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Embryonic germ cells induce epigenetic reprogramming of somatic nucleus in hybrid cells

1997; Springer Nature; Volume: 16; Issue: 21 Linguagem: Inglês

10.1093/emboj/16.21.6510

1460-2075

Masako Tada,

Chromosomal and Genetic Variations

Article1 November 1997free access Embryonic germ cells induce epigenetic reprogramming of somatic nucleus in hybrid cells Masako Tada Masako Tada Search for more papers by this author Takashi Tada Takashi Tada Search for more papers by this author Louis Lefebvre Louis Lefebvre Wellcome/CRC Institute of Cancer and Developmental Biology, and Physiological Laboratory, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QR UK Search for more papers by this author Sheila C. Barton Sheila C. Barton Wellcome/CRC Institute of Cancer and Developmental Biology, and Physiological Laboratory, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QR UK Search for more papers by this author M. Azim Surani Corresponding Author M. Azim Surani Wellcome/CRC Institute of Cancer and Developmental Biology, and Physiological Laboratory, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QR UK Search for more papers by this author Masako Tada Masako Tada Search for more papers by this author Takashi Tada Takashi Tada Search for more papers by this author Louis Lefebvre Louis Lefebvre Wellcome/CRC Institute of Cancer and Developmental Biology, and Physiological Laboratory, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QR UK Search for more papers by this author Sheila C. Barton Sheila C. Barton Wellcome/CRC Institute of Cancer and Developmental Biology, and Physiological Laboratory, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QR UK Search for more papers by this author M. Azim Surani Corresponding Author M. Azim Surani Wellcome/CRC Institute of Cancer and Developmental Biology, and Physiological Laboratory, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QR UK Search for more papers by this author Author Information Masako Tada2, Takashi Tada2, Louis Lefebvre1, Sheila C. Barton1 and M. Azim Surani 1 1Wellcome/CRC Institute of Cancer and Developmental Biology, and Physiological Laboratory, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QR UK 2CREST/Department of Molecular and Cell Genetics, School of Life Science, Faculty of Medicine, Tottori University, Nishimachi 86, Yonago, Tottori, 683 Japan *Corresponding author. E-mail: [email protected] The EMBO Journal (1997)16:6510-6520https://doi.org/10.1093/emboj/16.21.6510 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Genomic reprogramming of primordial germ cells (PGCs), which includes genome-wide demethylation, prevents aberrant epigenetic modifications from being transmitted to subsequent generations. This process also ensures that homologous chromosomes first acquire an identical epigenetic status before an appropriate switch in the imprintable loci in the female and male germ lines. Embryonic germ (EG) cells have a similar epigenotype to PGCs from which they are derived. We used EG cells to investigate the mechanism of epigenetic modifications in the germ line by analysing the effects on a somatic nucleus in the EG-thymic lymphocyte hybrid cells. There were striking changes in methylation of the somatic nucleus, resulting in demethylation of several imprinted and non-imprinted genes. These epigenetic modifications were heritable and affected gene expression as judged by re-activation of the silent maternal allele of Peg1/Mest imprinted gene in the somatic nucleus. This remarkable change in the epigenotype of the somatic nucleus is consistent with the observed pluripotency of the EG-somatic hybrid cells as they differentiated into a variety of tissues in chimeric embryos. The epigenetic modifications observed in EG-somatic cell hybrids in vitro are comparable to the reprogramming events that occur during germ cell development. Introduction Development of primordial germ cells (PGCs) is accompanied by major epigenetic modifications of the genome, including genome-wide demethylation in mice (Monk et al., 1987). This is partly to ensure that no aberrant epigenetic modifications are transmitted to subsequent generations and to obtain an equivalent epigenetic state in the germ line of male and female embryos. Indeed, this is probably the only stage in the life of a mammal when the homologous chromosomes are indistinguishable as far as their epigenetic status is concerned. The subsequent germ line-specific epigenetic modifications that result in the differences between parental alleles are responsible for the preferential expression of one parental allele of imprinted genes throughout development and in adulthood (Efstratiadis, 1994). DNA methylation that occurs primarily but not exclusively from gastrulation onwards, is an important component of the allele-specific parental imprints (Li et al., 1993) and of regulation of cell type-specific gene expression (Yeivin and Razin, 1993). Genome-wide demethylation, on the other hand, is evident in pre-implantation embryos and in PGCs (Monk et al., 1987; Kafri et al., 1992; Ueda et al., 1992). During pre-implantation development, genome-wide demethylation may be essential for the restoration of pluripotency in the developing epiblast cells. However, while genome-wide demethylation and bi-allelic expression of some imprinted genes are features of pre-implantation development (Monk et al., 1987; Kafri et al., 1992; Latham et al., 1994), this is crucially without incurring loss of underlying parental imprints (Brandeis et al., 1993; Kafri et al., 1993; Tremblay et al., 1995). The loss of allele-specific imprints does occur in cells or embryos with a null mutation of the DNA methyltransferase gene (Li et al., 1993). However, loss of allele-specific parental imprints also occurs naturally during demethylation in PGCs. Consistent with the loss of allele-specific methylation imprints (Brandeis et al., 1993), at least some imprinted genes have the potential for bi-allelic expression in PGCs (Szabo and Mann, 1995). Recently, embryonic germ (EG) cells have been derived directly from PGCs at different stages of development (Matsui et al., 1992; Stewart et al., 1994). There are notable similarities in the epigenotype of EG cells and PGCs (Labosky et al., 1994; T.Tada, M.Tada, K.Hilton, S.C.Barton, T.Sado, N.Takagi and M.A.Surani, in preparation). It seemed therefore that EG cells could be used to investigate the mechanism of epigenetic modifications that occur in PGCs. Furthermore, there are advantages in their use as EG cells can be manipulated easily and cultured in vitro, and used to generate chimeric embryos. In this context, our recent study showed that erasure of parental imprints occurs at several imprinted loci in EG cells from 11.5–12.5 days post-coitum (dpc) male and female embryos, with the process being almost if not entirely complete in the latter (T.Tada, M.Tada, K.Hilton, S.C. Barton, T.Sado, N.Takagi and M.A.Surani, in preparation). To examine the nature of epigenetic changes in germ cells, we investigated the fate of the somatic nucleus in a model system consisting of hybrid cells between EG cells from female embryos and thymic lymphocytes. We demonstrate that the EG cells induce reprogramming of the somatic nucleus in hybrid cells resulting in an extensive demethylation of a number of loci. Furthermore, demethylation of a silent maternal allele of an imprinted gene resulted in its re-activation. Consequently, the EG-somatic hybrid cells are pluripotential as they differentiate into many cell types in developing chimeric embryos. The experimental system we describe here should provide a valuable adjunct to the understanding of the mechanism of epigenetic reprogramming in germ cells. Results Cloning of female EG-somatic hybrid cells The TMA-58G EG cells were derived from PGCs of female 12.5 dpc embryos heterozygous for the Rb(X.2)2Ad translocation and characterized extensively (T.Tada et al., in preparation). Here we studied the consequences of cell fusion between the EG cells and somatic cells. The somatic cells were thymic lymphocytes from (129/Sv×129/Sv–ROSA26)F1 female mice carrying a neo/lacZ transgene that is expressed ubiquitously (Friedrich and Soriano, 1991). Therefore it was possible to isolate hybrid clones expressing neo by their resistance to G418 selection following electrofusion (see Figure 1A and Materials and methods). As a control, when thymic lymphocytes alone were subjected to a similar procedure, no colonies were detected. The morphology of EG-lymphocyte hybrid cells was similar to that of the EG cells and remained so without any detectable morphological changes. Figure 1.Experimental strategy for generating EGF×T hybrid cells. (A) In vitro system to study: (I) the demethylation/reprogramming of lymphocyte genome from transgenic mice carrying the ROSA26 transgene; (II) the erasure of parental imprints from the lymphocyte genome using the maternally inherited mutant allele of Peg1/Mest. (B) G-banded metaphase chromosome spread and karyotype of the EGF×T2 hybrid clone. The translocated chromosome between chromosome 2 and X chromosome is indicated (X in A, arrow in B). Download figure Download PowerPoint Ten metaphases in each hybrid clone were subjected to cytogenetic analysis by the G-banding method. This clearly demonstrated that there was a full tetraploid complement of chromosomes in these hybrid cells at least at early passage (Figure 1B). Furthermore, synchronous replication of four X chromosomes was revealed by the R-banding method, indicating that all four X chromosomes were active in hybrid cells (data not shown). Demethylation of maternally expressed imprinted genes First, we examined the H19 gene (Bartolomei et al., 1991; Ferguson-Smith et al., 1993), and in particular, the HhaI sites clustered in a 3.8 kb SacI fragment upstream to the transcription start site that are the key paternal-allele-specific methylation imprints (Tremblay et al., 1995). A 10 kb and a 2.7 kb BamHI fragment represent methylated HhaI sites, whereas ∼8 kb, 0.7 kb and 0.4 kb bands appeared when these sites were unmethylated (Figure 2A). As expected, a methylated paternal and a demethylated maternal allele was obvious in the DNA samples from thymic lymphocytes. However, in both the EG cells and hybrid cell clones, only the unmethylated DNA bands were prominent. Note that for comparison with samples from hybrid cell clones, we have always used a 1:1 mixture of DNA from the EG cells and lymphocytes (1:1 DNA mixture). These results clearly indicate that in the DNA from hybrid cells, the paternally inherited H19 allele in the somatic nucleus was demethylated at these critical sites. Figure 2.Demethylation of maternally expressed imprinted genes in EGF×T hybrid cells. DNA was extracted from thymus (T), female EG cells (EGF) and their 5–independent fusion clones (EGF×T–1, 2, 4, 5 and 7). Methylated DNA fragments are indicated with arrows and digested fragments with methylation-sensitive restriction enzymes are indicated with circles. (A) H19; DNA was digested with BamHI and the methylation-sensitive restriction enzyme HhaI (H), and hybridized with 3.8 kb SacI fragment. (B) p57Kip2; DNA was digested with BamHI and the methylation-sensitive restriction enzyme EagI, and hybridized with the 0.5 kb XhoI–EagI fragment in the 5′ region of mouse cDNA. The fully methylated fragment is detected at ∼3 kb (arrow). (C) Igf2r region 2; DNA was digested with PvuII and the methylation-sensitive restriction enzyme MluI, and hybridized with a 330 bp fragment (Stüger et al., 1993). (D) Igf2r region 1; DNA was digested with StuI and the methylation-sensitive restriction enzyme NotI, and hybridized with a 190 bp fragment (Stoger et al., 1993). Uncut fragment with NotI is detected at 1.2 kb (arrow) and cut fragment is at 0.5 kb (circle). Download figure Download PowerPoint Similar results were obtained with the p57kip2 gene, that is maternally expressed and paternally methylated (Hatada and Mukai, 1995). Using a 0.53 kb XhoI–EagI fragment of the 5′ region of mouse p57Kip2 cDNA as a probe, the EagI sites were undermethylated in the EG cells and in hybrid cell clones. The degree of undermethylation was similar to that observed in the MatDpDist7 embryo, which has two maternal copies and no paternal copy of the gene (Figure 2B). Finally, we examined the Igf2r gene, particularly the intronic CpG island 27 kb downstream of the transcription start site that is methylated only on the expressed maternal allele (Brandeis et al., 1993; Stoger et al., 1993). In this region, methylated (2.9 kb) and unmethylated (2.0 kb) DNA fragments were detected following restriction enzyme digestion with PvuII and the methylation-sensitive enzyme MluI. Both bands representing the maternal and paternal alleles respectively, were detected in samples from thymic lymphocytes. However, the 2.0 kb band was predominant in EG and hybrid cell clones, indicating dominant demethylation of the MluI site (Figure 2C). Quantitative densitometric analysis of the 2.0 kb and 2.9 kb bands indicated >90% demethylation at this site in the EG and hybrid cell clones instead of the 50% found in thymic lymphocytes. Analysis of the NotI site in the promoter region again showed that it is unmethylated in the EG cells and hybrid cell clones (Figure 2D). Demethylation of paternally expressed imprinted genes The preferential expression of the paternal Igf2 allele is accompanied by methylation of several HpaII sites ∼3 kb upstream of the first exon (Sasaki et al., 1992). The thymic lymphocyte DNA showed the fully methylated paternal 2.2 kb DNA fragment, and partially methylated maternal DNA fragments between 2.0–0.5 kb, with EcoRI and HpaII digestion. The fully methylated 2.2 kb band was diminished in the EG cells and hybrid cell clones (Figure 3A). Figure 3.Demethylation of paternally expressed imprinted genes in EGF×T hybrid cells. (A) Igf2; DNA was digested with EcoRI and the methylation-sensitive restriction enzyme HpaII (M), and hybridized with the 3.1 kb EcoRI fragment. Hypermethylated DNA fragment at 2.2 kb (arrow) was obvious only in the DNA from thymus and the 1:1 DNA mixture. (B) Peg3; DNA was digested with KpnI and the methylation-sensitive restriction enzyme SacII, and hybridized with a 1.3 kb NheI fragment. (C) Peg1/Mest; DNA was digested with XbaI and the methylation-sensitive restriction enzyme SmaI, and hybridized with the 3.0 kb XbaI fragment. Download figure Download PowerPoint Peg3, with preferential expression of the paternal allele (Kuroiwa et al., 1996) has a methylated maternal allele (L.–L.Li, E.B.Keverne, S.Aparicio, S.Viville, S.C.Barton, F.Ishino and M.A.Surani, in preparation). A SacII site of Peg3 is methylated on the maternal allele (represented by the 20 kb band) and unmethylated on the paternal allele (represented by the 9.0 kb band) in thymic lymphocytes. Methylation status at the SacII site was similar in both the EG cells and hybrid cell clones. Again, demethylation of the maternal allele in lymphocyte DNA was evident as shown by a single 9.0 kb band in the EG cells and hybrid cell clones (Figure 3B). Finally, Peg1/Mest, which shows expression of the paternal allele (Ishino-Kaneko et al., 1995), has a SmaI site in the promoter region that is only methylated on the maternal allele (L.Lefebvre, S.Viville, S.C.Barton and M.A.Surani, in preparation). Methylated and unmethylated bands were detected at 3.0 and 2.1 kb respectively, in thymic lymphocytes. As indicated by the dominant 2.1 kb band in hybrid cell clones and the EG cells, the maternal allele in the lymphocyte nucleus became unmethylated (Figure 3C). Although a faint 3.0 kb band was detected in hybrid cells, densitometric analysis revealed that ∼80% of DNA was demethylated in the hybrid cell clones again indicating that demethylation was dominant at this site after fusion of lymphocytes with EG cells. Demethylation of non-imprinted genes In addition to the analysis of genes that are subject to parental imprinting, we examined four well-characterized non-imprinted genes that were previously investigated during gametogenesis and early embryogenesis (Singer-Sam et al., 1990; Kafri et al., 1992). The CpG sites of Aprt, Pgk-2 and β globin are fully methylated in post-implantation embryos but unmethylated in PGCs of 12.5 dpc embryos (Kafri et al., 1992). Pgk-1 is an X–linked gene that is methylated on the inactive X chromosome (Singer-Sam et al., 1990). The HhaI site in the 3′ region of the Pgk-2 gene was hypermethylated in thymic lymphocytes. This was indicated by a 10 kb band, but unmethylated in the EG cells and hybrid cell clones (note the ∼5.0 kb bands in Figure 4A). The HhaI site in an intron between exon 2 and 3 of β globin was methylated in thymic lymphocytes (8.0 kb band) and undermethylated in the EG cells and in hybrid cell clones (2.0 kb band in Figure 4B). In the analysis of the X–linked Pgk-1 gene, a HpaII site in the 5′ region of exon 1 showed that in female thymic lymphocytes, random X–inactivation was accompanied by methylated (at ∼2.0 kb) and unmethylated (at ∼0.8 kb) DNA fragments. The unmethylated DNA band was predominant not only in the EG cells but also in hybrid cell clones (Figure 4C). This demethylation is consistent with the cytogenetic data showing the re-activation of the inactivated X chromosome derived from the lymphocyte in hybrid cells. Finally, a HpaII site in exon 4 of the Aprt gene was substantially methylated in the DNA from lymphocytes, whereas DNA from both the EG and hybrid cell clones was unmethylated as shown by a band of 20 kb. However, the DNA from hybrid cell clones showed <2.5 kb ladder bands that were similar to DNA from EG cells (Figure 5). These ladder bands were more obvious in DNA from hybrid cells than in 1:1 DNA mixture. Nevertheless, there was residual DNA methylation in hybrid cell clones as judged by comparison with the methylation-insensitive isoschizomer MspI digestion. Figure 5.Demethylation of mouse repetitive DNA in EGF×T hybrid cells. DNA (2 μg) was diluted and divided equally. One half was digested with the methylation-sensitive restriction enzyme HpaII and the other half was digested with the methylation insensitive isoschizomer MspI. The minor satellite sequence MR150 was oligo-synthesized and end-labelled as a probe. Download figure Download PowerPoint Reprogramming of a silent imprinted allele and its expression To investigate if the observed demethylation can cause re-activation of a silent allele, we used thymic lymphocytes from mice in which the Peg1/Mest gene was mutated so that exons were deleted and replaced with a IRES/β-geo cassette (henceforth called Peg1 βgeo). The mutant Peg1βgeo paternal allele is active but the maternal allele is methylated and repressed (Lefebvre et al., 1997). As before, hybrid cells were made by fusion between the EG cells and thymic lymphocytes (Figure 1A). First, we examined the reproducibility of the Peg1βgeo re-activation in hybrid cells. For this purpose, we compared lymphocytes carrying the Peg1 βgeo allele inherited from either the father (active) or the mother (inactive) after fusion with the EG cells and cultured under identical conditions for 7 days without selection. These cells were then used to produce embryoid bodies (EBs) which should be chimeric as they consist of normal EG cells and hybrid cells. No differences in the number of EBs or their phenotype were detected, and both sets of EBs were positive to an equal extent for X-gal staining (data not shown). This experiment suggests that not only the active paternal Peg1βgeo allele showed expression as expected but the silent maternal Peg1βgeo allele was also evidently re-activated to an equal extent. This latter expression could most readily be accounted for by postulating re-activation of the silent maternal allele in hybrid cells following fusion of lymphocyte with EG cell. The fate of the silent maternal Peg1 βgeo allele derived from lymphocytes in hybrid cells was further characterized. Following electrofusion between the EG cells and lymphocytes carrying the maternal Peg1 βgeo allele, the cultures were subjected to selection with G418 for 7–8 days, starting 2 days after cell fusion. This was done to allow for re-activation of the mutant allele and therefore of the neo gene. Individual colonies were examined cytogenetically to confirm 1:1 fusion product of the EG cell and thymic lymphocyte. The cells were also examined for the presence of four intact chromosomes 6 (on which Peg1/Mest is located) which was observed in a hybrid clone. The maternally inherited silent Peg1 βgeo allele was methylated in lymphocytes (Figure 7C). However, in the hybrid clone, EGF×TP1−/+ at passage 4, Southern hybridization analysis showed that the two SmaI sites of Peg1βgeo allele were demethylated, consistent with our findings described above (Figure 3C). Moreover, this allele was expressed appropriately following differentiation of the hybrid cells in chimeric embryos in vivo (see below). Developmental potential of hybrid cells The EG-somatic hybrid cells were phenotypically similar to the EG cells. To examine if the hybrid cells were capable of contributing to normal embryonic development as seen with the EG cells alone (M.Tada, T.Tada and N.Takagi, in preparation), we introduced the hybrid cells (EGF×T2) carrying the ROSA26 transgene into host diploid blastocysts and examined the embryos at 9.5 and 10.5 dpc as shown in Figure 1A(I). Since the lymphocytes contained the ubiquitously expressed ROSA26 transgene, we could assess the contribution of the hybrid cells following X-gal staining. Three out of the 52 (6%) embryos analysed were positive for X-gal staining (Table I). The degree of contribution of the tetraploid hybrid cells was modest, as previously observed when tetraploid and diploid cells were present in chimeras (James et al., 1995). Nevertheless, EGF×T2 hybrid cells did colonize a variety of tissues (Figure 6A). To increase the contribution of the hybrid cells, they were also injected into tetraploid host blastocysts. Fourteen out of the 44 embryos (32%) were positive for X-gal staining at 7.5–9.5 dpc (Table I), and the contribution of hybrid cells was less severely affected in this case. In the 8.5 dpc chimeric egg-cylinder, hybrid cells contributed significantly to the embryonic ectoderm and visceral endoderm (Figure 6B). In more advanced 9.5 dpc chimeras, X-gal positive cells were found in the yolk-sac mesoderm and embryonic ectoderm and mesoderm (Figure 6C). Figure 6.The contribution of EGF×T hybrid cells carrying the ROSA26 transgene in chimeric embryos. (A) A 10.5 dpc chimeric embryo formed with diploid host embryo and tetraploid EGF×T2 hybrid cells. (B) A 8.5 dpc egg-cylinder embryo formed with tetraploid host embryo and tetraploid EGF×T2 hybrid cells. (C) A 9.5 dpc chimeric embryo and the yolk sac formed with tetraploid host embryo and tetraploid EGF×T2 hybrid cells. Download figure Download PowerPoint Figure 7.Demethylation and gene re-activation of a silent maternal allele of Peg1/Mest (Peg1 βgeo) in EGF×TP1−/+ hybrid cells. (A) Abnormal 9.5 dpc chimeric embryo and the yolk sac from the tetraploid host embryo and tetraploid EGF×TP1−/+ hybrid cells. (B) The inside view of exocoelom (Exc) of a 9.5 dpc abnormal chimeric embryo. Strong expression of the Peg1 βgeo mutant allele was detected with X-gal within the first 3 h of staining time. Staining was seen in mesodermal tissues, inside layer of yolk sac (YsMe) and allantois (Al), but not in embryonic ectoderm (EmE) and endoderm layer of yolk sac (End). (C) Southern blot analysis of the methylation pattern of the Peg1/Mest and Peg1 βgeo alleles in EGF×TP1−/+ cells. DNA was extracted from thymus of m−/p+ heterozygote for Peg1/Mest (T−/+), EG cells (EGF) and their fusion clone (EGF×TP1−/+). DNA was digested with HindIII and the methylation-sensitive restriction enzyme SmaI and hybridized with the 3.0 kb XbaI fragment of the upstream region including exon 1. One of the two SmaI sites in the HindIII fragment is identical to that analysed in Figure 4C. The 13.2 kb and 10.6 kb HindIII fragments represent the wild-type Peg1/Mest (wt) and the mutant Peg1 βgeo (KO) alleles respectively. The methylated fragment is detected only at 10.6 kb in thymus DNA, and SmaI digested fragments are marked with circles. Download figure Download PowerPoint Table 1. Early post-implantation development of diploid/tetraploid and tetraploid/tetraploid chimeras with female EG cell × thymic lymphocyte hybrid cells Hybrid cell clone-passage (Blastocysts) Recipients Age of embryos (days) No. of embryos transfected No. of chimeric (non-chimeric) embryos Transferred Implanted Normal Retarded Abnormal Resorbeda EGF×T2–5 1 9.5 9 8 0 (6) 0 0 0 (2) (Diploid blastocysts) 2 9 9 1 (8) 0 0 0 3 10.5 9 8 0 (6) 0 0 0 (2) 4 8 7 0 (7) 0 0 0 5 9 9 1 (5) 1 0 1 (1) 6 8 7 0 (6) 0 0 0 (1) EGF×T2–4 1–1 7.5 8 6 0 0 1 (3) 0 (2) (Tetraploid blastocysts) 1–2 8.5 8 7 0 0 3 (3) 0 (1) 1–3 8 7 0 0 2 (3) 0 (2) 1–4 9.5 8 6 0 1 0 (2) 0 (3) EGF×T2–6 2–1 8.5 6 6 0 1 3 (1) 0 (1) (Tetraploid blastocysts) 2–2 6 5 0 0 3 (1) 0 (1) EGF×TP1−/+–2 1 9.5 8 3 0 0 0 3 (Tetraploid blastocysts) 2 9 8 0 0 2 6 3 9 2 0 0 1 1 4 9 3 0 0 0 3 a Chimerism in resorbed embryos was assessed by the contribution of EG hybrid cells in the yolk sac. Similar experiments were carried out with the hybrid cells (EGF×TP1−/+) carrying the maternally inherited Peg1βgeo allele as shown in Figure 1A(II). When these hybrid cells were injected into tetraploid host blastocysts, 3 out of 35 embryos transferred to foster mothers gave some embryonic development on 9.5 dpc, and a further 16 yolk-sac membranes were obtained. The contribution of EGF×T2 hybrid cells carrying ROSA26 transgene to the three primary germ layers of developing embryos was evident when injected into tetraploid blastocysts (Figure 6B and C). By contrast, Peg1βgeo was re-activated and expressed appropriately in the differentiated derivatives of hybrid cells in the mesoderm, the yolk-sac mesoderm and allantois in chimeric embryos (Figure 7A and B). This expression pattern is comparable to that of Peg1/Mest in early post-implantation embryos (Sado et al., 1993; Ishino-Kaneko et al., 1995). Discussion This study demonstrates that the EG-lymphocyte hybrid cells display phenotypic properties that are similar to those of EG cells, including pluripotency, as they contribute to many tissues in chimeric embryos. Perhaps their full developmental potential is only restricted because they are tetraploid. This phenotype contrasts markedly with that of non-dividing, non-adhesive thymic lymphocytes. There are at least two, although not mutually exclusive, possibilities to explain the observed phenotypic properties of our hybrid cells. First, there could be repression of lymphocyte-specific gene activity by trans-acting factors originating from EG cells, implying that the EG cell nucleus must be present continuously to maintain the phenotype of hybrid cells. Second, lymphocyte-specific properties may be lost after fusion with EG cells because of the extensive epigenetic changes of the lymphocyte genome. If the latter occurs, it is possible that a new heritable epigenetic modification of the somatic nucleus was induced by the EG cell in hybrid cells. We propose that both of these events probably occur sequentially following EG-lymphocyte cell fusion. The primary focus of our studies was to examine methylation changes in the somatic nucleus. Previous studies have already demonstrated the repression of lymphocyte-specific gene expression following fusion with embryonal carcinoma (EC) cells (Miller and Ruddle, 1977; Martin et al., 1984). This phenomenon, sometimes referred to as extinction, is well recognized even in hybrid cells between two somatic cells (reviewed by Boshart et al., 1993). In an EC-lymphocyte hybrid clone, it appeared that there was virtual suppression of lymphocyte-specific gene expression as judged by the analysis of proteins using two-dimensional gels (Forejt et al., 1984). A similar phenomenon probably occurs in the EG-lymph