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The paternally expressed imprinted Retrotransposon-like 1 (Rtl1/Peg11) is a retrotransposon-derived gene that has evolved a function in eutherian placentation. Seven miRNAs, including miR-127, are processed from a maternally expressed antisense Rtl1 transcript (Rtl1as) and regulate Rtl1 levels through RNAi-mediated post-transcriptional degradation. To determine the relative functional role of Rtl1as miRNAs in Rtl1 dosage, we generated a mouse specifically deleted for miR-127. The miR-127 knockout mice exhibit placentomegaly with specific defects within the labyrinthine zone involved in maternal-fetal nutrient transfer. Although fetal weight is unaltered, specific Rtl1 transcripts and protein levels are increased in both the fetus and placenta. Phenotypic analysis of single (ΔmiR-127/Rtl1 or miR-127/ΔRtl1) and double (ΔmiR-127/ΔRtl1) heterozygous miR-127 and Rtl1 deficient mice indicate that Rtl1 is the main target gene of miR-127 in placental development. Our results demonstrate that miR-127 is an essential regulator of Rtl1 mediated by a trans-homologue interaction between reciprocally imprinted genes on the maternally and paternally inherited chromosomes.

The junctional zone (Jz) of the mouse placenta consists of two main trophoblast populations, spongiotrophoblasts and glycogen cells (GCs), but the development and function of both cell types are unknown. We conducted a quantitative analysis of GC size, number, and invasion of cells into the decidua across gestation. Furthermore, we identified markers of GC function to investigate their possible roles in the placenta. While the spongiotrophoblast cell volume doubles, and cell number increases steadily from E12.5 to E16.5, there is a remarkable 80-fold increase in GC numbers. This finding is followed by a notable decrease by E18.5. Surprisingly, the accumulation of GCs in the decidua did not fully account for the decrease in GC number in the Jz, suggesting loss of GCs from the placenta. Glucagons were detected on GCs, suggesting a steady glucose release throughout gestation. Connexin31 staining was shown to be specific for GCs. GC migration and invasion may be facilitated by temporally regulated expression of matrix metalloproteinase 9 and the imprinted gene product, Decorin. Expression of the clearance receptor for type II insulin-like growth factor (IGF-II), IGF2R, in a short developmental window before E16.5 may be associated with regulating the growth effects of IGF-II from glycogen cells and/or labyrinthine trophoblast on the expansion of the Jz. Thus stereology and immunohistochemistry have provided useful insights into Jz development and function of the glycogen cells.

Imprinted genes have prominent effects on placentation; however, there is limited knowledge about the manner in which the genes controlled by two paternally methylated regions on chromosomes 7 and 12 contribute to placentation. In order to clarify the functions of these genes in mouse placentation, we examined transcription levels of the paternally methylated genes, tissue differentiation and development and the circulatory system in placentae derived from three types of bi-maternal conceptuses that contained genomes of non-growing (ng) and fully grown (fg) oocytes. The genetic backgrounds of the ng oocytes were as follows: one was derived from the wild-type (ngWT) and another from mutant mice carrying a 13 kb deletion in the H19 transcription unit including the germline-derived differentially methylated region (H19-DMR) on chromosome 7 (ngDeltach7). Another set of oocytes was derived from mutant mice carrying a 4.15 kb deletion in the intergenic germline-derived DMR (IG-DMR) on chromosome 12 (ngDeltach12). Although placental mass was lower in the ngWT/fg placentae compared with that in the WT placentae, it was recovered in the ngDeltach7/fg placentae, but not in the ngDeltach12/fg placentae. The ngDeltach7/fg placental growth improvement was associated with severe dysplasia such as an expanded spongiotrophoblast layer and a malformed labyrinthine zone. In contrast, the ngDeltach12/fg placentae retained the layer structures with expanded giant cells, but their total masses were smaller with a normal circulatory system in order. Our findings demonstrate that the genes controlled by the two paternally methylated regions, H19-DMR and IG-DMR, complementarily organize placentation.

Distal mouse chromosome 12 is imprinted. Phenotypic analysis of mouse embryos with maternal or paternal uniparental disomy for the whole of chromosome 12 has characterized the developmental defects associated with the altered dosage of imprinted genes on this chromosome. Here we conduct a characterization of maternal and paternal Dp(dist12) mice using the reciprocal translocation T(4;12)47H. This limits the region analysed to the chromosomal domain distal to the T47H breakpoint in B3 on mouse chromosome 12. Both MatDp(dist12)T47H and PatDp(dist12)T47H conceptuses are non-viable and the frequency of recovery of Dp(dist12) conceptuses by 10.5 days post coitum (dpc) was lower than expected after normal adjacent-1 disjunction. A subset of MatDp(dist12) embryos can survive up to one day post partum. In contrast to paternal uniparental disomy 12 embryos, no live PatDp (dist12) embryos were recovered after 16.5 days of gestation. Other phenotypes observed in maternal and paternal chromosome 12 uniparental disomy mice are recapitulated in the Dp(dist12) mice and include placental, muscle and skeletal defects. Additional defects were also noted in the skin of both MatDp(dist12) and maternal uniparental disomy 12 embryos. This study shows that the developmental abnormalities associated with the altered parent of origin for mouse chromosome 12 can be attributed to the genomic region distal to the T47H breakpoint.

The mouse is an extremely useful experimental model for the study of human disease owing to the ease of genetic and physiological manipulation. A more detailed knowledge of murine placental development will, we hope, increase our understanding of the pathogenesis of placentally related complications of human pregnancy. The murine placenta consists of two main fetally derived compartments: the labyrinthine zone and the junctional zone. Exchange in the labyrinthine zone takes place across an interhaemal membrane comprising an outer layer of cytotrophoblast cells and two inner layers of syncytial trophoblast. The cytotrophoblast layer thins as gestation advances, and in addition becomes highly perforated after embryonic day (E)12.5. Furthermore, as gestation advances cytotrophoblast nuclear volume and DNA content increase, suggesting the formation of labyrinthine trophoblast giant cells. The syncytial layers become increasingly microvillous, enlarging the surface area for exchange. Separate basement membranes support the syncytium and the fetal capillary endothelium throughout gestation, although these appear to fuse where the capillaries are closely approximated to the trophoblast. The junctional zone consists of two principal trophoblast cell types, spongiotrophoblasts and invasive glycogen cells, yet the functions of each remain elusive. Spongiotrophoblasts vary in their appearance even when not fully differentiated, but a striking feature is the extensive endoplasmic reticulum of the more mature cells. Early glycogen cells are distinguished by the presence of electron-dense glycogen granules, and large amounts of surrounding extracellular matrix. Later the accumulations of glycogen granules occupy almost all the cytoplasm and there are few organelles. This is the first study to use both scanning and transmission electron microscopy in an ultrastructural description of murine placental development and is complementary to contemporary genetic investigations.

L3mbtl encodes a member of the Polycomb group of proteins, which function as transcriptional repressors in large protein complexes. The Drosophila D-l(3)mbt protein is considered a tumor suppressor since its inactivation results in brain tumors. The human L3MBTL gene lies in a region of chromosome 20 frequently deleted in patients with myeloid malignancies and has been proposed as a candidate 20q tumor suppressor gene. Recently we have shown that L3MBTL undergoes monoallelic methylation in hematopoietic tissues and is transcribed from the paternally derived allele. The mouse L3mbtl gene is located on chromosome 2, a region of syntenic homology with human chromosome 20, and in a region containing a number of genes subject to epigenetic regulation. Here we analyze the genomic structure and alternative splicing of L3mbtl and assess its imprinting status in mouse. L3mbtl displays a complex pattern of alternative splicing involving both 5' noncoding and coding exons and is transcribed from two promoters. Unlike its human counterpart, L3mbtl escapes imprinting and there is no differential methylation of its CpG island.

The mouse is an excellent model for studying the genetic basis of placental development, but analyses are restricted by the lack of quantitative data describing normal murine placental structure. This study establishes a technique for generating such data, applies stereological techniques on systematic uniform random sections of placentas between E12.5-E18.5 of gestation (E1.0 = day of the vaginal plug), and considers the results in the context of development of the labyrinth zone. Half of each placenta was wax embedded and exhaustively sectioned to determine absolute volumes of the labyrinth zone (Lz), junctional zone (Jz), and decidua using the Cavalieri principle. The other half was resin embedded and 1-microm sections were used to generate all volume, surface, and length densities within the Lz. Maximum placental volume is reached by E16.5, whereas the Lz volume fraction increases until E18.5 at the expense of the Jz and decidua. Within the Lz, the absolute volume and surface area of maternal blood spaces (MBS) expand rapidly between E14.5 and E16.5, with no increase thereafter. In contrast, fetal capillary development is linear and continues for longer than that of the MBS. The interhemal membrane separating maternal and fetal circulations undergoes thinning prior to expansion of maternal and fetal surface areas, achieving a harmonic mean thickness of 4.39 microm by E18.5. The specific diffusion capacity for oxygen of the interhemal membrane is maximal by E16.5, which may be necessary to support rapid fetal growth until the end of gestation.

A subset of mammalian genes is controlled by genomic imprinting. This process causes a gene to be expressed from only one chromosome homologue depending on whether it originally came from the egg or the sperm. Parental origin-specific gene regulation is controlled by epigenetic modifications to DNA and chromatin. Genomic imprinting is therefore a useful model system to study the epigenetic control of genome function. Here we consider the value of the mouse as an experimental organism to address questions about the role of imprinted genes, about the regulation of mono-allelic gene expression and about the evolution of the imprinting function and mechanism.

Many genes subject to genomic imprinting function in a number of endocrine/paracrine pathways that are important for normal mammalian development. Here, we show that an endocrine/paracrine pathway involving thyroid hormone metabolism is also regulated by imprinting. Thyroid hormone action depends on thyroid hormone receptors and their predominant ligand, 3,5,3′-triiodothyronine (T3). In vivo, thyroid hormone levels are maintained within the physiological range through the interaction of three iodothyronine deiodinases, D1, D2, and D3. D3 inactivates thyroxine (T4) and T3 by 5-deiodination, and the gene for this enzyme, Dio3 , lies in the imprinted domain on human chromosome 14q32/distal mouse chromosome 12. Here, we report the imprinting of Dio3 , which is expressed preferentially from the paternal allele. No differentially methylated region was identified in the CpG-island promoter, which is completely unmethylated. Localization of transcripts suggests that Dio3 may be exerting its function in both endocrine and autocrine/paracrine manners. An assay was developed for T3, and we show that its levels in maternal and paternal uniparental disomy (UPD) 12 fetuses are reciprocally affected. These results demonstrate that disruption of the imprinting status of Dio3 results in abnormal thyroid hormone levels and may contribute to the phenotypic abnormalities in UPD12 mice and UPD14 humans.

Introduction: The syncytiotrophoblast (STB) epithelial covering of the villous tree in the human placenta is a multi-nucleated syncytium that is sustained by continuous incorporation of differentiating cytotrophoblast (CTB) cells. STB nuclei display a variety of morphologies, but are generally more condensed in comparison to CTB nuclei. Here, we consider whether this condensation is a feature of epigenetic regulation of chromatin structure. Methods: Semi-quantitative immunohistochemical investigations of a panel of histone modifications were performed to determine the relative proportions in CTB and STB nuclear populations. We also investigated the patterns of DNA methylation and distribution of DNA methyltransferases enzymes in these populations. Results: Unexpectedly DNA methylation, and H3K9me3 and H3K27me3, which are modifications associated with heterochromatin, are present at lower levels in STB nuclei compared to CTB, despite the intensive condensation in the former nuclear population and the progenitor state of the latter. By contrast, STB nuclei are enriched for H4K20me3, which is also associated with repressive states. 5'hydroxymethylcytosine immunoreactivity is higher in STB, with intense staining observed in the highly condensed nuclei within syncytial knots. Discussion: Cell-type specific epigenetic states exist within the trophoblast populations potentially regulating their different functions and developmental properties and suggesting non-canonical epigenetic states associated with the properties of these cells.

Although a small number of the vast array of animal long non-coding RNAs (lncRNAs) have known effects on cellular processes examined in vitro, the extent of their contributions to normal cell processes throughout development, differentiation and disease for the most part remains less clear. Phenotypes arising from deletion of an entire genomic locus cannot be unequivocally attributed either to the loss of the lncRNA per se or to the associated loss of other overlapping DNA regulatory elements. The distinction between cis- or trans-effects is also often problematic. We discuss the advantages and challenges associated with the current techniques for studying the in vivo function of lncRNAs in the light of different models of lncRNA molecular mechanism, and reflect on the design of experiments to mutate lncRNA loci. These considerations should assist in the further investigation of these transcriptional products of the genome. DOI: http://dx.doi.org/10.7554/eLife.03058.001

The nutritional sins of the mother… Prenatal exposures of a mother can affect the health of her offspring, but how? Radford et al. found that the male progeny of undernourished pregnant mice had altered DNA chemistry in their sperm. In addition, the offspring displayed compromised metabolic health. The specific affected genes not only lost DNA methylation but also lacked the normal sperm DNA packaging factors (protamines) and instead were enriched in nucleosomes. Thus, when subjected to a suboptimal prenatal environment, offspring feel the effects of the maternal assault. Science , this issue p. 10.1126/science.1255903

Adverse prenatal environments can promote metabolic disease in offspring and subsequent generations. Animal models and epidemiological data implicate epigenetic inheritance, but the mechanisms remain unknown. In an intergenerational developmental programming model affecting F2 mouse metabolism, we demonstrate that the in utero nutritional environment of F1 embryos alters the germline DNA methylome of F1 adult males in a locus-specific manner. Differentially methylated regions are hypomethylated and enriched in nucleosome-retaining regions. A substantial fraction is resistant to early embryo methylation reprogramming, which may have an impact on F2 development. Differential methylation is not maintained in F2 tissues, yet locus-specific expression is perturbed. Thus, in utero nutritional exposures during critical windows of germ cell development can impact the male germline methylome, associated with metabolic disease in offspring.

The complete developmental expression pattern of the cyclin dependent kinase inhibitor (CDKI) p57 kip2 has not been reported, here we report a detailed study of the localization of p57 kip2 protein during mouse organogenesis. We show that p57 kip2 is coincident with key stages of differentiation of several organs, some but not all of which are affected in Beckwith-Weidermann syndrome, a human congenital syndrome characterized by foetal overgrowth and childhood tumours.

The role of epigenetic modification of gene expression is becoming increasingly important in how we understand the loss of tumour suppressor gene function in a variety of tumours and tumour predisposing syndromes. This review explores the importance of epimutation in Beckwith–Wiedemann syndrome and Wilms’ tumour and focuses on genomic methylation in both imprinted and non-imprinted genes as a key mechanism in the development of cancer.

Genetic analysis has shown that the distal portion of mouse chromosome 12 is imprinted; however, the developmental roles of imprinted genes in this region are not known. We have therefore generated conceptuses with uniparental disomy for chromosome 12, in which both copies of chromosome 12 are either paternally or maternally derived (pUPD12 and mUPD12, respectively). Both types of UPD12 result in embryos that are non-viable and that exhibit distinct developmental abnormalities. Embryos with pUPD12 die late in gestation, whereas embryos with mUPD12 can survive to term but die perinatally. The mUPD12 conceptuses are invariably growth-retarded while pUPD12 conceptuses exhibit placentomegaly. Skeletal muscle maturation defects are evident in both types of UPD12. In addition, embryos with paternal UPD12 have costal cartilage defects and hypo-ossification of mesoderm-derived bones. In embryos with mUPD12, the development of the neural crest-derived middle ear ossicles is defective. Some of these anomalies are consistent with those seen with uniparental disomies of the orthologous chromosome 14 region in humans. Thus, imprinted genes on chromosome 12 are essential for viability, the regulation of prenatal growth, and the development of mesodermal and neural crest-derived lineages.

Parental origin specific congenital anomalies have been noted in patients with uniparental disomy of the long arm of human chromosome 14 (UPD14). This suggests the presence of imprinted genes, consistent with observations of imprinting in the region of syntenic homology in the mouse. It is not known whether the distinct defects reported for paternal and maternal UPD14 are the result of biallelic expression or absence of expression of imprinted genes. Furthermore, identification of the genes responsible would be facilitated by a higher resolution map of the imprinted region(s) involved. Subjects with partial trisomy for chromosome 14 (Ts14) have been reported and hence also have an alteration in the dosage of their parental chromosomes. In this study, we have carried out genotype-phenotype correlations considering the parental origin of the extra chromosome in previously reported cases of maternal and paternal partial Ts14. The analysis has provided evidence of a correlation between distal maternal Ts14 and anomalies including low birth weight, short philtrum, and small hands. The clinical features found in the maternal and paternal trisomies are compared with those associated with maternal and paternal UPD14 and their significance is discussed in relation to genomic imprinting on chromosome 14.

We have isolated a novel mouse gene (Gtl2) from the site of a gene trap integration (Gtl2lacZ) that gave rise to developmentally regulated lacZ expression, and a dominant parental-origin-dependent phenotype. Heterozygous Gtl2lacZ mice that inherited the transgene from the father showed a proportionate dwarfism phenotype, whereas the penetrance and expressivity of the phenotype was strongly reduced in Gtl2lacZ mice that inherited the transgene from the mother. Gtl2 expression is highly similar to the beta-galactosidase staining pattern, and is down-regulated but not abolished in mice carrying the Gtl2lacZ insertion. In early postimplantation embryos, Gtl2 is expressed in the visceral yolk sac and embryonic ectoderm. During subsequent development and organogenesis, Gtl2 transcripts are abundant in the paraxial mesoderm closely correlated with myogenic differentiation, in parts of the central nervous system, and in the epithelial ducts of developing excretory organs. The Gtl2 gene gives rise to various differentially spliced transcripts, which contain multiple small open reading frames (ORF). However, none of the ATG codons of these ORFs is in the context of a strong Kozak consensus sequence for initiation of translation, suggesting that Gtl2 might function as an RNA. Nuclear Gtl2 RNA was detected in a temporally and spatially regulated manner, and partially processed Gtl2 transcripts were readily detected in Northern blot hybridizations of polyadenylated RNA, suggesting that primary Gtl2 transcripts are differently processed in various cell types during development. Gtl2 transcript levels are present in parthenogenic embryos but may be reduced, consistent with the pattern of inheritance of the Gtl2lacZ phenotype.

We examined the allele-specific expression of six imprinted genes and the methylation profiles of three imprinting control regions to assess the epigenetic status of human embryonic stem cells. We identified generally monoallelic gene expression and normal methylation patterns. During prolonged passage, one cell line became biallelic with respect to H19, but without loss of the gametic methylation imprint. These data argue for a substantial degree of epigenetic stability in human embryonic stem cells.

In developed societies, high-sugar and high-fat (HSHF) diets are now the norm and are increasing the rates of maternal obesity during pregnancy. In pregnant rodents, these diets lead to cardiovascular and metabolic dysfunction in their adult offspring, but the intrauterine mechanisms involved remain unknown. This study shows that, relative to standard chow, HSHF feeding throughout mouse pregnancy increases maternal adiposity (+30%, P

The first mouse adult-repopulating hematopoietic stem cells emerge in the aorta-gonad-mesonephros region at embryonic day (E) 10.5. Their numbers in this region increase thereafter and begin to decline at E12.5, thus pointing to the possible existence of both positive and negative regulators of emerging hematopoietic stem cells. Our recent expression analysis of the aorta-gonad-mesonephros region showed that the Delta-like homologue 1 (Dlk1) gene is up-regulated in the region of the aorta-gonad-mesonephros where hematopoietic stem cells are preferentially located. To analyze its function, we studied Dlk1 expression in wild-type and hematopoietic stem cell-deficient embryos and determined hematopoietic stem and progenitor cell activity in Dlk1 knockout and overexpressing mice. Its role in hematopoietic support was studied in co-culture experiments using stromal cell lines that express varying levels of Dlk1. We show here that Dlk1 is expressed in the smooth muscle layer of the dorsal aorta and the ventral sub-aortic mesenchyme, where its expression is dependent on the hematopoietic transcription factor Runx1. We further demonstrate that Dlk1 has a negative impact on hematopoietic stem and progenitor cell activity in the aorta-gonad-mesonephros region in vivo, which is recapitulated in co-cultures of hematopoietic stem cells on stromal cells that express varying levels of Dlk1. This negative effect of Dlk1 on hematopoietic stem and progenitor cell activity requires the membrane-bound form of the protein and cannot be recapitulated by soluble Dlk1. Together, these data suggest that Dlk1 expression by cells of the aorta-gonad-mesonephros hematopoietic microenvironment limits hematopoietic stem cell expansion and is, to our knowledge, the first description of such a negative regulator in this tissue.

Background Genomic imprinting is an epigenetically regulated process wherein genes are expressed in a parent-of-origin specific manner. Many imprinted genes were initially identified in mice; some of these were subsequently shown not to be imprinted in humans. Such discrepancy reflects developmental, morphological and physiological differences between mouse and human tissues. This is particularly relevant for the placenta. Study of genomic imprinting thus needs to be carried out in a species and developmental stage-specific manner. We describe here a new strategy to study allele-specific DNA methylation in the human placenta for the discovery of novel imprinted genes. Results Using this methodology, we confirmed 16 differentially methylated regions (DMRs) associated with known imprinted genes. We chose 28 genomic regions for further testing and identified two imprinted genes (DNMT1 and AIM1). Both genes showed maternal allele-specific methylation and paternal allele-specific transcription. Imprinted expression for AIM1 was conserved in the cynomolgus macaque placenta, but not in other macaque tissues or in the mouse. Conclusions Our study indicates that while there are many genomic regions with allele-specific methylation in tissues like the placenta, only a small sub-set of them are associated with allele-specific transcription, suggesting alternative functions for such genomic regions. Nonetheless, novel tissue-specific imprinted genes remain to be discovered in humans. Their identification may help us better understand embryonic and fetal development.

We performed a genetic screen in mice to identify candidate genes that are associated with leukaemogenesis in the context of Trp53 heterozygosity. To do this we generated Trp53 heterozygous mice carrying the T2/Onc transposon and SB11 transposase alleles to allow transposon-mediated insertional mutagenesis to occur. From the resulting leukaemias/lymphomas that developed in these mice, we identified nine loci that are potentially associated with tumour formation in the context of Trp53 heterozygosity, including AB041803 and the Jun dimerization protein 2 (Jdp2). We show that Jdp2 transcriptionally regulates the Trp53 promoter, via an atypical AP-1 site, and that Jdp2 expression negatively regulates Trp53 expression levels. This study is the first to identify a genetic mechanism for tumour formation in the context of Trp53 heterozygosity.

Large regions of recurrent genomic loss are common in cancers; however, with a few well-characterized exceptions, how they contribute to tumor pathogenesis remains largely obscure. Here we identified primate-restricted imprinting of a gene cluster on chromosome 20 in the region commonly deleted in chronic myeloid malignancies. We showed that a single heterozygous 20q deletion consistently resulted in the complete loss of expression of the imprinted genes L3MBTL1 and SGK2, indicative of a pathogenetic role for loss of the active paternally inherited locus. Concomitant loss of both L3MBTL1 and SGK2 dysregulated erythropoiesis and megakaryopoiesis, 2 lineages commonly affected in chronic myeloid malignancies, with distinct consequences in each lineage. We demonstrated that L3MBTL1 and SGK2 collaborated in the transcriptional regulation of MYC by influencing different aspects of chromatin structure. L3MBTL1 is known to regulate nucleosomal compaction, and we here showed that SGK2 inactivated BRG1, a key ATP-dependent helicase within the SWI/SNF complex that regulates nucleosomal positioning. These results demonstrate a link between an imprinted gene cluster and malignancy, reveal a new pathogenetic mechanism associated with acquired regions of genomic loss, and underline the complex molecular and cellular consequences of "simple" cancer-associated chromosome deletions.

The ‘Genes and Development’ workshop focused on recentadvances in our understanding of the molecular control ofplacental development, focusing on the mouse model. JayCross gave an introduction to mouse placental developmentthat outlined the major structures and cell types. He alsoreviewed the considerable progress that has been made indefining molecular pathways controlling development usingmutant and transgenic mice as well as increasing knowledge ofthe biology underlying implantation and establishment of themature placenta [1,2]. One of the important general findings toemerge in recent years is that development and function of thedifferent sub-structures of the placenta can be interdependentand able to adapt to defects in other parts. This can complicatethe analysis of a phenotype in that some types of changes cansimply represent secondary changes reflecting attempts tocompensate, and investigators need to take this into consider-ation before making conclusions. The remaining speakers wereselected to discuss different aspects of placental developmentand highlight the use of new experimental approaches forstudying mouse placental development and function.

Dosage compensation of X-linked genes in female mammals occurs by inactivating one of the two X chromosomes. Two recent studies have asked when X inactivation first occurs, with different answers that leave open the question: does X inactivation occur before or after fertilisation?

The development of a placenta as an organ of foetal–maternal gas and nutrient exchange is characteristic of eutherian mammals. Mammalian development is also governed by a non-equivalence of the two parental genomes, a phenomenon known as genomic imprinting. It has been proposed that these characteristics are functionally linked, in that genomic imprinting plays a vital role particularly for placental development (Haig, 1993). Consistent with this hypothesis is the finding that alterations in the normal expression of imprinted genes result in growth abnormalities, the majority of which are seen in utero. This is of clinical relevance because of the increased interest in exploring any genetic basis for common problems of pregnancy, including intra-uterine growth restriction and pre-eclampsia. Over the last few years, much progress has been made in unravelling the specific functions of individual imprinted genes and of large imprinted chromosomal regions during placental development. Some of the latest results were reported during the ‘Imprinting and Growth Congress’ held in London from 11–13 April 2002. The first half of the meeting described work that utilized animal models, particularly the mouse, in the study of the functional relationship between imprinted genes and the control of prenatal growth. One of the first genes discovered to be subject to genomic imprinting is the paternally expressed insulin-like growth factor II (Igf2). Igf2 critically controls foetal growth rate, in particular in the second half of gestation when placental function becomes limiting for further embryonic growth (DeChiara et al., 1991; Ferguson-Smith et al., 1991; Sun et al., 1997). A prominent role for Igf2 in the placenta was suggested by the discovery of a placenta-specific promoter (P0) and two placenta-specific exons (U1 and U2) in the 5 -region of the Igf2 gene (Moore et al., 1997). Miguel Constância (The Babraham Institute, Cambridge) presented work on discerning the function of this placenta-specific Igf2 transcript that is confined to the labyrinthine region of the

Syncytiotrophoblast is the multinucleated epithelium of the placenta. Although many nuclei are dispersed within the syncytioplasm, others are aggregated into specializations referred to as true and false syncytial knots, and syncytial sprouts. Nuclei within true knots display highly condensed chromatin and are thought to be aged and effete. True knots increase in frequency with gestational age. Excessive formation (Tenney-Parker change) is associated with placental pathology, and a knotting index is used to assess severity. However, this index is potentially confounded by the creation of artifactual appearances (false knots) through tangential sectioning. In addition, knots must be distinguished from syncytial sprouts, which are markers of trophoblast proliferation. Here, we distinguish between sprouts, true knots, and false knots using serial sections and perform IHC for proliferating cell nuclear antigen, upstream binding factor, RNA polymerase II, and 8-oxo-deoxyguanosine as markers of recent incorporation, transcriptional activity, and oxidative damage. Villous explants were exposed to hydrogen peroxide to test the relationship between transcriptional activity and oxidative damage. Sprouts and false knots were found to contain recently incorporated and transcriptionally active nuclei. By contrast, most nuclei within true knots are negative for transcriptional markers but positive for 8-oxo-deoxyguanosine. In vitro, we observed a negative correlation between transcriptional activity and oxidative damage. These findings demonstrate that true knots contain effete damaged nuclei and provide IHC markers for their identification.

Trimprinting the Genome Reprogramming the parental genomes during the oocyte-to-embryo transition requires highly controlled epigenetic mechanisms. Although resetting the genome to a ground state is essential, conservation of inheritable marks is equally important. Now, Messerschmidt et al. (p. 1499 ) demonstrate that maternal deletion of the epigenetic modifier Trim28 in mice results in a strongly variable, yet ultimately embryonic, lethal phenotype. Aberrant loss of DNA methylation at imprinting control regions and thus partial loss of epigenetic memory was responsible for the phenotype. The stochastic time and mode of embryonic death reflect the exquisitely balanced interplay of maternal and zygotic factors in the early mammalian embryo.

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Environmental factors during early life are critical for the later metabolic health of the individual and of future progeny. In our obesogenic environment, it is of great socioeconomic importance to investigate the mechanisms that contribute to the risk of metabolic ill health. Imprinted genes, a class of functionally mono-allelic genes critical for early growth and metabolic axis development, have been proposed to be uniquely susceptible to environmental change. Furthermore, it has also been suggested that perturbation of the epigenetic reprogramming of imprinting control regions (ICRs) may play a role in phenotypic heritability following early life insults. Alternatively, the presence of multiple layers of epigenetic regulation may in fact protect imprinted genes from such perturbation. Unbiased investigation of these alternative hypotheses requires assessment of imprinted gene expression in the context of the response of the whole transcriptome to environmental assault. We therefore analyse the role of imprinted genes in multiple tissues in two affected generations of an established murine model of the developmental origins of health and disease using microarrays and quantitative RT–PCR. We demonstrate that, despite the functional mono-allelicism of imprinted genes and their unique mechanisms of epigenetic dosage control, imprinted genes as a class are neither more susceptible nor protected from expression perturbation induced by maternal undernutrition in either the F1 or the F2 generation compared to other genes. Nor do we find any evidence that the epigenetic reprogramming of ICRs in the germline is susceptible to nutritional restriction. However, we propose that those imprinted genes that are affected may play important roles in the foetal response to undernutrition and potentially its long-term sequelae. We suggest that recently described instances of dosage regulation by relaxation of imprinting are rare and likely to be highly regulated., Author Summary Environmental perturbations during early life are known to affect one's risk of metabolic disease many years later. Furthermore, that risk can be inherited by future generations, although the mechanisms responsible are poorly understood. Imprinted genes are unusual as only one of the two copies is expressed in a parent-of-origin–specific manner. As only one copy is active, imprinted gene dosage has been hypothesised to be uniquely vulnerable to environmental change. Therefore, it has been suggested that imprinted genes may play an important role in the developmental origins of health and disease. Alternatively, the opposite may be true—imprinted genes may be more tightly safeguarded from perturbation. To test these two hypotheses, we analysed the expression of imprinted genes in the context of all active genes in two affected generations of a mouse model of the developmental origins of health and disease. Our data show that imprinted genes as a class are neither more nor less susceptible to expression change, but a subset of imprinted genes may be involved in the adaptation of the conceptus. Furthermore, imprints in the developing germline are not affected and imprinted genes are largely stable in the second generation. This is important, as it is the first time that this hypothesis has been tested in an unbiased fashion.

Diploid parthenogenetic or androgenetic mouse conceptuses produce characteristic and opposite mutant phenotypes and are non-viable, presumably due to different contributions from the maternal and paternal genomes. This is likely to be the result of the preferential expression of only one parent's copy of certain genes in the offspring. So far, four such endogenous imprinted genes are known: the paternal alleles of Igf2 and Snrpn and the maternal alleles of Igf2r and H19 are active, while their opposite parental alleles are inactive. Here we demonstrate that the expression patterns of the Igf2 and Igf2r genes in androgenetic and parthenogenetic conceptuses correlate with which parental alleles normally express them, implying that the imprint can be maintained in the absence of the other parent's genome for these genes. This also indicates that both types of uniparental conceptuses are lacking developmentally important gene products. We did find, however, that the H19 gene was highly expressed not only in the parthenogenetic conceptus, but also in giant trophoblasts and secondary giant cells in the androgenetic placenta, in spite of the imprinting of the H19 gene in normal mouse extra embryonic tissues. We discuss these observations with respect to the non-viability of uniparental conceptuses and the reciprocal imprinting patterns of the Igf2 and H19 genes.

Epigenetic modifications to DNA and chromatin programme important genome functions including gene expression, chromosomal architecture and stability, and the maintenance of developmental states. Recent findings further implicate epigenetic modifications in the control of allelic choice in the immune system.

Ascent to high altitude is associated with a fall in the partial pressure of inspired oxygen (hypobaric hypoxia). For oxidative tissues such as skeletal muscle, resultant cellular hypoxia necessitates acclimatization to optimize energy metabolism and restrict oxidative stress, with changes in gene and protein expression that alter mitochondrial function. It is known that lowlanders returning from high altitude have decreased muscle mitochondrial densities, yet the underlying transcriptional mechanisms and time course are poorly understood. To explore these, we measured gene and protein expression plus ultrastructure in muscle biopsies of lowlanders at sea level and following exposure to hypobaric hypoxia. Subacute exposure (19 d after initiating ascent to Everest base camp, 5300 m) was not associated with mitochondrial loss. After 66 d at altitude and ascent beyond 6400 m, mitochondrial densities fell by 21%, with loss of 73% of subsarcolemmal mitochondria. Correspondingly, levels of the transcriptional coactivator PGC-1α fell by 35%, suggesting down-regulation of mitochondrial biogenesis. Sustained hypoxia also decreased expression of electron transport chain complexes I and IV and UCP3 levels. We suggest that during subacute hypoxia, mitochondria might be protected from oxidative stress. However, following sustained exposure, mitochondrial biogenesis is deactivated and uncoupling down-regulated, perhaps to improve the efficiency of ATP production.

MicroRNAs (miRs) recently emerged as prominent regulators of cancer processes. In the current study we aimed at elucidating regulatory pathways and mechanisms through which miR-494, one of the miR species found to be down-regulated in cholangiocarcinoma (CCA), participates in cancer homeostasis. miR-494 was identified as down-regulated in CCA based on miR arrays. Its expression was verified with quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR). To enforce miR expression, we employed both transfection methods, as well as a retroviral construct to stably overexpress miR-494. Up-regulation of miR-494 in cancer cells decreased growth, consistent with a functional role. mRNA arrays of cells treated with miR-494, followed by pathway analysis, suggested that miR-494 impacts cell cycle regulation. Cell cycle analyses demonstrated that miR-494 induces a significant G1/S checkpoint reinforcement. Further analyses demonstrated that miR-494 down-regulates multiple molecules involved in this transition checkpoint. Luciferase reporter assays demonstrated a direct interaction between miR-494 and the 3′-untranslated region of cyclin-dependent kinase 6 (CDK6). Last, xenograft experiments demonstrated that miR-494 induces a significant cancer growth retardation in vivo. Conclusion: Our findings demonstrate that miR-494 is down-regulated in CCA and that its up-regulation induces cancer cell growth retardation through multiple targets involved in the G1-S transition. These findings support the paradigm that miRs are salient cellular signaling pathway modulators, and thus represent attractive therapeutic targets. miR-494 emerges as an important regulator of CCA growth and its further study may lead to the development of novel therapeutics. (HEPATOLOGY 2011)

DNA demethylation processes are important for reproduction, being central in epigenetic reprogramming during embryonic and germ cell development. While the enzymes methylating DNA have been known for many years, identification of factors capable of mediating active DNA demethylation has been challenging. Recent findings suggest that cytidine deaminases may be key players in active DNA demethylation. One of the most investigated candidates is activation-induced cytidine deaminase (AID), best known for its role in generating secondary antibody diversity in B cells. We evaluate evidence for cytidine deaminases in DNA demethylation pathways in vertebrates and discuss possible models for their targeting and activity regulation. These findings are also considered along with alternative demethylation pathways involving hydroxymethylation.

The gene for the atypical NOTCH ligand delta-like homologue 1 (Dlk1) encodes membrane-bound and secreted isoforms that function in several developmental processes in vitro and in vivo. Dlk1, a member of a cluster of imprinted genes, is expressed from the paternally inherited chromosome. Here we show that mice that are deficient in Dlk1 have defects in postnatal neurogenesis in the subventricular zone: a developmental continuum that results in depletion of mature neurons in the olfactory bulb. We show that DLK1 is secreted by niche astrocytes, whereas its membrane-bound isoform is present in neural stem cells (NSCs) and is required for the inductive effect of secreted DLK1 on self-renewal. Notably, we find that there is a requirement for Dlk1 to be expressed from both maternally and paternally inherited chromosomes. Selective absence of Dlk1 imprinting in both NSCs and niche astrocytes is associated with postnatal acquisition of DNA methylation at the germ-line-derived imprinting control region. The results emphasize molecular relationships between NSCs and the niche astrocyte cells of the microenvironment, identifying a signalling system encoded by a single gene that functions coordinately in both cell types. The modulation of genomic imprinting in a stem-cell environment adds a new level of epigenetic regulation to the establishment and maintenance of the niche, raising wider questions about the adaptability, function and evolution of imprinting in specific developmental contexts.

The mouse insulin-like growth factor II gene (Igf 2), located on distal chromosome 7, is parentally imprinted such that the paternal allele is expressed while the maternal allele is transcriptionally silent. We derived a cell line from a mouse embryo maternally disomic and paternally deficient for distal chromosome 7 (MatDi7) to determine the stability of gene repression in culture. MatDi7 cells maintained Igf2 in a repressed state even after immortalization, except for one randomly picked clone which spontaneously expressed the gene. Igf 2 was expressed in a cell culture derived from a normal littermate; this expression was growth regulated, with Igf 2 mRNA levels increasing in the stationary phase of growth. Analysis of the methylation status of 28 sites distributed over 10 kb of the gene did not show consistent differences associated with expression level in the normal and MatDi7 cell lines, and the CpG island in the Igf 2 promoter remained unmethylated in all of the cell lines. Only with an oncogenically transformed cell line did the promoter become extensively methylated. We attempted to derepress the imprinted gene in MatDi7 cells by treatments known to alter gene expression. Expression of the Igf 2 allele in MatDi7 cells was increased in a dose-dependent manner by treatment with 5-aza-2'-deoxycytidine or bromodeoxyuridine, agents known to change DNA methylation patterns or chromatin conformation. Treatment of the cells with 1-beta-D-arabinofuranosylcytosine, 2'-deoxycytidine, calcium ionophore, heat shock, cold shock, or sodium butyrate did not result in increases in the levels of Igf 2 expression. It seems likely that the mechanism of the Igf 2 imprint involves subtle changes in the methylation or chromatin conformation of the gene which are affected by 5-aza-2'-deoxycytidine and bromodeoxyuridine.

Parental genomes have reciprocal phenotypic effects during development in the mouse because they are programmed (imprinted) with germ line-specific epigenetic modifications. These epigenetic modifications are inherited after fertilisation and they determine whether the maternal or the paternal allele of an 'imprinted' gene is expressed. Four such imprinted genes have so far been identified; the paternal genes of Igf2, and Snrpn, and the maternal genes of Igf2r and H19 are preferentially expressed during development. Igf2 and H19 are closely linked on chromosome 7 and show remarkably similar temporal and spatial patterns of expression. A mechanistic, and possibly a functional link may exist in the reciprocal imprinting of H19 and Igf2. The paternal H19 gene is apparently repressed by DNA methylation in the promoter region. This modification is not inherited from sperm but introduced after fertilisation. The nature of the primary germ line imprint therefore remains to be determined.

Parental genomes in mammals are programmed in the germline with heritable epigenetic modifications that exert control on the expression of specific (imprinted) genes. DNA methylation is one form of epigenetic modification which shows marked genome-wide variations in the germline and during early development. Certain transgene loci also demonstrate (reversible) germline-specific methylation imprints that are heritable in somatic tissues during development. Recently, four endogenous genes have been identified whose expression is dependent on their parental origin. The mechanism of genomic imprinting and the role of imprinted genes during development is beginning to be analysed. Three of these genes map to the mouse chromosome 7. Human chromosomes 11p13, 11p15, and 15ql 1-13 are associated with disorders exhibiting parental origin effects in their patterns of inheritance. These regions share syntenic homology with mouse chromosome 7. The relationship between parental imprints, germ line-dependent epigenetic inheritance and totipotency is also under investigation using embryonic stem cells derived from the epiblast. These cells are pluripotent or totipotent and evidence indicates the presence of at least the primary parental imprints. However, imprints inherited from the paternal germline in androgenetic cells are apparently more stable than those from the female germline in parthenogenetic cells.

The field of epigenetics provides neurobiologists with candidate mechanisms for experience-dependent changes in gene transcription. The ability to realize the potential of epigenetics in defining the causal pathways lying between environmental signals, transcriptional regulation and neural function will depend on moving beyond correlational studies focusing on individual epigenetic marks. Here we attempt to provide a conceptual framework for integrative research on nucleotide sequence, chromatin modifications, RNA signaling and their interactions in understanding experience-dependent phenotypic plasticity. Studies in genomic imprinting may serve as an existing model for such approaches.

X chromosome dosage compensation in female eutherian mammals is regulated by the noncoding Xist RNA and is associated with the differential acquisition of active and repressive histone modifications, resulting in repression of most genes on one of the two X chromosome homologs. Marsupial mammals exhibit dosage compensation; however, they lack Xist, and the mechanisms conferring epigenetic control of X chromosome dosage compensation remain elusive. Oviparous mammals, the monotremes, have multiple X chromosomes, and it is not clear whether they undergo dosage compensation and whether there is epigenetic dimorphism between homologous pairs in female monotremes. Here, using antibodies against DNA methylation, eight different histone modifications, and HP1, we conduct immunofluorescence on somatic cells of the female Australian marsupial possum Trichosurus vulpecula , the female platypus Ornithorhynchus anatinus , and control mouse cells. The two marsupial X's were different for all epigenetic features tested. In particular, unlike in the mouse, both repressive modifications, H3K9me3 and H4K20Me3, are enriched on one of the X chromosomes, and this is associated with the presence of HP1 and hypomethylation of DNA. Using sequential labeling, we determine that this DNA hypomethylated X correlates with histone marks of inactivity. These results suggest that female marsupials use a repressive histone-mediated inactivation mechanism and that this may represent an ancestral dosage compensation process that differs from eutherians that require Xist transcription and DNA methylation. In comparison to the marsupial, the monotreme exhibited no epigenetic differences between homologous X chromosomes, suggesting the absence of a dosage compensation process comparable to that in therians.

Uniparental disomy (UPD) refers to the situation in which both homologues of a chromosomal region/segment have originated from only one parent. This can involve the entire chromosome or only a small segment. As a consequence of UPD, or uniparental duplication/deficiency of part of a chromosome, there are two types of developmental risk: aberrant dosage of genes regulated by genomic imprinting and homozygosity of a recessive mutation. UPD models generated by reciprocal and Robertsonian translocation heterozygote intercrosses have been a powerful tool to investigate genomic imprinting in mice, whereas novel UPD patients such as those with cystic fibrosis and Prader-Willi syndrome, triggered the clarification of recessive diseases and genomic imprinting disorders in human. Newly developed genomic technologies as well as conventional microsatellite marker methods have been contributing to the functional and mechanistic investigation of UPD, leading to not only the acquisition of clinically valuable information, but also the further clarification of diverse genetic processes and disease pathogenesis.

Summary Neonatal survival in mammals is crucially dependent upon maintenance of body temperature. Neonatal body temperature is largely maintained by thermogenesis in brown adipose tissue (BAT). BAT develops perinatally in mice requiring integration of adipogenic and thermoregulatory gene pathways. We describe a regulatory mutation in the imprinted gene cluster on mouse chromosome 12 resulting in early postnatal lethality. Maternal inheritance of this mutation impairs the ability of young mice to maintain body temperature. While mechanisms of perinatal BAT development are well understood, our work highlights a second phase of BAT recruitment necessary to support small animals newly independent of the nest. We show that the imprinted delta-like homolog 1/preadipocyte factor (Dlk1/Pref1) and iodothyronine deiodinase type 3 (Dio3) functions converge on the development of brown fat at the transition to independent life. This shows that appropriate dosage control at imprinted loci can act as a critical determinant in postnatal survival during phases of physiological adaptation., Graphical Abstract Highlights ► Correct dosage of chromosome 12-imprinted genes is essential for postnatal survival ► Disruption of Dlk1 and Dio3 imprinting causes failure to adapt to independent life ► Maximal brown adipose activity is required as animals first leave the nest ► Dlk1 and Dio3 regulate maturation of brown adipose tissue

Human chromosome 14q32.2 harbors the germline-derived primary DLK1-MEG3 intergenic differentially methylated region (IG-DMR) and the postfertilization-derived secondary MEG3-DMR, together with multiple imprinted genes. Although previous studies in cases with microdeletions and epimutations affecting both DMRs and paternal/maternal uniparental disomy 14-like phenotypes argue for a critical regulatory function of the two DMRs for the 14q32.2 imprinted region, the precise role of the individual DMR remains to be clarified. We studied an infant with upd(14)pat body and placental phenotypes and a heterozygous microdeletion involving the IG-DMR alone (patient 1) and a neonate with upd(14)pat body, but no placental phenotype and a heterozygous microdeletion involving the MEG3-DMR alone (patient 2). The results generated from the analysis of these two patients imply that the IG-DMR and the MEG3-DMR function as imprinting control centers in the placenta and the body, respectively, with a hierarchical interaction for the methylation pattern in the body governed by the IG-DMR. To our knowledge, this is the first study demonstrating an essential long-range imprinting regulatory function for the secondary DMR., Author Summary Genomic imprinting is a process causing genes to be expressed in a parent-of-origin specific manner—some imprinted genes are expressed from maternally inherited chromosomes and others from paternally inherited chromosomes. Imprinted genes are often located in clusters regulated by regions that are differentially methylated according to their parental origin. The human chromosome 14q32.2 imprinted region harbors the germline-derived primary DLK1-MEG3 intergenic differentially methylated region (IG-DMR) and the postfertilization-derived secondary MEG3-DMR, together with multiple imprinted genes. Perturbed dosage of these imprinted genes, for example in patients with paternal and maternal uniparental disomy 14, causes distinct phenotypes. Here, through analysis of patients with microdeletions recapitulating some or all of the uniparental disomy 14 phenotypes, we show that the IG-DMR acts as an upstream regulator for the methylation pattern of the MEG3-DMR in the body but not in the placenta. Importantly, in the body, the MEG3-DMR functions as an imprinting control center. To our knowledge, this is the first study demonstrating an essential function for the secondary DMR in the regulation of multiple imprinted genes. Thus, the results provide a significant advance in the clarification of underlying epigenetic features that can act to regulate imprinting.

Background The field of epigenetics is developing rapidly, however we are only beginning to comprehend the complexity of its influence on gene regulation. Using genomic imprinting as a model we examine epigenetic profiles associated with different forms of gene regulation. Imprinting refers to the expression of a gene from only one of the chromosome homologues in a parental-origin-specific manner. This is dependent on heritable germline epigenetic control at a cis-acting imprinting control region that influences local epigenetic states. Epigenetic modifications associated with imprinting regulation can be compared to those associated with the more canonical developmental regulation, important for processes such as differentiation and tissue specificity. Here we test the hypothesis that these two mechanisms are associated with different histone modification enrichment patterns. Results Using high-throughput data extraction with subsequent analysis, we have found that particular histone modifications are more likely to be associated with either imprinting repression or developmental repression of imprinted genes. H3K9me3 and H4K20me3 are together enriched at imprinted genes with differentially methylated promoters and do not show a correlation with developmental regulation. H3K27me3 and H3K4me3, however, are more often associated with developmental regulation. We find that imprinted genes are subject to developmental regulation through bivalency with H3K4me3 and H3K27me3 enrichment on the same allele. Furthermore, a specific tri-mark signature comprising H3K4me3, H3K9me3 and H4K20me3 has been identified at all imprinting control regions. Conclusion A large amount of data is produced from whole-genome expression and epigenetic profiling studies of cellular material. We have shown that such publicly available data can be mined and analysed in order to generate novel findings for categories of genes or regulatory elements. Comparing two types of gene regulation, imprinting and developmental, our results suggest that different histone modifications associate with these distinct processes. This form of analysis is therefore a useful tool to elucidate the complex epigenetic code associated with genome function and to determine the underlying features conferring epigenetic states.