Imprinting and X chromosome counting mechanisms determine Xist expression in early mouse development

Cell, Vol. 77, 639-650,

June

3, 1994, Copyright

0 1994 by Cell Press

Imprinting and X Chromosome Determine Xist Expression in Early Mouse Development Gmham F. Kay,*+ Sheila C. Barton,* M. Azim Surani,S and Sohaiia Rastan’ *Section of Comparative Biology Medical Research Council Clinical Research Centre Watford Road Harrow HA1 3UJ England *Weiicome/Cancer Research Campaign institute of Cancer and Developmental Biology Tennis Court Road and Physiological Laboratory University of Cambridge Cambridge CB2 1OR England

Summary in mice, X inactivation is preceded by in cis Xisf exprea sion. initiaiiy, normal female embryos express the paternal Xlsf allele exclusively, preceding imprinted X inactivation in the trophectoderm. Later expression of Xisf alleles is random, preceding random X inactivation in the epibiast lineage. in this study using uniparental embryos, we demonstrate that Xlsf expression is initially dictated solely by parental imprinting, causing expression of ail paternal alleles. Maternal alleles remain repressed, irrespective of X chromosome number. At the compacting moruia stage, this parental imprint is emsed, and the mechanism counting the X chromosomes imposes appropriate Xkf expression with respect to chromosome number. Our results also suggest that Xlsf expression may itself be regulated by a novel imprinted maternally expressed gene. introduction X chromosome inactivation (X inactivation) in female mammals, a dosage compensation system that ensures equal dosage of X-linked gene products in XX females and XY males (Lyon, 1961), is dependent on a major cis-acting switch gene or X inactivation center (Xic) (Rastan and Brown, 1990). in a diploid ceil, one X chromosome must remain active for correct X chromosome dosage; supernumerary X chromosomes are inactivated. Thus, whatever the sex chromosome constitution (e.g., XY, XX, XXY, XXX etc.), in a diploid cell there are always n - 1 inactive X chromosomes (n = total number of Xs). A cellular mechanism must therefore act to inactivate all but one X chromosome. it has previously been shown that the ceil counts the number of X chromosomes by counting the number of Xics (Rastan, 1983; Rastan and Robertson, 1985). X inactivation is considered to consist of three stages: initia*Present Bancroft

address: Queensland Institute of Medical Research, The Centre, 300 Herston Road, Brisbane, Q4029, Australia.

Counting Mechanisms

tion, when the number of Xics are counted and one X chromosome is marked to remain active by “blocking” its Xic; spread of the inactivating signal from any unblocked Xics in cis in both directions along the X chromosome; and stable maintenance of the inactive state clonaliy through subsequent cell divisions (Lyon, 1991). During early cleavage stages, both X chromosomes are active (Epstein et al., 1978), and X inactivation occurs as cells differentiate from the totipotent lineage (Monk and Harper, 1978). The first X inactivation occurs in the extraembryonic trophectoderm and primitive endoderm lineages and is imprinted, with exclusive inactivation of the paternal X chromosome (Takagi and Sasaki, 1975; West et al., 1977; Takagi et al., 1978). X inactivation in the embryonic lineage occurs later, at around the timeof gastrulation, and in this lineage either X chromosome in a ceil may be inactivated at random. Thus, the imprint on X inactivation is considered to be‘ labile. The Xist locus is a candidate for the Xic, since it maps to the Xic in both mouse and human and is expressed exclusively from the inactive X chromosome (Brown et al., 1991; Brockdorffetal., 1991; Borsanietal., 1991).TheXist gene product is an untranslated, though highly conserved, transcript (Brockdorff et al., 1992; Brown et al., 1992), and its possible role as the cis-acting effector of the spread of X inactivation is currently under intensive study. it has recently been shown that continued Xist expression may not be required for the maintenance of X inactivation in cell lines in which X inactivation is already established (Brown and Willard, 1994). There is, however, a compeiling case for the early involvement of Xist in the initiation of X inactivation, since Xist expression precedes X inactivation early in development and the earliest Xist expression is imprinted with exclusive expression of the paternal allele (Kay et al., 1993). Expression of the maternal Xist allele is not seen until shortly before gastrulation in the egg cylinder embryo (Kay et al., 1993), consistent with the random X inactivation that occurs in cells of the embryonic lineage as these cells start to differentiate. Since in XX female embryos imprinted paternal Xist expression precedes imprinted paternal X inactivation and random Xist expression precedes random 8 inactivation, it has been suggested that Xist expression may have a significant role in subsequent X inactivation (Kay et al., 1993). It has also been suggested that the number of X chromosomes in a cell are counted by counting Xist loci (Kay et al., 1993). We have recently obtained an insight into the nature of the imprint on Xist by showing that the paternal Xist allele becomes demethylated at the onset of meiosis and enters the zygote hypomethylated and poised to be expressed and that, in extraembryonic tissues that undergo imprinted paternal X inactivation, the paternal Xist allele is always unmethylated and the maternal allele is totally methylated (Norris et al., 1994). Based on these results, we would predict that the paternal Xist allele would be preprogrammed to be expressed early in preimplantation devel-

Cell 640

A

Normal

embryos

#l

#I

#2

#2

#l #2

#l #2

#I #2 H,O Remove

GYNOGENETIC

Xist (578bp) Remove

Hpn (352bp)

B

Parthenogenetic

embryos

ANDROGENETIC

B --m--

#I

#2

#l

#2

#I #2

#l

#2

#l #2 Hz0

13, 129 (578bp)

MX23b +75bp

12,

Xist (578bp) PCK(S7Xbp)

MX23b *

Hprt (352bp) Figure 1. Xist Expression Starts at the 4-Cell Stage in Normal Embryos but Is Delayed until the Compacting Morula Stage in Parthenogenetic Embryos cDNA was prepared from RNA from pools of 20 2-cell, and morulae stages and pools of IO blastocysts of each Xist cDNA was amplified by two rounds of amplification PCR primers. The coamplification control in the first round tion was Hod cDNA. (A) Xist expression profile in normal embryos. (B) Xi.9 expression profile in parthenogenetic embryos.

4-cell, E-cell, embryo type. using nested of amplifica-

opment and that the maternal Xist allele would initially be preprogrammed to be silent. If the expression of paternal and maternal Xist alleles is initially preprogrammed, what is the relationship between imprinted expression and the later random expression? Is the imprint temporally labile or lineage-specific? If the imprints are temporally labile, when exactly are they erased? What is the relationship between imprinting and the X chromosome counting mechanism? Normal XY male embryos do not express X&t, consistent with the paradigm that two Xics are necessary for the initiation of the X inactivation process; however, normal XY male embryos carry a maternal X chromosome, so lack of Xist expression could be due to the parental origin of the X rather than the fact that there is only one X chromosome. To address these questions, we have analyzed Xist expression in parthenogenetic and gynogenetic embryos with only maternal chromosomes and androgenetic embryos with only paternal chromosomes. Our analysis of Xist expression in these embryos tests the prediction that expression of Xist alleles is initially preprogrammed by parental origin and addresses the relationship among imprinting and the counting mechanism and the time of erasure of parental imprints on Xist. These studies provide new insights into the mechanisms gov-

32flbp

I ,b”d 111

183bp I Hl”d 111

&x20

11)

l lhd 111 (21

Figure 2. Production of Androgenetic and Gynogenetic Genetically Distinguishable Xist Alleles

503bp * MIX20

Embryos

with

(A) Schematic diagram to show the genetics and mechanics of production of androgenetic and gynogenetic embryos, (8) Site of Hindlll polymorphism within the Xist RT-PCR product used for allele-specific RT-PCR. Hindlll site (1) is present in most Mus musculus domesticus strains tested. This site is abolished in the PGK strain by point mutations (Kay et al., 1993). Hindlll site (2) is present in all strains tested and acts as a control for the completion of Hindlll digestion. The sizes of the product DNA fragments after Hindlll digestion are indicated.

erning expression velopment.

of this locus during early mouse de-

Results Xist Expression Starts at the 4-Cell Stage in Normal Fertilized Female Embryos We had previously shown that in normal fertilized XmXP female embryos Xist is expressed by the 8-cell stage with exclusive expression of the paternal allele and that normal fertilized X”Y male embryos never express Xi.9 (Kay et al., 1993); we had not previously examined 4-cell stage embryos. We now show that Xi.9 expression is initiated as early as the 4-cell stage in normal fertilized female embryos. Figure 1A shows a reverse transcriptase polymerase chain reaction (RT-PCR) analysis of pools of 20 normal fertilized preimplantation embryos. As previously reported, Xist is not expressed in 2cell embryos (Kay et al., 1993) but expression is clearly detectable at the 4-cell stage. Allele-specific RT-PCR showed that from the start of expression there is exclusive expression of the paternal allele (data not shown). Thus, in normal fertilized female embryos, expression of the paternal Xist allele starts very

Imprinting 641

of Xisf

Xist (578bp)

Figure 3. Xist Expression in Gynogenetic Embryos Is Delayed until the Blastocyst Stage When Random Expression of Xi.9 Alleles Occurs (A) Xist expression profile in individual 4-&l, &cell, morula, and blastocyst stage gyncgenetic embryos after two rounds of amplification with nested primers. Hprf cDNA is coamplified in the first round of amplification as a control. Gynogenettc embryos were produced according to the scheme shown in Figure 2. (B) Allele-specific RT-PCR for embryos from (A) that are positive for Xist expression. At the blastocyst stage, there is random expression of PGK and Fl alleles. The exceptional individual earlier stage embryos that expressed Xist in (A) only express one or other allele, consistent with contamination from a maternal cell.

Hprt (352bp)

a 4

early at a stage when the blastomeres are developmentally totipotent (Kelly, 1977; McLaren, 1976). Initiation of Xisf Expression Is Delayed In Parthenogenetic and Gynogenetic Embryos Since the earliest Xist expression in preimplantation embryos is imprinted to be expressed exclusively from the paternal allele, it was of interest to determine if and when Xist was expressed in female embryos that have only maternal X chromosomes. We initially examined pools of XmXm parthenogenetic embryos produced by suppression of second polar body formation (Barton et al., 1965). For each preimplantation stage studied, the experiment was repeated twice on pools of 20 parthenogenetic embryos. Figure 16 shows that Xist can indeed be expressed in

% 320

4

183

4

75

XmXm parthenogenetic embryos, which lack a paternal X chromosome. However, theonset of expression isdelayed compared with normal fertilized X”Xpembryos. The 4-and 6-tell parthenogenetic embryos do not express Xist, and expression does not start until the compacting morula stage (Figure 16). The single pool of 4cell embryos that shows a signal for Xist (pool 2 in Figure 1B) is likely to result from maternal cell contamination, since the detection system is very sensitive. The conclusion that Xist expression in preimplantation development is delayed in the absence of a paternal X chromosome is supported by results from a similar analysis of XmXm gynogenetic embryos. Gynogenetic embryos are produced by removing the male pronucleus from a fertilized egg and replacing it with the female pronucleus

Cell 642

Frgure 4. Xist Expression Starts Stage in Androgenetic Embryos sion Is Lost from Most Embryos velop through to Blastocysts

at the 4-Cell but Expresas They De-

(A) Xis? expression profile in individual 4-cell, &cell, morula. and blastocyst stage androgenetic embryos after two rounds of amplification with nested primers. Hpr7 cDNA is coamplified in the first round of amplification as a control. Table 1 summarizes the full set of results. Androgenetic embryos were produced according to the scheme outlined in Figure 2. (6) Allele-specific RT-PCR analysis of some 4-cell embryos from (A) that are positive for Xist expression showing that there are two types of androgenones. one that expresses only one allele, i.e., PGK (e.g., a23, a29) or 129 (e.g., a22, a26) and the other that expresses both alleles, i.e., PGK and 129 (e.g., a24, a27). Table 2 summarizes the full set of allele-specific RT-PCR results for all the stages studied.

Hprt (352bp)

of another fertilized egg (McGrath and Salter, 1984; Barton et al., 1984). Figure 2A shows the scheme by which the gynogenetic embryos were produced. The resulting gynogenetic embryos contained one (66 x CBA)FI female pronucleus and one PGK strain female pronucleus. The PGK strain of mouse carries a Hindlll restriction fragment length polymorphism (RFLP) in the Xi.9 cDNA detectable by RT-PCR (Kay et al., 1993; Figure 28). It is thus possible to distinguish between expression of the two maternal alleles in the gynogenetic embryos. Figure 3A shows an analysis of Xi.9 expression in individual gynogenetic embryos at different preimplantation stages. Like the parthenogenetic embryos, Xistexpression is delayed in the gynogenetic embryos. No consistent Xist expression is seen

in these embryos until the blastocyst stage. The detection of an Xistsignal in the odd 4-cell embryo and &cell embryo is most probably the result of maternal contamination. Allele-specific RT-PCR of the gynogenetic samples expressing Xist showed that the two Xist alleles were expressed in approximately equal measure in all the gynogenetic blastocysts (Figure 38). This expression could be biallelic or be from roughly equal numbers of cells expressing one or the other Xist allele. The latter situation, i.e., random expression of Xist alleles, is by far the most likely since individual blastocysts in Figure 3B vary slightly in the relative proportion of the two alleles expressed. Also, it is known that one X chromosome is active and that one is inactive in postimplantation diploid parthenogenetic em-

Imprinting 643

of Xist

bryos (Rastan et al., 1980; Mann and Lovell-Badge, 1988). Furthermore, diploid parthenogenetic embryonic stem (ES) cells with cytogenetically distinguishable X chromosomes undergo random inactivation of one or the other X chromosome upon differentiation even when the X chromosomes are of identical origin, as in haploid-derived ES cells (Rastan and Robertson, 1985). Expression of only one Xist allele was seen in the occasional anomalous 4-tell, &ell, or morula that expressed Xist, three expressing only the PGK allele and two expressing only the Fl allele (Figure 38). This could be rogue expression in a single blastomere;but it is also consistent with contamination from a single maternal cell. These results show that in both XmXm parthenogenetic and XmXm gynogenetic embryos Xist expression is considerably delayed. The results support the prediction that the maternal allele is initially preprogrammed to be silent and show that the imprint must have been erased, at least in some cells of the embryo, by the compacting morulal blastocyst stage. Erasure of the silencing imprint on maternal Xist alleles and operation of the X chromosome counting mechanism would allow expression of Xist for the first time in these embryos with exclusively maternally derived X chromosomes. We consider it most likely that Xist expression at this time is from cells of the determined trophectoderm lineage (see below; Figure 6). Preprogrammed Expression of All Paternal Xisf Alleles In XX and XY Androgenetic Embryos Is Followed by Extlnction of Expression by the Blastocyst Stage We next analyzed Xist expression in androgenetic (McGrath and Solter, 1984; Barton et al., 1984) embryos with only paternal chromosomes. Androgenetic embryos were produced by removing the female pronucleus from a fertilized egg and replacing it with the male pronucleus of another fertilized egg. Figure 2A shows the scheme we used. The resulting androgenetic embryos contained one PGK paternal pronucleus and one 129 paternal pronucleus to enable us to distinguish the Xist alleles (see Figures 2A and 26). Although all gynogenetic embryos are XX, androgenetic embryos can be XX, XY, or YY and, before any selection, should be present in the fvlendelian ratios of lXX:2XY:lYY. Figure 4Ashows the RT-PCR analysis of Xist expression in individual androgenetic embryos. Here, Xi.3 expression starts at the 4-cell stage, i.e., the same time as in normal fertilized embryos, but by the blastocyst stage, Xistexpression is largely switched off. Table 1 shows the complete results for all the individual androgenetic embryos analyzed in this experiment. At the 4cell stage, 23 of 30 or 77% of androgenetic embryos expressed Xist. One would expect 25% of the samples at this stage to be genotypically YY (see below), and therefore incapable of expressing X-linked Xist. Amplification of control Hprt in such YY embryos at the 4-cell stage would be from persisting maternal message. Since the remaining androgenetic embryo population at the 4cell stage should consist of 50% XPY embryos and 25% XpXpembryos and our analysis shows that 77% of the 4-cell androgenetic embryos expressed Xist,

Table 1. Proportion of Androgenetic Various Stages of Development

Staae

XisvNprt

3-tell 4cell 6- to 7-tell Early morula Blastocyst

O/6 23130 IO/11 13/27 3/19

(0%) (77%) (91%) (46%) (16%)

Embryos

Expressing

Xisf at

Failed PCR

Total

4 a 2 13 1

10 30 13 40 20

The XisvHprt wlumn shows the number of embryos that expressed Xist but of the total that expressed Hprt. Embryos in the failed PCR column are those for which no signal was detectable for both Xisfand Hcxt.

this suggests that both the XPXP and XPY androgenetic embryos are expressing Xi&. At the 5- to 7-tell stage, the proportion of embryos expressing Xist increases to 91%, but this is most likely due to the elimination by selection of most of the YY embryos. By the compacting &cell stage, however, the proportion of embryos expressing Xi.9 has dropped to 46%, and the proportion drops even further to 16% by the blastocyst stage. Thus, it appears that from the 4-tell stage to the 8cell stage all androgenetic embryos, whether of the genotype XPXP or XPY, express Xist but at the compacting morula stage down-regulation of Xist expression starts to occur (see below). To confirm our inference that both the XPY as well as XPXP androgenetic embryos were expressing Xist prior to compaction, we performed allele-specific RT-PCR analysis on the samples. As before, the PGK Xi.9 allele can be distinguished from the other (129) allele by the absence of the Hindlll site in the cDNA (see Figure 28). Figure 48 shows an example of the results from androgenetic embryos, showing that at the 4-tell stage, when Xist expression starts, these embryos consist of two types, one type that expresses both paternal alleles (e.g., a24 and a27) and a second type that expresses only one Xist allele, either the PGK allele (e.g., a23 and a29) or the 129 allele (e.g., a22 and a26). A total of 121 preimplantation androgenetic embryos were analyzed, and Table 2 summarizes the results. Embryos expressing both Xist alleles must of necessity be genotypically XPXP, and it seems likely that the embryos expressing only one Xist allele are the majority XPY population. If this is so, it means that the X chromosome counting mechanism is not yet functional. Although the embryos expressing only one Xi.9 allele were most likely to be the majority XPY population, which by Mendelian ratios should outnumber the XPXP embryos by 2 to 1, it was formally possible (though highly unlikely) that all these embryos were XX embryos that were only expressing one allele. To address this question, we analyzed individual androgenetic embryos at the 8-cell and blastocyst stage, by sexing individual embryos using X- and Y-specific genomic PCR on one half of the sample and performing allele-specific RT-PCR analysis of Xist expression on the other half of the sample. Figure 5 shows the results of the sexing of this series of embryos using PCR coamplification of the X-linked Ott gene and the Y-linked Zfy gene. The two genes act as amplification controls for each other and will distinguish XX, XY, and W

Cdl 644

Table

2. Allele-Specific

Xist RT-PCR

Stage

Embryos Xist

4-cell 5- to 7-cell Early morula Blastocyst

23 IO 13 3

Results

for Individual

Expressing

Androgenetic

Embryos

That Scored

Xist Allele Expressed

Positive

for Xist Expression

(Genotype)

Both (XX)

PGK (XV?)

129 (XV?)

5 2 2 1

7 5 5 2

11 3 6 0

Embryos expressing Xist (Table 1) fall into two classes: those expressing both PGK and 129 alleles and those expressing only one allele, either PGK or 129. Embryos expressing both PGK and 129 alleles must be XPXP embryos; those expressing either the PGK or 129 allele may be XPY, but they could also represent XpXp embryos with nonrandom Xist allele expression. Since in this series of androgenetic embryos the genotype was not unequivocally determined by genomic PCR amplification for X- and Y-linked genes, the genotype for these embryos is marked (XV?).

embryos. The results show that by the a-cell stage XPY embryos outnumber XPXP embryos by about 3 to 1, suggesting that there is already some selection against XPXp embryos. Surprisingly, this analysis shows that in the absence of an X chromosome some YY embryos can persist to the a-cell stage (e.g., a67 and a96), presumably surviving this far on product from maternal transcripts for essential X-linked genes. The results from the allele-specific RTPCR performed on the other half sample is summarized in Table 3. By the late a-cell stage, the down-regulation of Xist expression previously noted is starting to occur, and many of the samples that express Hprt do not express Xist, but six embryos that were definitively sexed as XPY are expressing their single Xist allele. Thus, there is inappropriate expression of the single paternal X chromosome in XPY embryos, i.e., the paternal Xist allele is preprogrammed to be expressed irrespective of the number of X chromosomes present. By the blastocyst stage, none of the XPY embryos tested expressed Xist. It should also be noted that Xist was not expressed in 5 of 7 late a-cell embryos and blastocysts that typed XPXP in this analysis. SinceXistexpression starts to be extinguished at the compacting morula stage in XPXpas well as XPY androgenones, we infer that androgenones lack a gene product required for continued Xist expression (see below).

8-cell

Discussion In normal fertilized X”XPembryos, the earliest Xistexpression is imprinted with exclusive expression of the paternal allele (Kay et al., 1993); the maternal Xist allele is not expressed until just prior to gastrulation (Kay et al., 1993). The present study on embryos that have only maternally derived or paternally derived chromosomes sheds light on the mechanisms governing expression of the Xist gene in early mouse development and the relationship between imprinted and random Xi& expression. We show that the initial expression of Xist is dictated by parental imprinting such that, until the compacting morula stage, all paternal alleles, but no maternal alleles, are expressed. Later, there is loss of parental imprints, and an X chromosome counting mechanism ensures that Xisf expression is appropriate for the number of X chromosomes. Additional features of Xist regulation are the lineage-specific regulation of expression and the likely involvement of a maternally expressed factor. DNA methylation is a heritable epigenetic modification that is inversely correlated with gene expression and implicated in genomic imprinting(Bird, 1993). We have recently shown that Xi&is imprinted in the germline by DNA methylation since the paternal Xist allele in the sperm enters the

Figure 5. Sexing of a Series of Compacting 6-Cell and Blastocyst Stage Androgenetic Embryos using PCR Amplification of X- and Y-Specific Genes A PCR

product

from

X-linked

oic

only is an

a57 + _

a60 + _

a61 + _

a96 + _

RT-PCR

Stage

Fl

al00 -I+

Results

PGK

a34 + +

XY

PGK

a56 + ‘+

: a59 + _

for Xist for a Series

a62 + Fl

a64 + +

of Individual

a66 + _

Sexed

a66 -I-

a97 + Fl

a99 + +

Androgenetic

PGK

a102 + +

Embryos

al 05 + -

a106 + _

al 07 + _

a106 + -

a67 -

YY

Individual embryos were analyzed for Xist expression by RT-PCR as in Figure 3 and sexed (Figure 5). Only compacting &cell and blastocyst stage embryos have insufficient cellular material to give a result for both genomic PCR for sexing and RT-PCR analysis of Xist expression. Xist expression previously noted (see text) is starting to occur in these embryos, several embryos that unequivocally sex XY also express

Xist allele

Hprt xjst

xx

S6X

Embryo

Late &Cell

Table 3. Allele-Specific

a109 -

PGK

a66 + +

xx a69 + -

a91 + -

XY

Stage

a92 + -

a93 + -

a95 + -

stage embryos were tested in this series, as earlier The results show that, although the modulation of their single Xisf allele.

a96 -

Blastocyst

Cell 646

zygote demethylated and poised to be expressed as a result of developmentally programmed demethylation in the male germline at the onset of meiosis (Norris et al., 1994). While the methylation status of the maternal allele in the zygote has yet to be determined unequivocally, preliminary evidence indicates that it is methylated (G. F. K. and S. R., unpublished data). We have also demonstrated that the maternal allele is totally methylated and that the paternal allele is always unmethylated in the extraembryonic lineages that are characterized by imprinted paternal Xist expression and paternal X inactivation (Norris et al., 1994). Our current results on Xist expression in androgenones and gynogenones confirms that the earliest Xist expression is dictated entirely by parental imprinting. Not only are both copies of X&expressed in XPXPandrogenetic embryos, but XPY androgenetic embryos also expressxist. This result is particularly striking, since Xist expression is never seen in the normal X”Y embryo (Kay et al., 1993), and tells us that early in development Xist expression is not dependent on the number of X chromosomes present, i.e., the X chromosome counting mechanism is not yet operative. In addition, the failure of early XmXm parthenogenones and gynogenones to express Xist shows that the maternal allele is initially programmed by imprinting to be silent. This situation persists until the compacting morula stage. It has been previously suggested that X chromosome inactivation in the extraembryonic lineages of parthenogenetic embryos may be delayed (Rastan et al., 1980; Mann and Lovell-Badge, 1988); the delay in onset of Xist expression reported here may indeed cause a corresponding delay in the time of X inactivation and contribute to the poor development of extraembryonic tissues in parthenogenones and gynogenones (Surani and Barton, 1983; Surani et al., 1986). It is also interesting that a paternal X chromosome can retard development in XPOembryos compared with XmO embryos (Thornhill and Burgoyne, 1993), and it is possible that the early preprogrammed expression of the paternal Xist allele contributes to this effect. Recent studies also using uniparental embryos indicate that expression of other imprinted genes, Igf2, Igf2r (Latham et al., 1994), and H79 (H. Sasaki, A. C. FergusonSmith, A. S. W. Shum, S. C. B., and M. A. S., unpublished data) is not affected during preimplantation development. However, after implantation, there is parent of origin specific expression of these genes observed in controls as well as in androgenetic and parthenogenetic embryos (Walsh et al., 1994); in the caseof H79 this is accompanied by methylation of the promoter region (H. Sasaki, A. C. Ferguson-Smith, A. S. W. Shum, S. C. B., and M. A. S., unpublished data). By contrast, expression of Xist is dictated by parental imprinting only until about the 8-cell stage (this study; Latham et al., 1994). Beginning at the compacting morula stage, a second mechanism regulating Xist expression becomes evident. This is shown by expression of Xist at this stage for the first time in XmXmparthenogenones and gynogenones. Our results indicate that the gynogenetic blastocysts contain roughly equal numbers of cells expressing one or other Xist allele, i.e., either maternal allele can be silenced or

expressed at random in each cell. Since the maternal alleles were previously repressed and in this case there are no parental origin differences between the two Xistalleles, we must infer that the imprint has been erased and the counting mechanism has become operative and is now responsible for the silencing of one allele and the expression of the other allele in each cell. In androgenetic embryos, however, we see silencing of the previous Xist expression when the counting mechanism comes into force. Our results suggest that the X chromosome counting mechanism starts at about the same time as the occurrence of the loss of the parental imprints that govern initial expression of Xist. It is known that a major genome-wide demethylation occurs during preimplantation development between the 8-cell and blastocyst stage (Monk, 1990; Monk et al., 1991; Kafri et al., 1992; Brandeis et al., 1993). We have previously suggested that at this time the Xist locus also loses its methylation imprints and becomes free from constraints on its expression imposed by parental imprinting (Norris et al., 1994); our current evidence that expression of the maternal allele in parthenogenones and gynogenones starts at this time suggests that this is, indeed, the case. The molecular basis of the counting mechanism remains obscure. However, the essential features of the counting mechanism in relation to Xisr must be the stable silencing of one Xi.3 locus in each diploid cell and the expression of the Xist locus from any other X chromosomes present. How such stable silencing is brought about remains a key question for the future, although methylation is likely to be involved (Norris et al., 1994). However, one intriguing finding from the present study that may throw some light on the second part of the counting mechanism is the lack of Xist expression from most of the XPXP androgenones after the late 8-cell stage (Table 3) despite the presence of two X chromosomes. We propose that one of the transacting factors required for Xist transcription is the product of a gene that is itself imprinted and expressed only when maternally inherited. We further propose that expression of this maternally expressed gene product is triggered by differentiation of totipotent cells (Johnson and Ziomek, 1981) thus resulting in the close relationship between differentiation and X inactivation. Maternal product in the oocyte would allow the early preprogrammed imprinted expression of the paternal allele in normal fertilized X”XPembryos and XPY and XPXPandrogenones. Exhaustion of this product by the time the counting mechanism becomes operative would not affect normal fertilized XmXP embryos with a set of maternal chromosomes, but would result in loss of Xist expression in both XPY and XPXP androgenones. This would not compromise the XPY androgenones but may lead to subsequent failure of X inactivation in XPXP androgenones. In this context, we show that there is already selection against XpXp androgenones during the preimplantation period, and in a study on postimplantation androgenetic embryos, all nine egg cylinders were found to be of the XPY genotype (Kaufman et al., 1989). We propose that in normal fertilized XmXP embryos the early imprinted paternal Xist expression is stabilized and

imprinting

of Xist

647

Figure 6. Diagrammatic Summary of Events Influencing Xist Expression in Early Mouse De velopment During early development, the expression of Xist is programmed first by imprinting that resuits in the expression of all paternal, but no maternal, Xist alleles irrespective of the number of X chromosomes (normal XY embryos do not express Xist initially because their single Imprinted Paternal Xist Expmssed Xist allele is maternally derived, not because they only have one X chromosome). Expression of Xist is dependent on an imxpxp: solr Rbmal a*, Expmrcl xpxm:mmmdxl8l~ printed maternally expressed factor. This fatMm: NoXbI Expm*on xpv : Pdnvl a* ExpnUd XmY : No XmExprnla tor is present in the oocyte cytoplasm, and exSIkftion ..pind A’ pression from the embryonic genome is linked I. to differentiation. The paternal Xistallele enters I the zygote hypomethylated and poised to be Loss of Pkentai Imprints & StarI of Counting Mechanism expressed using the maternally encoded factor already present in the oocyte cytoplasm. At the compacting morula stage, a second mechanism that counts the number of X chromosomes and imposes appropriate Xist expression becomes operative. At around this time, parental imprints on Xist’are erased and one allele is silenced at random, defining the future active X. By this stage, the oocytederived maternally encoded factor has been depleted; expressionof theXistallelefrom anyotherxchromosomes present now becomes dependent on zygotic expression of this factor from the maternally inherited genome. In normal fertilized X”Xpembryos, the earlier imprinted expression of the paternal Xist allele, which is appropriate in terms of X chromosome number, is stabilized by this factor in the trophectoderm; erasure of the imprint does not become visible as Xist expression in the epiblast lineage until the imprinted maternally encoded factor is produced as cells differentiate from the totipotent lineage. In XmXm gynogenones, expression of this factor from the determined trophectoderm lineage allows expression of one or the other Xist allele at random for the first time. Absence of this factor in androgenones by the compacting morula stage results in extinction of Xist expression in both XPY and XPXP embryos.

maintained throughout development in cells that become determined first and are destined to become trophectoderm (Johnson and Ziomek, 1981) if the imprinted Xist expression is appropriate in terms of X chromosome number. This leads to imprinted paternal X inactivation in the extraembryonic lineage. Once the imprint has been erased and the counting mechanism has come into force, random expression of Xist alleles can occur in parthenogenones and gynogenones when the first zygotic expression of the proposed transcription factor from the maternally inherited genome occurs in the determined trophectoderm lineage. Although in normal fertilized embryos the erasure of parental imprints and the choice of one Xist allele to be preemptively silenced by the counting mechanism may have already occurred in the inner cell mass (ICM) lineage at the morulalblastocyst stage, we do not see expression of the maternal Xist allele indicative of random Xist expression until just prior to gastrulation (Kay et al., 1993). This is presumably because expression of the proposed transcription factor from the maternally inherited genome only starts in the epiblast lineage as it starts to differentiate. Normal XY male embryos do not express Xistinitially because their X chromosome is maternally derived; later, they do not express Xist because they only have one X chromosome. The main events that we

now propose act to influence Xist expression during early mouse development are summarized in Figure 6. There is growing evidence that nuclear-cytoplasmic interactions can induce epigenetic modifications with phenotypic consequences during development and even in adulti (Latham and Solter, 1991; Reik et al., 1993; Surani et al., 1990). For example, effects of such interactions are seen in early androgenetic development (Latham and Solter, 1991) and particularly in the DDK strain of mice (Babinet et al., 1990). B&d on studies on the imprinting of transgenes, the postzygotic control of imprinting and expression of parental alleles has tong been suspected (Sapienza et al., 1989; Allen et al., 1990; Engler et al., 1991), and the possibinty that factors responsible for postzygotic modificMon$‘bf parenial imijrints are themselves subject to parental origin effe’cts tia,s been considered. Indeed, it was demonstrated that methylation and repression of one speoific transgene locus occurs in response to a modifier gene that must b&maternally inherited (Allen et al., 1990; Surani et al., 1990). Xist expression particularly lends itself to the study of control of imprinting as the imprint on this gene is developmentally labile. Our evidence suggests that, after the late &cell stage, expression of Xi.9 is no longer controlled by the parental origin of the gene itself, but rather by the parental origin of a

Cell 648

factor that positively sustains or induces Xist expression. The postulated factor is expressed from the maternally derived genome only when the primary lineages differentiate from the totipotential state. The identification of this gene will be important not only for understanding the control of Xi.3 expression and X inactivation but also in the wider context of interactions between parental genomes in imprinting. Although the paternal Xist allele is expressed at the 4-cell stage in normal fertilized female embryos, a mature inactive X chromosome cannot be detected until the blastocyst stage in the trophectoderm. This suggests that Xisr expression is not in itself sufficient to effect X inactivation and that other interacting factors are required. It has been shown experimentally that at the 4-cell stage all the blastomeres are totipotent (Kelly, 1977). Since it appears that Xist expression does not instantly and inevitably cause inactivation and, in any case, the evidence indicates that the imprint on Xisr expression is erased and superseded by the counting mechanism, we consider it likely that all four blastomeres at the 4-cell stage are initially expressing paternal Xisr. However, it is formally possible that, even at this early stage, Xistexpression is mosaic and that blastomeres that express Xisr will normally form the trophectoderm lineage and that blastomeres that do not will normally form the ICM lineage. In situ hybridization analysis of early cleavagestage embryos, although technically difficult, will be useful in testing this intriguing possibility. Experimental

Procedures

Production of Parthenogenetic Embryos Preparation of parthenogenetic embryos was as previously described (Barton et al., 1965). Unfertilized eggs were obtained by superovulating (C57BU6J x CBAfCa)FI females. The eggs were first treated with hyaluronidase to remove cumulus cells and then activated at room temperature in T6 plus BSA medium containing 7% ethanol for 4-5 min. The eggs were washed six times in T6 plus BSA medium and cultured in this medium containing 5 pglml cytochalasin B for 3-4 hr at 37.8OC. After this time, the eggs were washed nine times and cultured for a further 2 hr. Diploid parthenogenetic zygoteswith two pronuclei produced as a result of the suppression of second polar body extrusion in the presence of cytochalasin B were identified and collected for subsequent culture in T6 plus BSA at 37.8OC in 5% CO2 in air. The zona pellucida of parthenogenetic and fertilized embryos was removed by digestion in 0.5% pronase, and the embryos were washed individually through a series of PBS drops containing 0.1% BSA (PBS/ BSA) to remove contaminating maternal cells. Pools of parthenogetic embryos and normal fertilized control embryos at the appropriate stages were then collected in a minimal volume, snap frozen in liquid nitrogen, and stored at -70°C until use. Production of Androgenones and Gynogenones Androgenones and gynogenones were prepared by micromanipulation and karyoplast fusion using Sendai virus exactly as previously described (Barton and Surani, 1993). In brief, superovulated females (C57BU6J x CBA/Ca)Fl (Fl) were mated with PGK males to produce recipient zygotes from which the female pronucleus was first removed. A second male pronucleus was then introduced in these zygotes from donor zygotes obtained by mating superovulated Fl females with 129/ Sv males. This resulted in androgenones with a 129IPGK genotype in Fl cytoplasm. The inclusion of the 129 genome in androgenones together with the Fl cytoplasm provides relatively better development of preimplantation embryos. For the preparation of gynogenones, recipient zygotes were prepared by mating superovulated Fl females with Fl males. The male Fl pronucleus (identified by the larger size of the male pronucleus) was removed from these zygotes, and a second female pronucleus was then introduced from donor zygotes obtained

by mating superovulated PGK females with the Fl males (Barton and Surani, 1993). This resulted in gynogenones with FllPGK genotype in Fl cytoplasm. All zygotes obtained following micromanipulation were subsequently cultured in T8 plus BSA at 37.8OC in 5% CO* in air up to the blastocyst stage. At appropriate stages throughout preimplantation development, embryos were then washed individually as above. After the final wash in PBSIBSA, the individual embryos were lysed in 10 ~1 of water with 0.01% diethyl pyrocarbonate (DEPC) as previously described (Kay et al., 1993). incubated at 37OC for 20 min then 95OC for 10 min, snap frozen in liquid nitrogen, and then stored at -70°C until use. AT-PCR on Pools of Embryos RNA was prepared from pools of embryos as previously described (Kay et al., 1993) and redissolved in 10 PI water. First strand cDNA prepared as previously described (Kay et al., 1993) in a total volume of 20 ~1 using a Superscript kit (BRL) with random hexamer priming of the cDNA synthesis. The cDNA was precipitated with glycogen as carrier and stored overnight at -7OV. PCR analysis using the cDNA as template was with PCR primers M/X10, MX20, M/X20, MX23, HPRTRNAF. HPRTRNAR, as previously described (Kay et al., 1993). A 20 ul sample of each PCR product (first and nested second round) was run out on 2% agarose gels and DNA visualized with ethidium bromide staining. RT-PCR on Individual Embryos First strand cDNA was synthesised as described (Kay et al., 1993) but with 50 ng each of specific primers for Xist and Hprt transcript rather than random hexamer priming. The primers used were XISTCDNA (5’-aat tag aca cat aga cca ag-3’) and HPRTCDNA (5’-tct tag get ttg tat ttg gc-3’). After cDNA synthesis, the samples were processed for PCR as described above. The PCR products (20 ~1) from the first round of amplification were run out on 2% agarose gels, Southern blotted, and hybridized with purified “P-labeled /-&VT PCR product to visualize the bands. Nested second round PCR products were visualized with ethidium bromide. Allele-Specific RT-PCR on Individual Embryos For allele-specific RT-PCR, the nested second round PCR products were subject to an extra cycle of amplification (denaturation 95OC for 5 min, primer annealing 55OC for 1 min, DNA synthesis 72’C for 15 min) after dilution with complete PCR mix (minus template) to minimize heterodimer formation. The PCR product was then purified by passage through Pharmacia S300 spincolumns and digested overnight with an excess of Hindlll. Digested DNA (5 ~1) was then fractionated on nondenaturing 5% polyacrylamide gels and visualized with ethidium bromide. Sexing of Embryos Individual embryos were processed as above but after lysis in waterDEPC, an extra 10 ~1 water was added and the sample mixed well. The sample was then divided into two 10 VI samples. One sample was used for RT-PCR analysis as described, the other used for sexing by genomic PCR with primer pairs specific for the X-linked Ott gene (OTCFOR and OTCREV) and the Y-linked Zfy-1 gene (YLNS.5 and Zfy7.8) as previously described (Kay et al., 1993). These PCR products were run out on 2% agarose gels, Southern blotted, and hybridized with purified J2P-labeled Oft and Zfy-7 PCR product to visualize the bands. PCRs All PCRs, using genomic or cDNA as target, viously described (Kay et al., 1993).

were carried

out as pre-

Mice The PGK and 129 strain mice were bred in-house in the Section of Comparative Biology at the Medical Research Council Clinical Research Centre. The (C57BlN x CBAICa)Fl mice were from the Wellcome/Cancer Research Campaign Institute. Acknowledgments Correspondence should be addressed to G. F. K. This work was supported by the United Kingdom Medical Research Council and by the

Imprinting 849

of Xist

Wellcome Trust (grant 038481 to M. A. S.). We thank Fay Shamanski and Anne FerguaonSmith as well as the members of our labs, in particular Dominic Norris and Graeme Penny, for useful discussions. Received

February

25, 1994; revised

April 14, 1994.

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