Skip to main content
NCBI home page
Scientific Reports logoLink to Scientific Reports
. 2026 Jan 7;16:4634. doi: 10.1038/s41598-025-34917-0

The FMRFamide-like peptide FLP-11 regulates the production and secretion of the TGF-β-like protein DAF-7 during Caenorhabditis elegans larval development

Riko Uegaki 1, Sho Maega 2, Masahiro Ono 1, Tomohiro Bito 1,2, Takashi Iwasaki 1,2, Akira Shiraishi 3, Honoo Satake 3, Tsuyoshi Kawano 1,2,✉
PMCID: PMC12867994  PMID: 41501349

Abstract

FMRFamide-like peptides (FLPs) are widely conserved in nematodes. Here, we conducted a molecular genetic study to confirm that the flp-11 gene regulates larval development in the free-living soil nematode model species Caenorhabditis elegans. We demonstrated that: (1) FLP-11 suppresses larval development, resulting in dauer diapause; (2) the production and secretion of FLP-11 by specific neurons is modulated by the availability of food (e.g., Escherichia coli) and by population density, both of which determine larval development/dauer diapause via transforming growth factor-β (TGF-β)-like and insulin-like signaling; (3) the FLP-11 peptide reduces the production and secretion of DAF-7, the sole TGF-β-like protein involved in larval development; (4) FLP-11 is not involved in insulin-like signaling; (5) FLP-11 modulates dauer diapause via neuropeptide receptor-22 (NPR-22), which regulates DAF-7 production and secretion. This study highlights the significance of a short neuropeptide that responds to the growth environment and directly regulates a TGF-β-like protein via its receptor candidate, thereby influencing larval development in C. elegans.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-34917-0.

Keywords: Caenorhabditis elegans, FMRFamide-like peptides, G protein-coupled receptor, Larval development, Transforming growth factor-β

Subject terms: Biotechnology, Molecular biology, Physiology

Introduction

In the animal kingdom, neuropeptides such as RFamide-family peptides modulate diverse physiological responses1. Moreover, neuropeptide families, such as the FMRFamides, are widely conserved in invertebrates, although not in vertebrates2. Nematodes, including the free-living soil nematode Caenorhabditis elegans, also possess FMRFamide-like peptides (FLPs; “flp” genes)3. C. elegans, in particular, is an excellent model system for studying FLPs due to the ease of experimentation and the depth of background knowledge available4. Consequently, the 31 flp genes in C. elegans have been intensively studied for their physiological functions. To date, the FLPs are reportedly involved in C. elegans behaviors, including sleep behavior, that remain the best studied5–12, while the involvement of FLPs in development is not fully understood. Previously, we have identified novel FLP-based molecular mechanisms involved in larval development13–16. However, detailed elucidation of the molecular mechanisms underlying C. elegans FLPs during larval development requires further research.

In C. elegans, head sensory neurons are the first to perceive environmental factors such as food, population, and temperature. After perception and subsequent signal integration, downstream signaling, including insulin-like and transforming growth factor-β (TGF-β)-like signaling, regulates steroid hormone signaling, which influences the final decision to continue larval development17. For example, the insulin-like peptide DAF-28 is the most prominent ligand of the sole insulin receptor-like protein DAF-218,19, while the sole ligand involved in TGF-β-like signaling relevant to larval development is DAF-720. Both ligands are produced in ASI neurons, which are primarily responsible for larval development21. In contrast, INS-35, mainly produced in the intestine, is a dominant insulin-like peptide responsible for both larval development and longevity22,23. Based on the presumption that neuropeptides, including FLPs, mediate the connection between environmental factors and downstream signaling, we attempted to elucidate the molecular mechanisms underlying C. elegans FLPs during larval development in this study. Previous studies have demonstrated that FLP-1 and FLP-2 modulate the insulin-like peptides DAF-28 and INS-35, respectively15,16. Moreover, FLP-3 and FLP-6 also modulate insulin-like peptides13,14. However, to date, no FLPs have yet been identified as involved in modulating the ligand of the TGF-β-like DAF-7 protein.

In this study, we selected the flp-11 gene for further elucidation. This selection was based on our prior screening of flp genes involved in larval development and/or DAF-7 secretion. Overall, we provide a comprehensive elucidation of the molecular mechanism by which flp-11 modulates larval development. Specifically, we report that FLP-11, via its strong receptor candidate, neuropeptide receptor-22 (NPR-22), suppresses both the production and secretion of DAF-7, resulting in dauer diapause. Moreover, although we have previously demonstrated that the orphan receptor NPR-15 is involved in DAF-7 production and secretion24, this study highlights how short peptides such as FLPs can directly regulate TGF-β-like proteins.

Results

Several flp genes regulate dauer diapause in C. elegans via modulation of DAF-7 secretion

Gene-disrupted nematodes were used to screen for flp genes potentially affecting reproductive development and/or dauer diapause. Several mutants exhibiting suppression or promotion of dauer diapause were identified (Supplementary Fig. 1; Supplementary Table 3A). Previous studies demonstrated that the flp-1, flp-2, flp-3, and flp-6 genes regulate larval development but are not involved in DAF-7 secretion13–16. Consequently, other flp genes, whose disruption suggested involvement in both larval development and DAF-7 secretion, were prioritized for further investigation. To identify such genes, tmIs4088 nematodes, which stably express daf-7p::DAF-7::mCherry (produced in a previous study), were crossed with corresponding gene-disrupted nematodes. mCherry fluorescence in coelomocytes was subsequently observed and compared with that in nematodes before crossing. Since coelomocytes take up a portion of secreted peptides and proteins fused to fluorescent markers25,26, mCherry fluorescence in coelomocytes was used as an indicator of fluorescent protein secretion. This screening process narrowed the focus to flp-8, flp-11, flp-21, and flp-26, whose disruption resulted in an increased uptake of fluorescent protein by coelomocytes (Supplementary Fig. 2). Among these genes, flp-11 has been reported to influence sleep and quiescent behaviors, as well as dauer diapause6,12. Therefore, flp-11 was selected for detailed analysis to determine its role in regulating sleep behavior and dauer diapause.

flp-11 is involved in larval development

The involvement of flp-11 in larval development was verified using flp-11(tm2706) nematodes. In this mutant line, a 154 bp deletion in the second exon of flp-11 (spanning Ala32 to Phe82) introduces a frameshift error, resulting in a null mutation (Fig. 1A and B). To ensure accuracy, flp-11(tm2706) nematodes were outcrossed with wild-type nematodes to eliminate unexpected mutations caused by chemicals and UV radiation27. The transgene flp-11p::flp-11, along with lin-44p::gfp (used as an injection marker), was then introduced. This transgene was subsequently fixed into the chromosome to ensure stable expression. Rescue nematodes (tkIs8) were generated by outcrossing wild-type nematodes with transgene-bearing lines, thereby eliminating additional mutations. Additionally, the transgene and injection marker were integrated into wild-type nematodes (tkIs9) to stably overexpress FLP-11. Rescue nematodes (tkIs8) and overexpression (OE) nematodes (tkIs9) were subjected to dauer diapause measurements, which were compared with those of wild-type and flp-11(tm2706) nematodes. As shown in Fig. 1D and Supplementary Table 3B, flp-11(tm2706) nematodes exhibited a significant decrease in dauer diapause compared to wild-type nematodes. In contrast, tkIs8 rescue nematodes showed dauer diapause levels nearly identical to wild-type nematodes. OE nematodes (tkIs9), however, displayed a significant increase in dauer diapause, differing markedly from flp-11(tm2706) mutants. Taken together, these findings confirm that flp-11 plays a critical role in larval development.

Fig. 1.

Fig. 1

Open in a new tab

The flp-11 is involved in larval development. (A) A schematic representation of the flp-11 gene structure. Black boxes and solid lines correspond to exons and introns, respectively. The dotted line represents the deletion site of flp-11(tm2706). (B) The amino acid sequence (110 amino acids) of the predicted precursor protein encoded by the flp-11 gene. The underlined sequence L(F)VRF is common. Bold sequences indicate predicted FLP-11 peptides before C-terminal amidation, while red residues represent putative proteolytic sites. Finally, the blue sequence represents the predicted signal sequence as determined by SignalP (https://services.healthtech.dtu.dk/service.php?SignalP-6.0). The precursor protein was predicted to yield three peptides with the C-terminal amide: FLP-11-1: AMRNALVRF-NH2, FLP-11-2: ASGGMRNALVRF-NH2, and FLP-11-3: NGAPQPFVRF-NH2. (C) Optical microscope image of dauer and non-dauer nematodes. The image depicts four dauer larvae indicated by open triangles, which are smaller and immobile, as well as two non-dauer larvae that reached the fourth stage of development. (D) Dauer diapause rates of nematode strains. The chart shows the actual values of larval diapause rate of mutant and wild-type nematodes in the presence of the dauer pheromone. The values represent three independent experiments (trials) using three NGM plates containing the dauer pheromone (n = 9). Multiple comparisons between groups were statistically assessed using Tukey’s HSD tests. The letters “a,” “b,” and “c” denote statistically significant differences between values. Detailed parameters, including numbers, trials, p-values, and mean ± SEM, are shown in Supplementary Table 3B.

FLP-11 modulates the production and secretion of DAF-7

The involvement of flp-11 in DAF-7 secretion was verified by analyzing daf-7p::DAF-7::mCherry fluorescence in coelomocytes across several nematode strains. The flp-11(tm2706), tkIs8 [flp-11p::flp-11; flp-11(tm2706)], and tkIs9(flp-11p::flp-11) nematodes were crossed with tmIs4088 nematodes, which stably express daf-7p::DAF-7::mCherry. The mCherry fluorescence levels in these strains were then compared to those of tmIs4088 nematodes. As shown in Fig. 2A, flp-11(tm2706); tmIs4088 nematodes exhibited a significant increase in mCherry fluorescence intensity in coelomocytes, indicating enhanced DAF-7 secretion. In contrast, tkIs9; tmIs4088 nematodes showed a significant decrease in fluorescence intensity. The fluorescence intensity in tkIs8; tmIs4088 nematodes was similar to that observed in tmIs4088 nematodes. These results align with the dauer diapause measurements described earlier (Fig. 1D). Collectively, these findings demonstrate that increased DAF-7 secretion suppresses dauer diapause, whereas reduced secretion promotes it.

Fig. 2.

Fig. 2

Open in a new tab

The flp-11 is involved in DAF-7 production and secretion. Multiple comparisons among groups were assessed for statistical significance using Tukey’s HSD tests. The chart shows the fluorescence intensity of five independent nematodes measured. The letters “a,” “b,” and “c” denote statistically significant differences between values. Detailed parameters, including numbers, trials, p-values, and mean ± SEM, are shown in Supplementary Tables 4A and B. (A) FLP-11 is involved in DAF-7 secretion. Shown is daf-7p::DAF-7::mCherry fluorescence taken up by coelomocytes of flp-11(tm2706); tmIs4088, flp-11(tm2706); tkIs8; tmIs4088, and tkIs9; tmIs4088 nematodes. Each group was compared with the tmIs4088 nematodes in a pairwise fashion. Open triangles indicate coelomocytes that took up fluorescent proteins. n = 5. (B) FLP-11 is involved in DAF-7 production. The daf-7p::GFP fluorescence levels of flp-11(tm2706); ksIs2, tkIs8; ksIs2, and tkIs9; ksIs2 nematodes were compared with ksIs2 nematodes. Open triangles indicate ASI neurons expressing daf-7p::GFP. n = 5.

The effect of flp-11 on DAF-7 production was also examined. To evaluate this, flp-11(tm2706), tkIs8, and tkIs9 nematodes were crossed with ksIs2 nematodes, which stably express daf-7p::GFP. GFP fluorescence in the resulting strains was compared to that in uncrossed ksIs2 nematodes (Fig. 2B). In flp-11(tm2706); ksIs2 nematodes, GFP fluorescence intensity significantly increased compared to ksIs2 nematodes. Conversely, tkIs9; ksIs2 nematodes exhibited a significant decrease in fluorescence intensity. The fluorescence intensity in tkIs8; ksIs2 nematodes was approximately the same as that in ksIs2 nematodes. These results are consistent with the mCherry fluorescence data, further confirming that DAF-7 production and secretion are modulated by flp-11.

Based on these findings, it is concluded that FLP-11 contributes to larval development by regulating both DAF-7 production and secretion. The levels of production were directly associated with the amounts of secretion.

FLP-11 modulates larval development via TGF-β-like signaling, not insulin-like signaling

The involvement of flp-11 in the TGF-β signaling pathway was verified through epistasis analysis. For this purpose, the daf-1(m40) and daf-3(e1376) mutants, both containing alterations in the TGF-β-like signaling pathway, were utilized. daf-1 encodes the receptor for the ligand DAF-7, and its loss of function results in constitutive dauer diapause (the daf-c phenotype)28. In contrast, daf-3 encodes a downstream transcription regulatory factor, and its loss of function results in defective dauer diapause (the daf-d phenotype)29. In addition, the daf-2(e1370) and daf-16(m27) mutants, which contain alterations in the insulin-like signaling pathway, were included. The daf-2 gene encodes the sole insulin receptor-like protein, and its loss of function also produces the daf-c phenotype19. Conversely, daf-16 encodes a downstream transcription factor, and its loss of function results in the daf-d phenotype30,31. flp-11(tm2706) mutants were crossed with daf-1 and daf-2 mutants, respectively, and dauer diapause phenotypes were measured relative to wild-type and flp-11(tm2706) nematodes. As shown in Fig. 3A, the daf-1 mutation completely abolished the reduction in dauer diapause observed in flp-11(tm2706) nematodes. Moreover, flp-11(tm2706); daf-1(m40) nematodes exhibited dauer diapause levels comparable to those of daf-1(m40) nematodes, indicating a significant increase in dauer diapause. These results indicate that daf-1 is epistatic to flp-11. In contrast, flp-11(tm2706); daf-2(e1370) nematodes displayed a decrease in dauer diapause even after crossing with daf-2(e1370) mutant nematodes (Fig. 3B). Taken together, these findings strongly suggest that flp-11 regulates dauer diapause through the TGF-β-like signaling pathway rather than the insulin-like signaling pathway.

Fig. 3.

Fig. 3

Open in a new tab

Epistasis analyses of flp-11 to mutations in TGF-β or insulin-like signaling, as measured by promotion or suppression of dauer diapause. The chart shows the actual values of dauer formation in mutant and wild-type nematode lines in the presence of dauer-inducing pheromone. The values represent three independent experiments (trials) using three NGM plates containing the dauer pheromone (n = 9). Multiple comparisons between groups were statistically assessed using Tukey’s HSD tests. The letters “a,” “b,” “c,” and “d” denote statistically significant differences between values. The total number of worms subjected to each assay is described. Data are presented as mean ± SEM, with error bars indicating the SEM. Detailed parameters, including numbers, trials, p-values, and mean ± SEM, can be found in Supplementary Table 3C–F. (A) The loss of function in daf-1 is epistatic to the function of flp-11. Shown are the actual values of dauer formation in each mutant nematode (i.e., tm2706, daf-1(m40), tm2706; daf-1(m40) and wild-type nematodes. n = 9. (B) The loss of function in daf-2 is not epistatic to the function of flp-11. Shown are the actual values of dauer formation in each mutant nematode (i.e., tm2706, daf-2(e1370), tm2706; daf-2(e1370) and wild-type nematodes. n = 9. (C) The loss of function in daf-3 is epistatic to the overexpression of flp-11. Shown are the actual values of dauer formation in each mutant nematode (i.e., tkIs9, daf-3(e1376), tkIs9; daf-3(e1376), and wild-type nematodes. n = 9. (D) The loss of function in daf-16 is not epistatic to the overexpression of flp-11. Shown are the actual values of dauer formation in each mutant nematode (i.e., tkIs9, daf-16(m27), tkIs9; daf-16(m27) and wild-type nematodes. n = 9.

Subsequently, a converse epistasis analysis was conducted to confirm these findings further. For this analysis, tkIs9 nematodes (overexpressing FLP-11) were crossed with daf-3 and daf-16 mutants, and dauer diapause phenotypes were measured relative to wild-type and tkIs9 nematodes. As shown in Fig. 3C, the daf-3 mutation completely suppressed the increase in dauer diapause observed in tkIs9 nematodes. In addition, tkIs9; daf-3(e1376) nematodes exhibited dauer diapause levels equivalent to those of daf-3(e1376) mutants, which show significantly reduced dauer diapause. These results indicate that daf-3 is epistatic to flp-11. In contrast, tkIs9; daf-16(m27) nematodes exhibited increased dauer diapause levels even after crossing with daf-16(m27) mutants (Fig. 3D). These results further support the conclusion that flp-11 regulates dauer diapause through TGF-β-like signaling rather than insulin-like signaling. By integrating these findings, it is concluded that flp-11 functions upstream of the TGF-β-like signaling pathway and does not regulate dauer diapause via the insulin-like signaling pathway.

FLP-11 production and secretion respond to food and dauer pheromone

FLP-11-producing neurons involved in the regulation of dauer diapause were identified, and the responsiveness of FLP-11 production and secretion to environmental factors, including food and population density (represented by dauer pheromone), was examined. NY2040 (ynIs40) nematodes, which stably express flp-11p::GFP, were utilized for this purpose. Previous studies have reported that FLP-11 is expressed in RIS neurons associated with sleep behavior6. GFP fluorescence was observed in several neurons, including RIS neurons (Fig. 4A and B). Analysis of single-cell RNA-seq datasets from CeNGEN (https://cengen.shinyapps.io/CengenApp/) and WormAtlas (https://www.wormatlas.org/) further identified URX and SAB neurons, in addition to RIS neurons, as FLP-11-producing cells.

Fig. 4.

Fig. 4

Open in a new tab

Effect of food and dauer pheromone on the production of FLP-11 in respective neurons. The FLP-11 production, as represented by the fluorescence created by flp-11p::GFP, was observed in the presence or absence of food and dauer pheromone. The chart shows the fluorescence intensity of five independent nematodes measured. The dot represents the value of each measurement. Comparisons between groups were made using Student’s t-tests. Data are expressed as mean ± SEM. *p < 0.05, ***p < 0.001. n.s., not significant. Detailed parameters, including p-values and mean ± SEM, are shown in Supplementary Table 4C. (A) The fluorescent intensity in RIS, URX, and SAB neurons in the presence or absence of food (E. coli). Arrowheads indicate corresponding neurons. n = 5. (B) Fluorescent intensity in RIS, URX, and SAB neurons in the absence or presence of dauer pheromones. Arrowheads indicate corresponding neurons. n = 5.

Subsequently, the effect of food on FLP-11 production was analyzed using ynIs40 nematodes by comparing flp-11p::GFP fluorescence intensity in the presence or absence of food (E. coli), as food promotes larval development and suppresses dauer diapause. As shown in Fig. 4A, fluorescence intensity in RIS neurons remained similar regardless of food availability. In contrast, significant increases in fluorescence intensity were observed in URX and SAB neurons when food was absent compared to when it was present. These results suggest that food suppresses FLP-11 production in URX and SAB neurons, but not in RIS neurons.

Next, to assess the effect of population density, represented by dauer pheromones, daf-22(ok693) mutant nematodes, which are defective in pheromone biosynthesis32, were crossed with ynIs40 nematodes. GFP fluorescence intensity was then compared in the presence and absence of exogenous crude pheromone extracts33. As expected, pheromone exposure resulted in increased fluorescence intensity in URX and SAB neurons compared to pheromone-free conditions, while fluorescence intensity in RIS neurons remained unchanged (Fig. 4B). These findings indicate that dauer pheromones promote FLP-11 production in URX and SAB neurons but not in RIS neurons. Overall, these results suggest that FLP-11 produced in URX and SAB neurons responds to environmental factors such as food and dauer pheromone, and is critical for modulating dauer diapause. In contrast, constitutive FLP-11 expression in RIS neurons may not contribute to dauer diapause regulation.

Additionally, the responsiveness of FLP-11 secretion to environmental factors was further evaluated using tkEx10 nematodes, which express flp-11p::FLP-11::mRFP. mRFP fluorescence in coelomocytes, representing FLP-11 secretion, was observed. As shown in Supplementary Fig. 4A, fluorescence intensity increased significantly in the absence of food compared to measurements taken in the presence of food. Similarly, tkEx10; daf-22(ok693) nematodes exposed to dauer pheromone exhibited increased mCherry fluorescence compared to pheromone-free conditions (Supplementary Fig. 4B). These results are consistent with the FLP-11 production data, indicating that increased FLP-11 production enhances its secretion.

FLP-11 production in RIS neurons is not necessary for dauer diapause

The transcription factor APTF-1 is reportedly expressed in AIB, RIB, and RIS neurons, where it regulates sleep behavior via FLP-11 production. In contrast, APTF-1 is not expressed in URX or SAB neurons6. To investigate the relevance of FLP-11 production in RIS neurons to dauer diapause, apft-1(tm3287) mutant nematodes were used. The ynIs40 nematodes, which stably express flp-11p::GFP, were crossed with apft-1(tm3287) mutants to generate the necessary lines. GFP fluorescence was then recorded in the absence of apft-1 to determine whether FLP-11 production in RIS neurons depends on this transcription factor. As expected, the fluorescent signal in RIS neurons disappeared in apft-1(tm3287) mutants (Fig. 5A). In contrast, GFP fluorescence in URX and SAB neurons was unaffected and appeared identical to that observed in ynIs40 nematodes. These findings indicate that FLP-11 production in URX and SAB neurons is independent of APTF-1, as anticipated. The relevance of RIS neurons to dauer diapause was further evaluated by crossing tkIs9 nematodes (which overexpress FLP-11) with apft-1(tm3287) mutants. Dauer diapause phenotypes were measured in apft-1(tm3287) mutants, tkIs9 nematodes, and tkIs9; apft-1(tm3287) lines, and were compared to wild-type nematodes. As shown in Fig. 5B, apft-1(tm3287) mutants exhibited dauer diapause levels comparable to wild-type nematodes. tkIs9 nematodes showed a significant increase in dauer diapause, and this phenotype remained unaffected in tkIs9; apft-1(tm3287) lines. These observations suggest that the loss of apft-1 has no significant effect on dauer diapause.

Fig. 5.

Fig. 5

Open in a new tab

The transcription factor APTF-1 is not involved in FLP-11 production in URX and SAB neurons during larval diapause. (A) Production of FLP-11 in RIS, URX, and SAB neurons in the presence or absence of aptf-1. Shown is the fluorescence intensity of flp-11p::GFP, which was used to represent FLP-11 production in each neuron. These intensities were then compared in the presence and absence of aptf-1. The chart shows the fluorescence intensity of five independent nematodes measured. Comparisons between group means were made using Student’s t-tests. All data shown are expressed as mean ± SEM. ***p < 0.001. n.s., not significant. n = 5. Detailed parameters, including p-values and mean ± SEM, are shown in Supplementary Table 4E. (B) The aptf-1 is not involved in larval diapause. Shown are the actual values of dauer formation in each mutant and wild-type nematodes in the presence of dauer-inducing pheromone. The values represent three independent experiments (trials) using three NGM plates containing the dauer pheromone. n = 9. All data are presented as mean ± SEM, with error bars indicating SEM. Multiple comparisons among groups were evaluated using Tukey’s HSD tests. The letters “a” and “b” indicate statistically significant differences between variables. Detailed parameters, including numbers, trials, p-values, and mean ± SEM, can be found in Supplementary Table 3G.

Taken together, these results demonstrate that FLP-11 production in URX and SAB neurons, rather than in RIS neurons, is the dominant factor influencing dauer diapause.

NPR-22 is a candidate for the FLP-11 receptor that plays a role in regulating dauer diapause

Finally, the identification of an FLP-11 receptor was pursued, as neuropeptide receptors (NPRs) in C. elegans are generally known to be G protein-coupled receptors that bind short neuropeptides such as FLPs3. Previous screening of npr genes associated with larval development and dauer diapause identified several candidates, including npr-2, npr-3, npr-4, npr-6, npr-10, npr-11, npr-12, npr-17, npr-22, npr-23, npr-24, npr-25, npr-27, npr-28, and npr-34. Disruption of these genes suppressed dauer diapause, a phenotype that closely resembles the effect of flp-11 disruption24. Subsequent machine-learning analyses that paired FLP-11 peptides with NPRs identified NPR-22 as the most likely receptor for FLP-11, as this pairing score was the highest (Supplementary Table 5). NPR-22 has also been speculated to be expressed in ASI neurons, which are responsible for DAF-7 production and secretion (WormBase). Additionally, Beets et al. demonstrated that synthetic FLP-11 binds to NPR-22 when expressed in CHO-K1 cells34. Based on these findings, NPR-22 was hypothesized to function as an FLP-11 receptor during dauer diapause, and its role was further investigated. To confirm the involvement of NPR-22, npr-22(tm13562) mutants were outcrossed with wild-type nematodes to eliminate unexpected mutations. The npr-22p::npr-22 transgene was subsequently introduced and integrated into the chromosome to generate tkIs10 nematodes for rescue experiments. Dauer diapause in npr-22(tm13562) mutants and in tkIs10 nematodes was measured and compared with that of wild-type nematodes. As shown in Fig. 6B, npr-22(tm13562) mutants exhibited decreased dauer diapause, similar to flp-11(tm2706) mutants. Rescue experiments with tkIs10 nematodes restored dauer diapause levels to those of wild-type nematodes.

Fig. 6.

Fig. 6

Open in a new tab

The npr-22, encoding a receptor candidate for FLP-11, is involved in larval development. (A) A schematic representation of npr-22 gene structure. Black boxes and solid lines correspond to exons and introns, respectively. The dotted line represents the deletion site of npr-22(tm13562). (B, C) Dauer diapause rates of nematode strains. The chart shows the actual values of dauer formation of mutant and wild-type nematodes in the presence of the dauer pheromone. The values represent three independent experiments (trials) using three NGM plates containing the dauer pheromone (n = 9). Multiple comparisons between groups were statistically assessed using Tukey’s HSD tests. The letters “a,” “b,” and “c” denote statistically significant differences between values. (B) The npr-22 gene is involved in larval development. The detailed parameters are shown in Supplementary Table 3H. n = 9. (C) The npr-22 gene is located downstream of the flp-11 gene to regulate larval development. The detailed parameters are shown in Supplementary Table 3I. n = 9.

To further investigate the genetic relationship between NPR-22 and FLP-11, an epistasis analysis was performed. Crosses between npr-22(tm13562) and tkIs9 nematodes (overexpressing FLP-11) were analyzed for dauer diapause phenotypes. As shown in Fig. 6C, the disruption of NPR-22 nullified the increase in dauer diapause observed in tkIs9 nematodes. Additionally, npr-22(tm13562) mutants exhibited a dauer diapause phenotype similar to that of npr-22(tm13562); tkIs9 nematodes. Similarly, npr-22(tm13562) and npr-22(tm13562); flp-11(tm2706) double mutants exhibited reduced dauer diapause at levels comparable to the respective single mutants (Fig. 6C). These findings suggest that the double mutation has no additive effect and indicate that NPR-22 is epistatic to FLP-11.

NPR-22 is expressed in ASI neurons and is involved in DAF-7 production and secretion

The influence of NPR-22 on DAF-7 production and secretion was examined, as ASI neurons are known to produce and secrete DAF-7 protein. To verify NPR-22 expression in ASI neurons, the npr-22p::npr-22::mrfp construct was introduced into wild-type nematodes and subsequently fixed, resulting in tkIs11 nematodes that stably express NPR-22::mRFP under the control of the npr-22 promoter. tkIs11 nematodes were then crossed with GR1455 nematodes, which stably express daf-28p::GFP in ASI and ASJ neurons. GFP and mRFP fluorescence were observed in the resulting nematodes, indicating the co-expression of daf-28p::GFP and npr-22p::NPR-22::mRFP. As shown in Fig. 7A, NPR-22::mRFP fluorescence was detected in both ASI and ASJ neurons, consistent with daf-28p::GFP expression. However, mRFP fluorescence intensity was notably higher in ASI neurons than in ASJ neurons, suggesting that NPR-22 is predominantly expressed in ASI neurons, which are responsible for DAF-7 production and secretion.　Subsequently, the role of NPR-22 in regulating DAF-7 production and secretion was further investigated by crossing npr-22(tm13562) mutants and tkIs10 rescue lines with ksIs2 nematodes that stably express daf-7p::GFP to detect DAF-7 production. In addition, npr-22(tm13562) mutants tkIs10 rescue lines were crossed with tmIs4088 nematodes (stably expressing daf-7p::DAF-7::mCherry) to analyze DAF-7 secretion. As shown in Fig. 7B, GFP fluorescence in the npr-22(tm13562) background was significantly higher than in the wild-type background, indicating increased DAF-7 production. However, GFP fluorescence levels in the tkIs10 background were similar to those observed in wild-type nematodes. Similarly, mCherry fluorescence in npr-22(tm13562) mutants was elevated compared to the wild-type background, whereas fluorescence in tkIs10 nematodes remained comparable to that of wild-type nematodes.

Fig. 7.

Fig. 7

Open in a new tab

NPR-22 modulates DAF-7 production and secretion by acting as an FLP-11 receptor. (A) NPR-22 is mainly expressed in ASI neurons. Shown are the fluorescence of daf-28p::GFP and npr-22p::NPR-22::mRFP. Arrowheads indicate ASI neurons. (B) NPR-22 is involved in DAF-7 production and secretion. Shown are the actual values of fluorescence intensity of daf-7p::GFP expressed in ASI neurons, which represents DAF-7 production. Data is shown for wild-type, npr-22-defect, and npr-22-rescued nematode lines (left). n = 5. Arrowheads indicate ASI neurons. Shown are the actual values of fluorescence intensity of the daf-7p::DAF-7::mCherry uptake in coelomocytes, which represents DAF-7 secretion. Data is provided for wild-type, npr-22 defect, and npr-22-rescued nematodes (right). n = 5. Arrowheads indicate coelomocytes. Multiple comparisons among groups were conducted using Tukey’s HSD tests. The letters “a” and “b” denote statistically significant differences between group means. Detailed parameters, including numbers, p-values, and mean ± SEM, are shown in Supplementary Table 4F.

These results indicate that NPR-22, in conjunction with FLP-11, suppresses DAF-7 production and secretion. Furthermore, these findings strongly suggest that NPR-22 acts as an FLP-11 receptor, regulating dauer diapause by modulating DAF-7 production and secretion. NPR-22 is not involved in DAF-28 secretion as FLP-11 (Supplementary Fig. 5).

Discussion

In this study, we determined that flp-11 is involved in dauer diapause by modulating DAF-7 production and secretion. DAF-7 is the sole ligand involved in TGF-β-like signaling and is involved in larval development together with other ligands linked to insulin-like signaling17. Our epistasis analyses revealed that FLP-11 suppressed larval development and promoted dauer diapause via TGF-β-like signaling, but not via insulin-like signaling. For insulin-like signaling, DAF-28 is the most dominant ligand among those relevant for larval development and/or dauer diapause. Moreover, both DAF-7 and DAF-28 are mainly produced in ASI neurons, which are the most important head neurons for regulating larval development21. Here, the molecular mechanism of FLP-11 is known to suppress DAF-7, but not DAF-28. Specifically, FLP-11 may regulate daf-7 transcription; this is supported by the observation that FLP-11 suppresses DAF-7 production (Fig. 2B). Moreover, FLP-11 does not seem to affect DAF-28 production or secretion (Supplementary Fig. 3). Further research should elucidate how flp-11 is involved in DAF-7 production, including which downstream transcription factors are involved. We previously demonstrated that FLP-1 is specifically involved in DAF-28 production in ASI neurons16 and that FLP-2 is specifically involved in INS-35 production in the intestine15. Combined with the findings of this study, we conclude that many FLPs have highly specific target molecules (e.g., a TGF-β-like or insulin-like molecule) to modulate larval development/dauer diapause. However, this study highlights that a short neuropeptide, such as FLP-11, is directly involved in TGF-β-like molecule production or secretion, thereby affecting larval development in C. elegans. In contrast, Hibert and Kim reported that the C. elegans neuropeptide Pigment Dispersing Factor-1, which is involved in male-specific behavior, regulates DAF-7 expression in ASJ neurons—not ASI neurons—in adult males35. This finding, along with our results, highlights the diverse physiological functions of DAF-7, which are modulated by distinct neuropeptides.

FLP-11 is reportedly involved in sleep behavior6, prompting us to focus on a putative link between the flp-11 gene and dauer diapause via increased DAF-7 production and secretion. Among the neurons expressing FLP-11, RIS neurons are the most responsible for sleep behavior, and these neurons have been found to express FLP-11 constantly. In these neurons, FLP-11 expression is regulated by the transcription factor APTF-1, which—according to CeNGEN data—is not expressed in URX or SAB neurons. We note that food and dauer pheromones showed no influence on FLP-11 expression in RIS neurons. In contrast, URX and SAB neurons expressing FLP-11 both responded to food and dauer pheromones, leading to changes in FLP-11 expression in these cells. Moreover, the absence of food induced dauer diapause and promoted FLP-11 production, as did the presence of dauer pheromones. Importantly, these results are consistent with the fact that FLP-11 is involved in dauer diapause. Via FLP-11, the RIS neurons modulate sleep behavior, whereas the URX and SAB neurons are involved in dauer diapause through DAF-7, a TGF-β-like molecule. Sleep behavior and dauer diapause induction through FLP-11 are triggered in a neuronally specific manner and are, therefore, distinguishable. The independence of RIS neurons in dauer diapause is supported by the fact that the disruption of aptf-1, which results in the loss of FLP-11 production, does not affect dauer diapause (Fig. 5B). Recently, Rossi et al. demonstrated that FLP-11 and its receptor candidate DroMyoSuppressin receptor-related 1 (DMSR-1), expressed in RIS neurons, are involved in regulating sleep behavior36. Therefore, FLP-11 may play a crucial role in not only sleep behavior but also dauer diapause through distinct GPCRs.

Additionally, we determined that NPR-22 is a strong candidate for the FLP-11 receptor that plays a role in regulating dauer diapause. Molecular genetic analyses revealed that NPR-22 is expressed in ASI neurons and that the GPCR, like FLP-11, regulates DAF-7 production and secretion, thereby modulating larval development. A previous study demonstrated that NPR-15, an orphan receptor, also regulates larval diapause by regulating DAF-7 production and secretion24. Interestingly, NPR-15, expressed in ASI neurons, promotes DAF-7 production and secretion, whereas NPR-22 suppresses it, indicating that these GPCRs regulate the same function in opposite directions. Overall, in this C. elegans model system, short neuropeptides coordinately regulate the production and secretion of a TGF-β molecule, thereby modulating development through specific receptors (Fig. 8).

Fig. 8.

Fig. 8

Open in a new tab

Short neuropeptides coordinately regulate TGF-β via the NPR-15 and NPR-22 receptors in C. elegans. FLP-11 suppresses DAF-7 production and secretion via its receptor NPR-22, which leads to larval diapause. An unidentified neuropeptide probably promotes DAF-7 production and secretion via the expected receptor NPR-15, thereby facilitating larval development.

In mammals, neuropeptide Y mediates in various physiological functions via different GPCRs37. This study suggests that FLP-11 may play a critical role in both sleep behavior and dauer diapause by acting on specific GPCRs, namely DMSR-1 and NPR-22, respectively. Such peptide/GPCR diversity is common not only in mammals but also in the model organism Caenorhabditis elegans. Although FLPs (FMRFamide-like peptides) are conserved among nematodes, not mammals3, this study may provide new insights into the function of RFamide-family peptides, which are widely conserved across the animal kingdom1.

Materials and methods

Nematode strains

We maintained Caenorhabditis elegans strains at 20 °C on nematode growth media (NGM) plates seeded with Escherichia coli OP5038. All strains were provided by the Caenorhabditis Genetics Center (CGC) and the National Bioresource Project for “C. elegans.” N2 (Bristol) was the primary wild-type strain used for all analyses—nematodes containing the alleles listed in Supplementary Table 1. Finally, transgenic nematodes carrying tmIs4088(daf-7p::daf-7::mCherry) and tmIs4106(daf-28p::daf-28::mCherry) were produced in our previous study.

Dauer formation assay

Dauer formation assays and all associated statistical analyses were performed as previously described15. Briefly, hatched nematodes were grown on NGM plates containing the dauer pheromone; for mutant nematodes, pheromone was added to NGM plates to avoid saturation of dauer formation. Next, both dauer and non-dauer larvae were counted. Larvae were categorized by assessing characteristic morphological features (e.g., the presence of dark pigment granules, constriction of body and pharynx, and loss of pharyngeal pumping) as well as by movement. Dauer larvae were then further categorized as full or partial dauer39. Finally, we obtained counts for all full-dauer larvae, which were motionless. The assay was conducted using more than three independent plates per strain as a single trial. At least three trials were conducted separately, except for the first screening described in Supplementary Fig. 1.

Plasmid construction

All plasmids described below were constructed as indicated and used to transform E. coli strain DH5α (TOYOBO, Osaka, Japan) for plasmid amplification. All PCR primers used during this study are listed in Supplementary Table 2. DNA amplification was performed by fidelity PCR, and the resulting plasmid sequences were verified by sequencing.

flp-11 transgene (flp-11p::flp-11)

We used PCR to amplify a genomic fragment containing the coding region of flp-11 from C. elegans genomic DNA; we also amplified sequences 4.2 kb upstream and 1.0 kb downstream of the gene itself. The amplified fragment was then inserted into the pGEM-T Easy vector (Promega, WI, USA) using a 10X A-attachment mix (TOYOBO) following dA attachment. This transgene was then introduced into flp-11-disrupted nematodes (i.e., for a rescue experiment) and wild-type nematodes (i.e., for an overexpression experiment), respectively.

flp-11 reporter transgene (flp-11p::flp-11::mrfp)

We amplified the flp-11 coding region, excluding the stop codon, as well as the sequence up to 4.2 kb upstream, by PCR from the verified flp-11p::flp-11 plasmid. The resulting amplified fragment was then introduced into the Sma I site of the pHK_mrfp vector using an In-Fusion kit (Clontech, CA, USA).

npr-22 transgene (npr-22p::npr-22)

A genomic fragment containing the npr22 coding region as well as sequences 4.3 kb upstream and 1.0 kb downstream was amplified by PCR from C. elegans genomic DNA. This fragment was then inserted into a pGEM-T Easy vector (Promega) following dA attachment using a 10X A-attachment mix (TOYOBO). The transgene-containing vector was then introduced into npr-22-disrupted nematodes to facilitate a rescue experiment.

npr-22 reporter transgene (npr-22p::npr-22::mrfp)

The npr-22 coding region, excluding the stop codon, as well as the sequence 4.3 kb upstream of the gene, was amplified by PCR from the npr-22p::npr-22 plasmid. The resulting amplified fragment was then introduced into the Sma I site of the pHK_mrfp vector using an In-Fusion kit (Clontech, CA, USA).

Transgenic worms

To conduct a rescue test for flp-11, the flp-11p::flp-11 transgene was first injected at 15 ng/µl into the gonads of flp-11-disrupted nematodes along with lin-44p::gfp (10 ng/µl), which acted as a transformation marker and did not influence larval development. To conduct an flp-11 overexpression experiment, the flp-11p::flp-11 transgene was injected at 15 ng/µl into the gonads of wild-type nematodes along with lin-44p::gfp (10 ng/µl). Transgenic nematodes were then subjected to ultraviolet (UV) irradiation for chromosomal insertion of the flp-11p::flp-11 transgene40. Transgenic nematodes were then outcrossed with wild-type nematodes five times to remove low-frequency mutations; these crosses yielded the tkIs8 and tkIs9 strains, respectively.

To conduct a rescue test of npr-22, the transgene npr-22p::npr-22 was injected at 15 ng/µl into the gonads of npr-22-disrupted nematodes along with lin-44p::gfp (10 ng/µl). Next, to assess the expression of npr-22p::NPR-22::mRFP, a reporter gene was injected at 15 ng/µl into the gonads of wild-type nematodes along with lin-44p::gfp (10 ng/µl). Transgenic nematodes were then subjected to UV irradiation before being subsequently outcrossed with wild-type nematodes to yield the tkIs10 and tkIs11 strains.

Microscopy

Fluorescence images and intensity profiles were obtained using an FV10i confocal laser-scanning microscope (Olympus Life Science, Japan), and images were analyzed using FV10-AWS software. These images are shown in Figs. 2, 4, 5 and 6, and 8 and Supplementary Figs. 2, 3, and 4. The statistical significance of mean differences in fluorescent intensity between groups was determined using Student’s t-tests as implemented by SPSS (IBM SPSS, USA). p < 0.05 was used as the threshold of statistical significance for all statistical tests.

Prediction of the pairing of FLP-11 peptides and NPRs

We previously developed a peptide-descriptor-incorporated support vector machine (PD-incorporated SVM) to use machine-learning procedures to predict peptide-GPCR pairs41. The predictive scores of the PD-incorporated SVM are normalized using logistic regression. Therefore, a difference of 0.01 in the score corresponds to a 1% difference in the probability of interaction. This method to was used to predict pairings among three predicted peptide amides (i.e., FLP-11-1 to FLP-11-3) and 12 focused NPRs. The three predicted FLP-11 peptides were deduced from the amino acid sequences of precursor proteins42.

Statistical analysis

Correlations were determined using Pearson’s correlation analysis, and data were fit with a linear regression. Differences between the two groups were analyzed by Student’s t-test with a two-tailed distribution, whereas differences among multiple groups were evaluated by Dunnett’s test or Tukey’s HSD test. All statistical analyses were conducted with KaleidaGraph Ver. 5.0.6 (Synergy Software, PA, USA). p-values < 0.05 were considered statistically significant.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary Material 1 (4.4MB, docx)

Acknowledgements

We acknowledge the strains provided by the Caenorhabditis Genetics Center funded by the National Institutes of Health National Center for Research Resources (NCRR), the International C. elegans Gene Knockout Consortium, and the National Bioresource Project for the Experimental Animal “Nematode C. elegans” of Japan. We are grateful to Ms. Masayo Nose for her technical assistance.

Abbreviations

C. elegans

Caenorhabditis elegans

daf

Dauer formation abnormal

FLP

FMRFamide-like peptide

GPCR

G protein-coupled receptor

NPR

Neuropeptide receptor

Author contributions

T.K. was responsible for the overall design of the research and supervised the experiments and analyses. R.U., S.M., and M.O. constructed the expression plasmids and performed the microinjection required to produce transgenic nematodes. R.U. and S.M. inserted the transgenes into the chromosomes and subsequently crossed the resulting nematodes with wild-type nematodes. They also conducted observations of dauer formation, performed statistical analyses, carried out molecular genetic analyses, and examined fluorescent proteins using confocal laser scanning microscopy (CLSM). M.O., T.B., and T.I. assisted with the experiments and critically analyzed the data. A.S. and H.S. conducted the machine learning for pairing FLP-11 with NPRs. T.K., R.U., and S.M. wrote the manuscript, while M.O., H.S., and T.I. reviewed it. All authors read and approved the final manuscript.

Funding

This work was supported in part by the JSPS KAKENHI Grant Numbers 21K05393 and 24K08701 (to TK).

Data availability

All data generated or analyzed during this study are included in this published article and its supplementary information files. The data in this article will be shared upon reasonable request to the corresponding author. Further information and requests for materials used in this study should be directed to the corresponding author and will be fulfilled upon reasonable consideration.

Declarations

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

  • 1.Quillet, R. et al. RF-amide neuropeptides and their receptors in mammals: Pharmacological properties, drug development and main physiological functions. Pharmacol. Ther.160, 84–132. 10.1016/j.pharmthera.2016.02.005 (2016). [DOI] [PubMed] [Google Scholar]
  • 2.Nagata, S. FMRFamides. In Comparative Endocrinology of Basic and Clinical Research. 432–433 (eds Ando, H., Ukena, K. & Nagata, S.) (Academic Press, 2016).
  • 3.Peymen, K., Watteyne, J., Frooninckx, L., Schoofs, L. & Beets, I. The FMRFamide-like peptide family in nematodes. Front. Endocrinol. (Lausanne). 5, 90. 10.3389/fendo.2014.00090 (2014). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Kaletta, T. & Hengartner, M. O. Finding function in novel targets: C. elegans as a model organism. Nat. Rev. Drug Discov.5, 387–399. (2006). [DOI] [PubMed]
  • 5.Chang, Y. J. et al. Modulation of locomotion and reproduction by FLP neuropeptides in the nematode Caenorhabditis elegans. PLoS One. 10, e0135164. 10.1371/journal.pone.0135164 (2015). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Turek, M., Besseling, J., Spies, J. P., König, S. & Bringmann, H. Sleep-active neuron specification and sleep induction require FLP-11 neuropeptides to systemically induce sleep. Elife5, e12499. 10.7554/eLife.12499 (2016). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Nath, R. D., Chow, E. S., Wang, H., Schwarz, E. M. & Sternberg, P. W. C. elegans stress-induced sleep emerges from the collective action of multiple neuropeptides. Curr. Biol.26, 2446–2455. 10.1016/j.cub.2016.07.048 (2016). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Lee, J. et al. (ed, S.) FMRFamide-like peptides expand the behavioral repertoire of a densely connected nervous system. Proc. Natl. Acad. Sci. U S A114 E10726–E10735 10.1073/pnas.1710374114 (2017). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Chew, Y. L. et al. An afferent neuropeptide system transmits mechanosensory signals triggering sensitization and arousal in C. elegans. Neuron99, 1233–1246. 10.1016/j.neuron.2018.08.003 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Marques, F., Falquet, L., Vandewyer, E., Beets, I. & Glauser, D. A. Signaling via the FLP-14/FRPR-19 neuropeptide pathway sustains nociceptive response to repeated noxious stimuli in C. elegans. PLoS Genet.17, e1009880. 10.1371/journal.pgen.1009880 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Reilly, D. K. et al. Distinct neuropeptide-receptor modules regulate a sex-specific behavioral response to a pheromone. Commun. Biol.4, 1018. 10.1038/s42003-021-02547-7 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Busack, I. & Bringmann, H. A sleep-active neuron can promote survival while sleep behavior is disturbed. PLoS Genet.19, e1010665. 10.1371/journal.pgen.1010665 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Nose, M. et al. Protein Research Foundation,. Regulatory mechanism of larval diapause by a C. elegans neuropeptide, FLP-3. In Peptide Science 2021 (ed. Hayashi, Y.) 91–94. (2022).
  • 14.Ono, M., Hori, Y., Matsunaga, Y., Iwasaki, T. & Kawano, T. Regulatory mechanism of larval diapause by a C. elegans neuropeptide, FLP-6 in the intestine. In Peptide Science 2021 (ed. Hayashi, Y.) 87–90. (Protein Research Foundation, 2022).
  • 15.Kageyama, N. et al. The FMRFamide-like peptide FLP-2 is involved in the modulation of larval development and adult lifespan by regulating the secretion of the insulin-like peptide INS-35 in Caenorhabditis elegans. Biosci. Biotechnol. Biochem.86, 1231–1239. 10.1093/bbb/zbac108 (2022). [DOI] [PubMed] [Google Scholar]
  • 16.Une, R. et al. The FMRFamide-like peptide FLP-1 modulates larval development by regulating the production and secretion of the insulin-like peptide DAF-28 in Caenorhabditis elegans. Biosci. Biotechnol. Biochem.87, 171–178. 10.1093/bbb/zbac187 (2023). [DOI] [PubMed] [Google Scholar]
  • 17.Hu, P. J. Dauer in WormBook (ed. The C. elegans Research Community). 10.1895/wormbook.1.144.1 (2007).
  • 18.Li, W., Kennedy, S. G. & Ruvkun, G. daf-28 encodes a C. elegans insulin superfamily member that is regulated by environmental cues and acts in the DAF-2 signaling pathway. Genes Dev.17, 844–848. 10.1101/gad.1066503 (2003). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Kimura, K. D., Tissenbaum, H. A., Liu, Y. & Ruvkun, G. daf-2, an insulin receptor-like gene that regulates longevity and diapause in Caenorhabditis elegans. Science277, 942–946. 10.1126/science (1997). [DOI] [PubMed] [Google Scholar]
  • 20.Ren, P. & et el. Control of C. elegans larval development by neuronal expression of a TGF-beta homolog. Science274, 1389–1391. 10.1126/science.274.5291.1389 (1996). [DOI] [PubMed] [Google Scholar]
  • 21.Bargmann, C. I. & Horvitz, H. R. Control of larval development by chemosensory neurons in Caenorhabditis elegans. Science251, 1243–1246. 10.1126/science.2006412 (1991). [DOI] [PubMed] [Google Scholar]
  • 22.Matsunaga, Y. et al. Diapause is associated with a change in the Polarity of secretion of insulin-like peptides. Nat. Commun.7, 10573. 10.1038/ncomms10573 (2016). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Honda, Y. et al. Genes down-regulated in spaceflight are involved in the control of longevity in Caenorhabditis elegans. Sci. Rep.2, 487. 10.1038/srep00487 (2012). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Ono, M. et al. The G protein-coupled receptor neuropeptide receptor-15 modulates larval development via the transforming growth factor-β DAF-7 protein in Caenorhabditis elegans. Biochem. Biophys. Res. Commun.660, 28–34. 10.1016/j.bbrc.2023.03.080 (2023). [DOI] [PubMed] [Google Scholar]
  • 25.Fares, H. & Greenwald, I. Genetic analysis of endocytosis in Caenorhabditis elegans: coelomocyte uptake defective mutants. Genetics159, 133–145. 10.1093/genetics/159.1.133 (2001). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Kao, G. et al. ASNA-1 positively regulates insulin secretion in C. elegans and mammalian cells. Cell128, 577–587. 10.1016/j.cell.2006.12.031 (2007). [DOI] [PubMed] [Google Scholar]
  • 27.Gengyo-Ando, K. & Mitani, S. Characterization of mutations induced by Ethyl methanesulfonate, UV, and trimethylpsoralen in the nematode Caenorhabditis elegans. Biochem. Biophys. Res. Commun.269, 64–69. 10.1006/bbrc.2000.2260 (2000). [DOI] [PubMed] [Google Scholar]
  • 28.Georgi, L. L., Albert, P. S. & Riddle, D. L. daf-1, a C. elegans gene controlling Dauer larva development, encodes a novel receptor protein kinase. Cell61, 635–645. 10.1016/0092-8674(90)90475-t (1990). [DOI] [PubMed] [Google Scholar]
  • 29.Patterson, G. I., Koweek, A., Wong, A., Liu, Y. & Ruvkun, G. The DAF-3 Smad protein antagonizes TGF-beta-related receptor signaling in the Caenorhabditis elegans Dauer pathway. Genes Dev.11, 2679–2690. 10.1101/gad.11.20.2679 (1997). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Lin, K., Dorman, J. B., Rodan, A. & Kenyon, C. daf-16: an HNF-3/forkhead family member that can function to double the life-span of Caenorhabditis elegans. Science278, 1319–1322. 10.1126/science.278.5341.1319 (1997). [DOI] [PubMed] [Google Scholar]
  • 31.Ogg, S. et al. The fork head transcription factor DAF-16 transduces insulin-like metabolic and longevity signals in C. elegans. Nature389, 994–999. 10.1038/40194 (1997). [DOI] [PubMed] [Google Scholar]
  • 32.Butcher, R. A. et al. Biosynthesis of the caenorhabditis elegans Dauer pheromone. Proc. Natl. Acad. Sci. U S A. 106, 1875–1879. 10.1073/pnas.0810338106 (2009). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Kawano, T. et al. Lifespan extending activity of substances secreted by the nematode Caenorhabditis elegans that include the dauer-inducing pheromone. Biosci. Biotechnol. Biochem.69, 2479–2481. 10.1271/bbb.69.2479.2005 (2005). [DOI] [PubMed] [Google Scholar]
  • 34.Beets, I. et al. System-wide mapping of peptide-GPCR interactions in C. elegans. Cell. Rep.42, 113058. 10.1016/j.celrep.2023.113058 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Hilbert, Z. A. & Kim, D. H. PDF-1 neuropeptide signaling regulates sexually dimorphic gene expression in shared sensory neurons of C. elegans. eLife7, e36547. 10.7554/eLife.36547 (2018). [DOI] [PMC free article] [PubMed]
  • 36.Rossi, L. et al. The neuropeptide FLP-11 induces and self-inhibits sleep through the receptor DMSR-1 in Caenorhabiditis elegans. Curr. Biol.35, 2183–2194. 10.1016/j.cub.2025.03.039 (2025). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Tan, C. M. J. et al. The role of neuropeptide Y in cardiovascular health and disease. Front. Physiol.9, 1281. 10.3389/fphys.2018.01281 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Brenner, S. The genetics of Caenorhabditis elegans. Genetics77, 71–94. 10.1093/genetics/77.1.71 (1974). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Zhang, M. G. & Sternberg, P. W. Both entry to and exit from diapause arrest in Caenorhabditis elegans are regulated by a steroid hormone pathway. Development149, dev200173. 10.1242/dev.200173 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Mariol, M. C., Walter, L., Bellemin, S. & Gieseler, K. A rapid protocol for integrating extrachromosomal arrays with high transmission rate into the C. elegans genome. J. Vis. Exp.82, e50773. 10.3791/50773 (2013). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Shiraishi, A. et al. Repertoires of G protein-coupled receptors for Ciona-specific neuropeptides. Proc. Natl. Acad. Sci. U S A. 116, 7847–7856. 10.1073/pnas.1816640116 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Li, C. & Kim, K. Family of FLP peptides in caenorhabditis elegans and related nematodes. Front. Endocrinol.5, 150. 0.3389/fendo.2014.00150 (2014). [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1 (4.4MB, docx)

Data Availability Statement

All data generated or analyzed during this study are included in this published article and its supplementary information files. The data in this article will be shared upon reasonable request to the corresponding author. Further information and requests for materials used in this study should be directed to the corresponding author and will be fulfilled upon reasonable consideration.

Articles from Scientific Reports are provided here courtesy of Nature Publishing Group

RESOURCES