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BackgroundPhosphoinositide-3 kinase (PI3K)/AKT signaling participates in cellular proliferation, survival and tumorigenesis. The activation of AKT signaling promotes the cellular reprogramming including generation of induced pluripotent stem cells (iPSCs) and dedifferentiation of primordial germ cells (PGCs). Previous studies suggested that AKT promotes reprogramming by activating proliferation and glycolysis. Here we report a line of evidence that supports the notion that AKT signaling is involved in TET-mediated DNA demethylation during iPSC induction.MethodsAKT signaling was activated in mouse embryonic fibroblasts (MEFs) that were transduced with OCT4, SOX2 and KLF4. Multiomics analyses were conducted in this system to examine the effects of AKT activation on cells undergoing reprogramming.ResultsWe revealed that cells undergoing reprogramming with artificially activated AKT exhibit enhanced anabolic glucose metabolism and accordingly increased level of cytosolic α-ketoglutarate (αKG), which is an essential cofactor for the enzymatic activity of the 5-methylcytosine (5mC) dioxygenase TET. Additionally, the level of TET is upregulated. Consistent with the upregulation of αKG production and TET, we observed a genome-wide increase in 5-hydroxymethylcytosine (5hmC), which is an intermediate in DNA demethylation. Moreover, the DNA methylation level of ES-cell super-enhancers of pluripotency-related genes is significantly decreased, leading to the upregulation of associated genes. Finally, the transduction of TET and the administration of cell-permeable αKG to somatic cells synergistically enhance cell reprogramming by Yamanaka factors.ConclusionThese results suggest the possibility that the activation of AKT during somatic cell reprogramming promotes epigenetic reprogramming through the hyperactivation of TET at the transcriptional and catalytic levels.

Retrotransposon Gag-like 5 [RTL5, also known as sushi-ichi-related retrotransposon homolog 8 (SIRH8)] and RTL6 (also known as SIRH3) are eutherian-specific genes presumably derived from a retrovirus and phylogenetically related to each other. They, respectively, encode a strongly acidic and extremely basic protein, and are well conserved among the eutherians. Here, we report that RTL5 and RTL6 are microglial genes with roles in the front line of innate brain immune response. Venus and mCherry knock-in mice exhibited expression of RTL5-mCherry and RTL6-Venus fusion proteins in microglia and appeared as extracellular dots and granules in the central nervous system. These proteins display a rapid response to pathogens such as lipopolysaccharide (LPS), double-stranded (ds) RNA analog and non-methylated CpG DNA, acting both cooperatively and/or independently. Experiments using Rtl6 or Rtl5 knockout mice provided additional evidence that RTL6 and RTL5 act as factors against LPS and dsRNA, respectively, in the brain, providing the first demonstration that retrovirus-derived genes play a role in the eutherian innate immune system. Finally, we propose a model emphasizing the importance of extra-embryonic tissues as the origin site of retrovirus-derived genes. This article has an associated ‘The people behind the papers’ interview.

Our understanding of the spatiotemporal regulation of cardiogenesis is hindered by the difficulties in modeling this complex organ currently by in vitro models. Here we develop a method to generate heart organoids from mouse embryonic stem cell-derived embryoid bodies. Consecutive morphological changes proceed in a self-organizing manner in the presence of the laminin-entactin (LN/ET) complex and fibroblast growth factor 4 (FGF4), and the resulting in vitro heart organoid possesses atrium- and ventricle-like parts containing cardiac muscle, conducting tissues, smooth muscle and endothelial cells that exhibited myocardial contraction and action potentials. The heart organoids exhibit ultrastructural, histochemical and gene expression characteristics of considerable similarity to those of developmental hearts in vivo. Our results demonstrate that this method not only provides a biomimetic model of the developing heart-like structure with simplified differentiation protocol, but also represents a promising research tool with a broad range of applications, including drug testing., Our understanding of the development of the heart has been limited by a lack of in vitro cellular models. Here, the authors treat mouse embryonic stem cell-derived embryoid bodies with laminin-entactin (to mimic the developing microenvironment) and FGF4 to form heart organoids, with atrial and ventricular-like parts.

ObjectiveAlthough immunoglobulin A (IgA) is abundantly expressed in the gut and known to be an important component of mucosal barriers against luminal pathogens, its precise function remains unclear. Therefore, we tried to elucidate the effect of IgA on gut homeostasis maintenance and its mechanism.DesignWe generated various IgA mutant mouse lines using the CRISPR/Cas9 genome editing system. Then, we evaluated the effect on the small intestinal homeostasis, pathology, intestinal microbiota, cytokine production, and immune cell activation using intravital imaging.ResultsWe obtained two lines, with one that contained a tm/tm) and the other that lacked the most constant region of the IgH α chain, which resulted in the deficiency of IgA production (IgA−/−). IgA−/− exhibited spontaneous inflammation in the ileum but not the other parts of the gastrointestinal tract. Associated with this, there were significantly increased lamina propria CD4+ T cells, elevated productions of IFN-γ and IL-17, increased ileal segmented filamentous bacteria and skewed intestinal microflora composition. Intravital imaging using Ca2+ biosensor showed that IgA−/− had elevated Ca2+ signalling in Peyer’s patch B cells. On the other hand, IgAtm/tm seemed to be normal, suggesting that the IgA cytoplasmic tail is dispensable for the prevention of the intestinal disorder.ConclusionIgA plays an important role in the mucosal homeostasis associated with the regulation of intestinal microbiota and protection against mucosal inflammation especially in the ileum.

Epigenetic properties of cultured embryonic stem cells (ESCs), including DNA methylation imprinting, are important because they affect the developmental potential. Here, we tested a variety of culture media, including knockout serum replacement (KSR) and fetal bovine serum (FBS) with or without inhibitors of Gsk3β and Mek1/2 (2i) at various time points. In addition to the previously known passage-dependent global changes, unexpected dynamic DNA methylation changes occurred in both maternal and paternal differentially methylated regions: under the widely used condition of KSR with 2i, a highly hypomethylated state occurred at early passages (P1-7) as well as P10, but DNA methylation increased over further passages in most conditions, except under KSR with 2i at P25. Dramatic DNA demethylation under KSR+2i until P25 was associated with upregulated Tet1 and Parp1, and their related genes, whereas 2i regulated the expressions of DNA methyltransferase-related genes for the change in DNA methylation during the cumulative number of passages. Although DNA methylation imprinting is more labile under KSR with and without 2i, it can be more faithfully maintained under condition of cooperative FBS and 2i. Thus, our study will provide the useful information for improved epigenetic control of ESCs and iPSCs in applications in regenerative medicine.

Paternally expressed 11/Retrotransposon-like 1 (Peg11/Rtl1) knockout (KO) mice show mid- to late fetal lethality or late fetal growth retardation associated with frequent neonatal lethality. The lethal phenotype is largely dependent on genetic background and becomes more severe with each succeeding generation in the course of backcross experiments to C57BL/6 (B6). We previously suggested that these lethal and growth phenotypes in the fetal stages were due to severe defects in placental fetal capillaries in the labyrinth layer. In this study, we re-examined KO fetuses and placentas and confirmed that the severe clogging of fetal capillaries was associated with KO fetuses showing mid-fetal lethality with internal bleeding. Importantly, the basal region of the fetal capillary network was specifically damaged, also leading to poor expansion of the labyrinth layer and placental size reduction in the later stage. An apparent down-regulation of transmembrane protein 100 (Tmem100), mesenchyme homeobox 2 (Meox2) and lymphatic vessel endothelial hyaluronan receptor 1 (Lyve1) expression were observed in earlier stage placentas even before apparent size reduction became, suggesting that these genes are involved in the maintenance of fetal capillaries associated with Peg11/Rtl1 during development.

In the human genome, there are approximately 30 LTR retrotransposon-derived genes, such as the sushi-ichi retrotransposon homologues (SIRH) and the paraneoplastic Ma antigen (PNMA) family genes. They are derivatives from the original LTR retrotransposons and each gene seems to have its own unique function. PEG10/SIRH1 as well as PEG11/RTL1/SIRH2 and SIRH7/LDOC1 play essential roles in placenta formation, maintenance of fetal capillaries and the differentiation/maturation of a variety of placental cells, respectively. All of this evidence provides strong support for their contribution to the evolution of viviparity in mammals via their eutherian-specific functions. SIRH11/ZCCHC16 is an X-linked gene that encodes a CCHC type of zinc-finger protein that exhibits high sequence identity to the LTR retrotransposon Gag protein and its deletion causes abnormal behavior related to cognition, including attention, impulsivity and working memory, possibly via the locus coeruleus noradrenaergic system in mice. Therefore, we have suggested that the acquisition of SIRH11/ZCCHC16 was involved in eutherian brain evolution. Interestingly, SIRH11/ZCCHC16 displays lineage-specific structural and putative species-specific functional variations in eutherians, suggesting that it contributed to the diversification of eutherians via increasing evolutionary fitness by these changes.

Sirh7/Ldoc1 [sushi-ichi retrotransposon homolog 7/leucine zipper, downregulated in cancer 1, also called mammalian retrotransposon-derived 7 (Mart7)] is one of the newly acquired genes from LTR retrotransposons in eutherian mammals. Interestingly, Sirh7/Ldoc1 knockout (KO) mice exhibited abnormal placental cell differentiation/maturation, leading to an overproduction of placental progesterone (P4) and placental lactogen 1 (PL1) from trophoblast giant cells (TGCs). The placenta is an organ that is essential for mammalian viviparity and plays a major endocrinological role during pregnancy in addition to providing nutrients and oxygen to the fetus. P4 is an essential hormone in the preparation and maintenance of pregnancy and the determination of the timing of parturition in mammals; however, the biological significance of placental P4 in rodents is not properly recognized. Here, we demonstrate that mouse placentas do produce P4 in mid-gestation, coincident with a temporal reduction in ovarian P4, suggesting that it plays a role in the protection of the conceptuses specifically in this period. Pregnant Sirh7/Ldoc1 knockout females also displayed delayed parturition associated with a low pup weaning rate. All these results suggest that Sirh7/Ldoc1 has undergone positive selection during eutherian evolution as a eutherian-specific acquired gene because it impacts reproductive fitness via the regulation of placental endocrine function.

The recent successful establishment of mouse parthenogenetic haploid embryonic stem cells (phESCs) and androgenetic haploid ESCs (ahESCs) has stimulated genetic research not only in vitro but also in vivo because of the germline competence of these cell lines. However, it is difficult to maintain the haploid status over time without a frequent sorting of the G1 phase haploid ESCs by fluorescence-activated cell sorting (FACS) at short intervals, because haploid cells tend to readily self-diploidize. To overcome this spontaneous diploid conversion, we developed a phESC culture condition using a small molecular inhibitor of Wee1 kinase to regulate the cell cycle by accelerating the G2/M phase transition and preventing re-entry into extra G1/S phase. Here, we demonstrate that, under this condition, phESCs maintained the haploid status for at least 4 weeks without the need for FACS. This method will greatly enhance the availability of these cells for genetic screening.

During mouse development, imprinted X chromosome inactivation (XCI) is observed in preimplantation embryos and is inherited to the placental lineage, whereas random XCI is initiated in the embryonic proper. Xist RNA, which triggers XCI, is expressed ectopically in cloned embryos produced by somatic cell nuclear transfer (SCNT). To understand these mechanisms, we undertook a large-scale nuclear transfer study using different donor cells throughout the life cycle. The Xist expression patterns in the reconstructed embryos suggested that the nature of imprinted XCI is the maternal Xist-repressing imprint established at the last stage of oogenesis. Contrary to the prevailing model, this maternal imprint is erased in both the embryonic and extraembryonic lineages. The lack of the Xist-repressing imprint in the postimplantation somatic cells clearly explains how the SCNT embryos undergo ectopic Xist expression. Our data provide a comprehensive view of the XCI cycle in mice, which is essential information for future investigations of XCI mechanisms.

Two major gene families derived from Ty3/Gypsy long terminal repeat (LTR) retrotransposons were recently identified in mammals. The sushi-ichi retrotransposon homologue (SIRH) family comprises 12 genes: 11 in eutherians including Peg10 and Peg11/Rtl1 that have essential roles in the eutherian placenta and 1 that is marsupial specific. Fifteen and 12 genes were reported in the second gene family, para-neoplastic antigen MA (PNMA), in humans and mice, respectively, although their biological functions and evolutionary history remain largely unknown. Here, we identified two novel candidate PNMA genes, PNMA-MS1 and -MS2 in marsupials. Like all eutherian-specific PNMA genes, they exhibit the highest homology to a Gypsy12_DR (DR, Danio rerio) Gag protein. PNMA-MS1 is conserved in both Australian and South American marsupial species, the tammar wallaby and grey short-tailed opossum. However, no PNMA-MS1 orthologue was found in eutherians, monotremes or non-mammalian vertebrates. PNMA-MS1 was expressed in the ovary, mammary gland and brain during development and growth in the tammar, suggesting that PNMA-MS1 may have acquired a marsupial-specific function. However, PNMA-MS2 seems to be a pseudogene. The absence of marsupial orthologues of eutherian PNMA genes suggests that the retrotransposition events of the Gypsy12_DR-related retrotransposons that gave rise to the PNMA family occurred after the divergence of marsupials and eutherians.

Pluripotent stem cells can be classified into two distinct states, naïve and primed, which show different degrees of potency. One difficulty in stem cell research is the inability to distinguish these states in live cells. Studies on female mice have shown that reactivation of inactive X chromosomes occurs in the naïve state, while one of the X chromosomes is inactivated in the primed state. Therefore, we tried to distinguish the two states by monitoring X chromosome reactivation. Thus far, X chromosome reactivation has been analysed using fixed cells; here, we inserted different fluorescent reporter gene cassettes (mCherry and eGFP) into each X chromosome. Using these knock-in “MOMIJI” mice, we detected X chromosome reactivation accurately in live embryos, and confirmed that the pluripotent states of embryos were stable ex vivo, as represented by embryonic and epiblast stem cells in terms of X chromosome reactivation. Thus, MOMIJI mice provide a simple and accurate method for identifying stem cell status based on X chromosome reactivation.

Cloning mammals by somatic cell nuclear transfer (SCNT) has become an established procedure, but the success rate remains low and gene expression abnormalities are also observed. In addition, SCNT pups exhibited an abnormal gene expression profile with a high degree of heterogeneity among individuals. Recently, we reported that somatic clones treated with trichostatin A (TSA) exhibited a significantly improved success rate, probably due to its effects on chromatin remodeling and histone modification in early embryos. Here we show that the TSA treatment also improves the long-term consistency of genome-wide gene expression regulation: the total number of genes commonly exhibiting up- or downregulation in the TSA clone pups decreased to half of the conventional SCNT pups, and the variation among individuals observed in the SCNT pups was also reduced to the level of the pups produced by the intracytoplasmic sperm injection (ICSI) method. Interestingly, the total gene expression profile of the TSA clones came to resemble that of the ICSI pups.

Differences between male and female mammals are initiated by embryonic differentiation of the gonad into either a testis or an ovary. However, this may not be the sole determinant. There are reports that embryonic sex differentiation might precede and be independent of gonadal differentiation, but there is little molecular biological evidence for this. To test for sex differences in early-stage embryos, we separated male and female blastocysts using newly developed non-invasive sexing methods for transgenic mice expressing green fluorescent protein and compared the gene-expression patterns. From this screening, we found that the Fthl17 (ferritin, heavy polypeptide-like 17) family of genes was predominantly expressed in female blastocysts. This comprises seven genes that cluster on the X chromosome. Expression analysis based on DNA polymorphisms revealed that these genes are imprinted and expressed from the paternal X chromosome as early as the two-cell stage. Thus, by the time zygotic genome activation starts there are already differences in gene expression between male and female mouse embryos. This discovery will be important for the study of early sex differentiation, as clearly these differences arise before gonadal differentiation.

Cloning Futures Cloning mammals by somatic cell nuclear transfer is a technique with many potential applications in regenerative medicine, agriculture, and pharmaceutics; however, it is inefficient because of the incidence of aberrant genomic reprogramming. Inoue et al. (p. 496 , published online 16 September) found that the gene product of Xist , which normally inactivates one of the two X chromosomes in females, was unexpectedly expressed ectopically from active X chromosomes in cloned mice. When Xist was deleted from the mice, gene expression returned to normal and the efficiency of somatic cell nuclear transfer increased about ninefold, offering promise for future nuclear transfer technology.

Mammalian androgenetic embryos can be produced by pronuclear exchange of fertilized oocytes or by dispermic in vitro fertilization of enucleated oocytes. Here, we report a new technique for producing mouse androgenetic embryos by injection of two round spermatid nuclei into oocytes, followed by female chromosome removal. We found that injection of round spermatids resulted in high rates of oocyte survival (88%). Androgenetic embryos thus produced developed into mid-gestation fetuses at various rates, depending on the mouse strain used. All the fetuses examined maintained paternally specific genomic imprinting memories. This technique also enabled us to produce complete heterozygous F1 embryos by injecting two spermatids from different strains. The best rate of fetal survival (12% per embryos transferred) was obtained with C57BL/6 x DBA/2 androgenetic embryos. We also generated embryonic stem cell lines efficiently with the genotype of Mus musculus domesticus x M. m. molossinus. Thus, injection of two round spermatid nuclei followed by maternal enucleation is an effective alternative method of producing androgenetic embryos that consistently develop into blastocysts and mid-gestation fetuses.

Mice with maternal duplication of proximal Chromosome 11 (MatDp(prox11)), where Meg1/Grb10 is located, exhibit pre- and postnatal growth retardation. To elucidate the responsible imprinted gene for the growth abnormality, we examined the precise structure and regulatory mechanism of this imprinted region and generated novel model mice mimicking the pattern of imprinted gene expression observed in the MatDp(prox11) by deleting differentially methylated region of Meg1/Grb10 (Meg1-DMR). It was found that Cobl and Ddc, the neighboring genes of Meg1/Grb10, also comprise the imprinted region. We also found that the mouse-specific repeat sequence consisting of several CTCF-binding motifs in the Meg1-DMR functions as a silencer, suggesting that the Meg1/Grb10 imprinted region adopted a different regulatory mechanism from the H19/Igf2 region. Paternal deletion of the Meg1-DMR (+/DeltaDMR) caused both upregulation of the maternally expressed Meg1/Grb10 Type I in the whole body and Cobl in the yolk sac and loss of paternally expressed Meg1/Grb10 Type II and Ddc in the neonatal brain and heart, respectively, demonstrating maternalization of the entire Meg1/Grb10 imprinted region. We confirmed that the +/DeltaDMR mice exhibited the same growth abnormalities as the MatDp(prox11) mice. Fetal and neonatal growth was very sensitive to the expression level of Meg1/Grb10 Type I, indicating that the 2-fold increment of the Meg1/Grb10 Type I is one of the major causes of the growth retardation observed in the MatDp(prox11) and +/DeltaDMR mice. This suggests that the corresponding human GRB10 Type I plays an important role in the etiology of Silver-Russell syndrome caused by partial trisomy of 7p11-p13.

Eutherian placenta, an organ that emerged in the course of mammalian evolution, provides essential architecture, the so-called feto-maternal interface, for fetal development by exchanging nutrition, gas and waste between fetal and maternal blood. Functional defects of the placenta cause several developmental disorders, such as intrauterine growth retardation in humans and mice. A series of new inventions and/or adaptations must have been necessary to form and maintain eutherian chorioallantoic placenta, which consists of capillary endothelial cells and a surrounding trophoblast cell layer(s). Although many placental genes have been identified, it remains unknown how the feto-maternal interface is formed and maintained during development, and how this novel design evolved. Here we demonstrate that retrotransposon-derived Rtl1 (retrotransposon-like 1), also known as Peg11 (paternally expressed 11), is essential for maintenance of the fetal capillaries, and that both its loss and its overproduction cause late-fetal and/or neonatal lethality in mice.

Ono, R., Ishii, M., Fujihara, Y. et al. Double strand break repair by capture of retrotransposon sequences and reverse-transcribed spliced mRNA sequences in mouse zygotes. Sci Rep 5, 12281 (2015). https://doi.org/10.1038/srep12281, The CRISPR/Cas system efficiently introduces double strand breaks (DSBs) at a genomic locus specified by a single guide RNA (sgRNA). The DSBs are subsequently repaired through non-homologous end joining (NHEJ) or homologous recombination (HR). Here, we demonstrate that DSBs introduced into mouse zygotes by the CRISPR/Cas system are repaired by the capture of DNA sequences deriving from retrotransposons, genomic DNA, mRNA and sgRNA. Among 93 mice analysed, 57 carried mutant alleles and 22 of them had long de novo insertion(s) at DSB-introduced sites; two were spliced mRNAs of Pcnt and Inadl without introns, indicating the involvement of reverse transcription (RT). Fifteen alleles included retrotransposons, mRNAs, and other sequences without evidence of RT. Two others were sgRNAs with one containing T7 promoter-derived sequence suggestive of a PCR product as its origin. In conclusion, RT-product-mediated DSB repair (RMDR) and non-RMDR repair were identified in the mouse zygote. We also confirmed that both RMDR and non-RMDR take place in CRISPR/Cas transfected NIH-3T3 cells. Finally, as two de novo MuERV-L insertions in C57BL/6 mice were shown to have characteristic features of RMDR in natural conditions, we hypothesize that RMDR contributes to the emergence of novel DNA sequences in the course of evolution.

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.

Somatic cell nuclear transfer technology has been applied to produce live clones successfully in several mammalian species, but the success rates are very low. In mice, about half of the nuclear transfer embryos undergo implantation, but very few survive to term. We undertook detailed histological analyses of placentas from cloned mouse embryos generated from cumulus cells at 10.5 dpc of pregnancy, by which stage most clones have terminated their development. At 10.5 dpc, the extraembryonic tissues displayed several defined histological patterns, each reflecting their stage of developmental arrest. The most notable abnormality was the poor development of the spongiotrophoblast layer of diploid cells. This is in contrast to the placental hyperplasia frequently observed in somatic clones at 12.5 dpc or later stages. A variety of structural abnormalities were also observed in the embryos. Both placental and embryonic defects likely contribute to the low success rate of the mouse clones.

We produced XX↔XY chimeras by using embryos whose X chromosomes were tagged with EGFP (X*), making the fluorescent green female (XX*) germ cells easily distinguishable from their nonfluorescent male (XY) counterparts. Taking advantage of tagging with EGFP, the XX* “prospermatogonia” were isolated from the testes, and the status of their genomic imprinting was examined. It was shown that these XX cells underwent a paternal imprinting, despite their chromosomal constitution. As previously indicated in sex-reversal XXsxr testes, we also found a few green XX* germ cells developed as “eggs” within the seminiferous tubules of XX*↔XY chimeric testes. These cells were indistinguishable from XX* prospermatogonia at birth but resumed oogenesis in a testicular environment. The biological nature of the “testicular eggs” was examined by recovering the eggs from chimeric testes. The testicular eggs not only formed an egg-specific structure, the zona pellucida, but also were able to fuse with sperm. The collected testicular eggs were indicated to undergo maternal imprinting, despite the testicular environment. The genomic imprinting did not always follow the environmental conditions of where the germ cells resided; rather, it was defined by the sex that was chosen by the germ cells at early embryonic stage.

Although germline cells can form multipotential embryonic stem (ES)/embryonic germ (EG) cells, these cells can be derived only from embryonic tissues, and such multipotent cells have not been available from neonatal gonads. Here we report the successful establishment of ES-like cells from neonatal mouse testis. These ES-like cells were phenotypically similar to ES/EG cells except in their genomic imprinting pattern. They differentiated into various types of somatic cells in vitro under conditions used to induce the differentiation of ES cells and produced teratomas after inoculation into mice. Furthermore, these ES-like cells formed germline chimeras when injected into blastocysts. Thus, the capacity to form multipotent cells persists in neonatal testis. The ability to derive multipotential stem cells from the neonatal testis has important implications for germ cell biology and opens the possibility of using these cells for biotechnology and medicine.

Mice with maternal duplication of proximal chromosome 6 die in utero at an early embryonic stage. Recently, two imprinted genes, paternally expressedSgceand maternally expressedAsb4, were identified in this region. This report analyzes the imprinting status of genes within a 1-Mb region containing these two genes.Peg10, which is next toSgce, shows complete paternal expression, likeSgce. Conversely,Neurabin, Pon2, and Pon3show preferential maternal expression at embryonic stages, although they all show biallelic expression in neonatal tissues. These results demonstrate that there is a large novel imprinted gene cluster in this region. 5′-RACE (Rapid Amplification of cDNA Ends) analysis ofPeg10revealed the existence of a novel first exon separate from the second exon, which encoded two putative ORFs similar to the viral Gag and Pol proteins. A differentially methylated region established in sperm and eggs is located just within the region containing the two first exons ofPeg10andSgce, and may play an important role in regulating the two paternally expressed genes:Peg10andSgce.

The human chromosome 15q11-q13 region is one of the most intriguing imprinted domains, and the abnormalities inherited are associated with neurological disorders including Prader-Willi syndrome (PWS), Angelman syndrome (AS) and autism. Recently we have identified a novel maternally expressed gene, ATP10C, that encodes a putative aminophospholipid translocase within this critical region, 200 kb distal to UBE3A in an imprinted domain on human chromosome 15. ATP10C, with UBE3A, displayed tissue-specific imprinting with predominant expression of the maternal allele in the brain. In this study, we demonstrated that the mouse homologue, Atp10c/pfatp, showed tissue-specific maternal expression in the hippocampus and olfactory bulb, which overlapped the region of imprinted Ube3a expression. These data suggest that the imprinted transcript of Atp10c in the specific region of CNS may be associated with neurological disorders including AS and autism.

The imprinted mouse gene Meg1/Grb10 is expres sed from maternal alleles in almost all tissues and organs, except in the brain, where it is expressed biallelically, and the paternal allele is expressed preferentially in adulthood. In contrast, the human GRB10 gene shows equal biallelic expression in almost all tissues and organs, while it is almost always expressed paternally in the fetal brain. To elucidate the molecular mechanisms of the complex imprinting patterns among the different tissues and organs of humans and mice, we analyzed in detail both the genomic structures and tissue-specific expression profiles of these species. Experiments using 5'-RACE and RT-PCR demonstrated the existence in both humans and mice of novel brain- specific promoters, in which only the paternal allele was active. The promoters were located in the primary differentially methylated regions. Interest ingly, CTCF-binding sites were found only in the mouse promoter region where CTCF showed DNA methylation-sensitive binding activity. Thus, the insulator function of CTCF might cause reciprocal maternal expression of the Meg1/Grb10 gene from another upstream promoter in the mouse, whereas the human upstream promoter is active in both parental alleles due to the lack of the corresponding insulator sequence in this region.

Human 11p15.5, as well as its orthologous mouse 7F4/F5, is known as the imprinting domain extending from IPL/Ipl to H19. OBPH1 and Obph1 are located beyond the presumed imprinting boundary on the IPL/Ipl side. We determined full-length cDNAs and complete genomic structures of both orthologues. We also investigated their precise imprinting and methylation status. The orthologues resembled each other in genomic structure and in the position of the 5' CpG island and were expressed ubiquitously. OBPH1 and Obph1 were predominantly expressed from the maternal allele only in placenta, with hypo- and not differentially methylated 5' CpG islands in both species. These results suggested that the imprinting domain would extend beyond the presumed imprinting boundary and that methylation of the 5' CpG island was not associated with the imprinting status in either species. It remains to be elucidated whether the gene is under the control of the KIP2/LIT1 subdomain or is regulated by a specific mechanism. Analysis of the precise genomic sequence around the region should help resolve this question.

Although a variety of phenotypes and epigenetic alterations have been reported in animals cloned from somatic cells, the exact nature and consequences of cloning remain unclear. We cloned mice using fresh or short-term cultures of donor cells (cumulus cells, immature Sertoli cells, and fetal or adult fibroblast cells) with defined genetic backgrounds, and then compared the phenotypic and epigenetic characteristics of the cloned mice with those of fertilization-derived control mice. Irrespective of the nucleus-donor cell type, about 50% of the reconstructed embryos developed to the morula/blastocyst stage, but about 90% of these clones showed arrested development between days 5 and 8, shortly after implantation. Most of the clones were alive at term, readily recovered respiration, and did not show any malformations or overgrowths. However, their placentas were two- to threefold larger than those of the controls, due to hyperplasia of the basal (or spongiotrophoblast) layer. Although there was significant suppression of a subset of both imprinted and non-imprinted placental genes, fetal gene suppression was minimal. The seven imprinted genes that we examined were all expressed correctly from the parental alleles. These findings were consistent for every cell type from the midgestation through term stages. Therefore, cloning by nuclear transfer does not perturb the parent-specific imprinting memory that is established during gametogenesis, and the phenotypic and epigenetic effects of cloning are restricted to placental development at the midgestation and term stages. Twelve male mice that were born in a normal manner following nuclear transfer with immature Sertoli cells (B6D2F1 genetic background) were subjected to long-term observation. They died earlier than the genotype-matched controls (50% survival point: 550 days vs. 1028 days, respectively), most probably due to severe pneumonia, which indicates that unexpected phenotypes can appear as a result of the long-term effects of somatic cell cloning.

In mice, the establishment of paternal genomic imprinting in male germ cells starts at midgestation, as suggested by DNA methylation analyses of differentially methylated regions (DMRs). However, this information is based on averages from mixed populations of germ cells, and the DNA methylation pattern might not always provide a full representation of imprinting status. To obtain more detailed information on the establishment of paternal imprinting, single prospermatogonia at Embryonic Days 15.5 (E15.5), E16.5, and E17.5 and at Day 0.5 after birth were cloned using nuclear transfer; previous reports suggested that cloned embryos reflected the donor's genomic imprinting status. Then, the resultant fetuses (E9.5) were analyzed for the DNA methylation pattern of three paternal DMRs (IG-DMR, H19 DMR, and Rasgrf1 DMR) and the expression pattern of imprinted genes therein. The overall data indicated that establishment of genomic imprinting in all paternally imprinted regions was completed by E17.5, following a short intermediate period at E16.5. Furthermore, comparison between the methylation status of DMRs and the expression profiles of imprinted genes suggested that methylation of the IG-DMR, but not the H19 DMR, solely governed the control of its imprinted gene cluster. The Rasgrf1 DMR seemed to be imprinted later than the other two genes. We also found that the methylation status of the Gtl2 DMR, the secondary DMR that acquires DNA methylation after fertilization, was likely to follow the methylation status of the upstream IG-DMR. Thus, the systematic analyses of prospermatogonium-derived embryos provided additional important information on the establishment of paternal imprinting.

X-chromosome inactivation (XCI) equalizes gene expression between the sexes by inactivating one of the two X chromosomes in female mammals. Xist has been considered as a major cis-acting factor that inactivates the paternally derived X chromosome (Xp) in preimplantation mouse embryos (imprinted XCI). Ftx has been proposed as a positive regulator of Xist. However, the physiological role of Ftx in female animals has never been studied. We recently reported that Ftx is located in the cis-acting regulatory region of the imprinted XCI and expressed from the inactive Xp, suggesting a role in the imprinted XCI mechanism. Here we examined the effects on imprinted XCI using targeted deletion of Ftx. Disruption of Ftx did not affect the survival of female embryos or expression of Xist and other X-linked genes in the preimplantation female embryos. Our results indicate that Ftx is dispensable for imprinted XCI in preimplantation embryos.

In mammalian primordial germ cells (PGCs), DNA demethylation is indispensible for parental imprint erasure, which is a reprogramming process essential for normal developmental potential. Thus, it is important to elucidate how DNA demethylation occurs in each imprinted region in PGCs and to determine which DNA demethylation pathway, passive or active, essentially contributes to the erasure of the imprint. Here, we report that active DNA demethylation via a putative Poly(ADP-ribose) polymerase (PARP) pathway is involved in H19-DMR imprint erasure in PGCs, as shown by an in vivo small molecule inhibitor assay. To the best of our knowledge, this is the first direct demonstration of a DNA replication-independent active DNA demethylation pathway in the erasure process of genomic imprinting in PGCs in vivo. The data also suggest that active DNA demethylation plays a significant role in the complete erasure of paternal imprinting in the female germ line.

A large imprinted gene cluster in human chromosome 11p15.5 has been implicated in Beckwith-Wiedemann syndrome and Wilms' tumor. We have identified a paternally expressed imprinted gene, PEG8/IGF2AS, in this locus. It is transcribed in the opposite direction to the IGF2 transcripts and some genomic regions are shared with the IGF2 gene, as in the case of the mouse imprinted Igf2as gene reported previously by T. Moore et al. As to the relationship between these genomic regions, the human and mouse genes are very similar but there is no homology in their middle parts. Interestingly, PEG8/IGF2AS and IGF2 were found to be overexpressed in Wilms' tumor samples, at levels over ten and a hundred times higher than that in normal kidney tissues neighboring the tumors, respectively. These findings indicate that PEG8/IGF2AS is a good marker of Wilms' tumor and also suggest the possibility of PEG8/IGF2AS being one of the candidate Wilms' tumor genes.

Peg3 is the first imprinted gene to be identified on mouse proximal chromosome 7; the human PEG3 homologue is on chromosome 19q13.4. Peg3 encodes a C(2)H(2)-type zinc finger protein that is expressed only from the paternal allele in embryos and adult brain. The gene has been shown to regulate maternal behavior and offspring growth and has been implicated in the TNF-NFkappaB signal pathway. Here we show that Peg3 consists of nine exons spanning 26 kb. The 5' region of the gene contains a region rich in repeated sequences and a CpG island. Analysis of expressed sequence tags revealed a transcript present upstream of the island and on the strand opposite to Peg3. These structural features and DNA sequences are conserved in mouse and human. The 5' region of Peg3 is preferentially methylated on the inactive maternal allele, as shown by comparing embryos with paternal (PatDp. prox7) and maternal (MatDp.prox7) duplication of proximal chromosome 7. Recently, a new maternally expressed Zim1 gene located downstream of Peg3 was identified, which suggested that another imprinted cluster is present on proximal chromosome 7.

Imprinted genes display parent-of-origin–dependent monoallelic expression that apparently regulates complex mammalian traits, including growth and behavior. The Peg3 gene is expressed in embryos and the adult brain from the paternal allele only. A mutation in the Peg3 gene resulted in growth retardation, as well as a striking impairment of maternal behavior that frequently resulted in death of the offspring. This result may be partly due to defective neuronal connectivity, as well as reduced oxytocin neurons in the hypothalamus, because mutant mothers were deficient in milk ejection. This study provides further insights on the evolution of epigenetic regulation of imprinted gene dosage in modulating mammalian growth and behavior.

We previously identified Peg1/Mest as a novel paternally expressed gene in the developing mouse embryo. The human PEG1 gene was recently assigned to 7q32 and shown to be imprinted and paternally expressed. Therefore, PEG1 deficiency could participate in the aetiology of pre- and post-natal growth retardation associated with maternal uniparental disomy 7 in humans. We have now initiated the characterization of the Peg1 locus in order to identify and dissect cis-acting elements implicated in its imprinted monoallelic expression. The genomic structure of Peg1 as well as the DNA sequence of the 5'-end of the gene, including 2.4 kb of promoter sequences and covering the first 2 exons, have been determined. Important sequence elements, such as a CpG island spanning exon 1 and direct repeats, are identified and discussed. To address the role of epigenetic modifications in the imprinting of Peg1, a methylation analysis of the Peg1 gene is presented. Partially methylated cytosine residues in 13.5 d.p.c. embryos and undifferentiated ES cells were identified. Using embryos carrying a targetted mutation at the Peg1 locus, we show that this partial promoter methylation pattern reflects a strict parent-of-origin-specific differential methylation: the expressed paternal allele is unmethylated, whereas the silenced maternal allele is fully methylated at the CpG sites studied. That the gametes carry the epigenetic information necessary to lay down this allele-specific methylation pattern is suggested by analysis of DNA isolated from sperm and parthenogenetic embryos.

We have established a systematic screen for imprinted genes using a subtraction-hybridization method with day 8.5 fertilized and parthenogenetic embryos. Two novel imprinted genes, Peg1/Mest and Peg3, were identified previously by this method, along with the two known imprinted genes, Igf2 and Snrpn. Recently three additional candidate imprinted genes, Peg5-7 , were detected and Peg5 is analyzed further in this study. The cDNA sequence of Peg5 is identical to Neuronatin, a gene recently reported to be expressed mainly in the brain. Two novel spliced forms were detected with some additional sequence in the middle of the known Neuronatin sequences. All alternatively spliced forms of Peg5 were expressed only from the paternal allele, confirmed using DNA polymorphism in a subinterspecific cross. Peg5/Neuronatin maps to sub-distal Chr 2, proximal to the previously established imprinted region where imprinted genes cause abnormal shape and behavior in neonates.

Embryogenesis in placental mammals is sustained by exquisite interplay between the embryo proper and placenta. UTF1 is a developmentally regulated gene expressed in both cell lineages. Here, we analyzed the consequence of loss of the UTF1 gene during mouse development. We found that homozygous UTF1 mutant newborn mice were significantly smaller than wild-type or heterozygous mutant mice, suggesting that placental insufficiency caused by the loss of UTF1 expression in extra-embryonic ectodermal cells at least in part contributed to this phenotype. We also found that the effects of loss of UTF1 expression in embryonic stem cells on their pluripotency were very subtle. Genome structure and sequence comparisons revealed that the UTF1 gene exists only in placental mammals. Our analyses of a family of genes with homology to UTF1 revealed a possible mechanism by which placental mammals have evolved the UTF1 genes.

In mice, one of the major epigenetic errors associated with somatic cell nuclear transfer (SCNT) is ectopic expression of Xist during the preimplantation period in both sexes. We found that this aberrant Xist expression could be impeded by deletion of Xist from the putative active X chromosome in donor cells. In male clones, it was also found that prior injection of Xist-specific siRNA could significantly improve the postimplantation development of cloned embryos as a result of a significant repression of Xist at the morula stage. In this study, we examined whether the same knockdown strategy could work as well in female SCNT-derived embryos. Embryos were reconstructed with cumulus cell nuclei and injected with Xist-specific siRNA at 6–7 h after oocyte activation. RNA FISH analysis revealed that siRNA treatment successfully repressed Xist RNA at the morula stage, as shown by the significant decrease in the number of cloud-type Xist signals in the blastomere nuclei. However, blastomeres with different sizes (from “pinpoint” to “cloud”) and numbers of Xist RNA signals remained within single embryos. After implantation, the dysregulated Xist expression was normalized autonomously, as in male clones, to a state of monoallelic expression in both embryonic and extraembryonic tissues. However, at term there was no significant improvement in the survival of the siRNA-injected cloned embryos. Thus, siRNA injection was largely effective in repressing the Xist overexpression in female cloned embryos but failed to rescue them, probably because of an inability to mimic consistent monoallelic Xist expression in these embryos. This could only be achieved in female embryos by applying a gene knockout strategy rather than an siRNA approach.

Since the discovery of oxidative demethylation of methylcytosine (mC) by Tet enzymes, an analytical method has been urgently needed that would enable the identification of mC and hydroxymethylcytosine (hmC) at the single base resolution level, because their roles in gene regulation are quite different from each other. However, the bisulfite sequencing method, the gold standard for DNA methylation analysis at present, does not distinguish them. Recently reported alternative methods, such as oxBS-seq and TAB-seq, are not even capable of determining mC and hmC simultaneously. Here, we report a novel method for the direct identification of mC, hmC and unmodified cytosine (C) at a single base resolution. We named this method the Enzyme-assisted Identification of Genome Modification Assay (EnIGMA), and it was demonstrated to indeed have a highly efficient and reliable analytic capacity for distinguishing them. We also successfully applied this novel method to the analysis of the maintenance of the DNA methylation status of imprinted H19-DMR. Importantly, hydroxymethylation plays an ambivalent role in the maintenance of the genome imprinting memory in parental genomes essential for normal development, shedding new light on the epigenetic regulation in ES cells.

Cloning mammals by somatic cell nuclear transfer (SCNT) is highly inefficient. Most SCNT-generated embryos die after implantation because of unidentified, complex epigenetic errors in the process of postimplantation embryonic development. Here we identify the most upstream level of dysfunction leading to impaired development of clones by using RNAi against Xist , a gene responsible for X chromosome inactivation (XCI). A prior injection of Xist -specific siRNA into reconstructed oocytes efficiently corrected SCNT-specific aberrant Xist expression at the morula stage, but failed to do so thereafter at the blastocyst stage. However, we found that shortly after implantation, this aberrant XCI status in cloned embryos had been corrected autonomously in both embryonic and extraembryonic tissues, probably through a newly established XCI control for postimplantation embryos. Embryo transfer experiments revealed that siRNA-treated embryos showed 10 times higher survival than controls as early as embryonic day 5.5 and this high survival persisted until term, resulting in a remarkable improvement in cloning efficiency (12% vs. 1% in controls). Importantly, unlike control clones, these Xist -siRNA clones at birth showed only a limited dysregulation of their gene expression, indicating that correction of Xist expression in preimplantation embryos had a long-term effect on their postnatal normality. Thus, contrary to the general assumption, our results suggest that the fate of cloned embryos is determined almost exclusively before implantation by their XCI status. Furthermore, our strategy provides a promising breakthrough for mammalian SCNT cloning, because RNAi treatment of oocytes is readily applicable to most mammal species.

Parent-of-origin-dependent expression of imprinted genes is mostly associated with allele-specific DNA methylation of the CpG islands (CGIs) called germ line differentially methylated regions (gDMRs). Although the essential role of gDMRs for genomic imprinting has been well established, little is known about how they evolved. In several imprinted loci, the CGIs forming gDMRs may have emerged with the insertion of a retrotransposon or retrogene. To examine the generality of the hypothesis that the CGIs forming gDMRs were novel CGIs recently acquired during mammalian evolution, we reviewed the time of novel CGI emergence for all the maternal gDMR loci using the novel data analyzed in this study combined with the data from previous reports. The comparative sequence analyses using mouse, human, dog, cow, elephant, tammar, opossum, platypus, and chicken genomic sequences were carried out for Peg13, Meg1/Grb10, Plagl1/Zac1, Gnas, and Slc38a4 imprinted loci to obtain comprehensive results. The combined data showed that emergence of novel CGIs occurred universally in the maternal gDMR loci at various time points during mammalian evolution. Furthermore, the analysis of Meg1/Grb10 locus provided evidence that gradual base pair-wise sequence change was involved in the accumulation of CpG sequence, suggesting the mechanism of novel CGI emergence is more complex than the suggestion that CpG sequences originated solely by insertion of CpG-rich transposable elements. We propose that acquisition of novel CGIs was a key genomic change for the evolution of imprinting and that it usually occurred in the maternal gDMR loci.

Faithful transcriptome regulation is important in development and also crucial for applications in reproductive and regenerative medicine. Intracytoplasmic sperm injection (ICSI), one of the human assisted reproductive technologies (ART), has long raised concerns about its influence on development. No clear consensus has been reached, however, in spite of many cohort studies carried out in the last two decades on the children conceived by ICSI and/or in vitro fertilization (IVF). In this study, the pre- and postnatal effects of ICSI were assessed using comprehensive transcriptome and phenotypic analyses in mice under strict conditions. Here we demonstrate that, in contrast to IVF, ICSI induces distinct long-lasting transcriptome change that remains at the neonatal stage. Importantly, no remarkable differences were observed in the ICSI adults in either the gene expression or phenotypic profiles, and there was no indication of transmission to the next generation via natural mating. Our results suggest there are no lifelong or transgenerational effects of ICSI, but the ICSI effects during neonatal period remain to be evaluated.

The evolution of two mammalian-specific traits, viviparous reproduction with a placenta and genomic imprinting, have been addressed by multiple studies of two retrotransposon derived, mammalian-specific genes. These gene targeting experiments in mice, together with recent comparative genomic analyses among three mammalian groups, suggest that extremely rare events; namely exaptations from retrotransposons, made crucial contributions to the establishment and diversification of mammals via placental formation. We propose that nearly neutral evolution, as well as Darwinian evolution (natural selection), plays an important role in the exaptation process. Comparative genomic analysis of various imprinted regions has also revealed that an imprinting control element essential for parent-of-origin specific monoallelic expression of imprinted genes emerged in each of the imprinted regions, possibly by the insertion of exogenous DNAs, such as retrotransposons. In both cases, DNA methylation in germ cells must have been of critical importance to repress the exogenous DNAs inserted into the genomes of mammalian ancestors. We propose that the ability of germ line DNA methylation enabled the emergence of certain mammalian-specific features during the course of evolution.

We assessed the possibility of C57BL/6-Tg (Meg1/Grb10)isn(Meg1 Tg) mice as a non-obese type 2 diabetes (2DM) animal model. Meg1 Tg mice were born normal, but their weight did not increase as much as normal after weaning and showed about 85% of normal size at 20 weeks of age. Body mass index of Meg1 Tg mice was also smaller than that of control mice. The glucose tolerance test and insulin tolerance test showed that Meg1 Tg mice had reduced ability to normalize the blood glucose level. Blood urea nitrogen (BUN) in Meg1 Tg mice (19.6 +/- 1.2 mg/dl) was significantly lower than in controls (22.0 +/- 0.8 mg/dl), while plasma triglyceride, insulin, adiponectin, and resistin levels were significantly higher (202.0 +/- 23.4 mg/dl vs 146.3 +/- 23.4 mg/dl, 152.4 +/- 16.3 pg/ml vs 88.1 +/- 16.9 pg/ml, 74.4 +/- 10.9 microg/ml vs 48.3 +/- 7.0 microg/ml, and 4.0 +/- 0.2 ng/ml vs 3.6 +/- 0.2 ng/ml, respectively). Body, visceral fat weight and liver weights were significantly lower (19.6 +/- 0.4 g vs 24.3 +/- 0.3 g, 376.7 +/- 29.6 mg to 507.5 +/- 23.0 mg, and 906.0 +/- 41.8 mg to 1,001.0 +/- 15.1 mg, respectively). Thus, hyperinsulinemia observed in Meg1 Tg mice indicates that their insulin signaling pathway is somehow inhibited. With high fat diet, the diabetes onset rate of Meg1 Tg mice increased up to 60%. These results suggest that Meg1 Tg mice resemble human 2DM.

Hyperplastic placentas have been reported in several experimental mouse models, including animals produced by somatic cell nuclear transfer, by inter(sub)species hybridization, and by somatic cytoplasm introduction to oocytes followed by intracytoplasmic sperm injection. Of great interest are the gross and histological features common to these placental phenotypes--despite their quite different etiologies--such as the enlargement of the spongiotrophoblast layers. To find morphological clues to the pathways leading to these similar placental phenotypes, we analyzed the ultrastructure of the three different types of hyperplastic placenta. Most cells affected were of trophoblast origin and their subcellular ultrastructural lesions were common to the three groups, e.g., a heavy accumulation of cytoplasmic vacuoles in the trophoblastic cells composing the labyrinthine wall and an increased volume of spongiotrophoblastic cells with extraordinarily dilatated rough endoplasmic reticulum. Although the numbers of trophoblastic glycogen cells were greatly increased, they maintained their normal ultrastructural morphology, including a heavy glycogen deposition throughout the cytoplasm. The fetal endothelium and small vessels were nearly intact. Our ultrastructural study suggests that these three types of placental hyperplasias, with different etiologies, may have common pathological pathways, which probably exclusively affect the development of certain cell types of the trophoblastic lineage during mouse placentation.

Recent cloning technology has been demonstrated successfully using nuclear transfer (NT) techniques to generate embryonic stem (ES) cells. Mice can be cloned from adult somatic cells or ES cells by NT, and such cloned embryos can be used to establish new NT-ES cell lines. However, ES cells derived from parthenogenetic embryos show epigenetic disorders and low potential for normal differentiation unless used to produce subsequent generations of NT-ES lines. Thus, enucleated oocytes can initialize epigenetic modification, but the extent and efficacy of this remain unclear. In this study, our goal was to clarify why the contribution rate of ES cells derived from parthenogenetic embryos (pES) cells appears to improve after NT. We compared gene expression profiles between pES and NT-pES cell lines using DNA microarray analysis and allele-specific DNA methylation analysis. Although changes in expression level were observed for 4% of 34,967 genes, only 81 (0.2%) showed common changes across multiple cell lines. In particular, the expression level of a paternally expressed gene, U2af1-rs1, was significantly increased in all NT-pES cell lines investigated. The methylation status at the upstream differentially methylated region of U2af1-rs1 was also changed significantly after NT. This was observed in NT-pES cells, but also in conventionally produced NT-ES cells, which has never been reported previously. These results suggest that NT affects the epigenetic status of a few gene regions in common and that a change in the methylation status of U2af1-rs1 could be used as a genetic marker to investigate the effects of NT. Disclosure of potential conflicts of interest is found at the end of this article.

Among mammals, only eutherians and marsupials are viviparous and have genomic imprinting that leads to parent-of-origin-specific differential gene expression. We used comparative analysis to investigate the origin of genomic imprinting in mammals. PEG10 (paternally expressed 10) is a retrotransposon-derived imprinted gene that has an essential role for the formation of the placenta of the mouse. Here, we show that an orthologue of PEG10 exists in another therian mammal, the marsupial tammar wallaby (Macropus eugenii), but not in a prototherian mammal, the egg-laying platypus (Ornithorhynchus anatinus), suggesting its close relationship to the origin of placentation in therian mammals. We have discovered a hitherto missing link of the imprinting mechanism between eutherians and marsupials because tammar PEG10 is the first example of a differentially methylated region (DMR) associated with genomic imprinting in marsupials. Surprisingly, the marsupial DMR was strictly limited to the 5′ region of PEG10, unlike the eutherian DMR, which covers the promoter regions of both PEG10 and the adjacent imprinted gene SGCE. These results not only demonstrate a common origin of the DMR-associated imprinting mechanism in therian mammals but provide the first demonstration that DMR-associated genomic imprinting in eutherians can originate from the repression of exogenous DNA sequences and/or retrotransposons by DNA methylation., Author Summary Genomic imprinting is a gene regulatory mechanism controlling parent-of-origin-dependent expression of genes. In eutherians, imprinting is essential for fetal and placental development and defects in this mechanism are the cause of several genetic disorders. In eutherian mammals, genomic imprinting is controlled by differential methylation of the DNA. However, no such methylation-dependent mechanism had been previously identified in association with marsupial imprinting. By comparing the genome of all three extant classes of mammals (eutherians, marsupials, and monotremes), we have investigated the evolution of PEG10 (paternally expressed 10), a retrotransposon-derived imprinted gene that is essential for the formation of the placenta in the mouse. PEG10 was present in a marsupial species, the tammar wallaby, but absent from an egg-laying monotreme species, the platypus. Therefore, PEG10 was inserted into the genome at the time when the placenta and viviparity were evolving in therian mammals. This study has shown that PEG10 is not only imprinted in a marsupial, but that its imprint is regulated by differential methylation, suggesting a common origin for methylation in the therian ancestor. These results provide direct evidence that retrotransposon insertion can drive the evolution of genomic imprinting in mammals.

Gene targeting of mouse S ushi- i chi-related r etrotransposon h omologue 11 / Z inc finger CCHC domain-containing 16 (Sirh11/Zcchc16) causes abnormal behaviors related to cognition, including attention, impulsivity and working memory. Sirh11/Zcchc16 encodes a CCHC type of zinc-finger protein that exhibits high homology to an LTR retrotransposon Gag protein. Upon microdialysis analysis of the prefrontal cortex region, the recovery rate of noradrenaline (NA) was reduced compared with dopamine (DA) after perfusion of high potassium-containing artificial cerebrospinal fluid in knockout (KO) mice. These data indicate that Sirh11/Zcchc16 is involved in cognitive function in the brain, possibly via the noradrenergic system, in the contemporary mouse developmental systems. Interestingly, it is highly conserved in three out of the four major groups of the eutherians, euarchontoglires, laurasiatheria and afrotheria, but is heavily mutated in xenarthran species such as the sloth and armadillo, suggesting that it has contributed to brain evolution in the three major eutherian lineages, including humans and mice. Sirh11/Zcchc16 is the first SIRH gene to be involved in brain function, instead of just the placenta, as seen in the case of Peg10, Peg11/Rtl1 and Sirh7/Ldoc1., Author Summary Retrotransposon-derived DNA sequences occupy approximately 40% of the mammalian genome, compared with only 1.5% of protein coding genes. They have been commonly considered “junk DNA” and even potentially harmful for host organisms. However, a series of knockout (KO) mouse analyses demonstrated that at least some of the LTR retrotransposon- and retrovirus-derived sequences play essential roles in the current mammalian developmental system as endogenous genes, such as Peg10, Peg11/Rtl1, Sirh7/Ldoc1, SYNCYTINs and FEMATRIN-1, which are active in multiple aspects of placental function. Here we demonstrate that another LTR retrotransposon-derived gene, Sirh11/Zcchc16, plays an important role in cognitive function in the brain. Sirh11/Zcchc16 KO mice exhibit abnormal behaviors related to cognition, including attention, impulsivity and working memory, possibly due to the locus coeruleus-noradrenaline (LC-NA) system, suggesting that human SIRH11/ZCCHC16 may be involved in X-linked intellectual disability and/or attention-deficit/hyperactivity disorder. Comparative genome analysis demonstrates that SIRH11/ZCCHC16 was acquired in a common eutherian ancestor, suggesting that it contributed to eutherian brain evolution because it confers a critically important advantage in the competition that occurs in daily life. This study provides further insight into the impact of LTR retrotransposon-derived genes on mammalian evolution.

Genomic imprinting is a mammalian specific epigenetic modification of the genome. Assessment of the integrity of the imprinting memory in somatic cell cloned animals is important not only for understanding of the "reprogramming" process during cloning by nuclear transfer, but also for the applications of this technique for therapeutic cloning in the future. In this chapter, we summarize the analytical methods for assessment of monoallelic expression of imprinting genes and expression analysis. From a practical point of view, the authors suggest the use of intersubspecific F1 hybrids between the laboratory mouse (Mus musculus musculus) and the JF1 strain (Mus musculus molossinus). We also list the sequence for PCR primers to detect the polymorphism of imprinted genes between musculus and molossinus.