Decoding sexual dimorphism of the sex-shared nervous system at single-neuron resolution

Abstract

Sex-specific behaviors are often attributed to differences in neuronal wiring and molecular composition, yet how genetic sex shapes the molecular architecture of the nervous system at the individual neuron level remains unclear. Here, we use single-cell RNA sequencing to profile the transcriptome of sex-shared neurons in adult Caenorhabditis elegans males and hermaphrodites. We uncover widespread molecular dimorphism across the nervous system, including in previously unrecognized neuron-types such as the touch receptors. Neuropeptides and signaling-related genes exhibit strong sex-biased expression, particularly in males, reinforcing the notion that neuropeptides are crucial for diversifying connectome outputs. Despite these differences, neurotransmitter identities remain largely conserved, indicating that functional dimorphism arises through modulatory, not identity-defining, changes. We show that sex-biased expression of neurotransmitter-related genes correlates with bias in outgoing synaptic connectivity and identify regulatory candidates for synaptic wiring, including both shared and sex-specific genes. This dataset provides a molecular framework for understanding how subtle regulatory differences tune conserved circuits to drive sex-specific behaviors.

Fig. 1.. scRNA-seq in both sexes of C. elegans.

(A) Schematic of the experimental design. Hermaphrodite and male C. elegans harboring the pan-neuronal marker rab-3p(prom1)::2xNLS::TagRFP were subjected to cell dissociation. RFP⁺ cells were purified by FACS. The single-cell RNA transcriptomes were obtained using 10x Genomics (www.10xgenomics.com). (B) UMAP representation of all cell clusters identified with their neuronal identity. Previously published (20) (CeNGEN) dataset or manual assignments were both used to determine the ID of the neurons. (C) UMAP representation of cell clusters with a shifted overlay to illustrate the overlap. Datasets are YA hermaphrodites (magenta), YA males (green), and CeNGEN L4 hermaphrodites (yellow). (D) Receiver operating characteristic (ROC) curve showing true-positive rate (TPR) versus false-positive rate (FPR) for hermaphrodites, males, hermaphrodites-males together, and CeNGEN compared to ground-truth expression data using the 65 cell types with nine or more cells in hermaphrodite and male clusters. (E) Precision-recall (PR) curve of recall (TPR) versus precision [1 − false discovery rate (FDR)] curve. Red dots represent thresholds 1 to 4. Gray shading represents 95% confidence intervals. (F) Schematic of AIM neurons neurotransmitter switch from glutamatergic identity (in hermaphrodites) to cholinergic identity (in males). (G) AIM representation in the UMAP projection of all cells (inset marked in orange). UMAP projections depict expression of unc-17, cho-1 cholinergic identity genes, eat-4 glutamatergic identity gene, and transcriptional factor lin-29 gene within AIM cluster. (H) High expression of ins-39 in males. AFD representation in the UMAP projection of all cells (inset marked in orange). UMAP projection depicts expression of ins-39 gene within AFD cluster. (I) Dimorphic expression of mab-3. Left: SMD representation in the UMAP projection of all cells (inset marked in orange). Right: UMAP projection depicts expression of mab-3 gene within SMD cluster. In (G) to (I), heatmaps represent expression levels (log₁₀). Created in BioRender. Oren, M. (2025) https://BioRender.com/swfhj3a.

Fig. 2.. Comparative analysis of sex-shared neurons reveals widespread molecular dimorphism.

(A) Number of DEGs in the sex-shared neuronal clusters. Clusters are divided to sensory (triangle), interneuron (hexagon), and motor (circle) neurons. A gene was considered differentially expressed with an average log fold change of >1, ≥9 cells per cluster in both sexes, P < 0.05, and an expression of threshold of ≥2 in at least one sex (see Materials and Methods). (B) Gene ontology (GO) term analysis for the 2215 DEGs in both sexes and in hermaphrodite and males separately (only genes meeting the criteria of log fold change of >1, ≥9 cells per cluster in both sexes, P < 0.05, and a threshold of ≥2 in at least one of the sexes are included) reveals specific enrichment for synaptic and signaling gene expression. WormCat P values are determined by one-sided Fisher’s test with FDR correction. NS, not significant; GTPase, guanosine triphosphatase; ATPase, adenosine triphosphatase. (C) Percentage of DEGs from each gene family as a fraction of the number of genes in the specific family. (D) The number of instances of DEGs in the gene families presented in (C) divided according to DEGs that are enriched in males (green) and hermaphrodites (magenta). Only significant comparisons are shown. A DEG that is observed in more than one cluster was counted accordingly (see Materials and Methods). For (D), we performed a two-sided Fisher’s exact test, followed by P value Bonferroni correction for multiple comparisons. (E) Number of DEGs across cells (light blue) and the number of binary genes (gray).

Fig. 3.. Sexual dimorphism of neuropeptides across the nervous system.

(A) Bubble plot of differentially expressed neuropeptide genes between sexes. Bubble size indicates average fold change in gene expression (only genes with a log fold change of >1, ≥9 cells per cluster, P < 0.05, and a threshold of ≥2 in at least one sex included), and bubble color shows sex enrichment (green, male; magenta, hermaphrodite). Rows represent neuron types (sensory, interneurons, and motor). Columns represent genes. (B) UMAP projection of AUA, AVE, RMH, DVC, AVB, AIB, PDB, URA, M2, DVB, SAB, PLM, SIB, and AVM clusters showing expression of nlp-11 gene in both sexes. Heatmap shows expression levels. Bubble plot representation of nlp-11 gene expression in clusters as in (A) of two sexes, arranged left to right by P value (most to least significant). Bubble size represents average log fold change, and bubble color (green, males; magenta, hermaphrodites) represents enrichment in each sex. (C and E) Representative confocal micrographs showing multicolored neuronal nuclei in the NeuroPAL strain in head (C) and tail (E), where neurons are identified by color barcode (88, 98). These images were used to identify neurons expressing the nlp-11(syb4759[nlp-11::SL2::GFP::H2B]) reporter in young adult hermaphrodites and males. Scale bars, 10 μm. (D) Quantification of nlp-11 fluorescent intensity from (C) in head neurons AUA, AVE, AIB, URAD, URAV, M2, SABV, SIB, and AVM. a.u., arbitrary units. (F) Quantification of nlp-11 fluorescent intensity from (E) in tail neurons DVC, PDB, PLML, PLMR, and DVB neurons in both sexes at YA stage. Number of worms (hermaphrodite, male) were as follows: AUA, AVE, URAD, URAV, PDB, PLML, and PLMR (14, 14); AIB (12, 12); M2 (12, 13); SABV and AVM (13, 13); SIB (9, 9); DVB (14, 12); and DVC (14, 13). n represents number of animals in each group in which neurons were identified and recorded. Box-and-whisker plots show median (center line), 25th to 75th percentiles (box), and minimum to maximum (whiskers), with dots representing data points. Two-sided Mann-Whitney test was used for comparisons.

Fig. 4.. Neurotransmitter identity is preserved across sex-shared neuronal clusters.

(A) Left and right bubble plots representing the mean expression values of neuronal identity genes in hermaphrodites and males, respectively. Neuronal clusters and genes are color coded according to their neurotransmitter identity. (B) Bubble plot representing differentially expressed neurotransmitter identity genes between the sexes. Only genes with significant log fold change when comparing the hermaphrodite and male mean expression values are shown. Bubble size in (A) corresponds to gene expression, and that in (B) corresponds to the average log fold change in gene expression. Bubble color indicates enrichment in either sex (green, male; magenta, hermaphrodite). Rows represent neuron types, grouped color coding for neurotransmitter identity. Columns represent individual genes. (C) Bar plot representing the difference in outgoing connectivity between males and hermaphrodites in each cluster (the number of outgoing connections in males minus the number of outgoing connections in hermaphrodites for each neuron that was examined). Numbers are represented as an absolute value (see Materials and Methods for more details). Magenta represents a higher value in hermaphrodites, while green represents a higher value in males. (D) Two-tailed Pearson correlation analysis between the difference in connectivity (outgoing connections, all synaptic connections, and incoming connections) and the average log fold change of neurotransmitter identity genes (see Materials and Methods for more details). The numbers representing log fold change were correlated with the number representing the difference in outgoing connectivity. The colors of the dots correspond to the neurotransmitter identity colors shown, except those that overlap and are labeled red (A). For (D), we performed a two-sided Pearson correlation test.

Fig. 5.. Sexually dimorphic properties of PLM neuron.

(A) Illustration of PLM sensory neuron (www.wormatlas.org/). (B) Bubble plot of nlp-11, flp-8, dop-3, sng-1, and mec-8 expression in PLM. Size, average log fold change; color: green, male; magenta, hermaphrodite enrichment. (C) Table showing gene functions from (B). (D to H) Representative confocal micrographs and quantification of gene expression in PLM (white dashed circle): (D) nlp-11::SL2::GFP::H2B (n = 16 hermaphrodites and 15 males), (E) flp-8p::GFP (n = 15 per group), (F) dop-3::RFP (n = 19 per group), (G) sng-1::GFP in PLM neurite (n = 10 per group), and (H) mec-8 smFISH probe with mec-4::gfp and 4′,6-diamidino-2-phenylindole (DAPI) (n = 27 hermaphrodites and 26 males). Scale bars, 10 μm. (I) Left: Illustration of gentle/harsh touch in C. elegans. Created in BioRender. Oren, M. (2025) https://BioRender.com/rvrqnms. Middle: Posterior gentle touch responses of both sexes and harsh tail-touch responses tested on the same animals (n = 20 per group). Right: Anterior and posterior gentle touch responses of both sexes (n = 15 per group). (J) Normalized GCaMP6s calcium responses in PLM (n = 13 animals per group). Heatmaps show calcium levels of individual worms. (K) Mean and SEM calcium traces from (J). (L) Mean traces showing normalized amplitudes of PLM, zoomed in at frequencies of 0 to 0.2 Hz. (M) AUC (area under the curve) measured from PLM amplitudes for chosen frequencies (0 to 2 Hz). (N) Posterior gentle touch responses of both sexes after RNAi: control (n = 15 each), nlp-11 (n = 14 hermaphrodites and 15 males), mec-8 (n = 15 hermaphrodites and 14 males), and mutants nlp-11(syb530), mec-8(ok2043) (n = 14 each). (O) Posterior gentle touch responses of hermaphrodites in wild-type (n = 17), mec-8 (ok2043) (n = 18), and mec-8 rescue in PLM (n = 18). Box plots: median (line), 25th to 75th percentile (box), minimum to maximum (whiskers), and dots = individual data points. In (D) to (I) and (M), we performed a two-sided Mann-Whitney test for each comparison. In (N) and (O), we performed a Kruskal-Wallis test, followed by a Dunn’s multiple comparison test.

Fig. 6.. Expression of DEGs in PHC.

(A) Illustration depicting the location of PHC sensory neurons and their neurite length (36) in both sexes, as outlined in WormAtlas. Created in BioRender. Oren, M. (2025) https://BioRender.com/vzo4swq. (B) Validation of previously reported DEGs. Left: PHC representation in the UMAP projection of all cells (inset marked in orange). UMAP projection depicts expression of dmd-3, eat-4, and flp-11 gene within PHC cluster. Heatmap represents expression level. (C) GO term enrichments for DEGs in PHC cluster, including the number of genes in each GO term. (D) Bubble plot representation of capa-1, F32B4.5, dop-3, and lad-2 DEGs in PHC cluster. Bubble size represents average log fold change, and bubble color (green, males; magenta, hermaphrodites) represents enrichment in each sex. (E) Table lists the established functions for genes from (D). (F to I) Representative confocal micrographs showing expression and quantification of various genes in PHC neurons (white dashed circle). (F) capa-1p::GFP; hermaphrodites, n = 14; males, n = 16. (G) F32B4.5p::GFP, n = 10 animals per group. (H) dop-3p::RFP, n = 14 animals per group. (I) lad-2p::GFP, n = 15 animals per group. (J and K) Representative confocal micrographs of eat-4p11::GFP expression in PHC neurons in wild-type and lad-2 (tm3056) mutant L4 males, n = 16 animals per group (J), and hermaphrodites, n = 10 animals per group (K). Scale bars, 10 μm. In the box-and-whisker graph, the center line in the box denotes the median, while the box contains the 25th to 75th percentiles of the dataset, and whiskers define the minimum and maximum value with dots showing all points. In (F) to (K), we performed a two-sided Mann-Whitney test for each comparison.

Fig. 7.. Identification of genes regulating connectivity.

(A) Schematic of correlation analysis. Rows: Number of outgoing connections calculated according to (37) across neurons. Columns: Mean expression of genes across neurons. Pearson correlation was calculated for connectivity numbers and mean expression per gene, separately for hermaphrodites and males. (B) Mean expression of VM106R.1 across outgoing connections in both sexes. (C) Venn diagram showing significantly correlated genes in hermaphrodites and in males and the number of neuronal genes within these groups (brown) (top). Heatmap of Pearson correlation coefficients of significantly correlated and overlapping genes (bottom). (D) Heatmaps of Pearson correlation coefficient of significantly correlated neuronal genes in hermaphrodites (top) and in males (bottom). (E) Expression of significant neuronal genes from (D). Size represents average log fold change, and color represents sex enrichment. (F) Mean expression of zig-1 (right) and ttx-7 (left) across outgoing connections in hermaphrodites and males. (G to I) Representative confocal micrographs and quantification of (G) glr-3p::SNB-1::venus in RIA in wild type (hermaphrodites, n = 15; males, n = 14) and ttx-7(nj40) (n = 15 per group) (left). Percentage of animals showing mislocalization of SNB-1 (SyNaptoBrevin related-1) (right). (H) glr-3p::SNB-1::venus expression in RIA in wild type (hermaphrodites, n = 15; males, n = 14) and zig-1(ok784) (n = 15 per group) (left). Quantification of venus signal (right). (I) dat-1p::novogfp::CLA-1 expression in CEP neurons in wild type and ttx-7(nj40) (left). Quantification of CLA-1 puncta in CEP (right). n = 15 per group. Scale bars, 10 μm. In the box-and-whisker graph, the center line in the box denotes the median, while the box contains the 25th to 75th percentiles of the dataset, and whiskers define the minimum and maximum value with dots showing all points. (C) Pearson correlation analysis with Benjamini-Hochberg correction for multiple comparisons was done. (G) Two-sided Fisher’s exact test was done for each comparison. [(H) and (I)] Two-sided Mann-Whitney test was done for each comparison. Magenta, hermaphrodites; green, males.