Preferential paternal X inactivation in extraembryonic tissues of early mouse embryos

In female eutherian mammals one of the two X chromosomes is inactivated early in development to produce adult somatic tissues that are mosaic with respect to X-linked alleles (Lyon, 1961: see reviews by Lyon, 1972; Gartler & Andina, 1976; Monk, 1978). Although X inactivation appears to be random in mouse foetal tissues, certain extraembryonic membranes display preferential expression of the maternally derived X chromosome. The two X chromosomes may be marked either cytologically in female embryos heterozygous for an X-chromosome translocation, or biochemically in females heterozygous for the two alleles of PGK-1 (phosphoglycerate kinase-1, E.C.2.7.2.3).

Takagi & Sasaki (1975) used Cattanach’s translocation to look at the distribution of the differentially staining, inactive X chromosome in postimplantation embryos. At days gestation 50 % of the late-replicating X chromosomes were paternal in the embryonic portion of the conceptus (consistent with random inactivation) whereas over 90 % of the inactive X chromosomes were paternal in the yolk sac and chorion, and 70 % in the allantois.

Studies of the X-linked electrophoretic variant of PGK-1 (Nielsen & Chapman, 1977) have supported the cytological work of Takagi’s group. West, Freis, Chapman & Papaioannou (1977) found that the maternal Pgk-1 allele is preferentially expressed in -day female mouse yolk sacs. It appears that the paternal allele is not expressed at all in the yolk-sac endoderm at this age thus suggesting inactivation of the paternal X chromosome. Using control crosses and embryo transfers these workers convincingly showed that the uneven expression of PGK-1 is not due to a selective pressure exerted by the phenotype of the female genital tract. From work on other tissues (West, Papaioannou, Freis & Chapman, 1978) it appears that all derivatives of trophectoderm and primary endoderm show preferential maternal X-chromosome expression.

Either cell selection or non-random inactivation could explain the uneven expression of the two X chromosomes. If cell selection is the cause then it must occur before days as Frels & Chapman (1980) have found preferential maternal expression of PGK-1 in the yolk sac and mural trophectoderm at this age with no evidence of the paternal form in the latter tissue. We therefore decided to look at an earlier stage.

At days blastocysts contain low levels of PGK-1 that appears largely maternal in origin, whereas by days the enzyme is embryonic and has risen over 100 fold (Kozak & Quinn, 1975). We employed cellogel electrophoresis to study the expression of PGK-1, which is a monomer, in dissected tissues from heterozygous -day females and in blastocysts and blastocyst outgrowths cultured in vitro. The results suggest that the paternal Pgk-1 allele may never be expressed in trophectoderm and primary endoderm and that the non-random expression of the X chromosome seen in extraembryonic tissues is due to preferential inactivation of the paternal X chromosome and not to cell selection.

Mice carrying the Pgk-1 variant allele, Pgk-1^a, were kindly provided by John West, these mice being derived from feral Mus musculus musculus from Denmark (Nielsen & Chapman, 1977). With the help of Dr Anne McLaren the Pgk-1^a allele was established on a C3H inbred background by repeated backcrossing. The stock now consists of females homozygous and males hemizygous for the Pgk-1^a allele, and is colony maintained. To produce heterozygous (Pgk-1^a/ Pgk-1^b) female embryos, reciprocal matings were set up with random-bred MF1 mice (Olac 1976 Ltd) as the source of the Pgk-1^b allele. Fertilization is assumed to occur at midnight and the day of detection of the vaginal plug is designated day 0.

Preimplantation embryos were collected from females that had been superovulated by intraperitoneal injection of 5 i.u. of PMS (pregnant mare serum) followed 48 h later by 5 i.u. of HCG (human chorionic gonadotrophin) and immediately caged with males. -day blastocysts were flushed from uteri of pregnant females into PB1 medium (Whittingham & Wales, 1969). If to be collected directly the embryos were passed through two washes of PB1/PVP (PB1 medium containing 4 mg/ml of polyvinylpyrrolidone, Sigma, in place of albumin) in watch glasses. Embryos were pooled and placed in 1 or 2 μl of medium in 10 μl Drummond microcaps. The ends of the microcaps were sealed in a small flame, and the samples were stored at – 70 °C. The numbers in each group are given in the results. Some blastocysts were cultured in vitro, as described in Monk & Ansell (1976), for up to 4 days to produce trophoblastic outgrowths and were harvested as above. -day postimplantation embryos were collected from normally mated females. The egg cylinders were dissected individually in PB1 /PVP as described by Monk & Harper (1979). The separated epiblast, extraembryonic ectoderm and primary endoderm (Fig. 1a) were washed and collected individually (each in 2 μl medium) as above.

Supernatant extracts were prepared by freeze thawing the sample three times, followed by centrifugation at 2000 rev./min at 4 °C. The samples were then analyzed for PGK-1 expression by running them on cellogel for h at 200 V. The electrophoresis and positive staining of PGK-1 were done according to B ü cher et al. (1981). Thymocyte extracts from hemizygous PGK-lA and PGK-1B males were used as controls for PGK-1 A and PGK-1B migration respectively.

To determine when the preferential expression of the maternal X chromosome can first be detected, tissues of -day embryos from crosses ♀ Pgk-1^a/Pgk-1^a X ♂ Pgk-1^b and ♀ Pgk-1^b/Pgk-1^b X ♂ Pgk-1^a were examined for PGK expression.

Individual samples of epiblast, extraembryonic ectoderm (e.e.e.) and primary endoderm from a single egg cylinder (Fig. 1 a) were run on cellogel. Figure 1 (b – d) shows representative gels from the reciprocal crosses and the results are recorded in Fig. 2.

Of the 33 embryos from the two reciprocal crosses 20 expressed only one PGK-1 band in the epiblast, and when 15 of these were tested for expression in the endoderm and e.e.e. only the same PGK-1 band was detected. These 20 embryos were therefore presumed to be hemizygous males.

Thirteen embryos expressed a heterozygous PGK-1A/PGK-1B phenotype in the epiblast and were therefore female. The expression of the two bands was fairly even although some female epiblasts showed slightly more PGK-1 A (see Fig. 1 c, d); others, slightly more PGK-IB. In most cases the e.e.e. and endoderm regions showed approximately equal activity of PGK-1 to the epiblast, but in these extraembryonic tissues all the activity was maternal. In four cases a very pale paternal band was seen in extraembryonic tissues. In one sample of e.e.e., from the cross ♀ Pgk-1^a/ Pgk-1^aX ♂ Pgk-1^b, the PGK-1B band was uneven and was almost certainly due to spillover from the standard PGK-IB sample run next to it. The other three cases of paternal bands in endoderm or e.e.e. were most likely due to contamination of the tissue with epiblast, as in two cases the embryo dissections had not been recorded as ‘clean’.

These results strongly suggest that cell selection is not the cause of the preferential expression of X^m (maternal X chromosome) seen in later derivatives of e.e.e. and primary endoderm, unless selection could occur even earlier than days gestation.

Embryo-coded Pgk-1 expression occurs after implantation (Kozak & Quinn, 1975). We investigated whether Pgk-1 expression occurs in vitro, together with the preferential inactivation of the paternal X chromosome in extraembryonic tissues as seen in vivo. Mixtures of heterozygous (female) and hemizygous (male) blastocysts were collected and cultured for up to four days to produce trophoblastic outgrowths. No paternal PGK-1 expression was seen in large numbers of day blastocysts (Fig. 3 a, b) or after one day of culture (data not shown). After 3 and 4 days of culture there was considerable increase in PGK-1 activity and the paternally derived PGK-1 band could be detected indicating expression of the embryonic genome (Fig. 3 a, b).

In both crosses the maternally derived enzyme band in 3- and 4-day outgrowths is far in excess of the paternal, and the activity of the former has increased considerably from that of the blastocysts as shown in Fig. 3a, b. Although males should make up 50 % of the embryo population their contribution alone is unlikely to account for the degree of imbalance between the bands. The preferential expression of the maternal enzyme is almost certainly due to synthesis by the trophoblastic cells (in line with the in vivo results), which constitute a large proportion of the outgrowths.

In Fig. 3b a dilution of the outgrowth sample shows that detection of the paternal PGK band could be diluted out and still leave more maternally derived activity than seen in the blastocyst band (Fig. 3 b). Therefore even if blastocysts expressed paternal PGK to the same proportion of total activity as in outgrowths, it would be undetectable due to the low PGK activity at this stage. A limitation for all samples showing a single PGK-1 band is the argument that if more activity were applied to the gel a very weak component might then be detected. However the approximate two-fold difference in X-linked PGK activity in blastocysts derived from XX and XO mothers (Kozak & Quinn, 1975) is a strong argument that the PGK-1 activity in blastocysts is predominantly maternally derived and not embryo-coded.

In Fig. 3 a there appears to be a third PGK-1 band migrating ahead of PGK-1 A in the outgrowth samples, which was also visible in a repeat of the gel. We do not know the explanation for this extra PGK-1 band. Figure 1 c shows the position of the autosome-coded PGK-2 from the testes.

Various workers have found that female tissues derived from the trophoblast and primary endoderm (cell lineages based on Gardner & Papaioannou, 1975) express only the maternal X whereas those derived from the epiblast have equal, or almost equal, expression of the two parental X chromosomes (Takagi & Sasaki, 1975; West et al. 1977, 1978; Frels & Chapman, 1980). Three different mechanisms (Takagi, 1976) could be proposed for this non-random expression of the X chromosome in certain tissues:

1. Random X inactivation with subsequent reversal to inactivate the paternal X chromosome,

2. Random X inactivation with subsequent cell selection in favour of the maternal X chromosome,

3. Preferential inactivation of the paternal X.

The first explanation appears unlikely, and Takagi (1976) has shown that there is no reversal of the allocyclic X in and day embryos by a doublelabelling technique. It is known that the differentiated state of the X chromosome is very stable, such that clones can be produced with the same X active through multiple generations (Hamerton et al. 1971 ; Chapman & Shows, 1976). Even very rigorous pressures have only succeeded in reactivating the individual selected gene on the inactive X (Kahan & DeMars, 1975).

If cell selection (2) does occur it is not through the phenotypic pressure of the maternal reproductive tract (West et al. 1977; Frels & Chapman, 1980). There does not appear to be anything intrinsically wrong with the paternal X in trophectoderm and primary endoderm as it is capable of being expressed in the membranes of XO embryos (Frels & Chapman, 1979). Frels & Chapman (1980) have studied the PGK-1 expression at days and found only the maternal band after electrophoresis of the mural trophoblast, limiting the possible paternal expression to less than 0·5 %. Takagi’s group (Takagi & Sasaki, 1975; Takagi, Wake & Sasaki, 1978) investigated even earlier stages. In the extraembryonic portion of the egg cylinder at days 90 % of the differentially staining X chromosomes are paternal, and even at days and days over 85 % of the allocyclic X chromosomes are derived from the father.

By days most, if not all, cells of the embryo have undergone X inactivation (Takagi & Oshimura, 1973; Monk & Harper, 1979). Our results show that at this stage both PGK-1 A and PGK-1B bands from heterozygous females appear of approximately equal intensity in the epiblast, suggesting that X inactivation is random in this tissue. Although Takagi & Sasaki (1975) found 65 % of allocyclic X chromosomes were paternal in the embryonic portion of the day egg cylinders they examined, this may be due to a skewing effect from primary endoderm as the authors do not say whether or not this layer was included in their cell spreads.

The work reported here shows that the extraembryonic ectoderm and the primary endoderm at days display marked, if not exclusive, expression of the maternal Pgk-1 allele, arguing against cell selection being the cause of nonrandom expression in certain older tissues. A faint paternal band is detected in some of these tissues which may be due either to a low level of paternal X-chromosome expression or, more likely, to contamination from the epiblast.

Blastocyst attachment and outgrowth in vitro may mimic implantation in vivo (Monk & Ansell, 1976; Monk & Petzoldt, 1977). The results presented show that embryo-coded Pgk-1 expression also occurs in outgrowths. The predominance of the enzyme form coded by the maternally inherited X chromosome is compatible with the expression of only the maternal allele in the trophoblast cells. It appears that the X chromosomes of the trophectoderm differentiate at the blastocyst stage (Monk & Kathuria, 1977; Kratzer & Gartler, 1978) and those of primary endoderm by 6 days gestation (Monk & Harper, 1979). It is possible that the paternal allele of Pgk-1 is never active in the trophectoderm or the primary endoderm and X-chromosome differentiation may occur in these tissues prior to the embryonic expression of PGK-1.

The results overall strongly support the third mechanism for non-random X-chromosome expression, namely preferential inactivation of the paternally derived X chromosome in trophectoderm (from which the extraembryonic ectoderm is derived) and in primary endoderm.

We wish to thank John West for the original PGK-1 A mice, Anne McLaren for help in establishing our present PGK-1 A colony, and Andy McMahon and John West for helpful discussion.