Ultrastructure of placental hyperplasia in mice: comparison of placental phenotypes with three different etiologies.

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.

Complementation hypothesis: the necessity of a monoallelic gene expression mechanism in mammalian development.

Gene expression from both parental alleles (biallelic expression) is beneficial in minimizing the occurrence of recessive genetic disorders in diploid organisms. However, imprinted genes in mammals display parent of origin-specific monoallelic expression. As some imprinted genes play essential roles in mammalian development, the reason why mammals adopted the genomic imprinting mechanism has been a mystery since its discovery. In this review, based on the recent studies on imprinted gene regulation we discuss several advantageous features of a monoallelic expression mechanism and the necessity of genomic imprinting in the current mammalian developmental system. We further speculate how the present genomic imprinting system has been established during mammalian evolution by the mechanism of complementation between paternal and maternal genomes under evolutionary pressure predicted by the genetic conflict hypothesis.

Aberrant regulation of imprinted gene expression in Gtl2lacZ mice.

The imprinted region on mouse distal chromosome 12 covers about 1 Mb and contains at least three paternally expressed genes (Pegs: Peg9/Dlk1, Peg11/Rtl1, and Dio3) and four maternally expressed genes (Megs: Meg3/Gtl2, antiPeg11/antiRlt1, Meg8/Rian, and Meg9/Mirg). Gtl2(lacZ) (Gene trap locus 2) mice have a transgene (TG) insertion 2.3 kb upstream from the Meg3/Gtl2 promoter and show about 40% growth retardation when the TG-inserted allele is paternally derived. Quantitative RT-PCR experiments showed that the expression levels of Pegs in this region were reduced below 50%. These results are consistent with the observed phenotype in Gtl2lacZ mice, because at least two Pegs(Peg9/Dlk1 and Dio3) have growth-promoting effects. The aberrant induction of Megs from silent paternal alleles was also observed in association with changes in the DNA methylation level of a differentially methylated region (DMR) located around Meg3/Gtl2 exon 1. Interestingly, a 60 approximately 80% reduction in all Megs was observed when the TG was maternally derived, although the pups showed no apparent growth or morphological abnormalities. Therefore, the paternal or maternal inheritance of the TG results in the down-regulation in cis of either Pegs or Megs, respectively, suggesting that the TG insertion influences the mechanism regulating the entire imprinted region.

No evidence of PEG1/MEST gene mutations in Silver-Russell syndrome patients.

Silver-Russell syndrome (SRS) is characterized by prenatal and postnatal growth retardation with morphologic anomalies. Maternal uniparental disomy 7 has been reported in some SRS patients. PEG1/MEST is an imprinted gene on chromosome 7q32 that is expressed only from the paternal allele and is a candidate gene for SRS. To clarify its biological function and role in SRS, we screened PEG1/MEST abnormalities in 15 SRS patients from various standpoints. In the lymphocytes of SRS patients, no aberrant expression patterns of two splice variants (alpha and beta) of PEG1/MEST were detected when they were compared with normal samples. Direct sequence analysis failed to detect any mutations in the PEG1/MEST alpha coding region, and there were no significant mutations in the 5'-flanking upstream region containing the predicted promoter and the highly conserved human/mouse genomic region. Differential methylation patterns of the CpG island for PEG1/MEST alpha were normally maintained and resulted in the same pattern as in the normal control, suggesting that there was no loss of imprinting. These findings suggest that PEG1/MEST can be excluded as a major determinant of SRS.

A retrotransposon-derived gene, PEG10, is a novel imprinted gene located on human chromosome 7q21.

A novel paternally expressed imprinted gene, PEG10 (Paternally Expressed 10), was identified on human chromosome 7q21. PEG10 is located near the SGCE (Sarcoglycan epsilon) gene, whose mouse homologue was recently shown to be imprinted. Therefore, it is highly possible that a new imprinted gene cluster exists on human chromosome 7q21. Analysis of two predicted open reading frames (ORF1 and ORF2) revealed that ORF1 and ORF2 have homology to the gag and pol proteins of some vertebrate retrotransposons, respectively. These data suggest that PEG10 is derived from a retrotransposon that was previously integrated into the mammalian genome. PEG10 is likely to be essential for understanding how exogenous genes become imprinted.

Tumour suppressor activity of human imprinted gene PEG3 in a glioma cell line.

BACKGROUND: Mouse imprinted gene Peg3 encodes a large C2H2 type zinc finger protein with unique characteristics. Peg3 knockout mice were found to show an impairment in maternal behaviour of the adult female. Mouse Peg3 is located on the proximal region of chromosome 7 which is syntenic to the long arm of human chromosome 19. It has been reported that a loss of heterozygosity (LOH) of chromosome 19q occurs frequently in several glioma types. RESULTS: We isolated human PEG3 cDNA. Both human and mouse PEG3 were strongly expressed in the adult brain and the Peg3 protein was localized in the nuclei of both neurones and glial cells. A significant decrease in PEG3 expression was more commonly observed in glioma cell lines as compared with that in primary cultures of astrocytes. Transfection of PEG3 cDNA in a glioma cell line resulted in a loss of tumorigenicity in nude mice. CONCLUSIONS: The human PEG3 gene is a paternally expressed imprinted gene. Introduction of PEG3 cDNA into the glioma cells suggests that human PEG3 protein functions as a tumour suppressor. Human PEG3 is located on 19q13.4 and is one of the candidates for tumour suppressor genes that are predicted to be sited in gliomas.

Subtraction-hybridization method for the identification of imprinted genes.

Imprinted genes show monoallelic expression from either the paternal or maternal genome (1,2), and their regulated expression is usually associated with the existence of parentally differentially methylated regions on genomic DNAs (3,4). Because of this, essentially two different approaches, using either cDNA or genomic DNA as starting material (5) have been developed for systematic isolation of imprinted genes. In this chapter, we describe a subtraction-hybridization method (6-8) as an example of the former approach. Both parthenogenetic embryos and androgenetic embryos (9,10) are the most suitable biological materials for the subtraction or detection of imprinted genes. However, it is difficult to obtain a large amount of such special materials because only a small number of these embryos develop to the d 10 stage (9,10). Thus, polymerase chain reaction (PCR)-based techniques, such as the differential display (11-13) and subtraction-hybridization methods, are necessary to accomplish this experiment. The subtraction-hybridization method has been successfully applied for isolation of both paternally expressed genes (Pegs) (6,14,15) and maternally expressed genes (Megs) (7), and it allows cDNA libraries to be made from a very small amount of biological material. We are convinced that this method can be applied in many fields of biological science.

Identification of an imprinted gene, Meg3/Gtl2 and its human homologue MEG3, first mapped on mouse distal chromosome 12 and human chromosome 14q.

BACKGROUND: The paternal duplication of mouse distal chromosome 12 leads to late embryonal/neonatal lethality and growth promotion, whereas maternal duplication leads to late embryonal lethality and growth retardation. Human paternal or maternal uniparental disomies of chromosome 14q that are syntenic to mouse distal chromosome 12 have also been reported to show some imprinting effects on growth, mental activity and musculoskeletal morphology. For the isolation of imprinted genes in this region, a systematic screen of maternally expressed genes (Megs) was carried out by our subtraction-hybridization method using androgenetic and normally fertilized embryos. RESULTS: We have isolated seven candidate clones of the mouse Meg gene. Among them, we identified a novel maternally expressed imprinted gene, Meg3, on mouse distal chromosome 12 and showed that it was identical to the Gtl2 gene. We also found that the human homologue MEG3 on chromosome 14q was also monoallelically expressed. CONCLUSIONS: This is the first identification of the imprinting gene, both on mouse distal chromosome 12 and on human chromosome 14q, respectively. Because there are no obvious open reading frames in either the mouse Meg3/Gtl2 or human MEG3, the function of these genes remains unclear. However, this result will provide a good basis for the further investigation of several important imprinted genes in this chromosomal region.

Expression and imprinting status of human PEG8/IGF2AS, a paternally expressed antisense transcript from the IGF2 locus, in Wilms' tumors.

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.

Mouse Peg9/Dlk1 and human PEG9/DLK1 are paternally expressed imprinted genes closely located to the maternally expressed imprinted genes: mouse Meg3/Gtl2 and human MEG3.

BACKGROUND: Genomic imprinting significantly influences development, growth and behaviour in mammals. Systematic screening of imprinted genes has been extensively carried out to identify the genes responsible for imprinted phenotypes and to elucidate the biological significance of this phenomenon. In this study, we applied DNA chip technology for isolating paternally expressed imprinted genes (Pegs). We compared the resulting expression profiles of parthenogenetic and fertilized control embryos to identify novel imprinted genes. RESULTS: A novel paternally expressed mouse imprinted gene, Peg9/Dlk1, was identified. Consistent with this finding, the paternal expression of its human homologue, PEG9/DLK1, was also confirmed. These two genes form imprinted gene clusters with the reciprocally imprinted mouse Meg3/Gtl2 and human MEG3 genes that we first identified on distal chromosome 12 and chromosome 14q32, respectively. CONCLUSIONS: As DNA chip technology allows us to quickly screen a large number of genes, using this technology to search for imprinted genes could accelerate the identification of genes responsible for human and mouse genetic diseases. Dlk1 and DLK1, which encode transmembrane proteins, have six EGF-like repeats and show homology to the Delta gene in Drosophila melanogaster. Because of its homology to mammalian Delta homologues, PEG9/DLK1 may contribute to the scoliosis phenotype observed in maternal uniparental disomy 14 (mUPD14) patients.

Disruption of primary imprinting during oocyte growth leads to the modified expression of imprinted genes during embryogenesis.

Parthenogenetic embryos, which contained one genome from a neonate-derived non-growing oocyte and the other from a fully grown oocyte, developed to day 13.5 of gestation in mice, 3 days longer than previously recorded for parthenogenetic development. To investigate the hypothesis that disruption of primary imprinting during oocyte growth leads to the modified expression of imprinted genes and this parthenogenetic phenotype, we have examined Peg1/Mest, Igf2, Peg3, Snrpn, H19, Igf2r and excess p57KIP2. We show that paternally expressed genes, Peg1/Mest, Peg3 and Snrpn, are expressed in the parthenotes, presumably due to a lack of maternal epigenetic modifications during oocyte growth. In contrast, the expression of Igf2, which is repressed in a competitive manner by transcription of the H19 gene, was very low. Furthermore, we show that the maternally expressed Igf2r and p57KIP2 genes were repressed in the alleles of the non-growing oocyte indicating maternal modifications during oocyte growth are necessary for its expression. Thus, our results show that primary imprinting during oocyte growth exhibits a crucial effect on both the expression and repression of maternal alleles during embryogenesis.

Identification of the Meg1/Grb10 imprinted gene on mouse proximal chromosome 11, a candidate for the Silver-Russell syndrome gene.

In a systematic screen for maternally expressed imprinted genes using subtraction hybridization with androgenetic and normal fertilized mouse embryos, seven candidate maternally expressed genes (Megs) have been isolated, including the H19 and p57(Kip2) genes that are known to be maternally expressed. Herein, we demonstrate that an imprinted gene, Meg1, is apparently identical to Grb10 (growth factor receptor-bound protein 10), which is located on mouse proximal chromosome 11. Grb10 protein was reported to bind to the insulin receptor and/or the insulin-like growth factor (IGF) I receptor via its src homology 2 domain and to inhibit the associated tyrosine kinase activity that is involved in the growth promoting activities of insulin and IGFs (IGF-I and -II). Thus, it is probable that Meg1/Grb10 is responsible for the imprinted effects of prenatal growth retardation or growth promotion caused by maternal or paternal duplication of proximal chromosome 11 with reciprocal deficiencies (MatDp.prox11 or PatDp.prox11), respectively. In the human, it has been reported that the maternal uniparental disomy 7 is responsible for the Silver-Russell syndrome (SRS) whose effects include pre- and postnatal growth retardation and other dysmorphologies. The human homologue GRB10 on chromosome 7q11.2-12 is a candidate gene for Silver-Russell syndrome.

Peg5/Neuronatin is an imprinted gene located on sub-distal chromosome 2 in the mouse.

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.

Human PEG1/MEST, an imprinted gene on chromosome 7.

The mouse Peg1/Mest gene is an imprinted gene that is expressed particularly in mesodermal tissues in early embryonic stages. It was the most abundant imprinted gene among eight paternally expressed genes (Peg 1-8) isolated by a subtraction-hybridization method from a mouse embryonal cDNA library. It has been mapped to proximal mouse chromosome 6, maternal duplication of which causes early embryonic lethality. The human chromosomal region that shares syntenic homology with this is 7q21-qter, and human maternal uniparental disomy 7 (UPD 7) causes apparent growth deficiency and slight morphological abnormalities. Therefore, at least one paternally expressed imprinted gene seems to be present in this region. In this report, we demonstrate that human PEG1/MEST is an imprinted gene expressed from a paternal allele and located on chromosome 7q31-34, near D7S649. It is the first imprinted gene mapped to human chromosome 7 and a candidate for a gene responsible for primordial growth retardation including Silver-Russell syndrome (SRS).

Peg3 imprinted gene on proximal chromosome 7 encodes for a zinc finger protein.

Genetic and embryological studies in the mouse demonstrated functional differences between parental chromosomes during development. This is due to imprinted genes whose expression is dependent on their parental origin. In a recent systematic screen for imprinted genes, we detected Peg3 (paternally expressed gene 3). Peg3 is not expressed in parthenogenones. In interspecific hybrids, only the paternal copy of the gene is expressed in the embryos, individual tissues examined in d9.5-13.5 embryos, neonates and adults. Peg3 mRNA is a 9 kb transcript encoding an unusual zinc finger protein with eleven widely spaced C2H2 type motifs and two groups of amino acid repeats. Peg3 is expressed in early somites, branchial arches and other mesodermal tissues, as well as in the hypothalamus. Peg3 maps to the proximal region of chromosome 7. Consistent with our findings, maternal duplication of the proximal chromosome 7 causes neonatal lethality. This region is syntenic with human chromosome 19q13.1-13.3 (refs 10,11), where the genes for myotonic dystrophy and a putative tumour suppressor gene are located.

Peg1/Mest imprinted gene on chromosome 6 identified by cDNA subtraction hybridization.

Parthenogenesis in the mouse is embryonic lethal partly because of imprinted genes that are expressed only from the paternal genome. In a systematic screen using subtraction hybridization between cDNAs from normal and parthenogenetic embryos, we initially identified two apparently novel imprinted genes, Peg1 and Peg3. Peg1 (paternally expressed gene 1) or Mest, the first imprinted gene found on the mouse chromosome 6, may contribute to the lethality of parthenogenones and of embryos with a maternal duplication for the proximal chromosome 6. Peg1/Mest is widely expressed in mesodermal tissues and belongs to the alpha/beta hydrolase fold family. A similar approach with androgenones can be used to identify imprinted genes that are expressed from the maternal genome only.

Fate of haploid parthenogenetic cells in mouse chimeras during development.

The developmental capability of haploid parthenogenetic cells was investigated by studies on haploid parthenogenetic in equilibrium fertilized mouse chimeras. Two chimeras were born. One female chimera was smaller at birth and grew slower than its littermates. The distribution of haploid-derived cells in the chimeras was analyzed 11 months after their birth. Cells derived from haploid embryos were found only in the brain, eyes, pigment cells in hair follicles, and spleen, in which they constituted 30%, 20%, 10%, and less than 5%, respectively, of the cells. The correlation between the parthenogenetic contribution to the brain and growth retardation is discussed. All of the cells examined in these chimeric organs (brain and eyes) contained a diploid amount of DNA, suggesting that diploidization of the haploid parthenogenetic cells occurred during development. Possibly, the haploid state is not sufficient for cell growth, even in chimeras with fertilized embryos.

Effect of c-myc gene expression on early inducible reactions required for erythroid differentiation in vitro.

By employing cell fusion between two genetically marked mouse erythroleukemia (MEL) cells in which an artificially introduced c-myc gene had been placed under the control of human metallothionein promoter, we investigated the mechanism of the suppressive action of c-myc gene expression in erythroid differentiation. The results indicated that the expression of the c-myc gene blocked the induction of dimethyl sulfoxide-inducible activity, one of the two early activities required for triggering the differentiation.