One-carbon metabolism is required for epigenetic stability in the mouse placenta

One-carbon metabolism, including the folate cycle, has a crucial role in fetal development though its molecular function is complex and unclear. The hypomorphic Mtrrgt allele is known to disrupt one-carbon metabolism, and thus methyl group availability, leading to several developmental phenotypes (e.g., neural tube closure defects, fetal growth anomalies). Remarkably, previous studies showed that some of the phenotypes were transgenerationally inherited. Here, we explored the genome-wide epigenetic impact of one-carbon metabolism in placentas associated with fetal growth phenotypes and determined whether specific DNA methylation changes were inherited. Firstly, methylome analysis of Mtrrgt/gt homozygous placentas revealed genome-wide epigenetic instability. Several differentially methylated regions (DMRs) were identified including at the Cxcl1 gene promoter and at the En2 gene locus, which may have phenotypic implications. Importantly, we discovered hypomethylation and ectopic expression of a subset of ERV elements throughout the genome of Mtrrgt/gt placentas with broad implications for genomic stability. Next, we determined that known spermatozoan DMRs in Mtrrgt/gt males were reprogrammed in the placenta with little evidence of direct or transgenerational germline DMR inheritance. However, some spermatozoan DMRs were associated with placental gene misexpression despite normalisation of DNA methylation, suggesting the inheritance of an alternative epigenetic mechanism. Integration of published wildtype histone ChIP-seq datasets with Mtrrgt/gt spermatozoan methylome and placental transcriptome datasets point towards H3K4me3 deposition at key loci. These data suggest that histone modifications might play a role in epigenetic inheritance in this context. Overall, this study sheds light on the mechanistic complexities of one-carbon metabolism in development and epigenetic inheritance

Cell lineage-specific mitochondrial resilience during mammalian organogenesis

Mitochondrial activity differs markedly between organs, but it is not known how and when this arises. Here we show that cell lineage-specific expression profiles involving essential mitochondrial genes emerge at an early stage in mouse development, including tissue-specific isoforms present before organ formation. However, the nuclear transcriptional signatures were not independent of organelle function. Genetically disrupting intra-mitochondrial protein synthesis with two different mtDNA mutations induced cell lineage-specific compensatory responses, including molecular pathways not previously implicated in organellar maintenance. We saw downregulation of genes whose expression is known to exacerbate the effects of exogenous mitochondrial toxins, indicating a transcriptional adaptation to mitochondrial dysfunction during embryonic development. The compensatory pathways were both tissue and mutation specific and under the control of transcription factors which promote organelle resilience. These are likely to contribute to the tissue specificity which characterizes human mitochondrial diseases and are potential targets for organ-directed treatments

RNA-Seq reveals changes in human placental metabolism, transport and endocrinology across the first-second trimester transition.

The human placenta is exposed to major environmental changes towards the end of the first trimester associated with full onset of the maternal arterial placental circulation. Changes include a switch from histotrophic to hemotrophic nutrition, and a threefold rise in the intraplacental oxygen concentration. We evaluated their impact on trophoblast development and function using RNA-sequencing (RNA-Seq) and DNA-methylation analyses performed on the same chorionic villous samples at 7-8 (n=8) and 13-14 (n=6) weeks of gestation. Reads were adjusted for fetal sex. Most DEGs were associated with protein processing in the endoplasmic reticulum (ER), hormone secretion, transport, extracellular matrix, vasculogenesis, and reactive oxygen species metabolism. Transcripts higher in the first trimester were associated with synthesis and ER processing of peptide hormones, and glycolytic pathways. Transcripts encoding proteins mediating transport of oxygen, lipids, protein, glucose, and ions were significantly increased in the second trimester. The motifs of CBX3 and BCL6 were significantly overrepresented, indicating the involvement of these transcription factor networks in the regulation of trophoblast migration, proliferation and fusion. These findings are consistent with a high level of cell proliferation and hormone secretion by the early placenta to secure implantation in a physiological low-oxygen environment

RNA-Seq reveals changes in human placental metabolism, transport and endocrinology across the first-second trimester transition.

The human placenta is exposed to major environmental changes towards the end of the first trimester associated with full onset of the maternal arterial placental circulation. Changes include a switch from histotrophic to hemotrophic nutrition, and a threefold rise in the intraplacental oxygen concentration. We evaluated their impact on trophoblast development and function using RNA-sequencing (RNA-Seq) and DNA-methylation analyses performed on the same chorionic villous samples at 7-8 (n=8) and 13-14 (n=6) weeks of gestation. Reads were adjusted for fetal sex. Most DEGs were associated with protein processing in the endoplasmic reticulum (ER), hormone secretion, transport, extracellular matrix, vasculogenesis, and reactive oxygen species metabolism. Transcripts higher in the first trimester were associated with synthesis and ER processing of peptide hormones, and glycolytic pathways. Transcripts encoding proteins mediating transport of oxygen, lipids, protein, glucose, and ions were significantly increased in the second trimester. The motifs of CBX3 and BCL6 were significantly overrepresented, indicating the involvement of these transcription factor networks in the regulation of trophoblast migration, proliferation and fusion. These findings are consistent with a high level of cell proliferation and hormone secretion by the early placenta to secure implantation in a physiological low-oxygen environment

Blastocyst transfer in mice alters the placental transcriptome and growth.

Assisted reproduction technologies (ARTs) are becoming increasingly common. Therefore, how these procedures influence gene regulation and foeto-placental development are important to explore. Here, we assess the effects of blastocyst transfer on mouse placental growth and transcriptome. C57Bl/6 blastocysts were transferred into uteri of B6D2F1 pseudopregnant females and dissected at embryonic day 10.5 for analysis. Compared to non-transferred controls, placentas from transferred conceptuses weighed less even though the embryos were larger on average. This suggested a compensatory increase in placental efficiency. RNA sequencing of whole male placentas revealed 543 differentially expressed genes (DEGs) after blastocyst transfer: 188 and 355 genes were downregulated and upregulated, respectively. DEGs were independently validated in male and female placentas. Bioinformatic analyses revealed that DEGs represented expression in all major placental cell types and included genes that are critical for placenta development and/or function. Furthermore, the direction of transcriptional change in response to blastocyst transfer implied an adaptive response to improve placental function to maintain foetal growth. Our analysis revealed that CpG methylation at regulatory regions of two DEGs was unchanged in female transferred placentas and that DEGs had fewer gene-associated CpG islands (within ~20 kb region) compared to the larger genome. These data suggested that altered methylation at proximal promoter regions might not lead to transcriptional disruption in transferred placentas. Genomic clustering of some DEGs warrants further investigation of long-range, cis-acting epigenetic mechanisms including histone modifications together with DNA methylation. We conclude that embryo transfer, a protocol required for ART, significantly impacts the placental transcriptome and growth.This work was supported by grants from the Centre for Trophoblast Research (CTR) (to EDW), Lister Institute for Preventative Medicine (to EDW), and Canadian Institutes for Health Research (to JCC). KM was funded by a Newnham College (Cambridge) studentship and A.G. Leventis scholarship. SJT was funded by a Next Generation Fellowship (CTR). GETB was funded by a Wellcome Trust PhD studentship in Developmental Mechanisms. RSH and MP were funded by the CTR

Trophoblast organoids as a model for maternal-fetal interactions during human placentation.

The placenta is the extraembryonic organ that supports the fetus during intrauterine life. Although placental dysfunction results in major disorders of pregnancy with immediate and lifelong consequences for the mother and child, our knowledge of the human placenta is limited owing to a lack of functional experimental models1. After implantation, the trophectoderm of the blastocyst rapidly proliferates and generates the trophoblast, the unique cell type of the placenta. In vivo, proliferative villous cytotrophoblast cells differentiate into two main sub-populations: syncytiotrophoblast, the multinucleated epithelium of the villi responsible for nutrient exchange and hormone production, and extravillous trophoblast cells, which anchor the placenta to the maternal decidua and transform the maternal spiral arteries2. Here we describe the generation of long-term, genetically stable organoid cultures of trophoblast that can differentiate into both syncytiotrophoblast and extravillous trophoblast. We used human leukocyte antigen (HLA) typing to confirm that the organoids were derived from the fetus, and verified their identities against four trophoblast-specific criteria3. The cultures organize into villous-like structures, and we detected the secretion of placental-specific peptides and hormones, including human chorionic gonadotropin (hCG), growth differentiation factor 15 (GDF15) and pregnancy-specific glycoprotein (PSG) by mass spectrometry. The organoids also differentiate into HLA-G+ extravillous trophoblast cells, which vigorously invade in three-dimensional cultures. Analysis of the methylome reveals that the organoids closely resemble normal first trimester placentas. This organoid model will be transformative for studying human placental development and for investigating trophoblast interactions with the local and systemic maternal environment.Centre for Trophoblast Reearch Royal Society Dorothy Hodgkin Fellowship Marie Curie Intra-European Fellowshi

Transcriptional profiling of Kiss1 neurons from arcuate and rostral periventricular hypothalamic regions in female mice

The neuropeptide kisspeptin is produced by Kiss1 neurons and is required for normal mammalian fertility. The two main populations of Kiss1 neurons are located in the arcuate (ARC) and the rostral periventricular area of the third ventricle (RP3V) of the hypothalamus. To define the molecular signature of these Kiss1 populations, transcriptomics profiling was performed using purified Kiss1 neurons from diestrous stage female mice. From a data set of 7026 genes, 332 differentially expressed transcripts were identified between the Kiss1ARC and Kiss1RP3V neurons. This data has uncovered novel transcripts and expanded the receptor expression, co-transmitter and transcription factor profiles of Kiss1 neurons. Validation by quantitative RT-PCR confirmed differential expression of Cartpt, Ddc, Gal, Gda, Npy2r, Penk, Rasp18, Rxfp3, Slc18a2, and Th in Kiss1RP3V neurons and Gpr83, Hctr2, Nhlh2, Nmn, Npr3, Nr4a2, Nr5a2, Olfm2, Tac2, and Tacr3 in Kiss1ARC neurons. Enriched pathways common to both Kiss1 populations included the NF-kB, mTor, endocannabinoid, GPCR, Wnt and estrogen signalling while some pathways (eg cytomegalovirus infection, dopaminergic and serotonergic biosynthesis) were specific to Kiss1RP3V neurons. Our gene expression dataset augments the existing data sets describing the transcriptional profiles of Kiss1 neuronal populations.</jats:p

One-carbon metabolism is required for epigenetic stability in the mouse placenta

Peer reviewed: TrueAcknowledgements: RNA library preparation and sequencing was performed by Cambridge Genomics Services, Department of Pathology, University of Cambridge, Cambridge, United Kingdom. Next generation sequencing of the meDIP libraries was performed at The Babraham Institute, Cambridge, United Kingdom.One-carbon metabolism, including the folate cycle, has a crucial role in fetal development though its molecular function is complex and unclear. The hypomorphic Mtrr gt allele is known to disrupt one-carbon metabolism, and thus methyl group availability, leading to several developmental phenotypes (e.g., neural tube closure defects, fetal growth anomalies). Remarkably, previous studies showed that some of the phenotypes were transgenerationally inherited. Here, we explored the genome-wide epigenetic impact of one-carbon metabolism in placentas associated with fetal growth phenotypes and determined whether specific DNA methylation changes were inherited. Firstly, methylome analysis of Mtrr gt/gt homozygous placentas revealed genome-wide epigenetic instability. Several differentially methylated regions (DMRs) were identified including at the Cxcl1 gene promoter and at the En2 gene locus, which may have phenotypic implications. Importantly, we discovered hypomethylation and ectopic expression of a subset of ERV elements throughout the genome of Mtrr gt/gt placentas with broad implications for genomic stability. Next, we determined that known spermatozoan DMRs in Mtrr gt/gt males were reprogrammed in the placenta with little evidence of direct or transgenerational germline DMR inheritance. However, some spermatozoan DMRs were associated with placental gene misexpression despite normalisation of DNA methylation, suggesting the inheritance of an alternative epigenetic mechanism. Integration of published wildtype histone ChIP-seq datasets with Mtrr gt/gt spermatozoan methylome and placental transcriptome datasets point towards H3K4me3 deposition at key loci. These data suggest that histone modifications might play a role in epigenetic inheritance in this context. Overall, this study sheds light on the mechanistic complexities of one-carbon metabolism in development and epigenetic inheritance

One-carbon metabolism is required for epigenetic stability in the mouse placenta

One-carbon metabolism, including the folate cycle, has a crucial role in fetal development though its molecular function is complex and unclear. The hypomorphic Mtrrgt allele is known to disrupt one-carbon metabolism, and thus methyl group availability, leading to several developmental phenotypes (e.g., neural tube closure defects, fetal growth anomalies). Remarkably, previous studies showed that some of the phenotypes were transgenerationally inherited. Here, we explored the genome-wide epigenetic impact of one-carbon metabolism in placentas associated with fetal growth phenotypes and determined whether specific DNA methylation changes were inherited. Firstly, methylome analysis of Mtrrgt/gt homozygous placentas revealed genome-wide epigenetic instability. Several differentially methylated regions (DMRs) were identified including at the Cxcl1 gene promoter and at the En2 gene locus, which may have phenotypic implications. Importantly, we discovered hypomethylation and ectopic expression of a subset of ERV elements throughout the genome of Mtrrgt/gt placentas with broad implications for genomic stability. Next, we determined that known spermatozoan DMRs in Mtrrgt/gt males were reprogrammed in the placenta with little evidence of direct or transgenerational germline DMR inheritance. However, some spermatozoan DMRs were associated with placental gene misexpression despite normalisation of DNA methylation, suggesting the inheritance of an alternative epigenetic mechanism. Integration of published wildtype histone ChIP-seq datasets with Mtrrgt/gt spermatozoan methylome and placental transcriptome datasets point towards H3K4me3 deposition at key loci. These data suggest that histone modifications might play a role in epigenetic inheritance in this context. Overall, this study sheds light on the mechanistic complexities of one-carbon metabolism in development and epigenetic inheritance.Centre for Trophoblast Research Lister Institute for Preventative Medicin

Prevention of the foreign body response to implantable medical devices by inflammasome inhibition.

SignificanceImplantable electronic medical devices (IEMDs) are used for some clinical applications, representing an exciting prospect for the transformative treatment of intractable conditions such Parkinson's disease, deafness, and paralysis. The use of IEMDs is limited at the moment because, over time, a foreign body reaction (FBR) develops at the device-neural interface such that ultimately the IEMD fails and needs to be removed. Here, we show that macrophage nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome activity drives the FBR in a nerve injury model yet integration of an NLRP3 inhibitor into the device prevents FBR while allowing full healing of damaged neural tissue to occur.Part of the RNA-Seq work was performed with the Genomics and Transcriptomics Core, which is funded by the UK Medical Research Council (MRC) Metabolic Disease Unit (MRC_MC_UU_00014/5) and a Wellcome Trust Major Award (208363/Z/17/Z), and guidance from Marcella Ma, whom the authors wish to thank. CEB was supported by a Wellcome Trust Investigator award (108045/Z/15/Z). This work was also supported by the UK Wellcome Trust (Translational Medicine and Therapeutics PhD Programme Fellowship 109511/Z/15/Z to DGB), the UK Health Education England and the National Institute for Health Research (HEE/ NIHR ICA Program Clinical Lectureship CL-2019-14-004 to DGB), the UK Medical Research Council (MRC) and the Sackler Foundation (doctoral training grant RG70550 to ACL), the Engineering and Physical Sciences Research Council (EPSRC) Cambridge NanoDTC (EP/L015978/1), the Centre for Trophoblast Research (MP and RSH), the Whitaker International Scholars Program (ALR), the European Commission’s Horizon 2020 (Marie Sklodowska-Curie Fellowship 797506 to ALR), the Bertarelli Foundation (SPL), the European Research Council (Consolidator Award 772426 to KF), the UK Biotechnology and Biological Sciences Research Council (Research Grant BB/N006402/1 to KF), and the Alexander von Humboldt Foundation (Humboldt Professorship to KF)