Reevaluation of whether a soma-to-germ-line transformation extends lifespan in Caenorhabditis elegans

Abstract

The germ lineage is considered to be immortal. In the quest to extend lifespan, a possible strategy is to drive germ-line traits in somatic cells, to try to confer some of the germ lineage's immortality on the somatic body. Notably, a study in Caenorhabditis elegans suggested that expression of germ-line genes in the somatic cells of long-lived daf-2 mutants confers some of daf-2's long lifespan. Specifically, mRNAs encoding components of C. elegans germ granules (P granules) were up-regulated in daf-2 mutant worms, and knockdown of individual P-granule and other germ-line genes in daf-2 young adults modestly reduced their lifespan. We investigated the contribution of a germ-line program to daf-2's long lifespan and also tested whether other mutants known to express germ-line genes in their somatic cells are long-lived. Our key findings are as follows. (i) We could not detect P-granule proteins in the somatic cells of daf-2 mutants by immunostaining or by expression of a P-granule transgene. (ii) Whole-genome transcript profiling of animals lacking a germ line revealed that germ-line transcripts are not up-regulated in the soma of daf-2 worms compared with the soma of control worms. (iii) Simultaneous removal of multiple P-granule proteins or the entire germ-line program from daf-2 worms did not reduce their lifespan. (iv) Several mutants that robustly express a broad spectrum of germ-line genes in their somatic cells are not long-lived. Together, our findings argue against the hypothesis that acquisition of a germ-cell program in somatic cells increases lifespan and contributes to daf-2's long lifespan.

Keywords: C. elegans; P granules; aging; daf-2; germ line.

Fig. 1.

daf-2 mutant worms do not accumulate germ-line proteins or transcripts in their somatic cells. (A) Wild-type, lin-13, daf-2(e1368), and daf-2(e1370) L1 larvae grown at 25 °C were immunostained for the P-granule protein PGL-3 (green), the meiotic protein HTP-3 (red), and DNA (blue). Primordial germ cells (Z2 and Z3) are indicated by arrows. (B) pgl-1p::pgl-1::gfp transgene expression in wild-type, lin-35(n745), and daf-2(e1370) adults upshifted to 25 °C for 8 h. To show entire worms, panels contain montages of spliced-together images: 12, 10, and 11 images in Top, Middle, and Bottom, respectively. Brackets indicate distal regions of the germ line, and asterisks indicate distal tips (when visible and not obstructed by the intestine). See Fig. S1B for brighter images. (C) Boxplots of normalized RNA-sequencing expression values [log₂(RPKM)] for ubiquitously expressed genes (green), genes with germ-line–enriched expression (light blue), germ-line–specific genes (dark blue), and soma-specific genes (red) in dissected germ lines, mes-1(bn84ts) germ-line–less mutant adults, and daf-2(e1370);mes-1(bn84ts) germ-line–less double-mutant adults. Expression values reflect three biological replicates for each sample. Each box extends from the 25th to 75th percentile of the log₂(RPKM) values. The whiskers extending from each box indicate the 2.5th and 97.5th percentiles. Wedges around the median indicate 95% confidence intervals for the medians.

Fig. S1.

daf-2 L1 larvae, L2/L3 larvae, and adults do not ectopically express a P-granule transgene in their soma (related to Fig. 1B). (A) Wild-type, lin-35(n745), and daf-2(e1370) L1 and L2/L3 larvae containing a pgl-1p::pgl-1::gfp transgene were upshifted to 25 °C for 8 h and then imaged. Primordial germ cells (Z2 and Z3) are indicated by an arrow in L1 larvae, and proliferated germ lines are indicated by brackets in L2/L3 larvae. Examples of nontransgene, autofluorescent gut granules are indicated by arrowheads in wild-type and daf-2 L2/L3 larvae. All worms were imaged at the same exposure. To show entire worms, figure contains montages of spliced-together images: four, two, three, seven, five, and eight images in the Top Left, Middle Left, Bottom Left, Top Right, Middle Right, and Bottom Right, respectively. (B) Brighter images of worms presented in Fig. 1B, better showing the germ-line GFP in wild-type and daf-2(e1370) adults. As noted in the legend to Fig. 1B, the figure contains montages of spliced-together images: 12, 10, and 11 images in the Top, Middle, and Bottom, respectively. Brackets indicate distal regions of the germ line, and asterisks indicate distal tips (when visible and not obstructed by the intestine). Examples of nontransgene, autofluorescent gut granules are indicated by arrowheads in wild-type and daf-2 adults.

Fig. S2.

Germ-line genes are not up-regulated in germ-line–less daf-2;mes-1 adults (related to Fig. 1C). (A and B) MA plots showing differential expression of all genes in daf-2;mes-1 vs. mes-1 adults. Gray dots show all genes. Significantly up- or down-regulated genes are indicated by red dots. Germ-line–specific genes (A) or germ-line–enriched genes (B) are indicated by blue dots. The total numbers of significantly up- or down-regulated genes are in red, and the numbers of significantly up- or down-regulated germ-line–specific (A) or germ-line–enriched (B) genes in daf-2;mes-1 are in blue. (C) Gene Ontology analysis of the 273 up-regulated genes in daf-2;mes-1 adults shows gene categories involved in aging and dauer formation. These analyses did not identify germ-line–associated processes.

Fig. 2.

Knockdown of P granules or inactivation of the germ-line program by loss of MES-4 does not decrease daf-2 lifespan at 20 °C. (A) Lifespan analysis of wild type (dashed lines) and daf-2(e1370) mutants (solid lines) treated with control RNAi [empty vector (EV) black] or P-granule (pgl-1, -3, glh-1, and -4) RNAi (red) from adulthood (day 3 after hatching). (B) Lifespan analysis of daf-2(e1370) mutants treated with control RNAi or P-granule (pgl-1, -3, glh-1, and -4) RNAi from hatching (day 0). (C) Lifespan analysis of wild type (black circles), mes-4(bn85) M−Z− (yellow squares), daf-2(e1370) (red triangles), daf-2(e1370);mes-4(bn85) M+Z− (blue inverted triangles), and daf-2(e1370);mes-4(bn85) M−Z− (purple diamonds) mutants. Replicates of lifespan analyses are in Dataset S1.

Fig. S3.

daf-2 worms do not rely on MES-4 or PGL-1 to extend lifespan (related to Fig. 2). Knockdown of pgl-1 (A) or mes-4 (B) from daf-2(e1370) adults did not alter lifespan at 25 °C. pgl-1 or mes-4 RNAi was initiated on day 3 after hatching (first day of adulthood).

Fig. S4.

Somatic expression of the pgl-1p::pgl-1::gfp transgene in lin-35 mutants is more effectively reduced if worms are treated with P-granule RNAi from hatching rather than from young adulthood (related to Fig. 2 A and B). (A) lin-35(n745) pgl-1p::pgl-1::gfp worms were fed from hatching on control (empty vector) RNAi bacteria. Some worms were transferred on day 3 after hatching (first day of adulthood) to P-granule (pgl-1, -3, glh-1, and -4) RNAi bacteria, and worms were imaged on subsequent days. Depletion of P granules starting in young adults resulted in a slight reduction of somatic expression of the P-granule transgene. (B) lin-35(n745) pgl-1p::pgl-1::gfp worms were fed from hatching on either control (empty vector) RNAi or P-granule RNAi bacteria. Depletion of P granules starting at L1 resulted in greater reduction of somatic expression of the P-granule transgene expression than depletion starting in young adults.

Fig. S5.

mes-4 M−Z− mutants possess somatic gonad cells but are defective in dafachronic acid signaling (related to Fig. 2C). (A) Germ-line–less mes-4(bn73) M−Z− worms do not have a significantly different lifespan than wild-type worms. (B) mes-4(bn85) M−Z− gonads stain positively for the somatic gonad marker CEH-18. To show an entire gonad arm, B contains a montage of three spliced-together images. Asterisks indicate the distal tip of the gonad. (C) Supplementing mes-4(bn85) M−Z− adults with Δ⁷-dafachronic acid (DA) slightly increases lifespan compared with ethanol control (vehicle). (D) daf-2(e1368);mes-4(bn85) M+Z− double-mutant lifespan is not significantly different from daf-2(e1368) single-mutant lifespan.

Fig. 3.

Somatic expression of germ-line genes in synMuv B mutants does not lead to longer lifespan at 20 °C or 24 °C. (A and B) synMuv B mutant L1 larvae grown at 20 °C (A) or 24 °C (B) were stained for the P-granule protein PGL-1 (green), the meiotic protein HTP-3 (red), and DNA (blue). Primordial germ cells (Z2 and Z3) are indicated by arrows. Boxed regions are expanded in the right panels. (C and D) Lifespans of synMuv B mutants at 20 °C (C) or 24 °C (D). Data are presented as the percent change in lifespan in mutant compared with wild type. Single points represent individual replicates; horizontal bars are the average lifespan of three replicates. Lifespan data are in Dataset S1, and representative lifespan curves are in Fig. S6.

Fig. S6.

Representative lifespan curves of synMuv B mutants grown at 20 °C (A) or 24 °C (B) (related to Fig. 3 C and D).