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Oct4 - Pluripotent but Not Totipotent

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Alongside Nanog, Oct4 is one of the quintessential pluripotency associated transcription factors; considered to be the genetic 'master switch' in the establishment of totipotency to pluripotency during the life cycle of mammals (Pesce et al) and presumed to be the most upstream gene in the molecular circuitry of pluripotency (Jaenisch and Young).  However, in a study published in Nature Cell Biology, researchers from the laboratory of Hans R. Schöler at the Max Planck Institute for Molecular Biomedicine, Münster, Germany have now provided strong proof that this may not be true (Wu et al); they find that while Oct4 is required for pluripotency, it is not required for totipotency.

Initial work used genetically modified mice allowing the generation of oocytes which lacked Oct4A mRNA and protein, but retained all other important factors.   Crossing of female Oct4A-null mice with wild type (WT) male gave an astounding result – a normal litter; suggesting that Oct4A-null mice are fertile and therefore Oct4A is dispensable for totipotency. Further crossing of an Oct4A-null female with a male with one disrupted Oct4A allele gave maternal- and zygotic-knockout blastocysts and, while they were smaller, at E4.5 exhibited normal cavitation and formed a distinct ICM.   Interestingly, at E3.5 the ICM of these embryos did not express the trophectoderm marker Cdx2 but did express Nanog, amongst other pluripotency associated factors, at a level similar to normal ICM.   Cdx2 expression was induced at day E4.5 and was co-expressed in Nanog positive cells, which was not seen in normal embryos. A similar cross, but this time adding GFP under the control of the Oct4 regulatory regions, found that maternal- and zygotic-knockout embryos activated Oct4-GFP expression at the same time as normal embryos. These results suggest that the first lineage separation of the ICM–trophectoderm is not determined by the reciprocal interaction between Oct4 and Cdx2, but rather it is required for the maintenance of the pluripotent state of the ICM. The authors state that most importantly "maternal Oct4A is not at the root of pluripotency as a determinant in initiating the pluripotent cell lineage".

So what is? The authors show that downregulation of the orphan receptor Nr5a2 in maternal Oct4A-deficient zygotes affected the expression of Oct4-GFP. This factor is known to maintain Oct4 expression at the epiblast stage of embryonic development but plays no evident role in ESC self-renewal (Gu et al), can induce epiblast stem cells towards naive pluripotency (Guo and Smith) and replace Oct4 in iPSC production (Heng et al). Further examination of this factor may allow a better understanding of the details surrounding the establishment of totipotency and may also improve reprogramming techniques.

When the authors went on to derive ESCs from these maternal–zygotic Oct4A-knockout embryos they found that the lines they derived were homozygous for the Oct4-knockout allele, indicating loss of pluripotency in Oct4A-null embryos, and outgrowths contained condensed and fragmented Cdx2 negative nuclei along with Cdx2 positive trophectoderm cells with a small amount of cytoplasmic Nanog. Furthermore, when these embryos were used to create chimeric embryos with WT embryos, Oct4A null cells contributed only to the placental tissue of trophoblast origin and not to the embryo proper. This was again seen when crosses were made with normal ICR (Imprinting Control Region) mice and no contribution of coat colour was seen by the Oct4A null embryos, supporting a role for Oct4 as the "gatekeeper" of pluripotency (Pesce & Scholer).

Nuclear reprogramming could also be achieved using Oct4A null oocytes, again showing the recapitulation of totipotency without Oct4A.   Furthermore, ESCs derived from these cells developed to the blastocyst stage in tetraploid complementation assays with ESCs integrated in the ICM and, for some lines, allowed the generation of multiple live pups containing Oct4A-null cells in the gonads, proving that reprogramming using oocytes does not require Oct4A.

In this extensive, detailed and fascinating study, researchers have demonstrated that reprogramming towards pluripotency which requires Oct4A, and reprogramming with an oocyte towards totipotency which does not require Oct4A (by fertilisation or cloning), are fundamentally different. This also suggests that Oct4 independent mechanisms exist behind in vivo reprogramming, key to embryonic cloning. The authors hint at Nr5a2 playing a potential role and further investigation of this factor may unveil the mysteries which lie behind the reprogramming processes which occur in vivo. If we can understand this process, we may be able to apply this to in vitro reprogramming, although this may entail moral and legal questions.

References

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)

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

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)

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

Pesce, M. et al.
Binding of Sp1 and Sp3 transcription factors to the Oct-4 gene promoter.
Cell Mol. Biol. 45, 709–716 (1999).

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

 

STEM CELLS correspondent Stuart P Atkinson reports on those studies appearing in current journals that are destined to make an impact on stem cell research and clinical studies.