SRD‐1 in AWA neurons is the receptor for female volatile sex pheromones in C. elegans males
2019; Springer Nature; Volume: 20; Issue: 3 Linguagem: Inglês
10.15252/embr.201846288
ISSN1469-3178
AutoresXuan Wan, Yuan Zhou, Chung Man Chan, Hainan Yang, Christine Yeung, King Lau Chow,
Tópico(s)Neurobiology and Insect Physiology Research
ResumoArticle21 February 2019free access Transparent process SRD-1 in AWA neurons is the receptor for female volatile sex pheromones in C. elegans males Xuan Wan Corresponding Author [email protected] orcid.org/0000-0002-6165-6340 Division of Life Science, The Hong Kong University of Science and Technology, Hong Kong, Hong Kong Search for more papers by this author Yuan Zhou Division of Life Science, The Hong Kong University of Science and Technology, Hong Kong, Hong Kong Search for more papers by this author Chung Man Chan Division of Life Science, The Hong Kong University of Science and Technology, Hong Kong, Hong Kong Search for more papers by this author Hainan Yang orcid.org/0000-0001-6864-1478 Division of Life Science, The Hong Kong University of Science and Technology, Hong Kong, Hong Kong Search for more papers by this author Christine Yeung Division of Life Science, The Hong Kong University of Science and Technology, Hong Kong, Hong Kong Search for more papers by this author King L Chow Corresponding Author [email protected] orcid.org/0000-0002-7722-813X Division of Life Science, The Hong Kong University of Science and Technology, Hong Kong, Hong Kong Department of Chemical and Biological Engineering, The Hong Kong University of Science and Technology, Hong Kong, Hong Kong Interdisciplinary Programs Office, The Hong Kong University of Science and Technology, Hong Kong, Hong Kong Search for more papers by this author Xuan Wan Corresponding Author [email protected] orcid.org/0000-0002-6165-6340 Division of Life Science, The Hong Kong University of Science and Technology, Hong Kong, Hong Kong Search for more papers by this author Yuan Zhou Division of Life Science, The Hong Kong University of Science and Technology, Hong Kong, Hong Kong Search for more papers by this author Chung Man Chan Division of Life Science, The Hong Kong University of Science and Technology, Hong Kong, Hong Kong Search for more papers by this author Hainan Yang orcid.org/0000-0001-6864-1478 Division of Life Science, The Hong Kong University of Science and Technology, Hong Kong, Hong Kong Search for more papers by this author Christine Yeung Division of Life Science, The Hong Kong University of Science and Technology, Hong Kong, Hong Kong Search for more papers by this author King L Chow Corresponding Author [email protected] orcid.org/0000-0002-7722-813X Division of Life Science, The Hong Kong University of Science and Technology, Hong Kong, Hong Kong Department of Chemical and Biological Engineering, The Hong Kong University of Science and Technology, Hong Kong, Hong Kong Interdisciplinary Programs Office, The Hong Kong University of Science and Technology, Hong Kong, Hong Kong Search for more papers by this author Author Information Xuan Wan *,1, Yuan Zhou1, Chung Man Chan1, Hainan Yang1, Christine Yeung1 and King L Chow *,1,2,3 1Division of Life Science, The Hong Kong University of Science and Technology, Hong Kong, Hong Kong 2Department of Chemical and Biological Engineering, The Hong Kong University of Science and Technology, Hong Kong, Hong Kong 3Interdisciplinary Programs Office, The Hong Kong University of Science and Technology, Hong Kong, Hong Kong *Corresponding author. Tel: +86 13434400602; E-mail: [email protected] *Corresponding author. Tel: +852 23587342; E-mail: [email protected] EMBO Rep (2019)20:e46288https://doi.org/10.15252/embr.201846288 See also: C Wang & O Hobert (March 2019) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Pheromones are critical cues for attracting mating partners for successful reproduction. Sexually mature Caenorhabditis remanei virgin females and self-sperm-depleted Caenorhabditis elegans hermaphrodites produce volatile sex pheromones to attract adult males of both species from afar. The chemoresponsive receptor in males has remained unknown. Here, we show that the male chemotactic behavior requires amphid sensory neurons (AWA neurons) and the G-protein-coupled receptor SRD-1. SRD-1 expression in AWA neurons is sexually dimorphic, with the levels being high in males but undetectable in hermaphrodites. Notably, srd-1 mutant males lack the chemotactic response and pheromone-induced excitation of AWA neurons, both of which can be restored in males and hermaphrodites by AWA-specific srd-1 expression, and ectopic expression of srd-1 in AWB neurons in srd-1 mutants results in a repulsive behavioral response in both sexes. Furthermore, we show that the C-terminal region of SRD-1 confers species-specific differences in the ability to perceive sex pheromones between C. elegans and C. remanei. These findings offer an excellent model for dissecting how a single G-protein-coupled receptor expressed in a dimorphic neural system contributes to sex-specific behaviors in animals. Synopsis The G-protein coupled receptor SRD-1 shows male-specific expression in AWA neurons and acts as receptor for volatile sex pheromones in Caenorhabditis elegans and Caenorhabditis remanei. Volatile sex pheromone perception depends on AWA neurons. The G-protein coupled receptor SRD-1 displays a sexually dimorphic expression profile in C. elegans. AWA-specific expression of SRD-1 confers responsiveness to sex pheromone. The C-terminal region of SRD-1 confers species-specific differences in the responsiveness to sex pheromones. Introduction Successful reproduction and maintenance of genetic diversity are vital to the survival of all species 1, 2. Numerous animals rely on chemical communication to locate members of the opposite sex and con-specific partners, and in these cases, the release of a chemical signal, frequently a sex pheromone, is an indication of sexual maturity. This signal conveys differential information for both sexes and the fertility status of an individual. Previous studies on this topic have engendered the hypothesis that communication by chemical signals and production of a potent and specific sex pheromone would enhance species viability, and thus, selection pressure would drive evolutionary convergence to this communication mode 2-10. Nematodes employ a similar chemoattraction scheme 4, 7, 11, and we found that nematode hermaphrodites and females use a chemical pheromone to attract males. In Caenorhabditis elegans, the chemosensory mechanism has been comprehensively elucidated at the molecular level from studies on hermaphrodites 12, and this provides a strong foundation for studying the poorly understood sex pheromone perception in males. Nematode sex pheromones can be classified broadly into volatile and non-volatile pheromones. In C. elegans, non-volatile sex pheromones are represented by the well-studied water-soluble ascarosides, which perform diverse biological functions, from short-range male attraction, hermaphrodite repulsion, olfactory plasticity, and aggregation to dauer formation 11, 13-17. Conversely, volatile sex pheromones in nematodes are defined as long-range chemical cues that help individuals locate mating partners from afar 4, 7. Little is currently known about the identity of these pheromones. Studies on better-characterized sex pheromones from other animals, including insects and rodents, frequently provide information regarding three crucial characteristics: (i) the existence of the pheromones as a mixture of chemical components; (ii) the key enzymes required for their biosynthetic process; and (iii) the unique receptors required for their perception 2-6, 8, 14, 18. Because these pheromones are volatile, they are commonly low-molecular-weight chemicals. In nematodes, the molecular mechanisms underlying the actions of volatile sex pheromones have been dissected to a limited extent. However, the findings of recent studies have offered an opportunity for further investigation. Con-specific nematode adult males were reported to be attracted by volatile sex pheromones produced by (i) sexually mature virgin Caenorhabditis remanei females, (ii) self-sperm-depleted virgin C. elegans hermaphrodites, or (iii) sexually mature virgin C. elegans females but with defective self-sperm 4, 7. Based on these findings, we hypothesized that closely related C. elegans and C. remanei might share evolutionarily conserved male-specific receptors that detect at least one shared component in the volatile pheromone mixtures from the two species. Most chemosensory odorant receptors in C. elegans are G-protein-coupled receptors (GPCRs), and all six non-volatile sex pheromone receptors reported thus far are also GPCRs 11, 19-22; consequently, we speculated that this volatile sex pheromone receptor in the male is a GPCR, which might share downstream components with other GPCRs in eliciting chemoattraction behaviors 12. This hypothesis helped us formulate our search for this candidate receptor and facilitated parallel interrogation of its downstream components by using both molecular genetics and cell biology approaches, which led to the identification of critical sensory neurons and a putative receptor required for sex pheromone perception in the male nematode. Results Amphid neurons are required for pheromone perception Given that C. elegans males differ from hermaphrodites by harboring several extra male-specific cells whose sex-specific functions remain poorly defined, the pheromone receptor was hypothesized to reside in one of these cells. The male-specific cephalic neuron (CEM) was suggested to function in volatile sex pheromone perception 4, but no chemoreceptor of volatile sex pheromone has been identified to date in CEMs. Our previous study revealed that males from neither CEM-defective mutant strains, ceh-30(sm130)X and unc-86(sm117)III, nor CEM-ablated strains, completely lose their chemoattraction response toward volatile sex pheromones. The findings suggest that other chemosensory neurons could be critical for perceiving the chemical cues that elicit this male-specific behavioral response. The functional components of sex pheromones are volatile and act in a sex- and stage-specific manner 4, and to study their action here, we used the chemoattraction behavioral assay described in Materials and Methods (Fig 1A). The pheromone solution deposited on the lid of a Petri dish evoked the same normal chemoattractive behavior in wild-type C. elegans males on the agar surface (Fig 1B), but after vaporization, the C. remanei female extract was no longer attractive to the males (Fig 1B). Therefore, the chemical likely acts through one or a subset of chemosensory amphid neurons required for detecting volatile attractants. Each amphid contains 12 sensory neurons whose neuronal endings are embedded in the sheath and socket cells. These neurons are responsible for diverse sensations, including physical and chemical sensation and nociception, as well as for navigation, lifespan regulation, and dauer formation. Mutants harboring defective amphids typically display aberrant chemosensation 12. We used the chemoattraction behavioral assay to test whether a mutant loses its chemoattraction to the applied sex pheromone; we tested mutants with known defects in specific amphid neurons that change their cell fate or specific cellular functions. Here, the attractiveness of animals toward a particular chemical is represented by a chemoattraction index (C.I.) (Fig 1A), the reduction in which implies the requirement of a cell or a molecular component functioning therein. Figure 1. Amphid neurons are required for pheromone perception In our chemoattraction-assay setup, nematode chemoattraction behavior is quantified using a measurable chemoattraction index (C.I.); the higher the C.I. measured in the assay, the stronger the attraction of the males to the sex pheromone. After vaporization (VAC) at 4°C for 24 h, the Caenorhabditis remanei female extract failed to attract wild-type Caenorhabditis elegans males. Conversely, a drop of the sex pheromone extract placed on the lid of a 60-mm Petri dish evoked chemoattractive behavior in wild-type C. elegans males (volatility test). Thus, the functional components in C. remanei female sex pheromone are volatile substances. Mutant males with established defects in amphid neurons responded to C. remanei sex pheromone to a lesser degree relative to wild-type, demonstrating that some or all of the amphid neurons are required for sex pheromone perception. Data information: We assayed 400 males from each strain by using the sex pheromone chemoattraction assay. Two biological replicates were combined into a single value. Significance was determined using one-way ANOVA with Bonferroni correction: ***P < 0.001. Means ± SEM (error bars) are shown. Download figure Download PowerPoint We determined whether defects in specific amphid neurons lead to a loss of ability in sex pheromone perception. In daf-6, daf-10, che-3, osm-6, and osm-3 mutant males exhibiting defective amphid-neuron functions, diverse chemotactic processes are affected, including chemotaxis, osmotaxis, navigation, lifespan regulation, nociception, nose touch, male mating, and dauer formation 12, 23-28 (Appendix Table S1). Accordingly, our results showed that sex pheromone attraction was reduced in the males of all these mutants (Fig 1C) and confirmed that amphid sensory neurons are required for sex pheromone perception. Notably, in osm-3 mutant animals, the cilia of amphid neurons are defective, which suggests that the hypothesized sex pheromone receptors might reside on the cilia of one or more of the amphid neurons in the males. AWA neurons are required for pheromone perception We next tested mutant C. elegans males exhibiting defects in specific amphid neurons. In these mutants, either terminal cell fates are altered or neuronal identities are changed in one or more amphid neurons, and in certain cases, atypical morphological features are present, and thus, the amphid-neuron functions are impaired. The tested mutants were lin-11, odr-7, ocr-2, mps-1, unc-3, ceh-37, ceh-36, ttx-1, che-1, eat-4, and ttx-3 (Appendix Table S1). Because ASJ- and ADF-defective mutants were not viable, we used caspase-1-based genetic ablation to eliminate ASJs and ADFs in worms (Fig EV1A). Males of all these strains were used in the same chemoattraction assay, and distinct mutants in which the same neuron was affected were then cross-checked for consistency of results and used to generate a holistic representation of the role played by each amphid neuron. Click here to expand this figure. Figure EV1. Males in which ASJ or ADF neurons are genetically ablated respond normally to sex pheromoneHuman caspase 1 (ICE, or CASP1) was transcriptionally driven by cell-specific promoters in Ptrx-1p::ICE (ASJ) and Psrh-142::ICE (ADF) in pPD95.77 vector. Pvha-6p::rfp was used as the injection marker and was expressed in the hypodermis and intestine. Ptrx-1p::gfp is specifically expressed in ASJ neurons in the head region of animals of both sexes (top figures). The transgenic animals displaying the injection-marker signal show no GFP signal at the ASJ position (bottom figures), which implies that ASJs were ablated. Arrowheads: ASJ neurons. Scale bars = 50 μm. Psrh-142::gfp is specifically expressed in ADF neurons in the head region of animals of both sexes (top figures). The transgenic animals displaying the injection-marker signal show no GFP signal at the ADF position (bottom figures), which indicates the removal of ADF neurons. Arrowheads: ADF neurons. Scale bars = 50 μm. Males in which ASJ and ADF neurons were genetically ablated responded normally to sex pheromone in the chemoattraction assays. ADF-specific rescue of srd-1 failed to restore wild-type sex pheromone response in males. We assayed 400 males from each transgenic line and tested three independent transgenic lines. Two biological replicates were combined into a single value. Significance was determined using one-way ANOVA: ***P < 0.001. Error bars: SEM. Download figure Download PowerPoint Most of the tested mutant strains responded well to the applied sex pheromone, with their C.I. values being close to that of wild-type C. elegans males (Fig 2A); these mutants were defective in AWB, AWC, ADF, ADL, AFD, ASE, ASG, ASH, ASI, ASK, and ASJ neurons. The only two exceptions were the lin-11 and odr-7 mutant males, which showed markedly attenuated attractive responses to the pheromone (Fig 2A). lin-11 plays an essential role in AWA cell-fate differentiation by activating the expression of the odr-7-encoded Zn-finger transcription factor. Subsequently, odr-7 expression is autoregulated in AWAs, where the respective differentiation program occurs 29-31. Therefore, AWA cell-specific differentiation and morphological features are abolished in odr-7(ky4)X mutants 29, 30, 32. Concurrently, odr-7 expression in AWAs inhibits AWC-specific olfactory receptor gene expression in AWA. Thus, our results identified AWA as a critical amphid neuron required for sex pheromone perception, and this was further corroborated by the finding that laser ablation of AWAs in wild-type males caused a loss of the sex pheromone perception ability in a single-worm chemoattraction assay (Fig 2B). Figure 2. AWA chemosensory neurons are responsible for pheromone detection Inspection of specific amphid-neuron-defective mutants revealed that AWA neurons are most likely responsible for pheromone perception. Here, an edge connects neurons to a genetic mutant if the neurons are documented to be affected in the mutant. The color code of edges represents the average C.I. of multiple genetic mutants with impaired neuronal functions or specified abnormal neuron identities. The region colored green shows the range of wild-type sex pheromone response in the male. We assayed 400 males from each strain in the sex pheromone chemoattraction assay. Two biological replicates were combined into a single value. Significance was determined using one-way ANOVA with Bonferroni correction: ***P < 0.001. Means ± SEM (error bars) are shown. AWA-laser-ablated males were not attracted by the sex pheromone in single-worm chemoattraction assays. Both AWA-defective mutant males (lin-11 and odr-7) and AWA-laser-ablated males failed to reach the sex pheromone within 30 min. Wild-type Caenorhabditis elegans males responded normally to the sex pheromone, and wild-type C. elegans hermaphrodites were not attracted by same-sex pheromone. The single-worm chemoattraction assay was performed as described in Materials and Methods. Sample size of males in AWA-laser-ablation experiment: 12; all other experiments: 100 worms. Download figure Download PowerPoint AWA role in pheromone perception depends on GPCR pathways The chemosensory function of AWA neurons is well documented 12, 33-35. Because numerous volatile odorants are sensed by GPCRs, we hypothesized that the receptors for sex pheromones are also GPCRs. In this scenario, the downstream signal transduction components of GPCRs might be required for the pheromone perception, and elimination of these components would impair or abolish sex pheromone perception ability in males. We selected four mutants, daf-11, odr-3, osm-9, and grk-2, which harbor defective GPCR pathway components, for examination in our chemoattraction assay (Fig 3A). The four genes encode these molecules: daf-11, a guanylate cyclase 36, 37; odr-3, a G-protein α-subunit. 38; grk-2, a G-protein-coupled receptor kinase 39, 40; and osm-9, a Ca2+-permeable transient receptor potential vanilloid (TRPV) channel protein 39, 40 (Appendix Table S1). daf-11 is required for cGMP-gated channel function, osm-9 and grk-2 affect TRPV channel function, and odr-3 is required for both functions. Males of all four mutant strains showed strong deficits in the perception of sex pheromones from C. elegans and C. remanei (Fig 3B), which implies that the sex pheromone signal transduction occurs through the cGMP-gated channel and the TRPV channel. Notably, AWA-specific rescue of odr-3 function in mutant males partially restored their sex pheromone response (Fig 3C), which strongly suggests that a GPCR-type receptor for sex pheromones is likely located in AWA neurons. Figure 3. GPCR signaling cascade components are required for pheromone perception If the sex pheromone receptor is a GPCR as hypothesized, it could elicit chemoattraction through the signal transduction pathways that control cGMP-gated channels and TRPV channels. Knocking out any key component in the two pathways should reduce or abolish chemoattraction behavior. Mutant males of four GPCR signaling cascade components failed to respond to Caenorhabditis remanei sex pheromone, suggesting that the sex pheromone receptor is a GPCR and that both aforementioned signaling pathways are involved in pheromone perception. We assayed 400 males from each strain in the sex pheromone chemoattraction assay. Two biological replicates were combined into a single value. Significance was determined using one-way ANOVA with Bonferroni correction: ***P < 0.001. Means ± SEM (error bars) are shown. In males from both AWA-specific-rescue odr-3 mutant strain and odr-3-rescued mutant strain, the sex pheromone response was significantly restored in single male worm arrival assays. However, males from the CEM-specific-rescue odr-3 mutant strain failed to reach the sex pheromone within 30 min. In odr-3 and him-5 strain experiments, the sample sizes were 12 and 20 worms, respectively; in all other experiments, 40 worms were used. Download figure Download PowerPoint Identification of the GPCR SRD-1 as the potential sex pheromone chemoreceptor that functions in AWA neurons We speculated that a GPCR expressed in AWA neurons would likely be used for pheromone perception. Thus, we profiled genes that are specifically expressed in male AWA neurons by using a poly-A-binding protein (PABP) mRNA pull-down approach 41, 42. For this analysis, we ectopically expressed the gene fem-3 in all neurons of wild-type hermaphrodites to masculinize their nervous system, including AWA neurons 43, 44. The successful pull-down of AWA-specific genes was confirmed by testing for the presence of dpy-13 (hypodermal gene), myo-3 (muscle gene), and odr-10 (gene specifically overexpressed in AWA) in total RNA samples: Whereas odr-10 was found to be highly enriched in the AWA-enriched samples, dpy-13 and myo-3 were depleted in these samples. The pull-down experiments were repeated independently four times for each experimental group. Three out of the four datasets were found to be highly consistent, and the mean ratio of each gene from the three consistent replicas was compiled. Comparison between transcriptome profiles revealed critical differences between the whole-worm and male AWA neurons, which allowed identification of male AWA-enriched transcripts, including those encoding GPCRs. From the obtained data, we identified 357 GPCRs that are enriched in male AWAs. We considered these data in combination with the data reported by another group regarding genes that are preferentially expressed in males 45, and by using 2× enrichment as the cutoff, we shortlisted 50 GPCR candidates potentially expressed in male AWAs. The results of transcriptional-reporter-expression assays confirmed that 9 of the 50 genes were highly expressed in male AWA neurons (Fig EV2A). Moreover, mutant and RNAi analyses for these GPCR genes revealed that one of these male AWA-specific GPCRs, SRD-1, is likely required for pheromone perception (Figs EV2B and 4A): The srd-1(eh1)II (hereafter "srd-1") mutant males lacked the ability to sense the sex pheromone in our chemoattraction assay (Fig 4A) but responded normally to diacetyl and pyrazine; this implies that the mobility and the chemosensory system of the srd-1 mutants were functional and that the impairment of chemoattraction behavior was solely due to the loss of function of a single GPCR encoded by srd-1. Click here to expand this figure. Figure EV2. Expression patterns of GPCR candidates and their functional verification Expression pattern of Psrx-76::gfp, Pstr-260::gfp, Psrt-12::gfp, Pstr-44::gfp, Psrt-7::gfp, Psrab-13::gfp, Pstr-116::gfp, and Pstr-164::gfp in adult males: (a–h) GFP images; (a′–h′) RFP images; (a″–h″) merged images. Podr-7::rfp served as a reporter of AWA neurons (arrowheads). GFP expression was observed in AWA neurons in all these reporter lines. Scale bar = 50 μm. Summary of all GPCR genes examined using transcriptional reporters, where mutant strains or RNAi-treated animals were analyzed. Psrd-1::gfp was expressed in ASI neurons in both males and hermaphrodites. Expression pattern of Psrd-1::gfp: (i–l) GFP images; (i′–l′) RFP images; (i″–l″) merged images. Pdaf-7::rfp and Pstr-3::rfp served as reporters of ASI neurons (arrowheads). Scale bar = 50 μm. Download figure Download PowerPoint Figure 4. Characteristics of SRD-1 as the receptor required for sex pheromone perception and expression pattern of srd-1 in Caenorhabditis elegans srd-1 mutant males were not attracted to the sex pheromone but were attracted to the positive-control chemoattractants, 1:1,000 diacetyl and 10 mg/ml pyrazine. This demonstrated that SRD-1 is required and specific for sex pheromone perception. Summary of srd-1 expression pattern. The transcriptional reporter Psrd-1::gfp was detected in both male and hermaphrodite ASI neurons in the head region and only in male AWA and ADF neurons. SRD-1::GFP fusion construct revealed predominant SRD-1 localization at the cilia of AWAs in C. elegans males. Scale bar = 5 μm. Examination of srd-1 transcriptional reporter revealed that srd-1 is expressed in AWA neurons in males but not hermaphrodites. A transcriptional reporter for odr-7 indicated the locations of AWAs (arrowheads). Scale bar = 50 μm. Schematic diagram of reprogramming of chemotaxis in C. elegans. Rescue-experiment results demonstrated the necessity of SRD-1 in sex pheromone perception; ectopic expression of SRD-1 in the chemorepulsion-responsible neuron, AWB, revealed that this SRD-1 expression is sufficient for altering sex pheromone preference in C. elegans. AWA-specific rescue of srd-1 restored sex pheromone perception ability in both males and hermaphrodites. Ectopic expression of srd-1 in AWB neurons elicited a distinct repulsive behavior toward the sex pheromone in both males and hermaphrodites. Data information: For each transgenic strain, three independent lines were examined. We assayed 400 males from each strain in the sex pheromone chemoattraction assay. Two biological replicates were combined into a single value. Significance was determined using one-way ANOVA with Bonferroni correction: ***P < 0.001. Means ± SEM (error bars) are shown. Download figure Download PowerPoint Sexually dimorphic expression pattern of srd-1 SRD-1 is a predicted chemosensory receptor, but its biological function remains undocumented 46. srd-1::GFP expression is not detected in che-3 mutants, which harbor defective sensory cilia, or in tax-2 mutants, in which sensory signal transduction is defective; srd-1 expression is repressed by developmental entry into the dauer stage 47. Troemel et al 46 also reported that SRD-1::GFP is expressed in both ADF and ASI neurons in males but only in ASI neurons in hermaphrodites (Fig 4B). After identifying SRD-1, we characterized its expression profile by using transcriptional reporters and the translational fusion-reporter GFP-tagged SRD-1, both of which were transcriptionally driven by the gene's own promoter (3-kb sequence upstream of the predicted translational start site), and SRD-1::GFP was found to be localized subcellularly at the cilia of male AWA neurons (Fig 4C). The cilia localization of SRD-1 also agreed with the morphological features of AWA neurons, which reconfirmed the previous finding of its AWA specificity 48. In the head region, the Psrd-1::gfp signal overlapped with that of the AWA-specific reporter Podr-7::rfp (Fig 4D) in all tested males and with that of the ASI reporters Pdaf-7::rfp and Pstr-3::rfp (Fig EV2C) in both sexes. No Psrd-1::gfp signal was detected in any tested hermaphrodite AWA neurons. Therefore, srd-1 displays a sexually dimorphic expression profile in C. elegans, being highly expressed in ASI, ADF, and AWA neurons in males and only expressed in ASI neurons in hermaphrodites (Fig 4B). unc-3, which encodes an Olf-1/EBF-family transcription factor, is required for promoting ASI-specific gene expression, and it represses alternative neuronal programs 49, 50. Because loss of unc-3 function did not result in defective sex pheromone perception (Fig 2), ASI neurons are unlikely to be necessary for sex pheromone sensing. By contrast, srd-1 exhibits a sexually dimorphic expression profile in ADFs and thus might be required in sex pheromone perception. Because ADF function was not entirely abolished in the mps-1 mutant strain, we performed genetic ablation with caspase-1 to eliminate ADFs. The ADF-ablated males responded well to the sex pheromone (Fig EV1C), and to further confirm that ADF neurons are not required for sex pheromone chemosensation, we performed ADF-specific rescue in srd-1 mutant animals. Restoring srd-1 only in ADF did not rescue defective sex pheromone chemotaxis (Fig EV1C). Collectively, these results suggest that srd-1 expressed in ADF neurons is insufficient for restoring sex pheromone perception, and no available evidence indicates that ASI and ADF neurons are required for volatile sex pheromone perception. AWA-specific expression of SRD-1 confers responsiveness to sex phero
Referência(s)