Somatic Donor Cell Type Correlates with Embryonic, but Not Extra-Embryonic, Gene Expression in Postimplantation Cloned Embryos

<div><p>The great majority of embryos generated by somatic cell nuclear transfer (SCNT) display defined abnormal phenotypes after implantation, such as an increased likelihood of death and abnormal placentation. To gain better insight into the underlying mechanisms, we analyzed genome-wide gene expression profiles of day 6.5 postimplantation mouse embryos cloned from three different cell types (cumulus cells, neonatal Sertoli cells and fibroblasts). The embryos retrieved from the uteri were separated into embryonic (epiblast) and extraembryonic (extraembryonic ectoderm and ectoplacental cone) tissues and were subjected to gene microarray analysis. Genotype- and sex-matched embryos produced by <i>in vitro</i> fertilization were used as controls. Principal component analysis revealed that whereas the gene expression patterns in the embryonic tissues varied according to the donor cell type, those in extraembryonic tissues were relatively consistent across all groups. Within each group, the embryonic tissues had more differentially expressed genes (DEGs) (>2-fold vs. controls) than did the extraembryonic tissues (<i>P</i><1.0×10^–26). In the embryonic tissues, one of the common abnormalities was upregulation of <i>Dlk1</i>, a paternally imprinted gene. This might be a potential cause of the occasional placenta-only conceptuses seen in SCNT-generated mouse embryos (1–5% per embryos transferred in our laboratory), because dysregulation of the same gene is known to cause developmental failure of embryos derived from induced pluripotent stem cells. There were also some DEGs in the extraembryonic tissues, which might explain the poor development of SCNT-derived placentas at early stages. These findings suggest that SCNT affects the embryonic and extraembryonic development differentially and might cause further deterioration in the embryonic lineage in a donor cell-specific manner. This could explain donor cell-dependent variations in cloning efficiency using SCNT.</p></div

List of samples from E6.5 cloned embryos analyzed by microarray.

<p>CC, cumulus cell-derived clone; FC, fibroblast-derived clone, SC: Sertoli cell-derived clone; IVF, <i>in vitro</i> fertilization-derived control embryos.</p

Expression levels of genes important for early placentation.

<p>Genes important for maintenance of undifferentiated trophoblast cells (<i>Cdx2</i>, <i>Esrrb</i> and <i>Eomes</i>) were downregulated while those essential for differentiation into giant cells (<i>Hand1</i>) were upregulated. * <i>P</i><0.05, ** <i>P</i><0.01 (compared with the corresponding IVF-derived controls). “†” indicates a tendency for down- or upregulation, but not with a statistical significance (<i>P</i><0.10).</p

Expression of LOCKs-related genes in SCNT-derived embryos.

<p>Raw intensity values of LOCKs-related genes at E3.5 (blastocysts) and E6.5. The data set at E3.5 was from a previous study <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076422#pone.0076422-Inoue1" target="_blank">[12]</a> (accession number: GSE23181). At E3.5, all the LOCKs genes examined were strongly repressed in SCNT-derived blastocysts compared with those in control IVF-derived blastocysts. At E6.5, after implantation, the genes were downregulated in the embryonic tissue and no significant differences were found between the SCNT- and IVF-derived embryos. By contrast, in the extraembryonic tissues, <i>Xlr</i> genes (e.g., <i>Xlr4b</i>, <i>Xlr3c</i>, <i>Xlr5a</i> and <i>Xlr5c</i>) remained active after implantation and in the SCNT-derived samples the expression levels were restored to nearly normal. Bla, blastocysts; Em, embryonic samples; Ex, extraembryonic samples; CC, cumulus cell-derived clone; SC, Sertoli cell-derived clone.</p

Principal component analysis (PCA) of global gene expression profiles.

<p>Visualization of gene expression similarities of samples clearly distinguishes donor cell-specific clusters of the embryonic samples (squares) from a donor cell-independent (i.e., SCNT-specific) overlapping cluster of extraembryonic samples (triangles). Red, green and blue represent cumulus cell-derived clone, fibroblast-derived clone and neonatal Sertoli cell-derived clone samples, respectively. Only the SCNT groups are presented based on the relative distance from the corresponding sex- and tissue type-matched IVF-derived control embryos. The result of conventional PCA, in which all IVF-derived samples were analyzed concomitantly, is shown in Figure S3.</p

Overlapping DEGs among the three SCNT groups.

<p>(<b>A</b>) Venn diagrams of the DEGs identified in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076422#pone-0076422-g003" target="_blank">Figure 3</a> (fold change >2.0), showing the numbers of shared and unique DEGs among three different SCNT groups. There were very few DEGs shared with all three groups in both embryonic and extraembryonic tissues. (<b>B</b>) List of overlapping DEGs among the three SCNT groups. A complete list of DEGs is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076422#pone.0076422.s003" target="_blank">Table S2</a>.</p

Preparation and validation of the samples.

<p>(<b>A</b>) Embryos were retrieved at E6.5 (left) and dissected into four parts (right). EPC, ectoplacental cone; VE, visceral endoderm; EXE, extraembryonic ectoderm; EPI, epiblast. Scale bar  = 100 µm. B) Raw signal values of <i>Pou5f1</i> (left), <i>Cdx2</i> (center) and <i>Pgk1</i> (right) genes extracted from microarray data. <i>Pou5f1</i> and <i>Cdx2</i> were detected exclusively in the embryonic and extraembryonic samples, respectively, while <i>Pgk1</i> was detected in both samples. These results confirmed the accuracy of sample preparation from the embryonic and extraembryonic tissues. Em, embryonic samples; Ex, extraembryonic samples; IVF, samples from <i>in vitro</i> fertilized control embryos; CC, cumulus cell-derived clone; FC, fibroblast-derived clone; SC, Sertoli cell-derived clone. See Figure S2 for further validation by several other marker genes.</p

Fold change analysis of gene expression profiles of embryonic and extraembryonic samples.

<p>Fold change analysis with a cutoff of f >2 identified the number of differentially expressed genes (DEGs) in SCNT-derived samples compared with IVF-derived controls. (<b>A</b>) Histogram showing the distribution of DEGs classified by the relative values compared with the IVF-derived controls. The embryonic samples included more DEGs (901–4642) than the extraembryonic samples (272–592). This tendency is depicted by the narrower and higher distribution patterns of the extraembryonic samples. (<b>B</b>) The cumulative numbers of up- and downregulated DEGs shown side-by-side based on fold changes. Dark and light bars represent up- and downregulated DEGs, respectively. In the embryonic tissues, large fold changes occurred predominantly with the upregulated DEGs, whereas in the extraembryonic tissues such changes occurred with the downregulated DEGs. *No corresponding gene.</p

DataSheet1_Suppression of endogenous retroviral enhancers in mouse embryos derived from somatic cell nuclear transfer.pdf

Endogenous retroviruses (ERVs) in the mammalian genome play diverse roles in embryonic development. These developmentally related ERVs are generally repressed in somatic cells and therefore are likely repressed in embryos derived from somatic cell nuclear transfer (SCNT). In this study, we sought to identify ERVs that are repressed in SCNT-derived morulae, which might cause previously unexplained embryonic deaths shortly after implantation. Our transcriptome analysis revealed that, amongst ERV families, ERVK was specifically, and strongly downregulated in SCNT-derived embryos while other transposable elements including LINE and ERVL were unchanged. Among the subfamilies of ERVK, RLTR45-int was most repressed in SCNT-derived embryos despite its highest expression in control fertilized embryos. Interestingly, the nearby genes (within 5–50 kb, n = 18; 50–200 kb, n = 63) of the repressed RLTR45-int loci were also repressed in SCNT-derived embryos, with a significant correlation between them. Furthermore, lysine H3K27 acetylation was enriched around the RLTR45-int loci. These findings indicate that RLTR45-int elements function as enhancers of nearby genes. Indeed, deletion of two sequential RLTR45-int loci on chromosome 4 or 18 resulted in downregulations of nearby genes at the morula stage. We also found that RLTR45-int loci, especially SCNT-low, enhancer-like loci, were strongly enriched with H3K9me3, a repressive histone mark. Importantly, these H3K9me3-enriched regions were not activated by overexpression of H3K9me3 demethylase Kdm4d in SCNT-derived embryos, suggesting the presence of another epigenetic barrier repressing their expressions and enhancer activities in SCNT embryos. Thus, we identified ERVK subfamily RLTR45-int, putative enhancer elements, as a strong reprogramming barrier for SCNT (253 words).</p

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