Fetal growth delay caused by loss of non-canonical imprinting is resolved late in pregnancy and culminates in offspring overgrowth

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   Version published to 10.7554/elife.81875 on eLife

   May 30, 2024
2. 

   eLife

   Jan 29, 2024

   Author response

   eLife assessment

   Using a genetically controlled experimental setting, the authors find that the lack of Polycomb-dependent epigenetic programming in the oocyte and early embryo influences the developmental trajectory through gestation in the mouse. By showing a two-phase outcome of early growth restriction followed by enhancement, the authors address previous inconsistencies in the field. However, the link with placenta function and gene misregulation is not yet fully supported.

   We thank the Reviewers for their constructive comments. In response we have added significantly more data to the study and substantially rewritten the manuscript. New data include analyses of glucose, amino acid and metabolite levels in fetal and maternal blood samples, more highly resolved fetal growth analyses, a more detailed study of the …

   Author response

   eLife assessment

   Using a genetically controlled experimental setting, the authors find that the lack of Polycomb-dependent epigenetic programming in the oocyte and early embryo influences the developmental trajectory through gestation in the mouse. By showing a two-phase outcome of early growth restriction followed by enhancement, the authors address previous inconsistencies in the field. However, the link with placenta function and gene misregulation is not yet fully supported.

   We thank the Reviewers for their constructive comments. In response we have added significantly more data to the study and substantially rewritten the manuscript. New data include analyses of glucose, amino acid and metabolite levels in fetal and maternal blood samples, more highly resolved fetal growth analyses, a more detailed study of the hyperplastic placenta including IF analyses of labyrinth area, labyrinth to placenta and capillary to labyrinth ratios. We have also added analyses of placental DNA methylation state in offspring from oocytes lacking EED, which reveals a range of DNA methylation changes at imprinted and non-imprinted genes in HET-hom offspring compared to HET-het or WT-wt controls.

   Reviewer #1 (Public Review):

   Oberin, Petautschnig et. al investigated the developmental phenotypes that resulted from oocyte-specific loss of the EED (Embryonic Ectoderm Development) gene - a core component of the Polycomb repressive complex 2 (PRC2), which possess histone methyltransferase activity and catalyses trimethylation of histone H3 at lysine 27 (H3K27). The PRC2 complex plays essential roles in regulating chromatin structure, being an important regulator of cellular differentiation and development during embryogenesis. As novel findings, the authors find that PRC2-dependent programming in the oocyte, via loss of the core component EE2, causes placental hyperplasia and propose that the increase of placental transplacental flux of nutrients leads to fetal and postnatal overgrowth. At the mechanistic level, they show altered expression of genes previously implicated in placental hyperplasia phenotypes. They also establish interesting parallelism with the placental hyperplasia phenotype that is frequently observed in cloned mice.

   Strengths:

   The mouse breeding experiments are very well designed and are powerful to exclude potential confounding genetic effects on the developmental phenotypes that resulted from the loss of EED in oocytes. Another major strength is the developmental profiling across gestation, from pre-implantation to late gestation.

   Weaknesses:

   The evidence for 'oocyte' programming is restricted to phenotypic and gene expression analysis, without measurements of epigenetic dysregulation. It would be an added value if the authors could show evidence for altered H3K27me3 or DNA methylation in the placenta, for example.

   In an earlier previous study we identified a large number of developmentally important genes that accumulated H3K27me3 in primary-secondary stage growing oocytes and were repressed by EED (Jarred et al., 2022 Clinical Epigenetics). However, H3K27me3 was removed from all from these genes during preimplantation development, indicating that maternal inheritance of H3K27me3 at a wide range of genes is unlikely (Jarred et al., 2022 Clinical Epigenetics). Consistent with this only a small number of genes, including Slc38a4 and C2MC, have been shown to be functionally important in H3K27me3-dependent imprinting (Matoba et al., 2022 Genes and Development). Moreover, a related study showed that deletion of Setd2 and consequent loss of H3K36me3 in oocytes led to spreading of H3K27me3 into regions that were otherwise marked by H3K36me3 and DNA methylation (Xu et al. 2019 Nature Genetics 51:844–56). Based on these studies, we proposed that loss of EED and H3K27me3 may result in the ectopic spreading of H3K36me3 and DNA methylation in oocytes and that altered DNA methylation may then be transmitted to offspring and affect developmental outcomes (Jarred et al., 2022 Clinical Epigenetics)

   Given this hypothesis we analysed DNA methylation rather than H3K27me3 in the placenta of WT-wt, HET- het and HET-hom offspring. This revealed differentially methylated regions (DMRs) in HET-hom placentas at two H3K27me3 imprinted genes Sfmbt2 (C2MC) and Mbnl2, five classically imprinted genes and at 74 DMRs not associated with imprinted loci. Together, our data supports the hypothesis from Jarred et al., 2022 Clinical Epigenetics that loss of EED in oocytes results in altered DNA methylation patterning at both imprinted and non-imprinted genes in offspring and that this is likely to affect offspring growth and development. However, whether these changes result from direct alteration of DNA methylation in oocytes remains unclear.

   These new data are now included in results (Lines 387-409), Figure 6I, Supplementary File H-J and Discussion Lines 569-581.

   Reviewer Comment 1. The claim that placental hyperplasia drives offspring catch-up growth is not supported by current experimental data. The authors do not address if transplacental flux is increased in the hyperplastic placentae, measure amino acids and glucose in fetal/maternal plasma, or perform tetraploid rescue experiments to ascertain the contribution of the placenta to growth phenotypes. Furthermore, it is unclear, from the current data, if the surface area for nutrient transport is actually increased in the hyperplastic placenta and the extent to which other cell populations (i.e. spongiotrophoblasts) are affected in addition to glycogen cells. In addition, one of the supporting conclusions that the placenta is a key contributor to fetal overgrowth is based on a very crude measurement - placenta efficiency - which the authors claim is increased in the homozygous mutants compared to controls. After analysing the data carefully, I find evidence for decreased placental efficiency instead. I believe that the authors mistakenly present the data as placenta to fetal weight ratios, which led to the misinterpretation of the 'efficiency' concept.

   We thank the reviewer for pointing out our error in the placental efficiency data and we have now corrected the placental efficiency graphs (fetal/placental weight ratios) and updated the text throughout the manuscript as required (Figure 3I-K). As requested and described below, we have also added significantly more data, which support the conclusion that placental function is not enhanced in HET-hom mice and is unlikely to support fetal growth recovery.

   The new data and analyses we have added include:

   1. Further analyses of glycogen-enriched and non-glycogen-enriched cell counts in the decidua and junctional zones (Figure 4F-J)

   2. Total glycogen cell counts for male and female placentas (Figure 4 – figure supplement 1F)

   3. New analyses of fetal blood glucose levels at E17.5 and E18.5 and matching data from the mothers of each litter (Figure 4M)

   4. New analyses of the circulating amino acid levels and metabolites in fetal blood of E17.5 offspring and matching data from the mothers of each litter (Figure 8)

   5. New IF analyses of CD31 (PECAM-1) and combined this with machine learning assisted quantitative analyses of labyrinth and capillary areas using HALO (Figure 5)

   6. Separated male and female offspring and placental weights at E14.5 and E17.5 and total areas of the placenta, decidua, junctional zone and labyrinth (Figure 3 – figure supplement 1) which provide more insight into potential sex-specific differences in HET-hom offspring and placenta

   We have significantly re-written the results and discussion to reflect our new data and interpretation.

   While we did not assess transplacental flux, our new data revealed: 1. HET-hom fetuses had lower blood glucose levels at E18.5; 2. Circulating levels of amino acids and a wide range of metabolites did not differ between HET-hom and control offspring, or between the mothers of these offspring; 3. HET-hom placentas had lower total labyrinth area, labyrinth/placenta and capillary/labyrinth ratios based on analysis of total capillary and labyrinth areas, indicating that the surface area for nutrient transfer is not increased

   Together these data strongly indicate that hyperplastic HET-hom placentas do not provide greater support to HET-hom fetuses than controls, and that increased placental function in HET-hom offspring is unlikely to explain the late gestation fetal growth recovery we observed in HET-hom offspring or how HET-hom offspring were able to attain normal weights by birth.

   While we have not directly counted the spongiotrophoblast populations, we have now included analyses of both the glycogen-enriched and non-glycogen cell populations in the junctional zone and the decidua (Figure 4H-K). This revealed an increased area of both glycogen-enriched and non-glycogen cells in the junctional zone and in the decidua of HET-hom placentas, consistent with the greater junctional zone/placenta ratio observed in HET-hom placentas (Figure 4D). Together with data in Figure 4C-F and Supp. Fig. 3, our observations demonstrate that the overall decidua and junctional zone areas were increased in HET-hom offspring, but there was a disproportionate expansion of the junctional zone that was caused by increased areas of both glycogen and non-glycogen-enriched cells.

   Tetraploid rescue experiments would require a very significant amount of time and investment and are technically very demanding. While creation of complementary tetraploid offspring would be informative, unfortunately these experiments are beyond the scope of this current study.

   Reviewer Comment 1 cont. The authors do not mention alternative explanations for the observed fetal catch-up and postnatal overgrowth. Why would oocyte epigenetic programming effects be restricted to the placenta, and not include fetal organs?

   Our intention was certainly not to convey a message that effects may be placenta specific. Indeed, our ongoing work beyond the scope of this study provides evidence for effects in other tissues (brain and bones) that will be published elsewhere. Our new data clearly show low placental efficiency, fetal blood glucose, low capillary/labyrinth ratio and no impact on circulating fetal amino acid or metabolite levels in HET-hom offspring. In light of these new data, we have reinterpreted the findings of this study and substantially updated the discussion.

   Given our observations that fetal growth rate markedly increased during late gestation, but placental efficiency was reduced, our data strongly indicate that the effects of altered epigenetic oocyte programming due to loss of Eed affect both the placenta and the fetus. While our findings are significant, the precise mechanism underlying this growth response in HET-hom fetuses remains unknown. Understanding this mechanism will require substantially more work that will be the subject of future studies.

   Reviewer #2 (Public Review):

   Consistent fetal growth trajectories are vital for survival and later life health. The authors utilise an elegant and novel animal model to tease apart the role of Eed protein in the female germline from the role of somatic Eed. The authors were able to experimentally attribute placental overgrowth - particularly of the endocrine region of the placenta - to the function of Eed protein in the oocyte. Loss of Eed protein in the oocyte was also associated with dynamic changes in fetal growth and prolonged gestation. It was not determined whether the reported catch-up growth apparent on the day of birth was due to enhanced fetal growth very late in gestation, a longer gestational time ie the P0 pups are effectively one day "older" compared to the controls, or the pups catching up after birth when consuming maternal milk.

   To understand if increased growth occurred in HET-hom fetuses prior to birth, we have now included analyses of offspring weight at E18.5 (Figure 2F), all pups collected with a verified E19.5 birth date (Figure 2J) and for pups from similar litter sizes (5-7 pups) at E19.5 (Figure 2K). Together with our existing data, these additional analyses provide average weights for fetuses at E14.5, E17.5, E18.5 and pups born on E19.5. This confirmed that HET-hom offspring undergo enhanced growth in the last few days of pregnancy, resulting in the progression of substantially growth and developmentally restricted HET-hom fetuses at E14.5, to pups with normal weight at birth within the 40% of pregnancies that were born on E19.5 in a normal gestational time.

   However, in addition, gestational length was increased by one to two days in 60% of pregnancies from hom oocytes, but not in control pregnancies from het or wt oocytes. As average weights were significantly greater in all surviving HET-hom offspring at P0 (i.e. surviving pups born on E19.5-E21.5; Figure 2G), it appears that this additional gestational time contributed to the offspring overgrowth. This is logical, however it does not explain how growth and developmentally delayed fetuses at E14.5 attained normal weight and developmental stage by E19.5 (Figure 2J-K).

   Together our data clearly show that HET-hom offspring undergo enhanced growth during the late stages of pregnancy, allowing them to resolve the developmental delay and growth insufficiency observed at E14.5 so that they were born at normal weight and stage at E19.5. In addition, increased gestational time contributes to weight of pups delivered on E20.5 or 21.5, partly explaining the overgrowth phenotype observed in this model.

   The idea that increased milk consumption may explain the overgrowth of HET-hom offspring is interesting. It is possible that the increased growth rate of HET-hom offspring continues after birth and contributes to overgrowth. However, examining this outcome in a tightly controlled manner is complicated given that we cannot predict the day of birth of HET-hom litters, and that these litters are generally small and would need to be fostered on the day of birth alongside control litters. Given these challenges and that our primary observation is that HET-hom offspring underwent fetal growth recovery during pregnancies of normal length and via extension of gestational length, we have not examined the possibility of increased milk consumption after birth.

   We have updated the results to reflect the new analyses and have provided relevant discussion to address these data. Our description of these data can be found in Results (lines 165-197) and in Figure 2.

   Reviewer #3 (Public Review):

   My understanding of the main claims of the paper, and how they are justified by the data are discussed below:

   Overall, loss of PRC2 function in the developing oocyte and early embryo causes:

   1. Growth restriction from at least the blastocyst stage with low cell counts and midgestational developmental delay.

   Strengths:

   • Live embryo imaging added an important dimension to this study. The authors were able to confirm an unquantified finding from a previous lab (reduced time to 2-cell stage in oocyte-deletion Eed offspring, Inoue 2018, PMID: 30463900) as well as identify developmental delay and mortality at the blastocyst- hatching transition.

   • For the weight and morphological analysis the authors are careful to provide isogenic controls for most of the experiments presented. This means that any phenotypes can be attributed to the oocyte genotype rather than any confounding effects of maternal or paternal genotype.

   • Overall, there is good evidence that oocyte deletion of Eed results in early embryonic growth restriction, consistent with previous observations (Inoue 2018, PMID: 30463900).

   Reviewer 3, Comment 1: Weaknesses: Gaps in the reporting of specific features of the methodology make it difficult to interpret/understand some of the results.

   While we are unsure exactly which methods Reviewer 3 would like expanded, we have updated parts that we thought required further detail and allow more informed interpretation of the results. These include methods for placental histology (Lines 650-669) and immuno- histochemistry (Lines 671-690), and new methods for CD31 immunofluorescence (Lines 692-714), glucose and metabolomics (Lines 752-769) and DNA methylation (RRBS; Lines 734-750) analyses.

   To clarify the approach taken for histology, immunohistochemical and immunofluorescent staining, sections were cut in compound series from the centre of each placenta, ensuring that we collected representative data for each sample. QuPath was used to quantify the decidual and junctional zone areas in one complete, fully intact midline section for each placenta as close to the midline as possible. This provided data from 10 placentas for each genotype. In addition, glycogen-enriched and non-glycogen-enriched cells were identified and quantified using machine learning assisted QuPath analyses of the whole placenta, decidua and junctional zone regions. We have also added quantitative analyses of the labyrinth and labyrinth capillary network using immunofluorescent CD31 staining and machine learning assisted HALO software. This new analysis of placental morphology is included in the methods section.

   Moreover, as there were no sex-specific differences in placental morphology or weight, we combined the samples from both sexes to provide greater numbers for analysis in each genotype. For example, as described for the analyses of labyrinth and capillaries using CD31 IF, 4 placentas of each sex were used for data collection. This provided data from a total of 8 placentas (4 male and 4 female) for each genotype from a total of 17 WT-wt (9 male and 8 female), 21 HET-het (9 male and 12 female) and 24 HET-hom (16 male and 8 female) sections (2-3 sections/placenta).

   Reviewer 3, Comment 2: Placental hyperplasia with disproportionate overgrowth of the junctional trophoblast especially the glycogen trophoblast (GlyT) cells.

   Strengths: • The authors provide a comprehensive description of how placental and embryo weight is affected by the oocyte-Eed deletion through mid-to-late gestation development. The case for placentomegaly is clear.

   Weaknesses:

   • The placental efficiency data presented in Figure 3G-I is incorrect. Placental efficiency is calculated as embryo mass/placental mass, and it increases over the late gestation period. For e14.5 for example (Fig3G), WT-wt embryo mass = ~0.3g, placenta mass = 0.11g (from Fig 3D) = placental efficiency 2.7; HET-hom = 0.25/0.12 = 2.1. The paper gives values: WT-wt 0.5, HET-hom 0.7. Have the authors perhaps divided placenta weight by embryo mass? This would explain why the E17.5 efficiencies are so low (WT-wt 0.11 rather than a more usual figure of 8.88. If this is the case then the authors' conclusion that placental efficiency is improved by oocyte deletion of Eed is wrong - in fact, placental efficiency is severely compromised.

   The authors have performed cell type counting on histological sections obtained from placentas to discover which cells are contributing to the placentomegaly. This data is presented as %cell type area in the main figure, though the untransformed cross-sectional area for each cell type is shown in the supplementary data. This presentation of the data, as well as the description of it, is misleading because, while it emphasises the proportional increase in the endocrine compartment of the placenta it downplays the fact that the exchange area of the mutant placentas is vastly expanded. This is important for two reasons.

   Firstly, the whole placenta is increased in size suggesting that the mechanism is not placental lineage- specific and instead acting on the whole organ. Secondly in relation to embryonic growth, generally speaking, genetic manipulations that modify labyrinthine volume tend to have a positive correlation with fetal mass whereas the relationship between junctional zone volume and embryonic mass is more complex (discussed in Watson PMID: 15888575, for example). The authors should reconsider how they present this data in light of the previous point.

   We thank the reviewer for pointing out our error in the placental efficiency analysis and apologise for this error. We have corrected the presentation and interpretation of these data and have described this in detail in our response to Reviewer 1, Comment 1.

   As discussed in our response to Reviewer 1, Comment 1, we have added a range of analyses to determine whether placental efficiency was enhanced in HET-hom offspring. These include measuring fetal and maternal circulating glucose levels (Figure 4K), individual amino acids and an extensive range of metabolites (Figure 8) and providing CD31 immunofluorescent analyses of labyrinth area, labyrinth/placental ratio and capillary/labyrinth ratio in HET-hom and control placentas (Figure 5).

   We also added analyses of glycogen enriched and non-glycogen-enriched cell counts in the decidua and junctional zones. As suggested by Reviewer 3, both glycogen-enriched and non-enriched cell populations are significantly increased in HET-hom placentas.

   Combined, these new analyses significantly expand the study and support the conclusion that placental efficiency in HET-hom offspring was either compromised or not different from controls, depending on the analysis. We find no evidence that placental efficiency was increased in HET-hom offspring and have reworked our results and discussion sections to reflect these new data and interpretation.

   Reviewer 3, Comment 2 cont: Again, some of the methods are not clearly reported making interpretation difficult - especially how they have estimated their GlyT number.

   As outlined in our response to Reviewer 3 Comment 1, in the methods section we have added further detail of how we counted glycogen-enriched and non-enriched cells in the decidua and junctional zone regions of sections for the middle of WT-wt, WT-het, HET-het and HET-hom placentas (Lines 650-669).

   Reviewer 3, Comment 3: Perinatal embryonic/pup overgrowth.

   Strengths:

   • The overgrowth exhibited by the oocyte-Eed-deleted pups is striking and confirms the previous work by this group (Prokopuk, 2018). This is an important finding, especially in the context of understanding how PRC2-group gene mutations in humans cause overgrowth syndromes. It is also intriguing because it indicates that genetic/environmental insults in the mother that affect her gamete development can have long-term consequences on offspring physiology.

   Weaknesses:

   • Is the overgrowth intrauterine or is it caused by the increase in gestation length? The way the data is reported makes it impossible to work this out. The authors show that gestation time is consistently lengthened for mothers incubating oocyte-Eed-deleted pups by 1-2 days. In the supplementary material, the mutant embryos are not larger than WT at e19.5, the usual day of birth. Postnatal data is presented as day post-parturition. It would probably be clearer to present the embryonic and postnatal data as days post coitum. In this way, it will be obvious in which period the growth enhancement is taking place. This is information really important to determine whether the increased growth of the mutants is due to a direct effect of the intrauterine environment, or perhaps a more persistent hormonal change in the mother that can continue to promote growth beyond the gestation period.

   We have used embryonic day (E) to denote embryo and fetal age throughout the study – this is the same as using DPC (i.e. E19.5 is equivalent to 19.5 DPC). As described in the Methods “Collection of post-implantation embryos, placenta and postnatal offspring”, mice were time mated for two-four nights, with females plug checked daily. Positive plugs were noted as day E0.5.

   To make the data presentation clearer, we have shown the data for surviving HET-hom pups born on E19.5 (Figure 2J) separately from all HET-hom surviving pups born on E19.5-E21.5. (Figure 2G). As discussed in our response to Reviewer 2, we have also included growth data for pregnancies at E14.5, E17.5, E18.5 (Fig. 2C-F) and E19.5 (Figure 2J,K), as well as P0 (combined data for surviving pups born E19.5-E21.5), and P3 (combined data for surviving pups born E19.5-E21.5, Figure 2G,H).

   These data clearly show that HET-hom fetuses are substantially growth and developmentally delayed at E14.5 (Figure 2D), but HET-hom pups born on E19.5 are the same weight as WT-wt, WT-het and HET-het control pups (Figure 2J). This demonstrates that weight of HET-hom fetuses is normalised in utero between E14.5 and day of birth on E19.5.

   Importantly, as requested by Reviewer 3, we have separated average weight for all surviving pups with a day of birth of E19.5-21.5 (Figure 2G) from average weight of pups born on E19.5 only (Figure 2J). These analyses revealed that the average weight of surviving pups born between E19.5-21.5 was significantly higher than for controls (Figure 2G), but the average weight of pups born on E19.5 only was not. It is therefore clear that extended gestation also contributed to increased HET-hom pup birth weight. We have updated these additional analyses in Results (Lines 165-197) and Figure 2

   As revealed in Figure 2H, it is also possible/likely that growth of HET-hom pups during the three days post- partum may have contributed to the offspring overgrowth we observed in this and our previous study (Prokopuk et al., 2018 Clinical Epigenetics). However, we cannot determine whether there is a contribution from a persistent maternal hormonal change that promotes post-natal offspring growth or whether there is an innate growth benefit in HET-hom pups. As this is very difficult to dissect, separating these possibilities is beyond the scope of our study.

   Reviewer 3, Comment 4: "fetal growth restriction followed by placental hyperplasia, .. drives catch-up growth that ultimately results in perinatal offspring overgrowth".

   Here the authors try to link their observations, suggesting that i) the increased perinatal growth rate is a consequence of placentomegaly, and ii) the placentomegaly/increased fetal growth is an adaptive consequence of the early growth restriction. This is an interesting idea and suggests that there is a degree of developmental plasticity that is operating to repair the early consequences of transient loss of Eed function.

   Strengths:

   • Discrepancies between earlier studies are reconciled. Here the authors show that in oocyte-Eed-deleted embryos growth is initially restricted and then the growth rate increases in late gestation with increased perinatal mass.

   Weaknesses:

   • Regarding the dependence of fetal growth increase on placental size increase, this link is far from clear since placental efficiency is in fact decreased in the mutants (see above).

   • "Catch-up growth" suggests that a higher growth rate is driven by an earlier growth restriction in order to restore homeostasis. There is no direct evidence for such a mechanism here. The loss of Eed expression in the oocyte and early embryo could have an independent impact on more than one phase of development.

   Firstly, there is growth restriction in the early phase of cell divisions. Potentially this could be due to depression of genes that restrain cell division on autosomes, or suppression of X-linked gene expression (as has been previously reported, Inoue, 2018 PMID: 30463900). The placentomegaly is explained by the misregulation of non-canonically imprinted genes, as the authors report (and in agreement with other studies, e.g. Inoue, 2020. PMID: 32358519).

   • Explaining the perinatal phase of growth enhancement is more difficult. I think it is unlikely to be due to placentomegaly. Multiple studies have shown that placentomegaly following somatic cell nuclear transfer (SCNT) is caused by non-canonically imprinted genes, and can be rescued by reducing their expression dosage. However, SCNT causes placentomegaly with normal or reduced embryonic mass (for example -Xie 2022, PMID: 35196486), not growth enhancement. Moreover, since (to my knowledge) single loss of imprinting models of non-canonically imprinted genes do not exist, it is not possible to understand if their increased expression dosage can drive perinatal overgrowth, and if this is preceded by growth restriction and thus constitutes 'catch up growth'.

   Reviewer 3 is correct in their assessment that placental efficiency was decreased in HET- hom offspring and we have corrected the placental efficiency analysis based on fetal/placental weight ratios (discussed in detail in our response to Reviewer 1 Comment 1). We have added substantially more data (glucose, amino acids, metabolites, labyrinth capillary area and density). These data support the conclusion that a placentally driven advantage for HET-hom fetal growth is unlikely, despite our observation that HET- hom fetuses are developmental delayed and underweight at E14.5, but are born at normal weight after a normal gestational length (19.5 days) (discussed in our responses to Reviewer 3, Comment 3 and Reviewer 2).

   This demonstrates that HET-hom fetuses are able to attain normal birth weight despite being initially growth restricted state at E14.5, and that this occurs despite low placental function. Moreover, as we compared isogenic offspring with heterozygous loss of Eed (Het-het compared to HET-hom offspring) the outcomes we observed in HET-hom offspring originate from loss of EED in the growing oocyte or loss of maternal EED in the zygote strongly suggesting that a non-genetic mechanism is involved.

   As pointed out by Reviewer 3, the initial developmental delay in HET-hom offspring may be due to increased expression of genes that regulate cell proliferation – this could clearly explain the lower number of cells we observed in the ICM and the growth delay at later stages of embryonic and fetal development. Another possibility is that maternal PRC2 provided by the oocyte promotes cell divisions in preimplantation embryos We have discussed these possibilities on Lines 467-476.

   In addition, Matoba et al 2022 demonstrated that deletion of maternal Xist together with Eed was able to rescue male-biased lethality in offspring from oocytes lacking Eed, revealing a clear role for X-linked genes in this phenotype (Matoba et al 2022, Genes and Development). However, deletion of maternal Xist did not properly normalise survival offspring from Eed null oocytes (i.e. Eed/Xist double maternal null litters were smaller than litters derived from wild type oocytes) strongly suggesting other mechanisms provide the capacity for HET-hom offspring to attain normal weight at birth. We have added further discussion of the Matoba study in the context of our study on of the Discussion (Lines 544-555)

   Finally, with respect to the outcomes for SCNT derived offspring, we extracted SCNT fetal growth and placental weight data from the supplementary data included in Matoba et al., 2018 Cell Stem Cell. 2018;23(3):343-54.e5 and compared it with data collected in our study (Figure 7). This analysis revealed that the weights of placentas and fetuses of offspring derived via SCNT were very similar to the HET-hom offpsring in our study and we have discussed the similarities and potential differences between HET-hom and SCNT offspring in the Discussion (Lines 478-500).

   As pointed out by Reviewer 3, deletion of maternal non-canonically imprinted genes partially or fully rescued the placental hyperplasia phenotype in both SCNT derived and offspring from oocyte lacking EED. However, as we have discussed, the mechanisms underlying other aspects of the offspring phenotype, such as fetal growth recovery of HET-hom offspring observed in our study, remain unknown. Moreover, the comparison we provide in Figure 7 strongly indicates that HET-hom and SCNT fetuses are similarly delayed at E14.5 and undergo similar fetal growth recovery before birth, but the mechanism also remains unknown. Together, it appears that offspring derived from either Eed-null oocytes or by SCNT have an innate ability to remediate fetal growth restriction during the late stages of pregnancy without a requirement to correct maternally inherited impacts mediated by Xist or H3K27me3-dependent imprinting.

   Read the original source
3. 

   Version published to 10.1101/2022.08.08.503175v3 on bioRxiv

   Feb 5, 2023
4. 

   eLife

   Oct 23, 2022

   eLife assessment

   Using a genetically controlled experimental setting, the authors find that the lack of Polycomb-dependent epigenetic programming in the oocyte and early embryo influences the developmental trajectory through gestation in the mouse. By showing a two-phase outcome of early growth restriction followed by enhancement, the authors address previous inconsistencies in the field. However, the link with placenta function and gene misregulation is not yet fully supported.

   Read the original source
5. 

   eLife

   Oct 23, 2022

   Reviewer #1 (Public Review):

   Oberin, Petautschnig et. al investigated the developmental phenotypes that resulted from oocyte-specific loss of the EED (Embryonic Ectoderm Development) gene - a core component of the Polycomb repressive complex 2 (PRC2), which possess histone methyltransferase activity and catalyses trimethylation of histone H3 at lysine 27 (H3K27). The PRC2 complex plays essential roles in regulating chromatin structure, being an important regulator of cellular differentiation and development during embryogenesis. As novel findings, the authors find that PRC2-dependent programming in the oocyte, via loss of the core component EE2, causes placental hyperplasia and propose that the increase of placental transplacental flux of nutrients leads to fetal and postnatal overgrowth. At the mechanistic level, they show altered …

   Reviewer #1 (Public Review):

   Oberin, Petautschnig et. al investigated the developmental phenotypes that resulted from oocyte-specific loss of the EED (Embryonic Ectoderm Development) gene - a core component of the Polycomb repressive complex 2 (PRC2), which possess histone methyltransferase activity and catalyses trimethylation of histone H3 at lysine 27 (H3K27). The PRC2 complex plays essential roles in regulating chromatin structure, being an important regulator of cellular differentiation and development during embryogenesis. As novel findings, the authors find that PRC2-dependent programming in the oocyte, via loss of the core component EE2, causes placental hyperplasia and propose that the increase of placental transplacental flux of nutrients leads to fetal and postnatal overgrowth. At the mechanistic level, they show altered expression of genes previously implicated in placental hyperplasia phenotypes. They also establish interesting parallelism with the placental hyperplasia phenotype that is frequently observed in cloned mice.

   Strengths:

   The mouse breeding experiments are very well designed and are powerful to exclude potential confounding genetic effects on the developmental phenotypes that resulted from the loss of EED in oocytes. Another major strength is the developmental profiling across gestation, from pre-implantation to late gestation.

   Weaknesses:

   The evidence for 'oocyte' programming is restricted to phenotypic and gene expression analysis, without measurements of epigenetic dysregulation. It would be an added value if the authors could show evidence for altered H3K27me3 or DNA methylation in the placenta, for example.

   The claim that placental hyperplasia drives offspring catch-up growth is not supported by current experimental data. The authors do not address if transplacental flux is increased in the hyperplastic placentae, measure amino acids and glucose in fetal/maternal plasma, or perform tetraploid rescue experiments to ascertain the contribution of the placenta to growth phenotypes. Furthermore, it is unclear, from the current data, if the surface area for nutrient transport is actually increased in the hyperplastic placenta and the extent to which other cell populations (i.e. spongiotrophoblasts) are affected in addition to glycogen cells. In addition, one of the supporting conclusions that the placenta is a key contributor to fetal overgrowth is based on a very crude measurement - placenta efficiency - which the authors claim is increased in the homozygous mutants compared to controls. After analysing the data carefully, I find evidence for decreased placental efficiency instead. I believe that the authors mistakenly present the data as placenta to fetal weight ratios, which led to the misinterpretation of the 'efficiency' concept.

   The authors do not mention alternative explanations for the observed fetal catch-up and postnatal overgrowth. Why would oocyte epigenetic programming effects be restricted to the placenta, and not include fetal organs?

   Read the original source
6. 

   eLife

   Oct 23, 2022

   Reviewer #2 (Public Review):

   Consistent fetal growth trajectories are vital for survival and later life health. The authors utilise an elegant and novel animal model to tease apart the role of Eed protein in the female germline from the role of somatic Eed. The authors were able to experimentally attribute placental overgrowth - particularly of the endocrine region of the placenta - to the function of Eed protein in the oocyte. Loss of Eed protein in the oocyte was also associated with dynamic changes in fetal growth and prolonged gestation. It was not determined whether the reported catch-up growth apparent on the day of birth was due to enhanced fetal growth very late in gestation, a longer gestational time ie the P0 pups are effectively one day "older" compared to the controls, or the pups catching up after birth when consuming …

   Reviewer #2 (Public Review):

   Consistent fetal growth trajectories are vital for survival and later life health. The authors utilise an elegant and novel animal model to tease apart the role of Eed protein in the female germline from the role of somatic Eed. The authors were able to experimentally attribute placental overgrowth - particularly of the endocrine region of the placenta - to the function of Eed protein in the oocyte. Loss of Eed protein in the oocyte was also associated with dynamic changes in fetal growth and prolonged gestation. It was not determined whether the reported catch-up growth apparent on the day of birth was due to enhanced fetal growth very late in gestation, a longer gestational time ie the P0 pups are effectively one day "older" compared to the controls, or the pups catching up after birth when consuming maternal milk.

   Read the original source
7. 

   eLife

   Oct 23, 2022

   Reviewer #3 (Public Review):

   My understanding of the main claims of the paper, and how they are justified by the data are discussed below:
   Overall, loss of PRC2 function in the developing oocyte and early embryo causes:

   1. Growth restriction from at least the blastocyst stage with low cell counts and midgestational developmental delay.

   Strengths:

   • Live embryo imaging added an important dimension to this study. The authors were able to confirm an unquantified finding from a previous lab (reduced time to 2-cell stage in oocyte-deletion Eed offspring, Inoue 2018, PMID: 30463900) as well as identify developmental delay and mortality at the blastocyst-hatching transition.
   • For the weight and morphological analysis the authors are careful to provide isogenic controls for most of the experiments presented. This means that any phenotypes can be …

   Reviewer #3 (Public Review):

   My understanding of the main claims of the paper, and how they are justified by the data are discussed below:
   Overall, loss of PRC2 function in the developing oocyte and early embryo causes:

   1. Growth restriction from at least the blastocyst stage with low cell counts and midgestational developmental delay.

   Strengths:

   • Live embryo imaging added an important dimension to this study. The authors were able to confirm an unquantified finding from a previous lab (reduced time to 2-cell stage in oocyte-deletion Eed offspring, Inoue 2018, PMID: 30463900) as well as identify developmental delay and mortality at the blastocyst-hatching transition.
   • For the weight and morphological analysis the authors are careful to provide isogenic controls for most of the experiments presented. This means that any phenotypes can be attributed to the oocyte genotype rather than any confounding effects of maternal or paternal genotype.
   • Overall, there is good evidence that oocyte deletion of Eed results in early embryonic growth restriction, consistent with previous observations (Inoue 2018, PMID: 30463900).

   Weaknesses:

   Gaps in the reporting of specific features of the methodology make it difficult to interpret/understand some of the results.

   1. Placental hyperplasia with disproportionate overgrowth of the junctional trophoblast especially the glycogen trophoblast (GlyT) cells.

   Strengths:

   • The authors provide a comprehensive description of how placental and embryo weight is affected by the oocyte-Eed deletion through mid-to-late gestation development. The case for placentomegaly is clear.
   Weaknesses:
   • The placental efficiency data presented in Figure 3G-I is incorrect. Placental efficiency is calculated as embryo mass/placental mass, and it increases over the late gestation period. For e14.5 for example (Fig3G), WT-wt embryo mass = ~0.3g, placenta mass = 0.11g (from Fig 3D) = placental efficiency 2.7; HET-hom = 0.25/0.12 = 2.1. The paper gives values: WT-wt 0.5, HET-hom 0.7. Have the authors perhaps divided placenta weight by embryo mass? This would explain why the E17.5 efficiencies are so low (WT-wt 0.11 rather than a more usual figure of 8.88. If this is the case then the authors' conclusion that placental efficiency is improved by oocyte deletion of Eed is wrong - in fact, placental efficiency is severely compromised.
   • The authors have performed cell type counting on histological sections obtained from placentas to discover which cells are contributing to the placentomegaly. This data is presented as %cell type area in the main figure, though the untransformed cross-sectional area for each cell type is shown in the supplementary data. This presentation of the data, as well as the description of it, is misleading because, while it emphasises the proportional increase in the endocrine compartment of the placenta it downplays the fact that the exchange area of the mutant placentas is vastly expanded. This is important for two reasons. Firstly, the whole placenta is increased in size suggesting that the mechanism is not placental lineage-specific and instead acting on the whole organ. Secondly in relation to embryonic growth, generally speaking, genetic manipulations that modify labyrinthine volume tend to have a positive correlation with fetal mass whereas the relationship between junctional zone volume and embryonic mass is more complex (discussed in Watson PMID: 15888575, for example). The authors should reconsider how they present this data in light of the previous point.
   • Again, some of the methods are not clearly reported making interpretation difficult - especially how they have estimated their GlyT number.

   1. Perinatal embryonic/pup overgrowth.

   Strengths:

   • The overgrowth exhibited by the oocyte-Eed-deleted pups is striking and confirms the previous work by this group (Prokopuk, 2018). This is an important finding, especially in the context of understanding how PRC2-group gene mutations in humans cause overgrowth syndromes. It is also intriguing because it indicates that genetic/environmental insults in the mother that affect her gamete development can have long-term consequences on offspring physiology.

   Weaknesses:

   • Is the overgrowth intrauterine or is it caused by the increase in gestation length? The way the data is reported makes it impossible to work this out. The authors show that gestation time is consistently lengthened for mothers incubating oocyte-Eed-deleted pups by 1-2 days. In the supplementary material, the mutant embryos are not larger than WT at e19.5, the usual day of birth. Postnatal data is presented as day post-parturition. It would probably be clearer to present the embryonic and postnatal data as days post coitum. In this way, it will be obvious in which period the growth enhancement is taking place. This is information really important to determine whether the increased growth of the mutants is due to a direct effect of the intrauterine environment, or perhaps a more persistent hormonal change in the mother that can continue to promote growth beyond the gestation period.

   1. "fetal growth restriction followed by placental hyperplasia, .. drives catch-up growth that ultimately results in perinatal offspring overgrowth".

   Here the authors try to link their observations, suggesting that i) the increased perinatal growth rate is a consequence of placentomegaly, and ii) the placentomegaly/increased fetal growth is an adaptive consequence of the early growth restriction. This is an interesting idea and suggests that there is a degree of developmental plasticity that is operating to repair the early consequences of transient loss of Eed function.

   Strengths:

   • Discrepancies between earlier studies are reconciled. Here the authors show that in oocyte-Eed-deleted embryos growth is initially restricted and then the growth rate increases in late gestation with increased perinatal mass.

   Weaknesses:

   • Regarding the dependence of fetal growth increase on placental size increase, this link is far from clear since placental efficiency is in fact decreased in the mutants (see above).
   • "Catch-up growth" suggests that a higher growth rate is driven by an earlier growth restriction in order to restore homeostasis. There is no direct evidence for such a mechanism here. The loss of Eed expression in the oocyte and early embryo could have an independent impact on more than one phase of development. Firstly, there is growth restriction in the early phase of cell divisions. Potentially this could be due to depression of genes that restrain cell division on autosomes, or suppression of X-linked gene expression (as has been previously reported, Inoue, 2018 PMID: 30463900). The placentomegaly is explained by the misregulation of non-canonically imprinted genes, as the authors report (and in agreement with other studies, e.g. Inoue, 2020. PMID: 32358519).
   • Explaining the perinatal phase of growth enhancement is more difficult. I think it is unlikely to be due to placentomegaly. Multiple studies have shown that placentomegaly following somatic cell nuclear transfer (SCNT) is caused by non-canonically imprinted genes, and can be rescued by reducing their expression dosage. However, SCNT causes placentomegaly with normal or reduced embryonic mass (for example -Xie 2022, PMID: 35196486), not growth enhancement. Moreover, since (to my knowledge) single loss of imprinting models of non-canonically imprinted genes do not exist, it is not possible to understand if their increased expression dosage can drive perinatal overgrowth, and if this is preceded by growth restriction and thus constitutes 'catch up growth'.

   Read the original source
8. 

   Version published to 10.1101/2022.08.08.503175v1 on bioRxiv

   Aug 11, 2022
9. 

   Version published to 10.1101/2022.08.08.503175v2 on bioRxiv

   Aug 11, 2022