Insulin-like signalling to the maternal germline controls progeny response to osmotic stress

Abstract

In 1893 August Weismann proposed that information about the environment could not pass from somatic cells to germ cells, a hypothesis now known as the Weismann barrier. However, recent studies have indicated that parental exposure to environmental stress can modify progeny physiology and that parental stress can contribute to progeny disorders. The mechanisms regulating these phenomena are poorly understood. We report that the nematode Caenorhabditis elegans can protect itself from osmotic stress by entering a state of arrested development and can protect its progeny from osmotic stress by increasing the expression of the glycerol biosynthetic enzyme GPDH-2 in progeny. Both of these protective mechanisms are regulated by insulin-like signalling: insulin-like signalling to the intestine regulates developmental arrest, while insulin-like signalling to the maternal germline regulates glycerol metabolism in progeny. Thus, there is a heritable link between insulin-like signalling to the maternal germline and progeny metabolism and gene expression. We speculate that analogous modulation of insulin-like signalling to the germline is responsible for effects of the maternal environment on human diseases that involve insulin signalling, such as obesity and type-2 diabetes.

Figure 1

Insulin-like signalling to the intestine regulates developmental arrest in response to osmotic stress. (a) Percent 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 after 48 hrs. Error bars, s.d. n = 3 experiments of >100 animals P < 0.01 for all genotypes (two-tailed t-test) (b) Percent of wild-type, daf-2(e1370), daf-16(mu86) and daf-2(e1370); daf-16(mu86) animals that resume development after 24 hours of exposure to osmotic stress. Error bars, s.d. n = 3 see Supplementary Table 6P < 0.01 for all genotypes (two-tailed t-test) (c) Percent of wild-type and daf-2(e1370) animals developing past the L1 larval stage at 300 mM NaCl after 48 hrs. Neuron-specific expression was 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 = 3 experiments of >100 animals (d) Confocal images of DAF-16::GFP after 6 hrs of exposure to 50 mM and 500 mM NaCl. Scale bar 10 μm. 3 experimental replicates of >100 animals. The quantified results are presented as mean ± s.d. using two-tailed t-test ****P < 0.0001 were considered significant. n.s., not significant. See Statistics Source Data in Supplementary Table 6

Figure 2

Insulin-like signalling to the maternal germline regulates progeny response to osmotic stress (a) Percent of wild-type, daf-2(e1370) and daf-2(e1370); daf-16(mu86) cross progeny failing to arrest development after 48 hrs at 500 mM NaCl. Males contained (Pegl-1::4xNLS::GFP); him-5(e1490); nIs349 (Pceh-28::4xNLS::mCherry) for the identification of cross progeny. The pie-1 promoter was used to drive germline specific expression 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) Percent of wild-type, gpdh-1(ok1558), and gpdh-2(ok1733) animals failing to arrest development at 500 mM NaCl after 48 hrs. Error bars, s.d. n = 3 experiments of >100 animals (c) Average fold change of 2 replicates of the 25 most upregulated genes in embryos in response to osmotic stress after 6 hrs. (d) Percent of wild-type, daf-2(e1370) and gpdh-2(ok1733) cross progeny failing to arrest development after 48 hrs at 500 mM NaCl. Males contained otIs39 (Punc-47::GFP); him-5(e1490) for the identification of cross progeny. Error bars, s.d. n = 3 experiments of >20 animals. The quantified results are presented as mean ± s.d. using ANOVA. *P < 0.05, ***P < 0.001, **** P < 0.0001 were considered significant. n.s., not significant. See Statistics Source Data in Supplementary Table 6

Figure 3

Insulin-like signalling to the maternal germline modifies progeny response to osmotic stress by regulating the RAS-ERK-like pathway. (a) Percent of wild-type and lin-45(n2018), mek-2(ku114), mpk-1(n5639) and lin-45(n2018); gpdh-2(ok1733) animals failing to arrest development at 500 mM NaCl after 48 hrs. Error bars, s.d. n = 3 experiments of >100 animals (b) Percent of wild-type and lin-45(n2018) cross progeny failing to arrest development at 500 mM NaCl after 48 hrs. Males contained otIs39 (Punc-47::GFP); him-5(e1490) for the identification of cross progeny. Error bars, s.d. n = 3 experiments of >20 animals. (c) Representative germlines dissected from wild-type animals exposed to either 50 mM NaCl or 300 mM NaCl and stained for DNA (DAPI, white) and diphosphorylated MPK-1 (dpMPK-1) (red) Each condition was replicated 16 times. Scale bar 50 μm (d) Relative expression of gpdh-2 mRNA in wild-type and lin-45(n2018) embryos measured by qRT-PCR and normalized to the expression of the histone his-24. Error bars, s.d. n = 3 experiments from pellets of >1000 embryos (e) Glycerol-to-glucose ratio in wild-type and lin-45(n2018) mutant embryos. Error bars, s.d. n = 3 experiments from pellets of >1000 embryos. The quantified results are presented as mean ± s.d. using ANOVA (a, b) and two-tailed t-test (d, e). *P < 0.05, **P < 0.01, ***P < 0.001 were considered significant. n.s., not significant. See Statistics Source Data in Supplementary Table 6

Figure 4

RAS-ERK signalling regulates C. elegans response to bacterial infection and starvation (a) Percent of animals expressing lin-4::YFP after 24 hrs of exposure to either E. coli OP50 or P. aeruginosa PA14. Error bars, s.d. n = 3 experiments of >100 animals (b) Percent of animals failing to arrest development after 24 hrs at 350 mM NaCl. Error bars, s.d. n = 3 experiments of >100 animals (c) Percent of wild-type, lin-45(n2018) and daf-16(mu86) mutants with a divided M-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 exposure to osmotic stress inhibits DAF-2 activity in the germline and affects progeny response to osmotic stress. See text for details. Color code: Red, embryo; green, germline; purple, intestine. The quantified results are presented as mean ± 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 considered significant. n.s., not significant. See Statistics Source Data in Supplementary Table 6