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. 2011 Dec 20;108(51):20621-6.
doi: 10.1073/pnas.1112664108. Epub 2011 Nov 7.

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

Shogo Matoba  1 , Kimiko Inoue, Takashi Kohda, Michihiko Sugimoto, Eiji Mizutani, Narumi Ogonuki, Toshinobu Nakamura, Kuniya Abe, Toru Nakano, Fumitoshi Ishino, Atsuo Ogura
Affiliations

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

Shogo Matoba et al. Proc Natl Acad Sci U S A. .

Abstract

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.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Transient repression of ectopic Xist in SCNT-generated embryos by Xist-siRNA injection. (A) Quantitative RT-PCR of Xist in cloned embryos injected with control or Xist-siRNA and cultured for 48 h (four-cell), 72 h (morula), or 96 h (blastocyst). The expression levels of Xist were significantly decreased by Xist-siRNA at 72 h (P < 0.001 by Student's t test), whereas no significant difference was observed at 48 or 96 h. (B) RNA-FISH analyses of Xist in siRNA-injected cloned embryos. After 72 h in culture (morula), ectopic Xist expression was observed as a cloud pattern in most nuclei of the control cloned embryos, whereas it was detected as small pinpoint signals in Xist-siRNA–treated cloned embryos (arrows). At 96 h (blastocyst), the majority of nuclei showed cloud signals of Xist RNA in both groups. (Scale bar, 50 μm.) (C) The ratios of blastomeres classified according to the cloud or pinpoint expression patterns of Xist analyzed by RNA FISH. Each column represents a single embryo. It is apparent that the siRNA against Xist strongly repressed spreading of the Xist RNA over the X chromosome at 72 h but not at 96 h. (D) Immunostaining for H3K27me3 (green) and Oct4 (red) in control or Xist-siRNA–treated cloned embryos at 96 h. Strong punctate signals of H3K27me3 were observed in more than 65% of trophectoderm cells (Oct4-negative; Upper Inset) and inner cell mass cells (Oct4-positive; Lower Inset) in both siRNA-treated groups. (Scale bar, 50 μm.)
Fig. 2.
Fig. 2.
Effects of Xist-siRNA injection on the global gene expression patterns of cloned embryos. (A) Relative expression levels of X-linked genes plotted on the X chromosome position in cloned embryos compared with IVF embryos at 72 h in culture (morula stage) and at 96 h (blastocyst stage) (n = 4 for each group). At the morula stage, X-linked genes were largely down-regulated over the entire chromosome in control cloned embryos (red), and the majority of them were increased in their expression levels by Xist-siRNA injection (green). By contrast, in blastocyst embryos, X-linked genes were down-regulated over the entire chromosome in both siRNA groups. Dotted lines represent a single embryo, and solid lines indicate their mean values. (B) Numbers of down-regulated genes (fold change >10) in cloned embryos compared with conventional IVF-generated embryos. At the morula stage, gene numbers were reduced by the injection of Xist-siRNA for genes not only on the X chromosome but also on autosomes, whereas they did not differ between the two groups at the blastocyst stage.
Fig. 3.
Fig. 3.
Effects of Xist-siRNA on the postimplantation development of cloned embryos. (A) Representative photomicrographs of siRNA-treated cloned embryos recovered at E5.5. Most Xist-siRNA embryos showed normal morphology (Left) with a distinct embryonic epiblast region (Epi) and extraembryonic ectoderm (EXE) or an ectoplacental cone (EPC), whereas most control siRNA-treated embryos showed abnormal morphology such as developmental retardation or ambiguous embryonic and extraembryonic regions (Center and Right). (Scale bar, 50 μm.) (B) The developmental rate of embryos assessed at E5.5 and at term. In some experiments, TSA was added at 5 or 50 nM in the culture medium. The numbers at the top of the bars indicate the rates of normal-shaped embryos (E5.5) and full-term births per embryos transferred. *P < 0.05, **P < 0.001 by Fisher's exact test. (C) A litter of cloned pups produced by SCNT from Sertoli cell nuclei, obtained following treatment with Xist-siRNA and 50 nM TSA. They were born at the best efficiency we observed in this series: Seven pups were born from 23 embryos transferred (30%) to a single recipient mother. (D) Gene expression profiles of livers in neonatal mice generated by SCNT with or without Xist-siRNA injection. The values indicate the mean fold changes from the control IVF level (=1). Red dots represent genes of which expression levels exceeded a twofold change in all of the individual clones. Xist-siRNA clones showed much fewer dysregulated genes compared with control clones. For data on each individual clone, see Fig. S2.
Fig. 4.
Fig. 4.
The XCI status of cloned embryos at E5.5. (A) RNA-FISH analyses of Xist (red) combined with immunostaining for Oct4 (green) in control or Xist-siRNA–injected cloned embryos. There were very few Xist signals in either Oct4-positive epiblast (Epi) (Middle Inset) or Oct4-negative extraembryonic ectoderm (EXE) (Top Inset) in either siRNA group. In these micrographs, only one Xist-positive cell was found within the epiblast (arrow; Bottom Inset, higher magnification). (Scale bar, 50 μm.) (B) Ratios of Xist-positive cells in the epiblast or extraembryonic ectoderm region analyzed by RNA FISH. Each column represents a single embryo. Only a few Xist-positive cells were observed in the regions irrespective of siRNA treatment. (C) Immunostaining for H3K27me3 (green), another marker for XCI, and Oct4 (red) in control or Xist-siRNA–treated cloned embryos. There were very few punctate H3K27me3 signals in either epiblast (Middle Inset) or extraembryonic ectoderm (Top Inset) in either siRNA group. The nuclei of some EXE cells showed relatively weak and diffuse staining for H3K27me3, which does not represent XCI. In these micrographs, only a single cell within the epiblast showed a punctate H3K27me3 signal (arrow; Bottom Inset). (Scale bar, 50 μm.) (D) Ratios of cells classified according to the punctate or diffuse patterns of H3K27me3 signals. Each column represents a single embryo. Consistent with the results of Xist RNA FISH, the punctate signals for H3K27me3 were rarely observed in cloned embryos in either group.
Fig. 5.
Fig. 5.
Schematic representation of siRNA-mediated Xist repression and its effect on the survival of male SCNT-generated embryos. (Upper) In control cloned embryos, ectopic Xist expression increased rapidly from 48 h through 72 h and maintained a high level until 96 h (black line; see also ref. 6). Injection of Xist-siRNA into pronuclear (PN)-stage cloned embryos at 6 h (dotted arrow) resulted in repression of the Xist level over 48–72 h, but this became ineffective at 96 h (magenta line). Thereafter, around implantation, ectopic Xist expression diminished spontaneously from all embryonic tissues in Xist-siRNA and control embryos. (Lower) Repression of Xist by siRNA had a remarkable effect on the survival of cloned embryos. As early as E5.5, more than 10 times as many Xist-siRNA embryos survived compared with control embryos, and this high survival persisted until term. These results suggest that the adverse effects of ectopic Xist expression in cloned embryos are confined to a short critical time window in the preimplantation period and that this can be reversed very efficiently by injecting Xist-siRNA into SCNT-derived embryos.
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Comment in

  • More with less Xist.
    Wells KD. Wells KD. Proc Natl Acad Sci U S A. 2012 Jan 10;109(2):349-50. doi: 10.1073/pnas.1118081109. Epub 2011 Dec 28. Proc Natl Acad Sci U S A. 2012. PMID: 22205767 Free PMC article. No abstract available.

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