FGF4 is required for lineage restriction and salt-and-pepper distribution of primitive endoderm factors but not their initial expression in the mouse

Abstract

The emergence of pluripotent epiblast (EPI) and primitive endoderm (PrE) lineages within the inner cell mass (ICM) of the mouse blastocyst involves initial co-expression of lineage-associated markers followed by mutual exclusion and salt-and-pepper distribution of lineage-biased cells. Precisely how EPI and PrE cell fate commitment occurs is not entirely clear; however, previous studies in mice have implicated FGF/ERK signaling in this process. Here, we investigated the phenotype resulting from zygotic and maternal/zygotic inactivation of Fgf4. Fgf4 heterozygous blastocysts exhibited increased numbers of NANOG-positive EPI cells and reduced numbers of GATA6-positive PrE cells, suggesting that FGF signaling is tightly regulated to ensure specification of the appropriate numbers of cells for each lineage. Although the size of the ICM was unaffected in Fgf4 null mutant embryos, it entirely lacked a PrE layer and exclusively comprised NANOG-expressing cells at the time of implantation. An initial period of widespread EPI and PrE marker co-expression was however established even in the absence of FGF4. Thus, Fgf4 mutant embryos initiated the PrE program but exhibited defects in its restriction phase, when lineage bias is acquired. Consistent with this, XEN cells could be derived from Fgf4 mutant embryos in which PrE had been restored and these cells appeared indistinguishable from wild-type cells. Sustained exogenous FGF failed to rescue the mutant phenotype. Instead, depending on concentration, we noted no effect or conversion of all ICM cells to GATA6-positive PrE. We propose that heterogeneities in the availability of FGF produce the salt-and-pepper distribution of lineage-biased cells.

Fig. 1.

Fgf4 is not required for initial expression of GATA6 but is crucial for its PrE lineage restriction. (A-J″) Localization and distribution of GATA6 (red) and NANOG (white) in wild-type (Fgf4^+/+) and Fgf4 mutant mouse embryos from the late morula to late/implanting blastocyst stage. Blue in merge, Hoechst. Cell numbers (c) for individual embryos are provided at the top right. Scale bars: 20 μm.

Fig. 2.

Co-expression of GATA6 and NANOG in embryos lacking zygotic and maternal/zygotic Fgf4. (A) Localization of GATA6 and NANOG in wild-type, maternal, maternal/zygotic and zygotic Fgf4 mutant embryos at the ∼16-cell stage. (B) Localization of GATA6 and NANOG in maternal and maternal/zygotic Fgf4 mutant embryos at the ∼32-cell stage and 32- to 64-cell stage. (C) Statistical analysis of the lineage composition of maternal, wild-type, maternal/zygotic and zygotic Fgf4 embryos at >80-cell stage. Wild-type, zFgf4^+/+ or zFgf4^+/–; maternal, mFgf4^–/–; zFgf4^+/–; maternal/zygotic, mFgf4^–/–; zFgf4^–/–; zygotic, zFgf4^+/+. EPI, epiblast; ICM, inner cell mass; PrE, primitive endoderm; TE, trophectoderm. Scale bars: 20 μm.

Fig. 3.

Absence of FGF4 affects the expression of transcription factors activated later in the PrE program. (A) Localization of SOX17 and NANOG in wild-type (Fgf4^+/+) and Fgf4^–/– embryos at E3.5 and E4.5. Arrowheads indicate SOX17-positive cells. (B) Localization of GATA4 and NANOG in wild-type and Fgf4^–/– embryos at E3.5 and E4.5. Scale bars: 20 μm.

Fig. 4.

Live imaging of PrE reporter dynamics in control and Fgf4 mutant embryos. The series of panels at the top depict single time points from a 3D time-lapse movie of two wild-type (WT) and two mutant (Mut) embryos over a 900-minute period. Each point represents a maximum intensity projection of 64 μm z-stacks. GFP intensity was quantified in individual cells in each embryo at five time points. Beneath is shown a plot of normalized GFP fluorescence intensity over time. The four embryos are imaged in this sequence. Individual dots represent GFP-positive cells. At early time points, wild-type and mutant embryos exhibit low GFP fluorescence (t=15 minutes). As they develop, GFP-positive cells in wild-type embryos exhibit an increase in fluorescence intensity (t=225, 450 minutes). In wild-type embryos, a wide range of GFP intensity is observed (min., 46.97; max., 361.02). GFP is downregulated in mutant embryos, becoming undetectable by the end of the movie (t=900 minutes).

Fig. 5.

Sustained exogenous FGF restores PrE cells in Fgf4 mutant embryos but fails to rescue the mutant phenotype. (A) The regime for exogenous FGF treatment experiments. KSOM refers to the culture medium employed. (B-D) Embryos from Fgf4^+/– intercrosses were recovered at E2.75-3.0 and cultured for 36 hours in KSOM (B), KSOM + 250 ng/ml FGF2 (C) and for 24 hours in KSOM + 500 ng/ml FGF2 followed by KSOM for 24 hours (D). (Ca) Fgf4^–/– embryo has all NANOG-positive ICM cells; (Cb) Fgf4^–/– embryo has all GATA6-positive ICM cells. Note that in 3D projection, GATA6 is detected in TE cells (as indicated by GATA6 staining in the outer TE cells). (E) Percentage of Fgf4^+/+ and Fgf4^–/– embryos with no NANOG-positive cells after treatment with FGF2 (bFGF) over a range of concentrations. Scale bars: 20 μm.

Fig. 6.

XEN cells representing the PrE lineage can be isolated from Fgf4 mutant blastocysts. Stem cell types isolated from Fgf4^+/– intercross embryos using various isolation protocols. (A) TS and XEN cells were derived from E2.5 embryos cultured for 2 days in the presence of FGF2 using TS cell medium. (B,C) ES and XEN cells were isolated from E3.5 (B) or E2.5 (C) embryos cultured for 2 days in the presence of FGF2 using ES cell derivation conditions. The bar chart shows the representation of TS (green), XEN (blue) and ES (red) cell lines of the indicated genotypes under the different derivation conditions. MEFS, murine embryonic fibroblasts.

Fig. 7.

Extra-embryonic endoderm differentiation of Fgf4 mutant ES cells. (A) Directed differentiation of Fgf4 heterozygous and mutant ES cells into extra-embryonic endoderm by misexpression of GATA4 and GATA6. Cells were immunostained with anti-SOX17, anti-FOXA2 and anti-DAB2 antibodies 2 days after transfection in serum-free culture conditions. Single optical sections are shown. (B) Cryosections of Fgf4 heterozygous and mutant embryoid bodies at 5 days differentiation (3D reconstructions). Insets show the boxed region at higher magnification, illustrating the outer cell layer comprising extra-embryonic endoderm. Hoechst is in gray. Scale bars: 50 μm.

Fig. 8.

Model for the role of FGF signaling in ICM lineage commitment. Differential expression levels of Fgf4 and Fgfr2 establish lineage biases within the ICM at the 32-cell stage. At the 64-cell stage, embryos exhibit a salt-and-pepper distribution of NANOG and GATA6 that represents the two lineages of NANOG-expressing epiblast (EPI) and GATA6-expressing primitive endoderm (PrE) lineage-biased cells. Continuous FGF4 signaling ensures lineage bias by (1) maintaining expression of the early PrE-specific factors GATA6, PDGFRα and SOX17, (2) activating the later PrE-specific factors GATA4 and SOX7, and (3) inhibiting EPI-specific factors such as NANOG. In the Fgf4 mutant, the early PrE-specific factors GATA6, PDGFRα and SOX17 are activated but fail to be maintained, leading to a failure in PrE formation.