Whether and how neurons that are present in both sexes of the same species can differentiate in a sexually dimorphic manner is not well understood. A comparison of the connectomes of the Caenorhabditis elegans hermaphrodite and male nervous systems reveals the existence of sexually dimorphic synaptic connections between neurons present in both sexes. Here we demonstrate sex-specific functions of these sex-shared neurons and show that many neurons initially form synapses in a hybrid manner in both the male and hermaphrodite pattern before sexual maturation. Sex-specific synapse pruning then results in the sex-specific maintenance of subsets of these connections. Reversal of the sexual identity of either the pre- or postsynaptic neuron alone transforms the patterns of synaptic connectivity to that of the opposite sex. A dimorphically expressed and phylogenetically conserved transcription factor is both necessary and sufficient to determine sex-specific connectivity patterns. Our studies reveal new insights into sex-specific circuit development.
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We thank Q. Chen for generating transgenic strains; J. White, J. Sulston and LMB/MRC for sharing their annotated electron microscopy images to D. Hall for curation, and http://www.wormimage.org where these annotated images have been made available by D. Hall; E. Yemini for advice on tracking experiments; S. Cook for help with Elegance software; M. Zhen for communicating unpublished results; P. Sengupta, I. Greenwald and members of the Hobert lab for comments on the manuscript. This work was supported by postdoctoral fellowships from the EMBO and HFSP (to M.O.-S.), the NIH (2R37NS039996) and the HHMI. M.O.-S. is an Awardee of the Weizmann Institute of Science, National Postdoctoral Award Program for Advancing Women in Science.
The authors declare no competing financial interests.
Extended data figures and tables
Four transmission electron microscopy prints from wild-type adult hermaphrodite ‘JSE’8 and four from adult male ‘N2Y’, showing adjacency of neuronal processes. These images were collected at MRC/LMB for ref. 8 and ref. 2, and the annotated images are available online at http://www.wormimage.org, courtesy of D. Hall. The set of processes directly adjacent to one another has been defined as the ‘neighbourhood’ of that process33, and the placement of processes into specific neighbourhoods is a major determinant of connectivity. Connections form only in one sex, although processes are adjacent in both sexes. a, Print 385, JSE series (JSE_122283; http://wormimage.org/image.php?i=122283&page=2) and print 620, N2Y series (PAG620; http://wormimage.org/image.php?id=103528&page=18) shows PHB–AVG adjacent processes, pseudo labelled in green (AVG) and red (PHB). b, Print 359, JSE series (JSE_122257; http://wormimage.org/image.php?id=122257&page=2) and print 500, N2Y series (PAG500; http://wormimage.org/image.php?id=103408&page=20) shows AVG–VD13 adjacent processes, pseudo labelled in green (AVG) and pink (VD13). c, Print 377, JSE series (JSE_122275; http://wormimage.org/image.php?id=122275&page=2) and print 800, N2Y series (PAG800; http://wormimage.org/image.php?id=103706&page=16) shows PHA–AVG adjacent processes, pseudo labelled in green (AVG) and orange (PHA). d, A table summarizing the number of electron microscopy sections in which direct adjacency of processes was observed. Over a 1000 PAG (preanal ganglion) serial sections were analysed for each sex.
a, The connectivity diagram shown in Fig. 1a, including dimorphic connections of the LUA and PHC connections. The GRASP data that we show in this figure as well as the pruning data, sexual reversal data and mutant data shown in Extended Figs 6, 8 and 9, supports the original LUA connectivity data reported in ref. 5 and summarized in this schematic. However, a recent reassessment of the tracing of electron micrographs suggests that the connectivity assignments of the PHC and LUA neurons may have been swapped with each other (S. Emmons, personal communication). b, Overview of LUA synaptic connections labelled in this paper. c, Visualizing LUA sexually dimorphic synapses. Quantification and fluorescent micrographs of GRASP trans-synaptically labelled puncta between LUA–AVG and LUA–AVA, in L1 and adult hermaphrodites and in males. M, merge; N, neurite; P, puncta. Region of neurite overlap and observed synaptic puncta are marked with white boxes. Gut, auto-fluorescence gut granules. Scale bars, 10 μm (adult) and 5 μm (L1). d, Fluorescent micrographs of the preanal ganglion region of transgenic animals expressing the presynaptic BirA::nrx-1 fusion in LUA (using the eat-4p9 promoter), and postsynaptic acceptor peptide::nlg-1 fusion in AVG. For more details see Extended Data Fig. 3. Scale bars, 10 μm. We performed nonparametric Mann–Whitney test (Wilcoxon rank sum test) with Bonferroni correction for multiple comparisons. ****P < 0.0001, ***P < 0.001, **P < 0.01; NS, not significant. Magenta horizontal bars represent the median.
a, Overview of synaptic connections labelled in this paper (Fig. 1 and this figure). Some connections were labelled with both GRASP and iBLINC, yielding similar results. We generally note that the number of synapses is roughly reproducible from animal to animal and the number of the fluorescent dots is roughly comparable to the number of synapses identified by the electron microscopy analysis. However, there is also some variance from animal to animal (quantified in Fig. 3), consistent with previous analysis34. b, Labelling data not shown in Fig. 1. Fluorescent micrographs of the preanal ganglion region of transgenic animals expressing the presynaptic BirA::nrx-1 fusion in PHB (using the gpa-6 promoter), AVG (using the inx-18 promoter) and PHA (using the srg-13 promoter), and postsynaptic acceptor peptide::nlg-1 fusion in AVG and DA9 (using the acr-2 promoter). Transgenic worms also express the streptavidin detector fused to 2× sfGFP from the coelomocytes (unc-122 promoter)10. Neuronal processes are labelled with cytoplasmic Cherry markers of the iBLINC pairs. Region of neurite overlap and observed synaptic puncta are marked with white boxes. Scale bars, 10 μM. Anterior is left and dorsal is up.
a, A 2.6 kb gpa-6 promoter fragment fused to GFP is expressed consistently in PHB at all stages. There is also faint and variable expression in AWA. b, The 3.1 kb flp-18 promoter fused to GFP is expressed consistently in AVA, and dimly and variably in AIY (85% of animals) and RIM (15% of animals), which were identified based on comparison to published flp-18 expression patterns35. c, The 1.8 kb inx-18 2nd intron fused to codon-optimized Cherry is expressed brightly and consistently in AVG, and dimly and variably in URXs. The AIY motif present in this fragment was deleted (see Methods). d, The 170 bp eat-4 promoter (eat-4p9) fused to GFP is expressed in LUAs and PVR.
a, PHB silenced hermaphrodites move slower forward (quantified as absolute mid-body speed) and as a result cover less of the plate (quantified as forward path range) compared to control hermaphrodites, whereas PHB-silenced males do not show any difference compared to control males. In addition, the tail-bending wave is affected in PHB-silenced hermaphrodites, but not in males (quantified as tail crawling frequency). Statistics were computed using Wilcoxon rank-sum test, and correction for multiple testing (q values) was computed across all measures (approximately 1404 tests) using the Benjamini–Hochberg procedure. b, Silencing of ASH neuronal activity using the histamine chloride channel 1(HisCl1) affects the animals’ chemosensory avoidance response. Males and hermaphrodites were assayed for effects of histamine on SDS avoidance behaviour. We used the him-5 mutant background (which gives a high incidence of male progeny) as wild type. There is no difference between worms assayed in the presence and absence of histamine (Fig. 2a). The avoidance index of single animals was calculated as the fraction of reversal responses in 10 or more assays, depicted as black dots. Magenta vertical bars represent the median. L4 animals carrying the kyEx5104 (pNP424 (sra-6::HisCl1::SL2::mCherry))11 transgene were grown on 10 mM histamine-containing NGM plates for 24 h. As a control, kyEx5104 animals were grown on NGM plates without histamine. ASH silencing reduces the head sensory response to SDS, thus in hermaphrodites the antagonizing activity of the PHBs inhibits the backward movement and the worms do not reverse. In males, no such antagonizing activity occurs and the worms reverse, albeit with reduced ability. c, Ablation of ASH neurons affects the animals’ chemosensory avoidance response in a similar manner to histamine-induced silencing. sra-6::gfp was used to identify the ASH neurons. d, Behavioural differences stem from dimorphic connectivity differences and not from amphid/phasmid sensory function. tax-4; ceh-14 double mutants behave in a similar manner in both sexes. PHB silencing (gpa-6p::HisCl1::SL2::gfp) does not affect behaviour in either sex. Silencing both the ASHs and PHBs in males showed no difference compared to ASH-silenced males. However, silencing ASHs and PHBs in hermaphrodites showed a significant difference compared to ASH silenced hermaphrodites, where we expect the PHBs to function in an antagonistic manner; thus in its absence, ASH-silenced hermaphrodites showed an increased ability to respond to SDS by reversing. e, PHB silencing in tax-4 mutant background. tax-4 is a subunit of a cyclic nucleotide gated channel expressed in chemosensory and thermosensory neurons36, see g. tax-4 animals show a strongly reduced avoidance response to SDS12. Silencing of PHBs in tax-4 hermaphrodites eliminated the antagonizing affect and animals were able to avoid SDS by backing. f, PHB silencing in tax-4 mutant background at the L1 stage. Lack of avoidance seen in tax-4 L1 males and hermaphrodites depends on PHB function. For all panels, we performed the nonparametric Mann–Whitney test with Bonferroni correction for multiple comparisons. ****P < 0.0001, **P < 0.01, *P < 0.05; NS, not significant. g, tax-4 expression pattern is identical in hermaphrodites and males. kyEx744 (tax-4p::TAX-4::gfp)36, was analysed in adult male and hermaphrodites. Amphid neurons in the head (ADL, ASH, ASI, ASJ, ASK, AWB) were stained using DiD to facilitate cell identification. Neurons identified, shown in the ‘Merge’ panel are identical in both sexes and match published data. All neurons are bilaterally symmetric left–right pairs, and for simplicity only left cells are shown. Scale bars, 10 μM.
a, Hermaphrodites in which LUAs have been ablated pause more frequently than mock-ablated hermaphrodites and LUA-ablated males. Error bars, s.e.m. b, LUA laser-ablated animals tested for the male’s vulva location efficiency. The behavioural data shown in a and b supports the reported connectivity data shown in Extended Data Fig. 2. We performed the nonparametric Mann–Whitney test (Wilcoxon rank sum test) with Bonferroni correction for multiple comparisons (a, b). Error bars, s.e.m. (b). ****P < 0.0001, ***P < 0.001, *P < 0.05; NS, not significant. c, Summary of dimorphic behaviours induced by PHB sensory neurons and AVG/LUA/AVA interneurons.
Hermaphrodites and males were analysed at the L1, L3, L4, young adult and gravid adult stages, and the number of synaptic puncta observed at each stage was plotted against developmental time points. Synaptic puncta in hermaphrodites are plotted in red, synaptic puncta in males are plotted in blue. a, PHB–AVG synapses are pruned in hermaphrodites at the L3 stage. b, PHB–AVA synapses are pruned in males at the L3 stage. c, LUA–AVG synapses are pruned earlier, starting at the L1 stage in hermaphrodites. Error bars, s.e.m.; for each time point depicted in graphs, at least 15 animals were analysed.
a, Cartoon summarizing sex-change effects on synapses. b, L1-stage connectivity is not affected by sex reversal. c, Simultaneous sex reversal of both AVA and AVG. d, Masculinization of the postsynaptic cell AVA is sufficient to induce LUA–AVA synaptic puncta in hermaphrodites. Masculinization of AVG was not sufficient to induce synapses between LUA and AVA in hermaphrodites. Feminizing LUA, AVA and AVG by expression of TRA-2IC was sufficient to induce ectopic LUA–AVA puncta in males. We performed the nonparametric Mann–Whitney test with Bonferroni correction for multiple comparisons. ****P < 0.0001, *P < 0.01; NS, not significant. Magenta horizontal bars represent the median.
a, Quantification of dimorphic expression of dmd-5 and dmd-11 in AVG. Expression in hermaphrodites was off or extremely faint. Expression of inx-18p::FEM-3 derepressed dmd-5 and dmd-11 gene expression in hermaphrodite AVGs. Statistics calculated using Fischer’s exact test. b, Quantification of the number of PHB–AVG synaptic puncta in L1 dmd-5(gk408945); dmd-11(gk552) double mutants, compared with wild-type L1 animals. At the L1 stage, dmd-5 and dmd-11 do not affect PHB–AVG synapses, suggesting they are required for maintenance of mature synapses. c, dmd-5 single mutants and dmd-5; dmd-11 double mutants display similar alterations in AVG synaptic wiring. d, dmd-5 mutation suppresses the ectopic PHB–AVG synapses in AVG-masculinized animals. e, The PHB–AVA connection is non-autonomously partially stabilized in dmd-5; dmd-11 mutants. f, dmd-11 genomic locus and gk552 deletion location. g, dmd-5 genomic locus and mutation description. ok1394 location was not curated, to determine location we used the following primers: forward primer: 5′-CAGAATGCCTGTTTCTCCGTC-3′; and reverse: 5′-CACTGCTTTTCCCGTTCAAAC-3′. ok1394 and tm1760 were both found to have an embryonic lethal phenotype that could not be rescued by the genomic locus (data not shown); thus we searched for single-point mutations of the ‘million mutation project’37. gk408945 is a missense substitution mutation of W54 to R, located in the second exon. Genomic analysis revealed that this mutation lies within the conserved DM domain (h.), with perfect conservation across evolution. h, DM-domain sequence conservation and location of gk408945 mutation. Conservation and multiple sequence alignment were performed using UCSC Genome Browser (http://genome.ucsc.edu) and ClustalW. The DM domain is an intertwined zinc-containing DNA binding module. The DM domain binds DNA as a dimer, allowing the recognition of pseudopalindromic sequences38. i, dmd-5 and dmd-11 are required for maintenance of AVG synapses. Fluorescent micrographs and quantification of synaptic puncta of LUA–AVG. Region of neurite overlap and observed synaptic puncta marked with white boxes. M, merge; N, neurite; P, puncta. Statistics were calculated using the nonparametric Mann–Whitney test (b, d, e, i) or Kruskal–Wallis test with Dunn’s multiple comparison test (c). ****P < 0.0001, ***P < 0.001, *P < 0.05; NS; not significant. Magenta horizontal bars represent the median. When using a parametric t-test, there is also a significant difference for the LUA–AVG synapse between dmd-5;dmd-11 mutant hermaphrodites and dmd-5;dmd-11 mutant hermaphrodites that overexpress DMD-5 (*P < 0.05). j, Summary of data. TRA-1 and DMD proteins are commonly thought to work as transcriptional repressors22. As dmd-5 and dmd-11 are already dimorphically expressed in AVG in embryos and L1 stage animals (not shown in this schematic), there must be other timer mechanisms that control the onset of pruning. For example, DMD-5 and DMD-11 may work together with a regulatory factor of the stage-specifically acting heterochronic pathway. Furthermore, we hypothesize that other neurons, such as the AVA neuron, may have its own complement of sex-specific dmd genes that control pruning.
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Oren-Suissa, M., Bayer, E. & Hobert, O. Sex-specific pruning of neuronal synapses in Caenorhabditis elegans. Nature 533, 206–211 (2016). https://doi.org/10.1038/nature17977
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