Sexually dimorphic architecture and function of a mechanosensory circuit in C. elegans

doi:10.1038/s41467-022-34661-3

. 2022 Nov 11;13(1):6825.

doi: 10.1038/s41467-022-34661-3.

Sexually dimorphic architecture and function of a mechanosensory circuit in C. elegans

Hagar Setty^#^{1

2}, Yehuda Salzberg^#^{1

2}, Shadi Karimi³, Elisheva Berent-Barzel^{1

2}, Michael Krieg³, Meital Oren-Suissa^{4

5}

Affiliations

¹ Department of Brain Sciences, Weizmann Institute of Science, Rehovot, 7610001, Israel.
² Department of Molecular Neuroscience, Weizmann Institute of Science, Rehovot, 7610001, Israel.
³ ICFO-Institut de Ciències Fotòniques, The Barcelona Institute of Science and Technology, 08860, Castelldefels, Barcelona, Spain.
⁴ Department of Brain Sciences, Weizmann Institute of Science, Rehovot, 7610001, Israel. meital.oren@weizmann.ac.il.
⁵ Department of Molecular Neuroscience, Weizmann Institute of Science, Rehovot, 7610001, Israel. meital.oren@weizmann.ac.il.

^# Contributed equally.

PMID: 36369281
PMCID: PMC9652301
DOI: 10.1038/s41467-022-34661-3

Sexually dimorphic architecture and function of a mechanosensory circuit in C. elegans

Hagar Setty et al. Nat Commun. 2022.

. 2022 Nov 11;13(1):6825.

doi: 10.1038/s41467-022-34661-3.

Authors

Hagar Setty^#^{1

2}, Yehuda Salzberg^#^{1

2}, Shadi Karimi³, Elisheva Berent-Barzel^{1

2}, Michael Krieg³, Meital Oren-Suissa^{4

5}

Affiliations

¹ Department of Brain Sciences, Weizmann Institute of Science, Rehovot, 7610001, Israel.
² Department of Molecular Neuroscience, Weizmann Institute of Science, Rehovot, 7610001, Israel.
³ ICFO-Institut de Ciències Fotòniques, The Barcelona Institute of Science and Technology, 08860, Castelldefels, Barcelona, Spain.
⁴ Department of Brain Sciences, Weizmann Institute of Science, Rehovot, 7610001, Israel. meital.oren@weizmann.ac.il.
⁵ Department of Molecular Neuroscience, Weizmann Institute of Science, Rehovot, 7610001, Israel. meital.oren@weizmann.ac.il.

^# Contributed equally.

PMID: 36369281
PMCID: PMC9652301
DOI: 10.1038/s41467-022-34661-3

Abstract

How sensory perception is processed by the two sexes of an organism is still only partially understood. Despite some evidence for sexual dimorphism in auditory and olfactory perception, whether touch is sensed in a dimorphic manner has not been addressed. Here we find that the neuronal circuit for tail mechanosensation in C. elegans is wired differently in the two sexes and employs a different combination of sex-shared sensory neurons and interneurons in each sex. Reverse genetic screens uncovered cell- and sex-specific functions of the alpha-tubulin mec-12 and the sodium channel tmc-1 in sensory neurons, and of the glutamate receptors nmr-1 and glr-1 in interneurons, revealing the underlying molecular mechanisms that mediate tail mechanosensation. Moreover, we show that only in males, the sex-shared interneuron AVG is strongly activated by tail mechanical stimulation, and accordingly is crucial for their behavioral response. Importantly, sex reversal experiments demonstrate that the sexual identity of AVG determines both the behavioral output of the mechanosensory response and the molecular pathways controlling it. Our results present extensive sexual dimorphism in a mechanosensory circuit at both the cellular and molecular levels.

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Conflict of interest statement

The authors declare no competing interests.

Figures

**Fig. 1. Sexually dimorphic perception of tail mechanosensation at the sensory level.**
a Predicted connectivity of the circuit for tail mechanosensation^,. Chemical synapses between sensory (triangles), inter- (hexagons) and motor (circles) neurons are depicted as arrows. Thickness of arrows correlates with degree of connectivity (number of sections over which *en passant* synapses are observed). b Tail-touch responses of *PHAp::HisCl1-*, *PHBp::HisCl1-* and *PHCp::HisCl1*-expressing animals of both sexes that were tested either on histamine or control plates (see *Methods*). Number of animals: *PHAp::HisCl1* hermaphrodites: n = 19 per group, *PHAp::HisCl1* control males: n = 18, *PHAp::HisCl1* histamine males: n = 19, *PHBp::HisCl1* control hermaphrodites: n = 16, *PHBp::HisCl1* histamine hermaphrodites: n = 18, *PHBp::HisCl1* males: n = 16 per group, *PHCp::HisCl1* control hermaphrodites: n = 15, *PHCp::HisCl1* control males: n = 12, *PHCp::HisCl1* histamine hermaphrodites: n = 20, *PHCp::HisCl1* histamine males: n = 16. c Table listing the genes selected for reverse genetic screen and their expression pattern at the sensory neurons PHA, PHB and PHC. Dark boxes represent gene expression. d Tail-touch responses of RNAi-silenced candidate genes in both sexes. Control hermaphrodites: n = 16, control males: n = 19, *mec-8* RNAi: n = 19 animals per group, *mec-12* RNAi hermaphrodites: n = 17, *mec-12* RNAi males: n = 20, *trpa-1* RNAi: n = 16 per group, *del-1* RNAi hermaphrodites: n = 18, *del-1* RNAi males: n = 17, *del-2* RNAi hermaphrodites: n = 19, *del-2* RNAi males: n = 17, *unc-1* RNAi hermaphrodites: n = 19, *unc-1* RNAi males: n = 20, *tmc-1* RNAi: n = 18 per group, *osm-10* RNAi hermaphrodites: n = 16, *osm-10* RNAi males: n = 15. e Tail-touch responses of mutant strains for candidate genes in both sexes and in *him-5(e1490)* animals (see *Methods*). *him-5(e1490)* hermaphrodites: n = 18, *him-5(e1490)* males: n = 15, *mec-12(e1605)* hermaphrodites: n = 17, *mec-12(e1605)* males: n = 15, *tmc-1(ok1859)* hermaphrodites: n = 16, *tmc-1(ok1859)* males: n = 18, *ocr-2(ak47)* hermaphrodites: n = 16, *ocr-2(ak47)* males: n = 15, *osm-9(ky10)* hermaphrodites: n = 12, *osm-9(ky10)* males: n = 15. f Schematic of the sensory neurons which function in each sex, with the respective suggested sites of action of *tmc-1* and *mec-12*. The response index represents an average of the forward responses (scored as responded or not responded) in five assays for each animal. In (b, d, e) we performed a Kruskal-Wallis test followed by Dunn’s multiple comparison test. Orange- hermaphrodites, cyan- males. All Bar graphs are a box-and-whiskers type of graph, min to max showing all points. The vertical bars represent the median.

**Fig. 2. *mec-12* and *tmc-1* function cell- and sex-specifically in tail mechanosensation.**
a Tail-touch responses of wild-type (n = 15), *mec-12(e1605)* (n = 15)*, mec-12(e1605);PHAp::mec-12* (n = 14) and *mec-12(e1605);PHBp::mec-12* (n = 11) males. b Tail-touch responses of wild-type (n = 15), *tmc-1(ok1859)* (n = 15) and *tmc-1(ok1859);PHAp::mec-12* (n = 13) hermaphrodites. c Tail-touch responses of wild-type (n = 16), *tmc-1(ok1859)* (n = 16) and *tmc-1(ok1859);PHCp::tmc-1* (n = 15) hermaphrodites. The response index represents an average of the forward responses (scored as responded or not responded) in five assays for each animal. We performed a Kruskal-Wallis test followed by a Dunn’s multiple comparison test for all comparisons. Orange- hermaphrodites, cyan- males. d Schematic of the sensory neurons which function in each sex, with the respective sites of action of *tmc-1* and *mec-12*. All Bar graphs are a box-and-whiskers type of graph, min to max showing all points. The vertical bars represent the median.

**Fig. 3. Sexually dimorphic integration of tail mechanosensation at the interneuron level.**
a Tail-touch responses of *AVGp::HisCl1-* and *DVAp::HisCl1*-expressing hermaphrodite and male worms that were tested either on histamine or control plates (see *Methods*). *AVGp::HisCl1* control hermaphrodites: n = 14, *AVGp::HisCl1* control males: n = 13, *AVGp::HisCl1* histamine hermaphrodites: n = 15, *AVGp::HisCl1* histamine males: n = 16, *DVAp::HisCl1* control: n = 13 animals per group, *DVAp::HisCl1* histamine hermaphrodites: n = 15, *DVAp::HisCl1* histamine males: n = 14. b Tail-touch responses of *AVGp::HisCl1* hermaphrodites (control and *AVGp::fem-3*) and males (control and *AVGp::tra-2*) that were tested on histamine plates. n = 20 animals per group. c Representative graph showing the head trajectory (head position) in a control male and ATR-supplemented male. The schematic represents the timeline of the experimental setup. d Speed (µm/s) of hermaphrodite and male worms grown on control and ATR plates at the time of light projection. Control hermaphrodites: n = 18, ATR hermaphrodites: n = 16, control males: n = 16, ATR males: n = 17. e Table listing the glutamate receptors selected for the reverse genetic screen, their expression pattern at the relevant interneurons and their predicted type. Dark boxes represent gene expression. f–h Tail-touch responses of gene candidates that were examined using mutant strains (f, g) or RNAi feeding (h) in both sexes. Each experiment was conducted with a control (*him-5(e1490)* (f)*, him-8(e1489)* (g) and *him-5(e1490)* fed with RNAi (h)). *him-5(e1490)* hermaphrodites: n = 18, *him-5(e1490)* males: n = 16, *glr-1(n2461)* hermaphrodites: n = 18, *glr-1(n2461)* males: n = 16, *glr-2(ok2342)*: n = 18 animals per group, *nmr-1(ak4)*: n = 20 animals per group, *him-8(e1489)* hermaphrodites: n = 20, *him-8(e1489)* males: n = 18, *nmr-2(ok3324)* hermaphrodites: n = 20, *nmr-2(ok3324)* males: n = 18, control for RNAi hermaphrodites: n = 13, control for RNAi males: n = 12, *glr-4/glr-5* RNAi: n = 14 animals per group. i Interneuron-level sexual dimorphism of tail mechanosensation with suggested mode of function for *nmr-1* and *glr-1*. The response index represents an average of the forward responses (scored as responded or not responded) in five assays for each animal. In (d), we performed a two-sided Mann-Whitney test for each comparison. In (a, b, f, g, h) we performed a Kruskal-Wallis test followed by a Dunn’s multiple comparison test. All Bar graphs are a box-and-whiskers type of graph, min to max showing all points. The vertical bars represent the median.

**Fig. 4. Cell-autonomous and sex-specific role for *nmr-1* in tail mechanosensation.**
a Tail-touch responses of wild-type (n = 14 hermaphrodites, n = 15 males), *nmr-1(ak4)* (n = 15 hermaphrodites, n = 17 males) and *nmr-1(ak4);AVGp::nmr-1* (n = 16 hermaphrodites, n = 15 males) in both sexes. b Representative confocal micrographs of *nmr-1::GFP* fosmid in the AVG interneuron, identified by the expression of mCherry in both sexes. Scale bar is 10 µm. c Quantification of (b). a.u., arbitrary units. n = 15 animas per group. d, e Tail-touch responses of hermaphrodites with AVG masculinized hermaphrodites (n = 20) (d) and AVG feminized males (n = 16) (e) in *nmr-1(ak4)* mutant background with respective controls: wild-type hermaphrodites: n = 18, *nmr-1(ak4)* hermaphrodites: n = 20, wild-type males: n = 16, *nmr-1(ak4)* males: n = 17. f Tail-touch responses of AVG masculinized hermaphrodites and AVG feminized males in *nmr-1(ak4)* mutant background expressing either *PHAp::HisCl1* or *PHCp::HisCl1* that were tested either on histamine or control plates (see *Methods*). Animals for each group were collected from two independent experiments. AVG masculinized hermaphrodites with *PHAp::HisCl1:* n = 23 for control group and n = 22 for histamine group, AVG feminized males with *PHAp::HisCl1:* n = 17 for control group and n = 15 for histamine group, AVG masculinized hermaphrodites with *PHCp::HisCl1:* n = 19 animals per group, AVG feminized males with *PHCp::HisCl1:* n = 19 animals per group. g Tail-touch responses of *nmr-1(ak4)* (n = 19 hermaphrodites, n = 18 males) and *nmr-1(ak4);DVAp::nmr-1* (n = 18 hermaphrodites, n = 12 males) in both sexes. h Receptor site-of-action for tail mechanosensation. The response index represents an average of the forward responses (scored as responded or not responded) in five assays for each animal. In (c and f), we performed a two-sided Mann-Whitney test for each comparison. In (a, d, e, g), we performed a Kruskal-Wallis test followed by a Dunn’s multiple comparison test for all comparisons. Orange- hermaphrodites, cyan- males. All Bar graphs are a box-and-whiskers type of graph, min to max showing all points. The vertical bars represent the median.

**Fig. 5. Sexually dimorphic responses of AVG to mechanical stimulation.**
a–c AVG GCaMP6s calcium responses of males (n = 13 recordings from 7 animals) and hermaphrodites (n = 17 recordings from 13 animals) to three consecutive tail mechanical stimulations (a, b) and of males to three consecutive mechanical stimulations anterior to the tail (n = 6 recordings from 6 animals) (c). Stacked kymographs represent the GCaMP intensity vs. time of individual recordings. Graphs represent average and SD traces of AVG calcium responses. Black vertical lines represent the time when a stimulus was applied. d, e AVG GCaMP6s calcium responses of *nmr-1(ak4)* mutant males (n = 13 recordings from 9 animals) (d) and hermaphrodites (n = 14 recordings from 8 animals) (e) to three consecutive tail mechanical stimulations. f, g AVG GCaMP6s calcium responses of *mec-12(e1605)* mutant males (n = 28 recordings form 16 animals) (f) and hermaphrodites (n = 12 recordings from 10 animals) (g) to three consecutive tail mechanical stimulations. Each 3.5 bar stimulus was applied for two seconds (see *Methods*). Full statistical analysis can be found in Supplementary Fig. 9.

**Fig. 6. Over-expression of TMC-1 in PHA can elicit a mechanosensory response in hermaphrodites.**
a AVG GCaMP6s calcium responses of hermaphrodites with *PHAp::tmc-1* to three consecutive tail mechanical stimulations. Stacked kymographs represent the GCaMP intensity vs. time of individual recordings. Graphs represent average and SD traces of AVG calcium responses. Black vertical lines represent the time when a stimulus was applied. n = 23 recordings from 15 animals. Full statistical analysis can be found in Supplementary Fig. 9. b Tail-touch responses of wild-type (n = 14), *tmc-1(ok1859)* (n = 15) and *tmc-1(ok1859)*; *PHAp::tmc-1* (n = 14) hermaphrodites. We performed a Kruskal-Wallis test followed by a Dunn’s multiple comparison test. Bar graph is a box-and-whiskers type of graph, min to max showing all points. The vertical bars represent the median.

**Fig. 7. *nmr-1* and *mec-12* function in the male mating behavior.**
a Quantification of response to contact in wild-type (n = 13) *nmr-1(ak4)* (n = 16), *mec-12(e1605)* (n = 15) and *nmr-1(ak4);mec-12(e1605)* (n = 14) males. b Quantification of vulva location efficiency (L.E., see Methods) in wild-type (n = 13) *nmr-1(ak4)* (n = 16), *mec-12(e1605)* (n = 15) and *nmr-1(ak4);mec-12(e1605)* (n = 14) males. c Quantification of the time until successful mating. in wild-type (n = 13) *nmr-1(ak4)* (n = 12), *mec-12(e1605)* (n = 15) and *nmr-1(ak4);mec-12(e1605)* (n = 11) males. We performed a two-sided Mann-Whitney test for each comparison. All Bar graphs are a box-and-whiskers type of graph, min to max showing all points. The vertical bars represent the median.

**Fig. 8. Sexually dimorphic cellular and molecular mechanisms controlling tail mechanosensation.**
A model depicting the cellular and molecular elements mediating tail mechanosensation in each sex. Neurons that function in each sex are highlighted in yellow. Labeling of TMC-1, MEC-12, NMR-1 and GLR-1 describes their suggested functional role in these cells. Figure was made by Weizmann Institute’s graphics unit.

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References

1. Ryan DA, et al. Sex, age, and hunger regulate behavioral prioritization through dynamic modulation of chemoreceptor expression. Curr. Biol. 2014;24:2509–2517. - PMC - PubMed
1. Hoke KL, Ryan MJ, Wilczynski W. Sexually dimorphic sensory gating drives behavioral differences in túngara frogs. J. Exp. Biol. 2010;213:3463–3472. - PMC - PubMed
1. Shen J-X, et al. Ultrasonic frogs show extraordinary sex differences in auditory frequency sensitivity. Nat. Commun. 2011;2:342. - PubMed
1. Fan Y, et al. Auditory perception exhibits sexual dimorphism and left telencephalic dominance in Xenopus laevis. Biol. Open. 2018;7:bio035956. - PMC - PubMed
1. Vihani A, et al. Semiochemical responsive olfactory sensory neurons are sexually dimorphic and plastic. Elife. 2020;9:e54501. - PMC - PubMed

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[1] Ryan DA, et al. Sex, age, and hunger regulate behavioral prioritization through dynamic modulation of chemoreceptor expression. Curr. Biol. 2014;24:2509–2517. - PMC - PubMed

[2] Ryan DA, et al. Sex, age, and hunger regulate behavioral prioritization through dynamic modulation of chemoreceptor expression. Curr. Biol. 2014;24:2509–2517. - PMC - PubMed

[3] Hoke KL, Ryan MJ, Wilczynski W. Sexually dimorphic sensory gating drives behavioral differences in túngara frogs. J. Exp. Biol. 2010;213:3463–3472. - PMC - PubMed

[4] Hoke KL, Ryan MJ, Wilczynski W. Sexually dimorphic sensory gating drives behavioral differences in túngara frogs. J. Exp. Biol. 2010;213:3463–3472. - PMC - PubMed

[5] Shen J-X, et al. Ultrasonic frogs show extraordinary sex differences in auditory frequency sensitivity. Nat. Commun. 2011;2:342. - PubMed

[6] Shen J-X, et al. Ultrasonic frogs show extraordinary sex differences in auditory frequency sensitivity. Nat. Commun. 2011;2:342. - PubMed

[7] Fan Y, et al. Auditory perception exhibits sexual dimorphism and left telencephalic dominance in Xenopus laevis. Biol. Open. 2018;7:bio035956. - PMC - PubMed

[8] Fan Y, et al. Auditory perception exhibits sexual dimorphism and left telencephalic dominance in Xenopus laevis. Biol. Open. 2018;7:bio035956. - PMC - PubMed

[9] Vihani A, et al. Semiochemical responsive olfactory sensory neurons are sexually dimorphic and plastic. Elife. 2020;9:e54501. - PMC - PubMed

[10] Vihani A, et al. Semiochemical responsive olfactory sensory neurons are sexually dimorphic and plastic. Elife. 2020;9:e54501. - PMC - PubMed

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Sexually dimorphic architecture and function of a mechanosensory circuit in C. elegans

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Sexually dimorphic architecture and function of a mechanosensory circuit in C. elegans

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