FGF-signalling in the differentiation of mouse ES cells towards ...

they were divided into three categories (Figure 2A; (Ornitz et al. 1996; Bottcher and Niehrs2005; Zhang et al. 2006)). FGF1, 2 and 9 activate a mixed population of both FGFRb- andFGFRc-isoforms, with a preference for the latter; FGF7 and 10 activate FGFRb-isoforms only;and FGF4, 5, 6, 8b, 8c, 8e and 16 activate one or more FGFRc-isoforms and/ or FGFR4(MacArthur et al. 1995; Ornitz et al. 1996; Olsen et al. 2006; Zhang et al. 2006; Mason 2007).FGFR4 is grouped with the FGFRc-type of receptors, based on the fact that it structurallyresembles this group of FGFRs (Vainikka et al. 1992). Importantly, most FGFs activatingFGFRc-isoforms also activate the FGFR4 (Mason 2007), making it also functionally anFGFRc-type. To evaluate the effect of the different FGFs in mesendodermal differentiation, wemonitored at the expression of PS and DE markers by means of reporter cell lines on days 3and 5.Accordingly, we induced mesoderm by 10 ng/ml BMP4 and added different FGFs to evaluatetheir effect on the PS marker T Gfp/ + cell line expression. FGF1, 2, 4, 6 and 9, binding a mixedpopulation of FGFRs or FGFRc-isoforms only, increased the number of T-GFP + cells on day 3by up to 20% compared to BMP4-treatment alone, i.e. 79 – 83 ± 6 – 10% and 69 ± 6%,respectively (mean % ± S.D., n=3; Figure 2B). FGF7 and 10 had no significant effect on T-GFP induction, nor did FGF8b, 8c, 8e or 16, but FGF5 slightly repressed T-induction (Figure2B). Looking at the same marker in a posterior streak/ mesoderm-inducing protocol, using 1ng/ml activin, we saw that FGF4 and 6 show a 31 – 42% increase in T-GFP induction on day 3(Figure 2C), while FGF5 and 10 show a smaller increase. FGF1, 2, 4, 6 and 9 induced numbersof T-GFP + cells by up to 34% on day 5. FGF7, 8b, 8c, 8e and 16 showed no effect on thenumbers of T-GFP + cells on either day 3 or 5. Thus, the largest effect was seen when addingFGFs binding FGFRc-isoforms or a mixed population of FGFRs, mediating an increase in T-GFP + cells in general and on day 5 in particular. The reason for this pronounced effect can bethrough either a delay in the response by some cells (in media containing BMP4 or activin only,it peaks on day 3), or maintaining the T-expression for a time period extending beyond day 3.Next, we looked at the effect of FGFs on anterior streak/ DE-induction by 30 ng/ml activin in amES cell line containing a GFP knock-in allele of the anterior streak marker Gsc, Gsc Gfp/ +(Tada et al. 2005). Addition of FGF1, 2, 4, 6, 8b and 9 increased the number of Gsc-GFP + cellsby 22 – 40% (Figure 2D). Activation of FGFRb-isoforms only, by FGF7 and 10, had no effectand nor did FGF5, 8c, 8e and 16. This finding was not due to the lack of receptors, as they werepresent in the cell population (Figure 1B). Looking at the DE marker Sox17, we saw up to a50% decrease of the Sox17-GFP Hi fraction, from 34 ± 4% to 17 ± 3% when adding FGFsactivating FGFRc-isoforms (Figure 2E). FGFs activating FGFRb-isoforms only, slightlyincreased the number of Sox17-GFP Hi cells or had no effect (FGF7 and FGF10, respectively).In summary, FGFs binding predominantly FGFR4/FGFRc-isoforms, i.e. FGF1, 2, 4, 6, 8b and9, promote differentiation towards a mesendoderm cell population expressing primitive andanterior streak markers.The mesendoderm population responds to FGFRc-isoform activation onlyNext we investigated whether the FGFs inhibiting DE formation would instead promote PS andmesoderm marker expression by analysing Sox17-GFP cells stained with antibodies against T,FLK1 and EpCAM. For analytical purposes, cell populations were divided into Sox17-GFP –/Loand Sox17-GFP Hi fractions. We limited the number of FGFs introduced in these experiments,focusing only on those showing the largest effect on PS and DE marker induction/ repression,namely FGF1, 2 and 9 (mixed FGFR-activation); FGF7 and 10 (FGFRb-activation only); andFGF4, 6 and 8b (FGFRc-activation only). As a control, we included differentiation by BMP4and saw that these cells expressed the most T in Sox17-GFP –/Lo cells as expected (Figures 3Aand B).In the Sox17-GFP Hi fraction, 12% of cells were T + on day 3 when treated with activin alone(Figure 3A). This number was increased by addition of FGF2, 4, 7 and 10, and decreased in thepresence of FGF6, 8b, 9 and especially FGF1. On day 5, numbers of T + cells were rather lowand an increase in the Sox17-GFP Hi /T + cell population was seen only when adding FGF2(Figure 3A). We also investigated the Sox17-GFP –/Lo fraction and saw that FGF7 and 10 had no64