Hypoblast Formation in Bovine Embryos Does Not Depend on NANOG.

Claudia Springer,Valeri Zakhartchenko,Kilian Simmet,Eckhard Wolf

doi:10.3390/cells10092232

Claudia Springer, Valeri Zakhartchenko + Show 2 more

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https://doi.org/10.3390/cells10092232

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Journal: Cells	Publication Date: Aug 28, 2021
Citations: 11	License type: CC BY 4.0

Affiliation: Ludwig-Maximilians-Universität München

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.

Highlights

IntroductionMammalian embryos undergo two consecutive lineage specifications
Before implantation, mammalian embryos undergo two consecutive lineage specifications
The percentage of SOX17 positive cells in the inner cell mass (ICM) did not increase in NANOG-KO embryos (Figure 3E), which is in contrast to mouse Nanog-KO embryos, where exogenous FGF4 induces SOX17 expression in most of the ICM cells [2]

Summary

Introduction

Mammalian embryos undergo two consecutive lineage specifications. Three distinct cell lineages arise: the EPI, which will give rise to the embryo proper, the PE/HB, which will form the yolk sac, and the TE, responsible for extraembryonic tissues and implantation [1,2,3,4,5,6,7,8], reviewed in [9]. While these landmarks of preimplantation embryonic development are conserved between mammalian species, fundamental differences exist regarding the regulation of the second lineage segregation. FGF4 signaling via the mitogen-activated protein kinase (MAPK)

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