Chemoreceptor co-expression in Drosophila melanogaster olfactory neurons
Curation statements for this article:-
Curated by eLife
Evaluation Summary:
A combination of methods. including a new method for tagging genes, demonstrates that the chemosensory co-receptors of Drosophila melanogaster (Orco, IR8a, IR25a, IR76b) are expressed widely and highly overlapping. These findings challenge a long-standing dogma in the field and suggest that different types of receptors, i.e. olfactory and ionotropic receptors, can be co-expressed in the same chemosensory neuron. Moreover, optogenetics and single sensillum recordings provide evidence that IR25a co-receptor might modulate the activity of typical Orco-dependent olfactory sensory neurons.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (eLife)
Abstract
Drosophila melanogaster olfactory neurons have long been thought to express only one chemosensory receptor gene family. There are two main olfactory receptor gene families in Drosophila , the odorant receptors (ORs) and the ionotropic receptors (IRs). The dozens of odorant-binding receptors in each family require at least one co-receptor gene in order to function: Orco for ORs, and Ir25a , Ir8a , and Ir76b for IRs. Using a new genetic knock-in strategy, we targeted the four co-receptors representing the main chemosensory families in D. melanogaster ( Orco, Ir8a, Ir76b, Ir25a ). Co-receptor knock-in expression patterns were verified as accurate representations of endogenous expression. We find extensive overlap in expression among the different co-receptors. As defined by innervation into antennal lobe glomeruli, Ir25a is broadly expressed in 88% of all olfactory sensory neuron classes and is co-expressed in 82% of Orco+ neuron classes, including all neuron classes in the maxillary palp. Orco , Ir8a , and Ir76b expression patterns are also more expansive than previously assumed. Single sensillum recordings from Orco-expressing Ir25a mutant antennal and palpal neurons identify changes in olfactory responses. We also find co-expression of Orco and Ir25a in Drosophila sechellia and Anopheles coluzzii olfactory neurons. These results suggest that co-expression of chemosensory receptors is common in insect olfactory neurons. Together, our data present the first comprehensive map of chemosensory co-receptor expression and reveal their unexpected widespread co-expression in the fly olfactory system.
Article activity feed
-
-
Author Response:
Reviewer #1 (Public Review):
In this manuscript, the authors challenge the long-standing conclusion that Orco and IR-dependent olfactory receptor neurons are segregated into subtypes such that Orco and IR expression do not overlap. First, the authors generate new knock-in lines to tag the endogenous loci with an expression reporter system, QF/QUAS. They then compare the observed expression of these knock-ins with the widely used system of enhancer transgenes of the same receptors, namely Orco, IR8a, IR25a, and IR76b. Surprisingly, they observe an expansion of the expression of the individual knock-in reporters as compared to the transgenic reporters in more chemosensory neurons targeting more glomeruli per receptor type than previously reported. They verify the expression of the knock-in reporters with antibody …
Author Response:
Reviewer #1 (Public Review):
In this manuscript, the authors challenge the long-standing conclusion that Orco and IR-dependent olfactory receptor neurons are segregated into subtypes such that Orco and IR expression do not overlap. First, the authors generate new knock-in lines to tag the endogenous loci with an expression reporter system, QF/QUAS. They then compare the observed expression of these knock-ins with the widely used system of enhancer transgenes of the same receptors, namely Orco, IR8a, IR25a, and IR76b. Surprisingly, they observe an expansion of the expression of the individual knock-in reporters as compared to the transgenic reporters in more chemosensory neurons targeting more glomeruli per receptor type than previously reported. They verify the expression of the knock-in reporters with antibody staining, in situ hybridization and by mining RNA sequencing data.
Finally, they address the question of physiological relevance of such co-expression of receptor systems by combining optogenetic activation with single sensillum recordings and mutant analysis. Their data suggests that IR25a activation can modulate Orco-dependent signaling and activation of olfactory sensory neurons.
The paper is well written and easy to follow. The data are well presented and very convincing due in part to the combination of complementary methods used to test the same point. Thus, the finding that co-receptors are more broadly and overlappingly expressed than previously thought is very convincing and invites speculation of how this might be relevant for the animal and chemosensory processing in general. In addition, the new method to make knock-ins and the generated knock-ins themselves will be of interest to the fly community.
We thank the reviewer for their enthusiasm and support of our work!
The last part of the manuscript, although perhaps the most interesting, is the least developed compared to the other parts. In particular, the following points could be addressed:
- It would be good to see a few more traces and not just the quantifications. For instance, the trace of ethyl acetate in Fig. 6C, and penthyl acetate for 6G.
Thank you for the suggestion. We have added a new figure supplement (Figure 6-Figure Supplement 3) with additional example traces for all odorants from Figure 6 for which we found a statistically significant difference between the two genotypes (Ir25a versus wildtype).
- In Fig. 4D, the authors show the non-retinal fed control, which is great. An additional genetic control fed with retinal would have been nice.
For these experiments, we followed a standard practice in Drosophila optogenetics to test the same experimental genotype in the presence or absence of the essential cofactor all-trans-retinal. This controls for potential effects from the genetic background. It is possible our description of these experiments was unclear (as also suggested by comments from Reviewer 2). As such, we have clarified our experimental design for the optogenetic experiments in the revised manuscript:
Modified text: “No light-induced responses were found in control flies, which had the same genotype as experimental flies but were not fed all-trans retinal (-ATR), a necessary co-factor for channelrhodopsin function (see Methods).” and “Bottom trace is control animal, which has the same genotype as the experimental animal but was not fed the required all-trans retinal cofactor (-ATR).”
Figure 4-Figure Supplement 1 legend: “In all optogenetic experiments, control animals have the same genotypes as the corresponding experimental animals but have not been fed all-trans retinal.”
Methods: “For all optogenetic experiments, the control flies were of the same genotype as experimental flies but had not been fed all-trans retinal.”
- It appears that mostly IR25a is strongly co-expressed with other co-receptors. The provided experiments suggest a possible modulation between IR25a and Orco-dependent neuronal activity. However, what does this mean? How could this be relevant? And moreover, is this a feature of Drosophila melanogaster after many generations in laboratories?
We share this reviewer’s excitement regarding the numerous questions our work now raises. While testing additional functional ramifications of chemosensory co-receptor expression is beyond the scope of this work (but will undoubtedly be the focus of future studies), we did expand on what this might mean in the revised Discussion section of the revised manuscript. Previously, we had raised the hypothesis that chemoreceptor co-expression could be an evolutionary relic of Ir25a expression in all chemoreceptor neurons , or a biological mechanism to broaden the response profile of an olfactory neuron without sacrificing its ability to respond to specific odors. We now extend our discussion to raise additional possible ramifications. For example, we suggest that modulating Ir25a coexpression could alter the electrical properties of a neuron, making it more (or possibly less) sensitive to Orco-dependent responses. We also suggest that Ir25a coexpression might be an evolutionary mechanism to allow olfactory neurons to adjust their response activities. That is, that most Orco-positive olfactory neurons are already primed to be able to express a functional Ir receptor if one were to be expressed. Such co-expression in some olfactory neurons might present an evolutionary advantage by ensuring olfactory responses to a complex but crucial biologically relevant odor, like human odors to some mosquitoes.
Reviewer #2 (Public Review):
In the present study, the authors:
- generated knock-in lines for Orco, Ir8a, Ir25a, and IR7ba, and examined their expression, with a main focus on the adult olfactory organs.
- confirmed the expression of these receptors using antibody staining.
- examined the innervation patterns of these knock-in lines in the nervous system.
- identified a glomerulus, VM6, that is divided into three subdivisions.
- examined olfactory responses of neurons co-expressing Orco and Ir25a
The results of the first four sets of experiments are well presented and support the conclusions, but the results of the last set of experiments (the electrophysiology part) need some details. Please find my detailed comments below.
We thank the reviewer for their support of our work and appreciating the importance of our findings. In the revised manuscript, we now provide the additional experimental details for the electrophysiology work as requested.
Major points
Line 167-171: I wonder if the authors also compared the Orco-T2A-QF2 knock-in with antibody staining of the antenna.
We did perform whole-mount anti-Orco antibody staining on Orco-T2A-QF2 > GFP antennae (example image below). We saw broad overlap between Orco+ and GFP+ cells, similar to the palps. However, we did not include these results since quantification of these tissues is challenging for the following reasons:
- There are ~1,200 olfactory neurons in each antenna, many of which are Orco+.
- The thickness of the tissue makes determinations of co-localization difficult in wholemount staining.
- Co-localization is further complicated by the sub-cellular localization of the signals: Orco antibodies preferentially label dendrites and weakly label cell bodies, while our GFP reporter is cytoplasmic and preferentially labels cell bodies. For these reasons, we focused on the numerically simpler palps for quantification. For the Ir8aT2A-QF2 and Ir76b-T2A-QF2 lines, palp quantification was not an option as neither knock-in drove expression in the palps (and the available antibodies did not work with the whole-mount staining protocol). This is why we performed antennal cryosections to validate these lines. Below is an example image of the antennal whole-mount staining in the Orco-T2A-QF2 knock-in line, illustrating the quantification challenges enumerated above.
*Co-staining of anti-Orco and GFP in Orco-T2A-QF2 > 10xQUAS-6xGFP antenna *
Lines 316-319 (Figure 4D): It would be better if the authors compare the responses of Ir25a>CsChrimson to those of Orco>CsChrimson.
The goal of the optogenetic experiments was to provide experimental support for Ir25a expression in Orco+ neurons in an approach independent to previous methods. Our main question was whether we could activate what was previously considered Orco-only olfactory neurons using the Ir25a knock-in. These experiments were not designed to determine if this optogenetic activation recapitulated the normal activity of these neurons. For these reasons, we did not attempt the optogenetic experiments with Orco>CsChrimson flies.
Line 324-326: Why the authors tested control flies not fed all-trans retinal? They should test Ir25a-T2A-QF2>QUAS-CsChrimson not fed all-trans retinal as a control.
We apologize for the confusion. The “control” flies we used were indeed Ir25a-T2AQF2>QUAS-CsChrimson flies not fed all-trans retinal as suggested by the reviewer. This detail was in the methods, yet likely was not clear. We have amended the main text in multiple locations to state the full genotype of the control fly more clearly:
Modified text: “No light-induced responses were found in control flies, which had the same genotype as experimental flies but were not fed all-trans retinal (-ATR), a necessary co-factor for channelrhodopsin function (see Methods).” and “Bottom trace is control animal, which has the same genotype as the experimental animal but was not fed the required all-trans retinal cofactor (-ATR).”
Figure 4-Figure Supplement 1 legend: “In all optogenetic experiments, control animals have the same genotypes as the corresponding experimental animals but have not been fed all-trans retinal.”
Methods: “For all optogenetic experiments, the control flies were of the same genotype as experimental flies but had not been fed all-trans retinal.”
Line 478-500: I wonder if the observed differences between the wildtype and Ir25a2 mutant lines are due to differences in the genetic background between both lines. Did the authors backcross Ir25a2 mutant line with the used wildtype for at least five generations?
Yes, the mutants are outcrossed into the same genetic background as the wildtypes for at least five generations. Please see Methods, revised manuscript: “Ir25a2 and Orco2 mutant fly lines were outcrossed into the w1118 wildtype genetic background for at least 5 generations.”
Line 1602-1603: Does the identification of ab3 sensilla using fluorescent-guided SSR apply for ab3 sensilla in Orco mutant flies. How does this ab3 fluorescent-guided SSR work?
In fluorescence guided SSR (fgSSR; Lin and Potter, PloS One, 2015), the ab3 sensilla is GFPlabelled (genotype: Or22a-Gal4>UAS-mCD8:GFP), which allows this sensilla to be specifically identified under a microscope and targeted for SSR recordings. We generated fly stocks for fgSSR identification of ab3 in all three genetic backgrounds (wildtype, Orco mutant, Ir25a mutant).
These three genotypes are described in the methods:
“Full genotypes for ab3 fgSSR were:
Pin/CyO; Or22a-Gal4,15XUAS-IVS-mcd8GFP/TM6B (wildtype),
Ir25a2; Or22a-Gal4,15XUAS-IVS-mcd8GFP/TM6B (Ir25a2 mutant),
Or22a-Gal4/10XUAS-IVS-mcd8GFP (attp40); Orco2 (Orco2 mutant).”
Line 1602-1604: There is no mention of how the authors identified ab9 sensilla.
Information on the identification of ab9 sensilla is under the optogenetics section of the methods: “Identification of ab9 sensilla was assisted by fluorescence-guided Single Sensillum Recording (fgSSR) (Lin and Potter, 2015) using Or67b-Gal4 (BDSC #9995) recombined with 15XUAS-IVS-mCD8::GFP (BDSC #32193).”
Line 1648: what are the set of odorants that were used to identify the different coeloconic sensilla?
We have added the specific odorants used for sensillar identification for coeloconic SSR in the Methods. The protocol and odorants used were:
*2,3-butanedione (BUT), 1,4-diaminobutane (DIA), Ammonia (AM), hexanol (HEX), phenethylamine (PHEN), and propanal (PROP) to distinguish coeloconic sensilla:
o Wildtype flies: Strong DIA and BUT responses identify ac2 and rule out ac4. Absence of strong AM response rules out ac1, absence of HEX response rules out ac3, absence of PHEN response further rules out ac4.
o Ir25a mutant flies (amine responses lost, so cannot use PHEN and DIA as diagnostics): Strong BUT response and moderate PROP response identify ac2 and rule out ac4. Absence of strong AM response rules out ac1, absence of HEX response rules out ac3. Ac4 is further ruled out anatomically based on sensillar location compared to ac2.
Revised text: “Different classes of coeloconic sensilla were identified by their known location on the antenna and confirmed with their responses to a small panel of diagnostic odorants: in wildtype flies, ac2 sensilla were identified by their strong responses to 1,4-diaminobutane and 2,3-butanedione. The absence of a strong response to ammonia was used to rule out ac1 sensilla, the absence of a hexanol response was used to rule out ac3 sensilla, and the absence of a phenethylamine response was used to rule out ac4 sensilla. In Ir25a mutant flies in which amine responses were largely abolished, ac2 and ac4 sensilla were distinguished based on anatomical location, as well as the strong response of ac2 to 2,3-butanedione and the moderate response to propanal (both absent in ac4). Ac1 and ac3 sensilla were excluded similarly in the mutant and wildtype flies. No more than 4 sensilla per fly were recorded. Each sensillum was tested with multiple odorants, with a rest time of at least 10s between applications.
-
Evaluation Summary:
A combination of methods. including a new method for tagging genes, demonstrates that the chemosensory co-receptors of Drosophila melanogaster (Orco, IR8a, IR25a, IR76b) are expressed widely and highly overlapping. These findings challenge a long-standing dogma in the field and suggest that different types of receptors, i.e. olfactory and ionotropic receptors, can be co-expressed in the same chemosensory neuron. Moreover, optogenetics and single sensillum recordings provide evidence that IR25a co-receptor might modulate the activity of typical Orco-dependent olfactory sensory neurons.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
-
Reviewer #1 (Public Review):
In this manuscript, the authors challenge the long-standing conclusion that Orco and IR-dependent olfactory receptor neurons are segregated into subtypes such that Orco and IR expression do not overlap. First, the authors generate new knock-in lines to tag the endogenous loci with an expression reporter system, QF/QUAS. They then compare the observed expression of these knock-ins with the widely used system of enhancer transgenes of the same receptors, namely Orco, IR8a, IR25a, and IR76b. Surprisingly, they observe an expansion of the expression of the individual knock-in reporters as compared to the transgenic reporters in more chemosensory neurons targeting more glomeruli per receptor type than previously reported. They verify the expression of the knock-in reporters with antibody staining, in situ …
Reviewer #1 (Public Review):
In this manuscript, the authors challenge the long-standing conclusion that Orco and IR-dependent olfactory receptor neurons are segregated into subtypes such that Orco and IR expression do not overlap. First, the authors generate new knock-in lines to tag the endogenous loci with an expression reporter system, QF/QUAS. They then compare the observed expression of these knock-ins with the widely used system of enhancer transgenes of the same receptors, namely Orco, IR8a, IR25a, and IR76b. Surprisingly, they observe an expansion of the expression of the individual knock-in reporters as compared to the transgenic reporters in more chemosensory neurons targeting more glomeruli per receptor type than previously reported. They verify the expression of the knock-in reporters with antibody staining, in situ hybridization and by mining RNA sequencing data.
Finally, they address the question of physiological relevance of such co-expression of receptor systems by combining optogenetic activation with single sensillum recordings and mutant analysis. Their data suggests that IR25a activation can modulate Orco-dependent signaling and activation of olfactory sensory neurons.
The paper is well written and easy to follow. The data are well presented and very convincing due in part to the combination of complementary methods used to test the same point. Thus, the finding that co-receptors are more broadly and overlappingly expressed than previously thought is very convincing and invites speculation of how this might be relevant for the animal and chemosensory processing in general. In addition, the new method to make knock-ins and the generated knock-ins themselves will be of interest to the fly community.
The last part of the manuscript, although perhaps the most interesting, is the least developed compared to the other parts. In particular, the following points could be addressed:
- It would be good to see a few more traces and not just the quantifications. For instance, the trace of ethyl acetate in Fig. 6C, and penthyl acetate for 6G.
- In Fig. 4D, the authors show the non-retinal fed control, which is great. An additional genetic control fed with retinal would have been nice.
- It appears that mostly IR25a is strongly co-expressed with other co-receptors. The provided experiments suggest a possible modulation between IR25a and Orco-dependent neuronal activity. However, what does this mean? How could this be relevant? And moreover, is this a feature of Drosophila melanogaster after many generations in laboratories?
-
Reviewer #2 (Public Review):
In the present study, the authors:
- generated knock-in lines for Orco, Ir8a, Ir25a, and IR7ba, and examined their expression, with a main focus on the adult olfactory organs.
- confirmed the expression of these receptors using antibody staining.
- examined the innervation patterns of these knock-in lines in the nervous system.
- identified a glomerulus, VM6, that is divided into three subdivisions.
- examined olfactory responses of neurons co-expressing Orco and Ir25a
The results of the first four sets of experiments are well presented and support the conclusions, but the results of the last set of experiments (the electrophysiology part) need some details. Please find my detailed comments below.
Major Points:
Line 167-171: I wonder if the authors also compared the Orco-T2A-QF2 knock-in with antibody staining of the …
Reviewer #2 (Public Review):
In the present study, the authors:
- generated knock-in lines for Orco, Ir8a, Ir25a, and IR7ba, and examined their expression, with a main focus on the adult olfactory organs.
- confirmed the expression of these receptors using antibody staining.
- examined the innervation patterns of these knock-in lines in the nervous system.
- identified a glomerulus, VM6, that is divided into three subdivisions.
- examined olfactory responses of neurons co-expressing Orco and Ir25a
The results of the first four sets of experiments are well presented and support the conclusions, but the results of the last set of experiments (the electrophysiology part) need some details. Please find my detailed comments below.
Major Points:
Line 167-171: I wonder if the authors also compared the Orco-T2A-QF2 knock-in with antibody staining of the antenna.
Lines 316-319 (Figure 4D): It would be better if the authors compare the responses of Ir25a>CsChrimson to those of Orco>CsChrimson.
Line 324-326: Why the authors tested control flies not fed all-trans retinal? They should test Ir25a-T2A-QF2>QUAS-CsChrimson not fed all-trans retinal as a control.
Line 478-500: I wonder if the observed differences between the wildtype and Ir25a2 mutant lines are due to differences in the genetic background between both lines. Did the authors backcross Ir25a2 mutant line with the used wildtype for at least five generations?
Line 1602-1603: Does the identification of ab3 sensilla using fluorescent-guided SSR apply for ab3 sensilla in Orco mutant flies. How does this ab3 fluorescent-guided SSR work?
Line 1602-1604: There is no mention of how the authors identified ab9 sensilla.
Line 1648: what are the set of odorants that were used to identify the different coeloconic sensilla?
-
-