| Summary: | The role of the pluripotency factor NANOG during the second embryonic lineage differentiation has been studied extensively in mouse, although species-specific differences exist. To elucidate the role of NANOG in an alternative model organism, we knocked out <i>NANOG</i> in fibroblast cells and produced bovine <i>NANOG</i>-knockout (KO) embryos via somatic cell nuclear transfer (SCNT). At day 8, <i>NANOG</i>-KO blastocysts showed a decreased total cell number when compared to controls from SCNT (NT Ctrl). The pluripotency factors OCT4 and SOX2 as well as the hypoblast (HB) marker GATA6 were co-expressed in all cells of the inner cell mass (ICM) and, in contrast to mouse <i>Nanog</i>-KO, expression of the late HB marker SOX17 was still present. We blocked the MEK-pathway with a MEK 1/2 inhibitor, and control embryos showed an increase in NANOG positive cells, but SOX17 expressing HB precursor cells were still present. <i>NANOG</i>-KO together with MEK-inhibition was lethal before blastocyst stage, similarly to findings in mouse. Supplementation of exogenous FGF4 to <i>NANOG</i>-KO embryos did not change SOX17 expression in the ICM, unlike mouse <i>Nanog</i>-KO embryos, where missing SOX17 expression was completely rescued by FGF4. We conclude that NANOG mediated FGF/MEK signaling is not required for HB formation in the bovine embryo and that another—so far unknown—pathway regulates HB differentiation.
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