Mouse Fetal Development Research Articles

The IMPDH cytoophidium couples metabolism and fetal development in mice

The cytoophidium is an evolutionarily conserved subcellular structure formed by filamentous polymers of metabolic enzymes. In vertebrates, inosine monophosphate dehydrogenase (IMPDH), which catalyses the rate-limiting step in guanosine triphosphate (GTP) biosynthesis, is one of the best-known cytoophidium-forming enzymes. Formation of the cytoophidium has been proposed to alleviate the inhibition of IMPDH, thereby facilitating GTP production to support the rapid proliferation of certain cell types such as lymphocytes, cancer cells and pluripotent stem cells (PSCs). However, past studies lacked appropriate models to elucidate the significance of IMPDH cytoophidium under normal physiological conditions. In this study, we demonstrate that the presence of IMPDH cytoophidium in mouse PSCs correlates with their metabolic status rather than pluripotency. By introducing IMPDH2 Y12C point mutation through genome editing, we established mouse embryonic stem cell (ESC) lines incapable of forming IMPDH polymers and the cytoophidium. Our data indicate an important role of IMPDH cytoophidium in sustaining a positive feedback loop that couples nucleotide biosynthesis with upstream metabolic pathways. Additionally, we find that IMPDH2 Y12C mutation leads to decreased cell proliferation and increased DNA damage in teratomas, as well as impaired embryo development following blastocoel injection. Further analysis shows that IMPDH cytoophidium assembly in mouse embryonic development begins after implantation and gradually increases throughout fetal development. These findings provide insights into the regulation of IMPDH polymerisation in embryogenesis and its significance in coordinating cell metabolism and development.

Transposable elements acquire time- and sex-specific transcriptional and epigenetic signatures along mouse fetal gonad development.

Gonadal sex determination in mice is a complex and dynamic process, which is crucial for the development of functional reproductive organs. The expression of genes involved in this process is regulated by a variety of genetic and epigenetic mechanisms. Recently, there has been increasing evidence that transposable elements (TEs), which are a class of mobile genetic elements, play a significant role in regulating gene expression during embryogenesis and organ development. In this study, we aimed to investigate the involvement of TEs in the regulation of gene expression during mouse embryonic gonadal development. Through bioinformatics analysis, we aimed to identify and characterize specific TEs that operate as regulatory elements for sex-specific genes, as well as their potential mechanisms of regulation. We identified TE loci expressed in a time- and sex-specific manner along fetal gonad development that correlate positively and negatively with nearby gene expression, suggesting that their expression is integrated to the gonadal regulatory network. Moreover, chromatin accessibility and histone post-transcriptional modification analyses in differentiating supporting cells revealed that TEs are acquiring a sex-specific signature for promoter-, enhancer-, and silencer-like elements, with some of them being proximal to critical sex-determining genes. Altogether, our study introduces TEs as the new potential players in the gene regulatory network that controls gonadal development in mammals.

Transcription factor NFYa controls cardiomyocyte metabolism and proliferation during mouse fetal heart development

Cardiomyocytes are highly metabolic cells responsible for generating the contractile force in the heart. During fetal development and regeneration, these cells actively divide but lose their proliferative activity in adulthood. The mechanisms that coordinate their metabolism and proliferation are not fully understood. Here, we study the role of the transcription factor NFYa in developing mouse hearts. Loss of NFYa alters cardiomyocyte composition, causing a decrease in immature regenerative cells and an increase in trabecular and mature cardiomyocytes, as identified by spatial and single-cell transcriptome analyses. NFYa-deleted cardiomyocytes exhibited reduced proliferation and impaired mitochondrial metabolism, leading to cardiac growth defects and embryonic death. NFYa, interacting with cofactor SP2, activates genes linking metabolism and proliferation at the transcription level. Our study identifies a nodal role of NFYa in regulating prenatal cardiac growth and a previously unrecognized transcriptional control mechanism of heart metabolism, highlighting the importance of mitochondrial metabolism during heart development and regeneration.

Maternal chronic caffeine intake impairs fertility, placental vascularization and fetal development in mice

Caffeine is commonly consumed by pregnant women to avoid fatigue or as a habit. However, it is not clearly determined its side effects to the conceptuses. This study evaluated placental morphofunctional alterations after maternal chronic caffeine intake and the effects on fetal growth. Female Swiss mice received, via gavage, caffeine doses (either 60, 120 or 240 mg/kg/day) seven days before mating until gestational days-(GD) 11.5 or 17.5. Fetal biometrical parameters were assessed, and placentae were either submitted to histomorphometrical or molecular evaluation of angiogenesis (placental growth factor-1[PlGF-1]), apoptosis (Caspase-3) and proliferation (Ki-67) markers (evaluated in Swiss dams) and to intravital microscopy (evaluated in C57BL/6 dams). Caffeine exposed fetuses exhibited intrauterine growth restriction in a sex-dependent manner, with greater commitment of female fetuses (P < 0.05). In addition, placentae from dams that received 120 mg/kg/day showed less irrigation by maternal blood and greater development of fetal vasculature, characterized by higher number of larger vessels (P < 0.05). Although no effects on apoptosis (Caspase-3) and angiogenesis (PlGF-1) were observed, dams treated with 60 mg/kg/day showed greater placental cell proliferation (Ki-67 staining) at GD 11.5 (P < 0.05). The group treated with 240 mg/kg/day exhibited only one pregnant dam for each gestational age, suggesting that this high caffeine consumption may compromise fertility. Taken together, even in the doses currently ingested by many pregnant women, caffeine has detrimental effects on placental vasculature and fetal development in mice. Therefore, our results strongly suggest that caffeine consumption in human pregnancies greater than the recommended doses should be avoided.

Effects of Gestational Arsenic Exposures on Placental and Fetal Development in Mice: The Role of Cyr61 .

Several epidemiological investigations demonstrated that maternal arsenic (As) exposure elevated risk of fetal growth restriction (FGR), but the mechanism remains unclear. This study aimed to investigate the effects of gestational As exposure on placental and fetal development and its underlying mechanism. Dams were exposed to 0.15, 1.5, and throughout pregnancy via drinking water. Sizes of fetuses and placentas, placental histopathology, and glycogen content were measured. Placental RNA sequencing was conducted. Human trophoblasts were exposed to () to establish an in vitro model of As exposure. The mRNA stability and protein level of genes identified through RNA sequencing were measured. () modification was detected by methylated RNA immunoprecipitation-quantitative real-time polymerase chain reason (qPCR). The binding ability of insulin-like growth factor 2 binding protein 2 to the gene of interest was detected by RNA-binding protein immunoprecipitation-qPCR. Intracellular S-adenosylmethionine (SAM) and methyltransferase activity were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and colorimetry, respectively. In vitro methyltransferase (As3MT) knockdown or SAM supplementation and invivo folic acid (FA) supplementation were used to evaluate the protective effect. A case-control study verified the findings. Sizes of fetuses (exposed to 1.5 and ) and placentas (exposed to ) were lower in As-exposed mice. More trophoblasts accumulated and the expression of markers of interstitial invasion was lower in the mouse group in comparison with control. Placental RNA sequencing identified cysteine-rich angiogenic inducer 61 (Cyr61) as a candidate gene of interest. Mechanistically, mice and cells exposed to As had lower protein expression of CYR61, and this was attributed to a lower incidence of Cyr61 . Furthermore, cells exposed to As had lower methyltransferase activity, suggesting that this could be the mechanism by which Cyr61 was affected. Depletion of intracellular SAM, a cofactor for methyltransferase catalytic domain, partially contributed to As-induced methyltransferase activity reduction. Either As3MT knockdown or SAM supplementation attenuated As-induced Cyr61 down-regulation. In mice, FA supplementation rescued As-induced defective trophoblastic invasion and FGR. In humans, a negative correlation between maternal urinary As and plasma CYR61 was observed in infants who were small for gestational age. Using in vitro and invivo models, we found that intracellular SAM depletion-mediated Cyr61 down-regulation partially contributed to As-induced defective trophoblastic invasion and FGR. https://doi.org/10.1289/EHP12207.

Editorial

Editorial

LPS Administration during Fertilization Affects Epigenetic Inheritance during Embryonic Development.

Intrauterine inflammation can cause infertility by disrupting reproductive function. The pathogenesis underlying this process may primarily involve endotoxins from lipopolysaccharides (LPS), which are produced by Gram-negative bacteria. However, the long-term effects of endotoxins in mammalian pregnancy following LPS exposure during fertilization have not been clarified. In this study, we performed experiments to analyze the influence of LPS on early embryonic development and fetal development in mice. Mice uteruses were examined for the expression of genes related to the inflammatory response. The expression of Il-1β and Il-6 increased following the administration of 200 and 1000 µg/kg LPS. Exposure to LPS using in vitro fertilization (IVF) significantly decreased the embryonic developmental rate. A concentration of 100 µg/kg LPS significantly increased the placental weight and fetal crown -rump length (CRL), whereas a concentration of 200 µg/kg LPS significantly decreased the placenta weight and fetal weight in vivo. These findings indicate that maternal LPS during fertilization affects fetal development until the late stage of pregnancy. Thus, maternal endotoxins may affect epigenetic inheritance during embryonic development from the early to late stages of pregnancy.

Maternal hyperglycemia inhibits pulmonary vasculogenesis during mouse fetal lung development by promoting GβL Ubiquitination-dependent mammalian target of Rapamycin assembly

BackgroundGestational diabetes mellitus (GDM) is associated with retarded lung development and poor lung health in offspring. Mammalian target of rapamycin (mTOR) is a key regulator of vasculogenesis and angiogenesis. The aim of this study was to investigate the role mTOR plays in pulmonary vasculogenesis during fetal lung development under maternal hyperglycemia.MethodsFirst, GDM was induced via streptozotocin injection in pregnant C57BL/6 mice before the radial alveolar count (RAC) in the fetal lungs was assessed using hematoxylin and eosin staining. The angiogenic ability of the cultured primary mouse fetal lung endothelial cells (MFLECs) was then assessed using the tube formation assay technique, while western blot and real-time polymerase chain reaction were performed to determine the expression of mTOR, regulatory-associated protein of mTOR (Raptor), rapamycin-insensitive companion of mTOR (Rictor), stress-activated protein kinase interacting protein 1 (Sin1), G protein beta subunit-like protein (GβL), Akt, tumor necrosis receptor associated factor-2 (TRAF2), and OTU deubiquitinase 7B (OTUD7B) in both the fetal lung tissues and the cultured MFLECs. Immunoprecipitation assays were conducted to evaluate the status of GβL-ubiquitination and the association between GβL and mTOR, Raptor, Rictor, and Sin1 in the cultured MFLECs.ResultsThe GDM fetal lungs exhibited a decreased RAC and reduced expression of von Willebrand factor, CD31, and microvessel density. The high glucose level reduced the tube formation ability in the MFLECs, with the mTOR, p-mTOR, p-Raptor, and TRAF2 expression upregulated and the p-Rictor, p-Sin1, p-Akt, and OTUD7B expression downregulated in both the GDM fetal lungs and the high-glucose-treated MFLECs. Meanwhile, GβL-ubiquitination was upregulated in the high-glucose-treated MFLECs along with an increased GβL/Raptor association and decreased GβL/Rictor and GβL/Sin1 association. Furthermore, TRAF2 knockdown inhibited the high-glucose-induced GβL-ubiquitination and GβL/Raptor association and restored the tube formation ability of the MFLECs.ConclusionMaternal hyperglycemia inhibits pulmonary vasculogenesis during fetal lung development by promoting GβL-ubiquitination-dependent mTORC1 assembly.

Anne Laura Dorinthea McLaren DBE. 26 April 1927—7 July 2007

Anne M c Laren DBE was born into a privileged family, which affected her future in two distinct ways. Her cut-glass accent and social confidence were advantageous reminders of this early privilege, but her unrelenting rejection of it was evident in her left-wing egalitarian political and social views and actions. Anne was among the pioneers of research into mammalian development—her experiments on embryonic and fetal development in mice led indirectly to ‘test tube’ babies and contributed to our understanding of the relative roles of soma and germ cells in generating the two sexes. However, it is not only her science for which she will be remembered; her social, educational, ethical and political activities in the cause of science were paramount. She was an articulate, logical and extraordinarily confident speaker, always unrelentingly focused and thoroughly prepared. These qualities made her a formidable member of the Warnock Committee of enquiry, which generated the internationally influential guidelines on how human-assisted reproduction should be regulated. She served for 10 years with distinction on the Human Fertilisation and Embryology Authority, the UK regulatory body that resulted from the Warnock enquiry. She was a founder member, and president, of the Association of Women in Science and Engineering. Her interests extended beyond human welfare to that of animals, and she became involved with conservation and biodiversity preservation programmes internationally. She was elected Foreign Secretary of the Royal Society, the first woman to hold this office in its 332 years.

Fibroblast Growth Factor 7 Suppresses Fibrosis and Promotes Epithelialization during Wound Healing in Mouse Fetuses.

Adult mammalian wounds leave visible scars, whereas skin wounds in developing mouse fetuses are scarless until a certain point in development when complete regeneration occurs, including the structure of the dermis and skin appendages. Analysis of the molecular mechanisms at this transition will provide clues for achieving scarless wound healing. The fibroblast growth factor (FGF) family is a key regulator of inflammation and fibrosis during wound healing. We aimed to determine the expression and role of FGF family members in fetal wound healing. ICR mouse fetuses were surgically wounded at embryonic day 13 (E13), E15, and E17. Expression of FGF family members and FGF receptor (FGFR) in tissue samples from these fetuses was evaluated using in situ hybridization and reverse transcription-quantitative polymerase chain reaction. Fgfr1 was downregulated in E15 and E17 wounds, and its ligand Fgf7 was upregulated in E13 and downregulated in E15 and E17. Recombinant FGF7 administration in E15 wounds suppressed fibrosis and promoted epithelialization at the wound site. Therefore, the expression level of Fgf7 may correlate with scar formation in late mouse embryos, and external administration of FGF7 may represent a therapeutic option to suppress fibrosis and reduce scarring.

Inducible knockout of syncytin-a leads to poor placental glucose transport in mice

IntroductionCell-cell fusion of cytotrophoblasts into the syncytiotrophoblast layer is a key process in placental development. Syncytin, an endogenous retroviral envelope protein, is expressed in placental trophoblasts and specifically mediates syncytiotrophoblast layer formation. Syncytin deficiency has been observed in fetal growth-restricted placentas. Abnormal fetal growth, especially fetal growth restriction, is associated with the decreased expression of glucose transporters. Here, we aimed to determine the role of syncytin in fetal growth restriction in placental glucose transport capacity. MethodsTo better explore the function of syncytin in fetal growth-restricted placenta, we generated an inducible knockout mouse model of syncytin-a gene. The expression levels of glucose transporters in BeWo cells were measured before and after HERV-W knockdown. ResultsSyncytin-A disruption was associated with significant abnormalities in placental and fetal development in mice. Syncytin-A destruction causes extensive abnormalities in the maternal-fetal exchange structures in the labyrinth, including an extremely reduced number and dramatically irregular distribution of fetal vessels. Moreover, glucose transporter 1, glucose transporters 3, and connexin 26 expression levels decreased after E14.5. Consistently, low glucose transporter 1, glucose transporter 3, and connexin 26 levels were observed in HERV-W-silenced BeWo cells. DiscussionSyncytin-A is crucial for both syncytiotrophoblast layer development and morphogenesis, suggesting that syncytin-A disruption leads to fetal growth restriction associated with abnormalities in the maternal-fetal exchange barrier and decreased glucose transport.

Disruption of Glucocorticoid Action on CD11c+ Dendritic Cells Favors the Generation of CD4+ Regulatory T Cells and Improves Fetal Development in Mice.

A wealth of innate and adaptive immune cells and hormones are involved in mounting tolerance towards the fetus, a key aspect of successful reproduction. We could recently show that the specific cross talk between the pregnancy hormone progesterone and dendritic cells (DCs) is significantly engaged in the generation of CD4+ FoxP3+ regulatory T (Treg) cells while a disruption led to placental alterations and intra-uterine growth restriction. Apart from progesterone, also glucocorticoids affect immune cell functions. However, their functional relevance in the context of pregnancy still needs clarification. We developed a mouse line with a selective knockout of the glucocorticoid receptor (GR) on DCs, utilizing the cre/flox system. Reproductive outcome and maternal immune and endocrine adaptation of Balb/c-mated C57Bl/6 GRflox/floxCD11ccre/wt (mutant) females was assessed on gestation days (gd) 13.5 and 18.5. Balb/c-mated C57Bl/6 GRwt/wtCD11ccre/wt (wt) females served as controls. The number of implantation and fetal loss rate did not differ between groups. However, we identified a significant increase in fetal weight in fetuses from mutant dams. While the frequencies of CD11c+ cells remained largely similar, a decreased expression of co-stimulatory molecules was observed on DCs of mutant females on gd 13.5, along with higher frequencies of CD4+ and CD8+ Treg cells. Histomorphological and gene expression analysis revealed an increased placental volume and an improved functional placental capacity in mice lacking the GR on CD11c+ DCs. In summary, we here demonstrate that the disrupted communication between GCs and DCs favors a tolerant immune microenvironment and improves placental function and fetal development.

Differential enrichment of H3K9me3 at annotated satellite DNA repeats in human cell lines and during fetal development in mouse

BackgroundTrimethylation of histone H3 on lysine 9 (H3K9me3) at satellite DNA sequences has been primarily studied at (peri)centromeric regions, where its level shows differences associated with various processes such as development and malignant transformation. However, the dynamics of H3K9me3 at distal satellite DNA repeats has not been thoroughly investigated.ResultsWe exploit the sets of publicly available data derived from chromatin immunoprecipitation combined with massively parallel DNA sequencing (ChIP-Seq), produced by the The Encyclopedia of DNA Elements (ENCODE) project, to analyze H3K9me3 at assembled satellite DNA repeats in genomes of human cell lines and during mouse fetal development. We show that annotated satellite elements are generally enriched for H3K9me3, but its level in cancer cell lines is on average lower than in normal cell lines. We find 407 satellite DNA instances with differential H3K9me3 enrichment between cancer and normal cells including a large 115-kb cluster of GSATII elements on chromosome 12. Differentially enriched regions are not limited to satellite DNA instances, but instead encompass a wider region of flanking sequences. We found no correlation between the levels of H3K9me3 and noncoding RNA at corresponding satellite DNA loci. The analysis of data derived from multiple tissues identified 864 instances of satellite DNA sequences in the mouse reference genome that are differentially enriched between fetal developmental stages.ConclusionsOur study reveals significant differences in H3K9me3 level at a subset of satellite repeats between biological states and as such contributes to understanding of the role of satellite DNA repeats in epigenetic regulation during development and carcinogenesis.

Loss of Mafb and Maf distorts myeloid cell ratios and disrupts fetal mouse testis vascularization and organogenesis†.

Testis differentiation is initiated when Sry in pre-Sertoli cells directs the gonad toward a male-specific fate. Sertoli cells are essential for testis development, but cell types within the interstitial compartment, such as immune and endothelial cells, are also critical for organ formation. Our previous work implicated macrophages in fetal testis morphogenesis, but little is known about genes underlying immune cell development during organogenesis. Here, we examine the role of the immune-associated genes Mafb and Maf in mouse fetal gonad development, and we demonstrate that deletion of these genes leads to aberrant hematopoiesis manifested by supernumerary gonadal monocytes. Mafb; Maf double knockout embryos underwent initial gonadal sex determination normally, but exhibited testicular hypervascularization, testis cord formation defects, Leydig cell deficit, and a reduced number of germ cells. In general, Mafb and Maf alone were dispensable for gonad development; however, when both genes were deleted, we observed significant defects in testicular morphogenesis, indicating that Mafb and Maf work redundantly during testis differentiation. These results demonstrate previously unappreciated roles for Mafb and Maf in immune and vascular development and highlight the importance of interstitial cells in gonadal differentiation.

Balanced Notch-Wnt signaling interplay is required for mouse embryo and fetal development

This study investigated the role of Notch and Wnt cell signaling interplay in the mouse early embryo, and its effects on fetal development. Developmental kinetics was evaluated in embryos in vitro cultured from the 8-16-cell to the hatched blastocyst stage in the presence of signaling inhibitors of Notch (DAPT) and/or Wnt (DKK1). An embryo subset was evaluated for differential cell count and gene transcription of Notch (receptors Notch1-4, ligands Dll1, Dll4, Jagged1-2, effectors Hes1-2) and Wnt (Wnt3a, Lrp6, Gsk3β, C-myc, Tcf4, β-catenin) components, E-cadherin and pluripotency and differentiation markers (Sox2, Oct4, Klf4, Cdx2), whereas a second subset was evaluated for implantation ability and development to term following transfer into recipients. Notch and Wnt blockades had significant opposing effects on developmental kinetics - Notch blockade retarded while Wnt blockade fastened development. This evidences that Notch and Wnt regulate the pace of embryo kinetics by respectively speeding and braking development. Blockades significantly changed the transcription profile of Sox2, Oct4, Klf4 and Cdx2, and Notch and double blockades significantly changed embryonic cell numbers and cell ratio. The double blockade induced more severe phenotypes than those expected from the cumulative effects of single blockades. Implantation ability was unaffected, but Notch and double blockades significantly decreased fetal development to term. Compared to control embryos, Notch blockade and Wnt blockade embryos originated, respectively, significantly lighter and heavier fetuses. In conclusion, Notch and Wnt signaling interplay in the regulation of the pace of early embryo kinetics, and their actions at this stage have significant carry-over effects on later fetal development to term.

Exposure to suboptimal ambient temperature during specific gestational periods and adverse outcomes in mice.

Exposure to suboptimal ambient temperature during pregnancy has been reported as a potential teratogen of fetal development. However, limited animal evidence is available regarding the impact of extreme temperatures on maternal pregnancy and the subsequent adverse pregnancy outcomes. Our objective in this study is to investigate the relationship between temperature and maternal stress during pregnancy in mice. This study used the Naval Medical Research Institute (NMRI) mice during the second and third pregnant weeks with the gestational day (GD) (GD 6.5-14.5 and GD 14.5-17.5). Mice were exposed to suboptimal ambient temperature (1 °C, 5 °C, 10 °C, 15 °C, 40 °C, 42 °C, 44 °C, 46 °C, and 48 °C for the experimental group and 23 °C for the control group) 1 h per day, 7 days a weekin each trimester. Measurements of placental development (placental weight [PW] and placental diameter [PD]) and fetal growth (fetal weight [FW] and crown-to-rump length [CRL]) between experimental and control groups were compared using analysis of variance (ANOVA). Data on the occurrence of preterm birth (PTB) and abnormalities were also collected. The results showed that exposure to both cold and heat stress in the second and third weeks of pregnancy caused significant decreases in measurements of placental development (PW and PD) and fetal growth (FW and CRL). For all temperature exposures, 15 °C was identified as the optimal temperature in the development of the embryo. Most PTB occurrences were observed in high-temperature stress groups, with the highest PTB number seen in the exposure group at 48 °C, whereas PTB occurred only at 1 °C among cold stress groups. In the selected exposure experiments, an approximate U-shaped relation was observed between temperature and number of abnormality occurrence. The highest percentage of these anomalies occurred at temperatures of 1 °C and 48 °C, while no abnormalities were observed at 15 °C and in the control group. Our findings strengthened the evidence that exposure to suboptimal ambient temperatures may trigger adverse pregnancy outcomes and worsen embryo and fetal development in mice.

Neural progenitors disrupted

Neurodevelopment Symptoms of Huntington's disease (HD) manifest in adulthood despite the aberrant protein being present much earlier in persons carrying the disease-causing mutation. Barnat et al. studied the cellular effects of the HD mutation on human and mouse fetal brain development (see the Perspective by DiFiglia). The authors found that neural progenitor cells at the brain's ventricular zone reach out to both the apical and basal surfaces of the neuroepithelial wall, and their cellular nuclei shuttle back and forth as the cell cycle progresses. With the aberrant protein, these epithelial junctions are disrupted, epithelial polarity is disturbed, and the cell cycle favors premature neuronal differentiation. Science , this issue p. [787][1]; see also p. [771][2] [1]: /lookup/doi/10.1126/science.aax3338 [2]: /lookup/doi/10.1126/science.abd6215

An atlas of dynamic chromatin landscapes in mouse fetal development

The Encyclopedia of DNA Elements (ENCODE) project has established a genomic resource for mammalian development, profiling a diverse panel of mouse tissues at 8 developmental stages from 10.5 days after conception until birth, including transcriptomes, methylomes and chromatin states. Here we systematically examined the state and accessibility of chromatin in the developing mouse fetus. In total we performed 1,128 chromatin immunoprecipitation with sequencing (ChIP–seq) assays for histone modifications and 132 assay for transposase-accessible chromatin using sequencing (ATAC–seq) assays for chromatin accessibility across 72 distinct tissue-stages. We used integrative analysis to develop a unified set of chromatin state annotations, infer the identities of dynamic enhancers and key transcriptional regulators, and characterize the relationship between chromatin state and accessibility during developmental gene regulation. We also leveraged these data to link enhancers to putative target genes and demonstrate tissue-specific enrichments of sequence variants associated with disease in humans. The mouse ENCODE data sets provide a compendium of resources for biomedical researchers and achieve, to our knowledge, the most comprehensive view of chromatin dynamics during mammalian fetal development to date.

Expanded encyclopaedias of DNA elements in the human and mouse genomes

The human and mouse genomes contain instructions that specify RNAs and proteins and govern the timing, magnitude, and cellular context of their production. To better delineate these elements, phase III of the Encyclopedia of DNA Elements (ENCODE) Project has expanded analysis of the cell and tissue repertoires of RNA transcription, chromatin structure and modification, DNA methylation, chromatin looping, and occupancy by transcription factors and RNA-binding proteins. Here we summarize these efforts, which have produced 5,992 new experimental datasets, including systematic determinations across mouse fetal development. All data are available through the ENCODE data portal (https://www.encodeproject.org), including phase II ENCODE1 and Roadmap Epigenomics2 data. We have developed a registry of 926,535 human and 339,815 mouse candidate cis-regulatory elements, covering 7.9 and 3.4% of their respective genomes, by integrating selected datatypes associated with gene regulation, and constructed a web-based server (SCREEN; http://screen.encodeproject.org) to provide flexible, user-defined access to this resource. Collectively, the ENCODE data and registry provide an expansive resource for the scientific community to build a better understanding of the organization and function of the human and mouse genomes.

Spatiotemporal DNA methylome dynamics of the developing mouse fetus

Cytosine DNA methylation is essential for mammalian development but understanding of its spatiotemporal distribution in the developing embryo remains limited1,2. Here, as part of the mouse Encyclopedia of DNA Elements (ENCODE) project, we profiled 168 methylomes from 12 mouse tissues or organs at 9 developmental stages from embryogenesis to adulthood. We identified 1,808,810 genomic regions that showed variations in CG methylation by comparing the methylomes of different tissues or organs from different developmental stages. These DNA elements predominantly lose CG methylation during fetal development, whereas the trend is reversed after birth. During late stages of fetal development, non-CG methylation accumulated within the bodies of key developmental transcription factor genes, coinciding with their transcriptional repression. Integration of genome-wide DNA methylation, histone modification and chromatin accessibility data enabled us to predict 461,141 putative developmental tissue-specific enhancers, the human orthologues of which were enriched for disease-associated genetic variants. These spatiotemporal epigenome maps provide a resource for studies of gene regulation during tissue or organ progression, and a starting point for investigating regulatory elements that are involved in human developmental disorders.