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

Most of the remaining cells are Sox17 –/Lo / FLK1 – / EpCAM – and these may represent amesoderm population which is no longer expressing FLK1, i.e. is not haematopoietic. Flk1 isnecessary for hematopoietic and endothelial development, but not for other mesodermallineages expressing the marker at an early stage (Ema et al. 2006).We have previously shown that FGF-signalling has a positive effect on differentiation towardsPS-type cells peaking on day 3 (T-GFP + and Gsc-GFP + ), but inhibitory effect on DE cells(Sox17-GFP + ; (Hansson et al. 2009)). In the present study we show that FGF activating severalFGFRc-isotypes elicit this effect and that FGFs activating only FGFRb-isotypes have no effect.This suggests that activation of FGFRc-isoforms is beneficial for early differentiation towardsan intermediary PS-type cell, primarily through increased proliferation of cells induced todifferentiate by activin or BMP4. Later, FGFRc-activation must be removed in order tooptimize differentiation into a DE cell, probably because FGFRc-activation drivesmesendodermal cells towards the mesodermal lineage. The FGFR-expression seen duringmesendoderm/ DE-induction supports this, as mainly FGFRc-isoforms are expressed in theculture. FGFR2b is expressed late during DE-induction suggesting that the forming epitheliumcan respond to FGFs activating this receptor. However, we failed to substantiate this hypothesisas the number of Sox17-GFP Hi cells were unaffected by FGF7 and 10. Possibly, this FGFR2bexpression renders the cells competent to respond to later signals during organogenesis.Prior to gastrulation, FGF4 and 5, activating FGFRc-isoforms only, are expressed in theembryonic ectoderm in the area of the later PS, and in the PS during gastrulation (Haub andGoldfarb 1991; Hebert et al. 1991; Niswander and Martin 1992). During gastrulation, FGF3,activating FGFRb-isoforms only, is expressed in the PS (Wilkinson et al. 1988). This supportsour suggestion that FGFRc-activation is important early in differentiation, although we do notsee a requirement for FGFRb-activation during either mesoderm or endoderm induction. Acombination of factors, activating FGFRc-isoforms early during differentiation and FGFRbisoformslater may be beneficial for future DE-induction.The FGFs having the largest effect on PS-marker induction were FGF4 and 6. FGF4 is animportant growth factor during gastrulation where it is responsible for the cell movementsthrough the PS (Bottcher and Niehrs 2005) and FGF4 knock-out mice die during gastrulation atE4-5 (Feldman et al. 1995). It has also been shown to be necessary for mESCs when leavingthe pluripotent state and differentiating into either ectoderm or mesoderm lineages (Kunath etal. 2007; Stavridis et al. 2007). Kunath and co-workers found that this knock-out cell line couldnot differentiate into either lineage, except when supplementing the growth medium with FGF4protein.Remarkably, using culture conditions similar to Kunath and co-workers, we found that ectopicFGF4 was dispensable when differentiating the FGF4 –/– cell line into SOX17 + /Ecadherin+ /FOXA2 + /SOX7 – DE cells expressing Sox17 and Cxcr4, but not Sox7 and Tdh. Thissupports our previous finding that induction of DE-formation is not dependent on early FGFsignalling,as cells readily become Sox17-GFP + in the presence of the FGFR-inhibitorPD173074 at early stages (Hansson et al. 2009). In the FGF4 –/– cell line, slightly more cells stayin a pluripotent state, i.e. more cells stain OCT4 + , than what is seen for wt and heterozygote celllines. Some of these cells can be induced to differentiate when FGF4 is added to thedifferentiation medium, but FGF4 supplement does not seem to alter the fate of thedifferentiated cells. We propose that ectopic administration of FGF4 is only necessary fordifferentiation into ectoderm and mesoderm lineages but not for leaving the pluripotent state(Kunath et al. 2007; Stavridis et al. 2007). Although FGF4 knockout mice have been shown tobe embryonic lethal at the stage of gastrulation (Feldman et al. 1995; Wilder et al.), theirdependence on FGF4 signalling may lie at an earlier time-point, namely in the area ofembryonic ectoderm where later the PS forms. This would render FGF4 necessary forformation of the PS rather than it’s function (Niswander and Martin 1992; Tam et al. 1993).This also shows that FGF4 is not necessary for cells to leave the pluripotent state when thedifferentiation protocol applied includes activin.68