Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Establishment of totipotency does not depend on Oct4A

Nature Cell Biology volume 15, pages 1089–1097 (2013)Cite this article

Subjects

Abstract

Oct4A is a core component of the regulatory network of pluripotent cells, and by itself can reprogram neural stem cells into pluripotent cells in mice and humans. However, its role in defining totipotency and inducing pluripotency during embryonic development is still unclear. We genetically eliminated maternal Oct4A using a Cre/loxP approach in mouse and found that the establishment of totipotency was not affected, as shown by the generation of live pups. After complete inactivation of both maternal and zygotic Oct4A expression, the embryos still formed Oct4–GFP- and Nanog-expressing inner cell masses, albeit non-pluripotent, indicating that Oct4A is not a determinant for the pluripotent cell lineage separation. Interestingly, Oct4A-deficient oocytes were able to reprogram fibroblasts into pluripotent cells. Our results clearly demonstrate that, in contrast to its role in the maintenance of pluripotency, maternal Oct4A is not crucial for either the establishment of totipotency in embryos, or the induction of pluripotency in somatic cells using oocytes.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Generation of oocytes lacking maternal Oct4A and their effect on embryo development.
Figure 2: Expression of Oct4 isoforms in Oct4A-knockout oocytes and embryos.
Figure 3: Gene expression of Oct4A-null blastocysts produced by crossing Oct4flox/flox–Zp3Cre/+ female mice with Oct4A+/Δ male mice.
Figure 4: Fate of Oct4A-null embryos in vitro and in vivo.
Figure 5: Oct4A-null oocytes reprogram somatic cells to full pluripotency as shown by generation of complete NT-ESC-derived mice through tetraploid complementation.

Similar content being viewed by others

References

  1. Mitalipov, S. & Wolf, D. Totipotency, pluripotency and nuclear reprogramming. Adv. Biochem. Eng. Biotechnol. 114, 185–199 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Scholer, H. R., Hatzopoulos, A. K., Balling, R., Suzuki, N. & Gruss, P. A family of octamer-specific proteins present during mouse embryogenesis: evidence for germline-specific expression of an Oct factor. EMBO J. 8, 2543–2550 (1989).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Pesce, M. & Scholer, H. R. Oct-4: gatekeeper in the beginnings of mammalian development. Stem Cells 19, 271–278 (2001).

    Article  CAS  PubMed  Google Scholar 

  4. Nichols, J. et al. Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4. Cell 95, 379–391 (1998).

    Article  CAS  PubMed  Google Scholar 

  5. Okumura-Nakanishi, S., Saito, M., Niwa, H. & Ishikawa, F. Oct-3/4 and Sox2 regulate Oct-3/4 gene in embryonic stem cells. J. Biol. Chem. 280, 5307–5317 (2005).

    Article  CAS  PubMed  Google Scholar 

  6. Li, L., Zheng, P. & Dean, J. Maternal control of early mouse development. Development 137, 859–870 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Chen, X. et al. Integration of external signaling pathways with the core transcriptional network in embryonic stem cells. Cell 133, 1106–1117 (2008).

    Article  CAS  PubMed  Google Scholar 

  8. Rodda, D. J. et al. Transcriptional regulation of nanog by OCT4 and SOX2. J. Biol. Chem. 280, 24731–24737 (2005).

    Article  CAS  PubMed  Google Scholar 

  9. Masui, S. et al. Pluripotency governed by Sox2 via regulation of Oct3/4 expression in mouse embryonic stem cells. Nat. Cell Biol. 9, 625–635 (2007).

    Article  CAS  PubMed  Google Scholar 

  10. Pesce, M., Marin Gomez, M., Philipsen, S. & Scholer, H. R. Binding of Sp1 and Sp3 transcription factors to the Oct-4 gene promoter. Cell Mol. Biol. 45, 709–716 (1999).

    CAS  PubMed  Google Scholar 

  11. Jaenisch, R. & Young, R. Stem cells, the molecular circuitry of pluripotency and nuclear reprogramming. Cell 132, 567–582 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Kehler, J. et al. Oct4 is required for primordial germ cell survival. EMBO Rep. 5, 1078–1083 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Lengner, C. J. et al. Oct4 expression is not required for mouse somatic stem cell self-renewal. Cell Stem Cell 1, 403–415 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Lee, J., Kim, H. K., Rho, J. Y., Han, Y. M. & Kim, J. The human OCT-4 isoformsdiffer in their ability to confer self-renewal. J. Biol. Chem. 281, 33554–33565 (2006).

    Article  CAS  PubMed  Google Scholar 

  15. Guo, C. L. et al. A novel variant of Oct3/4 gene in mouse embryonic stem cells. Stem Cell Res. 9, 69–76 (2012).

    Article  CAS  PubMed  Google Scholar 

  16. Farashahi Yazd, E. et al. OCT4B1, a novel spliced variant of OCT4, generates a stable truncated protein with a potential role in stress response. Cancer Lett. 309, 170–175 (2011).

    Article  PubMed  Google Scholar 

  17. Asadi, M. H. et al. OCT4B1, a novel spliced variant of OCT4, is highly expressed in gastric cancer and acts as an antiapoptotic factor. Int. J. Cancer 128, 2645–2652 (2011).

    Article  CAS  PubMed  Google Scholar 

  18. Zuccotti, M. et al. Maternal Oct-4 is a potential key regulator of the developmental competence of mouse oocytes. BMC Dev. Biol. 8, 97–110 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  19. Foygel, K. et al. A novel and critical role for Oct4 as a regulator of the maternal-embryonic transition. Plos One 3, e4109 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  20. Chambers, I. et al. Nanog safeguards pluripotency and mediates germline development. Nature 450, 1230–1234 (2007).

    Article  CAS  PubMed  Google Scholar 

  21. Yoshimizu, T. et al. Germline-specific expression of the Oct-4/green fluorescent protein (GFP) transgene in mice. Dev. Growth Differ. 41, 675–684 (1999).

    Article  CAS  PubMed  Google Scholar 

  22. Niwa, H., Miyazaki, J. & Smith, A. G. Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat. Genet. 24, 372–376 (2000).

    Article  CAS  PubMed  Google Scholar 

  23. Niwa, H. et al. Interaction between Oct3/4 and Cdx2 determines trophectoderm differentiation. Cell 123, 917–929 (2005).

    Article  CAS  PubMed  Google Scholar 

  24. Yuan, P. et al. Eset partners with Oct4 to restrict extraembryonic trophoblast lineage potential in embryonic stem cells. Genes Dev. 23, 2507–2520 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Dietrich, J. E. & Hiiragi, T. Stochastic patterning in the mouse pre-implantation embryo. Development 134, 4219–4231 (2007).

    Article  CAS  PubMed  Google Scholar 

  26. Lu, C. W. et al. Ras-MAPK signaling promotes trophectoderm formation from embryonic stem cells and mouse embryos. Nat. Genet. 40, 921–926 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Blij, S., Frum, T., Akyol, A., Fearon, E. & Ralston, A. Maternal Cdx2 is dispensable for mouse development. Development 139, 3969–3972 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Strumpf, D. et al. Cdx2 is required for correct cell fate specification and differentiation of trophectoderm in the mouse blastocyst. Development 132, 2093–2102 (2005).

    Article  CAS  PubMed  Google Scholar 

  29. Wu, G. et al. Initiation of trophectoderm lineage specification in mouse embryos is independent of Cdx2. Development 137, 4159–4169 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Ralston, A. et al. Gata3 regulates trophoblast development downstream of Tead4 and in parallel to Cdx2. Development 137, 395–403 (2010).

    Article  CAS  PubMed  Google Scholar 

  31. Kuroda, T. et al. Octamer and Sox elements are required for transcriptional cis regulation of Nanog gene expression. Mol. Cell Biol. 25, 2475–2485 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Szabo, P. E., Hubner, K., Scholer, H. & Mann, J. R. Allele-specific expression of imprinted genes in mouse migratory primordial germ cells. Mech. Dev. 115, 157–160 (2002).

    Article  CAS  PubMed  Google Scholar 

  33. Adachi, K. & Scholer, H. R. Directing reprogramming to pluripotency by transcription factors. Curr. Opin. Genet. Dev. 22, 1–7 (2012).

    Article  Google Scholar 

  34. Heng, J. C. et al. The nuclear receptor Nr5a2 can replace Oct4 in the reprogramming of murine somatic cells to pluripotent cells. Cell Stem Cell 6, 167–174 (2010).

    Article  CAS  PubMed  Google Scholar 

  35. Takahashi, K. & Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–676 (2006).

    Article  CAS  PubMed  Google Scholar 

  36. Gu, P. et al. Orphan nuclear receptor LRH-1 is required to maintain Oct4 expression at the epiblast stage of embryonic development. Mol. Cell Biol. 25, 3492–3505 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Guo, G. & Smith, A. A genome-wide screen in EpiSCs identifies Nr5a nuclear receptors as potent inducers of ground state pluripotency. Development 137, 3185–3192 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Chapman, D. L. et al. Expression of the T-box family genes, Tbx1-Tbx5, during early mouse development. Dev. Dyn. 206, 379–390 (1996).

    Article  CAS  PubMed  Google Scholar 

  39. Yagi, H. et al. Role of TBX1 in human del22q11.2 syndrome. Lancet 362, 1366–1373 (2003).

    Article  CAS  PubMed  Google Scholar 

  40. Campbell, K. H., McWhir, J., Ritchie, W. A. & Wilmut, I. Sheep cloned by nuclear transfer from a cultured cell line. Nature 380, 64–66 (1996).

    Article  CAS  PubMed  Google Scholar 

  41. De Vries, W. N. et al. Expression of Cre recombinase in mouse oocytes: a means to study maternal effect genes. Genesis 26, 110–112 (2000).

    Article  CAS  PubMed  Google Scholar 

  42. Lan, Z. J., Xu, X. & Cooney, A. J. Differential oocyte-specific expression of Cre recombinase activity in GDF-9-iCre, Zp3cre, and Msx2Cre transgenic mice. Biol. Reprod. 71, 1469–1474 (2004).

    Article  CAS  PubMed  Google Scholar 

  43. El-Hashemite, N., Wells, D. & Delhanty, J. D. Single cell detection of beta-thalassaemia mutations using silver stained SSCP analysis: an application for preimplantation diagnosis. Mol. Hum. Reprod. 3, 693–698 (1997).

    Article  CAS  PubMed  Google Scholar 

  44. Hogan, B. Manipulating The Mouse Embryo : A Laboratory Manual 2nd edn (Cold Spring Harbor Laboratory Press, 1994).

    Google Scholar 

  45. Palmieri, S. L., Peter, W., Hess,, H. & Scholer, H. R. Oct-4 transcription factor is differentially expressed in the mouse embryo during establishment of the first two extraembryonic cell lineages involved in implantation. Dev. Biol. 166, 259–267 (1994).

    Article  CAS  PubMed  Google Scholar 

  46. Boiani, M. et al. Variable reprogramming of the pluripotent stem cell marker Oct4 in mouse clones: distinct developmental potentials in different culture environments. Stem Cells 23, 1089–1104 (2005).

    Article  CAS  PubMed  Google Scholar 

  47. Ho, Y., Wigglesworth, K., Eppig, J. J. & Schultz, R. M. Preimplantation development of mouse embryos in KSOM: augmentation by amino acids and analysis of gene expression. Mol. Reprod. Dev. 41, 232–238 (1995).

    Article  CAS  PubMed  Google Scholar 

  48. Nagy, A. Manipulating The Mouse Embryo : A Laboratory Manual 3rd edn (Cold Spring Harbor Laboratory Press, 2003).

    Google Scholar 

  49. Long, J. Z., Lackan, C. S. & Hadjantonakis, A. K. Genetic and spectrally distinct in vivo imaging: embryonic stem cells and mice with widespread expression of a monomeric red fluorescent protein. BMC Biotechnol. 5, 20–30 (2005).

    Article  PubMed  PubMed Central  Google Scholar 

  50. Tachibana, M. et al. Mitochondrial gene replacement in primate offspring and embryonic stem cells. Nature 461, 367–372 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Bryja, V., Bonilla, S. & Arenas, E. Derivation of mouse embryonic stem cells. Nat. Protoc. 1, 2082–2087 (2006).

    Article  CAS  PubMed  Google Scholar 

  52. Kim, J. B. et al. Pluripotent stem cells induced from adult neural stem cells by reprogramming with two factors. Nature 454, 646–650 (2008).

    Article  CAS  PubMed  Google Scholar 

  53. Wu, G. et al. Efficient derivation of pluripotent stem cells from siRNA-mediated Cdx2-deficient mouse embryos. Stem Cells Dev. 20, 485–493 (2011).

    Article  CAS  PubMed  Google Scholar 

  54. Wu, G. et al. Generation of healthy mice from gene-corrected disease-specific induced pluripotent stem cells. PLoS Biol. 9, e1001099 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank J. Mueller-Keuker, M. Preusser and N. Stengel for assistance in preparing the manuscript and A. Malapetsas for proofreading the manuscript. We thank K. Huebner for her technical help on immunocytochemistry and B. Scháfer for her assistance on histology work. The authors of this manuscript bear sole responsibility for the content presented, which does not necessarily represent the official views of the Eunice Kennedy Shriver National Institute of Child Health & Human Development or the National Institutes of Health. This research was supported by the Max Planck Society, DFG grants DFG SI 1695/1-2 (SPP1356) and SCHO 340/7-1, and grant NIH R01HD059946-01 from the Eunice Kennedy Shriver National Institute of Child Health & Human Development.

Author information

Author notes

  1. Vittorio Sebastiano & Nishant Singhal

    Present address: Present addresses: Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, 1050 Arastradero Road, Palo Alto, California 94304, USA (V.S.); Department of Neurosciences, University of California San Diego, 9500 Gilman Drive, La Jolla, California 92093, USA (N.S.),

Authors and Affiliations

  1. Department of Cell and Developmental Biology, Max Planck Institute for Molecular Biomedicine, Röntgenstrasse 20, 48149 Münster, Germany

    Guangming Wu, Dong Han, Yu Gong, Vittorio Sebastiano, Luca Gentile, Nishant Singhal, Kenjiro Adachi, Gerrit Fischedick, Claudia Ortmeier, Martina Sinn, Martina Radstaak & Hans R. Schöler

  2. Russian Academy of Science, Institute of Cytology, 4 Tikhoretski Avenue, 194064, St Petersburg, Russia

    Alexey Tomilin

  3. University of Münster, Medical Faculty, Domagkstr. 3, 48149 Münster, Germany

    Hans R. Schöler

Authors
  1. Guangming Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Dong Han
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yu Gong
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Vittorio Sebastiano
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Luca Gentile
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Nishant Singhal
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Kenjiro Adachi
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Gerrit Fischedick
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Claudia Ortmeier
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Martina Sinn
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Martina Radstaak
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Alexey Tomilin
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Hans R. Schöler
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

G.W. designed and executed experiments as well as writing the manuscript. D.H., Y.G., V.S., L.G., N.S., K.A., G.F., C.O., M.S., M.R. and A.T. executed experiments, collected data and prepared reagents. H.R.S. provided the study concept and funding, and edited the manuscript.

Corresponding author

Correspondence to Hans R. Schöler.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Elimination of maternal Oct4A did not show any effect on the oocyte’s developmental competence.

(a) Schematic representation of the Oct4A targeting DNA construct and position of the genotyping primer sets modified from Kehler et al. (2004). Filled arrowheads: loxP sites; Oval box: Oct4 promoter; filled rectangles: Oct4 exons 1–5. (b) Mating strategy to generate maternal Oct4A-null oocytes. The Oct4flox/flox mice were mated with ZP3Cre/Cre mice to produce offspring with the Oct4flox/+/ZP3Cre/+ genotype. Then the Oct4flox/+/ZP3Cre/+ male mice were backcrossed with Oct4flox/flox female mice to obtain Oct4flox/flox/ZP3Cre/+ mice. The male mice of this genotype were backcrossed with Oct4flox/flox mice again to obtain Oct4flox/flox/ZP3Cre/+ female mice that would produce Oct4A- null oocytes. (c)The tails of offspring were cut and genotyped with primer pair B to detect for the presence of the Oct4 floxed allele (449 bp), Wt allele (415 bp), and a primer pair for Cre (373 bp). The last lane was used as negative control without adding any DNA. (d) Validation of elimination of maternal Oct4A at the protein level by Western blot analysis. Oocyte samples comprised extracts from 400 or more germinal vesicle oocytes. A monclonal Oct4A antibody detected a weak band of Oct4 protein in wild-type oocytes (WT) and very strong band in the ES cell sample (ES) of approximately 45 kDa, but not in the Oct4-knockout oocytes (KO) and cumulus cells (CM). (e) Gene expression of single oocytes at the germinal vesicle stage, analyzed by Fluidigm qRT–PCR using the Biomark 48.48 Dynamic Array system (Fluidigm) further confirmed the elimination of the Oct4A transcript without a significant impact on the expression of oocyte- and lineage-specific genes examined. The number (1, 2, 3, or 4) right after the abbreviation (Ctr, for wild-type control, or KO, for knockout) refers to the biological replicates. (f) Oct4A-knockout oocytes can support establishment of totipotency, which is necessary for full-term development, as shown by the normal litter size from the crossing of Oct4flox/flox/ZP3Cre/+ female mice with CD1 wild-type male mice. (g) PCR genotyping of the offspring from the above crossing confirmed that the Oct4A allele had been deleted. The bars represent the means from 3 technical replicates, a result representative of each biological replicate in e. The uncropped version of d is shown in Supplementary Fig. S5.

Supplementary Figure 2 Phenotype of Oct4A-null embryos.

(a) Genotyping with nested PCR on single biopsied blastomeres from embryos obtained by crossing Oct4flox/flox/ZP3Cre/+ female mice with Oct4+/− male mice. (b) Quantitative RT–PCR on single genotyped eight-cell embryos with triplicates shows that Oct4 elimination does not delay activation of Nanog gene transcription. Ctr: Oct4A+/Δ, KO, maternal and zygotic Oct4A-knockout. (c) Immunocytochemistry of E2.5 embryos for Nanog (green) and Oct4 (red), M-Z KO, maternal and zygotic Oct4A knockout. (d) Immunocytochemistry of E3.5 blastocysts for Troma-1 (red), another trophectoderm marker, and Oct4 (green) localized the protein to the trophectoderm, which further confirmed the lineage separation of ICM/trophectoderm in Oct4A-null embryos. (e) Average cell numbers of Nanog- and Cdx2-positive cells per E4.5 embryo were counted on confocal immages of immnostained embryos. KO, Oct4A-knockout; M-Z KO, maternal and zygotic Oct4A knockout. The scale bars represent 25 μm in c and d. Value represents mean±S.D. of 3 biological replicates in b and mean±s.d. of 61 and 41 embryo samples for wildtype and Oct4A KO, respectively in e.

Supplementary Figure 3 Oct4-GFP expression is activated in Oct4A-null embryos.

(a) At the end of the time-lapse observation on Oct4–GFP expression, each individual embryo was marked by a number with its genotype (maternal/zygotic) as determined by c. (b) Generated by crossing Oct4flox/flox/ZP3Cre/+ female mice with OG2-GFP+/−–Oct4+/Δ male mice, GFP-expressing E4.5 embryos were selected as shown. Genotyping of these embryos revealed that half (17/36) were maternal/zygotic knockout and suggested that OG2–GFP was still activated in Oct4A-null embryos. (c) Genotype was determined by nested PCR with corresponding numbers to a. (d) Oct4-GFP expression was not affected in E2.5 (left) and E3.5 (right) embryos following injection of siSall4 into zygotes obtained by crossing Oct4flox/flox/ZP3Cre/+ female mice with GOF18-GFPmale mice. Embryos without the Oct4-GFP transgene were used as negative control and siRNA targeting GFP (siGFP) was used as positive control. Fluorescence intensity was quantified by ImageJ software. (e) Efficient knockdown of Tpt1, Zscan4, Esrrb and Utf1 at the mRNA levelsby injection of siRNA duplexes as assessed by real-time RT–PCR. No significant effect on Oct4 expression was observed. Expression levels of Hprt1 were used as the internal control to normalize the data and siGFP-injected embryos were used as calibrators. Scale bars represent 50 μm in a and b. The error bars represent mean±s.e.m. of 8–16 biological replicates in d and mean±s.d. of 3 biological replicates in e. The uncropped version of c is shown in Supplementary Fig. S5.

Supplementary Figure 4 Oct4A-null oocytes reprogrammed somatic cell nuclei to pluripotent status.

(a) Immunocytochemistry of E4.0-NT blastocysts show activation of Nanog and Oct4 expression by Oct4A-knockout (Oct4A KO) oocytes. WT, wild type; NT: Nuclear transferred; PA: parthenogenic. (b) Gene expression profiling of NT blastocysts revealed activation of expression of the pluripotent genes Oct4 and Nanog without maternal Oct4A expression. The gene expression levels were obtained with pools of 3 blastocysts with triplicates and presented in comparison with ES cells (ES). Ctr: NT embryos using wild-type oocytes; KO, NT embryos using Oct4A-knockout oocytes; PA: parthenogenic embryos using Oct4-knockout oocytes. The number (1, 2 or 3) right after the abbreviation (Ctr and KO) refers to the biological replicates. (c) Morphology of NT-ES cells grown on MEFs expressing CAG–mRFP and Oct4-GFP.(d) Histology of teratoma from NT-ES cell line RG6 4 weeks after injection into SCID mice as assessed by haematoxylin and eosin staining. The teratoma contained cells of all 3 embryonic germ layers. Upper left panel: keratinized stratified squamous epithelial cells (ectodermal); upper right panel: neural rosettes (ectodermal); lower left panel: striated muscle (mesodermal); lower right: ciliated columnar epithelial cells adjacent to pancreatic acinar cells (both endodermal). (e) A litter of neonatal NT-ES cell-derived pups delivered by cesarean section on E19.0. In this particular litter, 8 pups showed normal full-term development, of which one was dead and one failed to initiate breathing (*). The scale bars represent 30 μm in a, 100 μm inc and 50 μm in d. Values represent mean±S.D. of 3 biological replicates.

Supplementary Figure 5 Uncropped figures for Figs 1a,2c,2e,4b and Supplementary Fig. 1d and 3c.

Supplementary Table 1 Sequencing results of RT-PCR amplicon using primers spanning exon 2 and exon 3 of the Oct4 gene in Oct4A-null oocytes and embryos.
Full size table
Supplementary Table 2 Sequencing results of RT-PCR amplicon using primers spanning exon 3 and exon 4 of Oct4 gene in Oct4A-null oocytes and embryos.
Full size table
Supplementary Table 3 Primers for gene expression study.
Full size table
Supplementary Table 4 siRNA target sequences
Full size table

Supplementary information

Supplementary Information

Supplementary Information (PDF 806 kb)

Supplementary Tables 1–4

Supplementary Information (XLS 50 kb)

Supplementary Table 5

Supplementary Information (XLS 1444 kb)

Time-lapse recording of in vitro development of Oct4A-null 8-cell embryo.

A biopsied and genotyped morula with maternal and zygotic Oct4A-null was cultured on MEFs in ESC medium and observed on the stage of a microscope with an incubation chamber (TOKAI HIT, Japan) filled with 5% CO2 in air and maintained at 37 °C. Brightfield pictures were taken every 5 min for 4 days and were compiled into a movie with 24 frames per second. The video demonstrates that Oct4A-null embryos initiated cavitation and formed grossly normal-looking blastocysts with distinct ICM. However, immunostaining of the outgrowth (Fig. 3a) showed cytoplasmic localization of Nanog as well as fragmentation of nuclei. (MOV 7661 kb)

Time-lapse confocal recording revealed activation of Oct4-GFP expression in maternal-knockout and maternal/zygotic-knockout embryos.

Twelve 2-cell embryos from the mating of Oct4flox/flox/ZP3Cre/+ female mice with Oct4A+/Δ/Oct4−GFP+/+ male mice and 4 embryos (#1, 3, 4 and 8) from the mating of Oct4flox/flox female mice with Oct4A+/Δ/Oct4−GFP+/+ male mice were placed in KSOMAA in a glass bottom dish with the same condition as Supplementary Video 1 for confocal examination with 488 nm laser. A confocal picture had been taken every 10 min for 3 days and was compiled into a movie with 24 frames per second. The video demonstrated that regardless of the genotype, all embryos activated Oct4-GFP at around E2.5in a timely fashion, as did wild-type embryos. The genotype of each embryo is shown in Fig. S3a. (MOV 3066 kb)

Brightfield time-lapse recording of the same embryos at the same time point as Supplementary Video 2.

This video was used to monitor the developmental stage of the embryos and to trace the position of individual embryos for genotype determination. (MOV 3047 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Wu, G., Han, D., Gong, Y. et al. Establishment of totipotency does not depend on Oct4A. Nat Cell Biol 15, 1089–1097 (2013). https://doi.org/10.1038/ncb2816

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ncb2816

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing