- Email: [email protected]
Epigenetic regulation in mammalian development and dysfunction: the effects of somatic cloning and genomic imprinting
International Congress Series 1246 (2002) 151 – 159
Epigenetic regulation in mammalian development and dysfunction: the effects of somatic cloning and genomic imprinting Takashi Kohda a,b, Jiyoung Lee a,b,c, Kimiko Inoue b,d, Natumi Ogonuki b,d, Noriko Wakisaka-Saito b,e, Tomoko Kaneko-Ishino b,e, Atsuo Ogura b,d, Fumitoshi Ishino a,b,* a
Gene Research Center, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori, Yokohama 226-8501, Japan b CREST, Japan Science and Technology Corporation (JST), 4-1-8 Hon-machi, Kawaguchi, Saitama 332-0012, Japan c Fellow for the Japan Society for the Promotion of Science, 5-3-1 Kojimachi, Chiyoda, Tokyo 102-0083, Japan d Bioresource Engineering Division, Riken BioResource Center, 3-1-1 Takanodai, Tsukuba, Ibaraki 305-0074, Japan e School of Health Sciences, Tokai University, Bohseidai, Isehara, Kanagawa 259-1193, Japan
Abstract Although somatic cell cloning has been accomplished in several mammalian species, its efficiency remains considerably low due to fetal mortality during the pre- and perinatal periods, which suggests incomplete initialization of epigenetic memories during the somatic cloning procedure. Genomic imprinting is an epigenetic mechanism that produces functional differences between the paternal and maternal genomes, and plays an essential role in mammalian development and growth. Therefore, it is very important to examine the genomic imprinting status of somatic clones. The placenta is one of the most commonly affected organs in the somatic clones. We confirmed that parental-origin-specific monoallelic expression of imprinted genes was maintained faithfully in cloned embryos and abnormal placentas. However, reduced expression was observed for several genes, including certain imprinted genes in both day 12.5 and term placentas. These results suggest that the development process in cloned mice is not identical to that in normal mice. We analyzed mouse clone embryos, which were produced from primordial germ cells (PGCs), and
Abbreviations: Peg, paternally expressed gene; Meg, maternally expressed gene; PGC, primordial germ cell; DMR, differentially methylated region. * Corresponding author. Gene Research Center, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori, Yokohama 226-8501, Japan. Tel.: +81-45-924-5812; fax: +81-45-924-5814. E-mail address: [email protected] (F. Ishino). 0531-5131/02 D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 5 3 1 - 5 1 3 1 ( 0 2 ) 0 11 3 8 - X
152
T. Kohda et al. / International Congress Series 1246 (2002) 151–159
characterized the initialization of the parental imprinted memories. Memory erasure proceeded in a step-wise manner and was coordinated specifically for each imprinted gene at embryonic day 11.5, followed by the establishment of default imprinting states that were common to both male and female germ lines. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Genomic imprinting; Somatic cloning; Imprinted genes; Epigenetics; Reprogramming
1. Background Somatic clones have been derived successfully from several mammalian species, including sheep, mice, cows, goats, pigs, cats, and rabbits [1,2]. However, several phenotypic anomalies, such as perinatal death, fetal overgrowth, and placental abnormalities, have been observed in these clones [1– 5]. The basis for these phenotypic alterations remains unclear, although a substantial epigenetic component has been proposed due to the fact that some of these phenotypes resemble those that are associated with aberrant imprinted gene expression [4]. Genomic imprinting is an epigenetic mechanism that produces functional differences between paternal and maternal genomes [6,7] by regulating the expression of paternally and maternally expressed genes (Pegs and Megs, respectively) [8 –14]. Imprinting plays an essential role in mammalian development and growth. Therefore, we examined the genomic imprinting status of the somatic clones to determine whether imprinting defects were responsible for the abnormal phenotypes. Parental imprinted memories persist in somatic cells after fertilization, whereas they should be erased and re-established during germ cell development in order to reflect the gender of the individual [15]. However, the initialization and reprogramming processes that occur during germ cell development are not fully understood. Somatic cloning provides us with a tool to examine the initialization process by enabling the production and analysis of mouse clones from primordial germ cells (PGCs).
2. Methods 2.1. Nuclear transfer Nuclei were removed from donor cells (Sertoli cells, cumulus cells, and PGCs) by gentle aspiration through an injection pipette (4 –5-Am inner diameter). The donor nuclei were injected deep inside the ooplasm using a Piezo-driven micropipette. 2.2. Embryo transfer Embryos that had developed to the morula or blastocyst stages after 72 h in culture were transferred into the uteri of day-2.5 pseudopregnant ICR females. Recipient females were killed on days 9.5– 11.5, and the uteri were examined for viable fetuses.
T. Kohda et al. / International Congress Series 1246 (2002) 151–159
153
2.3. Hybridization to the atlas mouse cDNA expression array Mouse cDNA fragments that encompassed 588 genes were immobilized on a nylon membrane (Clontech). The 32P-labeled cDNA probes were generated by reverse transcription from 1 Ag of each poly A(+) RNA species. Each cDNA probe was then hybridized to the membrane and washed with a high-stringency buffer, as recommended by the manufacturer. The hybridization patterns were detected by autoradiography and quantified by phosphoimaging with a BAS2000 system (Fuji). 2.4. RT-PCR Placental RNA was extracted using ISOGEN (Nippon Gene), as described previously. The cDNA was synthesized from 1 Ag of total RNA using the Superscript II reverse transcriptase (Life Technologies) with an oligo(dT) primer. The levels of gene expression were measured using the ABI PRISM 7700 system and SYBR Green PCR Core Reagents (Applied Biosystems). 2.5. PGC preparation Shortly before nuclear transfer, donor PGCs were collected from fetal gonads (days 11.5– 13.5). Two or three fetal gonads were placed in a 3-Al drop of HEPES-CZB that contained 10% polyvinylpyrrolidone in a micromanipulation chamber, and punctured using a disposable fine needle to allow the PGCs to spread into the medium.
3. Results 3.1. Analysis of somatic clones 3.1.1. Genomic imprinting in mouse somatic clones Although parental memory of genomic imprinting is erased in PGCs (see Section 2) and re-established during oocyte maturation or spermatogenesis, it is maintained in somatic cells even after several passages [15]. This suggests that somatic cells, in addition to having a complete set of genetic information, have adequate parental memory and may thus be used as donors in the production of animal clones. Is genomic imprinting memory erased during the somatic cloning procedure or is it retained as in the donor somatic cells? ES cell-derived clones showed high neonatal lethality and abnormality, whereas almost all of the Sertoli and cumulus clones (93%) were born normally, although the mouse somatic clones, as well as the ES and tail-tip clones, had enlarged placentas [16]. We examined the expression profiles of seven imprinted genes in both the embryos and placentas of Sertoli clones at day 9.5 of pregnancy [16]. DNA polymorphism analysis showed that these genes had parental-origin-specific monoallelic expression patterns that were identical to those observed in normal fertilized embryos and placentas (Fig. 1 and Table 1).
154
T. Kohda et al. / International Congress Series 1246 (2002) 151–159
Fig. 1. Imprinting of Igf 2 in Sertoli clones at day 9.5. RFLP experiment showed that only paternally derived B6 alleles of Igf 2 (B6; 584 bp fragment) were expressed in both embryos (E) and placentas (P) obtained from F1 crosses between JF1 mother and B6 father (JF1 B6) and vice versa (paternal JF1; 375- and 109-bp fragments) in those from reciprocal F1 crosses.
3.1.2. Abnormal gene expression in cloned placentas We examined the gene expression profiles of the cloned placentas [16]. Using cDNA macroarray filters (Clontech) that contained 588 mouse genes, the gene expression profiles Table 1 Faithful genomic imprinting in Sertoli clones Imprinted genes
Expression patterns in nomal embryos
Embryos B6 JF1 (#1)
B6 JF1 (#2)
B6 JF1 (#4)
JF1 B6 (#1)
Placentas B6 JF1 (#1)
B6 JF1 (#2)
B6 JF1 (#3)
B6 JF1 (#4)
JF1 B6 (#4)
Meg1/ Grb10 H19 Meg3 Igf2r Kip2 Peg1/Mest Igf2
Mat
Mat
Mat
Mat
Mat
Mat
Mat
Mat
Mat
Mat
Mat Mat Mat Mat Pat Pat
Mat Mat Mat Mat Pat Pat
Mat Mat Mat Mat Pat Pat
Mat Mat Mat Mat Pat Pat
Mat Mat Mat Mat Pat Pat
Mat Mat Mat Mat Pat Pat
Mat Mat Mat Mat Pat Pat
Mat Mat Mat Mat Pat Pat
Mat Mat Mat Mat Pat Pat
Mat Mat Mat Mat Pat Pat
We examined expression profiles of seven imprinted genes (5 Megs and 2 Pegs) in both embryos and placentas of Sertoli clones at day 9.5 of pregnancy. All imprinted genes showed parental-origin-specific expression patterns identical to those observed in normal fertilized embryos and placentas.
T. Kohda et al. / International Congress Series 1246 (2002) 151–159
155
of four Sertoli clone placentas and two normal placentas were measured at term and compared. Significant signals were detected for 230 genes, and changes in gene expression were observed for a total of 50 genes. Among the latter group, the genes for Igfbp2 (insulin-like, growth-factor-binding protein 2), Igfbp6, and Vegfr2/Flk1 (vesicular endothelial growth factor receptor 2/fetal liver kinase 1) exhibited reduced expression in all four clones. Since Esx1 knockout mice have placentas that are enlarged by 20 – 30% and have histological abnormalities that are similar to those in our clones, we measured more precisely the expression levels of these four genes using quantitative PCR. We examined four clone placentas that were associated with abnormal delivery, and seven clone placentas from apparently normal pups. The expression levels of Igfbp2, Igfbp6, and Vegfr2/Flk1 were decreased by 70 – 90% in the majority of the clone samples, while only moderate decreases in expression were seen in the other clones. Interestingly, Esx1 expression was decreased by more than 80% in all of the clones. Similar analyses were carried out on 10 additional imprinted genes from eight different chromosomal regions. We found that expression of the imprinted genes Peg1/Mest, Meg1/ Grb10 and Meg3/Gtl2, decreased variably among the clones. However, the parental-originspecific monoallelic expression of these imprinted genes was maintained even in the enlarged placentas. This result suggests that the regulation of genomic imprinting is not affected in somatic clones [16]. Although the clone placentas ranged in weight from 0.18 to 0.45 g, there was no significant correlation between placental size and the expression levels of the seven genes examined. Furthermore, no trends were apparent in the expression profiles of these genes. Since we analyzed only about 600 genes, it is possible that many additional genes could be affected in the clone placentas. Immunohistochemical analysis using anti-Igfbp2, -Igfbp6 and -Meg1/Grb10 antibodies showed that the expression levels of these proteins were decreased in several kinds of trophoblast cells (data not shown). 3.2. Analysis of cloned mouse embryos produced from PGCs 3.2.1. Development of PGC clones Since imprinted genomic memories cannot be perturbed (even by reprogramming) during nuclear transfer (see Section 1), the clones produced from PGCs should each have the genomic status of an individual PGC nucleus. Therefore, we analyzed the PGC clones to map precisely germ cell development. We produced mouse PGC clones from embryos at days 11.5 –13.5 using somatic cloning technology [17]. Somatic clones from Sertoli cells were derived consistently at 3% efficiency [1– 5,16]. However, when the day-12.5 – 13.5 PGCs were used as donor cells, the clones showed early embryonic lethality and did not develop to term [17]. With respect to early embryonic lethality, the PGC clone embryos resembled the so-called ‘germ cell embryos’ that were produced previously by Kato et al. [18] using nuclear transplantation of day-14.5 – 16.5 male PGCs. A modification of the somatic cloning process to incorporate an ‘initiation step’ did not improve embryonal development in the PGC clones. Cloned embryos from day-11.5 PGCs showed extended growth compared with embryos from day-12.5 and day-13.5 PGC clones. We observed they survived at least up to the day-11.5 embryonic stage. Therefore, it seems likely that clones do not develop to term when PGCs are used as donor cells, which suggests that gene expression in PGC cells is radically different from that in somatic cells.
156
T. Kohda et al. / International Congress Series 1246 (2002) 151–159
3.3. Genomic imprinting in PGC clones Twelve imprinted genes (six Pegs and six Megs) were analyzed in the cloned embryos. Surprisingly, the imprinted patterns were dramatically altered in the day-11.5 PGC clone embryos and completely lost in the day-12.5 and day-13.5 PGC clones [17]. Nine embryos, which were produced from the day-11.5 male PGCs and from female PGCs, were aligned in descending order of the number of imprinted genes that maintained monoallelic expression patterns (Fig. 2). In the day-11.5 PGC clones E1 and E2, monoallelic expression of 11/12 imprinted genes was preserved; the corresponding value for E3 was 10/12. These results indicate that gene expression in embryos resembles the normal imprinted gene expression profile observed in somatic cells. Residual imprinted memories in these embryos could explain the extended growth of the day-11.5 PGC clones. On the other hand, almost no imprinting was detected in clones E8 and E9, except for the Peg10 gene. The imprinting of between three and nine genes was maintained in the E4 –E7 intermediate-state clones. Conversion from monoallelic to biallelic expression was observed for the Peg1/Mest, Peg3, Peg5/Nnat, H19, and Meg3/Gtl2 genes, while the Igf2, Peg9/Dlk1, Igf2r, p57Kip2, Meg1/Grb10 and Mash2 genes showed patterns of nonexpression (see Fig. 3). Importantly, the day-12.5 female PGC clones resembled the day-12.5 male PGC clones, which indicates the existence of a common default state for genomic imprinting. We used DNA polymorphism analysis of each imprinted gene to demonstrate that these embryos had lost the expression of imprinted genes. We also analyzed the DNA methylation status of the differential methylated regions (DMRs) of three imprinted genes in the PGCs. The
Fig. 2. Genomic imprinting in day-11.5 PGC clones. Number of parental-origin-specific expression of imprinted genes remained in day-11.5 PGC clones was represented. In all day-12.5 PGC clones and 13.5 PGC clones, imprinted genes lost their monoallelic expression patterns completely, indicating they were in the default state. Interestingly, the day-11.5 PGC clones showed different imprinted status of each other, suggesting erasing process of genomic imprinting memory proceeded in day-11.5 PGCs.
T. Kohda et al. / International Congress Series 1246 (2002) 151–159
157
Fig. 3. Erasing process of genomic imprinting memory. It is highly possible that erasing process of genomic imprinting memory occurs just after PGCs enter genital ridges. In the default state, expressions of the imprinted genes become either biallelic or silenced. Black lines represent erasure time courses of Pegs and gray lines represent those of Megs.
results clearly show that the degree of DNA demethylation differed among the day-10.5 – 11.5 PGCs, which is consistent with the expression profiles seen for imprinted genes in day-11.5 PGC clones. Therefore, we conclude that what we observed in the day-11.5 PGC clones was the erasing of genomic imprinting, and the sequence shown in Fig. 2 represents the time course of the erasure process (Fig. 3).
4. Conclusions Our results indicate that the expression of a substantial number of genes was affected in the clone placentas. Recent analyses of global gene expression profiles in clone placentas using GeneChips support this view (data not shown). The abnormalities in Igfbp2 and Igfbp6 expression in clone placentas were not found in placentas that were produced by in vitro fertilization or intracytoplasmic sperm injection (data not shown). Therefore, these aberrant patterns are apparently due to a condition that is specific for somatic cell clones and are not due to the in vitro manipulation of the early embryos. Enlarged placentas have also been observed in mice that were cloned from different donor cells, i.e., cumulus cells, tail-tip fibroblast cells, and ES cells. Thus, this phenotype might be due to reprogramming of reconstituted oocytes rather than any effect of the donor nuclei. Therefore, we conclude that placentas are the most affected organs in the somatic clones, and although the regulation of monoallelic expression of imprinted genes is normal, the development of somatic clones is not identical to that of normal fertilized embryos. In contrast to the findings with clones produced from ES cells [4], we did not observe a high frequency of neonatal lethality or abnormality in either Sertoli or cumulus cellderived clones [5,16]. Abnormal expression of imprinted genes also occurs in ES clones [4]. It is likely that epigenetic abnormalities in the donor ES cells are represented in the aberrant phenotypes of ES clones because such abnormal expression of imprinted genes occurred already in the ES cells [19]. In addition to nuclear transfer, many other experimental factors, such as the properties of the donor cells and the in vitro culture conditions for both donor cells and nuclear-transferred cloned embryos, can affect the
158
T. Kohda et al. / International Congress Series 1246 (2002) 151–159
phenotypes of somatic clones. Therefore, care must be exercised in discussions concerning cloning-associated problems. We showed that PGC clones never developed to term [17]. Since PGC clones are considered to represent the developmental potential of PGCs, this finding gives rise to the novel paradox that cloned animals can be born from somatic cells but not from germ cells. Thus, we need to reconsider the role of genomic imprinting as an epigenetic mechanism in mammalian development [5,6]. We also demonstrated the potential of PGC cloning in the elucidation of reprogramming during genomic imprinting [17]. Assuming that the process starts when the PGCs enter the genital ridges, the variable imprinting observed in these embryos could represent the temporal stages of donor PGC immigration. Since it is known that PGCs arrive at, and begin to enter, the genital ridges around day 10.5 and that immigration is completed by day 11.5 [20], the development periods of individual PGCs in the genital ridges must differ by no more than 24 h. Recently, Y. Matsui et al. (personal communication) showed that the DNA methylation status of the DMR of Igf2r was maintained in migrating PGCs and that demethylation occurred at day 11.5. These findings are consistent with our results and indicate that PGC clones are excellent models for studying PGC behavior.
References [1] I. Wilmut, et al., Viable offspring derived from fetal and adult mammalian cells, Nature 385 (1997) 810 – 813. [2] T. Wakayama, et al., Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei, Nature 394 (1998) 369 – 374. [3] K. Eggan, et al., Hybrid vigor, fetal overgrowth, and viability of mice derived by nuclear cloning and tetraploid embryo complementation, Proc. Natl. Acad. Sci. U. S. A. 98 (2001) 6209 – 6214. [4] D. Humpherys, et al., Epigenetic instability in ES cells and cloned mice, Science 293 (2001) 95 – 97. [5] A. Ogura, et al., Production of male cloned mice from fresh, cultured, and cryopreserved immature Sertoli cells, Biol. Reprod. 63 (2000) 1579 – 1584. [6] M.A. Surani, S.C. Barton, M.L. Norris, Development of reconstituted mouse egg, Nature 308 (1984) 548 – 550. [7] J. McGrath, D. Solter, Completion of mouse embryogenesis requires both the maternal and paternal genomes, Cell 37 (1984) 179 – 183. [8] T. Kaneko-Ishino, et al., Peg1/Mest imprinted gene on Chromosome 6 identified by cDNA subtraction hybridization, Nat. Genet. 11 (1995) 52 – 59. [9] Y. Kuroiwa, et al., Peg3 imprinted gene on proximal chromosome 7, encodes for a zinc finger protein, Nat. Genet. 12 (1996) 186 – 190. [10] N. Miyoshi, et al., Identification of the Meg1/Grb10 imprinted gene on mouse proximal chromosome 11, a candidate for the Silver – Russell syndrome gene, Proc. Natl. Acad. Sci. U. S. A. 95 (1998) 1102 – 1107. [11] T.M. DeChiara, et al., Parental imprinting of the mouse insulin-like growth factor II gene, Cell 64 (1991) 849 – 859. [12] D.P. Barlow, et al., The mouse insulin-like growth factor type-2 receptor is imprinted and closely liked to the Tme locus, Nature 349 (1991) 84 – 87. [13] A.C. Ferguson-Smith, et al., Embryological and molecular investigation of parental imprinting on mouse chromosome 7, Nature 351 (1991) 667 – 670. [14] M.S. Bartolomei, S. Zemel, S.M. Tilghman, Parental imprinting of the mouse H19 gene, Nature 351 (1991) 153 – 155. [15] W. Reik, W. Dean, J. Walter, Epigenetic modification in mammalian development, Science 293 (2001) 1089 – 1093.
T. Kohda et al. / International Congress Series 1246 (2002) 151–159
159
[16] K. Inoue, et al., Faithful expression of imprinted genes in cloned mice, Science 295 (2002) 297. [17] J. Lee, et al., Erasing genomic imprinting memory in mouse clone embryos produced from day 11.5 primordial germ cells, Development 129 (2002) 1807 – 1817. [18] Y. Kato, et al., Developmental potential of mouse primordial germ cells, Development 126 (1999) 823 – 1832. [19] W. Dean, et al., Altered imprinted gene methylation and expression in completely ES cell-derived mouse fetuses: association with aberrant phenotypes, Development 125 (1998) 2273 – 2282. [20] K. Molyneaux, et al., Time-lapse analysis of living mouse germ cell migration, Dev. Biol. 240 (2001) 488 – 498.
Epigenetic regulation in mammalian development and dysfunction: the effects of somatic cloning and genomic imprinting Takashi Kohda a,b, Jiyoung Lee a,b,c, Kimiko Inoue b,d, Natumi Ogonuki b,d, Noriko Wakisaka-Saito b,e, Tomoko Kaneko-Ishino b,e, Atsuo Ogura b,d, Fumitoshi Ishino a,b,* a
Gene Research Center, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori, Yokohama 226-8501, Japan b CREST, Japan Science and Technology Corporation (JST), 4-1-8 Hon-machi, Kawaguchi, Saitama 332-0012, Japan c Fellow for the Japan Society for the Promotion of Science, 5-3-1 Kojimachi, Chiyoda, Tokyo 102-0083, Japan d Bioresource Engineering Division, Riken BioResource Center, 3-1-1 Takanodai, Tsukuba, Ibaraki 305-0074, Japan e School of Health Sciences, Tokai University, Bohseidai, Isehara, Kanagawa 259-1193, Japan
Abstract Although somatic cell cloning has been accomplished in several mammalian species, its efficiency remains considerably low due to fetal mortality during the pre- and perinatal periods, which suggests incomplete initialization of epigenetic memories during the somatic cloning procedure. Genomic imprinting is an epigenetic mechanism that produces functional differences between the paternal and maternal genomes, and plays an essential role in mammalian development and growth. Therefore, it is very important to examine the genomic imprinting status of somatic clones. The placenta is one of the most commonly affected organs in the somatic clones. We confirmed that parental-origin-specific monoallelic expression of imprinted genes was maintained faithfully in cloned embryos and abnormal placentas. However, reduced expression was observed for several genes, including certain imprinted genes in both day 12.5 and term placentas. These results suggest that the development process in cloned mice is not identical to that in normal mice. We analyzed mouse clone embryos, which were produced from primordial germ cells (PGCs), and
Abbreviations: Peg, paternally expressed gene; Meg, maternally expressed gene; PGC, primordial germ cell; DMR, differentially methylated region. * Corresponding author. Gene Research Center, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori, Yokohama 226-8501, Japan. Tel.: +81-45-924-5812; fax: +81-45-924-5814. E-mail address: [email protected] (F. Ishino). 0531-5131/02 D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 5 3 1 - 5 1 3 1 ( 0 2 ) 0 11 3 8 - X
152
T. Kohda et al. / International Congress Series 1246 (2002) 151–159
characterized the initialization of the parental imprinted memories. Memory erasure proceeded in a step-wise manner and was coordinated specifically for each imprinted gene at embryonic day 11.5, followed by the establishment of default imprinting states that were common to both male and female germ lines. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Genomic imprinting; Somatic cloning; Imprinted genes; Epigenetics; Reprogramming
1. Background Somatic clones have been derived successfully from several mammalian species, including sheep, mice, cows, goats, pigs, cats, and rabbits [1,2]. However, several phenotypic anomalies, such as perinatal death, fetal overgrowth, and placental abnormalities, have been observed in these clones [1– 5]. The basis for these phenotypic alterations remains unclear, although a substantial epigenetic component has been proposed due to the fact that some of these phenotypes resemble those that are associated with aberrant imprinted gene expression [4]. Genomic imprinting is an epigenetic mechanism that produces functional differences between paternal and maternal genomes [6,7] by regulating the expression of paternally and maternally expressed genes (Pegs and Megs, respectively) [8 –14]. Imprinting plays an essential role in mammalian development and growth. Therefore, we examined the genomic imprinting status of the somatic clones to determine whether imprinting defects were responsible for the abnormal phenotypes. Parental imprinted memories persist in somatic cells after fertilization, whereas they should be erased and re-established during germ cell development in order to reflect the gender of the individual [15]. However, the initialization and reprogramming processes that occur during germ cell development are not fully understood. Somatic cloning provides us with a tool to examine the initialization process by enabling the production and analysis of mouse clones from primordial germ cells (PGCs).
2. Methods 2.1. Nuclear transfer Nuclei were removed from donor cells (Sertoli cells, cumulus cells, and PGCs) by gentle aspiration through an injection pipette (4 –5-Am inner diameter). The donor nuclei were injected deep inside the ooplasm using a Piezo-driven micropipette. 2.2. Embryo transfer Embryos that had developed to the morula or blastocyst stages after 72 h in culture were transferred into the uteri of day-2.5 pseudopregnant ICR females. Recipient females were killed on days 9.5– 11.5, and the uteri were examined for viable fetuses.
T. Kohda et al. / International Congress Series 1246 (2002) 151–159
153
2.3. Hybridization to the atlas mouse cDNA expression array Mouse cDNA fragments that encompassed 588 genes were immobilized on a nylon membrane (Clontech). The 32P-labeled cDNA probes were generated by reverse transcription from 1 Ag of each poly A(+) RNA species. Each cDNA probe was then hybridized to the membrane and washed with a high-stringency buffer, as recommended by the manufacturer. The hybridization patterns were detected by autoradiography and quantified by phosphoimaging with a BAS2000 system (Fuji). 2.4. RT-PCR Placental RNA was extracted using ISOGEN (Nippon Gene), as described previously. The cDNA was synthesized from 1 Ag of total RNA using the Superscript II reverse transcriptase (Life Technologies) with an oligo(dT) primer. The levels of gene expression were measured using the ABI PRISM 7700 system and SYBR Green PCR Core Reagents (Applied Biosystems). 2.5. PGC preparation Shortly before nuclear transfer, donor PGCs were collected from fetal gonads (days 11.5– 13.5). Two or three fetal gonads were placed in a 3-Al drop of HEPES-CZB that contained 10% polyvinylpyrrolidone in a micromanipulation chamber, and punctured using a disposable fine needle to allow the PGCs to spread into the medium.
3. Results 3.1. Analysis of somatic clones 3.1.1. Genomic imprinting in mouse somatic clones Although parental memory of genomic imprinting is erased in PGCs (see Section 2) and re-established during oocyte maturation or spermatogenesis, it is maintained in somatic cells even after several passages [15]. This suggests that somatic cells, in addition to having a complete set of genetic information, have adequate parental memory and may thus be used as donors in the production of animal clones. Is genomic imprinting memory erased during the somatic cloning procedure or is it retained as in the donor somatic cells? ES cell-derived clones showed high neonatal lethality and abnormality, whereas almost all of the Sertoli and cumulus clones (93%) were born normally, although the mouse somatic clones, as well as the ES and tail-tip clones, had enlarged placentas [16]. We examined the expression profiles of seven imprinted genes in both the embryos and placentas of Sertoli clones at day 9.5 of pregnancy [16]. DNA polymorphism analysis showed that these genes had parental-origin-specific monoallelic expression patterns that were identical to those observed in normal fertilized embryos and placentas (Fig. 1 and Table 1).
154
T. Kohda et al. / International Congress Series 1246 (2002) 151–159
Fig. 1. Imprinting of Igf 2 in Sertoli clones at day 9.5. RFLP experiment showed that only paternally derived B6 alleles of Igf 2 (B6; 584 bp fragment) were expressed in both embryos (E) and placentas (P) obtained from F1 crosses between JF1 mother and B6 father (JF1 B6) and vice versa (paternal JF1; 375- and 109-bp fragments) in those from reciprocal F1 crosses.
3.1.2. Abnormal gene expression in cloned placentas We examined the gene expression profiles of the cloned placentas [16]. Using cDNA macroarray filters (Clontech) that contained 588 mouse genes, the gene expression profiles Table 1 Faithful genomic imprinting in Sertoli clones Imprinted genes
Expression patterns in nomal embryos
Embryos B6 JF1 (#1)
B6 JF1 (#2)
B6 JF1 (#4)
JF1 B6 (#1)
Placentas B6 JF1 (#1)
B6 JF1 (#2)
B6 JF1 (#3)
B6 JF1 (#4)
JF1 B6 (#4)
Meg1/ Grb10 H19 Meg3 Igf2r Kip2 Peg1/Mest Igf2
Mat
Mat
Mat
Mat
Mat
Mat
Mat
Mat
Mat
Mat
Mat Mat Mat Mat Pat Pat
Mat Mat Mat Mat Pat Pat
Mat Mat Mat Mat Pat Pat
Mat Mat Mat Mat Pat Pat
Mat Mat Mat Mat Pat Pat
Mat Mat Mat Mat Pat Pat
Mat Mat Mat Mat Pat Pat
Mat Mat Mat Mat Pat Pat
Mat Mat Mat Mat Pat Pat
Mat Mat Mat Mat Pat Pat
We examined expression profiles of seven imprinted genes (5 Megs and 2 Pegs) in both embryos and placentas of Sertoli clones at day 9.5 of pregnancy. All imprinted genes showed parental-origin-specific expression patterns identical to those observed in normal fertilized embryos and placentas.
T. Kohda et al. / International Congress Series 1246 (2002) 151–159
155
of four Sertoli clone placentas and two normal placentas were measured at term and compared. Significant signals were detected for 230 genes, and changes in gene expression were observed for a total of 50 genes. Among the latter group, the genes for Igfbp2 (insulin-like, growth-factor-binding protein 2), Igfbp6, and Vegfr2/Flk1 (vesicular endothelial growth factor receptor 2/fetal liver kinase 1) exhibited reduced expression in all four clones. Since Esx1 knockout mice have placentas that are enlarged by 20 – 30% and have histological abnormalities that are similar to those in our clones, we measured more precisely the expression levels of these four genes using quantitative PCR. We examined four clone placentas that were associated with abnormal delivery, and seven clone placentas from apparently normal pups. The expression levels of Igfbp2, Igfbp6, and Vegfr2/Flk1 were decreased by 70 – 90% in the majority of the clone samples, while only moderate decreases in expression were seen in the other clones. Interestingly, Esx1 expression was decreased by more than 80% in all of the clones. Similar analyses were carried out on 10 additional imprinted genes from eight different chromosomal regions. We found that expression of the imprinted genes Peg1/Mest, Meg1/ Grb10 and Meg3/Gtl2, decreased variably among the clones. However, the parental-originspecific monoallelic expression of these imprinted genes was maintained even in the enlarged placentas. This result suggests that the regulation of genomic imprinting is not affected in somatic clones [16]. Although the clone placentas ranged in weight from 0.18 to 0.45 g, there was no significant correlation between placental size and the expression levels of the seven genes examined. Furthermore, no trends were apparent in the expression profiles of these genes. Since we analyzed only about 600 genes, it is possible that many additional genes could be affected in the clone placentas. Immunohistochemical analysis using anti-Igfbp2, -Igfbp6 and -Meg1/Grb10 antibodies showed that the expression levels of these proteins were decreased in several kinds of trophoblast cells (data not shown). 3.2. Analysis of cloned mouse embryos produced from PGCs 3.2.1. Development of PGC clones Since imprinted genomic memories cannot be perturbed (even by reprogramming) during nuclear transfer (see Section 1), the clones produced from PGCs should each have the genomic status of an individual PGC nucleus. Therefore, we analyzed the PGC clones to map precisely germ cell development. We produced mouse PGC clones from embryos at days 11.5 –13.5 using somatic cloning technology [17]. Somatic clones from Sertoli cells were derived consistently at 3% efficiency [1– 5,16]. However, when the day-12.5 – 13.5 PGCs were used as donor cells, the clones showed early embryonic lethality and did not develop to term [17]. With respect to early embryonic lethality, the PGC clone embryos resembled the so-called ‘germ cell embryos’ that were produced previously by Kato et al. [18] using nuclear transplantation of day-14.5 – 16.5 male PGCs. A modification of the somatic cloning process to incorporate an ‘initiation step’ did not improve embryonal development in the PGC clones. Cloned embryos from day-11.5 PGCs showed extended growth compared with embryos from day-12.5 and day-13.5 PGC clones. We observed they survived at least up to the day-11.5 embryonic stage. Therefore, it seems likely that clones do not develop to term when PGCs are used as donor cells, which suggests that gene expression in PGC cells is radically different from that in somatic cells.
156
T. Kohda et al. / International Congress Series 1246 (2002) 151–159
3.3. Genomic imprinting in PGC clones Twelve imprinted genes (six Pegs and six Megs) were analyzed in the cloned embryos. Surprisingly, the imprinted patterns were dramatically altered in the day-11.5 PGC clone embryos and completely lost in the day-12.5 and day-13.5 PGC clones [17]. Nine embryos, which were produced from the day-11.5 male PGCs and from female PGCs, were aligned in descending order of the number of imprinted genes that maintained monoallelic expression patterns (Fig. 2). In the day-11.5 PGC clones E1 and E2, monoallelic expression of 11/12 imprinted genes was preserved; the corresponding value for E3 was 10/12. These results indicate that gene expression in embryos resembles the normal imprinted gene expression profile observed in somatic cells. Residual imprinted memories in these embryos could explain the extended growth of the day-11.5 PGC clones. On the other hand, almost no imprinting was detected in clones E8 and E9, except for the Peg10 gene. The imprinting of between three and nine genes was maintained in the E4 –E7 intermediate-state clones. Conversion from monoallelic to biallelic expression was observed for the Peg1/Mest, Peg3, Peg5/Nnat, H19, and Meg3/Gtl2 genes, while the Igf2, Peg9/Dlk1, Igf2r, p57Kip2, Meg1/Grb10 and Mash2 genes showed patterns of nonexpression (see Fig. 3). Importantly, the day-12.5 female PGC clones resembled the day-12.5 male PGC clones, which indicates the existence of a common default state for genomic imprinting. We used DNA polymorphism analysis of each imprinted gene to demonstrate that these embryos had lost the expression of imprinted genes. We also analyzed the DNA methylation status of the differential methylated regions (DMRs) of three imprinted genes in the PGCs. The
Fig. 2. Genomic imprinting in day-11.5 PGC clones. Number of parental-origin-specific expression of imprinted genes remained in day-11.5 PGC clones was represented. In all day-12.5 PGC clones and 13.5 PGC clones, imprinted genes lost their monoallelic expression patterns completely, indicating they were in the default state. Interestingly, the day-11.5 PGC clones showed different imprinted status of each other, suggesting erasing process of genomic imprinting memory proceeded in day-11.5 PGCs.
T. Kohda et al. / International Congress Series 1246 (2002) 151–159
157
Fig. 3. Erasing process of genomic imprinting memory. It is highly possible that erasing process of genomic imprinting memory occurs just after PGCs enter genital ridges. In the default state, expressions of the imprinted genes become either biallelic or silenced. Black lines represent erasure time courses of Pegs and gray lines represent those of Megs.
results clearly show that the degree of DNA demethylation differed among the day-10.5 – 11.5 PGCs, which is consistent with the expression profiles seen for imprinted genes in day-11.5 PGC clones. Therefore, we conclude that what we observed in the day-11.5 PGC clones was the erasing of genomic imprinting, and the sequence shown in Fig. 2 represents the time course of the erasure process (Fig. 3).
4. Conclusions Our results indicate that the expression of a substantial number of genes was affected in the clone placentas. Recent analyses of global gene expression profiles in clone placentas using GeneChips support this view (data not shown). The abnormalities in Igfbp2 and Igfbp6 expression in clone placentas were not found in placentas that were produced by in vitro fertilization or intracytoplasmic sperm injection (data not shown). Therefore, these aberrant patterns are apparently due to a condition that is specific for somatic cell clones and are not due to the in vitro manipulation of the early embryos. Enlarged placentas have also been observed in mice that were cloned from different donor cells, i.e., cumulus cells, tail-tip fibroblast cells, and ES cells. Thus, this phenotype might be due to reprogramming of reconstituted oocytes rather than any effect of the donor nuclei. Therefore, we conclude that placentas are the most affected organs in the somatic clones, and although the regulation of monoallelic expression of imprinted genes is normal, the development of somatic clones is not identical to that of normal fertilized embryos. In contrast to the findings with clones produced from ES cells [4], we did not observe a high frequency of neonatal lethality or abnormality in either Sertoli or cumulus cellderived clones [5,16]. Abnormal expression of imprinted genes also occurs in ES clones [4]. It is likely that epigenetic abnormalities in the donor ES cells are represented in the aberrant phenotypes of ES clones because such abnormal expression of imprinted genes occurred already in the ES cells [19]. In addition to nuclear transfer, many other experimental factors, such as the properties of the donor cells and the in vitro culture conditions for both donor cells and nuclear-transferred cloned embryos, can affect the
158
T. Kohda et al. / International Congress Series 1246 (2002) 151–159
phenotypes of somatic clones. Therefore, care must be exercised in discussions concerning cloning-associated problems. We showed that PGC clones never developed to term [17]. Since PGC clones are considered to represent the developmental potential of PGCs, this finding gives rise to the novel paradox that cloned animals can be born from somatic cells but not from germ cells. Thus, we need to reconsider the role of genomic imprinting as an epigenetic mechanism in mammalian development [5,6]. We also demonstrated the potential of PGC cloning in the elucidation of reprogramming during genomic imprinting [17]. Assuming that the process starts when the PGCs enter the genital ridges, the variable imprinting observed in these embryos could represent the temporal stages of donor PGC immigration. Since it is known that PGCs arrive at, and begin to enter, the genital ridges around day 10.5 and that immigration is completed by day 11.5 [20], the development periods of individual PGCs in the genital ridges must differ by no more than 24 h. Recently, Y. Matsui et al. (personal communication) showed that the DNA methylation status of the DMR of Igf2r was maintained in migrating PGCs and that demethylation occurred at day 11.5. These findings are consistent with our results and indicate that PGC clones are excellent models for studying PGC behavior.
References [1] I. Wilmut, et al., Viable offspring derived from fetal and adult mammalian cells, Nature 385 (1997) 810 – 813. [2] T. Wakayama, et al., Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei, Nature 394 (1998) 369 – 374. [3] K. Eggan, et al., Hybrid vigor, fetal overgrowth, and viability of mice derived by nuclear cloning and tetraploid embryo complementation, Proc. Natl. Acad. Sci. U. S. A. 98 (2001) 6209 – 6214. [4] D. Humpherys, et al., Epigenetic instability in ES cells and cloned mice, Science 293 (2001) 95 – 97. [5] A. Ogura, et al., Production of male cloned mice from fresh, cultured, and cryopreserved immature Sertoli cells, Biol. Reprod. 63 (2000) 1579 – 1584. [6] M.A. Surani, S.C. Barton, M.L. Norris, Development of reconstituted mouse egg, Nature 308 (1984) 548 – 550. [7] J. McGrath, D. Solter, Completion of mouse embryogenesis requires both the maternal and paternal genomes, Cell 37 (1984) 179 – 183. [8] T. Kaneko-Ishino, et al., Peg1/Mest imprinted gene on Chromosome 6 identified by cDNA subtraction hybridization, Nat. Genet. 11 (1995) 52 – 59. [9] Y. Kuroiwa, et al., Peg3 imprinted gene on proximal chromosome 7, encodes for a zinc finger protein, Nat. Genet. 12 (1996) 186 – 190. [10] N. Miyoshi, et al., Identification of the Meg1/Grb10 imprinted gene on mouse proximal chromosome 11, a candidate for the Silver – Russell syndrome gene, Proc. Natl. Acad. Sci. U. S. A. 95 (1998) 1102 – 1107. [11] T.M. DeChiara, et al., Parental imprinting of the mouse insulin-like growth factor II gene, Cell 64 (1991) 849 – 859. [12] D.P. Barlow, et al., The mouse insulin-like growth factor type-2 receptor is imprinted and closely liked to the Tme locus, Nature 349 (1991) 84 – 87. [13] A.C. Ferguson-Smith, et al., Embryological and molecular investigation of parental imprinting on mouse chromosome 7, Nature 351 (1991) 667 – 670. [14] M.S. Bartolomei, S. Zemel, S.M. Tilghman, Parental imprinting of the mouse H19 gene, Nature 351 (1991) 153 – 155. [15] W. Reik, W. Dean, J. Walter, Epigenetic modification in mammalian development, Science 293 (2001) 1089 – 1093.
T. Kohda et al. / International Congress Series 1246 (2002) 151–159
159
[16] K. Inoue, et al., Faithful expression of imprinted genes in cloned mice, Science 295 (2002) 297. [17] J. Lee, et al., Erasing genomic imprinting memory in mouse clone embryos produced from day 11.5 primordial germ cells, Development 129 (2002) 1807 – 1817. [18] Y. Kato, et al., Developmental potential of mouse primordial germ cells, Development 126 (1999) 823 – 1832. [19] W. Dean, et al., Altered imprinted gene methylation and expression in completely ES cell-derived mouse fetuses: association with aberrant phenotypes, Development 125 (1998) 2273 – 2282. [20] K. Molyneaux, et al., Time-lapse analysis of living mouse germ cell migration, Dev. Biol. 240 (2001) 488 – 498.