Trophectoderm-specific expression of the X-linked Bex1/Rex3 gene in preimplantation stage mouse embryos

The Bex1/Rex3 gene was recently identified as an X-linked gene that is differentially expressed between parthenogenetic and normal fertilized, preimplantation stage mouse embryos. The Bex1/Rex3 gene appears to be expressed preferentially from the maternal X chromosome in blastocysts, but from either X chromosome in later stage embryonic tissues and adult tissues. To investigate whether differential expression of the Bex1/Rex3 gene between normal and parthenogenetic blastocyst stage embryos reflects genomic imprinting at the Bex1/Rex3 locus itself, or instead is the result of preferential inactivation of the paternal X chromosome or differences in timing of cellular differentiation, we examined in detail the expression pattern of the Bex1/Rex3 mRNA in normal preimplantation stage embryos, and compared its expression between androgenetic, gynogenetic, and normal fertilized embryos. Expression data reveal that the Bex1/Rex3 gene is initially transcribed at the 2-cell stage, transiently induced at the 8-cell stage, and then increases in expression again at the blastocyst stage. Very little expression is observed in isolated inner cell masses, indicating selective expression in the trophectoderm. Comparisons of Bex1/Rex3 mRNA expression between male and female androgenetic and control embryos and gynogenetic embros failed to reveal any significant difference in expression between the different classes of embryos at the 8-cell stage, or the expanding blastocyst stage (121 hr post-hCG). At the late blastocyst stage (141 hr post-hCG), expression was significantly lower in XY control embryos as compared with XX controls. Bex1/Rex3 mRNA expression did not differ between XX and XY androgenones at the blastocyst stage or between gynogenones and XX control embryos. Thus, the Bex1/Rex3 gene does not appear to be regulated directly by genomic imprinting during the preimplantation period, just as it is not regulated by imprinting at later stages. Apparent differences in gene expression may arise through the effects of trophectoderm-specific expression coupled with differences in timing of trophectoderm differentiation between the different classes of embryos and effects of preferential paternal X chromosome inactivation (XCI).     

The dynamics of imprinted X inactivation during preimplantation development in mice

In the mouse, there are two forms of X chromosome inactivation (XCI), random XCI in the fetus and imprinted paternal XCI, which is limited to the extraembryonic tissues. While the mechanism of random XCI has been studied extensively using the in vitro XX ES cell differentiation system, imprinted XCI during early embryonic development has been less well characterized. Recent studies of early embryos have reported unexpected findings for the paternal X chromosome (Xp). Imprinted XCI may not be linked to meiotic silencing in the male germ line but rather to the imprinted status of the Xist gene. Furthermore, the Xp becomes inactivated in all cells of cleavage-stage embryos and then reactivated in the cells of the inner cell mass (ICM) that form the epiblast, where random XCI ensues.     

Disruption of a conserved region of Xist exon 1 impairs Xist RNA localisation and X-linked gene silencing during random and imprinted X chromosome inactivation

In XX female mammals a single X chromosome is inactivated early in embryonic development, a process that is required to equalise X-linked gene dosage relative to XY males. X inactivation is regulated by a cis-acting master switch, the Xist locus, the product of which is a large non-coding RNA that coats the chromosome from which it is transcribed, triggering recruitment of chromatin modifying factors that establish and maintain gene silencing chromosome wide. Chromosome coating and Xist RNA-mediated silencing remain poorly understood, both at the level of RNA sequence determinants and interacting factors. Here, we describe analysis of a novel targeted mutation, Xist(INV), designed to test the function of a conserved region located in exon 1 of Xist RNA during X inactivation in mouse. We show that Xist(INV) is a strong hypomorphic allele that is appropriately regulated but compromised in its ability to silence X-linked loci in cis. Inheritance of Xist(INV) on the paternal X chromosome results in embryonic lethality due to failure of imprinted X inactivation in extra-embryonic lineages. Female embryos inheriting Xist(INV) on the maternal X chromosome undergo extreme secondary non-random X inactivation, eliminating the majority of cells that express the Xist(INV) allele. Analysis of cells that express Xist(INV) RNA demonstrates reduced association of the mutant RNA to the X chromosome, suggesting that conserved sequences in the inverted region are important for Xist RNA localisation.     

Loss of Xist imprinting in diploid parthenogenetic preimplantation embryos.

We have analysed Xist expression patterns in parthenogenetic and control fertilised preimplantation embryos by using RNA FISH. In normal XX embryos, maternally derived Xist alleles are repressed throughout preimplantation development. Paternal alleles are expressed as early as the 2-cell stage. In parthenogenetic embryos, we observed Xist RNA expression and accumulation from the morula stage onwards, indicating loss of maternal imprinting. In the majority of cells, expression was from a single allele, indicating that X chromosome counting occurs to establish appropriate monoallelic Xist expression. We discuss these data in the context of models for regulation of imprinted and random X inactivation.     

An X-linked GFP transgene reveals unexpected paternal X-chromosome activity in trophoblastic giant cells of the mouse placenta

A GFP transgene has been integrated on the proximal part of the mouse X chromosome just distal of Timp and Syn1. During development, this X-linked GFP transgene exhibits widespread green fluorescence throughout the embryonic and adult life of male mice but displays mosaic expression in tissues as a result of X-inactivation in females. In living female embryos, inactivation of the transgene is imprinted in extraembryonic regions and random in the embryo proper, demonstrating that this reporter is behaving in a similar fashion to the majority of X-linked loci, and so provides a vital readout of X chromosome activity. This is observation is further supported in T16H/X female mice harboring the GFP transgene on the normal X chromosome where reporter inactivation is observed in somatic cells. The differential expression of GFP activity facilitates fluorescence activated cell sorting for the purification of GFP+ vs. GFP- cells from female embryonic tissues, thereby allowing access to populations of cells that have kept active a particular X chromosome. By tracking the activity of this X-linked GFP transgene, we discovered that the primary and secondary giant cells of the X/X placenta maintain an active paternal copy of this transgene on the presumed silenced paternal X-chromosome. This finding implies that the imprint on the paternal X chromosome may be relaxed in these trophectodermal derivatives.     

Disruption of imprinted X inactivation by parent-of-origin effects at Tsix

In marsupials and in extraembryonic tissues of placental mammals, X inactivation is imprinted to occur on the paternal chromosome. Here, we find that imprinting is controlled by the antisense Xist gene, Tsix. Tsix is maternally expressed and mice carrying a Tsix deletion show normal paternal but impaired maternal transmission. Maternal inheritance occurs infrequently, with surviving progeny showing intrauterine growth retardation and reduced fertility. Transmission ratio distortion results from disrupted imprinting and postimplantation loss of mutant embryos. In contrast to effects in embryonic stem cells, deleting Tsix causes ectopic X inactivation in early male embryos and inactivation of both X chromosomes in female embryos, indicating that X chromosome counting cannot override Tsix imprinting. These results highlight differences between imprinted and random X inactivation but show that Tsix regulates both. We propose that an imprinting center lies within Tsix.     

Expression of X-linked genes in androgenetic,gynogenetic, and normal mouse preimplantation embryos

A quantitative RT-PCR approach has been used to examine the expression of a number of X-linked genes during preimplan-tation development of normal mouse embryos and in androgenetic and gynogenetic mouse embryos. The data reveal moderately reduced expression of the Prps1, Hprt, and Pdha1 mRNAs in androge-netic eight-cell and morula stage embryos, but not in androgenetic blastocysts. Pgk1 mRNA abundance was severely reduced in androgenones at the eight-cell and morula stages and remained reduced, but to a lesser degree, in androgenetic blastocysts. These data indicate that paternally inherited X chromosomes are at least partially repressed in androgenones, as they are in normal XX embryos, and that the degree of this repression is chromosome position-dependent or gene-dependent. Gynogenetic embryos expressed elevated amounts of some mRNAs at the morula and blas-tocyst stages, indicative of a delay in dosage compensation that may be chromosome position-dependent. The Xist RNA was expressed at a greater abundance in androgenones than in gynogenones at the eight-cell and morula stages, consistent with previous studies. Xist expression was observed in both and rogenones and gynogenones at the blas-tocyst stage. We conclude that the developmental arrest in early androgenones may be, in part, due to reduced expression of essential X-linked genes, particularly those near the X inactivation center, where as the developmental defects of gyno-genones and parthenogenones, by contrast, may be partially due to overexpression of X-linked genes in extraembryonic tissues, possibly those far-thest away from the X inactivation center. © 1995 Wiley-Liss, Inc.     

The region 3' to Xist mediates X chromosome counting and H3 Lys-4 dimethylation within the Xist gene

A counting process senses the X chromosome/autosome ratio and ensures that X chromosome inactivation (XCI) initiates in the female (XX) but not in the male (XY) mouse embryo. Counting is regulated by the X-inactivation centre, which contains the Xist gene. Deleting 65 kb 3' to Xist in XO embryonic stem (ES) cells affects counting and results in inappropriate XCI upon differentiation. We show here that normal counting can be rescued in these deleted ES cells using cre/loxP re-insertion, and refine the location of elements controlling counting within a 20 kb bipartite domain. Furthermore, we show that the 65 kb deletion also leads to inappropriate XCI in XY differentiated ES cells, which excludes the involvement of sex-specific mechanisms in the initiation of XCI. At the chromatin level, we have found that the Xist gene corresponds to a peak of H3 Lys-4 dimethylation, which is dramatically and specifically affected by the deletion 3' to Xist. Our results raise the possibility that H3 Lys-4 dimethylation within Xist may be functionally implicated in the counting process.     

Micromanipulating dosage compensation: understanding X-chromosome inactivation through nuclear transplantation

Nuclear transfer (NT) studies have provided insight into the functional importance of epigenetic alteration of the X chromosomes during X-inactivation. Uniparental embryos created by NT have been informative as to the time and location at which the imprint controlling extraembryonic X-inactivation is established. Experiments with female somatic cells, have demonstrated that the inactive X chromosome (Xi) is reactivated after NT, leading to random X-inactivation in the embryonic lineages of cloned embryos. However, in the extraembryonic lineages of clones, epigenetic information from the donor cell nucleus persists, leading to preferential inactivation of the donor cell's inactive X in the placenta of cloned animals. These results suggest epigenetic information established during embryonic X-inactivation is functionally equivalent to the gametic imprint.