X chromosome inactivation revealed by the X-linked lacZ transgene activity in periimplantation mouse embryos

Using H253 mouse stock harboring X-linked HMG-lacZ transgene, we examined X chromosome inactivation patterns in sectioned early female embryos. X-gal staining patterns were generally consistent with the paternal X inactivation in the trophectoderm and the primitive endoderm cell lineages and random inactivation in the epiblast lineages. The occurrence of embryonic visceral endoderm cells apparently at variance with the paternal X chromosome inactivation in 7.5 dpc embryos was explained by the replacement of visceral endoderm cells with cells of epiblast origin. The frequency of cells negative for X-gal staining in 4.5-5.5 dpc XmXp* embryos fluctuated considerably especially in the extraembryonic ectoderm and the primitive endoderm, whereas it was less variable in the embryonic ectoderm. We could not, however, determine whether it is a normal phenomenon revealed for the first time by the use of HMG-lacZ transgene or an abnormality caused by the multicopy transgene.     

X chromosome regulation of autosomal gene expression in bovine blastocysts

Although X chromosome inactivation in female mammals evolved to balance the expression of X chromosome and autosomal genes in the two sexes, female embryos pass through developmental stages in which both X chromosomes are active in somatic cells. Bovine blastocysts show higher expression of many X genes in XX than XY embryos, suggesting that X inactivation is not complete. Here, we reanalyzed bovine blastocyst microarray expression data from a network perspective with a focus on interactions between X chromosome and autosomal genes. Whereas male-to-female ratios of expression of autosomal genes were distributed around a mean of 1, X chromosome genes were clearly shifted towards higher expression in females. We generated gene coexpression networks and identified a major module of genes with correlated gene expression that includes female-biased X genes and sexually dimorphic autosomal genes for which the sexual dimorphism is likely driven by the X genes. In this module, expression of X chromosome genes correlates with autosome genes, more than the expression of autosomal genes with each other. Our study identifies correlated patterns of autosomal and X-linked genes that are likely influenced by the sexual imbalance of X gene expression when X inactivation is inefficient.     

Detrimental effects of two active X chromosomes on early mouse development

Matings between female mice carrying Searle's translocation, T(X;16)16H, and normal males give rise to chromosomally unbalanced zygotes with two complete sets of autosomes, one normal X chromosome and one X16 translocation chromosome (XnX16 embryos). Since X chromosome inactivation does not occur in these embryos, probably due to the lack of the inactivation center on X16, XnX16 embryos are functionally disomic for the proximal 63% of the X chromosome and trisomic for the distal segment of chromosome 16. Developmental abnormalities found in XnX16 embryos include: (1) growth retardation detected as early as stage 9, (2) continual loss of embryonic ectoderm cells either by death or by expulsion into the proamniotic cavity, (3) underdevelopment of the ectoplacental cone throughout the course of development, (4) very limited, if any, mesoderm formation, (5) failure in early organogenesis including the embryo, amnion, chorion and yolk sac. Death occurred at 10 days p.c. Since the combination of XO and trisomy 16 does not severely affect early mouse development, it is likely that regulatory mechanisms essential for early embryogenesis do not function correctly in XnX16 embryos due to activity of the extra X chromosome segment of X16.     

The late-replicating X chromosome in digynous mouse triploid embryos

Using BrdU-labeling and acridine orange staining, the behavior of X-chromosome replication was studied in 28 XXX and 19 XXY digynous mouse triploids. In some of these the paternal and maternal X chromosome could by cytologically distinguished. Such embryos were obtained by mating chromosomally normal females with males carrying Cattanach's X chromosome which contains an autosomal insertion that substantially increases the length of this chromosome. In the XXX triploids there were two distinct cell lines, one with two late-replicating X chromosomes, and the other with only one late-replicating X. The XXY triploids were also composed of two cell populations, one with a single late-replicating X and the other with no late replicating X chromosome. Assuming that the late-replicating X is genetically inactive, in both XXX and XXY triploids, cells from the embryonic region tended to have only one active X chromosome, whereas those from the extra-embryonic membranes tended to have two active X chromosomes. The single active X chromosome was either paternal or maternal in origin, but two active X chromosomes were overwhelmingly maternal in origin, suggesting paternal X-inactivation in extra-embryonic tissues.     

Correlation between X-chromosome inactivation and cell differentiation in female preimplantation mouse embryos

By means of a cytological method involving BrdU incorporation and acridine orange fluorescence staining in combination with embryo manipulation, we studied X-chromosome activity in female preimplantation mouse embryos with special reference to the correlation between X-chromosome inactivation and cell differentiation. There was no sign of asynchronous replication between the two X chromosomes from the one-cell to intermediate blastocyst stage. The allocyclic X chromosome, first detected in late blastocysts, was paternal in origin, mostly replicating early in the S phase and limited to the trophectoderm. Subsequent X-chromosome inactivation occurring in the primary endoderm was also characterized by the involvement of the paternal X and early replication. Both X chromosomes continued to replicate synchronously in the embryonic ectoderm or epiblast at this stage. It was evident that overt cell differentiation preceded the appearance of the asynchronously replicating X chromosome in the trophectoderm and primary endoderm. This finding seems to support the view that cell differentiation is an important correlate of X-chromosome inactivation.     

Imprint switching for non-random X-chromosome inactivation during mouse oocyte growth

In mammals, X-chromosome inactivation occurs in all female cells, leaving only a single active X chromosome. This serves to equalise the dosage of X-linked genes in male and female cells. In the mouse, the paternally derived X chromosome (X(P)) is imprinted and preferentially inactivated in the extraembryonic tissues whereas in the embryonic tissues inactivation is random. To investigate how X(P) is chosen as an inactivated X chromosome in the extraembryonic cells, we have produced experimental embryos by serial nuclear transplantation from non-growing (ng) oocytes and fully grown (fg) oocytes, in which the X chromosomes are marked with (1) an X-linked lacZ reporter gene to assay X-chromosome activity, or (2) the Rb(X.9)6H translocation as a cytogenetic marker for studying replication timing. In the extraembryonic tissues of these ng/fg embryos, the maternal X chromosome (X(M)) derived from the ng oocyte was preferentially inactivated whereas that from the fg oocyte remained active. However, in the embryonic tissues, X inactivation was random. This suggests that (1) a maternal imprint is set on the X(M) during oocyte growth, (2) the maternal imprint serves to render the X(M) resistant to inactivation in the extraembryonic tissues and (3) the X(M) derived from an ng oocyte resembles a normal X(P).     

Nonrandom X chromosome inactivation in mouse embryos carrying Searle's T(X;16)16H translocation visualized using X-linked LACZ and GFP transgenes

Only the morphologically normal X chromosome is inactivated in female mice heterozygous for Searle's X-autosome translocation, T(X;16)16H. Here we performed a visual study of the primary and secondary events that culminate in the completely nonrandom inactivation of the X in female embryos having this translocation. The data we have obtained so far indicate that the initial choice of the future inactive X chromosome is biased, with the degree of skewing somewhere between 70:30% and 90:10% in favor of the morphologically normal X chromosome. The majority of genetically unbalanced cells that inactivate a translocated X chromosome are quickly eliminated from the embryo proper by E8.5, although the survival of such cells is sporadically observed thereafter. The initial nonrandom choice demonstrated in this study supports the contention that the T(X;16)16H translocation disrupts one of the loci involved in the randomness of the choice of the future inactive X chromosome. Although the HMG-LACZ transgene in H253 stock mice is an excellent marker of X chromosome inactivation, the present study suggests that it is infrequently de-repressed on the inactive X chromosome.     

An X1X1X2X2/X1X2Y mechanism of sex determination in a South American rodent, Deltamys kempi (Rodentia, Cricetidae)

Chromosome studies on 14 specimens of Deltamys kempi disclosed six males with 2n = 37, NF = 38, six females with 2n = 38, NF = 38, and two females with 2n = 37, NF = 38. G- and C-band analyses revealed a Y-autosome translocation in the males leading to a multiple chromosome system of sex determination of the type X1X1X2X2/X1X2Y, this being the second case of such a mechanism described in rodents. At meiosis the males presented a trivalent in which C-banding studies showed an alternate orientation of the sex chromosomes due to end-to-end association of the X1 and Y chromosomes, the Y and the X2 being held together by interstitial chiasmata. At metaphase II both n = 17 + Y and n = 18 + X1 are regularly observed. The two females with 2n = 37, NF = 38, are heterozygous for an autosomal centric fusion involving chromosomes 1 and 13. The product of the Y-autosome translocation constitutes the largest element of the karyotype (9.4% of the haploid set); the X1 chromosome amounts to 7.8% of this set, including a large heterochromatic block. When only its euchromatic region is considered, this percentage decreases to 4.6%. From two to seven NORs were observed at the telomeres, with a mean of 4.4 +/- 1.1 per cell.     

X chromosome inactivation in X autosome translocation carrier cows.

The pattern of X chromosome inactivation in X autosome translocation carries in a herd of Limousin-Jersey crossbred cattle was studied using the reverse banding technique consisting of 5-bromodeoxyuridine incorporation and acridine orange staining and autoradiography on cultures of solid tissues and blood samples exposed to tritiated thymidine. The late-replicating X chromosome was noted to be the normal X in strikingly high proportions of cells in cultures of different tissues from all translocation carriers. It is suggested that the predominance of cells in which the normal X is inactivated may be the result of a post-inactivation selection process. Such a selection process during the prenatal life favouring cells in which the genes of the normal X chromosome remain unexpressed in translocation carrier females may be the mechanism that helps these conceptuses escape the adverse effects of functional aneuploidy. Based on the observation that the translocation carriers of this line of cattle are exclusively females and that there is a higher than expected rate of pregnancy loss, it is also postulated that the altered X chromosome may be lethal to all male conceptuses and to some of their female counterparts.     

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.     

Construction of a DNA Library Enriched with Mouse 4xChromosome of T(X;4)37H Translocation

Normal mouse chromosomes are routinely separated into only 5 peaks by the current flow cytometry. Since this limited resolution hindered isolation of the normal mouse X chromosome with an appropriate purity, we attempted to sort the mouse 4^x chromosome, a larger translocation chromosome of T(X;4)37H, consisting of nearly the entire chromosome 4 and chromosome X by flow cytometry. To obtain a large number of cells having 4^x chromosome for flow sorting, we isolated a somatic hybrid cell line MHH-1 formed between S194 myelome cell line and normal splenocytes from a male mouse carrying T(X;4)37H. Flow karyotyping of propidium iodide-stained chromosomes from MHH-1 cell line revealed an additional peak containing 4^x chromosomes at about 80%. DNA purified from sorted 4^x chromosomes was cloned into phage lambda gtWES after complete digestion with EcoRl restriction endonuclease. Thus a 4^x chromosome-enriched library of about 4.4 × 10⁴ recombinant phages was made and 13 single copy DNA clones specific to the X chromosome were isolated from the library so far.     

Studies on the activity of the X chromosomes in female teratocarcinoma cells in culture

Embryonal carcinoma cells derived from murine teratocarcinomas are able to differentiate into the same variety of tissue types as early embryonic cells. Because embryonal carcinoma cells resemble those of the embryo at a stage before X chromosome inactivation has occurred in females embyronal carcinoma cells containing two X chromosomes were examined to determine whether both are genetically active. The specific activities of X-linked enzymes were measured in embryonal carcinoma cells containing either one or two X chromosomes. The activities in both cell types were similar, suggesting that only one X chromosome was active in the female cells. Further support for this conclusion came from experiments in which azaguanine-resistant mutants were recovered with similar frequencies from embryonal carcinoma cell lines containing one and two X chromosomes. Late replication of an X chromosome DNA was detected in one embryonal carcinoma cell line with two X chromosomes but not in another. This suggests that cells of these two lines were arrested at different developmental stages, and that late DNA replication may not be a necessary adjunct of X inactivation. Evidence is presented which suggests that X chromosome reactivation does not occur during differentiation of the cells in vitro.     

Presence of an Allocyclic Early Replicating X Chromosome in Female Embryos of the Rat, Chinese Hamster and Rabbit

Previous studies on early female mouse embryos revealed the presence of two kinds of inactive X chromosomes, one replicating late and the other early in the DNA synthetic period. The X chromosome that replicates early is of special interest because of its paternal origin, preferential occurrence in trophectoderm and primitive endoderm derivatives, and programmed shift to the late replicator. This study by BrdU labeling and acridine orange fluorescence staining was undertaken to examine whether the inactive X chromosome behaves in a similar manner in other laboratory mammals. In rat embryos the paternal X chromosome was found to show the same behavior in extraembryonic tissues. Early replicating chromosomes were also found in the extraembryonic regions of Chinese hamster and rabbit embryos, although their parental origin could not be determined due to the absent of X chromosome polymorphism in these species. Probably the early replicating X chromosome occurs commonly in mammals. Its functional significance is unknown.     

An extra maternally derived X chromosome is deleterious to early mouse development

An extra copy of the X chromosome, unlike autosomes, exerts only minor effects on development in mammals including man and mice, because all X chromosomes except one are genetically inactivated. Contrary to this contention, we found that an additional maternally derived X (XM) chromosome, but probably not a paternally derived one (XP), consistently contributes to early death of 41,XXY and 41,XXX embryos in mice. Because of imprinted resistance to inactivation, two doses of XM remain active in the trophectoderm, and seem to be responsible for the failure in the development of the ectoplacental cone and extraembryonic ectoderm, and hence, from early embryonic death. Discordant observations in man indicating viability of XMXMXP and XMXMY individuals suggest that imprinting on the human X chromosome is either weak, unstable or erased before the initiation of X-inactivation in progenitors of extraembryonic membranes.