Pheromone sensing in Drosophila requires support cell-expressed Osiris 8 - PubMed (original) (raw)

Pheromone sensing in Drosophila requires support cell-expressed Osiris 8

Marta Scalzotto et al. BMC Biol. 2022.

Abstract

Background: The nose of most animals comprises multiple sensory subsystems, which are defined by the expression of different olfactory receptor families. Drosophila melanogaster antennae contain two morphologically and functionally distinct subsystems that express odorant receptors (Ors) or ionotropic receptors (Irs). Although these receptors have been thoroughly characterized in this species, the subsystem-specific expression and roles of other genes are much less well-understood.

Results: Here we generate subsystem-specific transcriptomic datasets to identify hundreds of genes, encoding diverse protein classes, that are selectively enriched in either Or or Ir subsystems. Using single-cell antennal transcriptomic data and RNA in situ hybridization, we find that most neuronal genes-other than sensory receptor genes-are broadly expressed within the subsystems. By contrast, we identify many non-neuronal genes that exhibit highly selective expression, revealing substantial molecular heterogeneity in the non-neuronal cellular components of the olfactory subsystems. We characterize one Or subsystem-specific non-neuronal molecule, Osiris 8 (Osi8), a conserved member of a large, insect-specific family of transmembrane proteins. Osi8 is expressed in the membranes of tormogen support cells of pheromone-sensing trichoid sensilla. Loss of Osi8 does not have obvious impact on trichoid sensillar development or basal neuronal activity, but abolishes high sensitivity responses to pheromone ligands.

Conclusions: This work identifies a new protein required for insect pheromone detection, emphasizes the importance of support cells in neuronal sensory functions, and provides a resource for future characterization of other olfactory subsystem-specific genes.

Keywords: Comparative transcriptomics; Drosophila melanogaster; Olfactory subsystem; Osiris 8; Pheromone detection; Sensory neuron; Support cell.

© 2022. The Author(s).

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1

Fig. 1

A transcriptomic screen for olfactory subsystem-specific genes. A Top: schematic of the D. melanogaster antennal olfactory subsystems. Bottom: schematic of the comparative antennal transcriptomics experiment of ato mutant (ato 1 /Df(3R)p13) and amos mutant (amos 3) animals. B Heatmap showing differential expression of chemosensory receptor gene families (odorant receptor (Or), ionotropic receptor (Ir), and gustatory receptor (Gr)) in ato and amos antennal transcriptomes. The enrichment (or non-enrichment) of genes is as expected in all cases (see “Results”), with a few exceptions: (i) Or33c and Or42a are thought to be expressed specifically in the maxillary palp [17, 28]; however, Or33c was previously detected in the antenna by qRT-PCR [16] and Or42a transcripts have been detected in some _Orco_-negative neurons in the Fly Cell Atlas [29]. Ir68a encodes a hygroreceptor that acts in sacculus neurons [30], although transcripts of this gene appear to be expressed at low levels [31]. C Bar chart comparing the classification of Ir and Or subsystem-enriched genes into the indicated categories (see Additional file 4: Table S1). D Left: _t_-distributed stochastic neighbor embedding (tSNE) representation of RNA-seq datasets from individual antennal cells from the Fly Cell Atlas (10× stringent dataset in this and all subsequent figures) [29], colored for expression of the neuronal marker N-syb, which is expressed in OSNs, Johnston’s organ auditory neurons, and a neuron population of unknown identity (marked “?”; these also express the mechanoreceptor NompC, suggesting that they are auditory/mechanosensory, rather than olfactory). Right: bar chart of the expression of olfactory subsystem-enriched genes in neuronal and non-neuronal antennal cell populations (see Additional file 4: Table S1). E Top: tSNE plot of antennal single-cell transcriptomes colored for expression of Or and Ir co-receptors, which demarcate the two olfactory subsystems (although Ir25a is expressed at low levels across both subsystems [26, 32]). The Gr21a/Gr63a cell cluster is also indicated; although these do not express Orco, they are considered part of the Or subsystem. Bottom left: tSNE plots colored for expression of the Or subsystem-enriched GstE4 and Ir subsystem-enriched Tsp47F. Bottom right: combined RNA FISH and immunofluorescence for GstE4 and Orco (top) or Tsp47F and Ir8a (bottom) on whole-mount antennae of control (w 1118) animals confirming the broad, subsystem-enriched neuronal expression of these genes. Scale bars, 20 μm

Fig. 2

Fig. 2

Diverse breadth and spatial location of non-neuronal olfactory subsystem-enriched genes. A tSNE plot of antennal single-cell transcriptomes highlighting non-neuronal classes in the antenna, as defined by expression of the indicated marker genes (based upon [29]). B–E tSNE plots of antennal single-cell transcriptomes and RNA FISH on whole-mount antennae of wild-type (Canton-S) animals illustrating non-neuronal expression patterns of various (B) Or subsystem-enriched and (C–E) Ir subsystem-enriched genes. Expression of a5 and a10 was previously described, but not related specifically to the Ir subsystem [80, 81]; the Jhedup RNA FISH expression pattern is consistent with observations of a transgenic promoter reporter for this gene [82]. The sacculus-specific Obp59a expression pattern—as previously described [54, 70]—is shown for comparison with novel, sacculus support cell-expressed genes. The bright-field channel is overlaid to reveal cuticle morphology; occasional fluorescence signal within sensillar hairs is likely artefactual. Scale bars, 20 μm. F Demarcation of antennal unannotated support cell clusters (I1-14, O1-7) through their expression of Ir and Or subsystem-enriched genes (shaded magenta and green boxes, respectively). Select genes illustrated in B–D are highlighted; see Additional file 5: Table S2 for the full dataset). Although expression was assessed qualitatively, not quantitatively, support cells could easily be categorized to each subsystem through their “fingerprint” of subsystem-specific gene expression. G tSNE plot of antennal single-cell transcriptomes in which antennal support cell clusters are assigned to the Or and Ir subsystems based upon their expression of subsystem-enriched genes. I11 likely represent sacculus support cells (see “Results” and D). Other antennal cell classes are also indicated (based upon [29]). _sv_-positive clusters (see A) labeled with “?” (e.g., near I10) were not reliably marked by Or or Ir subsystem-specific genes; these may represent support cells in the Johnston’s organ (or first antennal segment). As Johnston’s organ development is _ato_-dependent [83], we cannot exclude that there are shared markers between support cells of these two segments and that some of the “Ir subsystem” support cell clusters correspond to cells of this auditory organ

Fig. 3

Fig. 3

Expression of Osi8 in the antenna. A Schematic of the protein domain structure of Osi8. B Histogram of expression levels of Osi family members in antennae of control (w 1118) and amos mutant (amos 3) animals, determined by bulk RNA-seq. Mean values ±SD of fragments per kilobase of transcript per million mapped reads (FPKM) are plotted; n = 3 biological replicates. Note that Osi10 values represent the combined counts of Osi10a and Osi10b. C Histogram of Osi8 expression levels in the indicated D. melanogaster tissues determined by bulk RNA-seq; mean FPKM values ±SD are plotted (n = 2–3 biological replicates; data are from the Fly Atlas 2.0 [88]). D tSNE plot of whole head single-cell transcriptomes (Fly Cell Atlas 10× stringent dataset [29]) highlighting the selective detection of Osi8 within a subset of _sv_-positive cells (most of which are likely to be antennal support cells (Fig. 2A)). E Histogram of expression levels of the Aedes aegypti Osi8 ortholog (AAEL004275) in the indicated tissues determined by bulk RNA-seq; mean values ±SD of transcripts per kilobase million mapped reads (TPM) are plotted (n = 3–8 biological replicates; data are from [89]). F Osi8 RNA FISH on a whole-mount antenna of a control (w 1118) animal; the bright-field channel is overlaid on the lower image to reveal cuticle morphology. Morphologically distinct proximal and distal populations of Osi8 RNA-expressing cells are indicated (see “Results”). Scale bar, 20 μm. G Top: schematic of the neuronal composition of the antennal trichoid (at) and antennal intermediate (ai) sensillar classes. Bottom: Osi8 RNA FISH and GFP immunofluorescence in whole-mount antennae of animals in which the distributions of the different sensillar classes are revealed with a representative Or neuron transgenic reporter. In the left-hand image, the arrowheads indicate _Osi8_-expressing cells that are found in close proximity to Or67d neurons (see “Results”). Genotypes (left-to-right): UAS-mCD8:GFP/+;Or67d Gal4#1 /+, Or88a-mCD8:GFP, Or83c-mCD8:GFP, Or2a-mCD8:GFP. Scale bar, 20 μm. H Top: schematic of an olfactory sensillum, illustrating the main cell types and other anatomical features. Bottom: Osi8 RNA FISH and GFP immunofluorescence on antennal cryosections of animals in which the tormogen (UAS-mCD8:GFP;ASE5-Gal4) or thecogen (nompA-Gal4;UAS-mCD8:GFP) support cells are labeled. Scale bars, 20 μm. I Immunofluorescence for GFP on antennal cryosections of Osi8-Gal4/+;UAS-SS:EGFP:Osi8/+ animals; the bright-field channel is overlaid on the right-hand image to reveal cuticle morphology. The open arrowheads point to prominent intracellular puncta of GFP signal. The filled arrowheads point to GFP signal within the lumen of the proximal region of trichoid sensillar hairs, which may represent extracellular vacuoles (see “Results”). Scale bar, 20 μm

Fig. 4

Fig. 4

Functional analysis of Osi8 reveals a selective role in pheromone sensing. A Schematic of the generation of the Osi8 1 mutant. Osi8 exons and UTRs are shaded dark and light green, respectively. B Osi8 RNA FISH on whole-mount antennae from control (w 1118) and Osi8 1 mutant animals. Scale bar, 20 μm. C Scanning electron micrographs of antennae from control (w 1118) and Osi8 1 mutants (2-day-old animals). Scale bars, 50 μm. The higher-magnification images (scale bars, 10 μm; samples from 20-day-old animals) highlight several trichoid sensilla on the distal edge of the antenna; the basal drums of these sensilla are indicated with yellow arrowheads. D Representative traces of electrophysiological responses of Or47b OSNs to palmitoleic acid (10−1 v/v) (0.5 s stimulus, gray bar) in control (w 1118), Osi8 mutant (Osi8 1), control rescue (UAS-CD4:tdTomato/+;Osi8 1 /Osi8 1-DsRed ,Osi8-Gal4), and Osi8 rescue (UAS-Osi8/+;Osi8 1 /Osi8 1-DsRed ,Osi8-Gal4) animals. Raster plots and peristimulus time histograms (PSTHs) of these responses are shown below each trace. Line width in the PSTH represents the SEM. E Dose-response curves of Or47b OSN responses to palmitoleic acid; genotypes are color-coded as in D. Mean responses ±SEM are plotted. n = 10 sensilla; 4–5 flies. Statistical comparisons between genotypes were performed by two-way ANOVA: NS P > 0.05, *** P < 0.001. Full statistical analyses are provided in Additional file 10: Data S2. **F** Representative traces, raster plots, and PSTHs of electrophysiological responses of Or88a OSNs to methyl palmitate (10−1 v/v) (0.5 s stimulus, gray bar) in the same genotypes as in **D**. **G** Dose-response curves of Or88a OSN responses to methyl palmitate; genotypes are color-coded as in **D**. Mean responses ±SEM are plotted; _n_ = 10 sensilla; 4–5 flies. Statistical analyses were performed as in **E**. **H** Representative traces, raster plots, and PSTHs of electrophysiological responses of Or67d OSNs to _cis_-vaccenyl acetate (10−1 v/v) (0.5 s stimulus, gray bar) in control (_w_ _1118_) and _Osi8_ mutant (_Osi8_ _1_) animals. **I** Dose-response curves of Or67d OSN responses to _cis_-vaccenyl acetate; genotypes are color-coded as in **H**. Mean responses ±SEM are plotted; _n_ = 12 sensilla; 4–6 flies. Statistical analyses were performed as in **E**. **J** Basal spiking frequency of the indicated OSNs for control (_w_ _1118_) and _Osi8_ mutant (_Osi8_ _1_) animals; _n_ = 12 sensilla, from 3 flies. _t_-test: NS _P_ > 0.05

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