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.     

High-resolution chromosomal microarray analysis of early-stage human embryonic stem cells reveals an association between X chromosome instability and skewed X inactivation

X chromosome inactivation (XCI) is a dosage compensation mechanism that silences the majority of genes on one X chromosome in each female cell via a random process. Skewed XCI is relevant to many diseases, but the mechanism leading to it remains unclear. Human embryonic stem cells (hESCs) derived from the inner cell mass (ICM) of blastocyst-stage embryos have provided an excellent model system for understanding XCI initiation and maintenance. Here, we derived hESC lines with random or skewed XCI patterns from poor-quality embryos and investigated the genome-wide copy number variation (CNV) and loss of heterozygosity (LOH) patterns at the early passages of these two groups of hESC lines. It was found that the average size of CNVs on the X chromosomes in the skewed group is twice as much as that in the random group. Moreover, the LOH regions of the skewed group covered the gene locus of either XIST or XACT, which are master long non-coding RNA (lncRNA) effectors of XCI in human pluripotent stem cells. In conclusion, our work has established an experimentally tractable hESC model for study of skewed XCI and revealed an association between X chromosome instability and skewed XCI.     

Relationship between Wnt-1 and En-2 expression domains during early development of normal and ectopic met-mesencephalon.

Grafting a met-mesencephalic portion of neural tube from a 9.5-day mouse embryo into the prosencephalon of a 2-day chick embryo results in the induction of chick En-2 (ChickEn) expression in cells in contact with the graft (Martinez et al., 1991). In this paper we investigate the possibility of Wnt-1 being one of the factors involved in En-2 induction. Since Wnt-1 and En-2 expression patterns have been described as diverging during development of the met-mesencephalic region, we first compared Wnt-1 and En-2 expression in this domain by in situ hybridization in mouse embryos after embryonic day 8.5. A ring of Wnt-1-expressing cells is detected encircling the neural tube in the met-mesencephalic region at least until day 12.5. This ring consistently overlapped with the En-2 expression domain, and corresponds to the position of this latter gene's maximal expression. We subsequently studied ChickEn ectopic induction in chick embryos grafted with various portions of met-mesencephalon. When the graft originated from the level of the Wnt-1-positive ring, ChickEn induction was observed in 71% of embryos, and in these cases correlated with Wnt-1 expression in the grafted tissue. In contrast, this percentage dropped significantly when the graft was taken from more rostral or caudal parts of the mesencephalic vesicle. Taken together, these results are compatible with a prolonged role of Wnt-1 in the specification and/or development of the met-mesencephalic region, and show that Wnt-1 could be directly or indirectly involved in the regulation of En-2 expression around the Wnt-1-positive ring during this time. We also provide data on the position of the Wnt-1-positive ring relative to anatomical boundaries in the neural tube, which suggest a more general role for the Wnt-1 protein as a positional signal involved in organizing the met-mesencephalic domain.     

Activity map of the tammar X chromosome shows that marsupial X inactivation is incomplete and escape is stochastic

Background  

X chromosome inactivation is a spectacular example of epigenetic silencing. In order to deduce how this complex system evolved, we examined X inactivation in a model marsupial, the tammar wallaby (Macropus eugenii). In marsupials, X inactivation is known to be paternal, incomplete and tissue-specific, and occurs in the absence of an XIST orthologue.     

Role of replication and CpG methylation in fragile X syndrome CGG deletions in primate cells

Instability of the fragile X CGG repeat involves both maternally derived expansions and deletions in the gametes of full-mutation males. It has also been suggested that the absence of aberrant CpG methylation may enhance repeat deletions through an unknown process. The effect of CGG tract length, DNA replication direction, location of replication initiation, and CpG methylation upon CGG stability were investigated using an SV40 primate replication system. Replication-dependant deletions with 53 CGG repeats were observed when replication was initiated proximal to the repeat, with CGG as the lagging-strand template. When we initiated replication further from the repeat, while maintaining CGG as the lagging-strand template or using CCG as the lagging-strand template, significant instability was not observed. CpG methylation of the unstable template stabilized the repeat, decreasing both the frequency and the magnitude of deletion events. Furthermore, CpG methylation slowed the efficiency of replication for all templates. Interestingly, replication forks displayed no evidence of a block at the CGG repeat tract, regardless of replication direction or CpG methylation status. Templates with 20 CGG repeats were stable under all circumstances. These results reveal that CGG deletions occur during replication and are sensitive to replication-fork dynamics, tract length, and CpG methylation.     

Immunological detection of changes in genomic DNA methylation during early zebrafish development.

DNA methylation reprogramming, the erasure of DNA methylation patterns shortly after fertilization and their reestablishment during subsequent early development, is essential for proper mammalian embryogenesis. In contrast, the importance of this process in the development of non-mammalian vertebrates such as fish is less clear. Indeed, whether or not any widespread changes in DNA methylation occur at all during cleavage and blastula stages of fish in a fashion similar to that shown in mammals has remained controversial. Here we have addressed this issue by applying the techniques of Southwestern immunoblotting and immunohistochemistry with an anti-5-methylcytosine antibody to the examination of DNA methylation in early zebrafish embryos. These techniques have recently been utilized to demonstrate that development-specific changes in genomic DNA methylation also occur in Drosophila melanogaster and Dictyostelium discoideum, both organisms for which DNA methylation was previously not thought to occur. Our data demonstrate that genome-wide changes in DNA methylation occur during early zebrafish development. Although zebrafish sperm DNA is strongly methylated, the zebrafish genome is not detectably methylated through cleavage and early blastula stages but is heavily remethylated in blastula and early gastrula stages.     

Global changes in genomic methylation levels during early development of the zebrafish embryo

We have examined the methylation status of the zebrafish genome during early embryogenesis and we find evidence that methylation fluxes do occur in that organism. The parental genetic contributions to the zygote are, initially, differently methylated with the genome of the sperm being hypermethylated relative to the genome of the oocyte. Post-fertilization there is an immediate decrease in methylation of the embryonic genome but the methylation begins to increase rapidly and is re-established by the gastrulation stage. These results are consistent with the results of Santos et al. (Dev Biol 241:172–182, 2002), who examined the methylation of early mouse embryos, and this conservation argues that demethylation/re-methylation is an important part of vertebrate development.Edited by D. Tautz     

Preferential X inactivation in human placenta membranes: Is the paternal X inactive in early embryonic development of female mammals?

Summary In placenta membranes of newborn girls carrying electrophoretically distinguishable G6PD alleles, the maternally derived isozyme is expressed preferentially. This phenomenon cannot be explained by allelic differences in enzyme activity or by somatic selection directed against cells with particular G6PD phenotypes. Instead, it may be that in this tissue X inactivation is nonrandom. Preferential expression of the maternal X chromosome, as has been shown in marsupials and in extraembryonic membranes of rodents and now in man, may reflect the state of activity of the X chromosomes in the early stages of female embryonic development.H.-H. R. is supported by the Deutsche Forschungsgemeinschaft