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Maternal age generates phenotypic variation in Caenorhabditis elegans

Abstract

Genetically identical individuals that grow in the same environment often show substantial phenotypic variation within populations of organisms as diverse as bacteria1, nematodes2, rodents3 and humans4. With some exceptions5,6,7, the causes are poorly understood. Here we show that isogenic Caenorhabditis elegans nematodes vary in their size at hatching, speed of development, growth rate, starvation resistance, fecundity, and also in the rate of development of their germline relative to that of somatic tissues. We show that the primary cause of this variation is the age of an individual’s mother, with the progeny of young mothers exhibiting several phenotypic impairments. We identify age-dependent changes in the maternal provisioning of the lipoprotein complex vitellogenin to embryos as the molecular mechanism that underlies the variation in multiple traits throughout the life of an animal. The production of sub-optimal progeny by young mothers may reflect a trade-off between the competing fitness traits of a short generation time and the survival and fecundity of the progeny.

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Figure 1: Gene expression profiling reveals inter-individual variation in soma–germline phasing.
Figure 2: Slow development, early starvation and young mothers are associated with relative germline acceleration and lower brood size, with early progeny also short and sensitive to starvation.
Figure 3: Vitellogenin expression and provisioning is variable between individuals and increases with age.
Figure 4: Increased yolk provisioning explains phenotypic differences between day 1 and day 2 progeny.

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Acknowledgements

This work was supported by a European Research Council Consolidator grant (616434), the Spanish Ministry of Economy and Competitiveness (BFU2011-26206 and SEV-2012-0208), the AXA Research Fund, the Bettencourt Schueller Foundation, Agència de Gestió d’Ajuts Universitaris i de Recerca (AGAUR, 2014 SGR 831), the EMBL-CRG Systems Biology Program, and the CERCA Program/Generalitat de Catalunya. M.F.P. was partially supported by a FPI-Severo Ochoa fellowship. Expression profiling was performed in the CRG Genomics core facility and microscopy in the CRG Advanced Light Microscopy Facility. Some strains were provided by the CGC, which is funded by National Institutes of Health Office of Research Infrastructure Programs (P40 OD010440).

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Authors

Contributions

M.F.P., M.F. and B.L. conceived the model, designed experiments and wrote the manuscript. M.F.P., M.F. and C.H.-C. performed experiments and analysed the data.

Corresponding author

Correspondence to Ben Lehner.

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The authors declare no competing financial interests.

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Reviewer Information Nature thanks D. Gems, O. Rechavi and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Figure 1 Spermatogenesis and oogenesis gene expression signatures extracted by independent component analysis.

a, Scatterplot showing gene loadings on the oogenesis component (x axis) and spermatogenesis component (y axis) extracted by independent component analysis. Spermatogenesis and oogenesis46 genes are highlighted. b, The oogenesis and spermatogenesis components in the reference time series46 ranked with principal curves. The three replicates per time point in the time series are considered separately. c, Early- and late-moulting signatures in the reference time series ranked according to germline rank. d, Enrichment (measured by odds) of oogenesis-46, spermatogenesis-46 and hypodermis-specific48 genes in the top 10% most variable genes among single worms judged by expression residuals after correcting for germline developmental stage. e, Enrichment (measured by log odds) for genes for which variance is better explained by germline (positive log odds) or hypodermal (negative log odds) rank in tissue-specific gene sets. Variation in other somatic tissues is best explained by hypodermal gene expression signatures. Error bars represent 95% confidence intervals. Gene sets are listed in Methods.

Extended Data Figure 2 Slow development, early starvation and young mothers are associated with altered temporal distribution of progeny production, with early progeny displaying enlarged gut granules, slow development and reduced resistance to larval starvation.

a, Schematic indicating the establishment of a synchronized population by allowing eggs to hatch for 1 h and transferring newly hatched larvae to a new plate. b, Progeny production per day for early-, mid- or late-moulting worms (n = 9, 11, 10 worms). c, Schematic indicating comparison of worms continuously fed after hatching with worms that underwent a transient 5-h starvation immediately after hatching. d, Progeny production per day for worms either continuously fed or transiently starved for 5 h immediately after hatching (n = 12, 13 worms). e, Schematic showing establishment of parallel cohorts of progeny from day 1, day 2 or day 3 mothers and subsequent phenotypic assays. f, Progeny production per day of offspring of day 1, day 2 or day 3 mothers. The start of the first day of egg laying for each cohort is counted from the point where a majority of the population have at least 1 embryo in utero (n = 18, 18, 19 worms). g, Confocal microscopy images of live L1 larvae from day 1, 2 or 3 mothers showing blue autofluorescence from intestinal lysosome-related organelles (‘gut granules’). Insets show expanded section of main image indicated by white box (shown in Fig. 2m). All images are maximum projections. Scale bars (main image and inset), 5 μm. h, i, Gut granule mean diameter (h) and count (i) in individual newly hatched L1 larvae from day 1, 2 or 3 mothers (n = 3, 3, 3 larvae). j, Intensity of blue autofluorescence from gut granules in segments along the anterior–posterior axis of digitally straightened newly hatched L1 larvae from day 1, 2 or 3 mothers (n = 32, 29, 16 larvae). Representative straightened brightfield and fluorescence images are positioned correspondingly along the x axis. Scale bar, 20 μm. k, Worm developmental stage determined by scoring vulval morphology of L4 larvae from day 1, day 2 or day 3 mothers after 42 h of feeding (n = 70, 61, 49 larvae). l, Length of larvae 40 h after recovery from a 7-day L1 starvation from day 1, day 2 or day 3 mothers (n = 26, 33, 26 larvae). m, Brightfield and epifluorescence images showing sterile progeny from day 1 mothers recovered for 96 h from a 14-day L1 starvation, or a worm that was continuously fed, with nuclei stained by DAPI. Gonads of worms recovered from starvation often exhibit severe abnormalities, such as failure to migrate (*), hyper-proliferation (+) and endomitotic oocytes51 (arrowhead). DAPI images are max intensity projection of Z-stacks following rolling ball background subtraction. Scale bars, 100 μm. n, Survival curves of progeny of day 1 or day 2 mothers upon recovery from L1 starvation for 12 h (day 1, n = 39, 66 worms, day 2, n = 52, 56, 55 worms), 1 week (day 1, n = 35, 40, 45 worms, day 2, n = 39, 47, 52 worms) or 2 weeks (day 1, n = 56, 65 worms; day 2, n = 51, 61, 68 worms). Each line represents a biological replicate. Boxplots represent median values, interquartile ranges and Tukey whiskers with individual data points superimposed. Error bars show s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Statistical tests used were Kruskal–Wallis test with Dunn’s multiple comparisons tests (k, l), generalized linear mixed model analysis (h, i), two-way ANOVA with Bonferroni multiple comparisons post-tests (j), log-rank test (n). Results were replicated at least 2 times independently. NS, not significant; YA, young adult.

Source data

Extended Data Figure 3 Maternal age affects progeny phenotypes in C. elegans wild isolates.

a, b, Boxplot showing length at hatching of L1 larvae from day 1 (grey), day 2 (blue) or day 3 (green) mothers of CB4856 (a, n = 51, 87, 53 larvae) and PB306 (b, n = 134, 237, 120 larvae). c, d, Boxplot showing length of larvae after 42 h of feeding from day 1, day 2 or day 3 mothers of CB4856 (c, n = 82, 66, 106 larvae) and PB306 (d, n = 168, 150, 93 larvae). e, f, Bar chart indicating worm developmental stage determined by scoring vulval morphology of L4 larvae after 42 h of feeding from day 1, day 2 or day 3 mothers of CB4856 (e, n = 81, 49, 101 larvae) and PB306 (f, n = 75, 85, 72 larvae). g, h, Boxplot showing length of larvae 48 h after recovery from a 7-day L1 starvation from day 1, day 2 or day 3 mothers of CB4856 (g, n = 174, 132, 185 larvae) and PB306 (h, n = 71, 62, 26 larvae). i, j, Confocal microscopy images of live L1 larvae from day 1, 2 or 3 mothers of CB4856 (i) and PB306 (j) showing blue autofluorescence from intestinal lysosome-related organelles (‘gut granules’). Insets show expanded section of main image indicated by white box. All images are maximum projections. Scale bars (main image and inset), 5 μm. Boxplots represent median values, interquartile ranges and Tukey whiskers with individual data points superimposed. *P < 0.05, **P < 0.01, ***P < 0.001. Statistical tests used were the Kruskal–Wallis test with Dunn’s multiple comparisons tests (ah). Results were replicated at least twice independently. NS, not significant; YA, young adult.

Source data

Extended Data Figure 4 Vitellogenin expression and loading into embryos increase with age.

a, Representative images of early embryos from day 1, day 2 and day 3 BCN9070 (vit-2::gfp) adults and wild-type N2 embryo for autofluorescence. Brightfield and epifluorescence images are shown. Scale bars, 20 μm. b, Average fluorescence intensity per embryo from immunostaining with an antibody against YP170, the protein encoded by vit-1 to vit-5, in 2–75-cell wild-type N2 embryos from day 1 or day 3 mothers. Duplicate stainings performed for each age group are shown (n = 41, 76; 58, 48 embryos). Fluorescence values are normalized to the mean of day 1 replicate A. c, Representative images of embryos extruded from day 1 or day 3 gravid hermaphrodites and stained with anti-YP170 antibody (green) and DAPI (blue). Scale bars, 20 μm. d, Total green autofluorescence of early embryos from wild-type N2 day 2 mothers treated with empty vector, RNAi against the constitutive intestinal gene ges-1 (ref. 52) as an additional control, combined vit-5 and vit-6 RNAi, and rme-2 RNAi (n = 58, 40, 22, 42 embryos). Fluorescence intensities are normalized to the mean of the empty vector control group. Representative images are shown above the corresponding group. e, Total green autofluorescence of early embryos from wild-type N2 day 1, day 2 or day 3 mothers (n = 92, 125, 70 embryos). Data shown are pooled from two trials analysed together. Fluorescence intensities are normalized to the mean of the day 1 group within each trial. Representative images from a single trial are shown above the corresponding group. f, mRNA transcripts quantified by qPCR of the yolk receptor rme-2 in day 1, day 2 and day 3 adults. Transcript levels were normalized to the geometric mean of two housekeeping genes (cdc-42 and rpl-27). Data shown are from three biological replicates. g, Representative images of day 1, day 2 and day 3 BCN9070 adults carrying an in-frame gfp knock-in at the C terminus of vit-2 and wild-type N2 worm for autofluorescence. Brightfield and epifluorescence images are shown. Worms have been digitally straightened. Scale bars, 100 μm. h, Representative lysates of day 1, 2 or 3 adult hermaphrodites run on an SDS–PAGE denaturing protein gel and stained with SYPRO Ruby fluorescent protein stain. Additionally, to confirm the identity of peaks corresponding to yolk proteins, samples are shown from hermaphrodites treated with empty vector control RNAi, rme-2 (yolk receptor) RNAi to induce yolk accumulation, and vit-5 and vit-6 RNAi to deplete yolk. All samples shown were run on the same gel. Myosin, YP170 (encoded by vit-1 to vit-5) and YP115 and YP88 (both encoded by vit-6) are indicated by arrows. For gel source data, see Supplementary Fig. 1. i, Estimate of the intestinal volume of adult hermaphrodites (n = 7, 9, 9 worms). Bar shows mean ± s.e.m. Boxplots represent median values, interquartile ranges and Tukey whiskers with individual data points superimposed. Error bars in barcharts and dot plots show s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, one-way ANOVA with Tukey’s multiple comparison tests (f, i). Statistical tests used were Kruskal–Wallis test with Dunn’s multiple comparisons test (d), generalized linear model analysis (b, e). NS, not significant; Ab, antibody.

Source data

Extended Data Figure 5 Maternal vitellogenin expression and embryonic lipid content increase with maternal age in C. elegans wild isolates.

a, b, Bar chart of total fluorescence of fixed embryos from day 1, 2 or 3 mothers of CB4856 (a, 719, 1,315, 249 embryos) and PB306 (b, n = 1,090, 839, 330 embryos) stained with BODIPY 493/503, a neutral lipid stain. Data shown are pooled from 4–5 biological replicates. c, d, Bar chart of mRNA transcripts quantified by qPCR of vit-2 and vit-6 in day 1, day 2 and day 3 adults of CB4856 (c) and PB306 (d). Transcript levels were normalized to the geometric mean of three housekeeping genes (cdc-42, rpl-27 and Y45F10D.4). Data shown are from three biological replicates. Error bars in bar charts show s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001. Statistical tests used are generalized linear mixed model analysis (a, b), one-way ANOVA with Tukey’s multiple comparison tests (panel c, d). NS, not significant.

Source data

Extended Data Figure 6 Progeny depleted of embryonic yolk by maternal vitellogenin knockdown recapitulate phenotypes observed in progeny of young mothers.

a, Schematic showing experimental method used to generate yolk-depleted progeny by maternal vit-5 and vit-6 RNAi treatment. b, mRNA transcripts quantified by qPCR of the 6 vit genes in day 2 adults treated with or empty vector control (blue), vit-5 RNAi (magenta), vit-6 RNAi (yellow) or a mixture of vit-5 and vit-6 RNAi (turquoise). Transcript levels were normalized to the geometric mean of three housekeeping genes (cdc-42, rpl-27 and Y45F10D.4). Data shown are from three biological replicates. c, The constitutive intestinal gene ges-1 (ref. 52) was used as an additional control. mRNA transcripts quantified by qPCR for ges-1, vit-2 and vit-6 in day 2 adults treated with empty vector control (blue) or ges-1 RNAi (brown). Transcript levels were normalized to the geometric mean of three housekeeping genes (cdc-42, rpl-27 and Y45F10D.4). Data shown are from three biological replicates. d, Length at hatching of L1 larvae from day 2 mothers treated with empty vector control (blue), ges-1 RNAi (brown) or vit-5 and vit-6 RNAi (turquoise) (n = 103, 130, 159 larvae). e, Confocal microscopy images of live L1 larvae from day 2 mothers treated with vit-5 and vit-6 RNAi, ges-1 RNAi or empty vector control showing blue autofluorescence from intestinal lysosome-related organelles (‘gut granules’). Insets show expanded section of main image indicated by white box. All images are maximum projections. Scale bars (main image and inset), 5 μm. f, Worm developmental stage determined by scoring vulval morphology of L4 larvae after 46 h of feeding from day 2 mothers treated with empty vector control (blue), ges-1 RNAi (brown) or vit-5 and vit-6 RNAi (turquoise) (n = 48, 36, 47 larvae). g, Time after hatching by which half the progeny of mothers treated with empty vector control, ges-1 RNAi or vit-5 and vit-6 RNAi had undergone the L4-to-adult moult (blue) or carried a fertilized embryo in utero (red). Each time point for each transition is calculated by sampling the population twice. (Sample sizes: empty vector, n = 199 and 198 for the L4-to-adult moult, n = 171 and 168 for the first embryo; ges-1, n = 204 and 206 for the L4-to-adult moult, n = 198 and 168 for the first embryo; vit-5 and vit-6, n = 201 and 202 for the L4-to-adult moult, n = 179 and 200 for the first embryo). h, Time between the L4-to-adult moult and the first embryo for the data shown in g. i, Length of larvae from day 2 mothers treated with empty vector control (blue), ges-1 RNAi (brown) or vit-5 and vit-6 RNAi (turquoise) after 46 h of feeding (n = 67, 56, 57 larvae). j, Length of larvae at early L4 vulval substages in progeny of day 2 mothers treated with empty vector control (blue), ges-1 RNAi (brown) or vit-5 and vit-6 RNAi (turquoise) (L4.1, n = 15, 15, 9 larvae; L4.2, n = 7, 8, 5 larvae, L4.3, n = 18, 22, 13 larvae). k, Total brood size of progeny of mothers treated with empty vector control (blue), ges-1 RNAi (brown) or vit-5 and vit-6 RNAi (turquoise) (n = 13, 14, 11 worms). l, Length 44 h after recovery from a 7-day L1 starvation of larvae from day 2 mothers treated with empty vector control (blue), ges-1 RNAi (brown) or vit-5 and vit-6 RNAi (turquoise) (n = 30, 33, 85 larvae). m, Percentage of adult progeny of day 2 mothers treated with empty vector control (blue), ges-1 RNAi (brown) or vit-5 and vit-6 RNAi (turquoise) displaying normal phenotypic outcomes 96 h after recovery from a 10-day L1 starvation (n = 610, 567, 1,135 worms). n, Percentage of adult progeny of day 2 mothers treated with empty vector control (blue), ges-1 RNAi (brown) or vit-5 and vit-6 RNAi (turquoise) that successfully produced offspring 168 h after recovery from a 11-day L1 starvation (n = 103, 116, 124 worms). Boxplots represent median values, interquartile ranges and Tukey whiskers with individual data points superimposed. Error bars in b and c represent s.e.m.; error bars in g, h, j, m and n represent 95% confidence intervals. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Statistical tests used were two-tailed t-tests (c), Kruskal–Wallis test with Dunn’s multiple comparisons tests (d, f, i, l), ratio tests (g, h), one-way ANOVA with Tukey’s multiple comparisons tests (k), two-way ANOVA with Bonferroni multiple comparisons tests (j), pairwise Fisher’s exact test with Bonferroni multiple corrections (m, n). Data shown are not all from the same trial but represent typical outcomes. All results were replicated at least twice. NS, not significant.

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Extended Data Figure 7 Increased yolk provisioning accounts for the differences in development and growth between day 1 and day 2 progeny.

a, Worm developmental stage determined by scoring vulval morphology of L4 larvae after 42 h of feeding from day 1 mothers (grey), day 2 mothers treated with empty vector control (blue) and day 2 mothers treated with 10% rme-2 RNAi (orange) (n = 69, 66, 52 embryo). b, Length 48 h after recovery from a 7-day L1 starvation of larvae from day 1 mothers (grey), day 2 mothers treated with empty vector control (blue) and day 2 mothers treated with 10% rme-2 RNAi (orange) (n = 295, 206, 293 larvae). Boxplots represent median values, interquartile ranges and Tukey whiskers with individual data points superimposed. **P < 0.01, ***P < 0.001, ****P < 0.0001. A Kruskal–Wallis test with Dunn’s multiple comparisons tests was used. Data shown (here and in Fig. 4) are from a single trial. Results were replicated twice independently.

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Extended Data Table 1 Sequences of qPCR primers used in this study

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Life Sciences Reporting Summary (PDF 72 kb)

Supplementary Figure 1

This file contains he uncropped source data for the gel shown in Extended Data Figure 4h. (PDF 506 kb)

Supplementary Tables 1 and 2

This file contains table 1 (supplementary to Fig. 1). Table including projection of single worm expression profiles onto the germline and somatic expression spaces and their germline and soma ranking. It also contains table 2 (supplementary to Extended Data Fig. 1). Table including residuals of single worm gene expression after correcting for germline and soma developmental stage. The table also indicates for each gene whether germline or somatic ranking better explains the observed variation and includes gene annotation for the tissue-specific gene sets. Both tables are combined as separate sheets in a single .xlsx file. (XLSX 16107 kb)

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Perez, M., Francesconi, M., Hidalgo-Carcedo, C. et al. Maternal age generates phenotypic variation in Caenorhabditis elegans. Nature 552, 106–109 (2017). https://doi.org/10.1038/nature25012

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