Loss of maternal EED results in postnatal overgrowth

Abstract

Background: Investigating how epigenetic information is transmitted through the mammalian germline is the key to understanding how this information impacts on health and disease susceptibility in offspring. EED is essential for regulating the repressive histone modification, histone 3 lysine 27 tri-methylation (H3K27me3) at many developmental genes.

Results: In this study, we used oocyte-specific Zp3-Cre recombinase (Zp3Cre) to delete Eed specifically in mouse growing oocytes, permitting the study of EED function in oocytes and the impact of depleting EED in oocytes on outcomes in offspring. As EED deletion occurred only in growing oocytes and females were mated to normal wild type males, this model allowed the study of oocyte programming without confounding factors such as altered in utero environment. Loss of EED from growing oocytes resulted in a significant overgrowth phenotype that persisted into adult life. Significantly, this involved increased adiposity (total fat) and bone mineral density in offspring. Similar overgrowth occurs in humans with Cohen-Gibson (OMIM 617561) and Weaver (OMIM 277590) syndromes, that result from de novo germline mutations in EED or its co-factor EZH2, respectively. Consistent with a role for EZH2 in human oocytes, we demonstrate that de novo germline mutations in EZH2 occurred in the maternal germline in some cases of Weaver syndrome. However, deletion of Ezh2 in mouse oocytes resulted in a distinct phenotype compared to that resulting from oocyte-specific deletion of Eed.

Conclusions: This study provides novel evidence that altering EED-dependent oocyte programming leads to compromised offspring growth and development in the next generation.

Keywords: EED; EZH2; Epigenetic inheritance; Germ; H3K27me3; Histone; Oocyte; Overgrowth; Polycomb; Weaver.

Fig. 1

Deletion of Eed significantly reduced H3K27me3 in growing oocytes: a Schematic of the study aims—germ cells commit to female development after E12.5 and new epigenetic information is established in growing oocytes after birth. H3K27me3 is enriched as oocytes grow, with strong enrichment in the maturing oocyte. Using an Eedfl-Zp3Cre mouse model, we investigated the impacts of deleting Eed in the growing oocyte on offspring weight and growth. As EED deletion occurs only in growing oocytes, this model allows the study of EED-dependent maternal programming without contributions from confounding factors such as in utero environment. b Representative confocal images of immunofluorescence in ovary sections from adult Eed^fl/fl and Eed^fl/fl;Zp3-Cre female mice producing Eed^fl/fl (wild type; wt) and Eed^del/del (homozygous; hom) oocytes, respectively. Merged channels: H3K27me3 (red) and DAPI (DNA; blue). Eed^fl/fl (wt) and Eed^del/del(hom) oocytes are shown within the white dashed line. Images are representative of four biological replicates. 10-μm scale bars. c Average litter sizes from mothers producing Eed^fl/fl (wt), Eed^wt/del (heterozygous; het) and Eed^del/del (hom) growing oocytes: n = 15, 20 and 13 litters per genotype, respectively, and 7 different mothers per genotype group. ****P < 0.0001. One-way ANOVA plus post hoc Tukey’s multiple comparisons test. Error bars ± SEM

Fig. 2

Schematic of experimental breeding to determine epigenetic differences in isogenic offspring from Eed floxed females: Wild type males were mated with Eed^fl/fl, Eed^fl/del;Zp3-Cre and Eed^fl/del;Zp3Cre female mice that produce Eed^del or Eed^wt haploid oocytes derived from Eed^fl/fl (wild type; wt), Eed^wt/del(heterozygous; het) or Eed^del/del (homozygous; hom) growing oocytes. Het oocytes grow and mature in the presence of functional EED, while hom oocytes grow and mature in the absence of EED function. Production of offspring by mating Eed^fl/del;Zp3-Cre and Eed^fl/del;Zp3Cre female mice with isogenic wild type males allowed the comparison of isogenic HET offspring from het and hom oocytes. As the resulting HET offspring were isogenic and carry identical heterozygous Eed deletion, differences detected could be ascribed to loss of epigenetic regulation by EED in the oocyte and before cavitation of paternal Eed in the preimplantation embryo. Similar comparisons were made between WT offspring produced from Eed^fl/fl and Eed^fl/del;Zp3-Cre females. Comparison of WT and HET offspring produced by Eed^fl/fl and Eed^fl/del;Zp3-Cre females provides an internal control identifying the contribution of Eed heterozygosity to the phenotype. Genetically identical offspring are shown in purple dashed box

Fig. 3

Offspring from Eed^del/del oocytes have increased weight and length that is independent of litter size. a Postnatal day (PND) 2 weights of WT and HET offspring produced from Eed^fl/fl (wt), Eed^wt/del (het) and Eed^del/del(hom) oocytes and wild type sperm (WT offspring from wt oocytes n = 118; WT offspring from het oocytes n = 37; HET offspring from het oocytes n = 52; HET offspring from hom oocytes n = 51). b Representative images showing two isogenic PND2 male pups. Left: HET offspring from a het growing oocyte; Right: HET offspring from a hom growing oocyte. c Crown to rump measurements of PND2 male and female pups. d Nose to rump measurements of PND2 male and female pups. c–d WT offspring from wt oocytes n = 29; WT offspring from het oocytes n = 10; HET offspring from het oocytes n = 10; HET offspring from hom oocytes n = 23) ****P < 0.0001. One-way ANOVA plus post hoc Tukey’s multiple comparisons test. Data represents mean ± SEM. e Relationship between PND2 weight and litter size: Litter size vs offspring weight (t_33.5 = − 1.32, P = 0.20; variance components: litter ID = 0.0398, residual = 0.0345). Accounting for litter size vs offspring weight: HET pups from Eed^del/del oocytes were heavier than HET pups produced from Eed^wt/del oocytes (t_34.8 = 3.44, P = 0.002), WT pups from either Eed^wt/del (t_36.3 = 2.81, P = 0.008) and Eed ^wt/wt (t_33.1 = 2.29, P = 0.03) oocytes

Fig. 4

Offspring from Eed^del/del oocytes have increased bone mineral density and fat content, but reduced lean muscle. a–d Postnatal day (PND) 2: bone mineral density (a), lean muscle content (b), fat content (c), ponderal index (d) in WT and HET offspring produced from Eed^fl/fl (wt), Eed^wt/del (het) and Eed^del/del(hom) oocytes and wild type sperm (a–d: WT offspring from wt oocytes n = 28; WT offspring from het oocytes n = 6; HET offspring from het oocytes n = 8; HET offspring from hom oocytes n = 19). *P < 0.05, **P < 0.01, one-way ANOVA plus post hoc Tukey’s multiple comparisons test. Error bars ± SEM

Fig. 5

Offspring from Eed^del/del oocytes have increased weight into adulthood. a–b Postnatal day (PND) 49 weights of female (a) and male (b) WT and HET offspring produced from Eed^fl/fl (wt), Eed^wt/del (het) and Eed^del/del(hom) oocytes and wild type sperm (WT offspring from wt oocytes a: n = 43, b 32; WT offspring from het oocytes a n = 9, b n = 10; HET offspring from het oocytes a n = 12, b n = 8; HET offspring from hom oocytes a n = 13, b n = 14). c Average growth trajectories calculated from average weights of female WT and HET offspring at PND2, 30 and 49 (WT offspring from wt oocytes PND2: n = 14, PND30: n = 14, PND49: n = 14; HET offspring from hom oocytes PND2: n = 10, PND30: n = 10, PND49: n = 10). d Average growth trajectories calculated from average weights of male WT and HET offspring at PND2, 30 and 49 (WT offspring from wt oocytes PND2: n = 12, PND30: n = 12, PND49: n = 12; HET offspring from hom oocytes PND2: n = 6, PND30: n = 6, PND49: n = 6). *P < 0.05, ****P < 0.0001; nsd represents no significant difference. One-way ANOVA plus post hoc Tukey’s multiple comparisons test. Error bars ± SEM

Fig. 6

De novo missense mutations in EZH2 are maternally or paternally inherited through the germline in Weaver patients: a PacBio sequencing was carried out to identify single nucleotide polymorphisms (SNPs) in DNA from each patient and their respective parental haplotypes. Informative SNPs (i.e., those that were specific to either parent) allowed linkage of the patient’s EZH2 mutation in the patient to either the maternal or paternal allele (example of experimental pipeline shown). b The EZH2 mutation detected in each patient is shown in the middle column and parent-of-origin shown on the right, based on genetic linkage to either the mother or the father

Fig. 7

Deletion of Ezh2 significantly reduced H3K27me3 in growing oocytes and weight in offspring: a representative confocal images of immunofluorescence in ovary sections from adult Ezh2^fl/fl and Ezh2^fl/fl;Zp3-Cre female mice. Left panel—merged H3K27me3 (red) and DAPI (DNA; blue); right panel H3K27me3 shown in greyscale. Oocyte nuclei are shown within the white dashed line. Images are representative of three biological replicates; 10 μm scale bars. b Postnatal day (PND) 2 weights of WT and HET offspring produced from Ezh2^fl/fl (wt), Ezh2^wt/del (het) and Ezh2^del/del(hom) oocytes and wild type sperm (WT offspring from wt oocytes n = 19; WT offspring from het oocytes n = 18; HET offspring from het oocytes n = 12; HET offspring from hom oocytes n = 19). *P < 0.05, **P < 0.01, one-way ANOVA plus post hoc Tukey’s multiple comparisons test. Error bars ± SEM