ABSTRACT
Using genetic and cytogenetic markers, we assessed early development and X-chromosome inactivation (X-inactivation) in XX mouse androgenones produced by pronuclear transfer. Contrary to the current view, XX androgenones are capable of surviving to embryonic day 7.5, achieving basically random X-inactivation in all tissues including those derived from the trophectoderm and primitive endoderm that are characterized by paternal X-activation in fertilized embryos. This finding supports the hypothesis that in fertilized female embryos, the maternal X chromosome remains active until the blastocyst stage because of a rigid imprint that prevents inactivation, whereas the paternal X chromosome is preferentially inactivated in extra-embryonic tissues owing to lack of such imprint. In spite of random X-inactivation in XX androgenones, FISH analyses revealed expression of stable Xist RNA from every X chromosome in XX and XY androgenonetic embryos from the four-cell to morula stage. Although the occurrence of inappropriate X-inactivation was further suggested by the finding that Xist continues ectopic expression in a proportion of cells from XX and XY androgenones at the blastocyst and the early egg cylinder stage, a replication banding study failed to provide positive evidence for inappropriate X-inactivation at E6.5.
INTRODUCTION
X-chromosome inactivation (X-inactivation) (Lyon, 1961) in female mouse embryos first occurs in the trophectoderm of expanded blastocysts and the primitive endoderm of implanting blastocysts (Sugawara et al., 1985). In these cells the paternally derived X chromosome (XP) is preferentially inactivated (Takagi and Sasaki, 1975; West et al., 1977). In the embryo proper, X-inactivation occurs randomly before gastrulation (Takagi et al., 1982; Rastan, 1982) with equal probability of either the maternal X (XM) or XP being inactivated. Lyon and Rastan (1984) hypothesized that XM is imprinted to remain active, and that this imprinting is erased in early postimplantation development, allowing random X-inactivation to occur in cells of the embryo proper. Monk and McLaren (1981), however, proposed that the XP chromosome is prone to inactivation in early development because it retains memory that it was previously inactive during spermatogenesis.
The XIST/Xist gene, which is mapped in the region of X-chromosome inactivation center and is exclusively expressed from the inactivated X chromosome of differentiated female somatic cells (Borsani et al., 1991; Brockdorff et al., 1991; Brown et al., 1991), is essential for X-inactivation in vivo and in vitro (Penny et al., 1996; Marahrens et al., 1997, 1998; Lee et al., 1996, 1999a; Lee and Jaenich, 1997; Herzing et al., 1997; Heard et al., 1999a,b; Wutz and Jaenisch, 2000). RT-PCR and RNA FISH (fluorescent in situ hybridization) analyses revealed two different levels of Xist gene expression in mice. Low expression corresponding to the spot or pinpoint signal detected in ES and early embryonic cells of both sexes, and high expression corresponding to the large paint signal found in female somatic cells and differentiated embryonic cells after X-inactivation (Panning et al., 1997; Sheardown et al., 1997). Prior to X-inactivation, unstable Xist RNA is expressed from all X chromosomes and accumulates only at the site of transcription; stabilization and spread of Xist RNA in cis from the inactivation center along the entire length correlate with genetic silencing of the X chromosome (Panning et al., 1997; Sheardown et al., 1997). Johnston et al. (1998) favor the promoter switch as a mechanism for the change in the stability of Xist RNA, but Lee et al. (1999b) speculate that Tsix RNA antisense to Xist RNA is involved in destabilization of the latter. The recent study by Warshawsky et al. (1999) does not uphold the promoter switch hypothesis.
Previous work has shown that early development of androgenones (two paternal sets of chromosomes) with two X chromosomes are more severely affected than that of XY androgenones. Kaufman et al. (1989) failed to find any XX embryo among 12 egg-cylinder-stage androgenones. Circumstantial evidence led Kay et al. (1994), and Latham and Rambhatla (1995) to speculate that XX androgenones were dead by the blastocyst stage. Two diverse causes are proposed for the early death of XX androgenones: two active X chromosomes, owing to the failure of X-inactivation (Kay et al., 1994); and no active X chromosome, owing to inactivation of both X chromosomes (Kaufman et al., 1989; Latham, 1996). So far as we are aware, no direct information is yet available that allow one to evaluate these possibilities. The present study was initiated to obtain more-relevant information from androgenetic mouse embryos produced by pronuclear transplantation. We made use of male mice carrying a Robertsonian-type translocation involving the X chromosome or exclusively autosomes, and an X-chromosome linked lacZ transgene that is subject to X-inactivation (Tan et al., 1993) as markers for verifying the androgenetic origin of each embryo.
The present histological and cytogenetic study provides compelling evidence that, contrary to the widely accepted view (Kaufman et al., 1989; Kay et al., 1994; Latham and Rambhatla, 1995), XX androgenones survive on embryonic (E) day 7.5, achieving basically random X-inactivation in all tissues including those derived from the trophectoderm and primitive endoderm. This finding supports the hypothesis that XM carries a rigid imprint that prevents inactivation until implantation, whereas XP is free of such imprint. Furthermore, Xist RNA FISH analysis suggested the occurrence of inappropriate X-inactivation resulting in a proportion of cells functionally nullisomic for X in XX and XY androgenetic embryos. A chromosome study, however, failed to provide positive evidence for it.
MATERIALS AND METHODS
Mice
Male mice from the transgenic H253 (Tan et al., 1993) and Robertsonian translocation stocks, Rb(X.2)2Ad (abbreviated to Rb2) (Adler et al., 1989) and Rb(X.9)6H (abbreviated to RX9) (Tease and Fisher, 1991) were mated with wild-type females (C57BL/6J×CBA/J) F1 (abbreviated to F1). The transgenic H253 stock carries E. coli lacZ gene with the promoter of a mouse housekeeping gene, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG CoA) and a SV40 T antigen nuclear localization signal sequence on the X chromosome (Tan et al., 1993). The HMG-lacZ transgene is subject to X inactivation in certain tissues, but residual β-galactosidase (β-gal) activity may persist for a little while after X-inactivation (Tan et al., 1993; Lebon et al., 1995). In addition to Rb2 and RX9 males, we mated F1 females with Rb(10;11)8Bnr (abbreviated to Rb8) (Gropp et al., 1972) and Rb(11;13)6Lub (abbreviated to Rb6) (Gropp et al., 1975) males to obtain fertilized eggs for generating androgenetic embryos used for the Xist RNA FISH analysis. Metacentric translocation chromosomes are easily identified in metaphase cells as all remaining chromosomes are acrocentric.
Production of androgenones
Female F1 mice were superovulated by injections of pregnant mare’s serum gonadotropin (PMSG, 10 IU) and human chorionic gonadotropin (hCG, 10 IU) 48 hours apart before mating. Fertilized eggs at the pronuclear stage were recovered from oviducts in M2 medium 18-19 hours after hCG injection and cultured in M16 medium (Whittingham, 1971). The female pronucleus was removed from recipient eggs and replaced with male pronucleus from donor eggs to yield F1+H253, H253+Rb2, H253+RX9, Rb2+RX9 and Rb6+Rb8 androgenetic eggs as previously described (McGrath and Solter, 1983; Barton et al., 1987). Fusion of the karyoplast with the egg was induced with inactivated Sendai virus (2700 hemagglutination U/ml). After cytoplasmic fusion, embryos were transferred to oviducts of pseudopregnant F1 foster mice. Some embryos used for FISH analysis were cultured in M16 medium under paraffin oil at 37°C in 5% CO2 in air up to the four- or 8-16-cell stage.
Histological analysis of X-inactivation
Embryos were recovered at E7.5 from foster mothers and fixed with 4% paraformaldehyde (5-10 minutes) in 0.1 M phosphate buffer (pH 7.2). They were washed briefly and stained in 0.1% 4-chloro-5-bromo-3-indolyl-β-D-galactopyranoside (X-gal), 2 mM MgCl2, 5 mM EGTA, 0.01% (w/v) sodium deoxycholate, 0.02% (w/v) Nonidet P-40, 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6.6H2O at 37°C overnight. Embryos were dehydrated with ethanol, embedded in JB-4 resin (Electron Microscopy Sciences, Ft Washington, PA) and sectioned at 2 μm. Sections were counterstained with Eosin or Nuclear Fast Red (Tan et al., 1993).
Chromosomal analysis of X-inactivation
Embryos recovered at E6.5-E7.5 were incubated in Eagle’s minimum essential medium supplemented with 10% fetal calf serum and 150 μg/ml 5-bromo-2-deoxyuridine (BrdU) at 37°C in 5% CO2 in air for 7.5 hours including the last hour of incubation in the presence of 1 μg/ml Colcemid. Chromosome slides were prepared according to the method described previously (Takagi et al., 1982). Cells were analyzed for the karyotype and chromosome replication pattern after staining with freshly prepared Acridine Orange solution.
Cytological preparation for FISH
Most androgenetic embryos recovered at E3.5 were at the morula stage and developed into fully expanded blastocysts after culture in M16 medium for 12 hours. Cytogenetic preparations were made from preimplantation embryos according to the methods described by Takagi et al. (1982) with minor modifications. Briefly, after hypotonic treatment with 1% sodium citrate for 10 minutes at room temperature, embryos were fixed with 3:1 mixture of methanol: glacial acetic acid on ice. Each embryo, together with a small volume of the fixative, was placed on a clean glass slide. A small drop of 3:1 mixture of glacial acetic acid and 25% lactic acid was applied immediately to the embryo to spread cells on the slide. Lactic acid was removed from the preparation by repeated application of the fixative. Preparations for RNA FISH were made from E6.5 embryos as mentioned above except for the omission of BrdU incorporation.
RNA FISH Analysis
Xist RNA was detected with the use of pBluescript-based plasmid clones, pR97E1, pR95B, pR91E and pR53E1 encompassing exon 1 to 6 (Sado et al., 1996). An equimolar mixture of these plasmids was labeled by nick translation with Cy3-dCTP (Amersham Pharmacia, Little Chalfort, UK). After addition of salmon sperm DNA and yeast tRNA, labeled probe was precipitated with ethanol, resuspended in deionized formamide, and denatured at 70°C for 10 minutes. About 250 ng of probe DNA was applied per slide in 10 μl of hybridization mixture (2×SSC, 1 mg/ml BSA, 20% dextran sulphate), incubated at 42°C overnight in a moist chamber. After hybridization, slides were washed twice in 50% formamide/2×SSC for 5 minutes at 42°C, and twice in 2×SSC/ 0.05% Tween-20 for 5 minutes at 42°C. Slides were mounted with antifading solution containing DAPI. Preparations used for RNA FISH were painted with X- and Y-specific probes to determine the sex chromosome constitution and to verify signals produced by Xist RNA. After removal of the cover slip, slides were washed three times in 2×SSC/0.05% Tween-20 for 10 minutes at room temperature and refixed with 3:1 methanol: glacial acetic acid twice for 10 minutes on ice and dried at room temperature. Preparations were hardened for 24 hours at 65°C. Biotin-labeled mouse X and Cy3-labeled mouse Y chromosome paint probes (Cambio, Cambridge, UK) were denatured for 10 minutes at 65°C and incubated for 1 hour at 37°C. Chromosome preparations were denatured in 70% formamide/ 2×SSC for 5 minutes at 70°C and dehydrated with ice-cold ethanol series. A mixture of 10 μl each of X and Y chromosome paint probe was applied onto the slide and hybridization was performed at 42°C overnight in a moist chamber. Slides were washed twice in 50% formamide in 2×SSC, and twice in 0.05% Tween-20 for 5 minutes at 42°C. Hybridization was detected with streptavidin-fluorescein isothiocyanate (Gibco BRL, Life Technologies, Rockville, MD). After incubation, slides were washed three times in 4×SSC/0.05% Tween-20 for 5 minutes at 42°C and mounted with antifading solution containing DAPI. Androgenetic origin of each embryo was ascertained by the presence of two Robertsonian translocation chromosomes. Slides were examined with an OLYMPUS fluorescence microscope, and images were captured with a Photometrics CCD camera coupled to IPLab software (Signal Analytics, Vienna, VA). Color channels were merged in Adobe PhotoShop.
RESULTS
Viability and X-inactivation in XX androgenones
To determine whether XX androgenones have a chance of surviving to the egg-cylinder stage, we first studied the genotype of androgenones at E7.5. Androgenetic embryos were produced by pronuclear transplantation between fertilized wild-type embryos and those carrying the lacZ transgene (Tan et al., 1993) on the XP. Seventeen out of 52 reconstituted androgenetic embryos transferred to pseudopregnant females were recovered at E7.5. They showed growth retardation with slightly underdeveloped embryonic region (Fig. 1) consistent with their androgenetic origin (Barton et al., 1984; Kaufman et al., 1989).
A part of the embryonic region cut with a fine glass needle was used for karyotyping, and the remaining part was subjected to X-gal staining of β-gal activity. Five embryos were XX, whereas 12 embryos were XY. In every XX embryo, all tissues including the chorion and yolk-sac endoderm were mosaic of X-gal-positive and X-gal-negative cells. This staining pattern has never been observed in fertilized female embryos hemizygous for HMG-lacZ substantiating androgenetic origin of these embryos. Although tissues of embryonic ectoderm origin are mosaic in every female hemizygous for HMG-lacZ, the extra-embryonic ectoderm is uniformly β-gal negative when the HMG-lacZ is inherited from father, and it is uniformly β-gal positive when the transgene is inherited from mother (Tam et al., 1994). We suggest that the mosaic pattern is the result of random X-inactivation, with the implication that XX androgenones can survive beyond early postimplantation stages and their X chromosomes are capable of undergoing X-inactivation. An example of XX androgenones thus verified is shown in Fig. 1C. Three of XY embryos were uniformly positive and remaining nine were negative for β-gal. Androgenetic origin is evident in β-gal-positive embryos, but it may not always be true in β-gal-negative embryos.
X-inactivation mosaicism in XX androgenones
Since the HMG-lacZ transgene alone is not enough to prove the androgenetic origin of manipulated embryos, we employed a Robertsonian translocation involving the X chromosome, Rb2 (Adler et al., 1989) or RX9 (Tease and Fisher, 1991) as an additional marker (Table 1). XX androgenetic embryos generated by pronuclear transplantation should have a metacentric translocation X chromosome and carry the lacZ transgene on the other X. A total of 47 presumptive H253+Rb androgenetic embryos were recovered from 57 implantation sites. Although most embryos were retarded for their age (Barton et al., 1984), 10 embryos were large and comparable in size and morphology to normal E7.5 embryos. The ectoplacental cone tissue from each embryo was assayed for β-gal activity to assess XlacZ transmission, and remaining tissues were used for analyzing karyotype and X chromosome replication patterns after they were divided into two (extra-embryonic and embryonic) or three (chorionic, yolk-sac and embryonic) parts depending on their sizes. Classification of all embryos obtained at E7.5 according to the sex chromosome and β-gal expression is shown in Table 1. As predicted from exceptionally good growth, contribution of both the maternal and the paternal genome was disclosed in 10 large embryos, suggesting technical errors during the micromanipulatory procedure in this series of experiments. Apparently the error was due to the fact that relatively low reproductive potential of Rb2 and RX9 males forced us to use eggs in which the parental origin of the pronucleus was not clearly defined. Out of 31 karyotypically verified androgenetic embryos 12 were XX and remaining 19 were XY consistent with the expected ratio of 1(XX): 2(XY): 1(YY). YY androgenones should have been lost before implantation as reported previously (Kaufman et al., 1989).
In these XX androgenetic embryos, either the morphologically normal X (XN) or the X chromosome arm of the Rb2 or RX9 translocation chromosome was replicating asynchronously in informative metaphase cells (Fig. 2). Although the metacentric X chromosome was inactivated more often than the XN (Table 2), the mean proportion of these two types of cell was not statistically different in comparison to the embryonic and the extra-embryonic regions by Cochran’s approximation of Behrens-Fisher test (t′=0.44, t0.05=2.45; 0.6<P<0.7). These results strongly suggest lack of nonrandom inactivation, which occurred in extra-embryonic tissues from fertilized female embryos (t′=6.86, t0.05=2.37; P<0.001).
The level of X-inactivation mosaicism was further studied by determining the proportion of β-gal positive cells in the chorionic ectoderm and embryonic ectoderm in nine XX androgenones that showed the androgenone-specific staining pattern (Table 3). Cochran’s approximation of Behrens-Fisher test again showed that the mean proportion of X-gal positive cells was not significantly different (t′=0.64, t0.05=2.37; 0.5<P<0.6) in these tissues. Taken together, our results point to the conclusion of random X-inactivation in XX androgenones, although further studies are required for clarifying the occasional deviation from apparent random inactivation in the extra-embryonic ectoderm (Table 3).
X-inactivation patterns revealed by Xist RNA FISH
Data obtained so far were consistent with no X-inactivation in XY and random inactivation of a single X chromosome in XX androgenones. However, they did not exclude the possibility that cells with aberrant inactivation patterns had occurred and were subjected to cell selection earlier, as suggested previously (Kay et al., 1994; Latham, 1996). To test this possibility and to elucidate the disagreement among the present and earlier works, we carried out RNA FISH experiments with Xist DNA probes on androgenetic embryos at preimplantation stages and at a postimplantation stage of E6.5.
Contrary to normally fertilized embryos, one and two strong Xist paint signals were observed in all nuclei of XY and XX putative androgenones, respectively, from the four-cell to the 16-cell stage (Fig. 3A,B,G,H). The sex chromosome constitution was determined by FISH with sex-chromosome-specific painting probes. As expected, YY embryos surviving at these stages did not show any Xist RNA signal (Fig. 3C). We believe that the unusual Xist RNA FISH pattern observed here proves the androgenetic origin of embryos under study, and we suggest that the paternal Xist allele is expressed in these embryos irrespective of the number of X chromosome in a cell. A single paint signal was still observed in about 10% of cells from XY androgenetic blastocysts (Fig. 3I; Table 4). Remaining cells showed a pinpoint signal or no signal at all. In XX androgenetic blastocysts, about 20% of cells had two Xist paint signals, 50% had a single paint signal and the remaining 30% had no such signal (Fig. 3M; Table 4). It was recently shown that transcription of stable Xist RNA for X-inactivation has not started in epiblast cell lineage of fully expanded blastocysts (Goto and Takagi, 2000). Thirty percent of cells from androgenetic XX blastocysts showing no Xist signal may correspond, therefore, to epiblast cells before X-inactivation. Cells having an ectopic paint signal were also found in E6.5 androgenetic embryos, though much less frequently than in blastocysts (Table 4). Androgenetic embryos at this stage were slightly larger than fertilized embryos at E5.5. X-inactivation is completed in normal female embryos by E5.5 to E6.0 (Rastan, 1982; Takagi et al., 1982). It is likely, therefore, that those cells with an ectopic paint signal are functionally nullisomic for the X chromosome, owing to inactivation of every XP chromosome, yet they still manage to survive in the embryos. A replication banding study of chromosomes in 20 E6.5 androgenones, however, failed to detect any cell that suggests inappropriate X-inactivation. A total of 132 XX and 108 XY androgenetic metaphase cells that incorporated suitable amount of BrdU for chromosome banding had one and no asynchronously replicating X chromosome, respectively. Androgenetic origin of the embryos older than blastocyst was verified by the presence of two Robertsonian metacentric chromosomes in cells at metaphase (Fig. 3R).
DISCUSSION
Several points of interest have emerged from this study. The most remarkable would be that, contrary to the earlier report (Kaufman et al., 1989), XX androgenones like XY counterparts, survive beyond implantation. During preparation of this paper, Obata et al. (2000) reported that mouse XX androgenones produced by in vitro fertilization of enucleated oocytes develop to E9.5. Data presented here and those reported by Obata et al. (2000) suggest that the failure by Kaufman et al. (1989) to find the XX androgenone at E7.5 was most probably a chance observation that was due to a small number of embryos they examined rather than factors such as differences in the genetic background of mice used for experiments. We obtained XX as well as XY androgenetic embryos from combinations of five different mouse stocks. The reported absence of XX androgenones had been corroborated by two opposing observations on Xist gene expression, both of which imply involvement of abnormal X-inactivation as a main cause. Kay et al. (1994) reported that Xist transcription that beginning at the four-cell stage is turned off by the morula-blastocyst stage resulting in failure of X-inactivation, whereas Latham and Rambhatla (1995) found that Xist transcription is maintained uninterruptedly culminating in inactivation of both X chromosomes.
Functional nullisomy and disomy for the X chromosome are extremely harmful to early embryonic development leading to preimplantation lethality or severely unbalanced growth after implantation (Morris, 1968; Takagi and Abe, 1990; Goto and Takagi, 1998). XX androgenones with well-defined embryonic and extra-embryonic structures, notwithstanding growth retardation, therefore predict that X-inactivation occurred successfully at least in a proportion of cells in trophectoderm and primitive endoderm as well as epiblast lineages. This prediction was fully substantiated by identification of an asynchronously replicating X chromosome in most informative cells from E7.5 androgenones. In addition to the embryonic tissues, X-inactivation was random in extra-embryonic tissues that are characterized by imprinted inactivation of the paternal X chromosome. However, the mode of Xist expression cast doubts on the consistent randomness of X-inactivation in XX androgenones.
In agreement with data provided by RT-PCR (Latham and Lambhatla, 1995), the present study revealed two paint signals in XX, and one paint signal in XY androgenetic embryos from the four-cell stage onward and they were still present at a considerable frequency in mature blastocysts and implanted embryos at E6.5. In normally fertilized embryos, Sheardown et al. (1997) consistently detected a single Xist paint signal in female E5.5 embryos, and never found any Xist paint signal in male embryos at the same stage. It is thus likely that X-inactivation or its initiation has finished in every cell of normal female embryos by E5.5 in agreement with earlier cytogenetic data (Rastan, 1982; Takagi et al., 1982). The ectopic Xist paint signal in E6.5 androgenetic embryos that are slightly larger than normal embryos at E5.5 probably indicates the occurrence of ectopic X-inactivation, unless it is delayed in androgenetic embryos. Correlation between high Xist expression and X-inactivation is suggested in mice with disrupted DNA methyltransferase (Dnmt1) genes (Beard et al., 1995; Panning and Jaenisch, 1996). In spite of our extensive effort, we could not find any XX androgenetic cell with two asynchronously replicating X chromosomes, nor any XY cell with a single asynchronously replicating X chromosome at E6.5. However, we can not rule out the possibility that such cells are present but mitotically inactive because of functional nullisomy for the X chromosome. Another intriguing possibility would be that those cells with an ectopic Xist paint signal at E6.5 correspond to cells in the reversible step of X-inactivation proposed by Wutz and Jaenisch (2000), and hence the late-replicating X chromosome has yet to be identified.
In fertilized embryos, extra-embryonic cells of trophectoderm origin do not inactivate the XM, indicating that the imprint on XM does not allow inactivation (Lyon and Rastan, 1984). Stringency of the imprinting was recently highlighted in mice carrying an Xist deletion (Marahrens et al., 1997). Female embryos that inherit the deleted Xist allele on the XM grow normally inactivating XP selectively, whereas those inherit the mutated Xist allele on the XP die soon after implantation. The defective conceptuses are characterized by poorly developed extra-embryonic tissues, as reported in embryos carrying two XM chromosomes (Shao and Takagi, 1990; Goto and Takagi, 1998) most probably owing to failure of inactivation. Our recent data (Tada et al., 2000) suggest that such imprint is established during the growth phase of oocytes. In view of the ectopic Xist paint signal in XY as well as in XX androgenones, however, it is difficult to rule out the possibility that XP is inactivated in extra-embryonic tissues of fertilized female embryos because it carries specific imprint that promotes Xist expression (Monk and McLaren, 1981).
Available data allow us to propose a sequence of events culminating in accomplishing X-inactivation in androgenetic embryos. Every XP chromosome, irrespective of the number in a diploid cell, transcribes stable Xist RNA in all cells from the four-cell to the 16-cell stage. Hence, it is likely that the initial Xist expression is turned on by a stage-specific cue to which only XP but not XM responds. Apparently, counting the number of X chromosomes in a cell is not involved in this process, because stable Xist RNA is transcribed even in XY androgenones. Decrease in the frequency of cells showing ectopic Xist expression by the blastocyst stage may be resulted from random extinction of Xist transcription based on counting the number of the X chromosome in both XX and XY androgenones. Probably, failure in terminating ectopic Xist expression may result in inappropriate X-inactivation and eventual cell selection. In fertilized embryos, another stage-specific cue may be necessary for erasing imprint to certify random inactivation in the epiblast lineage of the inner cell mass origin.
It is tempting to postulate that random inactivation in the androgenetic trophectoderm cell is the consequence of choosing an X chromosome that discontinues transcription of stable Xist RNA. Models for random X-inactivation proposed for embryonic tissues (Panning et al., 1997; Sheardown et al., 1997; Lee et al., 1999b) also postulate selection of a single X chromosome that turns off Xist transcription or destabilize Xist RNA. It may be reasonable to assume that the choice is made in a similar manner in both occasions of random inactivation. A series of recent studies has suggested critical roles played by sequences 3′ to Xist (Clerc and Avner, 1998) including Tsix gene antisense to Xist (Lee et al., 1999b) and DXPas34 locus (Debrand et al., 1999) in the control of Xist gene activity, hence X-inactivation. However, we are still ignorant of the roles of various players in the control of X-inactivation. Further analysis of the specific imprint imposed on XM may contribute to achieve a breakthrough in this interesting problem.
ACKNOWLEDGEMENTS
We thank Drs Tomohiro Kono and Yuji Goto for their invaluable technical advice, and Drs Ilse-Dore Adler and Charles Tease for their generous supply of Rb(X.2)2Ad and the Rb(X.9)6H mice. C57BL/6J and CBA/J were kindly supplied by Dr Toshihiko Shiroishi. Mice used for this study were bred at the Center for Experimental Plants and Animals, Hokkaido University. I. Okamoto is a Research Fellow of the Japan Society for the promotion of Science. This study was supported by Grants-in-aid for Scientific Research from the Ministry of Education, Science and Culture, Japan. Seong-Seng Tan is funded by a NH & MRC block grant to the Howard Florey Institute, and by a project grant from the Bushell Foundation. We are grateful to anonymous reviewers for helpful suggestions to improve our paper.