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Medaka Oct4 is Essential for Pluripotency in Blastula Formation and ES Cell Derivation

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Abstract

The origin and evolution of molecular mechanisms underlying cellular pluripotency is a fundamental question in stem cell biology. The transcription factor Oct4 or Pou5f1 identified in mouse features pluripotency expression and activity in the inner cell mass and embryonic stem (ES) cells. Pou2 identified in zebrafish is the non-mammalian homolog prototype of mouse Oct4. The genes oct4 and pou2 have reportedly evolved by pou5 gene duplication in the common ancestor of vertebrates. Unlike mouse oct4, however, zebrafish pou2 lacks pluripotency expression and activity. Whether the presence of pluripotency expression and activity is specific for mammalian Oct4 or common to the ancestor of vertebrate Oct4 and Pou2 proteins has remained to be determined. Here we report that Oloct4, the medaka oct4/pou2, is essential for early embryogenesis and pluripotency maintenance. Oloct4 exists as a single copy gene and is orthologous to pou2 by sequence and chromosome synteny. Oloct4 expression occurs in early embryos, germ stem cells and ES cells like mouse oct4 but also in the brain and tail bud like zebrafish pou2. Importantly, OlOct4 depletion caused blastula lethality or blockage. We show that Oloct4 depletion abolishes ES cell derivation from midblastula embryos. Thus, Oloct4 has pluripotency expression and is essential for early embryogenesis and pluripotency maintenance. Our results demonstrate the conservation of pluripotency expression and activity in vertebrate Oct4 and Pou2 proteins. The finding that Oloct4 combines the features of mouse oct4 and zebrafish pou2 in expression and function suggests that Oloct4 might represent the ancestral prototype of vertebrate oct4 and pou2 genes.

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Abbreviations

Dpc or hpc:

Day (s) or hour (s) post culture

Dpf or hpf:

Day (s) or hour (s) post fertilization

ES:

Embryonic stem

FISH:

Fluorescence in situ hybridization

IF:

Immunofluorescence

MBE:

Midblastula embryo

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Acknowledgments

We thank J. Deng for fish breeding, Choy Mei Foong and Veronica Wong for laboratory management. This work was supported by the National Research Foundation Singapore (NRF-CRP7-2010-03).

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No competing financial interests exist.

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Authors and Affiliations

  1. Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore, 117543, Singapore

    Rong Liu, Mingyou Li, Zhendong Li, Ni Hong, Hongyan Xu & Yunhan Hong

  2. College of Fisheries and Life Science, Shanghai Ocean University, 999 Hucheng Huan Road, Shanghai,, 201306, People’s Republic of China

    Mingyou Li & Yunhan Hong

  3. Genetics of Development and Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 10 Center Drive, Bethesda, MD, 20892,, USA

    Ni Hong

  4. Key Laboratory of Tropical & Subtropical fishery Resource application & cultivation, Ministry of Agriculture, Pearl River Fishery Research Science, Chinese Academic of Fishery Science, Guangzhou, 510380, People’s Republic of China

    Hongyan Xu

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  1. Rong Liu
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Correspondence to Yunhan Hong.

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Fig. S1

Cloning and sequence of Oloct4. (A) Cloning strategy and steps. arrowheads, positions and directions of primers. (B) Sequence. Start codon and stop codon are shown in bold. Primer sequences are underlined with arrows depicting their directions. Amino acid sequences for degenerate primers Pou5 and Pou3 are framed. POUs, POU-specific domain; POUh, POU homeodomain. Primers PR53 and Psb were used for RT-PCR, while PR51 and Psb define the sequence of DNA and RNA probes used in Southern and in situ hybridization. (TIFF 1260 kb)

High Resolution (GIF 286 kb)

Fig.S2

Amino acid sequence alignment. Two POU subdomains and the linker between them are defined by arrowheads. The antigenic sequence within the linker used for the production of a polyclonal antibody (αOct4) is underlined. At the end of the alignment are species common names, percentage identity values for full length sequences (full) and partial sequences spanning POU domains (POU). Accession numbers are AY639946.1 for Ol (Oryzias latipes, medaka), NP_571187.1 for Dr (Danio rerio, zebrafish), NP_001081342.1 for Xl (Xenopus laevis), AAT09163.1 for Am (Ambystoma mexicanum, axolotl), ABK27428.1 for Gg (Gallus gallus, chicken), NP_038661 for Mm (Mus musculus, mouse) and CAA77951 for Hs (Homo sapiens, human). (TIFF 940 kb)

High Resolution (GIF 111 kb)

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Liu, R., Li, M., Li, Z. et al. Medaka Oct4 is Essential for Pluripotency in Blastula Formation and ES Cell Derivation. Stem Cell Rev and Rep 11, 11–23 (2015). https://doi.org/10.1007/s12015-014-9523-2

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