br Results br Discussion We have previously shown

Results
Discussion We have previously shown that Fgf signaling augments differentiation toward PS-like Atractyloside Dipotassium Salt and is required at Days 3–5 for efficient differentiation of mESC into DE in response to activin treatment. However, addition of Fgf2 attenuated formation of activin-induced Sox17-GFP+ DE cells (Hansson et al., 2009)), suggesting that levels of signaling must be tightly regulated. Recently, Fgf4 was shown to be necessary for mESCs to leave the pluripotent state and differentiate into either ectoderm or mesoderm lineages (Kunath et al., 2007; Stavridis et al., 2007). Fgf4−/− mESCs could not differentiate into either lineage, except when supplementing the growth medium with exogenous Fgf4 protein. Fgf4 is important during gastrulation where it is responsible for movement of gastrulating cells through the PS (Bottcher and Niehrs, 2005) and Fgf4 knock-out mice die during gastrulation (Feldman et al., 1995). Using culture conditions similar to those Kunath and co-workers, we found that addition of exogenous Fgf4 was dispensable when differentiating Fgf4−/− cells into DE by treatment with activin. We readily obtained Sox17+/E-cadherin+/Foxa2+/Sox7– DE cells which by qPCR were shown to express the additional DE markers Cxcr4, Cldn6, and Foxa3, but not the VE markers Sox7 and Tdh. This supports our previous finding that induction of DE formation is not dependent on early Fgf signaling, as cells readily become Sox17-GFP+ when the FGFR-inhibitor PD173074 is present at early stages of differentiation (Hansson et al., 2009). We propose that exogenous Fgf4 is only necessary for differentiation into ectoderm and mesoderm lineages but not for leaving the pluripotent state per se (Kunath et al., 2007; Stavridis et al., 2007). Although Fgf4 knockout mice have been shown to be embryonic lethal at the stage of gastrulation (Feldman et al., 1995; Wilder et al., 1997), their dependence on Fgf4 signaling may lie at an earlier time point, namely in the area of embryonic ectoderm where later the PS forms. This would render Fgf4 necessary for the formation of the PS rather than its function (Tam et al., 1993; Niswander and Martin, 1992). This substantiates that Fgf4 is not necessary for cells to leave the pluripotent state when the differentiation protocol applied includes activin. Most studies on endoderm formation from mES cells rely on culturing conditions using either embryoid bodies as starting material or high cell densities (Willems and Leyns, 2008; Funa et al., 2008; Morrison et al., 2008). Compared to Kunath and co-workers, we seed cells at a low density. Possibly, a high cell density to some degree inhibits differentiation, a common phenomenon seen in ES cell differentiation systems. Indeed, when applying the ectoderm differentiation protocol as described by Kunath and co-workers to cells at low density, we saw an increase in neural differentiation. We speculate that high cell densities retain mES cells in the pluripotent state to a higher degree than cells at low densities and that Fgf4 signaling may be necessary for leaving the pluripotent state at high cell densities only. Nevertheless, formation of Sox17+ DE from mESC in response to activin treatment is influenced by Fgf signaling. Treatment with the FGFR inhibitor PD173074 at Days 3–5 prevents differentiation of Sox17+ DE but addition of exogenous Fgf2 also inhibits formation of these cells (Hansson et al., 2009). Here we find that activation of FGFRc but not FGFRb isoforms changes the fraction of EpCAM+ cells that coexpress Sox17. While activin alone inducs a nearly uniform population of EpCAM+Sox17Hi cells addition of, e.g., Fgf2 reduces the fraction of EpCAM+ cells that coexpress Sox17. This shift may be caused by a selective expansion of an EpCAM+Sox17− population developing in response to activin treatment, without affecting the total number of EpCAM+Sox17+ DE cells. Alternatively, Fgf2 may inhibit Sox17 expression in a subset of the EpCAM+ cells. During embryonic development, epithelial tissues generally express b but not c isoforms while mesenchymal tissues express mainly c isoforms (Ornitz and Itoh, 2001). Fgfs specifically activating FGFRb isoforms (e.g., Fgf7 and 10) are mainly expressed in the mesenchyme and Fgfs activating FGFRc isoforms (e.g., Fgf4, 8, and 9) are mainly expressed in epithelia, resulting in specificity during reciprocal epithelial-mesenchymal signaling in developing organs such as the lung, cecum, salivary glands, and pancreas (Ornitz and Itoh, 2001; Orr-Urtreger et al., 1993; Elghazi et al., 2002; Stark et al., 1991; Colvin et al., 2001; Manfroid et al., 2007). In the present report we show that both FGFRb and -c isoforms are expressed in the Sox17-GFPHi fraction after activin treatment. The expression of FGFRb as well as c isoforms in the Sox17-GFPHi fraction may be explained if the Sox17-GFPHi fraction is heterogeneous and contains a pool of cells slated to become DE but not yet committed to an epithelial fate or cells that are undergoing mesodermal differentiation. However, the Sox17-GFPHi fraction is also positive for EpCAM, indicating that they represent epithelial endoderm. When factors capable of activating the c isoforms for the FGFRs are added to the activin induction we still observe the formation of EpCAM+ cells but the percentage of these coexpressing Sox17-GFPHi is reduced. These cells may represent undifferentiated ES cells, but we consider this unlikely as activin-induced expression of the early mesendoderm markers T and Gsc is enhanced by FGFR c-isoform ligands and the concomitant reduction in Oct4 expression is not affected. Alternatively, the EpCAM+Sox17− cells may represent endoderm which has yet to activate Sox17 expression or alternatively have passed through the transient Sox17-expressing phase. Regardless of their true identity, this EpCAM+Sox17− population appears to be selectively expanded by Fgfs that activate the c isoforms for the FGFRs, although the precise mechanism causing this change remains to be determined. Examining EpCAM and Sox17-GFP at later time points as well as qPCR analyses of purified cell populations could determine if Fgf signaling is delaying the kinetics of development of EpCAM+Sox17Hi DE cells.