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.

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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.

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Author notes

    • Vittorio Sebastiano
    •  & Nishant Singhal

    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.)

Affiliations

  1. Max Planck Institute for Molecular Biomedicine, Department of Cell and Developmental Biology, 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

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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.

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Correspondence to Hans R. Schöler.

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Videos

  1. 1.

    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.

  2. 2.

    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/Oct4GFP+/+ 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.

  3. 3.

    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.

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DOI

https://doi.org/10.1038/ncb2816

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