Hypoblast Formation in Bovine Embryos Does Not Depend on NANOG

Abstract

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 NANOG in fibroblast cells and produced bovine NANOG-knockout (KO) embryos via somatic cell nuclear transfer (SCNT). At day 8, NANOG-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 Nanog-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. NANOG-KO together with MEK-inhibition was lethal before blastocyst stage, similarly to findings in mouse. Supplementation of exogenous FGF4 to NANOG-KO embryos did not change SOX17 expression in the ICM, unlike mouse Nanog-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.

Pluripotent Core in Bovine Embryos: A Review

“…This is consistent with studies showing that isolated trophoblast cells did not require an active FGF/MEK signaling to survive, proliferate, and maintain CDX2 expression [61,[63][64][65]. In agreement with Kuijk et al [28], under MEK inhibition (PD0325901 0.5 and 2.5  M) the embryonic development or cell numbers were not affected but the proportion of NANOG positive cells was markedly increased, while the expression of GATA6 was reduced but not completely switched off [33].…”

“…Thus, disruption of NANOG gene did not affect blastocyst rate but resulted in reduced total cell number [33] and an ICM composed mostly of hypoblast cells [34]. In the nascent epiblast, NANOG mediated repression of hypoblast markers, such as SOX17, which is dependent on MEK signaling, but FGF4-induced expression of SOX17 depends on NANOG, therefore the establishment of hypoblast lineage depends on epiblast mediated FGF/MEK signaling [33]. In relation to other markers, absence of NANOG resulted in lower expression of the epiblast cell marker SOX2, and hypoblast marker GATA6; without affecting the trophectoderm [34].…”

“…Functionally, NANOG is not required for proper segregation of TE and ICM, but it is required to derive and maintain the pluripotent epiblast and during the second lineage commitment [33,34]. Moreover, it seems to be implicated in cell proliferation, probably depending on FGF4 signaling (also involved in fate decision and patterning events in the early embryo) from EPI precursor cells [33]. Thus, disruption of NANOG gene did not affect blastocyst rate but resulted in reduced total cell number [33] and an ICM composed mostly of hypoblast cells [34].…”

“…Moreover, it seems to be implicated in cell proliferation, probably depending on FGF4 signaling (also involved in fate decision and patterning events in the early embryo) from EPI precursor cells [33]. Thus, disruption of NANOG gene did not affect blastocyst rate but resulted in reduced total cell number [33] and an ICM composed mostly of hypoblast cells [34]. In the nascent epiblast, NANOG mediated repression of hypoblast markers, such as SOX17, which is dependent on MEK signaling, but FGF4-induced expression of SOX17 depends on NANOG, therefore the establishment of hypoblast lineage depends on epiblast mediated FGF/MEK signaling [33].…”

OCT4/POU5F1 is indispensable for the lineage differentiation of the inner cell mass in bovine embryos

“…Although there was no reduction in expression of the early HB marker GATA6, expression of SOX17 failed in the absence of OCT4. As expression of SOX17 is not dependent on NANOG, 32 loss of SOX17 can be linked directly to the OCT4 KO phenotype. In mouse embryos, Oct4 KO prevents the differentiation of the PE, not only because FGF4‐MEK signaling is reduced 33 but also because OCT4 is required cell autonomously 7,8 …”