Epigenetic clocks reveal a rejuvenation event during embryogenesis followed by aging

Abstract

The notion that the germ line does not age goes back to the 19th-century ideas of August Weismann. However, being metabolically active, the germ line accumulates damage and other changes over time, i.e., it ages. For new life to begin in the same young state, the germ line must be rejuvenated in the offspring. Here, we developed a multi-tissue epigenetic clock and applied it, together with other aging clocks, to track changes in biological age during mouse and human prenatal development. This analysis revealed a significant decrease in biological age, i.e., rejuvenation, during early stages of embryogenesis, followed by an increase in later stages. We further found that pluripotent stem cells do not age even after extensive passaging and that the examined epigenetic age dynamics is conserved across species. Overall, this study uncovers a natural rejuvenation event during embryogenesis and suggests that the minimal biological age (ground zero) marks the beginning of organismal aging.

Fig. 1. A rejuvenation event during early embryogenesis revealed by aging clocks.

(A) Overview of the model, which posits that germline cells age during development and adulthood and are rejuvenated in the offspring after conception. The model also suggests that there is a time point corresponding to the lowest biological age (ground zero). (B) Multi-tissue and blood rDNA clocks applied to five datasets spanning the first 8 days of mouse embryogenesis (Table 1, datasets 1 to 5). We rescaled epigenetic age of each dataset to the interval [0,1] for comparison (“relative rDNA age”). 0 represents the lowest epigenetic age, and 1 represents the highest epigenetic age of each dataset. Blue lines indicate the mean of each group; P values of two-sided t test comparing the means of the two groups (before and after E6) are displayed. (C) Application of four genome-wide epigenetic aging clocks to two available mouse RRBS datasets. (D) Epigenetic age of human ESCs and iPSCs as a function of passage number. The Horvath human multi-tissue clock was applied.

Fig. 2. Organismal aging begins in mid-embryonic development in mice and humans.

(A) Epigenetic age (multi-tissue and blood rDNA clocks) analysis of the dataset that contains both early and late mouse embryo samples (E13.5 samples are based on primordial germline cells). (B) Application of genome-wide epigenetic clocks to later stages of mouse embryogenesis (r, Pearson correlation coefficient; P, P value of the correlation). (C) The same data as above but separated by tissue. An increasing trend is observed for almost all tissues, with few nonsignificant exceptions. (D) Epigenetic age dynamics of four independent prenatal human 450K methylation array datasets based on the Horvath human multi-tissue clock. (E) The same data as above but separated by tissue (five significant increases, nine nonsignificant increases, four nonsignificant decreases, and zero significant decreases).

Fig. 3. Epigenetic age of mouse ESCs during early passaging under different culture conditions.

(A) Epigenetic age (by rDNA clocks) of mouse ESCs after outgrowth (passage 0) and passage 5 under three different culture conditions (2i, both self-renewal supporting inhibitors used; PD, only one inhibitor; mES, no inhibitor). (B) Application of genome-wide mouse epigenetic clocks to the same data. (C) Principal components analysis (PCA) of RRBS methylation profiles of embryo (circles) and ESC (crosses) sample groups after passage 0 (left) and passage 5 (right). Convex hull around the ESC samples in the same group (if n > 2) is displayed to help distinguish ESC samples from embryo samples.

Fig. 4. Localization of the epigenetic age minimum (ground zero) during mouse embryonic development.

(A) We concatenated results for the entire period of embryogenesis by using the genome-wide mouse epigenetic clocks indicated. (B) We concatenated results for the entire period of embryogenesis by using the indicated mouse rDNA epigenetic clocks. (C) Application of genome-wide mouse epigenetic clocks to dataset 20 that contains mid-embryonic stages from E3.5 to E11.5. (D) Application of rDNA mouse epigenetic clocks to dataset 20.

Fig. 5. Role of TET enzymes in the rejuvenation event.

(A) Application of rDNAm clocks to TET triple KO (TET KO) and wild-type (WT) E6.5 epiblast samples (dataset 17). (B) Application of our recently developed single-cell clock [scAge; (49)] to TET KO and WT ESCs at days 2 and 5 of differentiation (dataset 18). Specific lineages and stages (Mapped lineage and Mapped E-day) were assigned by mapping the RNA expression profiles of the in vitro cells to an in vivo gastrulation atlas. Two-sided t tests were calculated (ns, P > 0.05; *, 1 × 10⁻² < P ≤ 5 × 10⁻²; **, 1 × 10⁻³ < P ≤ 1 × 10⁻²; ***, 1 × 10⁻⁴ < P ≤ 1 × 10⁻³; ****P ≤ 1 × 10⁻⁴).

Fig. 6. Role of DNMTs in the rejuvenation event.

(A) We applied rDNA clocks to single KO of DNMT1 (Dnmt1^−/−), double KO of DNMT3A and DNMT3B (DKO), wild type (WT), and heterozygous control (3a^−/+ and 3a^−/+ 3b^−/+) embryos at E8.5 (dataset 19). (B) Application of four genome-wide epigenetic aging clocks to the same dataset. Two-sided t tests were calculated (ns, P > 0.05; *, 1 × 10⁻² < P ≤ 5 × 10⁻²; **, 1 × 10⁻³ < P ≤ 1 × 10⁻²; ***, 1 × 10⁻⁴ < P ≤ 1 × 10⁻³; ****P ≤ 1 × 10⁻⁴).