The somatic reproductive tissues of C. elegans promote longevity through steroid hormone signaling

Abstract

In Caenorhabditis elegans and Drosophila melanogaster, removing the germline precursor cells increases lifespan. In worms, and possibly also in flies, this lifespan extension requires the presence of somatic reproductive tissues. How the somatic gonad signals other tissues to increase lifespan is not known. The lifespan increase triggered by loss of the germ cells is known to require sterol hormone signaling, as reducing the activity of the nuclear hormone receptor DAF-12, or genes required for synthesis of the DAF-12 ligand dafachronic acid, prevents germline loss from extending lifespan. In addition to sterol signaling, the FOXO transcription factor DAF-16 is required to extend lifespan in animals that lack germ cells. DAF-12/NHR is known to assist with the nuclear accumulation of DAF-16/FOXO in these animals, yet we find that loss of DAF-12/NHR has little or no effect on the expression of at least some DAF-16/FOXO target genes. In this study, we show that the DAF-12-sterol signaling pathway has a second function to activate a distinct set of genes and extend lifespan in response to the somatic reproductive tissues. When germline-deficient animals lacking somatic reproductive tissues are given dafachronic acid, their expression of DAF-12/NHR-dependent target genes is restored and their lifespan is increased. Together, our findings indicate that in C. elegans lacking germ cells, the somatic reproductive tissues promote longevity via steroid hormone signaling to DAF-12.

Figure 1. Dafachronic acid extends the lifespan of germ cell (−); somatic gonad (−) animals.

(A) Laser ablation of the Z1 and Z4 somatic gonad precursor cells in young larve results in animals that lack both the germ cells and the somatic gonad, since development of the germline requires the somatic gonad. germ cell (−); somatic gonad (−) animals, obtained by ablation of Z1 and Z4, lived longer on media containing Δ⁴-dafachronic acid (DA). Thus, increased dafachronic acid can substitute for loss of the somatic gonad. (B) This lifespan increase required daf-12/NHR. (C) No further increase in lifespan was observed when germ cell (−) animals, obtained by laser ablation of Z2 and Z3, the germline precursor cells of young larve, were grown on media containing Δ⁴-dafachronic acid. (D) Additionally, no increase was observed when intact-gonad animals were grown on Δ⁴-dafachronic acid containing media. This suggests that loss of the germ cells is required for dafachronic acid to extend lifespan. Details including means and p values for all experiments represented in this figure as well as replicates are listed in Table S1.

Figure 2. Overexpression of DAF-9/CYP450 in the XXX cells or hypodermis can extend the lifespan of germ cell (−); somatic gonad (−) animals.

A GFP-tagged DAF-9/CYP450 protein, which catalyzes the final step of the synthesis of dafachronic acids, was overexpressed under the control of various promoters using multi-copy transgene arrays. Use of a daf-9(e1406) mutant background limited overexpression to specific tissue types. (A) Removal of the somatic gonad plus the germ cells of animals expressing daf-9::GFP under the control of the daf-9 promoter extended lifespan. Somatic-gonad plus germ cell removal also extended the lifespan of animals expressing daf-9::GFP in the XXX cells using the sdf-9 promoter (B) and in animals expressing daf-9::GFP in the hypodermis using the dpy-7 promoter (C). [However, expression of DAF-9::GFP in the hypodermis of germine-deficient animals lacking the somatic gonad using the col-12 promoter did not extend lifespan (Table S2). GFP fluorescence was visible in these animals, and the construct rescued the constitutive dauer formation phenotype of daf-9(e1406) animals suggesting DAF-9/CYP450 was active in these transgenic animals. It is possible that the level of expression was not sufficient to rescue the longevity of these animals.] (D) Somatic gonad plus germ cell removal also extended the lifespan of animals expressing daf-9::GFP under the control of the che-2 promoter, which drives expression in sensory neurons, which do not normally express daf-9/CYP450. Details including means and p values for all experiments are listed in Table S2.

Figure 3. The somatic gonad modulates the expression of cdr-6 in a DAF-12/NHR- dependent fashion.

(A) Increased cdr-6 expression in germ cell (−) animals requires the somatic gonad. GFP driven by the cdr-6 promoter was observed in the intestine of animals with intact gonads. In germ cell (−) animals, GFP levels increased in the intestine, whereas in germ cell (−); somatic gonad (−) animals, the level of GFP dropped to levels similar to that of intact animals. Original images were rotated and placed on a flat black background. (B) The increase in expression of cdr-6 in germ cell (−) animals requires functional DAF-12/NHR. No increase was observed in daf-12(rh61rh411) mutants. Activation of DAF-12/NHR by addition of dafachronic acid increased the expression of cdr-6 in intact and germ cell (−); somatic gonad (−) to levels similar to those observed in germ cell (−) animals in a daf-12/NHR dependent fashion. Means and p values as determined by the Student's t test for the experiments shown in panel (B) as well as replicate experiments are listed in Table S4.

Figure 4. daf-12/NHR and daf-16/FOXO have distinct effects in germ cell (−) animals.

daf-12/NHR and daf-16/FOXO are both required for germ cell removal to extend lifespan. We examined whether the activity of one of these two transcription factors required the other. (A) daf-12/NHR is at most partially required for the transcriptional activity of DAF-16/FOXO. In a daf-12(rh61rh411) mutant, expression of sod-3, a direct target of DAF-16/FOXO still increased in germ cell (−) animals and decreased when the somatic gonad was removed in germ cell (−); somatic gonad (−) animals. In two experiments, expression of Psod-3::GFP was not lower in germ cell (−) daf-12(rh61rh411) mutants compared to germ cell (−) daf-12(+) animals, whereas in a third experiment, its expression did decrease somewhat. Consistent with this decrease, daf-12/NHR was identified as a “weak hit” in an unbiased screen looking for reduced Psod-3::GFP expression in germ cell (−) animals (MSK and CK, unpublished data). Means and p values are listed in Table S6. (B) daf-16/FOXO is partially required for the activity of DAF-12/NHR. In a daf-16(mu86) mutant, expression of cdr-6 still increased with germ cell removal, but to a lesser extent. Removal of both the somatic gonad and germ cells returned the level of expression to a similar level as that seen in intact animals. Means and p values for this experiment as well as additional replicates are listed in Table S7. (C) mRNA levels of sod-3 measured by quantitative RT-PCR in glp-1(e2141) mutants, which lack germ cells, decrease with mutation of daf-16/FOXO, but not with mutation of daf-12/NHR. Conversely, mRNA levels of cdr-6 decrease with mutation of daf-12/NHR, but not with mutation of daf-16/FOXO. *** p<0.0001, ** p<0.001, * p<0.05, n.s. p>0.05. Means and p values for this experiment as well as additional replicates are listed in Table S9. (D) Activation of DAF-12/NHR by Δ⁴-dafachronic acid treatment did not extend the lifespan of daf-16(mu86) mutants, suggesting that (E) DAF-12/NHR and DAF-16/FOXO are both required for the increased lifespan of germ cell (−) animals. Means and p values are listed in Table S1.