Previous studies have shown that C. elegans cannot digest SS, which prevents their development (Figure 1A). In natural environments, C. elegans rely on various bacteria for nutrition and growth. However, SS is not a viable food source for C. elegans. Larval arrest, such as the dauer stage, serves as an adaptive mechanism for survival under unfavorable conditions, including limited food availability or extreme temperatures (Baugh, 2013; Baugh and Hu, 2020). We hypothesize that C. elegans can sense or evaluate inedible food, such as SS, and subsequently shut down their digestion to arrest development as a protective survival strategy (Figure 1B).

Our previous research demonstrated that C. elegans can assess and avoid low-quality food, like heat-killed E. coli, to adapt to nutrient-deficient conditions (Liu et al., 2024; Qi et al., 2017). To determine if C. elegans also detect SS as an unfavorable food source, we conducted two behavioral assays: food dwelling/avoidance and food choice (Qi et al., 2017). In the food dwelling/avoidance assay, larval-stage animals exhibited strong discrimination against SS compared to the standard food, OP50 (Figure 1C). In the food choice assay, the animals preferred OP50 over low-quality food such as heat-killed E. coli (Figure 1—figure supplement 1A) or SS (Figure 1D). However, they could not distinguish between heat-killed E. coli and SS (Figure 1—figure supplement 1B). These results suggest that SS acts as an unfavorable food that C. elegans can detect and avoid.

In response to starvation, L1 larvae can enter a state of developmental arrest, pausing their growth to survive (Baugh and Sternberg, 2006). To test whether C. elegans shut down digestion of SS as a protective strategy upon sensing unfavorable food, we performed a survival assay on L1 larvae fed with SS. We found that larvae unable to digest SS still survived under SS feeding conditions (Figure 1—figure supplement 1C), similar to larvae under L1 starvation. This suggests that shutting down digestion may be a protective mechanism in larvae under SS feeding conditions.

Previously, we observed that activating larval digestion with heat-killed E. coli or E. coli cell wall peptidoglycan (PGN) enabled the digestion of SS as food (Hao et al., 2024). Additionally, when animals reached the L2 stage by feeding normal OP50 diet, they could utilize SS as a food source to support growth (Figure 1—figure supplement 1D). These findings suggest that once digestion is activated (via E. coli components or L2-stage maturation), worms gain the capacity to process SS as a viable food source, abolishing SS-induced growth impairment (Hao et al., 2024; Figure 1—figure supplement 1D).

If SS is indeed an unfavorable or toxic food for C. elegans, digesting it could result in physiological defects. We measured the lifespan of L4 stage animals fed with SS or OP50. We found that SS consumption shortened their lifespan (Figure 1E and F), indicating a cost associated with digesting unfavorable or toxic food.

In conclusion, our data suggest that larval-stage C. elegans can sense and evaluate SS as an unfavorable food source, leading to the shutdown of digestion to avoid consumption, thereby protecting them and allowing adaptation to an unfavorable food environment.

We speculated that key factors in C. elegans are involved in sensing SS and shutting down its digestion. If these factors are mutated, the animals would fail to detect SS as an unfavorable food source and would utilize it (Figure 2—figure supplement 1A). To identify these factors, we conducted an unbiased forward genetic screen to find mutant animals that cannot sense SS, thereby allowing its digestion and supporting growth. One of the mutant alleles identified, ylf6, could digest SS and exhibited a growth phenotype under SS feeding conditions (Figure 2—figure supplement 1B). Whole-genome deep sequencing revealed that ylf6 carries two mutations in the nsy-1 gene (H929Y, Q1191 ^{[stop codon}*^]) (Figure 2—figure supplement 1C).

To confirm that nsy-1 is essential for shutting down SS digestion, we used an independent nsy-1 mutant allele, ag3, and found that nsy-1(ag3) mutants also digested SS (Figure 2A). To determine whether nsy-1 is crucial for sensing SS, we performed two behavioral assays: food dwelling/avoidance and food choice. In the food dwelling/avoidance assay, larval stage nsy-1 mutant exhibited significantly impaired avoidance responses at both 4 h and 6 h but not at 8 h, suggesting that NSY-1 is essential for sustained aversion to SS food in the early response (Figure 2B). In contrast to wild-type N2 animals, nsy-1 mutants preferred SS when given a choice between two poor-quality foods, heat-killed E. coli and SS (Figure 2C, Figure 1—figure supplement 1B). These results suggest that nsy-1 is essential for C. elegans to sense and avoid SS.

Next, we examined whether larvae that cannot sense SS and do not shut down digestion can adapt to SS environments. We measured the survival rate of nsy-1 mutants under SS feeding conditions and found that these mutants had a higher mortality rate (Figure 2D).

Overall, these results indicate that C. elegans sense and detect unfavorable food, such as SS, through nsy-1 and subsequently shut down digestion to protect themselves and enhance survival.

The nsy-1 gene in C. elegans encodes a MAP kinase kinase kinase (MAPKKK) that operates in the AWC neurons (Kim et al., 2002; Sagasti et al., 2001), which are essential for chemotaxis and odor sensation. The primary role of nsy-1 in AWC neurons is to regulate the asymmetric expression of odorant receptors, contributing to neuronal asymmetry and diversity (Chuang et al., 2007; Sagasti et al., 2001). We hypothesized that the shutdown of SS digestion in C. elegans is mediated by nsy-1 function in AWC neurons.

Firstly, we constructed a Pnsy-1::GFP reporter strain and confirmed that nsy-1 is expressed in the AWC neurons (Figure 3A). Secondly, we expressed nsy-1 in the AWC neurons of nsy-1 mutant animals and observed that the transgenic animals rescued the indigestion phenotype (Figure 3B). This indicates that nsy-1 functions in AWC neurons to shut down SS digestion. Thirdly, we used CRISPR to construct a mutant strain that knocks out nsy-1 specifically in AWC neurons (Figure 3—figure supplement 1). We found that nsy-1 knockout in AWC neurons also resulted in the shutdown of SS digestion (Figure 3C). These results collectively suggest that NSY-1 is functional in AWC neurons and is crucial for shutting down SS digestion in C. elegans.

Beyond its established role in AWC neurons, we detected NSY-1 expression in the intestine (Figure 3—figure supplement 2A). To assess intestinal NSY-1 function, we performed tissue-specific rescue experiments in nsy-1 mutants using the intestinal-specific vha-6 promoter. Intestinal expression of NSY-1 significantly suppressed the enhanced SS digestion phenotype in nsy-1 mutants (Figure 3—figure supplement 2B), demonstrating functional involvement of gut-localized NSY-1 in regulating digestive responses. We propose intestinal NSY-1 mediates this effect through innate immune signaling, consistent with its known pathway components. As previously established (Geng et al., 2022), the canonical PMK-1/p38 MAPK pathway functions downstream of NSY-1, with both sek-1 and pmk-1 knockdown enhancing SS digestion through immune modulation. This indicates intestinal NSY-1 suppresses digestion may act through PMK-1-mediated immune responses. Since neuronal NSY-1’s role in digestive control was previously undefined, we prioritized mechanistic analysis of its neuronal function in digestion regulation.

To determine whether NSY-1 in AWC neurons mediates SS sensory perception, we performed dwelling (avoidance) and food-choice assays using AWC-specific nsy-1 knockout and AWC-rescued strains (nsy-1(ag3); Podr-1::nsy-1). In dwelling assays, AWC-specific nsy-1 KO mutants exhibited significantly impaired SS avoidance at 6 h (Figure 3—figure supplement 3A), while AWC-rescued strains restored avoidance capacity at 2–6 h (Figure 3—figure supplement 3B). Food-choice assays further revealed that AWC nsy-1 KO mutants preferentially migrated toward SS (Figure 3—figure supplement 3C), whereas AWC-rescued showed no preference between SS and HK-E. coli (Figure 3—figure supplement 3D). These data conclusively demonstrate that NSY-1 acts in AWC neurons to mediate SS recognition and aversion behaviors.

In C. elegans, the expression of the str-2 gene in AWC neurons indicates the ON state (AWC^ON) (Sagasti et al., 2001), which is associated with high cGMP levels and lower calcium activity, enabling the neuron to respond to specific odors. Conversely, the absence of str-2 expression marks the OFF state (AWC^OFF), characterized by different odor responses, low cGMP levels, and higher calcium activity (Troemel et al., 1999). We aimed to investigate (1) whether AWC neurons exhibit different states under normal food (E. coli OP50) versus unfavorable food (SS) conditions and (2) whether the state of AWC neurons affects the ability of C. elegans to digest SS.

Using str-2::GFP as a marker for AWC neuron states (Troemel et al., 1999), we found that wild-type animals feeding on normal OP50 food typically exhibit one AWC^OFF and one AWC^ON neuron. However, when feeding on SS, the proportion of animals with the AWC^OFF state increased, with approximately 50% of animals exhibiting two AWC^OFF neurons (Figure 3D–F).

In nsy-1 mutant animals feeding on OP50, str-2::GFP is expressed in both AWC neurons (2AWC^ON), consistent with previous studies (Chuang and Bargmann, 2005; Chuang et al., 2007; Sagasti et al., 2001; Troemel et al., 1999; Figure 3D and E). Notably, both AWC neurons remained in the AWC^ON state in nsy-1 mutants feeding on SS (Figure 3D and E). These results suggest that SS feeding induces an AWC^OFF state in wild-type animals, while the nsy-1 mutation promotes an AWC^ON state even under SS feeding conditions. This implies that the AWC^OFF state may inhibit SS digestion, whereas the AWC^ON state promotes it.

The TIR-1-NSY-1-SEK-1-MAPK pathway plays a crucial role in regulating the asymmetric AWC cell fate decision. Previous studies have shown that str-2::GFP expression in both AWC cells (2AWC^ON phenotype) occurs in tir-1, nsy-1, and sek-1 mutant animals (Chuang and Bargmann, 2005; Sagasti et al., 2001; Troemel et al., 1999). We found that tir-1 mutant can grow under SS feeding conditions (Figure 3G), indicating that tir-1 mutant can digest SS similarly to nsy-1 mutants. This demonstrates that the AWC^ON state promotes SS digestion.

To confirm the importance of AWC state in SS digestion, we performed AWC-specific neuron ablation experiments using previously validated transgenic strain that expresses cleaved caspase under the AWC-specific promoter, ceh-36 (ceh-36p::caspase). Critically, worms with ablated AWC neurons completely failed to digest SS food (Figure 3—figure supplement 4), phenocopying the non-digesting state of wild-type worms on SS. This result directly confirms that functional AWC neurons are essential for initiating SS digestion, aligning with our model where the AWC-OFF state (induced by SS) inhibits digestion while the AWC-ON state promotes it.

Overall, our data suggest that the state of AWC neurons is critical for sensing food in C. elegans. When sensing SS food, animals exhibit an AWC^OFF state, which shuts down digestion. Conversely, the AWC^ON state promotes the digestion of SS.

We have demonstrated that NSY-1 in AWC neurons detects unfavorable food and shuts down digestion (Figure 3). To investigate the genes regulated by NSY-1 in response to SS and their impact on SS digestion, we conducted a transcriptomic analysis on L1 larval animals fed with normal food (OP50) or unfavorable food (SS) for a short duration (4 h). We speculated that some genes induced by SS food are dependent on NSY-1 (Figure 4A), and their induction aids in shutting down SS digestion.

RNA-seq data analysis revealed 304 NSY-1-dependent candidate genes responding to SS (Figure 4B, Supplementary file 1). Enrichment analysis (Figure 4—figure supplement 1A, Supplementary file 2) of these candidate genes showed mainly associations with biotic stimulus, defense responses, xenobiotic stimulus, suggesting that NSY-1 positively regulates stress response pathways to protect animals under harmful food, SS, feeding conditions. Moreover, we found that sensory perception-related genes (sra-32, str-87, str-112, str-130, str-160, str-230) (Figure 4—figure supplement 1A, Supplementary file 2) were also enriched, with many genes being G protein-coupled receptors (GPCRs), which mediate odor sensing (Buck and Axel, 1991).

We further analyzed the dependence of these enriched GPCRs on NSY-1 under SS feeding conditions (Figure 4—figure supplement 1B) and found that str-130 is significantly upregulated in response to SS, with its function strongly being NSY-1 dependent (Figure 4C).

Using RNAi knockdown and the SS growth assay, we observed that RNAi of str-130, str-230, str-87, or str-112 significantly enhanced SS growth (Figure 4—figure supplement 2A), with str-130 RNAi exhibiting the most robust phenotype—phenocopying nsy-1(ag3) mutants. Crucially, none of these GPCR knockdowns further enhanced growth in nsy-1(ag3) mutants (Figure 4—figure supplement 2B), confirming their position downstream of NSY-1. These data establish str-130 as the dominant effector of NSY-1-mediated SS response regulation, while suggesting minor contributions from other GPCRs (str-230, str-87, str-112).

It has been shown that str-130 is expressed in AWC^OFF neurons, based on transgenic GFP reporter strains, str-130p::GFP (Vidal et al., 2018). Our data also show that str-130 expression is induced in wild-type animals fed with SS (Figure 4C), where AWC neurons exhibit the AWC^OFF state (Figure 3D and E). Therefore, it is possible that the high expression of str-130, regulated by nsy-1, alters the AWC state and inhibits SS digestion.

Firstly, we found that knockdown of str-130 in wild-type animals promoted SS digestion, thereby supporting animal growth (Figure 4D), and the proportion of animals with two AWC^OFF neurons decreased (Figure 4E). Secondly, we found that overexpression of str-130 in nsy-1 mutant animals inhibited SS digestion, thereby slowing animal growth (Figure 4F), and the proportion of animals with two AWC^OFF neurons increased (Figure 4G). These results demonstrate that NSY-1 promotes the AWC^OFF state by inducing str-130 expression, which in turn inhibits SS digestion in C. elegans.

To definitively establish the epistatic relationship between NSY-1 and STR-130, we performed RNAi knockdown of str-130 in the nsy-1(ag3) mutant background and assessed development on SS food. We found that the str-130 RNAi did not further enhance the developmental capacity of nsy-1(ag3) mutant animals on SS (Figure 4—figure supplement 3). This epistasis confirms STR-130 functions strictly downstream of NSY-1 within the same genetic pathway. Together with our overexpression data (Figure 4F and G) showing neuronal str-130 rescue suppresses SS digestion in nsy-1 mutants, these results establish a linear signaling axis where NSY-1 primarily achieves functional inhibition of SS digestion through induction of the GPCR str-130.

NSY-1 mutation promotes the digestion of unfavorable food, such as SS, and supports C. elegans growth (Figure 2A). We hypothesize that, in addition to upregulating certain genes, such as str-130, to inhibit SS digestion, NSY-1 may also suppress certain genes to prevent nematodes from utilizing SS (Figure 5A). One possibility is that some genes, induced by the nsy-1 mutation under SS feeding conditions, could facilitate SS digestion in the nsy-1 mutant (Figure 5A).

Our RNA-seq analysis identified 308+46 = 354 genes that are induced by the nsy-1 mutation under SS feeding conditions (Figure 5B, Supplementary file 3). However, among these 354 genes, 46 genes can also be induced in wild-type animals fed with SS, suggesting that these 46 genes may not be involved in digesting SS in nsy-1 mutants. Therefore, the 308 candidate genes induced by the nsy-1 mutation under SS feeding conditions could potentially promote animals to digest SS.

Enrichment analysis revealed that genes related to extracellular functions, such as insulin-related genes, are induced in nsy-1 mutant animals (Figure 5—figure supplement 1A, Supplementary file 4). Further analysis of insulin-related genes from the RNA-seq data showed that ins-23 is predominantly induced in nsy-1 mutant animals (Figure 5—figure supplement 1B), suggesting its potential role in promoting SS digestion.

To determine if insulin-like peptide genes were functionally responsible for the enhanced SS growth observed in nsy-1(ag3) mutants, we performed functional phenotypic screening using the SS growth assay (worm length assay). We individually knocked down each of these candidates (ins-22, ins-23, ins-24, ins-27) in the nsy-1(ag3) mutant background. Among these, only RNAi targeting ins-23 significantly suppressed the enhanced development of the nsy-1(ag3) mutant on SS (Figure 5—figure supplement 2, Figure 5D). This targeted functional screening revealed that ins-23 has the most robust and specific role in mediating the enhanced digestion phenotype downstream of NSY-1 loss, providing the critical justification for our subsequent focus on this particular insulin-like peptide.

Given that INS-23 is expressed in AWC neurons (Figure 5—figure supplement 3A, from CeNGEN), this suggests increased production and likely enhanced release of INS-23 from AWC neurons in the nsy-1(ag3) mutant background, which promotes SS digestion.

The insulin/insulin-like growth factor signaling (IIS) pathway, particularly through the DAF-2 receptor, integrates nutritional signals to regulate various behavioral and physiological responses related to food (Kodama et al., 2006; Ryu et al., 2018). It has been shown that INS-23 acts as an antagonist for the DAF-2 receptor to promote larval diapause (Matsunaga et al., 2018). To test whether ins-23 induction in nsy-1(ag3) mutants promotes SS digestion through its receptor, DAF-2, we constructed a nsy-1; daf-2 double mutant. We found that the SS digestion ability of the nsy-1 mutant was inhibited by the daf-2 mutation (Figure 5E). This suggests that the nsy-1 mutation induces the insulin peptide ins-23, which promotes SS digestion through its potential receptor, DAF-2.

To investigate whether DAF-2 acts as the gut-localized receptor for neuronal INS-23 signaling, we performed tissue-specific rescue experiments in the nsy-1(ag3);daf-2(e1370) double mutant. When DAF-2 was re-introduced specifically in the intestine (using the ges-1 promoter), we observed a significant suppression of SS digestion (Figure 5—figure supplement 3B), but not rescue digestive defect. This indicates that INS-23 induction in nsy-1 mutants promotes digestion independently of intestinal DAF-2 function.

As established in our prior work (Geng et al., 2022), SS exposure triggers phosphorylation of PMK-1 (P-PMK-1) in C. elegans, and pmk-1 mutants exhibit enhanced growth on SS. This confirms that PMK-1-mediated innate immune signaling actively regulates SS responsiveness and digestion. To address whether PMK-1 functions downstream of NSY-1 within our proposed model, we performed critical epistasis analyses. While we observed that nsy-1 mutation elevates ins-23 (indicating NSY-1 suppression of ins-23), knockdown of pmk-1 did not alter ins-23 expression levels (Figure 5—figure supplement 3C). This demonstrates that PMK-1 does not operate through the INS-23 pathway to regulate SS digestion. Thus, although both pathways respond to SS, the PMK-1-mediated innate immune response and the NSY-1/INS-23 axis constitute distinct regulatory mechanisms governing digestive adaptation.

In our previous study, we found that heat-killed E. coli promotes SS digestion in C. elegans (Geng et al., 2022), a process requiring intestinal BCF-1 (Hao et al., 2024). In the absence of BCF-1, the digestive capability of the animals is significantly reduced (Hao et al., 2024). This led us to investigate whether NSY-1 in AWC neurons regulates intestinal bcf-1 expression.

Firstly, we used a BCF-1::GFP reporter to measure bcf-1 expression in nsy-1 mutant animals. We found that mutation of nsy-1 induced bcf-1 expression in animals fed either SS or OP50 food (Figure 6A). Additionally, we confirmed that nsy-1 mutation in AWC neurons also induced intestinal bcf-1 expression under SS feeding conditions (Figure 6B). This data indicates that NSY-1 in AWC neurons inhibits intestinal bcf-1 expression, implying that nsy-1 mutation promotes SS digestion through BCF-1.

Next, we constructed a nsy-1; bcf-1 double mutant and analyzed the growth of these animals on SS. We found that the digestive ability of nsy-1 mutants was inhibited by the mutation of bcf-1 (Figure 6C). Together, our data suggest that the increased digestion ability in nsy-1 mutant animals is dependent on the intestinal digestion factor BCF-1.

We then asked how nsy-1 regulates intestinal bcf-1 expression. Since nsy-1 mutation induces the insulin peptide ins-23, which promotes SS digestion, we tested whether the induction of intestinal bcf-1 by nsy-1 mutation is also mediated through INS-23. We found that the bcf-1::GFP level decreased in nsy-1 mutant animals following ins-23 RNAi treatment (Figure 6D). This suggests that nsy-1 mutation activates bcf-1 expression in the intestine, which requires INS-23.

To test whether INS-23 acts in AWC neurons to regulate intestinal BCF-1, we generated AWC-specific knockdown strains, which were achieved by rescuing sid-1 cDNA expression under the ceh-36 promoter in a sid-1(qt9);BCF-1::GFP background.

We found that AWC-restricted ins-23 knockdown significantly reduced intestinal BCF-1::GFP expression (Figure 6—figure supplement 1A). This confirms that INS-23 functions within AWC sensory neurons to activate intestinal BCF-1, consistent with NSY-1’s upstream inhibition of INS-23 in this neuronal subtype.

NSY-1 promotes the AWC^OFF state through STR-130 to suppress SS digestion. To determine whether AWC-expressed STR-130 regulates intestinal BCF-1 expression, we observed that AWC neuron-specific RNAi of str-130 elevated intestinal BCF-1::GFP expression (Figure 6—figure supplement 1B). This demonstrates that STR-130 functions in AWC neurons to repress BCF-1 expression in the intestine.

Nematode stocks were maintained on nematode growth medium (NGM) plates seeded with bacteria (E. coli OP50) at 20°C.

The following strains/alleles were obtained from the Caenorhabditis Genetics Center (CGC) or as indicated:

E. coli-OP50 (from CGC) and SS (from ATCC) were cultured at 37℃ in LB medium. A standard overnight cultured bacteria was then spread onto each Nematode growth media (NGM) plate.

To construct the C. elegans plasmid for knockout of nsy-1 in AWC neuron, 600 bp promoter of eft-3 was replaced by 1348 bp promoter of odr-1 and nsy-1 sgRNA was also inserted into the same CRISPR-Cas9-sgRNA vector pDD162 (Dickinson et al., 2013). The Cas9 target sites were designed via CRISPR design tool (http://crispor.tefor.net/) and the sgRNA sequence was 5’-GAATTTACGCGTTCGAGAAATGG-3’. Knockout strains were generated by injecting 25 ng/μl Cas9-sgRNA plasmid, 2 μM repair template, co-injection markers include 20 ng/μl dpy-10 Cas9-sgRNA plasmid and 2 μM dpy-10 repair template.

Worms were picked into 10 μl of worm lysis buffer (50 mM KCl, 10 mM Tris–HCl pH 8.0, 2.5 mM MgCl₂, 0.45% NP40, 0.45% Tween-20, 0.01% gelactin, 0.2 mg/ml proteinase K), quickly freeze–thaw three times using liquid nitrogen, incubated it at 60°C for 90 min and 95°C for 20 min. 1 µl supernatant was taken and performed for PCR analysis with the following primers: nsy-1: forward 5′-CAAGAGGCAAGTGCAGCATA-3′, reverse 5′-TGACTGTCCCATGCTCTCAC-3′, then digested with NheI endonuclease overnight and identified by DNA agarose electrophoresis.

SS preparation was followed by our published protocol (Geng et al., 2022; Liu and Qi, 2023). Briefly, a standard overnight culture of SS (37℃ in LB broth) was diluted into fresh LB broth (1:100 ratio). SS was then spread onto each NGM plate when the diluted bacteria grew to OD600=0.5.

The standard overnight cultured SS was then spread onto 60 mm NGM plate. Worms were grown on E. coli - OP50, and eggs were collected by bleaching and then washing in M9 buffer. Synchronized L1 larvae were obtained by allowing the eggs to hatch in M9 buffer for 12 h. Synchronized L1s were seeded to plates prepared for the specific assay and incubated at 20℃ for 4 days. The developmental conditions were determined by body length.

Synchronized L1 animals were grown to the L4 stage on OP50 and then subjected to a 4 h treatment with 0.5% ethyl methanesulfonate (EMS). After treatment, the P0 generation animals were thoroughly washed with M9 solution and transferred to OP50 plates to grow and produce the F1 generation. Groups of 3–4 F1 animals were picked and placed onto new OP50 plates (3–4 F1 worms per plate) to generate the F2 generation through self-fertilization. Once the F2 generation reached adulthood, all the F2 animals were bleached to obtain the next generation of eggs. Synchronized L1 larvae were obtained by allowing the eggs to hatch in M9 buffer for 12 h. The synchronized L1s were then seeded onto SS plates and incubated at 20℃ for 4 days. Candidate mutants that could grow on SS plates were identified. To confirm the suspected mutants, each candidate mutant was picked singly onto OP50 for passaging and growth to adulthood. These adult animals were then bleached to obtain synchronized L1 mutants. The synchronized L1 mutants were seeded onto SS plates to confirm their ability to grow on SS.

DNA isolation, library construction, and whole-genome sequencing (WGS) with gene identification were carried out according to the published protocol (Joseph et al., 2018).

RNA-seq was done with three biological replicates that were independently generated, collected, and processed. Adult wild-type (N2) or nsy-1 mutant worms were bleached and then the eggs were incubated in M9 for 18 h to obtain synchronized L1 worms. Synchronized L1 worms were cultured in the NGM plate seeded with OP50 or SS for 4 h at 20°C. L1 worms were then collected for sequencing.

Slides for imaging were prepared by making a fresh flattened 5% agarose pad. Worms were mounted on 5% agar pads in M9 buffer with 5 mM levamisole then sealed beneath a 22 × 22 mm coverglass. Imaging was done using an Olympus BX53 microscope with a DP80 camera. str-2::gfp or bcf-1::gfp expression was measured through imaging.

The fluorescence photographs were taken using an Olympus BX53 microscope with a DP80 camera. Development statistics were taken using Olympus MVX10 dissecting microscope with a DP80 camera.