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RESEARCH |
diploid and trisomy-3diploid mouse chimeras as models for investigating how lineage restriction occurs in confined placental mosaicismGenes and Development Group, Division of Reproductive and Developmental Sciences, University of Edinburgh, Hugh Robson Building, George Square, Edinburgh EH8 9XD, Scotland, UK
Correspondence should be addressed to J D West; Email: john.west{at}ed.ac.uk
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Abstract |
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diploid chimeras provide a model for the development of one type of CPM, but animal models for other types of restricted mosaicism are needed. The objective of the present study was to evaluate triploiddiploid and trisomy-3diploid chimeric mouse conceptuses as new models for investigating the development of restricted mosaicism. Novel stocks of mice were generated to produce triploid and trisomy-3 embryos that could be identified by DNA in situ hybridisation to a chromosome 3 transgenic marker. Triploiddiploid and trisomy-3diploid mouse chimeras were produced byembryo aggregation, and the contribution of triploid or trisomy-3 cells was analysed in the fetus and extraembryonic tissues. Only two trisomy-3diploid chimeras were analysed but trisomy-3 cells contributed well to all lineages, so these chimeras did not show restricted mosaicism. In contrast, triploid cells usually contributed poorly to all lineages in the ten 3n2n chimeras analysed. They contributed more to the primitive endoderm derivatives than other lineages and were present in the primitive endoderm derivatives of all ten chimeras, but excluded from fetuses and trophectoderm derivatives in some cases. This pattern of restricted mosaicism differs from that reported for tetraploid cells in tetraploiddiploid chimeras, and triploiddiploid chimeras may provide a useful model for the development of some types of restricted mosaicism in human conceptuses. |
Introduction |
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Human CPM usually involves mosaicism for a trisomic (Ts) cell line, but not all trisomies are equally associated with CPM (Wolstenholme 1996) and CPM involving polyploid mosaicism also occurs (Tuerlings et al. 1993, Kalousek 1994, Wolstenholme et al. 1994, Wilkins-Haug et al. 2006). CPM is a clinically significant condition that affects ~2% of conceptuses. It can produce a false-positive result after prenatal diagnosis by chorionic villus sampling and may be associated with prenatal or perinatal complications (Bennett et al. 1992, Kalousek & Vekemans 1996). There is a need to develop a range of realistic animal models of human CPM and other forms of restricted mosaicism to help understand how different types may arise.
Mouse tetraploiddiploid (4n2n) chimeras provide one useful animal model because tetraploid (4n) cells contribute poorly to derivatives of the fetus and other epiblast derivatives but well to the primitive endoderm and trophectoderm lineages (Tarkowski et al. 1977, 2001, Nagy et al. 1990, James et al. 1995, Goto et al. 2002) (In the mouse, the primitive endoderm tissue produces the endoderm layer of the visceral yolk sac and the parietal endoderm associated with Reichert’s membrane. The equivalent lineage in human conceptuses is usually called the hypoblast). The 4n2n animal model has been used to investigate the mechanisms responsible for the restricted distribution of chromosomally abnormal cells (James et al. 1995, Everett & West 1996, 1998, Goto & Takagi 1998, Everett et al. 2000, Tang et al. 2000, Goto et al. 2002, Eakin et al. 2005, MacKay & West 2005). However, human 4n/2n CPM is relatively uncommon, so new animal models are required. The aim of this study was to evaluate two other possible models of restricted mosaicism involving, respectively, another class of polyploid cells (triploid) and a class of aneuploid cells (trisomy-3) in mouse chimeras.
The origin of human triploid/diploid mosaics has been discussed by several authors (e.g. Daniel et al. 2003, Golubovsky 2003). Although most die, their fate is variable and may depend on whether triploidy is diandric or digynic, the relative proportions and distributions of triploid and diploid cells, the genetic background and the presence of other chromosomal anomalies. Some human triploid/diploid mosaics show CPM (Sarno et al. 1993, Tuerlings et al. 1993, Daniel et al. 2003) but others survive postnatally as mosaic individuals (van de Laar et al. 2002). The previous reports of mouse triploiddiploid chimeras showed that 3n cells can contribute to extraembryonic, fetal and adult tissues (Azuma et al. 1991, Suwinska et al. 2005), but it is not yet known whether 3n cells contribute less readily to the fetus (and other epiblast derivatives) than to the primitive endoderm and trophectoderm derivatives.
The ideal model of human CPM would be mosaicism for a mouse trisomy that is equivalent to a human trisomy that is commonly involved in type-I CPM. Spontaneous trisomic/diploid (Ts/2n) mosaicism is very rare in mammals other than humans, so experimental trisomicdiploid (Ts2n) mouse chimeras offer a better chance of developing a realistic model for CPM. Specific trisomic mouse embryos can be produced from mice heterozygous for particular Robertsonian translocations (Gropp et al. 1974, Gropp 1976), but because mouse and human chromosomes are not syntenic, it is difficult to decide which mouse trisomies are likely to be most relevant to the trisomies commonly involved in type-I human CPM.
The production of mouse Ts2n chimeras is technically demanding and relatively few have been produced (Magnuson et al. 1982, Cox et al. 1984, Epstein et al. 1984, Fundele et al. 1985, Epstein 1986). In Ts2n chimeras studied so far (Ts12, Ts15, Ts16, Ts17 and Ts19), there was little or no selection against Ts cells in the fetus. These previous studies were not intended to model CPM and the extraembryonic tissues were usually ignored or pooled for analysis. For the current study, we investigated the distribution of trisomy-3 cells in mouse Ts32n chimeras. Mouse Ts3 fetuses die at early stage (10–11 days) with hypoplasia and developmental retardation (Gropp et al. 1974, 1983).
For this study, we developed special strains of mice to generate either digynic 3n or Ts3 embryos carrying a transgenic lineage marker on chromosome 3, which is highly reiterated and so readily detectable by DNA in situ hybridisation (Lo 1986, Lo et al. 1987, Katsumata & Lo 1988, Keighren & West 1993, Everett et al. 1994). DNA in situ hybridisation was used to detect chromosome 3, such that two hybridisation signals were produced in interphase nuclei of 3n or Ts3 cells but only one signal was seen in 2n nuclei. We have used this novel approach to assess the contribution of 3n and Ts3 cells to different developmental lineages of postimplantation stage mouse chimeras. Our results suggest that 3n2n chimeras may provide a useful new model system for investigating the development of restricted mosaicism.
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2n chimeras2n chimeras. The frequency of 3n2n chimeras (Table 1
) was higher in series TrB and TrC (9/22 chimeras; 40.9%) than in series TrA (2/27 chimeras; 7.4%). Different frequencies were expected because of the nature of the strains used, but the frequency of 3n2n chimeras in TrA was lower than that expected. TRIP is an LT/SvKau congenic and so expected to produce about 40% 3n embryos, whereas derived strain PO2 was expected to produce ~23% 3n embryos (West et al. 1993, Everett et al. 2004). The frequency of non-chimeric conceptuses varied among series and this may also partly reflect differences in strain combination. All were entirely NIH or FVB genotype (embryo 2) and some may have been 3n2n chimeras in which 3n cells were depleted to below the detectable level.
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2n chimeras are shown in Table 2. Although 3n2n chimera TrA-16 had reached the average developmental stage (hind limb morphology index) for its age, it was well below the control average for conceptus weight, fetal weight, placental weight and crown-rump length, and the placental weight was below the minimum for the control group. Triploid2n chimera TrA-26 was within the control range for all physical parameters measured.
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shows examples of sections of E9.5 3n2n and 2n2n chimeras and the hybridisation signals used to distinguish between chimeras containing Tg/Tg/–3n cells and those containing Tg/– 2n cells. Abnormalities were seen in two of the E9.5 3n2n chimeras. Chimera TrC-26 was an empty gestational sac, without a fetus or amnion, and chimera TrB-3 had an abnormal allantoic vesicle, which had not fused normally with the chorion (Fig. 1F).
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2n chimeras for the two 3n2n chimeras in series TrA together with their 2n2n controls. Overall, the controls showed a balanced chimeric composition with the means for each tissue being ~50%. Triploid cells made no contribution to the fetus in either of the E12.5 3n2n chimeras, but contributed to the primitive endoderm lineage (visceral yolk sac endoderm and parietal endoderm) of both chimeras and also contributed to the extraembryonic epiblast tissues (amnion and visceral yolk sac mesoderm) and the trophectoderm (trophoblast cells overlying Reichert’s membrane and placenta) of chimera TrA-16.
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shows the contribution of 3n cells to the fetus and different extraembryonic tissues of eight E9.5 3n2n chimeric conceptuses in series TrB and TrC and five of the E9.5 2n2n control chimeras (ordered by contribution to the fetus). 3n2n chimera TrC-25 was damaged and so was not analysed and TrC-26 was an incomplete conceptus with no fetus.
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2n chimeras. Grouping the different tissues into four lineages (excluding the allantois and chorion, which were only scored for TrB-3) showed that 3n cells contributed 7.8 ± 3.9% to the fetus, 7.9 ± 3.1% to the extraembryonic epiblast (amnion plus visceral yolk sac mesoderm), 16.8 ± 4.6% to the primitive endoderm and 9.5 ± 4.7% to the trophectoderm. The contribution of 3n cells varied significantly among these four lineages (P = 0.01 by the Kruskal–Wallis test). Although the contribution of 3n cells was lowest in the fetus, Mann–Whitney U-tests revealed no significant differences between the fetus and the extraembryonic epiblast (P = 0.75), the primitive endoderm (P = 0.09) or the trophectoderm (P = 0.09). However, the contribution of 3n cells was significantly lower in the epiblast as a whole (fetus plus extraembryonic epiblast) than the primitive endoderm (P = 0.03) but not the trophectoderm lineage (P = 0.09).
The contribution of 3n cells to the trophectoderm derivatives was significantly lower than in the primitive endoderm (P = 0.002 by Mann–Whitney U-test) but variable. Two of the eight E9.5 3n2n chimeras had mean 3n contributions greater than 70% in one of the trophectoderm derivatives sampled but most had low 3n levels, and no 3n cells were detected in any trophectoderm derivatives of three out of the eight chimeras. Chimera TrB-3 had relatively high 3n contributions to all lineages, which might be associated with its apparently abnormal placental development (see above).
The histograms shown in Fig. 2 indicate that 3n contributions in the fetus did not differ markedly among the three different tissues scored (brain, heart and somites).
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summarises the 3n contribution to the fetus and the mean contribution to each of the three extraembryonic lineages for both E12.5 and E9.5 3n2n chimeras. It is clear that 3n cells usually contributed poorly to all lineages of these 10 3n2n chimeras. Although 3n cells were only excluded completely from three out of the nine E9.5 and E12.5 fetuses in 3n2n chimeric conceptuses, only three out of the nine had more than 2% 3n cells in the fetus. This was significantly fewer than that for the combined tissues of the primitive endoderm lineage (9/10; P = 0.02) but not significantly different from either the extraembryonic epiblast (4/10; P > 0.99) or the trophectoderm lineage (5/10; P = 0.65), by Fisher’s exact test.
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2n fetuses contained some 3n cells at E9.5 (6/7), none did at E12.5 (0/2), but this difference was not significant by Fisher’s exact test (P = 0.08). Also, the fetuses of three other E9.5 3n2n chimeras contained < 2% 3n cells, and this may be below the level detectable by GPI electrophoresis method used to analyse E12.5 chimeras (West & Green 1983). There was less difference in the frequency of fetuses with > 2% 3n cells at E9.5 and E12.5 (3/7 vs 0/2; P = 0.50).
Both of the E12.5 and one of the E9.5 3n2n chimeras showed restricted chimerism where 3n cells were absent from the fetus but present in extraembryonic tissues. In TrA-16, 3n cells were absent from the fetus but present in the extraembryonic epiblast, trophectoderm and primitive endoderm lineages, and in TrA-26 and TrC-11 they were absent from the fetus, extraembryonic epiblast and trophectoderm but present in the hypoblast.
Ts32n chimeras
One series of Ts32n and 2n2n control chimeras (TS) were produced and analysed at E9.5 (Table 1
). Ts32n chimeras were identified retrospectively by DNA in situ hybridisation to the transgenic sequence (Tg/Tg/– Ts3 cells produce two hybridisation signals per nucleus). Only 2 out of 34 chimeras proved to be Ts3diploid (5.9%) but this is close to the expected frequency (see the Discussion section). The composition of the two E9.5 Ts32n chimeras and two of the control 2n2n chimeras were analysed by DNA in situ hybridisation (Table 5). Figure 4 shows examples of sections of E9.5 Ts32n and 2n2n chimeras and the hybridisation signals used to distinguish between chimeras containing Tg/Tg/–Ts3 cells and those containing Tg/–2n cells. In contrast to the 3n2n chimeras, both Ts32n chimeras had a high Ts3 cell contribution in all lineages (Table 5; Fig. 3C). Ts32n chimera TS-10 was an incomplete conceptus, comprising an empty gestational sac without a fetus or amnion but the trophectoderm lineage and parietal endoderm consisted entirely of Ts3 cells. Ts32n chimera TS-46 had a high contribution of Ts3 cells in all tissues analysed. The overall contribution to the fetus was 74.7% and high levels were seen in all the three fetal tissues scored (76%, 72% and 58% in the brain, heart and somites respectively). The high Ts3 fetal contribution indicated that Ts32n chimeras are unlikely to provide a useful model of CPM.
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Discussion |
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2n and 3n2n chimeras and quantifying the Ts3 and 3n contributions in histological sections. The yield of embryos from TRIP strain females was poor and hampered the production of 3n2n chimeras. The breeding performance was improved by further genetic crosses but only at the expense of a lower proportion of triploid embryos. Unless higher yields of triploid embryos can be obtained for future experiments (e.g. by selective breeding), it may be worth evaluating the use of cytochalasin B to induce triploidy (Niemierko 1975) in another strain carrying the chromosome 3 transgene.
Only two E9.5 series TS chimeric conceptuses proved to be Ts32n, but this frequency (2/34; 5.9%) is close to that expected if meiotic non-disjunction in Rb1 heterozygotes produces ~19% aneuploid zygotes (Cattanach & Moseley 1973, Gropp et al. 1974, Everett et al. 1996), comprising about 5% each of trisomy-3, monosomy-3, trisomy-1 and monosomy-1. If monosomic cells contribute infrequently to postimplantation stage chimeras (Magnuson et al. 1982), the expected frequency of Ts32n chimeras is ~5.6% (five Ts32n chimeras per 90 overt Tg-positive–/– chimeras).
Although only one of these two Ts32n chimeric conceptuses had a fetus, this fetus and the extraembryonic tissues of both chimeras had high levels of Ts3 cells, implying that Ts3 cells can survive well in all tissues of chimeric conceptuses. The absence of the fetus in one Ts32n chimera with a very high Ts3 contribution may have been a result of too few diploid cells to correct the abnormal Ts3 phenotype, which is normally lethal around E10–11 days (Gropp et al. 1974, 1983). As Ts3 cells contributed well to the only Ts32n chimeric fetus and to the extraembryonic epiblast lineage in both chimeras, Ts32n chimeras are unlikely to provide a useful model of CPM. This follows the pattern established by earlier studies on other trisomydiploid chimeras, involving Ts12, Ts15, Ts16, Ts17 and Ts19, where Ts cells contributed reasonably well to embryos and adults (Magnuson et al. 1982, Cox et al. 1984, Epstein et al. 1984, Fundele et al. 1985, Epstein 1986). None of the trisomydiploid chimeras studied so far has been identified as a good model of human CPM.
Some digynic triploiddiploid chimeras showed morphological abnormalities. E9.5 chimera TrC-26 had no fetus or amnion, but this may not be attributable to the presence of 3n cells as they were not particularly abundant in this chimera. The chimera with the highest contribution of 3n cells (E9.5 chimera TrB-3) had an abnormal allantoic vesicle that was not fused normally with the chorion. This also occurs in some fully digynic triploids (Wróblewska 1971), parthenogenetictetraploid chimeras (Spindle et al. 1996) and several mutants and genetic knockouts (Copp 1995). E12.5 chimera TrA-16 had a small placenta which may reflect poor placental trophoblast growth associated with digynic imprinting abnormalities (Surani et al. 1986).
Unlike Ts3 cells in Ts32n chimeras, digynic triploid cells generally contributed poorly to all lineages of 3n2n chimeras. This cannot be attributable to the marker transgene as both Ts3 and 3n cells had an additional copy of chromosome 3 carrying the marker transgene. Only five out of nine (56%) of the 3n2n chimeras had any 3n cells in the fetus, and this contribution only exceeded 2% in three out of nine (33%) cases. All of the non-chimeras in the 3n2n series were derived from the diploid Gpi1b/b (FVB or NIH strain) embryo, and it seems likely that the 3n cell population was preferentially lost because the 2n2n control series showed no such bias in non-chimeric genotype.
Two other studies have demonstrated that 3n cells can contribute to adult tissues of 3n2n chimeras. Azuma et al. (1991) identified triploid cells in 24 out of 89 (27%) potential adult 3n2n chimeras in two reciprocal series, where digynic triploidy was induced by suppressing extrusion of the second polar body with cytochalasin B. The frequency and genotype of non-chimeras was consistent with loss of 3n cells from the fetal lineage. Suwinska et al. (2005) produced 3n2n mouse chimeras by two methods and showed that 3n cells could survive at postimplantation stages and in some adults. The low overall level of 3n contribution estimated by in situ hybridisation in the present study was similar to the cytogenetic results for adult 3n2n chimeras reported by Azuma et al. (1991), but rather lower than estimates made by GPI electrophoresis for embryonic, fetal and adult 3n2n chimeras reported by Suwinska et al. (2005). Differences in genetic background and methods of production and analysis could each contribute to these differences.
In our 3n2n chimeras, 3n cells contributed less to the epiblast (including fetus) and trophectoderm derivatives than to the primitive endoderm derivatives. Triploid cells were present in the primitive endoderm derivatives of all ten 3n2n chimeras, but their contribution to the trophectoderm was variable, being absent from three out of ten chimeras but contributing over 70% in one chimera. This variability is consistent with previous analyses of 2n2n chimeric placentas (West et al. 1995). Suwinska et al. (2005) also found that 3n cells tended to contribute more to the visceral yolk sac than to the fetus or placenta of 3n2n chimeras, but it was unclear whether 3n cells were specifically more abundant in the yolk sac endoderm layer because it was not analysed separately.
In two of our chimeras, 3n cells were absent from the fetus and confined to the primitive endoderm lineage. Although this is not equivalent to CPM, it is not known whether this class of restricted mosaicism occurs spontaneously in humans because the primitive endoderm (hypoblast) lineage is rarely investigated in human conceptuses. While some 3n2n chimeras showed some sort of restricted chimerism, 3n cells were not rigorously excluded from the fetus and the extra-embryonic epiblast to the extent that tetraploid cells are excluded from these lineages in 4n2n chimeras (James et al. 1995). Instead, the low contribution of 3n cells more closely resembles the consistently low contribution of one cell population in genotypically unbalanced 2n2n chimeras, where cells of one strain or genotype tend to predominate in all lineages (West & Flockhart 1994). It seems likely that the cases of restricted chimerism, where 3n cells were excluded from the fetus but not from the other tissues, may have arisen partly by chance following a more generalised depletion of 3n cells from all lineages and partly because of a tendency for 3n cells to survive better in the primitive endoderm. It is not yet known how lineage restriction in human CPM occurs, but different mechanisms may apply to different types of restricted mosaicism and different chromosome abnormalities (Wolstenholme 1996). For some types of human restricted mosaicism, 3n2n mouse chimeras may provide a more appropriate model than 4n2n chimeras.
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Materials and Methods |
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). One of each pair of aggregated embryos carried a highly reiterated ß-globin transgene (abbreviated to Tg) that is located on chromosome 3 (Lo et al. 1992, Everett et al. 1994) and can be identified readily by DNA in situ hybridisation in histological sections (Lo et al. 1987, Katsumata & Lo 1988, Keighren & West 1993). The pairs of aggregated embryos also differed at the albino locus (Tyr) and Gpi1 (glucose phosphate isomerase). Genetic crosses with specially developed strains of mice were designed to produce some chromosomally abnormal embryos with either a complete extra set of maternal chromosomes (digynic triploid) or an extra paternal copy of chromosome 3 (trisomy-3) carrying the chromosome 3 transgenic marker. All animal work was carried out in accordance with the UK Home Office regulations. Mice were bred and maintained under conventional conditions in the Centre for Reproductive Biology, University of Edinburgh. (For cross-referencing, TrA, TrB, TrC and TS were originally designated XU, C9B, C9H and C9I respectively.) The female is shown first in each genetic cross. Strains TRIP, PO2, TGA and RBT are all pigmented (Tyr+/+), Gpi1a/a and homozygous for the reiterated ß-globin marker transgene, Tg/Tg (Everett et al. 1994, Keighren & West 1994). Inbred strains NIH and FVB are albino (Tyrc/c), Gpi1b/b and Tg negative; (BALB/c x A/J)F1 hybrids are Tyrc/c, Gpi1a/a and Tg negative. Strains TRIP and PO2 were derived from inbred strain LT/SvKau and designed to produce Tg/Tg/– triploid embryos (see below). RBT is homozygous for both the Rb(1.3)1Bnr Robertsonian translocation (abbreviated to Rb1) and the reiterated transgene: Rb1/Rb1, Tg/Tg (Everett et al. 1994). Heterozygous Rb1/+ males undergo meiotic non-disjunction and produce elevated frequencies of sperm with aneuploidy for chromosomes 1 or 3, and so produce some Ts-1 and Ts-3 embryos (Gropp et al. 1974). Hybrid (TGAxRBT)F1 males are Rb1/+, Tg/Tg and their trisomy-3 offspring are Tg/Tg/–.
Production of 3n2n chimeras
LT/SvKau females spontaneously ovulate up to 40% of oocytes as diploid primary oocytes which, on fertilisation, produce digynic triploid zygotes (Kaufman & Howlett 1986, Kaufman & Speirs 1987, O’Neill & Kaufman 1987, Speirs & Kaufman 1988). TRIP is an LT/SvKau congenic strain but is homozygous for the chromosome 3 transgenic marker (Tg/Tg) and was developed to generate Tg/Tg/–, digynic 3n embryos for chimera production. However, TRIP females produced a low yield of embryos, so a different strategy was used to produce Tg/Tg/– triploid embryos for the first series of triploiddiploid chimeras (series TrA). Tg/Tg TRIP males were outcrossed to Tg/Tg TGA females and the Tg/Tg F1 hybrid females (designated PO1: ‘primary oocyte ovulation stock 1’) were backcrossed to Tg/Tg TRIP males to produce Tg/Tg PO2 mice with 75% TRIP (~75% LT/Kau) genetic background. PO2 females produced a better yield of embryos but a lower proportion were triploid, which proved to be even more of a problem. Thus, Tg/Tg TRIP females were used to produce Tg/Tg/– triploid embryos in series TrB and TrC.
TRIP and PO2 females were superovulated at 3–4 weeks to maximise the yield of digynic, Tg/Tg/– triploid embryos (Speirs & Kaufman 1990) using a standard protocol (West & Flockhart 1994). Other females were superovulated at 6–8 weeks old. Eight-cell stage embryos were collected at E2.5 (vaginal plug date designated E0.5) in M2 handling medium (Quinn et al. 1982). A mixture of Tg/Tg/– 3n and Tg/– 2n embryos were produced from both the PO2x(BALB/cxA/J) and TRIPx(BALB/cxA/J) crosses and aggregated (without identifying the 3n embryos) with either FVB or NIH embryos using methods described previously (James et al. 1993, West & Flockhart 1994), based on the original method of Tarkowski (1961). NIH strain embryos were used as partner 2n embryos for series TrA and TrB, but FVB embryos were used for series TrC because FVB females responded better to our superovulation protocol. Homozygous Gpi1c/c females (hybrid stock ‘CF1’; West & Flockhart 1994) were used as recipients at E2.5 and pregnancies timed according to the recipient female. Series TrA was analysed at E12.5 but series TrB and TrC were analysed at E9.5. Each series produced its own diploiddiploid control chimeras. DNA in situ hybridisation was used to identify Tg/Tg/–cells in triploid2n chimeras and distinguish them retrospectively from control 2n2n chimeras.
Production of Ts32n Chimeras
Female (BALB/cxA/J)F1 mice were superovulated and mated to (TGAxRBT)F1 males and embryos were collected and aggregated with FVB embryos as described above. Aggregates were cultured and transferred to pseudopregnant females and conceptuses were analysed at E9.5. Ts32n and 2n2n chimeras were distinguished retrospectively by DNA in situ hybridisation to the transgene. Tg/Tg/– Ts3 cells produce two hybridisation signals per nucleus but Tg/– 2n cells produce only one. Ts1 and Ms1 cells that were disomic for chromosome 3 (Tg/–) would also produce single hybridisation signals, whereas Ms3 cells (–) would produce no hybridisation signal.
Analysis of E12.5 chimeras
E12.5 conceptuses were dissected to provide seven tissue samples: placenta, trophoblast overlying Reichert’s membrane (plus maternal decidual tissue), parietal endoderm, yolk sac endoderm, yolk sac mesoderm, amnion and fetus (West & Flockhart 1994). The whole conceptus, placenta and fetus were weighed, crown-rump length measured and morphological index assessed by hind limb development (McLaren & Buehr 1990, Palmer & Burgoyne 1991). Tissue preparation, electrophoresis, GPI1 staining and densitometry are described elsewhere (James et al. 1993, West & Flockhart 1994).
The proportions of the two cell populations in the chimeric tissue were estimated from the proportions of GPI1-A and GPI1-B allozymes. The recipient females were Gpi1c/c and produced only GPI1-C enzyme; so any maternal contamination could be excluded from the analysis. The percentage GPI1-A contribution was also corrected to allow for the expectation that triploid cells would produce 1.5 times the activity of diploid cells. The corrected value was calculated as 2/3 Ax100/(2/3 A+B), where A = %GPI1-A and B = %GPI1-B, and this probably provides a closer estimate of the percentage 3n cells. (However, triploid cells do not always produce the predicted 1.5-fold increase in gene product (Epstein 1986), so both uncorrected and corrected GPI1-A levels are presented.) A piece of yolk sac was fixed in acetic alcohol (3 ethanol: 1 acetic acid) and used to distinguish E12.5 3n2n and 2n2n chimeras by DNA in situ hybridisation on 7 µm histological sections (see below). 3n2n chimeras were identified by the presence of Tg/Tg/– triploid cells with two hybridisation signals per nucleus.
Analysis of E9.5 chimeras
E9.5 conceptuses were fixed in acetic alcohol and sections were cut at 7 µm. Probe pMß
2 was labelled using digoxygenin-labelled deoxyuridine triphosphate (non-radioactive DNA Labelling and Detection Kit; Boehringer Mannheim), DNA–DNA in situ hybridisation was performed and hybridisation signals scored as described previously (Keighren & West 1993). In situ hybridisation of Ts32n and 3n2n chimeras produced two hybridisation signals in most Tg-positive nuclei, so they were distinguishable from 2n2n control chimeras with one signal per nucleus. Conceptuses with no hybridisation signals were excluded from the analysis, and considered to be non-chimeric, although in series TS some may have been Ms32n chimeras.
Three longitudinal sections of each conceptus were scored (middle plus two flanking sections, approximately midway from the middle to the sides). Fetal brain, heart, somites, amnion, yolk sac mesoderm, yolk sac endoderm, parietal endoderm, placental trophoblast and giant cells were analysed separately and a mean fetal value was calculated from the results for the brain, heart and somites. (The term ‘fetus’ is used at both E9.5 and E12.5 to avoid confusion with the preimplantation embryo, which produces both the fetus and extraembryonic tissues.) The percentage of Tg-positive nuclei was estimated for each tissue by counting positive and negative nuclei in a sample of ~250–300 cells. Not all 2n nuclei produce the expected (single) hybridisation signal and not all 3n and Ts3 nuclei produce two signals because only part of the nucleus may be included in the section (Keighren & West 1993). Thus, once a chimera had been identified as Ts32n or 3n2n, all nuclei with hybridisation signals were scored as Tg positive (and therefore Ts or 3n) irrespective of whether one or two signals were present. The percentages of Tg-positive cells in Tg/Tg/––/– (Ts32n or 3n2n) and Tg/––/– (2n2n) chimeras were corrected using tissue-specific means from two homozygous Tg/Tg and two hemizygous Tg/– control conceptuses respectively.
Statistical analysis
Statistical tests were performed on an Apple Macintosh computer using StatView software (Abacus Concepts Inc., Berkeley, CA, USA).
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Received 19 June 2007
First decision 2 August 2007
Accepted 4 October 2007
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References |
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