Influence of cloning by chromatin transfer on placental gene expression at Day 45 of pregnancy in cattle

Poor success rates in somatic cell cloning are often attributed to abnormal early embryonic development as well as late abnormal fetal growth and placental development. Although promising results have been reported following chromatin transfer (CT), a novel cloning method that includes the remodeling of the donor nuclei in vitro prior to their transfer into enucleated oocytes, animals cloned by CT show placental abnormalities similar to those observed following conventional nuclear transfer. We hypothesized that the placental gene expression pattern from cloned fetuses was ontologically related to the frequently observed placental phenotype. The aim of the present study was to compare global gene expression by microarray analysis of Day 44–47 cattle placentas derived from CT cloned fetuses with those derived from in vitro fertilization (i.e. control), and confirm the altered mRNA and protein expression of selected molecules by qRT-PCR and immunohistochemistry, respectively. The differentially expressed genes identified in the present study are known to be involved in a range of activities associated with cell adhesion, cell cycle control, intracellular transport and proteolysis. Specifically, an imprinted gene, involved with cell proliferation and placentomegaly in humans (CDKN1C) and a peptidase that serves as a marker for non-invasive trophoblast cells in human placentas (DPP4), had mRNA and protein altered in CT placentas. It was concluded that the altered pattern of gene expression observed in CT samples may contribute to the abnormal placental development phenotypes commonly identified in cloned offspring, and that expression of imprinted as well as trophoblast invasiveness-related genes is altered in cattle cloned by CT.     

Influence of murine maternal diabetes on placental morphology, gene expression, and function

Maternal diabetes causes placental and foetal abnormalities in both rat and humans; however, its effect is less well documented in the mouse. We used a standard approach to induce manifest diabetes in pregnant mice and assessed morphology, function and gene expression in the placentas isolated from these females. We found that diabetic placentas exhibit a consistent abnormal phenotype characterized by increased junctional zone cross sectional area. Lipid profiling of diabetic foetuses and placentas showed that the placental phenotypes do not compromise the lipid transport function of this organ. In a genome-wide survey of mRNA expression by using cDNA micro-arrays, we identified 118 ESTs, corresponding to 59 annotated genes, with differential expression in the diabetic placentas. A significant proportion of these known is involved in metabolism, immunity and defence, and signal transduction. In addition, we found two imprinted genes, Igf2 and Gatm, which exhibited altered expression. The expression of other imprinted genes, Peg1, Gtl2, Peg3, Igf2r and Grb10, was determined by quantitative RT-PCR. For all of these genes, slight changes in gene expression were observed between diabetic placentas and control placentas. Our study thus provides the basis for future work that will address gene action in the diabetic mouse placenta.     

Disruption of imprinting and aberrant embryo development in completely inbred embryonic stem cell-derived mice

The completely embryonic stem (ES) cell-derived mice (ES mice) produced by tetraploid embryo complementation provide us with a rapid and powerful approach for functional genome analysis. However, inbred ES cell lines often fail to generate ES mice. The genome of mouse ES cells is extremely unstable during in vitro culture and passage, and the expression of the imprinted genes is most likely to be affected. Whether the ES mice retain or repair the abnormalities of the donor ES cells has still to be determined. Here we report that the inbred ES mice were efficiently produced with the inbred ES cell line (SCR012). The ES fetuses grew more slowly before day 17.5 after mating, but had an excessive growth from day 17.5 to birth. Five imprinted genes examined (H19, Igf2, Igf2r, Peg1, Peg3) were expressed abnormally in ES fetuses. Most remarkably, the expression of H19 was dramatically repressed in the ES fetuses through the embryo developmental stage, and this repression was associated with abnormal biallelic methylation of the H19 upstream region. The altered methylation pattern of H19 was further demonstrated to have arisen in the donor ES cells and persisted on in vivo differentiation to the fetal stage. These results indicate that the ES fetuses did retain the epigenetic alterations in imprinted genes from the donor ES cells.     

Growth restricted in vitro culture conditions alter the imprinted gene expression patterns of mouse embryonic stem cells

Embryonic stem (ES) cell-derived clones and chimeras are often associated with growth abnormalities during fetal development, leading to the production of over/under-weight offspring that show elevated neonatal mortality and morbidity. Due to the role played by imprinted genes in controlling fetal growth, much of the blame is pointed at improper epigenetic reprogramming of cells used in the procedures. We have analyzed the expression pattern of two growth regulatory imprinted genes, namely insulin like growth factor II (Igf2) and H19, in mouse ES cells cultured under growth restricted conditions and after in vitro aging. Culture of cells with serum-depleted media (starvation) and at high cell density (confluence) increased the expression of both imprinted genes and led to aberrant methylation profiles of differentially methylated regions in key regulatory sites of Igf2 and H19. These findings confirm that growth constrained cultures of ES cells are associated with alterations to methylation of the regulatory domains and the expression patterns of imprinted genes, suggesting a possible role of epigenetic factors in the loss of developmental potential.     

Neuronal development of embryonic stem cells: a model of GABAergic neuron differentiation

Neural cultures derived from differentiating embryonic stem (ES) cells are a potentially powerful in vitro model of neural development. We show that neural cells derived from mouse ES cells express mRNAs characteristic of GABAergic neurons. The glutamate decarboxylase genes (Gad1 and Gad2), required for GABA synthesis and the vesicular inhibitory amino acid transporter (Viaat) gene, required for GABA vesicular packaging are activated in the ES-derived cultures. Nearly half of the ES-derived neurons express the GAD67 protein, the product of the Gad1 gene. Building on these results we show that Gad1-lacZ "knockin" reporter ES cell lines can be used to easily monitor Gad1 expression patterns and expression levels during ES differentiation. We also demonstrate that the ES-derived neural progenitors can be infected with retroviruses or transfected with plasmids via lipofection. These experiments outline the basic strategies and methods required for studies of GABAergic gene expression and regulation in ES-derived neuronal cultures.     

Role of histone methyltransferase G9a in CpG methylation of the Prader-Willi syndrome imprinting center

Imprinted genes in mammals are often located in clusters whose imprinting is subject to long range regulation by cis-acting sequences known as imprinting centers (ICs). The mechanisms by which these ICs exert their effects is unknown. The Prader-Willi syndrome IC (PWS-IC) on human chromosome 15 and mouse chromosome 7 regulates imprinted gene expression bidirectionally within an approximately 2-megabase region and shows CpG methylation and histone H3 Lys-9 methylation in somatic cells specific for the maternal chromosome. Here we show that histone H3 Lys-9 methylation of the PWS-IC is reduced in mouse embryonic stem (ES) cells lacking the G9a histone H3 Lys-9/Lys-27 methyltransferase and that maintenance of CpG methylation of the PWS-IC in mouse ES cells requires the function of G9a. We show by RNA fluorescence in situ hybridization (FISH) that expression of Snrpn, an imprinted gene regulated by the PWS-IC, is biallelic in G9a -/- ES cells, indicating loss of imprinting. By contrast, Dnmt1 -/- ES cells lack CpG methylation of the PWS-IC but have normal levels of H3 Lys-9 methylation of the PWS-IC and show normal monoallelic Snrpn expression. Our results demonstrate a role for histone methylation in the maintenance of parent-specific CpG methylation of imprinting regulatory regions and suggest a possible role of histone methylation in establishment of these CpG methylation patterns.     

Partial Loss of Genomic Imprinting Reveals Important Roles for Kcnq1 and Peg10 Imprinted Domains in Placental Development

Mutations in imprinted genes or their imprint control regions (ICRs) produce changes in imprinted gene expression and distinct abnormalities in placental structure, indicating the importance of genomic imprinting to placental development. We have recently shown that a very broad spectrum of placental abnormalities associated with altered imprinted gene expression occurs in the absence of the oocyte–derived DNMT1o cytosine methyltransferase, which normally maintains parent-specific imprinted methylation during preimplantation. The absence of DNMT1o partially reduces inherited imprinted methylation while retaining the genetic integrity of imprinted genes and their ICRs. Using this novel system, we undertook a broad and inclusive approach to identifying key ICRs involved in placental development by correlating loss of imprinted DNA methylation with abnormal placental phenotypes in a mid-gestation window (E12.5-E15.5). To these ends we measured DNA CpG methylation at 15 imprinted gametic differentially methylated domains (gDMDs) that overlap known ICRs using EpiTYPER-mass array technology, and linked these epigenetic measurements to histomorphological defects. Methylation of some imprinted gDMDs, most notably Dlk1, was nearly normal in mid-gestation DNMT1o-deficient placentas, consistent with the notion that cells having lost methylation on these DMDs do not contribute significantly to placental development. Most imprinted gDMDs however showed a wide range of methylation loss among DNMT1o-deficient placentas. Two striking associations were observed. First, loss of DNA methylation at the Peg10 imprinted gDMD associated with decreased embryonic viability and decreased labyrinthine volume. Second, loss of methylation at the Kcnq1 imprinted gDMD was strongly associated with trophoblast giant cell (TGC) expansion. We conclude that the Peg10 and Kcnq1 ICRs are key regulators of mid-gestation placental function.     

Stability of genomic imprinting in embryonic stem cells: lessons from assisted reproductive technology

Imprinted genes are expressed predominantly or exclusively from one allele only. This mode of gene expression makes the regulation of imprinted genes susceptible to epigenetic insults, which may in turn lead to disease. There is compelling experimental evidence that certain aspects of assisted reproductive technology (ART) such as in vitro cell culture may have adverse effects on the regulation of epigenetic information in mammalian embryos, including the disruption of imprinted genes and epigenetic regulators. Moreover, in humans, disorders of genomic imprinting have been reported in children conceived by ART. The derivation and in vitro culture of embryonic stem (ES) cells are potential points of origin for epigenetic abnormalities. There is evidence that defects of genomic imprinting occur in mouse embryonic stem cells, with similar data now emerging in related studies in non-human primate and human ES cells. It is therefore pertinent to rigorously assess the epigenetic status of all stem cells and their derivatives prior to their therapeutic use in humans. Focusing on the stability of genomic imprinting, this review discusses the current evidence for epigenetic disruption in mammalian embryonic stem cells in light of the epigenetic disruption observed in ART-derived mammalian embryos.     

Androgenetic mouse embryonic stem cells are pluripotent and cause skeletal defects in chimeras: implications for genetic imprinting

The inviability of diploid androgenetic and parthenogenetic embryos suggests imprinting of paternal and maternal genes during germ cell development, and differential expression of loci depending on parental inheritance appears to be involved. To facilitate identification of imprinted genes, we have derived diploid androgenetic embryonic stem (ES) cell lines. In contrast to normal ES cells, they form tumors composed almost entirely of striated muscle when injected subcutaneously into adult mice. They also form chimeras following blastocyst injection, although many chimeras die at early postnatal stages. Surviving chimeras develop skeletal abnormalities, particularly in the rib cartilage. These results demonstrate that androgenetic ES cells are pluripotent and point to stage- and cell-specific expression of developmentally important imprinted genes.     

A haplolethal locus uncovered by deletions in the mouse T complex

Proper levels of gene expression are important for normal mammalian development. Typically, altered gene dosage caused by karyotypic abnormalities results in embryonic lethality or birth defects. Segmental aneuploidy can be compatible with life but often results in contiguous gene syndromes. The ability to manipulate the mouse genome allows the systematic exploration of regions that are affected by alterations in gene dosage. To explore the effects of segmental haploidy in the mouse t complex on chromosome 17, radiation-induced deletion complexes centered at the Sod2 and D17Leh94 loci were generated in embryonic stem (ES) cells. A small interval was identified that, when hemizygous, caused specific embryonic lethal phenotypes (exencephaly and edema) in most fetuses. The penetrance of these phenotypes was background dependent. Additionally, evidence for parent-of-origin effects was observed. This genetic approach should be useful for identifying genes that are imprinted or whose dosage is critical for normal embryonic development.     

Non-germ Line Restoration of Genomic Imprinting for a Small Subset of Imprinted Genes in Ubiquitin-like PHD and RING Finger Domain-Containing 1 (Uhrf1) Null Mouse Embryonic Stem Cells

The underlying mechanism for the establishment and maintenance of differential DNA methylation in imprinted genes is largely unknown. Previous studies using Dnmt1 knock-out embryonic stem (ES) cells demonstrated that, although re-expression of DNMT1 restored DNA methylation in the non-imprinted regions, the methylation patterns of imprinted genes could be restored only through germ line passage. Knock-out of Uhrf1, an accessory factor essential for DNMT1-mediated DNA methylation, in mouse ES cells also led to impaired global DNA methylation and loss of genomic imprinting. Here, we demonstrate that, although re-expression of UHRF1 in Uhrf1^−/− ES cells restored DNA methylation for the bulk genome but not for most of the imprinted genes, it did rescue DNA methylation for the imprinted H19, Nnat, and Dlk1 genes. Analysis of histone modifications at the differential methylated regions of the imprinted genes by ChIP assays revealed that for the imprinted genes whose DNA methylation could be restored upon re-expression of UHRF1, the active histone markers (especially H3K4me3) were maintained at considerably low levels, and low levels were maintained even in Uhrf1^−/− ES cells. In contrast, for the imprinted genes whose DNA methylation could not be restored upon UHRF1 re-expression, the active histone markers (especially H3K4me3) were relatively high and became even higher in Uhrf1^−/− ES cells. Our study thus supports a role for histone modifications in determining the establishment of imprinting-related DNA methylation and demonstrates that mouse ES cells can be a valuable model for mechanistic study of the establishment and maintenance of differential DNA methylation in imprinted genes.     

Differential genomic imprinting and expression of imprinted microRNAs in testes-derived male germ-line stem cells in mouse

Background

Testis-derived male germ-line stem (GS) cells, the in vitro counterpart of spermatogonial stem cells (SSC), can acquire multipotency under appropriate culture conditions to become multipotent adult germ-line stem (maGS) cells, which upon testicular transplantation, produce teratoma instead of initiating spermatogenesis. Consequently, a molecular marker that can distinguish GS cells from maGS cells would be of potential value in both clinical and experimental research settings.

Methods and Findings

Using mouse as a model system, here we show that, similar to sperm, expression of imprinted and paternally expressed miRNAs (miR-296-3p, miR-296-5p, miR-483) were consistently higher (P<0.001), while those of imprinted and maternally expressed miRNA (miR-127, miR-127-5p) were consistently lower (P<0.001) in GS cells than in control embryonic stem (ES) cells. DNA methylation analyses of imprinting control regions (ICR), that control the expression of all imprinted miRNAs in respective gene clusters (Gnas-Nespas DMR, Igf2-H19 ICR and Dlk1-Dio3 IG-DMR), confirmed that imprinted miRNAs were androgenetic in GS cells. On the other hand, DNA methylation of imprinted miRNA genes in maGS cells resembled those of ES cells but the expression pattern of the imprinted miRNAs was intermediate between those of GS and ES cells. The expression of imprinted miRNAs in GS and maGS cells were also altered during their in vitro differentiation and varied both with the differentiation stage and the miRNA.

Conclusions

Our data suggest that GS cells have androgenetic DNA methylation and expression of imprinted miRNAs which changes to ES cell-like pattern upon their conversion to maGS cells. Differential genomic imprinting of imprinted miRNAs may thus, serve as epigenetic miRNA signature or molecular marker to distinguish GS cells from maGS cells.     

Cdk1 is required for the self-renewal of mouse embryonic stem cells

Cyclin-dependent kinase 1 (Cdk1) is indispensible for the early development of the embryo. However, its role in maintaining the undifferentiated state of the embryonic stem (ES) cells remains unknown. In this study, we dissected the function of Cdk1 in mouse ES cells by RNA-interference and gene expression analyses. Cdk1 expression is tightly correlated with the undifferentiated state of the ES cells. Upon differentiation, Cdk1 expression reduced drastically. Cdk1 knock-down by RNA interference resulted in the loss of proliferation and colony formation potential of the ES cells. Consequentially, expression of self-renewal genes was reduced while differentiation markers such as Cdx2 were induced. Our results suggest a role for Cdk1 in maintaining the unique undifferentiated and self-renewing state of the mouse ES cells.