2.2 Transgenic strains

SEH306 pdrEx80 (sre-1p::gfp; unc-122p::dsRed) was made by injecting N2 adults with a plasmid containing 3 kb of the sre-1 promoter fused upstream of gfp (sre-1p::gfp) and a unc-122p::dsRed reporter as the co-injection marker. The sre-1p::gfp; unc-122p::dsRed extrachromosomal array was integrated using a UV crosslinker and backcrossed 6x with our N2 laboratory strain to create SEH318 pdrIs9 (sre-1p::gfp; unc-122p::dsRed). SEH318 was crossed into YY158 nrde-3(gg66) X to build SEH321 nrde-3(gg66) X; pdrIs9 (sre-1p::gfp; unc-122p::dsRed). All mutant strains expressing pdrIs9 (sre-1p::gfp; unc-122p::dsRed) were built by genetically crossing SEH318 into each mutant strain and confirmed by PCR or Sanger sequencing (Supplementary Table S1).

2.3 Adult neuronal isolation

Isolation of adult ADL neurons of SEH318 pdrIs9 (sre-1p::gfp; unc-122p::dsRed) CON and PD_Phe adults and SEH321 nrde-3(gg66) X; pdrIs9 (sre-1p::gfp; unc-122p::dsRed) CON and PD_Phe adults were done as described by (Kaletsky et al., 2016). Briefly, staged 1-day old adult animals grown at 20°C on 100 mm NGM plates seeded with 20x concentrated OP50 were harvested and washed with M9 buffer until the supernatant was clear. 750 μL of Neuronal Isolation Lysis Buffer (200 mM DTT; 0.25% SDS; 20 mM HEPES pH 8.0; 3% sucrose) was added to a packed pellet of worms (up to 250 μL) and rotated at room temperature for 6.5 min. The worm mixture was then washed with M9 and 500 μL of a freshly made pronase (Sigma P6911) solution (20 mg/mL) was added and vigorous pipetted for 100 times (~40 s) in 2 min intervals until ADL head neurons dissociated from the worm body as monitored using fluorescence microscopy. Once worm heads disintegrated (~14–16 min), the pronase reaction was stopped with 250 μL of 1x PBS buffer supplemented with 2% Fetal Bovine Serum (Fisher Scientific 10082139) and iced. The worm mixture was then filtered through 5 μM filter, stored on ice, and immediately transported to the cell sorting facility (~15 min interval) where it was sorted upon arrival.

2.4 Fluorescence activated cell sorting (FACS)

ADL chemosensory neurons were collected from a neuronal population obtained from 1-day old adults using with a Becton Dickinson FACS Aria III Cell Sorter. Neurons collected from 1-day old wild-type N2 adults were used to set the FACS gate. The average total FACS event for a strain and condition ranged between 128,229 and 846,079. FACS events were collected directly into TRIzol (Life Technologies) and immediately frozen in dry ice. All frozen samples were stored at −80°C until ready for processing.

2.5 RNA extraction, mRNA-Seq library preparation, and data analyses

Total RNA extraction of FACS events was done using NEB Monarch Total RNA Miniprep (New England Biolabs [NEB] T2010). Four biological independent RNA-Seq libraries for a strain and a condition were prepared using NEBNext^® Poly(A) mRNA Magnetic Isolation Module (NEB E7490S) and NEBNext^® Ultra™ II RNA Library Prep with Sample Purification Beads (NEB E7775S) following the recommendations of the manufacturer. Indexing of libraries was done using NEBNext^® Multiplex Oligos for Illumina^® (Index Primers Set 1) (NEB E7335S) and NEBNext^® Multiplex Oligos for Illumina^® (Index Primers Set 2) (NEB E7500S) according to the manufacturer’s instructions. Data analysis was performed on the CLC Genomics Workbench (Qiagen) with differential expression assessed using EdgeR (Robinson, McCarthy, and Smyth, 2010) as previously described (Ow et al., 2018). Raw data can be accessed through NCBI GEO GSE268801.

2.6 Gene ontology (GO) term analyses

GO terms enrichment was determined using Gorilla (Eden et al., 2009).

2.7 Statistical significance

Statistical significance was determined using GraphPad Prism version 9.5 (San Diego, CA, United States). The significance of hypergeometric distributions was determined using a hypergeometric p-value software calculator (https://systems.crump.ucla.edu/hypergeometric/index.php).

2.8 Bioinformatics analysis of the PD motif

To identify genes whose upstream regulatory sequence contain the DAF-3/SMAD binding site and the conserved sequence of the PD motif, a custom BLAST database including 500 bp upstream of ATG translational start sites for all genes in the C. elegans genome was created using NCBI. This database was used to search for genes whose 5′UTR contained “GTCTA” or the “CT[AT]TAA[AT]TTN(0,3)[AC]AN(0,2)TTTTG[CT]CATAA[TA]C[TC]” pattern, where “N” could be any nucleotide and the numbers signify the range of possible number of nucleotides. The overlap of the genes in each list were genes that contained the full PD motif. The python script used for this analysis can be found at https://github.com/mnishiguchi7/Hall_Lab/blob/main/script.

2.9 Single molecule fluorescence in situ hybridization (smFISH)

One-day old adults expressing the sre-1p::gfp transgene were used for smFISH (Ji and Oudenaarden, 2012) and carried out as previously described (Sims et al., 2016). Custom Stellaris™ smFISH probe sets consisting of 27 probes for msp-19 (Quasar^® 570) and 32 probes for gfp (Quasar^® 670) were designed using the Stellaris™ probe designer website (LGC Biosearch Technologies). Probe sequences will be made available upon request. Images were acquired on a Leica DM5500 B microscope equipped with a Leica CTR5500 electronic box with a Hamamatsu Digital Camera C10600 ORCA R2 using maximally projected images of z-stacks as optimized by the Leica LAS AF 3.1.0 software followed by quantification on ImageJ. SmFISH experiments were done using two independent biological samples.

2.10 Immunofluorescence

One-day old adults expressing the sre-1p::gfp transgene were used for immunofluorescence using the method reported by Serrano-Saiz et al. (2017). In brief, 1-day old adults were fixed overnight with 1x PBS supplemented with 4% paraformaldehyde (Thermo Fisher AA433689M) followed by washing in PT Buffer (1x PBS, 0.5% Triton X-100). Fixed worms were reduced for ~18–20 h at 37°C with 5% ß-mercaptoethanol in 1% Triton X-100 and 0.1 M Tris-HCl pH 7.5 followed by washing with TT Buffer (1% Triton X-100, 0.1 M Tris-HCl pH 7.5) and CTT Buffer (1 mM CaCl₂, 1% Triton X-100, 0.1 M Tris-HCl pH 7.5). Worms were pelleted and shaken at room temperature for ~30 min in CTT Buffer supplemented with 1 mg/ml of collagenase Type IV (Millipore Sigma C5138) until they fragmented (20%–50%). Fragmented worm samples were rinsed with PT Buffer and incubated with 1x PBS supplemented with 1 mg/mL of NaBH4 (Millipore Sigma 71320) for 1 h at 4°C with rotation. Samples were washed with PT Buffer and blocked in 0.2% gelatin solution [1x PBS, 0.5% Triton X-100, 0.2% gelatin (Millipore Sigma G7041)] supplemented with 2% donkey serum (Jackson Immunoresearch 102644-006) at room temperature for 2 h followed by an overnight rotation with mouse anti-MSP (Developmental Studies Hybridoma Bank, University of Iowa, United States; 4A5) (1:5) or chicken anti-GFP (Thermo Fisher A10262) (1:500) at 4°C in 0.2% gelatin solution supplemented with 0.5% donkey serum. Next day, samples were rinsed with PT Buffer followed by rotation in PT Buffer for 2 h at room temperature. Samples were incubated with goat anti-mouse Alexa Fluor 568 (Thermo Fisher A11031) (1:100) or goat anti-chicken Alexa Fluor Plus 647 (Thermo Fisher A32933) (1:1000) and rotated in the dark overnight at 4°C in 0.2% gelatin solution supplemented with 0.5% donkey serum. Next day, samples were washed with PT Buffer followed by rotation in PT Buffer for 1.5 h at room temperature in the dark. The DNA counterstain DAPI (Thermo Fisher D1306) was added and rotated for an additional 30 min followed by rinsing with PT Buffer. Worm samples were pelleted and VectaShield H1000 antifade mounting medium (Vector Laboratories) was added. Samples were imaged on a Leica DM5500 B microscope fitted with a Leica CTR5500 electronic box and a Hamamatsu Digital Camera C10600 ORCA R2 with maximally projected images of z-stacks optimized by the Leica LAS AF 3.1.0 software followed by quantification on ImageJ (NIH). Immunofluorescence was done using two to three independent biological replicates.

2.11 Expression of osm-9p::gfp

Imaging of osm-9p::gfp expression in ADL was assessed as previously done (Sims et al., 2016) except that animals were dye filled with either DiD (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt, Thermo Fisher D7757) (for 2–4 h) or DiI (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate, Sigma Aldrich 42364) (for 30 min) (Wallace et al., 2016).

2.12 Octanol avoidance assays

Avoidance assays to the odorant 1-octanol (TCI O0036) by 1-day old adults was done as detailed by Troemel et al. (1997). In short, square petri dishes (Fisher Scientific FB0875711A) containing 2% BD Difco Bacto agar (Fisher Scientific DF0140154) in 5 mM potassium phosphate, 1 mM CaCl₂, and 1 mM MgSO₄ was divided into six equally sized sectors (A-F) where the two most distal sectors (A and F) from the center served as the sites for the deposition of 1-octanol and the control (100% ethanol) with 1 μL 1 M NaN₃ anesthetic pipetted equidistantly near the plate’s edge. One-day adults grown at 20°C in NGM plates seeded with OP50 were collected and washed with S-basal buffer (0.1 M NaCl, 0.05 M potassium phosphate pH 6.0) until the supernatant was clear followed by one rinse in dH₂O. Worms (~200) were placed onto the center (between sectors C and D) of the petri dish equidistantly to sectors A and F where 1 μL of 100% ethanol (sector F) and 1-octanol (sector A) was immediately pipetted. Excess liquid was rapidly removed using a kimwipe without disturbing the animals or puncturing the agar. Assays were placed an undisturbed location at room temperature for 1 h after which worms were assessed for their reaction to octanol using the following formula: Avoidance Index = (A + B)-(E + F)/N; where A, B, E and F are the number of animals in these sectors and N is the total number of animals in all the six sectors.

2.13 Ascr#3 avoidance assays

Avoidance of ascr#3 was determined off-food using 1-day old adults grown at 20°C on NGM plates seeded with OP50 and performed essentially as previously done (Sims et al., 2016) except that forward-moving adults were first tested for their reaction to a drop of benign M13 buffer (30 mM Tris-HCl pH 7.0, 100 mM NaCl, 10 mM KCl) deposited near their tail and migrating up to their nose. 10–12 adults that were indifferent upon encountering M13 buffer were then tested for their behavior when presented with 100 nM ascr#3 and 1 M glycerol. A backward-moving motion consisting of 1.5 body lengths in 4 s was considered an avoidance response. Only animals that were tested in the same timeframe were subjected to statistical comparisons to the wild-type N2 control.

2.14 Chemotaxis assays

Chemotaxis assays for the benzaldehyde and diacetyl attractants were performed on 1-day adults as described in (Bargmann et al., 1993; Hart, 2006). Assays plates consisting of 2% BD Difco Bacto agar (Fisher Scientific DF0140154) in 5 mM potassium phosphate, pH 6.0, 1 mM CaCl₂, and 1 mM MgSO₄ were prepared on 100 mm round petri dishes. Two marks (one for the attractant and one for the 100% ethanol control), 180^o degrees opposite each other, were indicated on the bottom of the plate near its edge where 1 μL of 1 M NaN₃ anesthetic was deposited. Worms grown at 20°C on OP50-seeded NGM plates were collected and washed with S-buffer (0.1 M NaCl, 0.05 M potassium phosphate pH 6.0) until the supernatant was cleared of their bacteria food followed by one wash with dH₂O. Pipette worms (~200) onto the center of the assay plate, equidistantly from the marks and slightly off center. Excess liquid was quickly soaked off using a kimwipe without disturbing the animals or puncturing the agar. Immediately place 1 μL of the attractant and the ethanol on their respective marks, assay plate was closed with the lid and placed in an undisturbed location at room temperature for 1 h. Worms that were within a 0.5 cm radius of the spot where the attractant and the ethanol were deposited were then counted and the chemotaxis index was calculated with the following formula: Chemotaxis Index (CI) = (number worms at the attractant after 1 h - number worms at control ethanol after 1 h)/total # of worms. Chemotaxis indexes of +1.0 to −1.0 indicated perfect attraction or repulsion, respectively.

3.2 NRDE-3 is required for differential expression of major sperm protein genes in ADL

We showed previously that osm-9 downregulation in PD_Phe ADL neurons requires components of the somatic nuclear RNAi pathway, including NRDE-3, NRDE-1, and NRDE-4 (Sims et al., 2016). To examine whether the somatic nuclear RNAi complex plays a broader role in programming an animal’s mRNA transcriptome in response to early-life environmental experience, we set out to identify additional targets of NRDE-3 AGO by performing RNA-Seq of control and PD_Phe ADL neurons collected from the nrde-3(gg66) mutant strain. Using an expression cutoff of RPKM >10, we identified 6001 and 6377 genes that are expressed in the ADL neurons of control and PD_Phe nrde-3 adults, respectively (Supplementary Table S11). Comparison of the transcriptomes from wild-type and nrde-3 control ADL neurons resulted in a 77.7% similarity (5206 genes) (p = 5.17e-3045) (Supplementary Table S12). Similarly, the transcriptomes of the wild-type and nrde-3 PD_Phe neurons shared an 87.5% similarity (5951 genes) (p = 4.07e-3939) (Supplementary Table S12). Differential expression analysis of ADL genes in the nrde-3 mutants revealed 105 genes that are downregulated and have functions in signal transduction, G protein-coupled receptor signaling, dauer entry, and detection of stimuli (Figures 2A,B; Supplementary Table S13). In addition, we found 96 genes that are upregulated in nrde-3 PD_Phe ADL neurons compared to controls with functions in endoplasmic reticulum stress response pathways, organic molecule transport, and histidine metabolism (Figures 2A,C; Supplementary Table S14).

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FIGURE 2

The NRDE-3 somatic nuclear RNAi pathway is required for differential expression of ADL genes. (A) Volcano plot representing nrde-3(gg66) CON and PD_Phe adult ADL RNA-Seq data. Dotted line indicates FDR cutoff of p < 0.05; red dots represent mRNAs that are either significantly up- or downregulated in PD_Phe compared to CON. (B,C) GO terms of genes that are most significantly (B) decreased or (C) increased based on FDR <0.05 in nrde-3 PD_Phe compared to nrde-3 CON. (D,E) Venn diagrams comparing WT and nrde-3 gene lists for (D) DE Up and (E) DE Down genes.

Next, we identified which genes were dependent on NRDE-3, either directly or indirectly, for their differential expression in wild-type ADL neurons. First, we compared the 116 genes upregulated in wild-type PD_Phe ADL neurons with the 96 genes that are upregulated in nrde-3 PD_Phe ADL neurons (Supplementary Tables S5, S15). We found only 15 genes overlapping between the two gene lists, indicating that 101 genes require NRDE-3 for their upregulation in PD_Phe ADL neurons in wild-type adults (Figure 2D; Supplementary Table S16). Amongst this gene set were 33 (32.7%) secreted protein genes, 26 (25.7%) transmembrane domain genes, 10 (9.9%) serpentine receptor genes involved in chemoreception, and 10 (9.9%) lipid metabolism genes (Suh and Hutter, 2012; Zhang et al., 2013). The lipid metabolism genes included the Rieske-like oxygenase daf-36 important for cholesterol metabolism, Δ9 desaturase fat-7 involved in lipid metabolism, and six vitellogenins, vit-1/-2/-3/-4/-5/-6, that have known functions to transport lipids into the germ line (Ow et al., 2021). We then compared the 595 downregulated ADL genes in wild-type PD_Phe adults to the 105 genes that are differentially downregulated in nrde-3 mutants. We found 53 genes overlapping between the two gene lists, indicating that 542 downregulated genes in wild type ADL neurons were dependent on NRDE-3 function for their differential expression (Figure 2E; Supplementary Tables S5, S15, S17). Notably, sperm-expressed genes were members of this gene set, including 20 msp genes and the two PP1 phosphatases responsible for sperm development and motility, gsp-3 and gsp-4 (Wu et al., 2012). Additional genes in this group include 81 collagen genes (14.9%), 273 genes (50%) predicted to encode secreted proteins, 77 genes (14.2%) encoding transmembrane domain proteins, and 58 endoplasmic reticulum (ER) genes (10.7%) (Suh and Hutter, 2012), the latter of which include those in ER unfolded protein response and are amongst the most significantly upregulated genes present in nrde-3 PD_Stv adults. Also included is the odr-7 gene, required for the function of the AWA chemosensory neurons in a germline-dependent neural circuitry response to the odorant diacetyl during development (Sengupta et al., 1994; Fujiwara et al., 2016). Together, these results show that NRDE-3 plays a significant role in the regulation of genes in ADL neurons, particularly after passage through dauer due to crowding. The NRDE-3-dependent differentially expressed genes included those with functions related to fertility, reproduction, odor response, and fat metabolism, which are phenotypic traits that we have previously observed to be significantly altered in adults that experienced dauer (Sims et al., 2016; Ow et al., 2018; Ow et al., 2021).

3.3 A conserved upstream regulatory sequence is enriched for genes with NRDE-3 dependent expression changes

In C. elegans, neuronal gene expression is modular in that the presence and arrangement of transcription factor binding sites in the upstream regulatory sequences can specify in which neurons that gene is transcribed (Stefanakis et al., 2015; Reilly et al., 2020; 2022; Hobert, 2021; Leyva-Díaz and Hobert, 2022). We next asked if ADL-expressed germline genes have the hallmarks of neuronal gene expression in their upstream regulatory sequences. C. elegans expresses over 40 MSP genes, and 20 of these genes exhibited differential expression in our ADL gene set (Supplementary Table S10). Previously, an ADL-enhancer element consisting of the CASCTG E-box motif (S=C or G) was found by analyzing promoters of ADL-expressed chemoreceptor genes (McCarroll et al., 2005). A similar motif of CACGTG was identified as overrepresented in msp promoter regions (McCarroll et al., 2005). While the modified E-box CACGTG motifs were present in 7 of the MSP genes expressed in ADL (msp-19, -36, -55, -56, -64, -142, -152), we also identified 1 to 3 copies of the ADL specific CASCTG E-box motif in the 5’ UTR sequences of 11 MSP genes (msp-10, -19, -31, -38, -45, -50, -51, -55, -77, -81, -113) (Figure 3A). These observations underscore the possibility that msp expression in ADL could be dictated at the transcriptional level for ADL specification.

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FIGURE 3

Genes with NRDE-3-dependent expression changes are enriched for a conserved sequence in their upstream regulatory regions. (A) Diagrams of upstream regulatory regions of osm-9, msp-19, and msp-36 genes. Names of sequence motifs, location relative to translational start sites, and known interactors are indicated. (B,C) Venn diagrams comparing the set of genes containing an MSP motif in their 5′ UTRs with (B) all WT and (C) NRDE-3 dependent DE Up and DE Down gene lists. p-values were determined using a hypergeometric distribution.

We next asked whether the differential regulation of gene expression in ADL is also dependent upon upstream regulatory sequences. We previously identified a cis-acting motif located in the osm-9 promoter, referred to as the “PD motif”, that was required for its transcriptional silencing in ADL of PD_Phe adults (Sims et al., 2016). This motif is composed of a DAF-3/SMAD binding site (GTCTA) and a ~30 base pair conserved sequence (CT[A/T]TAA[A/T]TTN_0-3[A/C]AN_0-2TTTTG[C/T]CATAA[T/A]C[T/C]) that is responsive to TGF-ß signaling (Sims et al., 2016; Pekar et al., 2017). While >6000 genes possess the DAF-3 binding site in their upstream regulatory regions, 1008 genes contain the conserved sequence, and 116 genes the full PD motif (Supplementary Table S18). Interestingly, nine genes, in addition to osm-9, that were downregulated in WT postdauer ADL neurons compared to controls contain a full PD motif: msd-3, msp-36, msp-51, msp-65, msp-81, msp-79, col-133, B0205.14, and T16G1.2 (Supplementary Tables S5, S18). Since five of these genes were MSP genes, we next examined the overlap of differentially expressed genes in ADL and the list of genes containing just the conserved sequence in their upstream regulatory regions. For all genes differentially expressed in ADL, we found an overlap of 4 and 39 genes for the up- and downregulated gene lists, respectively (Figure 3B). A comparison of genes that exhibit NRDE-3-dependent differential expression in ADL with the list of genes with conserved sequences revealed a similar overlap of 4 and 36 genes for the up- and downregulated gene set, respectively (Figure 3C; Supplementary Tables S16–S18). The downregulated genes with conserved sequences included 19 MSP genes (msp-3, -10, -19, -31, -36, -38, -45, -49, -50, -51, -55, -56, -65, -77, -78, -79, -81, -113, -152). Future work will determine if the downregulation of MSP genes in postdauer ADL neurons may be attributed to binding of an unidentified factor to the conserved sequence in their promoter (Figure 3A). Given the enrichment of the conserved sequence in MSP gene regulatory sequences, we will henceforth refer to this ~30 base pair conserved sequence element in the PD motif as the “MSP motif”.

3.4 Major sperm proteins are expressed in ADL neurons

Since the expression of spermatogenic genes in wild-type ADL neurons was unexpected, we sought to validate the expression of msp genes in ADL. First, we performed single molecule fluorescence in situ hybridization (smFISH) to detect the presence of msp mRNAs in wild-type and nrde-3 control adults carrying the sre-1p::gfp transgene expressed in ADL neurons (Supplementary Figure S1). Our RNA-Seq results showed that wild-type control adults express msp-19 at an 11-fold higher level than in nrde-3 control ADL neurons (RPKM = 129.91 vs. RPKM = 11.61, respectively) (Supplementary Table S19); thus, we chose msp-19 as the template for smFISH probes design. However, because of the high sequence homology of the msp genes (Kasimatis and Phillips, 2018), it is likely that most, if not all, msp mRNAs were detected using our probes. Using smFISH, we detected msp-19 mRNAs in wild-type control ADL neurons (Figures 4A,B; Supplementary Figure S2). Consistent with our RNA-Seq results, the msp-19 signal was significantly decreased in nrde-3 mutants, validating that NRDE-3 is required for the expression of msp mRNA in control ADL neurons (Figures 4A,B; Supplementary Figure S2). As a positive control, we also performed smFISH with probes detecting gfp mRNA, and we observed no significant differences in gfp fluorescence intensity in ADL neurons of wild-type and nrde-3 control adults (Figure 4A; Supplementary Figure S2).

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FIGURE 4

Major sperm proteins are expressed in ADL neurons. (A) Representative images of smFISH of ADL neurons with probes against msp (red) and gfp (magenta) mRNAs using 1-day old wild-type and nrde-3(gg66) CON adults. sre-1p::gfp transgene fluorescence shown in green. Images of animal heads shown in Supplementary Figure S2. (B) Quantification of msp relative to gfp smFISH intensity in ADL; a.u. are arbitratry units. ** p < 0.01, Student’s t-test of three independent experiments. (C) Representative images of immunostainings using antibodies against MSP (magenta) and GFP (yellow) on 1-day old wild-type CON and PD_Phe adults. sre-1p::gfp transgene fluorescence shown in cyan. Images of animal heads are shown in Supplementary Figure S3. (D) Quantification of MSP immunofluorescence intensity relative to the intensity of GFP immunofluorescence. **** p < 0.0001, Student’s t-test of two independent experiments. (E) Representative images of immunostainings using antibodies against MSP (magenta) and GFP (yellow) on 1-day old wild-type and gsp-3(tm1647) gsp-4(y148) CON adults. sre-1p::gfp transgene fluorescence shown in cyan. Images of animal heads are shown in Supplementary Figure S4. (F) Quantification of MSP immunofluorescence intensity relative to the intensity of GFP immunofluorescence. **** p < 0.0001, Student’s t-test of three independent experiments. Number of animals are indicated by n. D, V, A, P represent the dorsal, ventral, anterior, and posterior orientation of the animal. Additional data included in Supplementary Table S20.

Next, we examined whether the msp mRNAs were being translated by performing indirect immunofluorescence in ADL neurons of wild-type control and PD_Phe adults expressing the sre-1p::gfp transgene. Using a monoclonal antibody that recognizes MSP-19, -31, -40-, 45, -50, -51, -53, -59, -61, -65, -81, -113, and -142 (Developmental Studies Hybridoma Bank, University of Iowa, United States; 4A5), we found that control ADL neurons expressed detectable MSPs, and PD_Phe ADL neurons exhibited a significant downregulation of MSP (Figures 4C,D; Supplementary Figures S3A, S3C). As a positive control, we also performed indirect immunofluorescence with anti-GFP antibody and observed similar levels of GFP fluorescence intensity between wild-type control and PD_Phe ADL neurons. As an additional control, we verified that MSP staining was visible in both control and PD_Phe adult gonads (Supplementary Figures S3B, S3D). Overall, these results demonstrate that MSPs are expressed in ADL neurons of continuously developed hermaphrodites in a NRDE-3 dependent manner and are downregulated specifically in ADL neurons of adults that experienced crowding-induced dauer.

3.5 Sperm-expressed phosphatase is required for attraction and avoidance behaviors

After verifying the expression of germline genes in ADL neurons, we next sought to determine what role they play in ADL function. The msp genes comprise a 48-membered gene family with highly conserved sequences that encode 14 kilodalton (kDa) proteins (Scott et al., 1989). MSPs are the most abundant protein in nematode sperm, comprising ~17% of their total protein and concentrating up to 3-fold in their sole locomotive structure, the pseudopod, compared to the cell body (Klass and Hirsh, 1981; Ward and Klass, 1982; King et al., 1992; Smith, 2014). Nematode sperm lack a flagellum, and MSPs substitute for a function that would otherwise be performed by structural proteins such as actin and myosin by forming a pseudopod (Nelson and Ward, 1981; Nelson et al., 1982; Roberts and Stewart, 1995; 2000; Bottino et al., 2002). MSPs polymerize into filaments at the leading edge of pseudopod in mature spermatozoa and disassemble at its trailing edge in an assembly-disassembly mechanism results in a treadmill-like form of locomotion (Sepsenwol et al., 1989; Sepsenwol and Taft, 1990; Roberts and King, 1991; King et al., 1994; Smith, 2014). In addition to enabling sperm movement, MSPs are also extracellular signaling molecules secreted by sperm that function as hormones to trigger oocyte maturation and ovulation, despite them lacking sequence motifs associated with secretory proteins (Miller et al., 2001; 2003; Kosinski et al., 2005). Although originally discovered in nematodes, the phylogenetic scope of proteins with MSP-like domains extends from plants to humans (Burke and Ward, 1983; Skehel et al., 1995; Laurent et al., 2000; Skehel et al., 2000).

Mature sperm are considered transcriptionally and translationally dormant on account of their extremely compacted protamine-bound haploid genome and cytoplasmic shedding during the terminal stage of spermatogenesis; thus, mechanisms involving post-translational modifications are important for regulating fertility (Hecht, 1998). In mammals, the serine/threonine PP1 phosphatases play critical roles in modulating sperm development and locomotion (Fardilha et al., 2011; Ferreira et al., 2022). C. elegans has two serine/threonine PP1 phosphatases, GSP-3 and GSP-4, which are required for MSP filament disassembly, and a double mutation in gsp-3 and gsp-4 results in sterility due to chromosome segregation defects and sperm immobility (Wu et al., 2012). Because of the technical difficulty to inactivate ADL-expressed msp genes simultaneously, we first investigated the potential of MSP function in ADL by characterizing the behavioral phenotypes of gsp-3(tm1647) and gsp-4(y148) mutants. Like the msp genes, gsp-3 and gsp-4 are only expressed in wild-type control ADL neurons in a NRDE-3 dependent manner (Supplementary Table S17). First, we examined MSP levels in a gsp-3 gsp-4 double mutant strain expressing the sre-1p::gfp transgene and found that MSPs continue to be detected in ADL neurons, albeit at significantly lower levels than wild-type adults (Figures 4E,F; Supplementary Figures S4A, S4C). Previous work showed that GSP-3 and GSP-4 do not affect the transcription of msp genes in sperm (Supplementary Figures S4B, S4D) (Wu et al., 2012); thus, the observed decrease in MSP in the gsp-3 gsp-4 mutant strain may be due to their effects on stability of the protein. Next, we asked whether GSP-3 and GSP-4 play a role in regulating the expression of additional genes by examining the fraction of ADL neurons that exhibit GFP due to expression an osm-9p::gfp transcriptional reporter transgene. We showed previously that in wild-type adults carrying the osm-9p::gfp transgene, GFP was expressed in ADL neurons (ADL^ON) of control adults but was significantly downregulated in PD_Phe adults (ADL^OFF), while the expression in AWA neurons remained unaffected (Figures 5A,B) (Sims et al., 2016). While gsp-4 control and PD_Phe adults exhibited similar GFP expression as wild-type ADL neurons, gsp-3 control adults had a significant reduction of animals expressing GFP, resulting in the loss of osm-9p::gfp expression difference between wild-type control and PD_Phe ADL neurons (Figure 5B). Our results are consistent with GSP-3, potentially through the action of MSPs, being required to regulate gene expression in ADL neurons of continuously developed hermaphrodites.

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FIGURE 5

Sperm genes are required for olfactory behaviors. (A) Images of osm-9p::gfp fluorescent expression in WT control and postdauer adults. Dashed ovals indicate location of ADL neurons. The dashed line delineates a part of the head. A, V, P, and D indicate the anterior, ventral, posterior, and dorsal orientation of the animal. (B) Fraction of animals with ADL^ON status in wild-type, gsp-3(tm1647), and gsp-4(y148) CON and PD_Phe 1-day adults expressing the osm-9p::gfp transgene reporter. **** and ^&&&& p < 0.0001; ns = not significant (Fisher’s exact test). Dots represent independent biological trials. (C) Fraction of 1-day old wild-type, osm-9 (ky10), gsp-3(tm1647), gsp-4(y148), and gsp-3(tm1647) gsp-4(y148) CON adults reversing in ascr#3 avoidance behavior assays. **** p < 0.0001; one-way ANOVA with Dunnett’s post-hoc test. (D) Avoidance index of 1-day old wild-type, osm-9 (ky10), gsp-3(tm1647), and gsp-3(tm1647) gsp-4(y148) CON adults in response to 1-octanol. Additional strains include expression of GSP-3 (gsp-3; sre-1p::gsp-3::gfp) or MSP-19 (gsp-3; sre-1p::msp-19::gfp lines 1 and 2) in ADL neurons in gsp-3 mutants. *** p < 0.001, **** p < 0.0001, one-way ANOVA with Tukey’s post-hoc test. (E) Fraction of 1-day old wild-type, osm-9 (ky10), gsp-3(tm1647), gsp-4(y148), and gsp-3(tm1647) gsp-4(y148) CON adults reversing upon exposure to 1M glycerol. **** p < 0.0001; one-way ANOVA with Dunnett’s post-hoc test. (F,G) Chemotaxis indexes of 1-day old wild-type, osm-9(ky10), nrde-3(gg66), gsp-3(tm1647), gsp-4(y148), and gsp-3(tm1647) gsp-4(y148) CON adults in response to (F) diacetyl and (G) benzaldehyde. Additional strains include expression of GSP-3 (gsp-3; sre-1p::gsp-3::gfp) or MSP-19 (gsp-3; sre-1p::msp-19::gfp lines 1 and 2) in ADL neurons in gsp-3 mutants. * p < 0.05, **** p < 0.0001; one-way ANOVA with Tukey’s post-hoc test. ns = not significant. Each dot in Figures 5B–F represents an independent biological trial. Additional data included in Supplementary Table S20.

Next, we sought to determine if the osm-9 gene expression changes in gsp-3 mutants altered ADL functions. Avoidance of the pheromone component ascr#3 by hermaphrodites is an OSM-9-dependent and ADL-mediated behavior, and disruption of OSM-9 levels or function result in failure to avoid ascr#3 (Jang et al., 2012; Sims et al., 2016). To examine whether gsp-3 mutants exhibit altered ascr#3 avoidance, we examined the fraction of gsp-3, gsp-4, and gsp-3 gsp-4 double mutant control adults exhibiting an avoidance response to ascr#3. As expected, the wild-type control exhibited avoidance and the osm-9(ky10) mutant showed reduced avoidance in response to ascr#3 (Figure 5C) (Jang et al., 2012). The gsp-3 and gsp-4 single mutant strains showed a small, but non-significant, decrease in their ascr#3 avoidance response, while the gsp-3 gsp-4 double mutant showed no significant difference compared to wild type (Figure 5C). These results suggest that GSP-3 and GSP-4 do not play a major role in regulation of ascr#3 avoidance behavior.

To further explore the function of ADL in the gsp-3 mutants, we tested the avoidance of volatile odorant, 1-octanol, a behavior mediated by the ADL, ASH, and AWB neurons in the absence of food (Chao et al., 2004). In the presence of 1-octanol, the wild-type controls exhibited the expected avoidance behavior (negative index indicates avoidance), which was significantly reduced in the osm-9 mutant strain (Figure 5D). Interestingly, the avoidance responses of the gsp-3 and gsp-3 gsp-4 mutant strains were eliminated (Figure 5D), suggesting that the function of other neurons may also be affected in the gsp-3 mutants. To test this possibility, we measured avoidance to 1 M glycerol, which is an OSM-9-dependent behavior mediated by the ASH sensory neurons required for avoidance behavior in response to volatile stimuli, mechanical stimulation, or osmotic shock (Kaplan and Horvitz, 1993; Troemel et al., 1995; Colbert et al., 1997; Hilliard et al., 2005). Again, the wild-type controls and osm-9 mutants exhibited the avoidance and lack of avoidance, respectively, that was expected in response to glycerol (Figure 5E). However, neither the gsp-3 or gsp-4 single mutants or the gsp-3 gsp-4 double mutant exhibited significant defects in glycerol avoidance compared to wild type (Figure 5E). These results suggest that GSP-3 phosphatase activity is required specifically for octanol avoidance mediated by nociceptive neurons.

ADL and ASH sensory neurons are closely related in their avoidance functions and synaptic connections (Cook et al., 2019). To test if other neuron classes require GSP-3 and GSP-4 for their function, we examined the chemotaxis behavior of the gsp-3 and gsp-4 single and gsp-3 gsp-4 double mutant strains in response to attractive odorants. Benzaldehyde and diacetyl are volatile odorants whose attraction to worms is mediated by the AWC and AWA neurons, respectively (Bargmann et al., 1993). As previously observed, wild-type adults showed robust attraction to diluted benzaldehyde (5/1000 dilution) and diacetyl (1/1000 dilution) (Colbert et al., 1997). In our assays, this attraction was abrogated by a mutation in the osm-9(ky10) gene, although OSM-9-dependent attraction to diluted benzaldehyde has not been reported previously (Figure 5F) (Colbert et al., 1997). We also tested nrde-3(gg66) mutant adults, which exhibited a small, but not significant, decrease in chemotaxis compared to wild-type adults. In contrast, the gsp-3 and gsp-3 gsp-4 mutant strains exhibited significant decreases in both diluted benzaldehyde and diacetyl attraction, while the gsp-4 mutant showed a similar chemotaxis index as wild type (Figure 5F). Overall, these results indicate that sperm-expressed serine/threonine PP1 phosphatase GSP-3 is required for both avoidance and attraction behaviors mediated by different sensory neurons in adult hermaphrodites.

We next tested the hypothesis that GSP-3 is required cell non-autonomously for chemosensory behaviors by regulating the secretion of MSPs from ADL neurons. In the gonad, gsp-3 gsp-4 double mutants have abnormal MSP localization in sperm and decreased MSP secretion promoting ovulation compared to wild-type animals (Wu et al., 2012). Our smFISH and immunofluorescence experiments suggest that MSPs are expressed only in ADL neurons (Supplementary Figures S2–S4); therefore, their secretion from ADL may impact the function of neighboring neurons. ADL, ASH, AWB, AWC, and AWA sensory neurons are located together in the same glial-like, amphid structure in the worm head (Ward et al., 1975). To test our hypothesis, we constructed a strain carrying a transgene that rescued the expression of gsp-3 in ADL neurons in a gsp-3(tm1647) mutant background (gsp-3; sre-1p::gsp-3::gfp). We compared the octanol avoidance, diacetyl attraction, and benzaldehyde attraction indexes exhibited by this strain to the gsp-3 mutant. For all three behaviors, we found that the expression of GSP-3 in ADL neurons was not sufficient to alter the behavior of the gsp-3 mutants (Figures 5D,F,G). Next, we tested the hypothesis that decreased levels of MSPs in the ADL neurons of gsp-3 gsp-4 mutants may play a role in altered chemosensory behaviors (Figures 4E, ​,F).F). To test this possibility, we created a strain that carries an extrachromosomal array with a transgene that over-expresses msp-19 under an ADL-specific promoter in the gsp-3(tm1647) background (gsp-3; sre-1p::msp-19::gfp). Using this strain, we tested the octanol avoidance, diacetyl attraction, and benzaldehyde attraction behaviors compared to gsp-3 mutants alone. Similar to the ADL-specific rescue of GSP-3, we found that over-expression of MSP-19 in ADL was also not sufficient to alter the behavior of the gsp-3 mutants (Figures 5D,F,G). Together, these results indicate that GSP-3 does not act in ADL to regulate the behavioral functions of other amphid neurons, suggesting the possibility that sperm genes may be expressed more widely in the C. elegans nervous system.

3.6 Loss of ADL neuronal function affects fecundity

Communication between neurons and the germ line is well documented in mammals, Drosophila, and C. elegans (Yoon et al., 2005; Häsemeyer et al., 2009; Hill and Elias, 2018; Wang et al., 2019; Gaddy et al., 2021). For example, in C. elegans, chemotaxis behavior in response to diacetyl by the activities of the AWA olfactory neurons and their downstream AIB and AIB interneurons is dependent on larval germline proliferation (Fujiwara et al., 2016). The neuronal activity of the ASJ temperature-sensing neurons is affected by a number of sperm genes, including gsp-4 (Sonoda et al., 2016). The expression of daf-7/TGF-ß in ASI neurons promotes lag-2 expression and activation of the GLP-1/Notch receptor to maintain the stem cell niche in the gonadal distal tip cells (Pekar et al., 2017). The detection of social pheromones by the worm nervous system directs the timing of post-embryonic germline development in the next-generation (Perez et al., 2021). Ectopic germline differentiation in worms due to endoplasmic reticulum (ER)-induced stress response is suppressed by a neuronal-germline circuit mediated by serotonin (Levi-Ferber et al., 2021). The male-enriched ascr#10 (asc-C9) sex pheromone, which serves in hermaphrodite attraction and mating success, also functions to shorten the lifespan and delay the reproductive senescence of its hermaphroditic mating partner in an ADL-dependent manner (Aprison and Ruvinsky, 2022).

Based on these observations, we asked whether the expression of germline genes in ADL could influence germline development in a manner that would affect fecundity. During hermaphrodite germline development, mitotically dividing germline stem cells develop into sperm during the L4 larval stage, then switch to oocyte production during adulthood (L’Hernault, 2006). Therefore, the brood size of a C. elegans hermaphrodite resulting from self-fertilization is sperm-limited (Byerly et al., 1976; Ward and Carrel, 1979; Kimble and Ward, 1988). To examine whether ADL influences hermaphrodite fecundity, we measured the brood size of hermaphrodites that have a loss of ADL cellular identity or function. The hlh-4 gene encodes a helix-loop-helix (bHLH) transcription factor that is required for ADL neuron fate and promotes expression of olfactory receptors that drive ADL-specific functions as a nociceptive sensory neuron (Masoudi et al., 2018). First, we verified that ADL identity is disrupted in hlh-4(tm604) mutants by showing that hlh-4 adults were severely defective in ADL-dependent ascr#3 avoidance, but they were unaffected in ASH-dependent glycerol avoidance behaviors (Figures 6A,B). However, loss of ADL identity did not significantly impact the fecundity of hlh-4 mutant hermaphrodites compared to wild type (Figure 6C).

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FIGURE 6

ADL chemosensory neurons contribute to fecundity. (A,B) Fraction of 1-day old wild-type and hlh-4(tm604) CON adults reversing in response to (A) ascr#3 and (B) 1M glycerol. **** p < 0.0001, Student’s t-test. Dots represent independent biological trials. (C) Brood size assays of wild-type and hlh-4(tm604) CON adults. ns = not significant; Student’s t-test. Data represents three independent biological replicates. (D) Brood size assays of CON adults with (+) or without (−) extra-chromosomal arrays consisting of tetanus toxin (TeTx) or a gain-of-function constitutively active protein kinase C isoform gene (pkc-1 (gf)) under the control of the sre-1 promoter (sre-1p). ** p < 0.01, ns = not significant; Student’s t-test. Data represents three independent biological trials. Additional data included in Supplementary Table S20.

Since HLH-4 is required for ADL neuronal identity but not for ADL birth (Masoudi et al., 2018), we next tested whether ablating ADL function would affect hermaphrodite fecundity. Expression of an extrachromosomal array expressing the light chain of the tetanus toxin (TeTx) under the control of the sre-1 promoter has been shown to effectively eliminate ADL neuronal function by preventing chemical synapses (Jang et al., 2012). The neurotoxin TeTx is a zinc protease that specifically cleaves synaptobrevin, an essential synaptic vesicle membrane protein, and inhibits neurotransmitter release (Schiavo et al., 1992). We therefore asked whether blocking synaptic transmission in ADL would affect hermaphrodite reproduction by comparing the brood size of adults expressing the TeTx gene under the control of the sre-1 promoter (sre-1p::TeTx) to adults of the same generation that lost the extrachromosomal array (Jang et al., 2012). We found that adults expressing the TeTx transgene exhibited a significant decrease in brood size compared to adults compared to the controls (Figure 6D). If ADL function is required to promote fecundity, we next asked whether hyperactive ADL would increase the brood size beyond controls. We increased ADL synaptic output by expressing an extrachromosomal array with a transgene carrying a constitutively active, gain-of-function allele of the protein kinase C isoform that promotes neuronal synapses, pkc-1(gf) (Okochi et al., 2005; Sieburth et al., 2007; Tsunozaki et al., 2008; Macosko et al., 2009; Jang et al., 2012). We observed no effect on brood size between adults carrying the array expressing pkc-1(gf) and adults from the same generation that lost the pkc-1(gf) transgene (Figure 6D). These results suggest that while eliminating ADL synaptic transmission slightly decreases hermaphrodite brood size, the germ line could be buffered from the consequences resulting from over-active ADL chemical synapses (Marchal and Tursun, 2021).

3.7 Germ cells are not required for ascr#3 avoidance behavior

Because we observed that loss of ADL function affects brood size (Figure 6D), we wondered whether loss of a functional germ line could result in defective ADL function. To test this hypothesis, we first performed ascr#3 avoidance assays using a glp-1(q224) mutant strain. The GLP-1/Notch receptor is necessary for the maintenance of the germline stem cell niche through promoting mitotic division of germ cells; thus, loss of GLP-1 causes the few present germline stem cells to prematurely enter meiosis and progress through spermatogenesis without germ stem cell renewal. At the restrictive temperature of 25°C, germline proliferation is prevented in the temperature-sensitive glp-1(q224) mutant (Austin and Kimble, 1987; Kimble and Crittenden, 2005). Glp-1 expression was below our threshold of 10 RPKM in ADL neurons of control adults; thus, our experiment should be measuring the contribution of GLP-1 in the germline. As a control, we showed that ascr#3 avoidance by osm-9(ky10) adults was reduced compared to wild-type as previously shown (Figures 5C, ​,7A)7A) (Jang et al., 2012; Sims et al., 2016). However, reversals by glp-1 mutants at the restrictive (25°C, no germ lines) temperature were not significantly different from wild-type despite having slightly lower responses on average (Figure 7A).

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FIGURE 7

Endogenous small RNAi pathways mediate ascr#3 avoidance response. (A–I) Fraction of 1-day old CON adults reversing in response to ascr#3 for (A) wild-type, osm-9 (ky10), glp-1(q224); (B) wild-type, fem-1(hc17), fog-2(q71); (C) wild-type, hrde-1(tm1200); (D) wild-type, wago-1(ok1074); (E) wild-type, wago-4(tm1019); (F) wild-type, prg-1(tm872); (G) wild-type N2, prg-2(tm1094); (H) wild-type, ppw-1(pk2505), ppw-2(tm1120); and (I) wild-type, alg-1(gk214), alg-2(ok304) strains. * p < 0.05, ** p < 0.01, ns = not significant; (A,I) Kruskal-Wallis test with Dunn’s post-hoc test, (B) RM one-way ANOVA with Dunnett’s post-hoc test, (C–G) Student’s t-test, (H) one-way ANOVA with Tukey’s post-hoc test. Each dot in all assays represents an independent biological trial. Glycerol avoidance assays for the same strains are shown in Supplementary Figure S5. Additional data included in Supplementary Table S20.

A previous study found that a number of sperm genes, including gsp-4, were required for temperature tolerance and were mediated by the ASJ neurons (Sonoda et al., 2016). Since glp-1(q224) mutants produce a few sperm at the restrictive temperature, we next tested whether signals from sperm specifically are required for ADL function. We examined ascr#3 avoidance in strains carrying mutations in somatic sex determination genes, fem-1 and fog-2, which result in hermaphrodite germline feminization and the production of only oocytes (Doniach and Hodgkin, 1984; Schedl and Kimble, 1988; Goodwin and Ellis, 2002; Ellis and Schedl, 2007). Reversals in response to ascr#3 by control female fem-1(hc17) and fog-2(q71) mutants were not statistically different from that of the wild-type strain. However, these strains exhibited the high variability in their avoidance behavior across trials, ranging from 10% to 80% and 22% to 80% of animals responding for fem-1 and fog-2 animals, respectively (Figure 7B). fem-1 expression is above, and fog-2 slightly below, our RPKM cutoff for ADL-expressed genes. In addition to promoting the production of sperm in hermaphrodites, noradrenergic signals required to maintain oocyte quiescence involve FEM-1 and FOG-2 functions (Kim et al., 2021); thus, we cannot rule out that their variable behavior is due to their function in ADL and not from the germ line. Collectively, our results are consistent with a model that ADL function contributes to hermaphrodite fecundity, but that germ cells are not required for ADL function.

3.8 Endogenous small non-coding RNA pathway genes mediate avoidance response to ascr#3

We have previously shown that, in addition to NRDE-3, the AGO ERGO-1 and components of the Mutator focus (MUT-16 and MUT-15) are critical for the regulation of osm-9 expression and modulating ascr#3 avoidance behavior as a result of developmental history. Additionally, control adults harboring a mutation in genes involved in endogenous RNAi pathways, including Mutator focus components (mut-14, mut-15, and mut-16), nrde-3, and ergo-1 exhibited statistically decreased ascr#3 avoidance when compared to wild-type (Sims et al., 2016). With the exception of ergo-1, these genes are lowly expressed in ADL neurons according to our RNA-seq data, beneath the 10 RPKM cutoff (Supplementary Table S2). We wondered whether lesions in additional endogenous small non-coding RNA pathways genes that are ADL-expressed would also affect ascr#3 avoidance behavior. First, HRDE-1/WAGO-9 is the germline-specific AGO counterpart of its NRDE-3 somatic paralog. HRDE-1 shares some of the same protein cofactors as NRDE-3 (NRDE-1, NRDE-2, and NRDE-4) to exert its heterochromatin formation and gene silencing roles (Guang et al., 2008; Burkhart et al., 2011; Ashe et al., 2012; Buckley et al., 2012; Shirayama et al., 2012; Billi et al., 2014). Unlike NRDE-3, HRDE-1 is critical for transgenerational epigenetic inheritance (TEI), a germline-dependent inheritance of a response to a trigger that persists for at least two to three generations beyond the original trigger (Ashe et al., 2012; Buckley et al., 2012; Gu et al., 2012; Shirayama et al., 2012; Rechavi and Lev, 2017; Perez and Lehner, 2019). We observed that hrde-1 mutants have a slight, but not significant, increase in ascr#3 avoidance compared to wild-type (Figure 7C).

Next, we examined for a role in ascr#3 avoidance of some additional siRNA associated AGOs expressed in ADL neurons. WAGO-1/R06C7.1 and WAGO-4/F58G1.1 are two germline-specific proteins belonging to the WAGO clade (Yigit et al., 2006). WAGO-1 was shown to localize to P granules, the largest constituent of RNA-protein perinuclear condensates (or nuage) that accumulate in the cytoplasmic side of the nuclear envelop and are believed to be hubs where siRNA amplification and AGO distribution occur (Gu et al., 2009; Shirayama et al., 2012; Wang et al., 2014; Aoki et al., 2021; Sundby et al., 2021; Ouyang and Seydoux, 2022). WAGO-4 localizes to the Z granule component in nuage and is a necessary factor for RNAi inheritance (Wan et al., 2018). Comparison between wild-type adults and the wago-1(ok1074) mutant did not result in a significant difference in avoidance behavior to ascr#3 (Figure 7D). The wago-4(tm1019) mutants, however, showed statistically greater avoidance to ascr#3 compared to wild-type (Figure 7E). This suggests that, in addition to the requirement of Mutator proteins for ascr#3 avoidance (Sims et al., 2016), proteins known to localize in the germline Z granules may also play a role. Although ascr#3 exposure has no identified effects in future generations, exposure to ascr#3 during early larval development can affect how animals react to ascr#3 upon reaching adulthood (Hong et al., 2017). Notably, formation of germ granules in somatic tissue has not been detected to date, suggesting that certain AGOs may have different molecular functions in the soma compared to the germ line (Seroussi et al., 2023).

Next, we examined genes we found expressed in ADL neurons that function in different small non-coding RNA pathways, including the PIWI pathway. PRG-1 is a PIWI class AGO that associates with piRNAs (also known as 21U-RNAs) to safeguard the germ line by silencing detrimental mobile genetic elements. PRG-1 shares 91% amino acid identity with a second PIWI protein, PRG-2. PRG-1 and PRG-2 function in the germ line but unlike a prg-1 mutant, mutation of prg-2 does not result in aberrant accumulation of piRNAs (Batista et al., 2008; Das et al., 2008). Ascr#3 avoidance assays conducted on control adult prg-1(tm872) and prg-2(tm1094) mutants showed slight increases in avoidance behavior that were not significantly different compared to wild-type control animals (Figures 7F,G). Furthermore, PPW-1/WAGO-7 and PPW-2/WAGO-3 are members of the PIWI clade of AGOs (Yigit et al., 2006). PPW-1 is expressed in the germ line, intestine, and somatic gonad and is required for exogenous and endogenous RNAi (Tijsterman et al., 2002; Vasale et al., 2010). Most pseudogenes, long intergenic non-coding RNAs (lincRNAs), and transposable elements are targeted by a small cohort of AGOs that include PPW-1 (Seroussi et al., 2023). Expression of germline-specific PPW-2 has been detected in the spermatheca and in sperm where it associates with sperm-specific germ granules and paternal 22G RNAs to promote paternal epigenetic inheritance (Schreier et al., 2022; Seroussi et al., 2023). In addition to targeting spermatogenic genes, PPW-2 also targets repetitive elements and silences transposons (Vastenhouw et al., 2003; Seroussi et al., 2023). Avoidance to ascr#3 was not significantly altered in the ppw-1(pk2505) or ppw-2(tm1120) mutant strain, although ppw-1 mutants exhibited slightly decreased avoidance compared to wild-type CON adults (Figure 7H).

Finally, we examined the role of paralogous ALG-1 and ALG-2 AGOs that associate with the miRNA class of small non-coding RNAs (Grishok et al., 2001; Yigit et al., 2006). Alg-1 and alg-2 mRNAs are expressed slightly below and above, respectively, our threshold cutoff for ADL expression. We measured the ascr#3 avoidance of each mutant strain given their relatedness with respect to their spatiotemporal expression patterns and overlapping function as miRNA-associated AGOs (Tops et al., 2006; Vasquez-Rifo et al., 2012; Aalto et al., 2018; Brosnan et al., 2021). ALG-1 and ALG-2 are ubiquitously expressed in somatic tissues with the latter also expressed in the germ line (Seroussi et al., 2023). Alg-1(gk214) mutant control adults exhibit a significant defect in ascr#3 avoidance indicating that ALG-1-associated miRNAs play a significant role in ADL function (Figure 7I). Although ALG-2 was found to be prevalently expressed in neurons over ALG-1 and is the chief direct interactor of pan-neuronal miRNAs (Maier et al., 2003; Redecker et al., 2003; Meizel, 2004; Ramírez-Reveco et al., 2017; Aalto et al., 2018; Brosnan et al., 2021), alg-2(ok304) adults showed no significant difference in ascr#3 response compared to wild-type (Figure 7I). Together, our results highlight the role that two major classes of endogenous small RNAs, namely, siRNAs and miRNAs, and their cognate AGOs play in ADL-dependent olfactory behavior.

4.1 Gene expression regulation of MSPs in ADL neurons

The questions raised by our study on how and why sperm genes are expressed in neurons is a long-standing question in biology. In humans, the brain and testes share a striking similarity when comparing their transcriptome and proteome, with one study finding that a surprising ~75% of sperm proteins are also expressed in neuronal cells (Matos et al., 2021). Consistent with this observation, mutations in genes expressed in neurons and sperm result in brain developmental defects and male infertility (Andersen et al., 2003). Some emphasis has been placed on understanding the role of neuronal genes in mammalian sperm function, particularly neuronal receptors and proteins that function in the calcium-dependent acrosome reaction for fertilization (Maier et al., 2003; Redecker et al., 2003; Meizel, 2004; Ramírez-Reveco et al., 2017). However, the function of sperm genes in neurons is less studied (Hu et al., 2016).

In multicellular organisms, cellular identity is determined by the set of genes expressed in that specific cell type. Related cell types derived from the same lineage often express transcription factors that drive gene expression, which defines that lineage (Bertrand et al., 2002; Nutt and Kee, 2007). Pan-neuronal gene expression, for example, is driven by DNA regulatory motifs in upstream regulatory regions that are bound by homeobox transcription factors (Leyva-Díaz and Hobert, 2002; Stefanakis et al., 2015; Reilly et al., 2020; 2022; Hobert, 2021). In C. elegans, Drosophila, and mice, identities of individual neurons are established and maintained via terminal selector transcription factors which, in conjunction with histone modification enzymes, promote expression of cell-specific neuropeptides, ion channels, and receptors and block expression of genes not part of the neuronal identity (Feng et al., 2020; Destain et al., 2024). Disruption of terminal selector genes results in a type of homeosis in the nervous system, where neurons change their identity to other types of neurons (Arlotta and Hobert, 2015).

In contrast, C. elegans hermaphrodite germline stem cells maintain totipotency to become either sperm or oocyte via mechanisms involving germline-specific perinuclear RNA granules (P granules), chromatin regulators, histone chaperones, and translation and transcription factors (Seydoux and Braun, 2006; Spickard et al., 2018; Fatima and Tursun, 2020; Marchal and Tursun, 2021). Germ cells that fail at totipotency are relegated to somatic fates with ensuing germline mortality and sterility (Updike et al., 2014). Oocytes that have disrupted P granules mis-appropriately express neuropeptide and sperm genes (Campbell and Updike, 2015; Rochester et al., 2022). The 3′ untranslated (UTR) regions of mRNAs are sufficient to regulate expression in most germ cells; however, sperm-expressed genes require 5′ UTRs to specify correct expression patterns (Shim, 1999; Merritt et al., 2008; del Castillo-Olivares et al., 2009; Kulkarni et al., 2012; Ragle et al., 2022). Msp gene expression requires a conserved GATAA motif in their 5’ UTRs that is bound by transcription factor, ELT-1, and the SET-17 H3K4me2 methyltransferase to maintain a permissive chromatin state at the msp gene cluster loci (Figure 3A) (del Castillo-Olivares et al., 2009; Engert et al., 2018).

In C. elegans, the 5′ UTR driven regulation of sperm genes is more similar to how neuron genes are regulated compared to other germline-expressed genes as described above. Our work here shows that MSP genes are not only expressed in wild-type control ADL neurons, but also that they are significantly differentially expressed in postdauer ADL neurons in a NRDE-3-dependent manner. We have shown previously that osm-9 is downregulated in postdauer ADL neurons, which required NRDE-3 and the PD motif sequence in the 5′ UTR (Sims et al., 2016). Unlike osm-9 expression, NRDE-3 is required for positive MSP expression in control adults instead of their downregulation in postdauer adults. Given the role of NRDE-3 in transcriptional silencing, these results suggest that NRDE-3 may be indirectly regulating MSPs (Supplementary Figure S6). Additionally, of the 21 msp genes differentially expressed in ADL, 19 genes were found to contain the ~30 base pair conserved sequence element in the PD motif, which we have named the “MSP motif” (Figure 3A), further supporting our hypothesis that expression and regulation of MSPs in ADL is driven by upstream regulatory sequences (Sims et al., 2016). Our previous osm-9 promoter analysis experiments demonstrated that the 5’ end of the MSP motif was required for osm-9 expression in ADL neurons (Sims et al., 2016); thus, the MSP motif may also drive expression of the msp genes in ADL. Further experimentation will be necessary to determine the role of the MSP and E-box motifs in regulating differential expression of msp genes in ADL neurons (Figure 3A).

4.2 Potential MSP function in ADL

MSPs have two functions in C. elegans hermaphrodites to promote reproduction. First, they function in amoeboid-like sperm movement by forming filaments that promote lamellipod formation (Roberts and Stewart, 1995). MSPs were first discovered in C. elegans sperm (Klass and Hirsh, 1981), but much of our knowledge about the dynamics in sperm movement comes from studies in the Acaris suum nematodes. MSP dimers are added to growing filaments in the lamellipod at the leading edge of the cell to push to membrane forward (Italiano et al., 1996). MSP polymerization requires a membrane protein called MSP Polymerization Organizing Protein (MPOP) and cytosolic protein, MSP fiber protein 1 (MFP1); in contrast, MSP fiber protein 2 (MFP2) antagonizes this process to slow polymerization (Sepsenwol et al., 1989; Buttery et al., 2003; Grant et al., 2005). The filaments “treadmill” towards the trailing nucleus where MSP filaments are depolymerized, allowing the sperm to crawl over a substrate (Roberts and King, 1991). Mutant screens in C. elegans hermaphrodites have identified genes encoding proteins that play a role in this process, including the SPE-6 kinase required for filament assembly and the PP1 phosphatases, GSP-3 and GSP-4, for disassembly (Wu et al., 2012; Price et al., 2021). Our RNA-Seq data indicate that C. elegans genes encoding GSP-3 and GSP-4 are expressed in ADL neurons, while MFP1/MSD-1, MFP2/NSPH-2, and SPE-6 are not, suggesting that MSPs are not forming fibers in this context (Morrison et al., 2021). Our behavioral assays in this study demonstrated that the phosphatases, particularly GSP-3, are required for avoidance and attraction responses mediated by nociceptive and attractive sensory neurons, and rescue of GSP-3 in ADL is not sufficient to rescue the mutant phenotypes (Figures 5D,F,G). However, whether GSP-3 is expressed in other neurons and the targets of GSP-3 in ADL remain to be determined.

The second function of MSPs is to promote oocyte maturation and ovulation in the hermaphrodite gonad. MSPs are secreted from sperm and travel distally through the gonad to bind as an antagonist to the VAB-1 Ephrin receptor on the surface of arrested oocytes to promote their maturation. Additionally, MSPs bind to VAB-1 receptors in the gonad sheath cells to promote ovulation of oocytes through spermatheca for fertilization (Miller et al., 2003; 2001). VAB-1 is also expressed in the nervous system, and its role in neuronal morphogenesis, neuroblast movement, and axon migration has been well characterized (George et al., 1998; Zallen et al., 1999; Ghenea et al., 2005; Boulin et al., 2006; Mohamed and Chin-Sang, 2006; Mohamed et al., 2012). Whether MSPs expressed in ADL can be secreted and bind to neuronal VAB-1 receptors is unknown. Other proteins containing domains with similar structures to MSPs also have established roles in the nervous system. For example, a MSP-domain containing protein was identified in a screen for genes with roles in C. elegans axon guidance (Schmitz et al., 2007). In humans, a clinically relevant protein with a MSP domain is the vesicle-associated membrane protein-associated protein B (VAP-B) expressed in the nervous system, which plays a role in endoplasmic reticulum stress and the unfolded protein response among other cellular processes (Lev et al., 2008). The N-terminal MSP domain of VAP-B (MSPd) is cleaved and secreted from neurons and can bind as a ligand to Ephrin receptors (Tsuda et al., 2008). The P56S mutation in the VAP-B MSPd is associated with familial amyotrophic lateral sclerosis 8 (ALS8), and it alters the function of VAP-B by inhibiting cleavage of the MSPd and promoting formation of protein aggregates in neurons (Kanekura et al., 2006; Teuling et al., 2007; Kim et al., 2010). Additionally, the VAP protein in Drosophila localizes to neuronal cell bodies, and not in neuromuscular junctions, yet functions in regulating the latter (Tsuda et al., 2008). Interestingly, the cleaved VAP-B MSPd of humans and Drosophila can bind to the C. elegans VAP-1 Ephrin receptor and rescue oocyte maturity and ovulation defects in the gonad of sperm-deficient animals, suggesting evolutionary conservation of MSPd function in metazoans (Tsuda et al., 2008). VPR-1 is the C. elegans homolog of VAP-B, and it functions similarly by secreting its cleaved MSPd to signal through Ephrin receptors to regulate neuronal positioning and muscle function (Mohamed and Chin-Sang, 2006; Tsuda et al., 2008). Given the evolutionary conservation of function between the VAP-B and sperm MSP domains, we are testing the hypothesis that expression and potential secretion of sperm MSPs from ADL neurons affects VPR-1 function and Ephrin signaling.

We are grateful to Cornelia Bargmann, Diana Chu, Eleanor Maine, Oliver Hobert, and Shohei Mitani for strains, and Eleanor Maine, Leanne Kelley, and Kyuhyung Kim for critical reading of this manuscript. Some strains were provided by the CGC, which is funded by the NIH Office of Research Infrastructure Programs (P40 OD010440). We thank Lisa Phelps at SUNY Flow Cytometry Facility for operating the FACS instrument.