Ftx is dispensable for imprinted X-chromosome inactivation in preimplantation mouse embryos

Soma, Miki; Fujihara, Yoshitaka; Okabe, Masaru; Ishino, Fumitoshi; Kobayashi, Shin

doi:10.1038/srep05181

Ftx is dispensable for imprinted X-chromosome inactivation in preimplantation mouse embryos

Article
Open access
Published: 05 June 2014

Volume 4, article number 5181, (2014)
Cite this article

Download PDF

You have full access to this open access article

Scientific Reports

Ftx is dispensable for imprinted X-chromosome inactivation in preimplantation mouse embryos

Download PDF

Miki Soma¹,
Yoshitaka Fujihara²,
Masaru Okabe²,
Fumitoshi Ishino¹ &
…
Shin Kobayashi^1,3

2636 Accesses
2 Altmetric
Explore all metrics

Abstract

X-chromosome inactivation (XCI) equalizes gene expression between the sexes by inactivating one of the two X chromosomes in female mammals. **st has been considered as a major cis-acting factor that inactivates the paternally derived X chromosome (Xp) in preimplantation mouse embryos (imprinted XCI). Ftx has been proposed as a positive regulator of **st. However, the physiological role of Ftx in female animals has never been studied. We recently reported that Ftx is located in the cis-acting regulatory region of the imprinted XCI and expressed from the inactive Xp, suggesting a role in the imprinted XCI mechanism. Here we examined the effects on imprinted XCI using targeted deletion of Ftx. Disruption of Ftx did not affect the survival of female embryos or expression of **st and other X-linked genes in the preimplantation female embryos. Our results indicate that Ftx is dispensable for imprinted XCI in preimplantation embryos.

Manipulation of **st Imprinting in Mouse Preimplantation Embryos

REX1 is the critical target of RNF12 in imprinted X chromosome inactivation in mice

Article Open access 12 November 2018

The role of maternal-specific H3K9me3 modification in establishing imprinted X-chromosome inactivation and embryogenesis in mice

Article Open access 14 November 2014

Introduction

Female mammals have a unique mechanism of gene dosage compensation called X-chromosome inactivation (XCI), which is an essential epigenetic process for development. In mice, inactivation of the paternally derived X chromosome (Xp), but not of the maternally derived X chromosome (Xm), is initiated in the preimplantation embryos (imprinted XCI)^1,2,3. This imprinted inactivation of the Xp is maintained in post-implantation extra-embryonic tissues. At the blastocyst stage, reactivation of the Xp occurs in the inner cell mass that will later form the embryo. Thus, the imprinted XCI is erased once, subsequently, random XCI of either the Xp or Xm chromosomes is initiated in the post-implantation embryo by a cis-acting region on the X chromosome termed the X-inactivation centre (** are shown as red arrows. (b) Validation of recombination on the chromosomal long and short arm target sequences of ES cells (cell lines #64 and #77). (c) Genoty** results for wild-type females (+_m/+_p, where m is the maternally derived X chromosome and p is the paternally derived X chromosome), heterozygous females (+_m/−_p), homozygous females (−_m/−_p), wild-type males (+_m/Y, where Y is the Y chromosome) and hemizygous males (−_m/Y). The PCR primers (233, 234, and330) used for genoty** are depicted in Fig2-a. (d) Expression of Ftx in Ftx-deficient blastocysts. PCR primers used for RT–PCR are shown in parentheses. Uncropped images of the full-length gels are presented in Supplementary Figure S1. (e) The expression levels of miR-374-5p and miR-421-3p were quantified by q-PCR in wild-type female (+_m/+_p), male (+_m/Y) and heterozygous female (+_m/−_p) blastocysts. The ratios of the number of cDNA copies for each miRNA to that of cDNA copies for miR-295 are indicated as expression levels. The bar graphs and lines show the mean ± standard deviation (SD) (n = 3).

Full size image

Expression of miRNAs in Ftx-deficient blastocysts

Two miRNAs, miR-374-5p and miR-421-3p, are located within the Ftx intron in the same orientation as the Ftx transcript (Figure 1a). We confirmed that these miRNAs were not expressed in female blastocysts (+_m/−_p) when the Ftx-targeted allele was derived from the father, whereas these miRNAs were predominantly expressed in wild-type female blastocysts (+_m/+_p) (Figure 2e). This finding indicated that the Ftx-deficient mice failed to express miR-374-5p and miR-421-3p simultaneously and suggested that both miRNAs were transcribed from the same promoter as Ftx.

Mating of Ftx-deficient mice

Paternal transmission of the ** gene, β-actin, are indicated as expression levels. The bar graphs and lines show the mean ± standard deviation (SD) (n = 3). (b) RNA FISH analysis for **st (red) expression in Ftx-deficient blastocysts. The nuclei of blastocysts were stained with DAPI (blue). Scale bar, 50 μm. (c) Summary of **st RNA FISH analysis. Three wild-type (+_m/+_p) and Three Ftx-deficient (+_m/−_p) blastocysts were examined. Cells with 2 **st signals were not observed. (d) Analysis of X-linked gene expression levels in Ftx-deficient blastocysts. No differences in the allelic expression patterns of X-linked genes between JF1 and C57BL/6N embryos were detected using RFLP. Uncropped images of the full-length gels are presented in Supplementary Figure S1.

Full size image

Effects of Ftx* disruption on imprinted XCI in preimplantation embryos*

Our results with crossing experiments among Ftx-deficient mice and normal **st expression in the Ftx-deficient blastocysts indicated that disruption of Ftx has no major role on imprinted XCI. However, the effect of Ftx-disruption on each X-linked gene was still uncertain. We next performed restriction enzyme fragment polymorphism (RFLP) analysis to examine the allelic expression patterns of **st and four X-linked genes, Fmr1, Rnf12, Fgd1 and Padh1, which are all subject to imprinted XCI. We took advantage of a single nucleotide polymorphism found in each gene between C57BL/6N and the Japanese wild mouse M. m. molossinus (JF1) and examined the expression patterns of all five genes in Ftx-deficient blastocysts. The expression patterns of all genes were similar to those of wild-type embryos (Figure 3d). Taken together, the results indicate that disruption of Ftx expression produced no obvious effects on imprinted XCI during preimplantation development in mice.

Discussion

In this study, we examined whether Ftx might be involved in the mechanisms of imprinted XCI at the preimplantation stage. Although **st deletion results in female-specific embryonic death when it is derived from the father because of the lack of XCI in extra-embryonic tissues⁶, disruption of Ftx did not affect survival rates or litter sizes in both sexes, demonstrating that this ncRNA is not necessary for the survival of female mice. Further studies indicated that Ftx disruption did not cause major deficiency in the expression of **st or the expression of X-linked genes in preimplantation embryos. These results are inconsistent with the hypothesis that Ftx is a positive regulator of **st in XCI, as proposed by Chureau et al.¹³. Because they analysed the expression of **st using Ftx-deficient XY male ES cells that had one active X chromosome and lacked XCI, their results might not reflect the situation for female cells in vivo. Chureau also reported that, in addition to **st, Ftx deletion decreased the expression levels of the adjacent imprinted genes, Jpx and, Zcchc13¹³. It would be of interest to examine whether these adjacent genes are also affected in female cells. We were also interested in whether Ftx is involved in the onset of the imprinted **st upregulation during early preimplantation stages because previous studies have shown that **st RNA upregulation is detected from the two- to four-cell stage onward^14,15. We used q-PCR to examine the Ftx expression at preimplantation stages and found that the expression begins at the blastocyst stage (Figure S1). This suggests that Ftx is not involved in the onset of this **st upregulation. As far as we examined, Ftx disruption had no apparent effects on imprinted XCI at the preimplantation stage. This indicates that Ftx is dispensable for imprinted XCI and that the remaining genes included in the 210 kb regulatory region of imprinted XCI—Tsix and/or Jpx—might play essential roles in the regulation of imprinted **st.

Tsix, an antisense transcript to **st, is another lncRNA that functions as a repressor of **st during the initiation of random XCI in post-implantation embryos^9,10. It inhibits the expression of **st on one female X chromosome to maintain the active state of the randomly chosen one. It is imprinted and expressed from maternally derived X chromosomes at the preimplantation stage¹⁰. However, whether Tsix is also required for the imprinted expression of **st in preimplantation embryos remains unclear. Tsix expression was reported to start later than **st expression from Xp alleles, implying that there were mechanisms other than Tsix that repress **st expression from Xm alleles¹⁶. Further study is required to reveal the role of Tsix in imprinted XCI at the preimplantation stage.

Jpx is located just downstream of the Ftx locus. Its deletion in XX female ES cells results in failure of accumulation of **st on the inactive X chromosome and XCI in differentiated ES cells, indicating that Jpx can upregulate **st expression in random XCI¹⁷. However, how Jpx regulates **st expression in vivo is not known. In the case of imprinted XCI, there are only three paternally expressed imprinted genes, **st, Ftx and Jpx, located in the 210 kb transgene. Apart from Ftx, Jpx is the only candidate gene that potentially upregulates **st. Based on its proposed role in ES cells¹⁷, Studies in Jpx-targeted mice will be of interest to delineate the necessity of this imprinted ncRNA in imprinted XCI.

In this study, we focused on imprinted XCI, but not on random XCI. Because the Tsix–Zcchc13 cluster is included within the **c region that plays a major role in random XCI, it is necessary to elucidate whether Ftx is involved in random XCI in post-implantation embryos. Further study is required to elucidate the mechanism of XCI, but the results presented in this paper will help clarify the answer.

Methods

Animals

All animal experiments were performed according to the guidelines of the Committee on the Use of Live Animals in Teaching and Research of Tokyo Medical and Dental University and animal protocols were reviewed and approved by the animal welfare committee of the Tokyo Medical and Dental University (Permit Number: 0130228A).

Embryo collection

For blastocyst collection, 8-week-old C57BL/6N female mice (Crea Inc., Tokyo, Japan) were superovulated with an intraperitoneal (i.p.) injection of 5 IU of pregnant mare serum gonadotropin (Aska Pharmaceutical Co. Ltd., Tokyo, Japan) followed by an i.p. injection of 5 IU of human chorionic gonadotropin (hCG; Aska Pharmaceutical Co. Ltd.) 48 h later. These superovulated female mice were mated with C57BL/6N(+/Y) or Ftx-deficient mice (−/Y) and blastocysts were collected from the uterus on 3.5 dpc from around 92–100 h post-hCG.

5′-RACE

Total RNA was prepared from male and female wild-type blastocysts using TRIzol (Invitrogen, Carlsbad, CA, USA). 5′-RACE was performed on RNA prepared from blastocysts with GSP1 and GSP2 (Supplementary Table S2) using SMARTer RACE cDNA Amplification kits (Clontech, Mountain View, CA, USA). To separate male and female blastocysts, we used a non-invasive sexing method by tagging X chromosomes with an enhanced green fluorescent protein (EGFP) transgene^18,19,20. With this method, the female (XX^EGFP) blastocysts can be separated from the non-fluorescent male (XY) blastocysts by mating X^EGFPY male mice to wild-type female (XX) mice. The RACE products were subsequently cloned into pCR4-TOPO (Invitrogen) for sequencing. Sequencing was performed using BigDye Terminator v3 Cycle Sequencing kits (ABI, Life Technologies, Carlsbad, CA, USA).

Construction of the Ftx-targeting vector

The targeting vector was designed to replace exons 1–4 of Ftx with a pNT1.1 Neo cassette. A 5.3 kb NotI–XhoI fragment as a short chromosomal arm and a 5.0 kb AscI–PacI fragment as a long arm were obtained by PCR using genomic DNA of a bacterial artificial chromosome clone (RP23, #239G16) as a template. Primers used for constructing the Ftx targeting vector are listed in Supplementary Table S2.

Generation of Ftx-deficient mice

The targeting vector was linearized with NotI digestion and electroporated into ES cells (EGR-G101) derived from C57BL/6N mice (Crea, Inc.). ES cell clones that were candidates for possessing the targeted allele were isolated by positive selection with G418. Correct targeting of the Ftx allele in ES cell clones by homologous recombination was confirmed by PCR. Positive ES cell clones were subsequently used to generate chimeras by injection into 8-cell stage ICR strain embryos. Injected embryos were transferred to ICR strain pseudopregnant foster mothers, resulting in the birth of chimeric mice. Male chimeric mice were crossed with C57BL/6N female mice to obtain F1 heterozygous female offspring and then crossed with CAG-FLPe transgenic mice (B6.Cg-Tg (CAG-FLPe)) to delete the Neo cassette. The Ftx-deficient mice used in the present study were from a C57BL/6N background. The primers used for confirming homologous recombination are listed in Supplementary Table S2.

q-PCR analysis

Total RNA of embryos was extracted using TRIzol (Invitrogen) and contaminating DNA was digested using RNase-free DNase (Nippon Gene, Tokyo, Japan). First-strand cDNA was synthesized using reverse transcriptase SuperScript III (Invitrogen). For miRNA expression analysis, total RNA was reverse transcribed using the TaqMan MicroRNA Reverse Transcription Kit (ABI). The TaqMan MicroRNA assays used (with identification numbers in parentheses), were as follows: mmu-miR-374-5p (001319), mmu-miR-421-3p (002700) and mmu-miR-295 (000189). Mature miRNAs were amplified with TaqMan Universal PCR Master Mix II (ABI) using a LightCycler 480 Instrument (Roche, Basel, Switzerland). The q-PCR reactions were performed using samples collected from three independent preparations.

FISH

**st-specific probes were prepared by nick translation with Cy3-dCTP (GE Healthcare, Little Chalfont, UK) from p**st 1986–9498. Blastocysts were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 10 min on ice and incubated in 0.1% Triton X-100 in PBS for 10 min on ice. Hybridisation was carried out at 42 °C overnight. The nuclei of embryos were stained with DAPI. **st signals were observed using a confocal scanning laser microscope (LSM 510; Carl Zeiss, Jena, Germany).

Polymorphic analysis

(J × B) or (J × KO) F1 embryos were obtained from mating 8- to 12-week-old wild-type JF1 (J) female mice with wild-type C57BL/6N (B) or Ftx-deficient male mice (KO). Polymorphisms of X-linked genes between JF1 and C57BL/6N were detected by RFLP analysis. Primers and restriction enzymes used for polymorphic analysis are summarized in Supplementary Table S2.

References
Huynh, K. D. & Lee, J. T. Inheritance of a pre-inactivated paternal X chromosome in early mouse embryos. Nature 426, 857–862, 10.1038/nature02222 (2003).
Article ADS CAS Google Scholar
Mak, W. et al. Reactivation of the paternal X chromosome in early mouse embryos. Science 303, 666–669, 10.1126/science.1092674 (2004).
Article ADS CAS PubMed Google Scholar
Okamoto, I., Otte, A. P., Allis, C. D., Reinberg, D. & Heard, E. Epigenetic dynamics of imprinted X inactivation during early mouse development. Science 303, 644–649, 10.1126/science.1092727 (2004).
Article ADS CAS Google Scholar
Rastan, S. Non-random X-chromosome inactivation in mouse X-autosome translocation embryos--location of the inactivation centre. J. Embryol. Exp. Morphol. 78, 1–22 (1983).
CAS PubMed Google Scholar
Rastan, S. & Robertson, E. J. X-chromosome deletions in embryo-derived (EK) cell lines associated with lack of X-chromosome inactivation. J. Embryol. Exp. Morphol. 90, 379–388 (1985).
CAS PubMed Google Scholar
Marahrens, Y., Panning, B., Dausman, J., Strauss, W. & Jaenisch, R. **st- deficient mice are defective in dosage compensation but not spermatogenesis. Genes Devel. 11, 156–166 (1997).
Article CAS Google Scholar
Namekawa, S. H., Payer, B., Huynh, K. D., Jaenisch, R. & Lee, J. T. Two-step imprinted X inactivation: repeat versus genic silencing in the mouse. Mol. Cell. Biol. 30, 3187–3205, 10.1128/MCB.00227-10 (2010).
Article CAS PubMed PubMed Central Google Scholar
Kobayashi, S. et al. Identification of an imprinted gene cluster in the X-inactivation center. PloS One 8, e71222, 10.1371/journal.pone.0071222 (2013).
Article ADS CAS PubMed PubMed Central Google Scholar
Lee, J. T. Disruption of imprinted X inactivation by parent-of-origin effects at Tsix. Cell 103, 17–27 (2000).
Article CAS Google Scholar
Sado, T., Wang, Z., Sasaki, H. & Li, E. Regulation of imprinted X-chromosome inactivation in mice by Tsix. Development 128, 1275–1286 (2001).
CAS PubMed Google Scholar
Okamoto, I. et al. Evidence for de novo imprinted X-chromosome inactivation independent of meiotic inactivation in mice. Nature 438, 369–373, 10.1038/nature04155 (2005).
Article ADS CAS PubMed Google Scholar
Schulz, E. G. & Heard, E. Role and control of X chromosome dosage in mammalian development. Curr. Opin. Genet. Devel. 23, 109–115, 10.1016/j.gde.2013.01.008 (2013).
Article CAS Google Scholar
Chureau, C. et al. Ftx is a non-coding RNA which affects **st expression and chromatin structure within the X-inactivation center region. Hum. Mol. Genet. 20, 705–718, 10.1093/hmg/ddq516 (2011).
Article CAS PubMed Google Scholar
Kay, G. F., Barton, S. C., Surani, M. A. & Rastan, S. Imprinting and X chromosome counting mechanisms determine **st expression in early mouse development. Cell 77, 639–650 (1994).
Article CAS Google Scholar
Zuccotti, M. et al. Mouse **st expression begins at zygotic genome activation and is timed by a zygotic clock. Mol. Reprod. Dev. 61, 14–20, 10.1002/mrd.1126 (2002).
Article CAS PubMed Google Scholar
Sado, T. & Sakaguchi, T. Species-specific differences in X chromosome inactivation in mammals. Reproduction 146, R131–139, 10.1530/REP-13-0173 (2013).
Article CAS PubMed Google Scholar
Tian, D., Sun, S. & Lee, J. T. The long noncoding RNA, Jpx, is a molecular switch for X chromosome inactivation. Cell 143, 390–403, 10.1016/j.cell.2010.09.049 (2010).
Article CAS PubMed PubMed Central Google Scholar
Nakanishi, T. et al. FISH analysis of 142 EGFP transgene integration sites into the mouse genome. Genomics 80, 564–574 (2002).
Article CAS Google Scholar
Kobayashi, S. et al. Comparison of gene expression in male and female mouse blastocysts revealed imprinting of the X-linked gene, Rhox5/Pem, at preimplantation stages. Curr. Biol. 16, 166–172, 10.1016/j.cub.2005.11.071 (2006).
Article CAS PubMed Google Scholar
Kobayashi, S. et al. The X-linked imprinted gene family Fthl17 shows predominantly female expression following the two-cell stage in mouse embryos. Nucl. Acids Res. 38, 3672–3681, 10.1093/nar/gkq113 (2010).
Article CAS PubMed Google Scholar
Download references
Acknowledgements
We thank Dr Kuniya Abe at RIKEN BioResource Center for providing the p**st 1986–9498 plasmid, Y. Esaki, S. Nishioka, Y. Koreeda and K. Kawata for technical assistance in generating Ftx-deficient mouse lines, Y. Hosoi for assistance in FISH analysis, Dr Takashi Kohda and Dr Daisuke Endo for critical reading and discussion. This work was supported by the Japan Science and Technology Agency, Precursory Research for Embryonic Science and Technology (PRESTO) and by a Grant- in-Aid for Scientific Research from The Ministry of Education, Culture, Sports, Science and Technology and the Sumitomo Foundation to S.K. The funders had no roles in the study design, data collection and analysis, decision to publish or preparation of the manuscript.
Author information
Authors and Affiliations
Department of Epigenetics, Medical Research Institute, Tokyo Medical & Dental University, 1-5-45 Yushima Bunkyo-ku, Tokyo, 113-8510, Japan
Miki Soma, Fumitoshi Ishino & Shin Kobayashi
Research Institute for Microbial Diseases, Osaka University, Yamadaoka 3-1, Suita, Osaka, 565-0871, Japan
Yoshitaka Fujihara & Masaru Okabe
Japan Science and Technology Agency, PRESTO, 4-1-8 Honcho Kawaguchi, Saitama, 332-0012, Japan
Shin Kobayashi
Authors
Miki Soma
View author publications
You can also search for this author in PubMed Google Scholar
Yoshitaka Fujihara
View author publications
You can also search for this author in PubMed Google Scholar
Masaru Okabe
View author publications
You can also search for this author in PubMed Google Scholar
Fumitoshi Ishino
View author publications
You can also search for this author in PubMed Google Scholar
Shin Kobayashi
View author publications
You can also search for this author in PubMed Google Scholar
Contributions
M.S. and S.K. performed most experiments, assisted by Y.F., M.O. and I.F.; S.K., Y.F. and M.O. generated mutant mice. M.S. and S.K. analysed the data. M.S. and S.K. wrote the manuscript and all authors discussed the results and commented on the manuscript.
Ethics declarations

Competing interests

The authors declare no competing financial interests.

Electronic supplementary material
Supplementary Information
Supplementary infomation
Rights and permissions

This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. The images in this article are included in the article's Creative Commons license, unless indicated otherwise in the image credit; if the image is not included under the Creative Commons license, users will need to obtain permission from the license holder in order to reproduce the image. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/3.0/

Reprints and permissions
About this article
Cite this article
Soma, M., Fujihara, Y., Okabe, M. et al. Ftx is dispensable for imprinted X-chromosome inactivation in preimplantation mouse embryos. Sci Rep 4, 5181 (2014). https://doi.org/10.1038/srep05181
Download citation
Received: 05 February 2014
Accepted: 13 May 2014
Published: 05 June 2014
DOI: https://doi.org/10.1038/srep05181
Springer Nature Limited

Advertisement

Ftx is dispensable for imprinted X-chromosome inactivation in preimplantation mouse embryos

Abstract

Similar content being viewed by others

Manipulation of **st Imprinting in Mouse Preimplantation Embryos

REX1 is the critical target of RNF12 in imprinted X chromosome inactivation in mice

The role of maternal-specific H3K9me3 modification in establishing imprinted X-chromosome inactivation and embryogenesis in mice

Introduction

Expression of miRNAs in Ftx-deficient blastocysts

Mating of Ftx-deficient mice

Effects of Ftx* disruption on imprinted XCI in preimplantation embryos*

Discussion

Methods

Animals

Embryo collection

5′-RACE

Construction of the Ftx-targeting vector

Generation of Ftx-deficient mice

q-PCR analysis

FISH

Polymorphic analysis

References

Acknowledgements

Author information

Authors and Affiliations

Contributions

Ethics declarations

Competing interests

Electronic supplementary material

Supplementary Information

Rights and permissions

About this article

Cite this article

Navigation

Ftx is dispensable for imprinted X-chromosome inactivation in preimplantation mouse embryos

Abstract

Similar content being viewed by others

Manipulation of **st Imprinting in Mouse Preimplantation Embryos

REX1 is the critical target of RNF12 in imprinted X chromosome inactivation in mice

The role of maternal-specific H3K9me3 modification in establishing imprinted X-chromosome inactivation and embryogenesis in mice

Introduction

Expression of miRNAs in Ftx-deficient blastocysts

Mating of Ftx-deficient mice

Effects of Ftx disruption on imprinted XCI in preimplantation embryos

Discussion

Methods

Animals

Embryo collection

5′-RACE

Construction of the Ftx-targeting vector

Generation of Ftx-deficient mice

q-PCR analysis

FISH

Polymorphic analysis

References

Acknowledgements

Author information

Authors and Affiliations

Contributions

Ethics declarations

Competing interests

Electronic supplementary material

Supplementary Information

Rights and permissions

About this article

Cite this article

Share this article

Search

Navigation

Effects of Ftx* disruption on imprinted XCI in preimplantation embryos*