Michihiko Sugimoto

Impeding Xist Expression from the Active X Chromosome Improves Mouse Somatic Cell Nuclear Transfer

Cloning Futures Cloning mammals by somatic cell nuclear transfer is a technique with many potential applications in regenerative medicine, agriculture, and pharmaceutics; however, it is inefficient because of the incidence of aberrant genomic reprogramming. Inoue et al. (p. 496, published online 16 September) found that the gene product of Xist, which normally inactivates one of the two X chromosomes in females, was unexpectedly expressed ectopically from active X chromosomes in cloned mice. When Xist was deleted from the mice, gene expression returned to normal and the efficiency of somatic cell nuclear transfer increased about ninefold, offering promise for future nuclear transfer technology. Efficiency of mouse nuclear transfer was improved by correcting aberrant gene expression on the active X chromosome. Cloning mammals by means of somatic cell nuclear transfer (SCNT) is highly inefficient because of erroneous reprogramming of the donor genome. Reprogramming errors appear to arise randomly, but the nature of nonrandom, SCNT-specific errors remains elusive. We found that Xist, a noncoding RNA that inactivates one of the two X chromosomes in females, was ectopically expressed from the active X (Xa) chromosome in cloned mouse embryos of both sexes. Deletion of Xist on Xa showed normal global gene expression and resulted in about an eight- to ninefold increase in cloning efficiency. We also identified an Xist-independent mechanism that specifically down-regulated a subset of X-linked genes through somatic-type repressive histone blocks. Thus, we have identified nonrandom reprogramming errors in mouse cloning that can be altered to improve the efficiency of SCNT methods.

RNAi-mediated knockdown of Xist can rescue the impaired postimplantation development of cloned mouse embryos

Cloning mammals by somatic cell nuclear transfer (SCNT) is highly inefficient. Most SCNT-generated embryos die after implantation because of unidentified, complex epigenetic errors in the process of postimplantation embryonic development. Here we identify the most upstream level of dysfunction leading to impaired development of clones by using RNAi against Xist, a gene responsible for X chromosome inactivation (XCI). A prior injection of Xist-specific siRNA into reconstructed oocytes efficiently corrected SCNT-specific aberrant Xist expression at the morula stage, but failed to do so thereafter at the blastocyst stage. However, we found that shortly after implantation, this aberrant XCI status in cloned embryos had been corrected autonomously in both embryonic and extraembryonic tissues, probably through a newly established XCI control for postimplantation embryos. Embryo transfer experiments revealed that siRNA-treated embryos showed 10 times higher survival than controls as early as embryonic day 5.5 and this high survival persisted until term, resulting in a remarkable improvement in cloning efficiency (12% vs. 1% in controls). Importantly, unlike control clones, these Xist-siRNA clones at birth showed only a limited dysregulation of their gene expression, indicating that correction of Xist expression in preimplantation embryos had a long-term effect on their postnatal normality. Thus, contrary to the general assumption, our results suggest that the fate of cloned embryos is determined almost exclusively before implantation by their XCI status. Furthermore, our strategy provides a promising breakthrough for mammalian SCNT cloning, because RNAi treatment of oocytes is readily applicable to most mammal species.

New Evidence Confirms That the Mitochondrial Bottleneck Is Generated without Reduction of Mitochondrial DNA Content in Early Primordial Germ Cells of Mice

In mammals, observations of rapid shifts in mitochondrial DNA (mtDNA) variants between generations have led to the creation of the bottleneck theory for the transmission of mtDNA. The bottleneck could be attributed to a marked decline of mtDNA content in germ cells giving rise to the next generation, to a small effective number of mtDNA segregation units resulting from homoplasmic nucleoids rather than the single mtDNA molecule serving as the units of segregation, or to the selective transmission of a subgroup of the mtDNA population to the progeny. We have previously determined mtDNA copy number in single germ cells and shown that the bottleneck occurs without the reduction in germline mtDNA content. Recently one study suggested that the bottleneck is driven by a remarkable decline of mtDNA copies in early primordial germ cells (PGCs), while another study reported that the mtDNA genetic bottleneck results from replication of a subpopulation of the mtDNA genome during postnatal oocyte maturation and not during embryonic oogenesis, despite a detected a reduction in mtDNA content in early PGCs. To clarify these contradictory results, we examined the mtDNA copy number in PGCs isolated from transgenic mice expressing fluorescent proteins specifically in PGCs as in the aforementioned two other studies. We provide clear evidence to confirm that no remarkable reduction in mtDNA content occurs in PGCs and reinforce that the bottleneck is generated without reduction of mtDNA content in germ cells.

Differences and similarities in the developmental status of embryo-derived stem cells and primordial germ cells revealed by global expression profiling.

Embryonic germ‐line cells are unipotent cells that give rise to either sperm or oocytes. However, pluripotent stem cells can be derived from primordial germ cells (PGCs) or spermatogonia, suggesting that germ‐line cells retain a capacity for pluripotency. Here, we made genome‐wide comparisons of the gene expression profiles of freshly isolated PGCs, in vitro‐formed PGCs (iPGCs), and other stem cell lines, including embryonic stem cells (ESCs), embryonic germ cells (EGCs) and germ‐line stem (GS) cells. Comparing PGC with ESC, 382 genes/transcripts were significantly up‐regulated in ESC, while 188 were elevated in PGC. This suggests that PGCs possess transcription program distinct from that of ESC, although both share expression of many pluripotency‐associated genes.

A simple and robust method for establishing homogeneous mouse epiblast stem cell lines by wnt inhibition.

Summary Epiblast stem cells (EpiSCs) are pluripotent stem cells derived from epiblasts of postimplantation mouse embryos, and thus provide a useful model for studying “primed” pluripotent states. Here, we devised a simple and robust technique to derive high-quality EpiSCs using an inhibitor of WNT secretion. Using this method, we readily established EpiSC lines with high efficiency and were able to use whole embryonic portions without having to separate the epiblast from the visceral endoderm (VE). Expression analyses revealed that these EpiSCs maintained a homogeneous, undifferentiated status, yet showed high potential for differentiation both in vitro and in teratomas. Unlike EpiSCs derived by the original protocol, new EpiSC lines required continuous treatment with the Wnt inhibitor, suggesting some intrinsic differences from the existing EpiSCs. The homogeneous properties of this new version of EpiSCs should facilitate studies on the establishment and maintenance of a “primed” pluripotent state, and directed differentiation from the primed state.

Molecular Identification of tw5: Vps52 Promotes Pluripotential Cell Differentiation through Cell–Cell Interactions

After implantation, pluripotent epiblasts are converted to embryonic ectoderm through cell-cell interactions that significantly change the transcriptional and epigenetic networks. An entrée to understanding this vital developmental transition is the t(w5) mutation of the mouse t complex. This mutation produces highly specific defects in the embryonic ectoderm before gastrulation, leading to death of the embryonic ectoderm. Using a positional cloning approach, we have now identified the mutated gene, completing a decades-long search. The gene, vacuolar protein sorting 52 (Vps52), is a mouse homolog of yeast VPS52 that is involved in the retrograde trafficking of endosomes. Our data suggest that Vps52 acts in extraembryonic tissues to support the growth and differentiation of embryonic ectoderm via cell-cell interactions. It is also required in the formation of embryonic structures at a later stage of development, revealing hitherto unknown functions of Vps52 in the development of a multicellular organism.

RNAi-mediated knockdown of Xist does not rescue the impaired development of female cloned mouse embryos

Abstract In mice, one of the major epigenetic errors associated with somatic cell nuclear transfer (SCNT) is ectopic expression of Xist during the preimplantation period in both sexes. We found that this aberrant Xist expression could be impeded by deletion of Xist from the putative active X chromosome in donor cells. In male clones, it was also found that prior injection of Xist-specific siRNA could significantly improve the postimplantation development of cloned embryos as a result of a significant repression of Xist at the morula stage. In this study, we examined whether the same knockdown strategy could work as well in female SCNT-derived embryos. Embryos were reconstructed with cumulus cell nuclei and injected with Xist-specific siRNA at 6–7 h after oocyte activation. RNA FISH analysis revealed that siRNA treatment successfully repressed Xist RNA at the morula stage, as shown by the significant decrease in the number of cloud-type Xist signals in the blastomere nuclei. However, blastomeres with different sizes (from “pinpoint” to “cloud”) and numbers of Xist RNA signals remained within single embryos. After implantation, the dysregulated Xist expression was normalized autonomously, as in male clones, to a state of monoallelic expression in both embryonic and extraembryonic tissues. However, at term there was no significant improvement in the survival of the siRNA-injected cloned embryos. Thus, siRNA injection was largely effective in repressing the Xist overexpression in female cloned embryos but failed to rescue them, probably because of an inability to mimic consistent monoallelic Xist expression in these embryos. This could only be achieved in female embryos by applying a gene knockout strategy rather than an siRNA approach.

Large-scale production of growing oocytes in vitro from neonatal mouse ovaries

Although fetal or neonatal mammalian ovaries contain many non-growing oocytes within primordial follicles, most degenerate and only a few contribute to the oocyte pool in the mature ovary. Here, we report a follicle-free culture system that allows a large number of these arrested oocytes to enter the growth phase in vitro. As many as 800 oocytes from a newborn mouse, corresponding to more than 10(4) oocytes in large animals, continued to develop, formed a zona pellucida, and were able to fuse with spermatozoa. Some oocytes reached the size of those in normal antral follicles and entered metaphase I, indicating the completion of the growth phase. The key to success was the sequential provision of essential nutrients and growth factors to the oocytes, while preventing the apoptosis that normally occurs in the majority of growing oocytes in vivo. Importantly, maternal genomic imprinting, which is necessary for normal embryonic development, was imposed correctly on their genomes autonomously. Thus, arrested primordial oocytes can be rescued effectively in vitro and can undergo the morphological and genomic modifications necessary for fertilization and subsequent embryonic development. This culture system may provide a significant impetus to the development of new techniques for the efficient production of oocytes from fetal or neonatal ovaries, for research, clinical, and zoological purposes.

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.

Large, Male Germ Cell-Specific Hypomethylated DNA Domains With Unique Genomic and Epigenomic Features on the Mouse X Chromosome

To understand the epigenetic regulation required for germ cell-specific gene expression in the mouse, we analysed DNA methylation profiles of developing germ cells using a microarray-based assay adapted for a small number of cells. The analysis revealed differentially methylated sites between cell types tested. Here, we focused on a group of genomic sequences hypomethylated specifically in germline cells as candidate regions involved in the epigenetic regulation of germline gene expression. These hypomethylated sequences tend to be clustered, forming large (10 kb to ∼9 Mb) genomic domains, particularly on the X chromosome of male germ cells. Most of these regions, designated here as large hypomethylated domains (LoDs), correspond to segmentally duplicated regions that contain gene families showing germ cell- or testis-specific expression, including cancer testis antigen genes. We found an inverse correlation between DNA methylation level and expression of genes in these domains. Most LoDs appear to be enriched with H3 lysine 9 dimethylation, usually regarded as a repressive histone modification, although some LoD genes can be expressed in male germ cells. It thus appears that such a unique epigenomic state associated with the LoDs may constitute a basis for the specific expression of genes contained in these genomic domains.