Sensory level sexual dimorphism in the tail mechanosensation circuit
By combining data from the published connectome maps of both C. elegans sexes26 with behavioral data from previous studies24,25, we revealed that some of the sensory cells in the tail mechanosensation circuit are connected differently in the two sexes (Figure 1A). To explore the contribution of each sensory neuron to tail mechanosensation, we silenced individual neurons by cell-specific expression of the inhibitory Drosophila histamine-gated chloride channel (HisCl1)29 and then tested both sexes for tail-touch response (see Methods). Silencing the PHB neuron diminished the tail-touch response equally in both sexes, suggesting it has a sex-independent role in touch sensitivity (Figure 1B). However, silencing PHA and PHC revealed that PHC is necessary only in hermaphrodites (corroborating previous findings)25, while PHA is required only in males (Figure 1B). These results show that, at the sensory level, each sex utilizes a different combination of sensory cells in tail-touch perception.
We next asked whether the molecular mechanisms that govern tail mechanosensation at the sensory level are, too, sexually dimorphic. We carried out a reverse genetic screen targeting ion channels and other proteins previously suggested to be involved in mechanosensation that are known to be expressed in the tail sensory cells21,30–36 (Figure 1C). Assaying tail-touch responses of RNA interference (RNAi)-fed or mutant animals led to the identification of two genes whose silencing caused sex-specific defects in tail mechanosensation.
First, targeting mec-12 by using both RNAi or a mec-12 mutant, reduced tail mechanosensation only in males (Figure 1D-E). mec-12 (expressed in PHA, PHB and PHC, Figure 1C) encodes an alpha-tubulin protein, is one of several genes required for touch receptor neuron (TRN) function in C. elegans and is specifically responsible for generating 15-protofilament microtubules in TRNs37.
Second, we found that RNAi of tmc-1 elicited a reduced response only in hermaphrodites, and tmc-1 mutant hermaphrodites exhibited a significantly reduced tail-touch response (Figure 1D-E). TMC-1 (expressed in PHA and PHC, Figure 1C) is a mechanosensitive sodium channel and an ortholog of the mammalian TMC proteins important for hair-cell mechanotransduction31,38. Taken together, our screen uncovered two molecules that play a role in mediating tail mechanosensation in a sex-dependent manner, possibly functioning through different types of sensory cells (Figure 1F). Overall, our findings demonstrate extensive cellular and molecular sexual dimorphism in mechanosensation at the sensory level.
Cell- and sex-specific function of mec-12 and tmc-1 in tail mechanosensation
Having established a role for mec-12 in mechanosensation in males, we turned to explore whether it functions sex-specifically through the phasmid neurons. Since PHA and PHB are required for tail mechanosensation in males (Figure 1B), we restored the expression of mec-12 in mutant animals under the che-12 promoter, which drives expression in ciliated amphid and phasmid neurons, including PHA and PHB39. We found that mec-12 expression in ciliated neurons in mec-12 mutant males is sufficient to rescue the tail-touch phenotype (Figure 2A), suggesting that mec-12 functions in PHA/PHB. Although mec-12 is required only in male PHA/PHB for tail mechanosensation, its expression in these cells was similar in the two sexes (Figure 2B-C).
tmc-1 has been shown to be expressed in PHA and PHC in hermaphrodites33 (Figure 1C). Since PHC is required for tail mechanosensation in hermaphrodites and not PHA (Figure 1B), we hypothesized that tmc-1 mediates tail mechanosensation through PHC in hermaphrodites. Indeed, expressing tmc-1 specifically in PHC rescued the defective tail-touch response of tmc-1 mutant hermaphrodites (Figure 2D). This finding is supported by the evident expression of a tmc-1::mKate2 transcriptional reporter in PHC in both sexes (Figure 2E). Taken together, our results demonstrate that not only do different sensory neurons participate in the processing of mechanosensory information in each sex, the molecules involved in this processing in the sensory neurons are, too, distinct between the sexes.
The tail mechanosensation circuit is sexually dimorphic at the interneuron level
The predicted sexually dimorphic connectivity of the sensory neurons (Figure 1A) suggests dimorphic activities for the downstream interneurons. The sex-shared interneuron AVG is predicted to possess a striking dimorphic connectivity pattern according to the published connectomes, receiving more inputs in males compared to hermaphrodites (Figure S1A-B). Given that the sensory cells connected to AVG in males are the ones with a suggested role in mechanosensation, we speculated that AVG might be involved in tail mechanosensation. Therefore, we silenced AVG using a cell-specific driver (Figure S2A-B) and tested animals for tail-touch responses in both sexes. We found that silencing AVG elicits a sexually dimorphic effect on tail mechanosensation, reducing only the male response (Figure 3A), in agreement with AVG’s predicted dimorphic connectivity.
If AVG connectivity to the sensory neurons plays a significant role, rewiring AVG’s connections should affect tail-mechanosensation responses. Namely, adding connections in hermaphrodites would elicit an effect, and vice-versa for males. We therefore introduced a sex-determining factor specifically in AVG to switch its sexual identity and connectivity to that of the opposite sex40–42. We found that sex-reversing AVG is sufficient to convert the phenotype of the AVG-silenced tail-touch response: In hermaphrodites with a masculinized AVG, the tail-touch response was impaired when AVG was silenced compared to wild-type hermaphrodites, and in males with feminized AVG, the tail-touch response was rescued when AVG was silenced compared to wild-type males (Figure 3B). These results suggest that the sexual identity of AVG and consequently its wiring pattern, can shape tail mechanosensory behavior.
Since the behavioral output of harsh touch applied to the tail is a forward movement, we asked whether optogenetic activation of AVG will result in forward movement only in males, as was shown for the sensory phasmid neurons in hermaphrodites24. Optogenetic activation of AVG did not affect the forward or total (forward+reverse) speed of the animals in both sexes (Figure S3). However, optogenetic inhibition of AVG reduced the total speed of males only (Figure 3C-D). Thus, AVG is required for locomotion in a sexually dimorphic manner.
We next tested whether the sex-shared interneurons DVA and PVC also have a dimorphic role in tail mechanosensation. As we were unable to generate a PVC-specific driver, in accordance with previous observations (Figure S443), we focused on the role of DVA. A tail-touch assay on DVA-silenced animals revealed that only hermaphrodites are affected (Figure 3A, Figure S2C), in agreement with the predicted connectivity (Figure 1A). Taken together, our results uncover the sex-specific use of different interneurons in the circuit for tail mechanosensation, and assign a novel functional role for AVG in locomotion and mechanosensation.
Since we uncovered sexually dimorphic functions of the interneuron level in the circuit, we explored potential molecular mechanisms that might govern these differences. To this end, we screened a list of glutamate receptor genes expressed in the relevant interneurons (AVG, DVA and PVC33,44; Figure 3E), as the sensory neurons required for tail mechanosensation are glutamatergic45. We found that two glutamate receptors are required for tail mechanosensation in a sexually dimorphic manner: glr-1 (AMPA type) is needed only in hermaphrodites, while nmr-1 (NMDA type), which operates in both sexes, has a stronger effect in males (Figure 3F-H). Taken together, our data suggest that the dimorphic nature of the tail mechanosensation circuit spans beyond mere neuronal connectivity to include also different receptor dependency (Figure 3I).
NMDA receptor nmr-1 is required specifically in AVG to mediate tail mechanosensation in males
We next sought to assess the cell-autonomous role of nmr-1 and glr-1 in tail mechanosensation in AVG. To do so, we overexpressed nmr-1 and glr-1 specifically in AVG and determined the tail-touch response in the respective mutants. We found that nmr-1 re-expression in AVG, but not glr-1 re-expression, rescues the defective tail-touch phenotype of mutant males and not of hermaphrodites (Figure 4A, Figure S5A), suggesting that nmr-1 functions cell autonomously in male AVG for this purpose. In line with this observation, the expression of nmr-1 fosmid in AVG was higher in males (Figure 4B-C). glr-1 expression pattern was observed in AVG in both sexes but at higher levels in males (Figure S5B-C), suggesting it mediates a different and possibly dimorphic function in AVG.
Since we observed that the sexual identity of AVG is sufficient to determine the behavioral outcome of the circuit when AVG is silenced (Figure 3B), we asked whether this is also true in nmr-1 or glr-1 mutant animals. Sex-reversing AVG in nmr-1 mutant animals switched the tail-touch phenotype to that of the opposite sex, i.e., it reduced the tail-touch response in hermaphrodites with masculinized AVG and enhanced it in males with feminized AVG (Figure 4D-E). This was not evident in glr-1 mutant animals, as sex-reversal of AVG had no effect in both sexes (Figure 4F-G). These results corroborate the observations of the cell-specific rescue experiments, and point to a cell-autonomous role for nmr-1 in AVG in males that mediates tail mechanosensation. Importantly, the sexual identity of AVG not only dictates the connectivity pattern40, but also the cell-autonomous molecular pathway that mediates the behavior.
Our results also show a slight defect in tail mechanosensation in nmr-1 mutant hermaphrodites (Figure 4A). This led us to search for the interneuron in hermaphrodites in which nmr-1 mediates tail mechanosensation. Since DVA is required for tail-touch response only in hermaphrodites (Figure 3A), and nmr-1 is known to be expressed in hermaphrodites in DVA (Figure 3E), we checked whether nmr-1 functions through DVA to mediate tail mechanosensation. Re-expressing nmr-1 in DVA did not rescue the response of nmr-1 mutant hermaphrodites (Figure 4H), suggesting nmr-1 might be required in a different interneuron, such as PVC, for tail mechanosensation (Figure 4I).
Mechanical stimulation of the tail elicits a sexually dimorphic neuronal response in AVG
Since AVG has a role in integrating mechanical information specifically in males, we asked whether it is activated in response to the application of mechanical force to the tail, and whether such activation occurs sex-specifically. We recorded the calcium traces of AVG in both sexes in response to tail mechanical stimulation using a microfluidic device that was adjusted to fit the male body46 (Figure S6). Similar to our previous observation in touch receptor neurons46, we found that AVG exhibits blue-light-evoked Ca2+ transients even in the absence of a mechanical stimulus, suggesting a previously uncharacterized stimulatory effect of LITE-1 on AVG (Figure S7A-D). We therefore measured the mechanosensitive activity of AVG under a lite-1 mutant background. Three consecutive tail mechanical stimulations, but not posterior stimulations (mock), elicited neuronal responses in AVG (Figure 5A-C). Importantly, these responses were sexually dimorphic, being significantly lower in hermaphrodites compared to males (Figure 5A-B; Figure S8A). These findings support our behavioral results, and further indicate that AVG integrates mechanosensory information in a dimorphic manner. In line with the cell-autonomous role we uncovered for nmr-1 in AVG in tail mechanosensation, we found that AVG responses to mechanical stimulations were reduced in nmr-1 mutant males compared to controls (Figure 5D, Figure S8B).
We next asked how the sensory processing of mechanical stimulation is translated at the interneuron level. To explore this issue, we recorded the calcium traces of AVG in response to tail mechanical stimulation in mec-12 mutant males, where sensory processing of mechanical stimulation is compromised (Figure 2). Interestingly, mec-12 mutant males showed significantly lower AVG responses compared to wild-type (Figure 5F, Figure S8C). This result indicates that proper sensory perception of mechanical stimulation through mec-12 is critical for the integration at the interneuron level. We also observed lower AVG responses both in nmr-1 and mec-12 mutant hermaphrodites, suggesting a role for the two genes in AVG integration of mechanical stimulation in hermaphrodites as well (Figure 5E, G). Taken together, AVG integrates tail mechanosensation in a sexually dimorphic manner, and requires mec-12-dependent inputs and nmr-1 for this purpose.
Mating behavior is compromised in nmr-1 mutant males
The male tail bears the copulatory apparatus and contains specialized sensory structures required for mating47. We thus wondered whether the male-specific use of particular neurons and genes for tail mechanosensation may reflect a broader role they have in the mating circuit. To test this, we performed mating assays on mutants for the male-specific genes we discovered, mec-12 and nmr-1. While mec-12 mutant males did not show any defects in mating, nmr-1 mutant males were indeed defective in their ability to locate the hermaphrodite vulva, a crucial step in the mating sequence. nmr-1 mutant males also displayed slight defects in their response to contact with hermaphrodites (Figure 6A-C). These findings suggest that at least some of the genes and mechanisms that mediate tail mechanosensation in males may have been “hijacked” from or by the mating circuit, where they serve additional functions.