Peptidergic gut-to-neuron FLP-2 signaling potentiates the oxidative stress response.

A (Top) Schematic showing the positions of AIY, intestine and coelomocytes of transgenic animals co-expressing FLP-1::Venus in the intestine and mCherry in coelomocytes. Representative image of the posterior coelomocyte that has taken up Venus into the endocytic compartment. Scale bar: 5μM. (Bottom) Schematic showing FLP-1 and FLP-2 peptides as inter-tissue signals in gut-intestine regulation of the antioxidant response.

B Representative images and quantification of average coelomocyte fluorescence of the indicated mutants expressing FLP-1::Venus fusion proteins in AIY following vehicle M9 or juglone treatment for 10min. Neuronal aex-5 denotes expression of aex-5 cDNA under the rab-3 promoter; intestinal aex-5 denotes expression of aex-5 cDNA under the ges-1 promoter. Unlined *** denotes statistical analysis compared to “wild type”; a denotes statistical analysis compared to “aex-5+juglone”, P < 0.001; b denotes statistical analysis compared to “intestinal aex-5; aex-5+juglone”, P < 0.01. n = 30, 30, 24, 30, 26, 30, 30 independent animals. Scale bar: 5μM.

C Average percentage of surviving young adult animals of the indicated genotypes after 16h recovery following 4h juglone treatment. Unlined ** denotes statistical analysis compared to “wild type”, a denotes statistical analysis compared to “flp-1”, P > 0.99; b denotes statistical analysis compared to “flp-2”, P > 0.99. n = 213, 156, 189, 195 independent biological samples over three independent experiments.

D Quantification of average coelomocyte fluorescence of the indicated mutants expressing FLP-1::Venus fusion proteins in AIY following M9 or juglone treatment for 10min. Neuronal flp-2 denotes expression of flp-2 gDNA under the rab-3 promoter; intestinal flp-2 denotes expression of flp-2 gDNA under the ges-1 promoter; intestinal flp-2(OE) denotes expression of flp-2 gDNA under the ges-1 promoter in wild type animals. Unlined *** denotes statistical analysis compared to “wild type”; a denotes statistical analysis compared to “flp-2+juglone”, P < 0.0001; b denotes statistical analysis compared to “wild type”, P > 0.99; c denotes statistical analysis compared to “wild type+juglone”, P < 0.01. n = 20, 20, 25, 20, 20, 20, 25, 22 independent animals.

E Representative images and quantification of average fluorescence in the posterior region of transgenic animals expressing Pgst-4::gfp after 4h vehicle M9 or juglone exposure. Asterisks mark the intestinal region used for quantification. Pgst-4::gfp expression in the body wall muscles, which appears as fluorescence on the edge animals in some images, was not quantified. Unlined *** and ns denote statistical analysis compared to “wild type”; a, b, and c denote statistical analysis compared to “wild type+juglone”, P < 0.01, P < 0.001, P < 0.01 respectively; unlined ns denotes statistical analysis compared to “wild type”. n = 25, 26, 25, 25, 25, 25, 25, 25 independent animals. Scale bar: 10μM.

F Representative images and quantification of average fluorescence in the posterior region of transgenic animals expressing Pgst-4::gfp after 4h M9 or juglone exposure. Asterisks mark the intestinal region for quantification. Pgst-4::gfp expression in the body wall muscles, which appears as fluorescence on the edge animals in some images, was not quantified. Unlined *** denotes statistical analysis compared to “wild type”; unlined ### denotes statistical analysis compared to “wild type+juglone”; a denotes statistical analysis compared to “intestinal flp-2(OE)”, P < 0.0001; b denotes statistical analysis compared to “intestinal flp-2(OE)+juglone”, P < 0.0001. n = 23, 25, 25, 26, 24, 25 independent animals. Scale bar: 10μM. Data are mean values ± s.e.m normalized to wild type controls. ns. not significant, ** and ## P < 0.01, *** and ### P < 0.001 by Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test.

FLP-2 secretion from the intestine is stress regulated.

A Schematic showing the positions of intestine and coelomocytes of transgenic animals co-expressing FLP-2::Venus in the intestine and mCherry in coelomocytes. Representative images of the posterior coelomocyte that have taken up Venus into the endocytic compartment (Scale bar: 5μM) and the posterior intestinal region showing the distribution of FLP-2::Venus in puncta in the intestine are shown (Scale bar: 15μM).

B Representative images of fluorescence distribution in the posterior intestinal region of transgenic animals co-expressing FLP-2::Venus and AEX-5::mTur2 fusion proteins. Arrowheads denote puncta marked by both fusion proteins. Scale bar: 5μM.

C Representative images and quantification of average coelomocyte fluorescence of the indicated mutants expressing FLP-2::Venus fusion proteins in the intestine following M9, juglone or H2O2 treatment for 10min. Unlined *** and ns denote statistical analysis compared to “wild type”. n = 29, 25, 24, 30, 23, 30, 25, 25, 25 independent animals. Scale bar: 5μM.

D Quantification of average coelomocyte fluorescence of transgenic animals expressing FLP-2::Venus fusion proteins in the intestine following treatment of vehicle (DMSO) or the indicated stressors for 10min. Unlined *** denotes statistical analysis compared to “M9”. n = 23, 25, 25 independent animals.

E Quantification of average coelomocyte fluorescence of the indicated mutants expressing FLP-2::Venus fusion proteins in the intestine following M9 or juglone treatment for 10min. Unlined ** denotes statistical analysis compared to “wild type”; unlined ## denotes statistical analysis compared to “flp-1”; a denotes statistical analysis compared to “wild type+juglone”, P > 0.90. n = 30, 30, 30, 30 independent animals. Data are mean values ± s.e.m normalized to wild type controls. ns. not significant, ** and ## P < 0.01, *** and ### P < 0.001 by Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test.

SOD-1/SOD-3 mediates endogenous H2O2 regulates FLP-2 release from the intestine.

A Quantification of average coelomocyte fluorescence of the indicated mutants expressing FLP-2::Venus fusion proteins in the intestine following M9 or juglone treatment for 10min. Intestinal sod-1 denotes expression of sod-1b cDNA under the ges-1 promoter. Unlined *** denotes statistical analysis compared to “wild type”. n = 25, 22, 24, 24, 25 independent animals.

B Quantification of average coelomocyte fluorescence of the indicated mutants expressing FLP-2::Venus fusion proteins in the intestine following M9 or juglone treatment for 10min. Intestinal sod-3 and sod-3(ΔMLS) denote intestinal expression of sod-3 cDNA and sod-3(ΔMLS) variants, which lacks the mitochondrial localization sequence, under the ges-1 promoter. Unlined *** denotes statistical analysis compared to “wild type”. n = 25, 25, 25, 25, 25, 25 independent animals.

C Quantification of average coelomocyte fluorescence of the indicated mutants expressing FLP-2::Venus fusion proteins in the intestine following M9 or juglone treatment for 10min. Unlined *** denotes statistical analysis compared to “wild type”. n = 25, 25, 22, 25 independent animals.

D Representative images of fluorescence distribution in the posterior intestinal region of transgenic animals expressing SOD-1b::GFP fusion proteins in contrast against auto-fluorescence of gut granules. Scale bar: 10μM.

E Representative images of fluorescence distribution in the posterior intestinal region of transgenic animals co-expressing SOD-3::GFP and TOMM-20::mCherry (to target mitochondria) fusion proteins. Scale bar: 15μM.

F Representative images of fluorescence distribution in the posterior intestinal region of transgenic animals co-expressing SOD-3(ΔMLS)::GFP and TOMM-20::mCherry fusion proteins. Scale bar: 15μM.

G Quantification of average coelomocyte fluorescence of the indicated mutants expressing FLP-2::Venus fusion proteins in the intestine following M9, juglone or H2O2 treatment for 10min. Unlined *** and ns denote statistical analysis compared to “wild type”. n = 29, 30, 25, 25, 25, 24, 25 independent animals.

H Quantification of average coelomocyte fluorescence of the indicated mutants expressing FLP-2::Venus fusion proteins in the intestine following M9 or H2O2 treatment for 10min. n = independent animals.

I Schematic showing that SOD-1 and SOD-3 mediate juglone-induced H2O2 production in promoting FLP-2 release, and the PRDX-2/TRX-3 system detoxifies excessive H2O2.

J Schematic, representative images and quantification of fluorescence in the posterior region of the indicated transgenic animals co-expressing mitochondrial matrix targeted HyPer7 (matrix-HyPer7) or mitochondrial outer membrane targeted HyPer7 (OMM-HyPer7) with TOMM-20::mCherry following M9 juglone or H2O2 treatment. Unlined *** and ns denote statistical analysis compared to “wild type; a and b denote statistical analysis compared to “wild type+juglone”, P < 0.01, P > 0.19. (top) n = 20, 20, 18, 20, 19,

19, 20, 20 independent animals. (bottom) n = 20, 20, 19, 20, 20, 20, 20, 20 independent animals. Scale bar: 5μM. Data are mean values ± s.e.m normalized to wild type controls. ns. not significant, * P < 0.05, *** P < 0.001 by Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test.

PRDX-2/PRDX and TRX-3/TRX regulate endogenous H2O2 and FLP-2 secretion.

A (Top) Schematic showing the PRDX/TRX system in H2O2 detoxification. (Bottom) Schematic showing the three isoforms of prdx-2 transcripts and vj380 allele of prdx-2b knockout.

B Quantification of average coelomocyte fluorescence of the indicated mutants expressing FLP-2::Venus fusion proteins in the intestine following M9 or juglone treatment for 10min. Intestinal prdx-2b denotes expression of prdx-2b cDNA under the ges-1 promoter. Intestinal trx-3 denotes expression of trx-3 cDNA under the ges-1 promoter. Unlined *** denotes statistical analysis compared to “wild type”; a denotes statistical analysis compared to “prdx-2”, P < 0.0001; b, c and d denote statistical analysis compared to “trx-3”, P < 0.001, P < 0.0001, P > 0.99 respectively. n= 25, 23, 25, 25, 25, 25, 25, 25, 25, 25, 25 independent animals.

C and D Quantification of fluorescence in the posterior region of the indicated transgenic animals co-expressing matrix-HyPer7 (C) or OMM-HyPer7 (D) with TOMM-20::mCherry following M9 or juglone treatment. Unlined *** and ns denote statistical analysis compared to “wild type. (C) n = 20, 20, 20, 20, 20 independent animals. (D) n = 20, 20, 20, 20, 20 independent animals.

E Quantification of average coelomocyte FLP-2::Venus fluorescence of transgenic animals fed with RNAi bacteria targeting the indicated genes following M9 treatment for 10min. Unlined *** denotes statistical analysis compared to “empty vector”. n = 25, 23, 24 independent animals.

F Representative images and quantification of average fluorescence in the posterior region of transgenic animals expressing Pgst-4::gfp after 4h M9 or juglone exposure. Asterisks mark the intestinal region for quantification. Pgst-4::gfp expression in the body wall muscles, which appears as fluorescence on the edge animals in some images, was not quantified. Unlined ** denotes statistical analysis compared to “wild type”, unlined ## denotes statistical analysis compared to “prdx-2b”. n = 25, 25, 25 independent animals. Scale bar: 10μM. Data are mean values ± s.e.m normalized to wild type controls. n.s. not significant, ** and ## P < 0.01, *** P < 0.001 by Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test.

PKC-2/PKCα/β activation by H2O2 promotes FLP-2 secretion from the intestine.

A Quantification of average coelomocyte fluorescence of the indicated mutants expressing FLP-2::Venus fusion proteins in the intestine following M9 or juglone treatment for 10min. Intestinal pkc-2 denotes expression of pkc-2b cDNA under the ges-1 promoter. Intestinal pkc-2b(K375R) denotes expression of pkc-2b(K375R) variants under the ges-1 promoter. Unlined *** and ns denote statistical analysis compared to “wild type”; ### denote statistical analysis compared to “intestinal pkc-2b;pkc-2+juglone”. n = 24, 24, 25, 25, 25, 25 independent animals.

B and C Quantification of fluorescence in the posterior region of the indicated transgenic animals co-expressing matrix-HyPer7 (B) or OMM-HyPer7 (C) with TOMM-20::mCherry following M9 or juglone treatment. Unlined *** denotes statistical analysis compared to “wild type”; unlined ### denotes statistical analysis compared to “pkc-2”. (B) n = 20, 20, 19, 20 independent animals, (C) n = 20, 20, 20, 20 independent animals.

D Quantification of average coelomocyte fluorescence of the indicated mutants expressing FLP-2::Venus fusion proteins in the intestine following M9 or H2O2 treatment for 10min. n = 23, 25, 25 independent animals. E Quantification of average coelomocyte fluorescence of the indicated mutants expressing FLP-2::Venus fusion proteins in the intestine following M9 treatment for 10min. Unlined *** denotes statistical analysis compared to “wild type”; unlined ### denotes statistical analysis compared to “prdx-2”. n = 25, 25, 25, 25 independent animals. Data are mean values ± s.e.m normalized to wild type controls. ns. not significant, *** and ### P < 0.001 by Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test

DAG promotes PKC-2 mediated FLP-2 secretion from the intestine.

A Schematic showing PLC and DGK mediates DAG metabolism and DAG functions in H2O2-mediated FLP-2 signaling.

B Quantification of average coelomocyte fluorescence of the indicated mutants expressing FLP-2::Venus fusion proteins in the intestine following M9 or juglone treatment for 10min. n = 25, 25, 25, 25 independent animals.

C and D Quantification of fluorescence in the posterior region of the indicated transgenic animals co-expressing matrix-HyPer7 (C) or OMM-HyPer7 (D) with TOMM-20::mCherry following M9 or juglone treatment. Unlined *** denotes statistical analysis compared to “wild type”; unlined ### denotes statistical analysis compared to “egl-8”. (C) n = 22, 20, 20, 21 independent animals, (D) n = 20, 20, 20, 20 independent animals.

E Quantification of average coelomocyte fluorescence of the indicated mutants expressing FLP-2::Venus fusion proteins in the intestine following M9 or juglone treatment for 10min. Intestinal dgk-2 denotes expression of dgk-2a cDNA under the ges-1 promoter. Unlined *** denotes statistical analysis compared to “wild type”; unlined ### denotes statistical analysis compared to “dgk-2/DGKε”. n = 25, 25, 25, 25, 24 independent animals.

F and G Quantification of fluorescence in the posterior region of the indicated transgenic animals co-expressing matrix-HyPer7 (F) or OMM-HyPer7 (G) with TOMM-20::mCherry following M9 treatment. (F) n = 20, 20 independent animals, (G) n = 20, 20 independent animals.

H Quantification of average coelomocyte fluorescence of the indicated transgenic animals fed with RNAi bacteria targeting the indicated genes in the intestine following M9 treatment for 10min. n = 25, 24, 25, 30 independent animals.

I (Top) Schematic showing the position of intestine and AIY neurons in FLP-1-FLP-2 mediated axis. (Bottom) Schematic showing endogenous H2O2 promotes PKC-2/AEX-4 mediated FLP-2 release from the intestine in FLP-1-FLP-2 regulated inter-tissue axis. Data are mean values ± s.e.m normalized to wild type controls. ns. not significant, ** P < 0.01, *** and ### P < 0.001 by Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test.

The effect of intestinal DCV secretion mutations on FLP-1 release from AIY. A

Schematic showing the locations of AEX-1/UNC13, AEX-3/MADD, AEX-4/SNAP25 and AEX-6/Rab27 relative to a DCV.

B Quantification of average coelomocyte fluorescence of the indicated mutants expressing FLP-1::Venus fusion proteins in AIY following vehicle (DMSO) or juglone treatment for 10min. Unlined *** and ### denotes statistical analysis compared to “wild type”. n = 30, 30, 29, 30, 30, 30 independent animals.

C Quantification of average coelomocyte fluorescence of the indicated mutants expressing FLP-1::Venus fusion proteins in AIY following vehicle (DMSO) or juglone treatment for 10min. Unlined *** and ns denote statistical analysis compared to “wild type”. n = 24, 24, 25, 25, 30, 30 independent animals. Data are mean values ± s.e.m normalized to wild type controls. ns. not significant, *** and ### P < 0.001 by Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test.

Specificity of juglone on intestinal peptide secretion, and FLP-2 and NLP-40 localization in the intestine.

A Quantification of average coelomocyte fluorescence of the indicated mutants co-expressing FLP-2::Venus in the intestine (under the ges-1 promoter) and mCherry in the coelomocytes (under the ofm-1 promoter) following M9 or juglone treatment for 10min. n = 23, 19 independent animals.

B Quantification of average coelomocyte fluorescence of transgenic animals expressing NLP-40::Venus fusion proteins in the intestine following M9 or juglone exposure for 10min. n = 25, 24 independent animals. C Quantification of average coelomocyte fluorescence of transgenic animals expressing NLP-27::Venus fusion proteins in the intestine following M9 or juglone exposure for 10min. n = 23, 25 independent animals. D Representative images of fluorescence distribution in the posterior intestinal region of transgenic animals co-expressing FLP-2::Venus fusion proteins (marked by arrowheads) and NLP-40::mTur2 fusion proteins (marked by arrows). Scale bar: 5μM. Data are mean values ± s.e.m normalized to wild type controls. ns. not significant by Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test.

SODs function in juglone induced FLP-2 release from the intestine and mitochondrial mCherry control.

A Quantification of average coelomocyte fluorescence of the indicated mutants expressing FLP-2::Venus fusion proteins in the intestine following M9 or juglone treatment for 10min. Unlined ***, ### and ns denote statistical analysis compared to “wild type+juglone”. n = 29, 27, 29, 27, 25, 26, 24 independent animals.

B and C Representative images and quantification of average fluorescence intensity of TOMM-20::mCherry proteins in transgenic animals co-expressing matrix-HyPer7 (B) or OMM-HyPer7 (C) following M9 or H2O2 treatment for 10min. (B) Scale bar: Scale bar: 5μM. n = 20, 20 independent animals. (C) Scale bar: Scale bar: 5μM. n = 20, 22 independent animals.

PRDX-2 intestinal rescue and mediates SOD-3 dependent regulation of FLP-2 release.

A Quantification of average coelomocyte fluorescence of the indicated mutants expressing FLP-2::Venus fusion proteins in the intestine following M9 treatment for 10min. n = 30, 29 independent animals.

B Quantification of average coelomocyte fluorescence of the indicated mutants expressing FLP-2::Venus fusion proteins in the intestine following M9 treatment for 10min. Intestinal prdx-2a denotes expression of prdx-2a cDNA under the ges-1 promoter. n = 30, 30, 25 independent animals.

C Quantification of average coelomocyte fluorescence of the indicated mutants expressing FLP-2::Venus fusion proteins in the intestine following M9 treatment for 10min. Intestinal prdx-2c denotes expression of prdx-2c cDNA under the ges-1 promoter. n = 25, 25, 25 independent animals.

D Quantification of average coelomocyte fluorescence of the indicated mutants expressing FLP-2::Venus fusion proteins in the intestine following M9 treatment for 10min. n = 25, 23, 22 independent animals. Data are mean values ± s.e.m normalized to wild type controls. Data are mean values ± s.e.m normalized to wild type controls. ns. not significant, *** and ### P < 0.001 by Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test.

Juglone promotes FLP-2 release in pkc-1 mutants and expulsion analysis.

A Quantification of average coelomocyte fluorescence of the indicated mutants expressing FLP-2::Venus fusion proteins in the intestine following M9 or juglone treatment for 10min. Unlined ns and ** denote statistical analysis compared to “wild type”. n = 24, 25, 20, 25 independent animals.

B Quantification of the number of expulsions (Exp) per defecation cycle in adult animals of the indicated genotypes. n = 30, 30, 30, 30 in three independent animals. Data are mean values ± s.e.m normalized to wild type controls. ns. not significant, ** P < 0.01, *** P < 0.001 by Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test.

Juglone promotes FLP-2 release in plc-2 mutants.

Quantification of average coelomocyte fluorescence of the indicated mutants expressing FLP-2::Venus fusion proteins in the intestine following M9 or juglone treatment for 10min. Unlined *** denotes statistical analysis compared to “wild type”; unlined ### denotes statistical analysis compared to “plc-2/PLCβ”. n = 25, 25, 23, 28 independent animals. Data are mean values ± s.e.m normalized to wild type controls. ns. not significant, *** and ### P < 0.001 by Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test.

Strains, transgenic lines and plasmids used in this study