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.     

Late replication patterns in adult and embryonic mice carrying Searle's X-autosome translocation

Bromodeoxyuridine-dye technique analysis of X chromosome DNA synthesis in female adult and fetal mice carrying the balanced form of the T(X; 16) 16H translocation demonstrated that the structurally normal X chromosome was late replicating (and hence presumably inactive) in 93% of the adult cells and 99% of the 9-day embryo cells, with the X¹⁶ chromosome late replicating in the remaining cells. We conclude from these results that in T16H/+ females either there is preferential inactivation of the normal X chromosome or that, if inactivation is random, cell selection takes place before 9 days of development. Two 9-day female embryos with an unbalanced karyotype were also studied; both had two late-replicating chromosomes in most of their cells, one being the chromosome 16^X, the other a normal X chromosome. These results, together with the presence of a late-replicating X¹⁶ chromosome in T16H/+ adult and fetal mice, support the concept that more than one inactivation center is present on the X chromosome of the mouse because the X¹⁶ and the 16^x chromosomes can be late replicating.     

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.     

Karyotypes and X chromosome inactivation in segregants of a murine X-autosome translocation, T(X;4)37H

Karyotypes and X chromosome inactivation were studied in embryos obtained from female mice carrying T(X;4)37H translocation on day 6 to 8 of gestation by a BrdU-acridine orange method. A total of 18 different karyotypes were found in 477 embryos examined: 90.0% embryos were products expected from 2:2 alternate or adjacent 1 disjunction. 3:1 and adjacent 2 disjunctions accounted for approximately 8.0% and 0.7% conceptuses, respectively. In the embryo proper of balanced T37H/ + conceptuses, inactivation was random with respect to the normal X and the larger translocation X (4x) chromosome. In all the cells with the 4x inactive, the late replication apparently did not spread to the attached autosomal portion, although black/brown coat variegation implies spreading of inactivation into the autosomal region. The X chromosome segment deprived of the inactivation center remained active in all the cells examined and it exerted deleterious effects on embryonic or fetal development. Observation in embryos having two maternally derived X chromosomes showed that they were indeed resistant to inactivation in early extraembryonic cell lineages, and two copies of active X chromosomes in the trophectoderm fatally affected embryonic development due to inability to form the extraembryonic ectoderm and ectoplacental cone from the polar trophectoderm. In unbalanced X aneuploids the X chromosomes with the deletion were preferentially inactivated due to strong selection against nullisomy X.     

Sex ratio and testis size in mice carrying Sxr and T(X;16)16H

Mice heterozygous for the T(X;16)16H translocation and carrying Sxr on their normal (inactive) X chromosome (ie, T16H/X Sxr individuals) may develop as males, females, or hermaphrodites. The proportion of males varied from 22% to 65% depending on the source of the normal X chromosome. A model is proposed, according to which relatively small variations in the spreading of inactivation from the X chromosome into the attached Sxr fragment produce large changes in the proportion of males. Testis weight in T16H/X Sxr males was found to be significantly smaller than in X/X Sxr males, irrespective of the source of the normal X chromosome.     

Fragile X expression and X inactivation

Summary The major concept of fragile X pathogenesis postulates that the fragile site at band Xq27.3 [fra(X)] represents the primary defect. The expression of fra(X) is predicted to be an intrinsic property of the mutated chromosome and, hence, should not be suppressed by X inactivation in females or induced by X-linked trans-acting factors. We made fibroblast clones of a fra(X)-positive female. Monoclonality was demonstrated using the DNA methylation assay at DXS255. The mutated X chromosomes and their states of genetic activity in the different clones were also defined by molecular methods. Five clones were selected to induce expression of fra(X) by 10^-7 M FUdR; two carried an active mutated X chromosome, in the other three the mutated X chromosome was inactivated. Fra(X) was found expressed in both types of clones. The percentages of positive cells were as high as 7–10%, regardless of the genetic activity of the mutated X chromosomes. DNA replicating patterns, obtained by BUdR labelling, demonstrated that expression occurred only on the mutated X chromosomes previously identified by molecular methods. The concept that the fragile site represents the primary mutation is now strongly supported by experimental evidence. The expression of fra (X) in females is independent of X inactivation and other trans-acting factors.     

Variable spread of X inactivation affecting the expression of different epitopes of the Hya gene product in mouse B-cell clones

Cloned B-cell lines from a female T16H/XSxr mouse in which Tdy expression was suppressed due to X inactivation and from a male X/XSxr mouse, both of the (kxb)F₁ haplotype, were examined for H-Y expression. This was determined both by their ability to act as targets for H-2^k and H-2^b-restricted H-Y-specific cytotoxic T cells and by their ability to stimulate the proliferation of H-2K^k, H-2D^b (class I) and A^b (class II)-restricted T-cell clones. In B-cell clones from the T16H/XSxr mouse, expression of H-Y/D^b exhibited partial X inactivation and only a proportion ( 30%) of the cells were targets for or stimulated H-2D^b-restricted H-Y-specific T cells. In contrast, H-Y eiptopes restricted by H-2^k (H-Y/K^k, H-Y/D^k) and A^b (H-Y/A^b) exhibited no X inactivation. Furthermore, no inactivation of H-Y/D^b, H-Y/A^b, or H-Y^k was observed in the male X/XSxr mouse. These results indicate that the T16H/XSxr female is a mosaic, as a result of the variable spread of X inactivation into the Sxr region. They further suggest that the H-Y antigen recognized in association with H-2^k and H-2D^b class I molecules and A^b class II molecules may be the product of more than one gene.     

Nonrandom X Chromosome Inactivation Is Influenced by Multiple Regions on the Murine X Chromosome

During the development of female mammals, one of the two X chromosomes is inactivated, serving as a dosage-compensation mechanism to equalize the expression of X-linked genes in females and males. While the choice of which X chromosome to inactivate is normally random, X chromosome inactivation can be skewed in F1 hybrid mice, as determined by alleles at the X chromosome controlling element (Xce), a locus defined genetically by Cattanach over 40 years ago. Four Xce alleles have been defined in inbred mice in order of the tendency of the X chromosome to remain active: Xce^a < Xce^b < Xce^c < Xce^d. While the identity of the Xce locus remains unknown, previous efforts to map sequences responsible for the Xce effect in hybrid mice have localized the Xce to candidate regions that overlap the X chromosome inactivation center (Xic), which includes the Xist and Tsix genes. Here, we have intercrossed 129S1/SvImJ, which carries the Xce^a allele, and Mus musculus castaneus EiJ, which carries the Xce^c allele, to generate recombinant lines with single or double recombinant breakpoints near or within the Xce candidate region. In female progeny of 129S1/SvImJ females mated to recombinant males, we have measured the X chromosome inactivation ratio using allele-specific expression assays of genes on the X chromosome. We have identified regions, both proximal and distal to Xist/Tsix, that contribute to the choice of which X chromosome to inactivate, indicating that multiple elements on the X chromosome contribute to the Xce.     

Inhibition of condensation in the late-replicating X chromosome induced by 5-azadeoxycytidine in human lymphocyte cultures

Summary The cytidine analogue 5-azadeoxycytidine (5-aza-dC) induces a very distinct inhibition of condensation in the genetically inactive, late-replicating X chromosome (X_L) when applied to human lymphocyte cultures. One of the two X chromosomes in cytogenetically normal female cells becomes dramatically longer than its homologous partner. The highest rate of metaphases with an undercondensed X_L chromosome is achieved when 5-aza-dC is added at a final concentration of 10^-5 M 2 h before cell harvesting. The interactions between 5-aza-dC and chromosomal DNA as well as the factors involved in X chromosome inactivation are discussed.     

An X‐linked Myh11‐CreERT2 mouse line resulting from Y to X chromosome‐translocation of the Cre allele

The Myh11‐CreER^T2 mouse line (Cre⁺) has gained increasing application because of its high lineage specificity relative to other Cre drivers targeting smooth muscle cells (SMCs). This Cre allele, however, was initially inserted into the Y chromosome (X/Y^Cre+), which excluded its application in female mice. Our group established a Cre⁺ colony from male ancestors. Surprisingly, genotype screening identified female carriers that stably transmitted the Cre allele to the following generations. Crossbreeding experiments revealed a pattern of X‐linked inheritance for the transgene (k > 1000), indicating that these female carries acquired the Cre allele through a mechanism of Y to X chromosome translocation. Further characterization demonstrated that in hemizygous X/X^Cre+ mice Cre activity was restricted to a subset arterial SMCs, with Cre expression in arteries decreased by 50% compared to X/Y^Cre+ mice. This mosaicism, however, diminished in homozygous X^Cre+/X^Cre+ mice. In a model of aortic aneurysm induced by a SMC‐specific Tgfbr1 deletion, the homozygous X^Cre+/X^Cre+ Cre driver unmasked the aortic phenotype that is otherwise subclinical when driven by the hemizygous X/X^Cre+ Cre line. In conclusion, the Cre allele carried by this female mouse line is located on the X chromosome and subjected to X‐inactivation. The homozygous X^Cre+/X^Cre+ mice produce uniform Cre activity in arterial SMCs.     

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).     

Non-random X-chromosome expression early in mouse development

We have used a sensitive electrophoretic technique for estimating the activity, or ratio, of two allozymes of the X-chromosome-linked enzyme phosphoglycerate kinase (PGK-1), in order to investigate the randomness of X-chromosome expression in the derivatives of the three primary cell lineages of the early mouse conceptus. The maternally derived Pgk-1 allele is preferentially expressed in the derivatives of the primitive endoderm and trophectoderm lineages at 6 1/2 days post coitum in Pgk-1^a/Pgk-1^b heterozygous conceptuses, and in the one informative 5 1/2-day heterozygous conceptus analysed. This evidence for preferential expression of the maternally derived X chromosome (X^m), so soon after the time of X-chromosome inactivation, favors the possibility that the preferential expression of X^m is a consequence of primary non-random X-chromosome inactivation, rather than a secondary selection phenomenon. The majority of embryos analysed at 4 1/2 and 5 1/2 days pc produced only a single PGK-1 band, corresponding to the allozyme produced by the Pgk-1 allele on X^m, although 50% of these embryos should have been heterozygous females. Possible explanations are discussed.     

Nucleosome unit repeat size in aneuploid mice

Male 8-day-old mice that have part of chromosome 7 translocated to an X chromosome [T(X;7)1Ct] and that are chromosomally unbalanced for chromosome 7, and consequently trisomic for that part of chromosome 7, were found to have a smaller nucleosome repeat unit size than normal littermate males (Rake, A. V., and Edwards, R. H.,Biochem. Genet. 25:671, 1987). This smaller nucleosome size is maintained in adult trisomic males. Males with a balanced chromosomal translocation [T(X;7)1Ct] had a normal nucleosome size compared to their littermates. The nucleosome unit size is not altered in two other types of aneuploid mice studied (XO vs XX, 2n=39 and 40, respectively; and Ts12¹⁷ vs normal, 2n=40 and 41, respectively).     

Mutations in USP9X Are Associated with X-Linked Intellectual Disability and Disrupt Neuronal Cell Migration and Growth

With a wealth of disease-associated DNA variants being recently reported, the challenges of providing their functional characterization are mounting. Previously, as part of a large systematic resequencing of the X chromosome in 208 unrelated families with nonsyndromic X-linked intellectual disability, we identified three unique variants (two missense and one protein truncating) in USP9X. To assess the functional significance of these variants, we took advantage of the Usp9x knockout mouse we generated. Loss of Usp9x causes reduction in both axonal growth and neuronal cell migration. Although overexpression of wild-type human USP9X rescued these defects, all three USP9X variants failed to rescue axonal growth, caused reduced USP9X protein localization in axonal growth cones, and (in 2/3 variants) failed to rescue neuronal cell migration. Interestingly, in one of these families, the proband was subsequently identified to have a microdeletion encompassing ARID1B, a known ID gene. Given our findings it is plausible that loss of function of both genes contributes to the individual''s phenotype. This case highlights the complexity of the interpretations of genetic findings from genome-wide investigations. We also performed proteomics analysis of neurons from both the wild-type and Usp9x knockout embryos and identified disruption of the cytoskeleton as the main underlying consequence of the loss of Usp9x. Detailed clinical assessment of all three families with USP9X variants identified hypotonia and behavioral and morphological defects as common features in addition to ID. Together our data support involvement of all three USP9X variants in ID in these families and provide likely cellular and molecular mechanisms involved.     

Allele‐specific expression of the MAOA gene and X chromosome inactivation in in vitro produced bovine embryos

During embryogenesis, one of the two X chromosomes is inactivated in embryos. The production of embryos in vitro may affect epigenetic mechanisms that could alter the expression of genes related to embryo development and X chromosome inactivation (XCI). The aim of this study was to understand XCI during in vitro, pre‐implantation bovine embryo development by characterizing the allele‐specific expression pattern of the X chromosome‐linked gene, monoamine oxidase A (MAOA). Two pools of ten embryos, comprised of the 4‐, 8‐ to 16‐cell, morula, blastocyst, and expanded blastocyst stages, were collected. Total RNA from embryos was isolated, and the RT‐PCR‐RFLP technique was used to observe expression of the MAOA gene. The DNA amplicons were also sequenced using the dideoxy sequencing method. MAOA mRNA was detected, and allele‐specific expression was identified in each pool of embryos. We showed the presence of both the maternal and paternal alleles in the 4‐, 8‐ to 16‐cell, blastocyst and expanded blastocyst embryos, but only the maternal allele was present in the morula stage. Therefore, we can affirm that the paternal X chromosome is totally inactivated at the morula stage and reactivated at the blastocyst stage. To our knowledge, this is the first report of allele‐specific expression of an X‐linked gene that is subject to XCI in in vitro bovine embryos from the 4‐cell to expanded blastocyst stages. We have established a pattern of XCI in our in vitro embryo production system that can be useful as a marker to assist the development of new protocols for in vitro embryo production. Mol. Reprod. Dev. 77: 615–621, 2010. © 2010 Wiley‐Liss, Inc.