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Insulin-like signalling to the maternal germline controlsprogeny response to osmotic stressNicholas O. Burton1, Tokiko Furuta2, Amy K. Webster3, Rebecca E. W. Kaplan3, L. Ryan Baugh3, Swathi Arur2and H. Robert Horvitz1,4

In 1893 August Weismann proposed that information about theenvironment could not pass from somatic cells to germ cells1, ahypothesis now known as the Weismann barrier. However, recentstudies have indicated that parental exposure to environmentalstress can modify progeny physiology2–7 and that parentalstress can contribute to progeny disorders8. The mechanismsregulating these phenomena are poorly understood. We reportthat the nematode Caenorhabditis elegans can protect itselffrom osmotic stress by entering a state of arrested developmentand can protect its progeny from osmotic stress by increasingthe expression of the glycerol biosynthetic enzyme GPDH-2in progeny. Both of these protective mechanisms are regulatedby insulin-like signalling: insulin-like signalling to the intestineregulates developmental arrest, while insulin-like signallingto the maternal germline regulates glycerol metabolismin progeny. Thus, there is a heritable link between insulin-likesignalling to the maternal germline and progeny metabolismand gene expression. We speculate that analogous modulationof insulin-like signalling to the germline is responsiblefor effects of the maternal environment on human diseases thatinvolve insulin signalling, such as obesity and type-2 diabetes8.

Maternal exposure to a wide variety of environmental stresses altersprogeny growth, development and physiology of diverse organisms2–7

and is thought to be a contributing factor to several humanpathologies, including obesity and diabetes8. The mechanisms bywhich the maternal environment can modify progeny biology arepoorly understood. Parental exposure of the nematodeCaenorhabditiselegans to mild osmotic stress can protect progeny from the effectsof strong osmotic stress9. This finding and similar observations ofother organisms2 suggest that besides the potentially deleterious effectsof maternal environmental stress on progeny, maternal exposure toenvironmental stress might epigenetically precondition progeny andprotect them from similar environmental insults in the future. How

maternal exposure to environmental stress can protect progeny fromfuture environmental stress remains largely unknown.

To determine how parental exposure to mild osmotic stress(300mM NaCl) protects progeny from the effects of strong osmoticstress (500mM NaCl) we first examined the effects of 500mM NaClon C. elegans. Embryos placed at 500mMNaCl completed embryonicdevelopment and hatched but arrested development immediately afterhatching (Fig. 1a and Supplementary Fig. 1a). These arrested animalswere unable to move, feed or respond to touch (SupplementaryFig. 1a). However, when arrested animals were returned to normalosmotic conditions (50mM NaCl), they regained mobility andresumed development (Fig. 1b). We made similar observations whenNaBr, KCl or sucrose was used to cause osmotic stress (SupplementaryFig. 1b–d). We conclude that young C. elegans larvae can enter a stateof immobile arrested development in response to osmotic stress.

C. elegans also arrests development in response to other environ-mental stresses, such as starvation10,11. These arrests are caused bythe loss of insulin-like signalling via the insulin receptor DAF-2 andthe consequent activation of the FOXO transcription factor DAF-16(refs 10–12). We tested whether the activation of DAF-16 is requiredfor developmental arrest in response to osmotic stress. Animals lackingDAF-16 were less likely than the wild type to arrest development inresponse to osmotic stress (Fig. 1a), whereas animals with increasedDAF-16 activation, such as daf-2mutants12, were more likely to arrestdevelopment in response to osmotic stress (Fig. 1a). Genes in otherstress response pathways, such as those that regulate the responses ofC. elegans to oxidative stress13 or infection14, were not required fordevelopmental arrest in response to osmotic stress (SupplementaryFig. 1e,f). In addition, DAF-2 activity in the intestine was requiredfor development at 300mM NaCl (Fig. 1c), and exposure to 500mMNaCl caused the translocation of DAF-16 from the cytoplasm to thenucleus (Fig. 1d). Like the developmental arrest caused by starva-tion10, the arrest caused by osmotic stress required dense-core vesiclerelease (Supplementary Fig. 1g); furthermore, we found that it is

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

1Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA. 2Department of Genetics,UT MD Anderson Cancer Center, Houston, Texas 77030, USA. 3Department of Biology Duke University, Durham, North Carolina 27708, USA.4Correspondence should be addressed to H.R.H. (e-mail: [email protected])

Received 16 November 2016; accepted 10 January 2017; published online 6 February 2017; DOI: 10.1038/ncb3470

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Figure 1 Insulin-like signalling to the intestine regulates developmental arrestin response to osmotic stress. (a) Percentage of wild-type, daf-2(e1370),age-1(hx546), pdk-1(sa709), daf-16(mu86) and daf-2(e1370);daf-16(mu86) animals developing past the L1 larval stage (L2+) after48 h. Error bars, s.d. n=3 experiments of >100 animals; P<0.01 for allgenotypes (two-tailed t-test). (b) Percentage of wild-type, daf-2(e1370),daf-16(mu86) and daf-2(e1370); daf-16(mu86) animals that resumedevelopment after 24 h of exposure to osmotic stress. Error bars, s.d. n=3;see Supplementary Table 6; P <0.01 for all genotypes (two-tailed t-test).(c) Percentage of wild-type and daf-2(e1370) animals developing past the

L1 larval stage at 300mM NaCl after 48 h. Neuron-specific expressionwas driven by Prgef-1; intestine-specific expression was driven by Pges-1;muscle-specific expression was driven by Pmyo-3. Error bars, s.d. n=3experiments of >100 animals. (d) Confocal images of DAF-16::GFP after6 h of exposure to 50mM and 500mM NaCl. Percentage values indicatethe percentage of embryos resembling the representative image. SeeSupplementary Table 6. 3 experimental replicates of >100 animals. Thequantified results are presented as mean ± s.d. using two-tailed t-test;∗∗∗∗P <0.0001 were considered significant. See Statistics Source Data inSupplementary Table 6.

dense-core vesicle release from sensory neurons that was required fordevelopmental arrest in response to osmotic stress (SupplementaryFig. 1g), and daf-16 mutants, which are resistant to developmentalarrest, showed an increased susceptibility to osmotic stress (Fig. 1b).These results indicate that like starvation, osmotic stress causes devel-opmental arrest by inhibiting insulin-like signalling and that arresteddevelopment correlates with enhanced survival.

In contrast to starvation-induced developmental arrest, we foundthat developmental arrest in response to osmotic stress is regulated by adifferent insulin-like peptide, INS-3 (Supplementary Fig. 1h,i and Sup-plementary Table 1), than arrest in response to starvation (INS-4 andDAF-28; ref. 15) and that animals that arrest development in responseto osmotic stress are immobile and unable to respond to touch, unlikearrest in response to starvation in which animals remain mobile10.In addition, we found that a majority (78%) of genes the expressionof which reproducibly changed in response to osmotic stress werenot affected by starvation (Supplementary Fig. 2 and SupplementaryTable 2). These results suggest that these two arrest phenotypes arecontrolled by partially overlapping but distinct pathways.

Frazier and Roth (2009) found that parental exposure of C. elegansto mild osmotic stress protects progeny from the effects of strongosmotic stress and that this protection required DAF-2 activation9.These authors described an apparently anomalous result: daf-2; daf-16 double mutants were significantly better at adaptation to osmotic

stress than both wild-type animals and either daf-2 or daf-16 singlemutants9. We hypothesized that this anomaly might be at least inpart caused by differing effects of maternal insulin-like signallingand progeny insulin-like signalling with respect to progeny responseto osmotic stress. Specifically, we suspected that DAF-2 activationin embryos was required for adaption to osmotic stress, consistentwith both the earlier observations9 and our findings (Fig. 1a). Wealso suspected that it was an inhibition rather than an activationof parental insulin-like signalling that resulted in the protection ofprogeny from osmotic stress, just as the inhibition of larval insulin-like signalling induced by osmotic stress protects larvae from osmoticstress by causing developmental arrest. To test this hypothesis, wecrossed wild-type animals with daf-2 and daf-2; daf-16 double-mutantanimals in normal osmotic conditions (50mM NaCl) and assayedthe response of their progeny to 500mM NaCl. Approximately 60%of the progeny from the cross of wild-type males with daf-2 mutanthermaphrodites hatched and developed at 500mM NaCl (Fig. 2a).By contrast, the reciprocal cross of daf-2 mutant males with wild-type hermaphrodites and the cross of wild-type males with daf-2;daf-16 double-mutant hermaphrodites did not produce any progenythat hatched and developed at 500mM NaCl (Fig. 2a). These resultsdemonstrate that like parental exposure to mild osmotic stress,reduced maternal insulin-like signalling can protect progeny from theeffects of strong osmotic stress. Importantly, these observations also

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Figure 2 Insulin-like signalling to the maternal germline regulates progenyresponse to osmotic stress. (a) Percentage of wild-type (WT), daf-2(e1370)and daf-2(e1370); daf-16(mu86) cross progeny failing to arrest develop-ment after 48h at 500mM NaCl. Males contained (Pegl-1::4xNLS::GFP);him-5(e1490); nIs349 (Pceh-28::4xNLS::mCherry) for the identificationof cross progeny. The pie-1 promoter was used to drive germline-specificexpression of DAF-2 and the mex-5 promoter was used to drive germline-specific expression of DAF-16. Error bars, s.d. n=7, 3, 6, 3, 3, 3 and 3;see Supplementary Table 6. (b) Percentage of wild-type, gpdh-1(ok1558)and gpdh-2(ok1733) animals failing to arrest development at 500mM NaCl

after 48 h. Error bars, s.d. n=3 experiments of >100 animals. (c) Averagefold change of 2 replicates of the 25most upregulated genes in embryos inresponse to osmotic stress after 6 h. FPKM, fragments per kilobase of tran-script per million mapped reads. (d) Percentage of wild-type, daf-2(e1370)and gpdh-2(ok1733) cross progeny failing to arrest development after 48 hat 500mM NaCl. Males contained otIs39 (Punc-47::GFP); him-5(e1490) forthe identification of cross progeny. Error bars, s.d. n=3 experiments of >20animals. The quantified results are presented as mean ± s.d. using ANOVA.∗P<0.05, ∗∗∗P<0.001, ∗∗∗∗P<0.0001 were considered significant. NS, notsignificant. See Statistics Source Data in Supplementary Table 6.

indicate that there is a previously undescribed link between maternalinsulin-like signalling and progeny physiology.

Parental exposure to osmotic stress has been hypothesized toprotect progeny from the effects of osmotic stress by increasing thedeposition of glycerol from mothers into embryos, since embryosfrom parents exposed to 300mM NaCl have more glycerol thanembryos from animals grown at normal osmotic conditions (50mMNaCl)9 and glycerol is known to be protective against variousenvironmental stresses16,17. We confirmed that exposure of parentsto 300mM NaCl resulted in progeny that are resistant to theeffects of 500mM NaCl (Fig. 2b,c and Supplementary Table 3),and we discovered that the glycerol biosynthetic enzyme GPDH-2 is required for parental exposure to 300mM NaCl to protectprogeny from 500mMNaCl (Fig. 2b) but does not affect the responseto osmotic stress of animals with parents grown at 50mM NaCl(Supplementary Fig. 3a). These observations are consistent withthe hypothesis that an increased level of glycerol is required for

adaptation to osmotic stress. However, it remained unclear how theseobservations relate to our finding that reduced maternal insulin-likesignalling can protect progeny from strong osmotic stress (Fig. 2a),since previous studies indicated that embryos from daf-2 mutanthermaphrodites contain the same amount of glycerol as embryosfrom wild-type animals9 and hence that daf-2mutant mothers do notdeposit more glycerol into embryos. We hypothesized that reducedmaternal insulin-like signalling protects progeny from the effectsof osmotic stress not by increasing deposition of glycerol frommothers into embryos but rather by increasing glycerol productionin embryos via GPDH-2. To test this hypothesis, we crossed gpdh-2mutant males with daf-2; gpdh-2 double-mutant hermaphrodites.GPDH-2 was required for reduced maternal insulin-like signallingto protect progeny from developmental arrest (Fig. 2d). To testwhether GPDH-2 functions maternally, we crossed wild-type maleswith daf-2; gpdh-2 double-mutant hermaphrodites. GPDH-2 was notrequired in mothers to protect progeny from developmental arrest in

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Figure 3 Insulin-like signalling to the maternal germline modifies progenyresponse to osmotic stress by regulating the RAS–ERK-like pathway. (a) Per-centage of wild-type and lin-45(n2018), mek-2(ku114), mpk-1(n5639) andlin-45(n2018); gpdh-2(ok1733) animals failing to arrest development at500mM NaCl after 48 h. Error bars, s.d. n=6, 6, 3, 3, and 3 experimentsof >100 animals. See Supplementary Table 6. (b) Percentage of wild-typeand lin-45(n2018) cross progeny failing to arrest development at 500mMNaCl after 48 h. Males contained otIs39 (Punc-47::GFP); him-5(e1490)for the identification of cross progeny. Error bars, s.d. n=3 experimentsof >20 animals. (c) Representative germlines dissected from wild-typeanimals exposed to either 50mM NaCl or 300mM NaCl and stained for DNA

(DAPI, white) and diphosphorylated MPK-1 (dpMPK-1) (red). Each conditionwas replicated 16 times. Asterisks, mitotic end of germline. (d) Relativeexpression of gpdh-2 mRNA in wild-type and lin-45(n2018) embryos at50mM NaCl measured by qRT–PCR and normalized to the expression of thehistone his-24. Error bars, s.d. n=3 experiments from pellets of >1,000embryos. (e) Glycerol-to-glucose ratio in wild-type and lin-45(n2018) mutantembryos at 50mM NaCl. Error bars, s.d. n=3 experiments from pellets of>1,000 embryos. The quantified results are presented as mean ± s.d. usingANOVA (a, b) and two-tailed t-test (d, e). ∗P<0.05, ∗∗P<0.01, ∗∗∗P<0.001were considered significant. NS, not significant. See Statistics Source Datain Supplementary Table 6.

response to osmotic stress (Fig. 2d). We conclude that the inhibitionof maternal insulin-like signalling does not result in the increaseddeposition of glycerol into embryos but rather results in increasedglycerol production in embryos. Importantly, these results reveal thatthere is a heritable link between maternal insulin-like signalling andprogeny metabolism.

We hypothesized that the heritable effects of maternal insulin-likesignalling on progeny might be mediated by insulin-like signallingto the germline. To test this hypothesis, we expressed rescuingcopies of the wild-type daf-2 or daf-16 genes specifically in thegermline. Germline-specific expression of either daf-2 or daf-16 wassufficient to rescue the effects of deficient maternal insulin-likesignalling on progeny response to osmotic stress (Fig. 2a). In addition,overexpression of daf-2 in the germline blocked the protective effectsof parental exposure to 300mMNaCl on progeny response to 500mMNaCl (Supplementary Fig. 3b). These data suggest that maternalexposure to 300mM NaCl inhibits insulin-like signalling to thegermline and that this loss of insulin-like signalling to the germlineprotects progeny from the effects of osmotic stress.

Insulin-like signalling to the C. elegans germline both inhibitsDAF-1618 and activates the RAS–ERK pathway19, which includes theRaf protein LIN-45, the Mek protein MEK-2 and the Erk proteinMPK-120. We found that partial loss-of-function mutants in lin-45,mek-2 or mpk-1 (null mutants are lethal) did not arrest developmentat 500mM NaCl (Fig. 3a and Supplementary Fig. 3c). In addition,treatment of wild-type animals with theMEK inhibitorU0126 (ref. 21)or RNAi knockdown of mek-2 prevented developmental arrest inresponse to 500mM NaCl (Supplementary Fig. 3d–f); RAS–ERKsignalling functioned maternally to regulate progeny response toosmotic stress (Fig. 3b); GPDH-2 was required for reduced RAS–ERKsignalling to protect animals from developmental arrest (Fig. 3a);and maternal exposure to osmotic stress inhibited MPK-1 activationin the germline (Fig. 3c and Supplementary Table 4), possibly byinhibiting the release of insulin-like peptides from sensory neurons(Supplementary Fig. 4a). We conclude that reduced insulin-likesignalling from the soma (probably from sensory neurons) to thematernal germline protects progeny from the effects of osmotic stressby both activating DAF-16 and inactivating MPK-1.



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Figure 4 RAS–ERK signalling regulates C. elegans response to bacterialinfection and starvation. (a) Percentage of animals expressing lin-4::YFPafter 24 h of exposure to either Escherichia coli OP50 or Pseudomonasaeruginosa PA14. Error bars, s.d. n= 3 experiments of >100 animals.(b) Percentage of animals failing to arrest development after 24h at 350mMNaCl. Error bars, s.d. n=3 experiments of >100 animals. (c) Percentageof wild-type, lin-45(n2018) and daf-16(mu86) mutants with a dividedM-cell after 7 days without food in S-basal at 20 ◦C. Error bars, s.e.m.

n=4 experiments of >100 animals. (d) Model for how maternal exposureto osmotic stress inhibits DAF-2 activity in the germline and affectsprogeny response to osmotic stress. See text for details. Red, embryo;green, germline; purple, intestine. The quantified results are presented asmean ± s.d. (a,b) and s.e.m. (c) using ANOVA (a) and two-tailed t-test(b,c). ∗P<0.05, ∗∗P<0.01, ∗∗∗P<0.001, ∗∗∗∗P<0.0001 were consideredsignificant. NS, not significant. See Statistics Source Data in SupplementaryTable 6.

To further examine how reduced maternal insulin-like signallingvia theRAS–ERKpathwaymodifies progeny physiologywe performedRNA-seq of wild-type and lin-45 mutant embryos. We identifieda total of 616 genes upregulated more than twofold and 1,310genes downregulated more than twofold in lin-45 mutant embryos(Supplementary Table 5). Among the 616 upregulated genes wasgpdh-2, and we confirmed that gpdh-2 messenger RNA expression isupregulated approximately threefold in lin-45 mutant embryos usingquantitative PCR with reverse transcription (qRT–PCR) (Fig. 3d).To test whether increased GPDH-2 expression results in increasedglycerol production, we compared the glycerol-to-glucose ratios inwild-type and lin-45 mutant embryos by mass spectrometry. Weobserved an 82% increase in the glycerol-to-glucose ratio in lin-45mutant embryos compared with that in wild-type embryos (Fig. 3e).These data are consistent with our hypothesis that reduced insulin-likesignalling to the maternal germline protects progeny from the effectsof osmotic stress by increasing the expression in embryos of the rate-limiting glycerol biosynthetic enzyme GPDH-2.

We note that previous studies found that adaptation to osmoticstress resulted in up to a 1,000% increase in glycerol in C. elegans9,

significantly higher than the increase we observed in lin-45 mutants.Our genetic data demonstrate that GPDH-2 is required for reducedRAS–ERK signalling in thematernal germline to protect progeny fromdevelopmental arrest (Fig. 3a). However, given the modest increasein glycerol levels in lin-45 mutants it remains possible, and perhapslikely, that one or more of the additional 615 genes upregulated inlin-45 mutants also contribute to the resistance of these animals todevelopmental arrest in response to osmotic stress.

We tested whether parental exposure to other environmentalstresses, such as bacterial infection or starvation, could similarly mod-ify progeny response to environmental stress via RAS–ERK signalling.Exposure of C. elegans to the opportunistic pathogen Pseudomonasaeruginosa PA14 slowed early larval development (Fig. 4a), parentalexposure to PA14 enhanced this slowing of larval development(Fig. 4a), and this heritable slowing of larval development in responseto bacterial infection requiredRAS–ERK signalling (Fig. 4a). However,maternal exposure to PA14 did not protect progeny fromdevelopmen-tal arrest at 500mMNaCl (Supplementary Fig. 4b) but rather resultedin progeny that were more sensitive to arrest in response to osmoticstress (Fig. 4b). In addition, we found that RAS–ERK signalling was

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required for L1 arrest in response to starvation (Fig. 4c). Collectively,these results suggest that maternal exposure to environmental stressmodifies progeny physiology via RAS–ERK signalling, but that theeffects of these stresses on progeny are different for different environ-mental stresses.

In conclusion, we propose a model in which the inhibition ofinsulin-like signalling to both the intestine and the germline canenhance C. elegans survival during osmotic stress but in which theeffects of inhibition of insulin-like signalling to these two tissues aredistinct. Specifically, the loss of insulin-like signalling to the intestineenhances resistance to osmotic stress by promoting developmentalarrest, whereas the loss of insulin-like signalling to the maternalgermline enhances progeny resistance to osmotic stress by increasingglycerol synthesis in embryos (Fig. 4d). In this model, informationabout the maternal environment is inherited via germ cells to enhanceprogeny resistance to future environmental stress.

The salt concentrations at which C. elegans arrests developmentin response to osmotic stress are approximately those of seawater,480mM Na+ and 559mM Cl− (ref. 22). We speculate that both thestate of immobile arrested development and the ability of parentsto confer progeny resistance to osmotic stress evolved to enhanceorganismal survival in response to osmotic stress caused by seawater.The insulin signalling pathway is broadly conserved among metazoa,and we postulate that insulin signalling to the germline plays a role inseveral human developmental and metabolic abnormalities known toresult from abnormal insulin signalling, such as intrauterine growthrestriction, obesity and type-2 diabetes, all of which have been linkedto maternal environmental stress8,23. Consistent with this hypothesis,a recent report found that feeding parental mice a high-fat diet causesepigenetic changes to oocytes that result in progeny that are moresusceptible to both obesity and diabetes7. These observations of amaternal high-fat diet modifying progeny physiology via oocytes inmice are similar to our observations of maternal exposure to osmoticstress modifying progeny physiology via oocytes in C. elegans. Wepropose that modified insulin-like signalling to the mouse germlinemight be the mechanism underlying these epigenetic changes inmouse oocytes. �

METHODSMethods, including statements of data availability and any associatedaccession codes and references, are available in the online version ofthis paper.

Note: Supplementary Information is available in the online version of the paper

ACKNOWLEDGEMENTSWe thank E. J. Hubbard, K. Ashrafi, S. Mitani and the Caenorhabditis GeneticCenter, which is funded by the NIH National Center for Research Resources(NCRR), for providing strains; N. An for strain management; and K. Burkhart,S. Luo, A. Doi, N. Paquin and A. Corrionero for helpful discussions. H.R.H. and

N.O.B. were supported by NIH grant GM024663 and NSF grant 1122374. T.F. andS.A. were supported by NIH grant GM98200 and ACS grant RSG014-044-DDC.L.R.B., A.K.W. and R.E.W.K. were supported by NIH grant GM117408. H.R.H. isan investigator of the Howard Hughes Medical Institute.

AUTHOR CONTRIBUTIONSN.O.B., T.F., A.K.W., R.E.W.K., S.A., L.R.B. and H.R.H. designed the experimentsand analysed the data. N.O.B., T.F., A.K.W., R.E.W.K. and S.A. performed theexperiments. N.O.B. and H.R.H. wrote the manuscript.

COMPETING FINANCIAL INTERESTSThe authors declare no competing financial interests.

Published online at http://dx.doi.org/10.1038/ncb3470Reprints and permissions information is available online at www.nature.com/reprints

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6. Carone, B. R. et al. Paternally induced transgenerational environmentalreprogramming of metabolic gene expression in mammals. Cell 143,1084–1096 (2010).

7. Huypens, P. et al. Epigenetic germline inheritance of diet-induced obesity and insulinresistance. Nat. Genet. 48, 497–499 (2016).

8. Gallo, L., Tran, M., Master, J., Moritz, K. & Wlodek, M. Maternal adaptations andinheritance in the transgenerational programming of adult disease. Cell Tissue Res.349, 863–880 (2012).

9. Frazier III, H. N. & Roth, M. B. Adaptive sugar provisioning controls survival ofC. elegans embryos in adverse environments. Curr. Biol. 19, 859–863 (2009).

10. Baugh, L. R. & Sternberg, P. W. DAF-16/FOXO regulates transcription of cki-1/Cip/Kip and repression of lin-4 during C. elegans L1 arrest. Curr. Biol. 16,780–785 (2006).

11. Hu, P. J. Dauer 1–19 (WormBook, 2007); https://doi.org/10.1895/wormbook.1.144.112. Murphy, C. & Hu, P. J. Insulin/insulin-like Growth Factor Signaling in C. elegans

(WormBook, 2013); https://doi.org/10.1895/wormbook.1.164.113. An, J. H. & Blackwell, T. K. SKN-1 links C. elegans mesendodermal specification to

a conserved oxidative stress response. Genes Dev. 17, 1882–1893 (2003).14. Kim, D. H. et al. A conserved p38 MAP kinase pathway in Caenorhabditis elegans

innate immunity. Science 297, 623–626 (2002).15. Chen, Y. & Baugh, L. R. ins-4 and daf-28 function redundantly to regulate C. elegans

L1 arrest. Dev. Biol. 394, 314–326 (2014).16. Lamitina, S. T., Morrison, R., Moeckel, G. W. & Strange, K. Adaptation of the

nematode Caenorhabditis elegans to extreme osmotic stress. Am. J. Physiol. 286,C785–C791 (2004).

17. Lamitina, T., Huang, C. G. & Strange, K. Genome-wide RNAi screening identifiesprotein damage as a regulator of osmoprotective gene expression. Proc. Natl Acad.Sci. USA 103, 12173–12178 (2006).

18. Michaelson, D., Korta, D. Z., Capua, Y. & Hubbard, E. J. A. Insulin signalingpromotes germline proliferation in C. elegans. Development 137, 671–680 (2010).

19. Lopez III, A. L. et al. DAF-2 and ERK couple nutrient availability tomeiotic progression during Caenorhabditis elegans oogenesis. Dev. Cell 27,227–240 (2013).

20. Sundaram, M. V. RTK/Ras/MAPK Signaling (WormBook, 2006);https://dx.doi.org/10.1895/wormbook.1.80.1

21. Favata, M. F. et al. Identification of a novel inhibitor of mitogen-activated proteinkinase kinase. J. Biol. Chem. 273, 18623–18632 (1998).

22. Pilson, E. Q. M. An Introduction to the Chemistry of the Sea 2nd edn, 67 (CambridgeUniv. Press, 2013).

23. Prada, J. A. & Tsang, R. C. Biological mechanisms of environmentally inducedcauses of IUGR. Eur. J. Clin. Nutr. 52, S21-7-28 (1998).



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https://doi.org/10.1895/wormbook.1.164.1

https://dx.doi.org/10.1895/wormbook.1.80.1

METHODS DOI: 10.1038/ncb3470

METHODSStrains.AllC. elegans strains were cultured as described previously24 andmaintainedat 20 ◦C unless noted otherwise. The Bristol strain N2 was the wild-type strain.Mutations used are:LGI: daf-16(mu86), mek-2(ku114), ins-18(ok1672), ins-18(ok2478), ins-26(tm1983),ins-28(ok2722), ins-29(tm1922), ins-30(ok2343), ins-33(tm2988), ins-36(tm6125),rrf-1(pk1417); gpdh-1(ok1558)LGII: ins-2(tm4467), ins-3(ok2488), ins-3(tm3608), ins-4(ok3534), ins-5(tm2560),ins-6(tm2416), ins-11(tm1053), ins-12(tm2918), ins-13(tm4856), ins-14(tm4886),ins-15(ok3444), ins-19(tm5155), ins-20(tm5634), ins-31(ok3543), ins-32(tm6109),ins-37(tm6268), age-1(hx546), knuSi379 [Pmex-5::daf-16cDNA::GFP::nos-2 3’UTR,unc-119(+)]LGIII: daf-2(e1370), mpk-1(n5639), ins-17(tm790), ins-21(tm5180), ins-22(tm4639);gpdh-2(ok1733)LGIV: lin-45(n2018), ins-1(nj32), ins-7(tm1907), ins-8(tm4144), ins-34(tm3095);unc-31(ft1); zIs356 (daf-16::GFP); pmk-1(km25); skn-1(zu67)LGV: daf-28(tm2308), ins-10(tm3498), ins-27(ok2474), ins-35(ok3297),him-5(e1490)LGX: ins-9(tm3618), pdk-1(sa709) nIs349[Pceh-28::4xNLS::mCherry; lin-15(+)];unknown linkage: vizIs23 [pie-1p::GFP::daf-2(WT)::pie-1 3’UTR + unc-119(+)],vizIs22 [pie-1p::GFP::daf-2(WT)::pie-1 3’UTR + unc-119(+)], nIs343 [Pegl-1::4xNLS::GFP; lin15(+)]; otIs39[unc-47p::GFP];Extrachromosomal arrays: naEx187[pGC467 (ins-3(+)), pRF4)rol-6(su1006)],hpEx2906 [Prgef-1::daf-2; Pmyo-2::mCherry], hpEx3369 [Pges-1::daf-2; Pmyo-2::mCherry], hpEx2905 [Pmyo-3::daf-2; Pmyo-2::mCherry] fx325 [Posm-6::unc-31a;Pmyo-3::mCherry; unc-119(+)]

Assay for developmental arrest. Approximately 200 developing eggs from mothersgrown at 50mM NaCl (unless otherwise noted) were collected and placed onstandard NGM plates containing varying concentrations of NaCl at 25 ◦C for 48 h.After 48 h, animals that remained immobile and were not feeding were scored asarrested. Mobile animals that were feeding were scored as developing. Percentageof L2+ is defined by the percentage of animals mobile and feeding and that havedeveloped past the L1 larval stage. Percentage failing to arrest is defined by thepercentage of animals mobile and feeding (unlike animals normally arrested inresponse to osmotic stress) but includes L1-stage larvae.

Assay for survival after arrest. Approximately 100 developing eggs from mothersgrown at 50mMNaClwere collected and placed on standardNGMplates containingvarying concentrations of NaCl at 20 ◦C for 24 h. After 24 h arrested animals werepicked onto plates containing 50mM NaCl and allowed to recover for 24 h. After24 h the fraction that regained mobility and resumed development were scored assurviving and the fraction that failed to resume development and did not respond totouch were assumed to be dead.

DAF-16::GFP localization. Confocal microscopy was performed using a Zeiss LSM800 instrument. The resulting imageswere prepared using ImageJ software (NationalInstitutes of Health). Image acquisition settings were calibrated to minimize thenumber of saturated pixels and were kept constant throughout the experiment.

mRNA expression analysis by RNA-seq and qRT–PCR. L4-stage wild-type andlin-45 animals were placed on standard NGM plates containing either 50mM or300mM NaCl for 24 h. Developing eggs from these animals were collected andplaced at either 50mM or 500mMNaCl for 6 h. After 6 h embryos were collected inM9, andRNAwas extracted usingTissueRuptor and theRNeasyMini kit (QIAGEN).For RNA-seq, RNA integrity and concentration were checked on a FragmentAnalyzer (Advanced Analytical). ThemRNAwas purified by polyA-tail enrichment,fragmented, and reverse transcribed into cDNA (Illumina TruSeq). cDNA sampleswere then end-repaired and adaptor-ligated using the SPRI-works Fragment LibrarySystem I (Beckman Coulter Genomics) and indexed during amplification. Librarieswere quantified using the Fragment Analyzer (Advanced Analytical) and qPCRbefore being loaded for paired-end sequencing using the Illumina NextSeq. ForqRT–PCR reverse transcription was performed using SuperScript III (Invitrogen),and quantitative PCR was performed using Applied Biosystems Real-Time PCRInstruments. All results are normalized to the mRNA levels of the histonegene his-24.Primersgpdh-2

forward: tttgatccaaccgtccgtat reverse: cgaattgatgtggaacaacghis-24

forward: atgatcaaggaggccatcaa reverse: tgagcattgatctggatgaca

Assay for wild-type adaptation to osmotic stress. Wild-type embryos were placedon standard NGM plates containing 300mM NaCl at 20 ◦C for 60 h and allowed

to develop to adulthood. Embryos from these adult animals were extracted usinga razor blade into M9 solution and pipetted onto plates containing 500mM NaCl.These animals were allowed to develop for 48 h at 20 ◦C. Percentage failing to arrestis defined by the fraction of animals that were mobile and feeding.

Cross progeny analysis. Approximately 20 L4 hermaphrodites were crossed withwild-type or daf-2(e1370)males for 24 h at 25 ◦C. After 24 h, embryos were dissectedfrom hermaphrodites using a razor blade and then placed on plates containing500mM NaCl at 20 ◦C for 24 h. Percentage of L2+ is defined by the percentageof animals that developed past the L1 larval stage. All males contained nIs343(Pegl-1::4xNLS::mCherry); nIs349 (Pceh-28::4xNLS::mCherry) for the identificationof cross progeny.

Photographs. Photographs of animals and embryos on standard NGM platescontaining either 300mM or 500mMNaCl were obtained using an AxioCamMRmcamera (Zeiss).

dpMPK-1 imaging.Wild-type animals at the L4 stage were placed on either 50mMor 300mM NaCl NGM plates seeded with OP50 at 20 ◦C for 24 h, and germlineswere extruded. Dissections were performed as described previously25,26. Briefly,dissections were performed within 5min of adding levamisole to achieve optimaldiphosphorylated MPK-1 (dpMPK-1) staining. The dissected germlines were thenfixed in 3%paraformaldehyde for 10min, followed by a post-fix in 100%methanol at−20 ◦C. The fixed germ lines were then processed for immunofluorescence stainingas described previously27–29. Anti-MAP kinase was used at a dilution of 1:200 (CloneMAPK-YT, Sigma). Secondary antibodies were donkey anti-mouse Alexa Fluor 594and used at a dilution of 1:400. Each gonad was photographed as a montage, witheach image taken as a 0.15 µ section and capturedwith overlapping cell boundaries at×63magnification. Imageswere takenusing aZeissAxio Imager uprightmicroscopewith AxioVs40 V4.8.2.0 microimaging software and an Axio MRm camera (Zeiss).Montages were then assembled using Adobe Photoshop CS5.1, and white levels wereuniformly adjusted using Affinity Illustrator to reduce background.

RNA-seq prep and data analysis. For Supplementary Tables 1 and 3 RNAintegrity and concentration were checked on a Fragment Analyzer (AdvancedAnalytical). The mRNA was purified by polyA-tail enrichment, fragmented, andreverse transcribed into cDNA (Illumina TruSeq). cDNA samples were then end-repaired and adaptor-ligated using the SPRI-works Fragment Library System I(Beckman Coulter Genomics) and indexed during amplification. Libraries werequantified using the Fragment Analyzer (Advanced Analytical) and qPCR beforebeing loaded for paired-end sequencing using the Illumina NextSeq. For QCpurposes, BEDTools30 (version: 2.25.0) was used to count the reads falling intogenes, coding regions, intronic regions, 5′ or 3′ UTRs, flanking 3-kb genic regionsand intergenic regions. Other basic statistics, including mapping rate, ratio ofsense versus anti-sense reads and rRNA percentages were also collected for eachsample. The reads were first cleaned up by removing reads aligned to rRNAs(BWA 0.6.131 and BEDTools 2.25.0 were used). RSEM32 (version 1.2.15) was usedto estimate gene levels based on ce10 ensembl annotations downloaded from UCSCgenome table browser33. The gene expression count table was then imported intoDESeq33 (version 1.10.1) for differential gene expression test. See the README tabin Supplementary Table 2 for detailed descriptions of RNA-seq and analysis forSupplementary Table 2.

Pathogen exposure and development assay. Wild-type embryos were placed ontoeither NGM plates seeded with Escherichia coli OP50 or slow-killing assay platesseeded with P. aeruginosa PA14 and allowed to grow at 25◦C for 72 h. After 72 hembryos were collected from adults and placed on new plates seeded with eitherOP50 or PA14 and placed at 25◦C.

M-cell division in response to starvation.M-cell division analysis was performed asdescribed previously10.

Statistics and reproducibility. ANOVA analysis with post hoc P-value calculationswas used for Fig. 2, Fig. 3a,b, Fig. 4a and Supplementary Figs 3b and 4b. Unpairedtwo-tailed Student’s t-test was used for Fig. 1, Fig. 3d,e, Fig. 4b,c and SupplementaryFigs 1, 3c–f; ∗ = P < 0.05, ∗∗ = P < 0.01, ∗∗∗ = P < 0.001, ∗∗∗∗P < 0.0001. Nostatistical method was used to predetermine sample size. The experiments were notrandomized. The investigators were not blinded to allocation during experimentsand outcome assessment.

Metabolite preparation for quantification. Approximately 100 µl of concentratedembryos were collected by egg preparation and placed on normal NGM agar platesfor 3 h to recover. After 3 h, embryos were collected in M9, pelleted, and frozen.Frozen embryos were resuspended in 400 µl PBS and homogenized by douncing.


NATURE CELL BIOLOGY

DOI: 10.1038/ncb3470 METHODS

Homogenized embryos were centrifuged at 800g for 2min to remove undouncedtissue and 200 µl of supernatant was mixed with 800 µl of methanol and dried toextract polar metabolites. Dried samples were stored at−80 ◦C.

Metabolite profiling. Liquid chromatography and mass spectrometry wereperformed as described previously33.

Assay for L2-, L3- and L4-stage developmental arrest. Approximately 50 L2-,L3- or L4-stage animals were placed onto standard NGM Petri plates containing500mM NaCl for 24 h. After 24 h animals that were immobile and not developingor responding to touch were moved to plates containing 50mM NaCl. Percentagedeveloping is defined by the percentage of animals that resumed development andmobility after returning to normal growth conditions.

MEK inhibitor exposure. The MEK inhibitor U0126 (U120 Sigma-Aldrich) wasresuspended in dimethylsulfoxide and added to standard NGMPetri plates at a finalconcentration of 100muupM. These plates were then seeded with Escherichia coliOP50.Wild-type embryos were placed on plates containing either dimethylsulfoxidealone or dimethylsulfoxide and the MEK inhibitor U0126. Animals were allowed togrow for 72 h at 25 ◦C. After 72 h, embryos from adult animals were collected andplaced onto Petri plates containing 500mM NaCl at 25 ◦C for 24 h and the fractionof animals mobile and developing was determined.

mek-2 RNAi exposure. Wild-type embryos fed Escherichia coli HT115 containingeither the empty vector L4440 or a mek-2 RNAi vector (Ahringer library—SourceBiosciences) were grown at 20 ◦C for 72 h. After 72 h, embryos were collected andplaced onto Petri plates containing 500mM NaCl for 24 h at 25 ◦C and the fractionof animals mobile and developing was determined.

Assay for daf-2 adaptation to osmotic stress. L4 animals were placed on standardNGM plates containing 300mM NaCl at 25 ◦C overnight. Embryos from resulting

adult animals were extracted with a razor blade into M9 solution and pipetted ontoplates containing 500mM NaCl. These animals were allowed to develop for 48 h at20 ◦C. Percentage of L2+ is defined by the percentage of animals that developed pastthe L1 larval stage.

Data availability. RNA-seq data that support the findings of this study have beendeposited in theGene ExpressionOmnibus (GEO) under accession codes GSE91073andGSE91039. Data supporting the findings of Figs 1b,c,d, 2a,b,d, 3a,b,d,e and 4a,b,cand Supplementary Fig. 1b,c,d,e,f,g, Supplementary Fig. 3b,d,e,f and SupplementaryFig. 4b,c,d are provided in Supplementary Table 6. All other relevant data areavailable from the authors on request and/or are included with the manuscript.

24. Brenner, S. The genetics of Caenorhabditis elegans. Genetics 77, 71–94 (1974).25. Arur, S. et al. MPK-1 ERK controls membrane organization in C. elegans oogenesis

via a sex-determination module. Dev. Cell 20, 677–688 (2011).26. Arur, S. et al. Multiple ERK substrates execute single biological processes in

Caenorhabditis elegans germ-line development. Proc. Natl Acad. Sci. USA 106,4776–4781 (2009).

27. Lee, M.-H. et al. Multiple functions and dynamic activation of MPK-1 extracellularsignal-regulated kinase signalling in Caenorhabditis elegans germline development.Genetics 177, 2039–2062 (2007).

28. Quinlan, A. R. BEDTools: the Swiss-army tool for genome feature analysis. Curr.Protoc. Bioinformatics 47, 11.12.1–11.12.34 (2014).

29. Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows-Wheelertransform. Bioinformatics 25, 1754–1760 (2009).

30. Li, B. & Dewey, C. N. RSEM: accurate transcript quantification from RNA-Seq datawith or without a reference genome. BMC Bioinformatics 12, 323 (2011).

31. Karolchik, D. et al. The UCSC Table Browser data retrieval tool. Nucleic Acids Res.32, D493–D496 (2004).

32. Anders, S. & Huber, W. Differential expression analysis for sequence count data.Genome Biol. 11, R106 (2010).

33. Birsoy, K. et al. An essential role of the mitochondrial electron transport chain in cellproliferation is to enable aspartate synthesis. Cell 162, 540–551 (2015).

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http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE91039

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Supplementary Figure 1 C. elegans arrests development in response to osmotic stress. (a) Representative images of wild-type animals after 72 hrs of exposure to 50 mM or 500 mM NaCl. Experiment replicated 12 times Scale bar 200 μm (b) Percent of wild-type animals developing past the L1 larval stage after 24 hrs of exposure to 50 mM NaCl and 450 mM KCl . Error bars, s.d. n = 3 experiments of >100 animals (c) Percent of wild-type animals developing past the L1 larval stage after 24 hrs of exposure to 50 mM NaCl and 450 mM NaBr. Error bars, s.d. n = 3 experiments of >100 animals (d) Percent of wild-type animals developing past the L1 larval stage after 24 hrs of exposure to 50 mM NaCl and 900 mM sucrose. Sucrose was added at twice the concentration of salts to compensate for the difference in osmolarity. Error bars, s.d. n = 3 experiments of >100 animals (e) Percent of wild-type, pmk-1(km25), or skn-1(zu67) animals developing past the L1 larval stage after 48 hrs of exposure to 300 mM NaCl. Error bars, s.d. n = 3 experiments of >100 animals (f) Percent of wild-type, pmk-1(km25), or skn-1(zu67) animals failing to

arrest development at 500 mM NaCl after 48 hrs. Error bars, s.d. n = 3 experiments of >100 animals (g) Percent of wild-type and unc-31(ft1) animals failing to arrest development after 48 hrs. The osm-6 promoter was used to drive the expression of UNC-31 specifically in sensory neurons. Error bars, s.d. n = 3 experiments of >100 animals (h) Percent of wild-type and ins-3(tm3608) mutant animals failing to arrest development at 400 mM NaCl after 48 hrs. Error bars, s.d. n = 3 experiments of >100 animals (i) Percent of wild-type, ins-3(ok2488), ins-3(ok2488);daf-2(e1370) and rescue animals failing to arrest development at 400 mM NaCl 48 hrs post-hatching. Error bars, s.d. n = 3 experiments of >100 animals The variation between the wild type in panels (a) and (b) is likely to be a result of variations in evaporation between different batches of Petri plates. The quantified results are presented as mean ± s.d. using two-tailed t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 were considered significant. n.s., not significant. See Statistics Source Data in Supplemental Table 6




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sulfoxide (DMSO) developing past the L1 larval stage after 48 hrs (500 mM NaCl). Error bars s.d. n = 3 experiments of >100 animals (e) Percent of progeny from animals exposed to either L4440 empty vector or mek-2 RNAi that developed past the L1 larval stage after 48 hrs (500 mM NaCl). Error bars s.d. n = 3 experiments of >100 animals (f) Percent of rrf-1(pk1417) progeny, which exhibit reduced RNAi silencing in somatic tissue but retain germline RNAi silencing1, from animals exposed to either L4440 empty vector or mek-2 RNAi that developed past the L1 larval stage after 48 hrs (500 mM NaCl). Error bars s.d. n = 3 experiments of >100 animals. The quantified results are presented as mean ± s.d. using ANOVA (b) and two-tailed t-test (d,e,f). **P < 0.01, ***P < 0.001, were considered significant. n.s., not significant. See Statistics Source Data in Supplemental Table 6




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bar 100 μm (b) Percent of animals mobile and feeding after 48 hrs at 500 mM NaCl. Error bars s.d. n = 3 experiments of >100 animals. We note that the observation that less wild-type animals adapted to 500 mM NaCl when parents were exposed to 300 mM NaCl than in Fig. 2a is likely due to the fact that in this experiment embryos had to be collected by bleaching to remove any contaminating PA14 and this likely added an extra stressor to the embryos.




Supplementary Table Legends

Supplementary Table 1 Most insulin peptides do not regulate development in response to osmotic stress. Mutant animals that each lacked one of 35 insulin-like peptides were assayed for the percent of animals that developed after exposure to 300 mM NaCl or 500 mM NaCl. WT at 300 mM NaCl: fewer than 1% of animals arresting in response to 300 mM NaCl. WT at 500 mM NaCl: 0% of animals mobile and feeding at 500 mM NaCl. WT, wild-type. The quantified results are presented as mean ± s.d. using two-tailed t-test ***P < 0.001 was considered significant. n.s., not significant. See Statistics Source Data in Supplemental Table 6

Supplementary Table 2 Profile of gene expression after 24 hrs of exposure to 500 mM NaCl and starvation. See README tab for detailed description of analysis.

Supplementary Table 3 Profile of mRNA expression in wild-type embryos at 50 mM NaCl and 500 mM NaCl.

Supplementary Table 4 Quantification of diphosphorylated MPK-1 levels in wild-type germlines exposed to either 50 mM or 300 mM NaCl for 24 hrs.

Supplementary Table 5 Profile of mRNAs upregulated and downregulated >2-fold in lin-45(n2018) mutant embryos when compared to wild-type embryos.

Supplementary Table 6 Statistics source data. Table contains individual values for all bar graphs and for quantification for Fig. 1d.

References:

1. Kumsta, C. & Hansen, M. C. elegans rrf-1 mutations maintain RNAi efficiency in the soma in addition to the germline. PLoS ONE 7(5): e35428. doi: 10.1371/journal.pone.00354282. Speese, S. et al. UNC-31 (CAPS) is required for dense-core vesicle but not synaptic vesicle exocytosis in Caenorhabditis elegans. J Neurosci 27, 6150–6162 (2007).

progeny response to osmotic stress -...

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