Name: Author Spotlight: Visualizing Olfactory Receptor Expression in Mosquitoes
Uploaded: 2023-11-17T13:40:00
Description: Watch this Scientific Journal Video about Visualizing Olfactory Receptor Expression in Mosquitoes at JoVE.com

Vi takker Margo Herre og Leslie Vosshall-laboratoriet for at dele deres in-situ hybridiseringsprotokol for Aedes aegypti olfaktoriske vedhæng. Dette arbejde blev støttet af tilskud fra National Institutes of Health til C.J.P. (NIAID R01Al137078), et HHMI Hanna Gray-stipendium til JIR, en Johns Hopkins Postdoctoral Accelerator Award til JIR og et Johns Hopkins Malaria Research Institute Postdoc-stipendium til JIR Vi takker Johns Hopkins Malaria Research Institute og Bloomberg Philanthropies for deres støtte.

1. Overvejelser og forberedelse af materialer
<ol><li>Beslut, om helmonteret eller kryo-sektion af væv vil være passende. Denne protokol er optimeret til helmonteret in situ-billeddannelse af RNA i Anopheles-myggeantennen og maksillær palp uden kryosektionering. Hvis prøverne er tykkere end 5 mm, anbefales kryosnit for at muliggøre sondeindtrængning.</li>
	<li>Identificer generne af interesse og kopier sekvenserne, herunder introner og exoner fra en passende database. Transskrib...

Chemosensory Gene Expression Mapping in Mosquito Olfactory Appendages

<ol>
	<li>Konopka, J. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfaction+in+Anopheles+mosquitoes.">Olfaction in Anopheles mosquitoes.</a> Chem Senses. 46, (2021).</li><li>Raji, J. I., Potter, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemosensory+ionotropic+receptors+in+human+host-seeking+mosquitoes.">Chemosensory ionotropic receptors in human host-seeking mosquitoes.</a> Curr Opin Insect Sci. 54, 100967 (2022).</li><li>Young, A. P., Jackson, D. J., Wyeth, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+technical+review+and+guide+to+RNA+fluorescence+in+situ+hybridization.">A technical review and guide to RNA fluorescence in situ hybridization.</a> PeerJ. 8, e8806 (2020).</li><li>Herre, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-canonical+odor+coding+in+the+mosquito.">Non-canonical odor coding in the mosquito.</a> Cell. 185 (17), 3104-3123.e28 (2022).</li><li>Raji, J. I., Konopka, J. K., Potter, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+spatial+map+of+antennal-expressed+ionotropic+receptors+in+the+malaria+mosquito.">A spatial map of antennal-expressed ionotropic receptors in the malaria mosquito.</a> Cell Rep. 42 (2), 112101 (2023).</li><li>Choi, H. M. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Programmable+in+situ+amplification+for+multiplexed+imaging+of+mRNA+expression.">Programmable in situ amplification for multiplexed imaging of mRNA expression.</a> Nat Biotechnol. 28 (11), 1208-1212 (2010).</li><li>Choi, H. M. T., Beck, V. A., Pierce, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Next-generation+in+situ+hybridization+chain+reaction:+higher+gain,+lower+cost,+greater+durability.">Next-generation in situ hybridization chain reaction: higher gain, lower cost, greater durability.</a> ACS Nano. 8 (5), 4284-4294 (2014).</li><li>Choi, H. M. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Third-generation+in+situ+hybridization+chain+reaction:+multiplexed,+quantitative,+sensitive,+versatile,+robust.">Third-generation in situ hybridization chain reaction: multiplexed, quantitative, sensitive, versatile, robust.</a> Development. 145 (12), dev165753 (2018).</li><li>Schwarzkopf, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hybridization+chain+reaction+enables+a+unified+approach+to+multiplexed,+quantitative,+high-resolution+immunohistochemistry+and+in+situ+hybridization.">Hybridization chain reaction enables a unified approach to multiplexed, quantitative, high-resolution immunohistochemistry and in situ hybridization.</a> Development. 148 (22), dev199847 (2021).</li><li>Choi, H. M. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+a+multiplexed+zoo+of+mRNA+expression.">Mapping a multiplexed zoo of mRNA expression.</a> Development. 143 (19), 3632-3637 (2016).</li><li>Pitts, R. J., Derryberry, S. L., Zhang, Z., Zwiebel, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variant+ionotropic+receptors+in+the+malaria+vector+mosquito+Anopheles+gambiae+tuned+to+amines+and+carboxylic+acids.">Variant ionotropic receptors in the malaria vector mosquito Anopheles gambiae tuned to amines and carboxylic acids.</a> Sci Rep. 7, 40297 (2017).</li><li>Rinker, D. C., Zhou, X., Pitts, R. J., Rokas, A., Zwiebel, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antennal+transcriptome+profiles+of+anopheline+mosquitoes+reveal+human+host+olfactory+specialization+in+Anopheles+gambiae.">Antennal transcriptome profiles of anopheline mosquitoes reveal human host olfactory specialization in Anopheles gambiae.</a> BMC Genomics. 14, 749 (2013).</li><li>Maguire, S. E., Afify, A., Goff, L. A., Potter, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odorant-receptor-mediated+regulation+of+chemosensory+gene+expression+in+the+malaria+mosquito+Anopheles+gambiae.">Odorant-receptor-mediated regulation of chemosensory gene expression in the malaria mosquito Anopheles gambiae.</a> Cell Rep. 38 (10), 110494 (2022).</li><li>Athrey, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemosensory+gene+expression+in+olfactory+organs+of+the+anthropophilic+Anopheles+coluzzii+and+zoophilic+Anopheles+quadriannulatus.">Chemosensory gene expression in olfactory organs of the anthropophilic Anopheles coluzzii and zoophilic Anopheles quadriannulatus.</a> BMC Genomics. 18 (1), 751 (2017).</li><li>Task, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemoreceptor+co-expression+in+Drosophila+melanogaster+olfactory+neurons.">Chemoreceptor co-expression in Drosophila melanogaster olfactory neurons.</a> eLife. 11, e72599 (2022).</li><li>Shah, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+RNA+detection+at+depth+by+hybridization+chain+reaction+and+tissue+hydrogel+embedding+and+clearing.">Single-molecule RNA detection at depth by hybridization chain reaction and tissue hydrogel embedding and clearing.</a> Development. 143 (15), 2862-2867 (2016).</li><li>Trivedi, V., Choi, H. M. T., Fraser, S. E., Pierce, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multidimensional+quantitative+analysis+of+mRNA+expression+within+intact+vertebrate+embryos.">Multidimensional quantitative analysis of mRNA expression within intact vertebrate embryos.</a> Development. 145 (1), dev156869 (2018).</li><li>Herre, M., Greppi, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+in+situ+hybridization+and+immunohistochemistry+to+visualize+gene+expression+in+peripheral+chemosensory+tissues+of+mosquitoes.">RNA in situ hybridization and immunohistochemistry to visualize gene expression in peripheral chemosensory tissues of mosquitoes.</a> Cold Spring Harb Protoc. 2023 (1), 48-54 (2023).</li><li>Marx, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+of+the+Year:+spatially+resolved+transcriptomics.">Method of the Year: spatially resolved transcriptomics.</a> Nat Methods. 18 (1), 9-14 (2021).</li></ol>

Den tredje generation af hybridiseringskædereaktion (HCR) er bemærkelsesværdig for sin følsomhed og robusthed til at visualisere flere RNA-mål8. HCR WM-FISH er med succes blevet brugt på embryoner af Drosophila, kylling, mus og zebrafisk samt larver af nematoder og zebrafisk 10,16,17. Myggeantenner og maksillære palper er typisk tilbøjelige til høj autofluorescens og svag sondepenetratio...

Mygvektorer som Anopheles gambiae er afhængige af et rigt repertoire af kemosensoriske gener udtrykt i deres perifere olfaktoriske vedhæng for at trives i en kompleks kemisk verden og identificere adfærdsmæssigt relevante lugte, der stammer fra menneskelige værter, detektere nektarkilder og lokalisere ovipositionssteder1. Mygantennen og den maksillære palp er beriget med kemosensoriske gener, der driver lugtdetektion i disse olfaktoriske vedhæng. Tre hovedklasser af ligand-gated ionkanaler driver lugtdetektion i mygs olfaktoriske vedhæng: lugtreceptorerne (OR'er), som fungerer med en obligatorisk lugtreceptor-co-receptor (Orco); de ionotropiske receptorer (IR'er), som interagerer med en eller flere IR-coreceptorer (IR8a, IR25a og IR76b); de kemosensoriske gustatoriske receptorer (GR'er), der fungerer som et kompleks af tre proteiner til påvisning af kuldioxid (CO2)1,2.
RNA-fluorescens in situ-hybridisering er et kraftfuldt værktøj til påvisning af ekspression af endogent mRNA3. Generelt anvender denne metode en fluoroformærket enkeltstrenget nukleinsyresonde med sekvens, der er komplementær til et mål-mRNA. Binding af den fluorescerende RNA-sonde til mål-RNA'et muliggør identifikation af celler, der udtrykker et transkript af interesse. Nylige fremskridt muliggør nu påvisning af transkripter i helmonteret mygvæv 4,5. Den første generation af hybridiseringskædereaktionsteknologi (HCR) anvendte en RNA-baseret HCR-forstærker; dette blev forbedret i en anden generations metode, der i stedet brugte konstrueret DNA til HCR-forstærkeren 6,7. Denne opgradering resulterede i en 10x stigning i signal, et dramatisk fald i produktionsomkostningerne og en betydelig forbedring af holdbarheden af reagenser 6,7.
I protokollen beskriver vi udnyttelsen af en tredje generations HCR helmonteret RNA fluorescens in situ hybridiseringsmetode (HCR RNA WM-FISH) metode designet til at detektere den rumlige lokalisering og ekspression af ethvert gen 8,9. Denne totrinsmetode anvender først nukleinsyreprober, der er specifikke for mRNA'et af interesse, men som også indeholder en initiatorgenkendelsessekvens; det andet trin bruger fluoroformærkede hårnåle, der binder til initiatorsekvensen for at forstærke det fluorescerende signal (figur 1). Denne metode giver også mulighed for multiplexing af to eller flere RNA-sonder og forstærkning af sondesignaler for at lette RNA-detektion og kvantificering8. Visualisering af transkriptionsoverflod og RNA-lokaliseringsmønstre af kemosensoriske gener udtrykt i de olfaktoriske vedhæng giver den første linje af indsigt i kemosensoriske genfunktioner og lugtkodning.

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hybridization chain reaction rna whole-mount fluorescence in situ hybridization of chemosensory genes in mosquito olfactory appendages

Myg er effektive vektorer af dødelige sygdomme og kan navigere i deres kemiske miljø ved hjælp af kemosensoriske receptorer udtrykt i deres olfaktoriske vedhæng. At forstå, hvordan kemosensoriske receptorer er rumligt organiseret i de perifere olfaktoriske vedhæng, kan give indsigt i, hvordan lugt er kodet i myggens olfaktoriske system og informere nye måder at bekæmpe spredningen af mygbårne sygdomme på. Fremkomsten af tredje generations hybridiseringskædereaktion RNA helmonteret fluorescens in situ hybridisering (HCR RNA WM-FISH) muliggør rumlig kortlægning og samtidig ekspressionsprofilering af flere kemosensoriske gener. Her beskriver vi en trinvis tilgang til udførelse af HCR RNA WM-FISH på Anopheles mygantenne og maksillær palp. Vi undersøgte følsomheden af denne teknik ved at undersøge ekspressionsprofilen for ionotropiske olfaktoriske receptorer. Vi spurgte, om den beskrevne HCR WM-FISH-teknik var egnet til multipleksede undersøgelser ved at binde RNA-sonder til tre spektralt forskellige fluoroforer. Resultaterne viste, at HCR RNA WM-FISH er robust følsom over for samtidig at detektere flere kemosensoriske gener i antennen og maksillære palp olfaktoriske vedhæng. Yderligere undersøgelser vidner om HCR WM-FISH's egnethed til co-ekspression profilering af dobbelt og tredobbelt RNA-mål. Denne teknik, når den anvendes med modifikationer, kunne tilpasses til at lokalisere gener af interesse i olfaktoriske væv af andre insektarter eller i andre vedhæng.

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&#39; 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 tissues4,5. 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 amplifier6,7. This upgrade resulted in a 10x increase in signal, a dramatic decrease in production cost, and significant improvement in the durability of reagents6,7.
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 gene8,9. 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.

Author Spotlight: Visualizing Olfactory Receptor Expression in Mosquitoes 

Hybridiseringskædereaktion RNA helmonteret fluorescens in situ hybridisering af kemosensoriske gener i myggeolfaktoriske vedhæng

Our group is interested in the chemo-sensory system of mosquitoes. We hope that by targeting a mosquito sense of taste and smell, we can stop it from finding us and biting us. Hybridization chain reaction fluorescent in situ hybridization is a recent development.This technology is highly sensitive, robust, and capable of multiplexing several gene targets such that we can visualize the expression and localization of these genes simultaneously within their native environment. Special transcriptomics is a cutting edge method used in the field of spatial biology. This technology is capable of multiplexing several thousands of genes, and you can visualize the expression of these genes simultaneously.Our protocol allows us to directly visualize which neurons in the antenna express any of the 50 or so possible ionotropic receptors. This provides us with a foundational roadmap to guide our understanding of how this classical factor receptors might mediate behaviors. Olfactory neurons within olfactory organs of an insect like the antenna, are typically buried inside a protective chitinous shell.This makes visualizing olfactory receptor expression in those neurons very challenging. Our protocol uses a combination of chitinase treatments and a state-of-the-art fluorescence and see-through protocols to overcome these challenges.

Robust påvisning af kemosensoriske gener i Anopheles-antennen Vi undersøgte følsomheden af HCR FISH-metoden (figur 1) til at detektere ekspressionen af kemosensoriske receptorer i myggeolfaktorisk væv. Vejledt af RNA-transkriptionsdata, der tidligere blev rapporteret om den kvindelige Anopheles-mygantenne, genererede vi sonder til at målrette mod en række IR'er. De gennemsnitlige transkriptionsværdier fra fire uafhængige antennetranskriptomunders...

Watch this Scientific Journal Video about Visualizing Olfactory Receptor Expression in Mosquitoes at JoVE.com

Artiklen beskriver de metoder og reagenser, der er nødvendige for at udføre hybridiseringskædereaktion RNA helmonteret fluorescens in situ hybridisering (HCR, RNA, WM-FISH) for at afsløre indsigt i den rumlige og cellulære opløsning af kemosensoriske receptorgener i mygantennen og maksillær palp.

Mosquitoes are effective vectors of deadly diseases and can navigate their chemical environment using chemosensory receptors expressed in their olfactory ...

Vores gruppe er interesseret i myggenes kemo-sensoriske system. Vi håber, at vi ved at målrette mod en myggesans for smag og lugt kan forhindre den i at finde os og bide os. Hybridisering kædereaktion fluorescerende in situ hybridisering er en nylig udvikling.Denne teknologi er meget følsom, robust og i stand til at multiplexere flere genmål, så vi kan visualisere ekspressionen og lokaliseringen af disse gener samtidigt i deres oprindelige miljø. Special transcriptomics er en banebrydende metode, der anvendes inden for rumlig biologi. Denne teknologi er i stand til at multiplexere flere tusinde gener, og du kan visualisere ekspressionen af disse gener samtidigt.Vores protokol giver os mulighed for direkte at visualisere, hvilke neuroner i antennen der udtrykker nogen af de omkring 50 mulige ionotropiske receptorer. Dette giver os en grundlæggende køreplan til at guide vores forståelse af, hvordan denne klassiske faktor receptorer kan formidle adfærd. Olfaktoriske neuroner i olfaktoriske organer af et insekt som antennen, er typisk begravet inde i en beskyttende chitinøs skal.Dette gør visualisering af olfaktorisk receptorekspression i disse neuroner meget udfordrende. Vores protokol bruger en kombination af chitinasebehandlinger og en avanceret fluorescens- og gennemsigtige protokoller til at overvinde disse udfordringer.