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

and 9 being the most effective ones (up to 20.400 cells/ cm 2 or a 3 times increase compared tothe activin-treated cells). By means of 5-ethynyl-2’-deoxyuridine (EdU)-incorporation (Salicand Mitchison 2008) we could quantify the proliferation of cells (see Figure S1 for gating andcontrols). We found that the absolute number of proliferating cells was higher at day 3 than day5, by an approximate 4 time increase for the activin-treated cells and a 4-7 times increase forthe conditions supplemented with FGFs. At day 3, the relative number of proliferating cells didnot differ much from the activin-treated sample (Figure 4A) except for a small reduction whenadding FGF1 or not adding any growth factors at all, i.e. the BSA control.On day 5, there was app. 80.000 cells/cm 2 in samples treated with activin alone. This numberincreased 1,6 times to app. 130.000 cells/ cm 2 when adding FGF2 (Figure 4B). This indicatesthat the effect of FGFs on cell growth is decreasing over time probably because more cells aredifferentiated at day 5. At this stage, especially FGF1 and 2 positively affect cell numbers.These have been shown also in other cell systems to have the largest mitogenic effect (Ornitz etal. 1996; Zhang et al. 2006). By EdU-incorporation, we saw a 50% higher proliferation rate inthe BSA control than with cells treated with activin, showing 25% and 15% total proliferation,respectively (Figure 4B). The FGFs most frequently reduced proliferation; FGF1, 2, 4 and 6 by40 – 50% and FGF8b, 9 and 10 showing moderate reductions. These data indicate that the maineffect seen by FGF1, 2, 4, 6 and 9 on proliferation occurs prior to day 3, and that most of thecells in these cultures have left the proliferative state by day 5.FGF4 is dispensable for the formation of endodermOf the FGFs tested, FGF4 and 6 binding FGFRc-isotypes only, increase BMP4 and activininducedexpression of T and Gsc the most. FGF4 is important during gastrulation where it isresponsible for the cell movements through the PS (Bottcher and Niehrs 2005). We wanted toinvestigate whether an FGF4 –/– cell line (Wilder et al. 1997) would be able to i) differentiateinto the endoderm lineage; and ii) promote differentiation of DE cell types, in that the latterwould not be inhibited by the FGFRc isoform-activating FGF4. When cells were maintained aspluripotent cells, the FGF4 –/– cell line showed a different cell morphology than the E14 andFGF4 +/– cell lines. Cells grew in small, very dense clusters indicative of pluripotent cells(Figure 5A) and growth rates were slower, confirming the mitogenic effect of FGF4. Cellsstained positive for the pluripotency marker OCT4 and negative for the endoderm markerSox17, similar to the wt and heterozygote cell lines.We subjected the wt E14, FGF4 +/– and FGF4 –/– cell lines to our DE induction-protocol.Through antibody staining of SOX17, E-cadherin (Ecad) and FOXA2 we identified cells of anendoderm origin. Foxa2 is expressed during embryonic development in the anterior primitivestreak, the newly formed definitive endoderm and is maintained throughout most matureendoderm-derived tissues (Kaestner et al. 1994; Weinstein et al. 1994). FGF4 +/– cells behavedmuch like E14 wt cells, showing vast numbers of OCT4 – /SOX17 + and SOX17 + /FOXA2 + /Ecadherin+ cells by day 5 (Figure 5B). In each cell line, differentiation was not 100% and smallclusters of tightly connected, undifferentiated OCT4 + cells persisted in the culture. Remarkably,FGF4 –/– cells readily differentiated along the endoderm lineage, showing mainly OCT4 –/SOX17 + cells and only a few more OCT4 + cells than the wt and heterozygote cell lines (Figure5A). When administrating FGF4 protein ectopically, the number of OCT4 + cells was reduced towt levels. There were comparable numbers of SOX17 + /FOXA2 + /E-cadherin + in the knock-outcell line and wt or heterozygote cell lines, and these did not change by the addition of FGF4 tothe medium (Figure 5B).Thus, we conclude that FGF4 is dispensable for differentiation of mES cells along theendoderm lineage and for cells to leave the pluripotent state when the differentiation protocolapplied includes activin.Definitive endoderm is formed in the absence of FGF4Looking for expression of Sox7 and Thermostable direct hemolysin gene (Tdh), markers ofvisceral endoderm (VE; (Sherwood et al. 2007)), we wanted to see whether the endodermformed was definitive or visceral. Staining for endoderm and VE markers in the66