The roles of FGF and MAP kinase signaling in the segregation of the epiblast and hypoblast cell lineages in bovine and human embryos

Abstract

At the blastocyst stage of mammalian pre-implantation development, three distinct cell lineages have formed: trophectoderm, hypoblast (primitive endoderm) and epiblast. The inability to derive embryonic stem (ES) cell lines in a variety of species suggests divergence between species in the cell signaling pathways involved in early lineage specification. In mouse, segregation of the primitive endoderm lineage from the pluripotent epiblast lineage depends on FGF/MAP kinase signaling, but it is unknown whether this is conserved between species. Here we examined segregation of the hypoblast and epiblast lineages in bovine and human embryos through modulation of FGF/MAP kinase signaling pathways in cultured embryos. Bovine embryos stimulated with FGF4 and heparin form inner cell masses (ICMs) composed entirely of hypoblast cells and no epiblast cells. Inhibition of MEK in bovine embryos results in ICMs with increased epiblast precursors and decreased hypoblast precursors. The hypoblast precursor population was not fully ablated upon MEK inhibition, indicating that other factors are involved in hypoblast differentiation. Surprisingly, inhibition of FGF signaling upstream of MEK had no effects on epiblast and hypoblast precursor numbers in bovine development, suggesting that GATA6 expression is not dependent on FGF signaling. By contrast, in human embryos, inhibition of MEK did not significantly alter epiblast or hypoblast precursor numbers despite the ability of the MEK inhibitor to potently inhibit ERK phosphorylation in human ES cells. These findings demonstrate intrinsic differences in early mammalian development in the role of the FGF/MAP kinase signaling pathways in governing hypoblast versus epiblast lineage choices.

Fig. 1.

Expression of NANOG, GATA6 and GATA4 in day 5-8 bovine embryos. (A) Immunofluorescent detection of NANOG and GATA6 in bovine pre-implantation embryos. Each image is a single optical section. NANOG and GATA6 expression is shown at consecutive stages in bovine development. The bottom panels depict a day 8 embryo in which GATA6-positive cells line the blastocoelic surface of the inner cell mass (ICM) and enclose the NANOG-positive population, which is indicative of physical segregation of the epiblast and hypoblast cell lineages. (B) The proportion of GATA6-positive cells as a function of the total number of cells. At day 8, GATA6 expression is localized to the ICM. (C) Expression of GATA4 in a day 8 bovine embryo. GATA4-expressing cells are present throughout the embryo. Scale bars: 50 μm.

Fig. 2.

Effect of MEK inhibition on NANOG and GATA6 expression in bovine embryos. (A) NANOG and GATA6 expression in bovine day 8 embryos that were cultured from the zygote stage to the blastocyst stage in the presence of the MEK inhibitor PD98059. All images are maximum image projections of z-stacks. (B) Box-whisker plot of the quantified proportion of NANOG-positive (green) and GATA6-positive (red) cells. (C) Maximum image projection of NANOG and GATA6 expression in bovine day 8 embryos that were cultured from the zygote stage (day 1) to the blastocyst stage (day 8) in the presence of the MEK inhibitor PD0325901. (D) Box-whisker plot of the quantified proportion of NANOG-positive (green) and GATA6-positive (red) cells. (E) Box-whisker plots of the numbers of NANOG-positive cells (green), GATA6-positive cells (red), ICM cells, and NANOG/GATA6 double-positive cells in bovine embryos cultured in the presence of MEK inhibitor added at different time points to the embryo culture. In the 1-5 and 3-5 conditions, the MEK inhibitor was washed away and embryos were cultured under regular embryo culture conditions from day 5 onwards. In all experiments, equal concentrations of solvent (DMSO) served as negative controls. Asterisks denote significant differences between the experimental and control groups: ^*, P<0.05; ^**, P<0.01; ^***, P<0.001. Error bars comprise the whiskers that extend to the maximum and minimum value data sets. Scale bars: 50 μm.

Fig. 3.

Effect of activated FGF signaling on NANOG and GATA6 expression in bovine embryos. (A) Immunofluorescence images for NANOG and GATA6 in bovine embryos that were cultured from the zygote stage (day 1) to the blastocyst stage (day 8) in the presence of FGF4 and heparin. (B) Box-whisker plot of the quantified proportion of NANOG-positive (green) and GATA6-positive (red) cells in the ICM. (C) Box-whisker plot of the total number of ICM cells in embryos that were cultured in FGF4 plus heparin or control conditions from day 5 to day 8. (D) Immunofluorescence images for NANOG and GATA6 in bovine embryos that were cultured from the morula stage (day 5) to the blastocyst stage (day 8) in the presence of FGF4 and heparin. (E) Box-whisker plot of the quantified proportion of NANOG-positive (green) and GATA6-positive (red) cells in the ICM. (F) Box-whisker plot of the total number of ICM cells of bovine embryos that were cultured in FGF4 plus heparin or control conditions from day 5 to day 8. (G) Box-whisker plot of the relative numbers of NANOG-positive (green) and GATA6-positive (red) cells in bovine embryos cultured in the presence of FGF4 or heparin. (H) Immunofluorescence images for CDX2 on bovine embryos that were cultured from day 1 until day 8 in the presence of FGF4 and heparin or in control conditions. (I) Box-whisker plot of the number of CDX2-positive cells in bovine embryos cultured from day 1 to day 8 in the presence of FGF4 and heparin or in control conditions. In each experiment, equal concentrations of solvent (PBS) served as negative controls. Asterisks denote significant differences between the experimental and control groups: ^*, P<0.05; ^**, P<0.01; n.s., not significant. Error bars comprise the whiskers that extend to the maximum and minimum value data sets. Scale bars: 50 μm.

Fig. 4.

Effects of FGF receptor inhibition or blocking of proteoglycan sulfation on NANOG and GATA6 expression in bovine embryos. (A) NANOG and GATA6 expression in day 8 bovine embryos that were cultured from the zygote stage to the blastocyst stage in the presence of the FGF receptor inhibitor PD173074, the MEK inhibitor PD98059, PD173074 and PD98059, or in the broad-range sulfation inhibitor NaClO₃. All images are maximum image projections of z-stacks. (B) Box-whisker plot of the quantified proportion of NANOG-positive (green) and GATA6-positive (red) cells in the ICMs of the embryos that were cultured in the presence of PD173074 and/or PD98059. (C) Box-whisker plot of the quantified proportion of NANOG-positive (green) and GATA6-positive (red) cells in the ICMs of the embryos that were cultured from day 1 to day 8 or from day 5 to day 8 in the presence of NaClO₃. In all experiments, equal concentrations of solvent (DMSO) served as negative controls. ND, not determined because n<5. Error bars comprise the whiskers that extend to the maximum and minimum value data sets. Scale bars: 50 μm.

Fig. 5.

Effect of GSK3β and ALK5 inhibition on NANOG and GATA6 expression in bovine embryos. (A) NANOG and GATA6 expression in day 8 bovine embryos that were cultured from the zygote stage to the blastocyst stage in the presence of the MEK inhibitor PD0325901, the GSK3β inhibitor CHIR99021 or the ALK5 inhibitor A-83-01. All images are maximum image projections of z-stacks. (B,C) Box-whisker plots of the quantified proportion of NANOG-positive (green) and GATA6-positive (red) cells in each condition. In all experiments, equal concentrations of solvent (DMSO) served as negative controls. Asterisks denote significant differences between the experimental and control groups: ^*, P<0.05; ^***, P<0.001; n.s., not significant. Error bars comprise the whiskers that extend to the maximum and minimum value data sets. Scale bars: 50 μm.

Fig. 6.

Expression of NANOG, GATA6 and GATA4 in human blastocyst stage embryos and the effect of MEK inhibition on this expression. (A) Immunofluorescent detection of NANOG, GATA6 and GATA4 in hatching human blastocysts. NANOG-positive cells are enclosed by a layer of GATA4/6-positive cells, indicating that physical segregation of the epiblast and hypoblast cell lineages has initiated. The boxed areas are enlarged in the bottom row. (B) Immunofluorescence for NANOG, GATA6 and GATA4 on human PGD embryos that were cultured from the 8-cell stage to the blastocyst stage in the presence of the MEK inhibitor PD0325901 or DMSO (negative control). (C) Box-whisker plots showing quantification of the number of NANOG-positive, GATA6-positive and GATA4-positive cells in PGD embryos were cultured from the 8-cell stage to the blastocyst stage in the presence of PD0325901 or DMSO. (D) Box-whisker plots showing the quantified proportion of NANOG-positive (green) and GATA6-positive (red) cells in the ICM of human embryos grown in PD0325901 or DMSO. (E) Immunoblot for ERK and phosphorylated ERK on lysates of human ES cells that were treated with decreasing concentrations of the MEK inhibitor PD0325901. In all experiments, equal concentrations of solvent (DMSO) served as negative controls. n.s., not significant. Error bars comprise the whiskers that extend to the maximum and minimum value data sets. Scale bars: 50 μm.

Fig. 7.

Model for the roles of FGF/MAP kinase signaling in the formation of NANOG-positive epiblast cells or GATA6-positive hypoblast/primitive endoderm cells. In mouse, FGF signaling activates MAPK, which leads to the expression of GATA6 and repression of NANOG. In cattle, FGF signaling represses NANOG expression via MAPK. As a result of NANOG expression, expression of GATA6 is inhibited. GATA6 expression is also activated via an as yet unknown (question mark) FGF/MAPK-independent pathway. In human, activation of NANOG and GATA6 expression is coordinated via unknown (question mark) mechanisms.