wash, with 400 µL of 0.1% PBS-Tween.

5. Probe hybridization

1. Incubate tissue in 400 µL of probe hybridization buffer for 5 min. Ensure tissue is completely submerged in the buffer by gently pipetting.
2. Prepare for the next step by heating an aliquot of probe hybridization buffer to 37 ˚ C for 30 min.
3. Remove buffer and pre-hybridize with 400 µL of pre-heated probe hybridization buffer for 30 min at 37 ˚ C.
4. Make probe solution for the target chemosensory receptors (IR8a, IR76b, IR25a, IR41t.1, IR75d, IR7t, IR64a or Orco) by adding 8 pM of probe. Add 8 µL of 1 µM probe stock to 500 µL pre-heated probe hybridization buffer.
5. Remove the pre-heated probe hybridization buffer and replace it with 400 µL of heated probe solution.
6. Incubate tissue at 37 ˚ C for two nights on a nutator placed inside an incubator and covered under a box.

6. Probe amplification

1. Heat up the probe wash buffer to 37 ˚ C. Remove excess probe solution by rinsing the tissue 5x, 10 min per wash, with 400 µL of probe wash buffer at 37 ˚ C, nutating in the incubator.
   NOTE: Preparation for step 6.4 may begin. See the note in step 6.4.
2. Wash samples 2x, 5 min per wash, with 400 µL of 5x saline-sodium citrate containing 10% Tween-20 (SSCT) at room temperature. Thaw amplification buffer to room temperature on a bench top.
3. Prepare tissue for amplification by incubating with 400 µL of amplification buffer for 10 min at room temperature. Due to the viscosity of the amplification buffer, tissues may not be completely submerged. Mix by gently pipetting the amplification buffer below the tissue and expelling the buffer on top of the tissue until they are submerged.
4. Separately prepare 18 pM of hairpin h1 and 18 pM of hairpin h2 by heating 6 µL of 3 µM stock at 95 °C for 90 s and cool to room temperature in a dark drawer for 30 min. Ensure PCR tubes are tightly capped to prevent evaporation of the hairpins while heating in a thermal cycler.
   NOTE: To save time, step 6.4 can be initiated while on the 4^th washing step in step 6.1. Hairpins should be separately heated to prevent cross reaction. Hairpins should not be diluted with any buffer in this step. The hairpins can be purchased from the probe manufacturer along with the probes.
5. Replace the amplification buffer added in step 6.3 with a mixture containing the heated hairpins (from step 6.4) together with 100 µL of amplification buffer. A typical reaction will contain 6 µL of h1, 6 µL of h2, and 100 µL of amplification buffer.
   NOTE: Hairpins h1 and h2 can be combined after cooling to room temperature and then added to 100 µL of amplification buffer. To avoid transferring the tissue samples from one tube to another, the amplification buffer in step 6.3 can be removed and replaced by the hairpins mixture diluted in amplification buffer.
6. Incubate tissue overnight and nutate in the dark at room temperature using the default speed of the nutator.

7. Mounting tissue sample

1. Dilute amplification buffer in the incubated tissue with 300 µL of 5x SSCT (sodium chloride-sodium citrate diluted in Tween-20).
   NOTE: The dilution is helpful to reduce the viscosity and makes it easier to remove the amplification buffer from the tissue. We used Triton-X 100 for the pre-fixative and Tween-20 for the post-fixative step because of the differences in their actions as agents to permeabilize cell membrane.
2. Wash tissue 5x with 400 µL of 5x SSCT at room temperature.
   NOTE: Store tissue temporarily at 4 °C until ready to be mounted, if needed. We have not exceeded 2 days before imaging.
3. Make 5 droplets of mounting solution on a glass slide. Cut the tip of a 200 µL pipette tip to make it wider and transfer the tissue to a new glass slide.
4. Grab the tissue samples by their base with forceps and gently immerse and rinse in a series of mounting solution droplets. Be careful not to break the tissues in this step.
5. Mount with mounting solution, place coverslip, and seal with nail polish. Image in situ tissue samples using a confocal microscope.

Representative Results

Robust detection of chemosensory genes in Anopheles antenna
We investigated the sensitivity of the HCR FISH method (Figure 1) to detect the expression of chemosensory receptors in mosquito olfactory tissues. Guided by the RNA transcript data reported earlier on the female Anopheles mosquito antenna, we generated probes to target a variety of IRs. The average transcript values from four independent antennal transcriptome studies revealed that Ir41t.1 (11 RPKM), Ir75d (12 RPKM), and Ir7t (13 RPKM) were less abundant in the antenna compared to co-receptors Ir25a (197 RPKM) and Ir76b (193 RPKM)^{5,11,12,13,14}. We generated probes to target Ir41t.1, Ir75d, and Ir7t. IR64a (31 RPKM) was also targeted given its transcript level is approximately 3x more abundant than the lowest transcript value among the candidate genes of interest. It must be noted here that RPKM values from transcriptomics studies represent a bulk measurement of transcripts from the entire antenna. As such, it could be that only a few neurons highly express an IR gene transcript, or it could be that an IR gene is lowly expressed across many cells. RPKM values may therefore not necessarily correspond to the abundance level of an IR within a neuron. In addition, the signal amplification afforded by the HCR method might also make it difficult to use this method to accurately gauge neuronal transcript levels based on fluorescent signals. Our recent publication discussed these concepts in more detail⁵. Data suggests that HCR WM-FISH is highly sensitive to detecting mRNA transcripts from antennal tissues as shown in Figure 2.

Multiplexed co-labeling of different RNA targets in chemosensory appendages
To examine the co-localization of RNA targets, we generated probes conjugated to different fluorophores. Double in situ hybridization targeting transcripts of the Odorant receptor co-receptor gene (Orco) and the most broadly expressed ionotropic receptor co-receptor gene (Ir25a) revealed colocalization of these distinct chemosensory receptor families in a subset of cell populations in the antenna (Figure 3A) and maxillary palp (Figure 3B). We also investigated the colocalization of transcripts of three IR-coreceptor genes (Ir8a, Ir25a, and Ir76b). Colocalization patterns suggest that Ir76b-positive cells express Ir25a, whereas Ir8a-positive cells partially co-localize with Ir76b and Ir25a (Figure 4). The co-expression analysis of the IR-coreceptors demonstrates the robustness of using HCR WM-FISH for multiplexed studies.

Figure 1: Schematic of the in situ hybridization chain reaction. This method works in two steps: detection and amplification. A probe set for in situ hybridization chain reaction comprises a split initiator and a nucleic acid sequence specific to the RNA target. Multiple pairs of probe sets can be designed to hybridize several regions in the RNA target; this defines the detection step. The amplification step requires fluorophore-tagged hairpins (h1 and h2) to bind specifically to the initiator conjugated to a probe set. Upon binding, the RNA signal labeled by the fluorophore is amplified by repeated binding of fluorophore-tagged hairpins. Please click here to view a larger version of this figure.

Figure 2: RNA localization of IR chemosensory genes. HCR WM-FISH of female Anopheles coluzzii antennae to target IRs. RNA probes include (A) IR41t.1, (B) IR75d, (C) IR7t, and (D) IR64a. The insets in white dashed boxes are magnified images from the tissue. Please click here to view a larger version of this figure.

Figure 3: Double in situ co-localization of chemosensory genes in the antenna and maxillary palp. Confocal Z-stack images of cells expressing Ir25a (green) and Orco (magenta) in the (A) antenna and (B) maxillary palp of female Anopheles coluzzii mosquitoes. The insets in white dashed boxes are magnified images from the tissue. The scale is 10 µm. Figure 3B has been modified from¹⁵. Please click here to view a larger version of this figure.

Figure 4: Mapping the spatial colocalization of multiple RNA targets. In situ images showing colocalization patterns of three IR coreceptors, IR25a (magenta), IR8a (green), and IR76b (blue) in the mosquito antenna. White arrows point to cells that express the three IR coreceptors (IR25a, IR8a, and IR76b). The scale is 20 µm. This figure has been modified from⁵. Please click here to view a larger version of this figure.

Table 1: Reagents for in situ hybridization chain reaction. Reagents needed to perform hybridization chain reaction whole-mount fluorescent in situ hybridization. Please click here to download this Table.

Discussion

The third generation of hybridization chain reaction (HCR) is remarkable for its sensitivity and robustness to visualize several RNA targets⁸. HCR WM-FISH has been successfully used on the embryos of Drosophila, chicken, mice, and zebrafish as well as the larvae of nematodes and zebrafish^10,16,17. Mosquito antennae and maxillary palps are typically prone to high autofluorescence and weak probe penetration which are particularly challenging when conducting traditional whole-mount in situ methods. These drawbacks have been diminished by the signal amplification step integrated into the HCR protocol which improves the signal-to-background ratio and allows for the detection of olfactory chemoreceptor mRNA transcripts (Figure 2). The HCR protocol design ensures that the initiating probes are tethered to the amplification polymers which allows for imaging RNA targets with low signal. In the multiplexed set-up, different orthogonal HCR amplifiers were tethered to spectrally unique fluorophores. This approach was critical to avoid spectral bleed-through from a neighboring imaging channel.

To optimize the HCR method for use with mosquito appendages, we made several observations. The HCR amplifiers are light-sensitive and should always be stored in a box in a -20 ˚ C freezer. In our experience, extensive incubation of tissues in CCD buffer, proteinase K, or short fixation time in paraformaldehyde could result in breakage of antennae or palps during the wash steps. Antennae and maxillary palps should also always be grabbed by the base to avoid breakage. At the early stage of adapting this protocol, we consistently lost tissues during the series of buffer exchanges (steps 5 and 6). To minimize such losses, the number of tissues were doubled at the start of the experiment, wash steps and buffer exchanges were performed under the microscope, and gel loading tips no wider than 0.5 mm were used to remove the buffers from the tissues.

The HCR WM-FISH has been successful in peripheral olfactory appendages of Anopheles gambiae^5,15 and Aedes aegypti^4,18 mosquito vectors, and the protocol could further be optimized and adapted to other insect tissue parts or different animals. The original protocol designed by the manufacturer does not incorporate incubation in CCD buffer. This step was integrated into the protocol to digest and permeabilize the chitinous cuticle. We extended the incubation time in the probe sets for two nights to give more time for probe penetration, a modification of the manufacturer's protocol that recommends a 16 h incubation time that works well for generic samples on a slide. Optimization of this protocol for other insect tissue parts would require varying the incubation time in the CCD buffer; peripheral tissue parts with thick cuticles will likely require extended incubation time. The concentration of proteinase K must also be experimentally adjusted while adapting this protocol for different tissue or developmental stages of animals. When working with tissues thicker than 5 mm, probe penetration becomes a challenge. In such a situation, additional efforts are needed to perform tissue cryosection or further alter the protocol described in this study.

HCR WM-FISH is limited to simultaneously visualizing only a few gene targets at a time compared to spatial transcriptomics which could potentially allow the simultaneous imaging of thousands of genes¹⁹. Commercial synthesis of RNA probes is expensive; the alternative would be to produce the probes and amplifiers in the laboratory, but this can be challenging for most groups and is labor-intensive and time-consuming. This protocol is also limited by difficulties encountered by RNA probe penetration through whole-mount thick samples, which could require alternations to the protocol (e.g., step 2 tissue preparation) or sectioning of the intact tissue. If the HCR WM-FISH method described here fails to detect an RNA transcript in an olfactory appendage, the gene might be expressed in only a few cells and require extensive surveying of the tissue, is possibly not transcribed, or might be transcribed at a level below the detection limit of this method. Quantitative RT-PCR or RNA seq could be performed to validate such findings.

Materials

Name	Company	Catalog Number	Comments
Amplification buffer	Molecular Instruments	Molecular Instruments, Inc. | In Situ Hybridization + Immunofluorescence	50 mL
Calcium Chloride (CaCl2) 1M	Sigma-Aldrich	21115-100ML
Chitinase	Sigma-Aldrich	C6137-50UN
Chymotrypsin	Sigma-Aldrich	CHY5S-10VL
Dimethyl sulfoxide (DMSO)	Sigma-Aldrich	472301
Eppendorf tube	VWR	20901-551	1.5 mL
Forceps	Dumont	11251	Number 5
Gel loading tip	Costar	4853	1-200 µL tip
Hairpins	Molecular Instruments	Molecular Instruments, Inc. | In Situ Hybridization + Immunofluorescence	h1 and h2 initiator splits
HEPES (1M)	Sigma-Aldrich	H0887
IR25a probe	Molecular Instruments	Probe Set ID: PRK149	AGAP010272
IR41t.1 probe	Molecular Instruments	Probe Set ID: PRK978	AGAP004432
IR64a probe	Molecular Instruments	Probe Set ID: PRK700	AGAP004923
IR75d probe	Molecular Instruments	Probe Set ID: PRK976	AGAP004969
IR76b probe	Molecular Instruments	Probe Set ID: PRI998	AGAP011968
IR7t probe	Molecular Instruments	Probe Set ID: PRL355	AGAP002763
IR8a probe	Molecular Instruments	Probe Set ID: PRK150	AGAP010411
LoBind Tubes	VWR	80077-236	0.5 mL DNA/RNA LoBind Tubes
Magnessium Chloride (MgCl2) 1M	Thermo Fisher	AM9530G
Methanol	Fisher	A412-500
Nuclease-free water	Thermo Fisher	43-879-36
Nutator	Denville Scientific	Model 135	3-D Mini rocker
Orco probe	Molecular Instruments	Probe set ID PRD954	AGAP002560
Paraformaldehyde (20% )	Electron Microscopy Services	15713-S
Phosphate Buffered Saline (10X PBS)	Thermo Fisher	AM9625
Probe hybridization buffer	Molecular Instruments	https://www.molecularinstruments.com/	50 mL
Probe wash buffer	Molecular Instruments	Molecular Instruments, Inc. | In Situ Hybridization + Immunofluorescence	100 mL
Proteinase-K	Thermo Fisher	AM2548
Saline-Sodium Citrate (SSC) 20x	Thermo Fisher	15-557-044
SlowFade Diamond	Thermo Fisher	S36972	mounting solution
Sodium Chloride (NaCl) 5M	Invitrogen	AM9760G
Triton X-100  (10%)	Sigma-Aldrich	93443
Tween-20 (10% )	Teknova	T0027
Watch glass	Carolina	742300	1 5/8" square; transparent