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

FGF4, 5, FGF8b and FGFR1, are expressed during PS-formation and gastrulation (Haub andGoldfarb 1991; Hebert et al. 1991; Deng et al. 1994; Yamaguchi et al. 1994; Sun et al. 1999).FGFR1 –/– or FGF8 –/– embryos are embryonic lethal at this time point, the latter failing toexpress Fgf4 in the streak. In mES cell cultures, Fgf5 is upregulated at the onset ofdifferentiation (Kunath et al. 2007). Interestingly, Kunath and co-workers could not rescueneural differentiation in the FGF4 –/– cell line when adding FGF5 (Kunath et al. 2007).However, we suggest a redundancy in FGF-signalling at the point of initiation of endodermdifferentiation taking place, in such that FGF5 or 8b, binding FGFRc-isoforms as does FGF4,facilitate the activation of key differentiation genes.Most studies on endoderm formation from mES cells rely on culturing conditions using eitherembryoid bodies as starting material or high cell densities (Funa et al. 2008; Morrison et al.2008; Willems and Leyns 2008). Compared to Kunath and co-workers, we seed cells at a lowerdensity. Possibly, an excess of cells to some degree inhibits differentiation, a commonphenomenon seen in many ES cell differentiation systems. Indeed, when applying the ectodermdifferentiation protocol as described by Kunath and co-workers to cells at low density, we sawa significant increase in differentiation (data not shown). It has been shown that only mES cellsgrown at high density loose their renewal properties after ROCK-inhibition (Chang et al. 2010).Cells grown at high densities have more cell-cell interactions and their β-catenin pool is partlylocated at the plasma membrane, activating the Wnt signalling pathway implicated inmaintaining the pluripotent state in both human and mouse ES cells. Thus, we speculate thathigh cell densities preferentially retain mES cells in the pluripotent state to a higher degree thancells at low density and that FGF4-signalling may be necessary for leaving the pluripotent stateat high cell densities only.AcknowledgementsWe are thankful to Drs. G. Keller, S. Nishikawa, S. J. Morrison and A. Rizzino/ T. Kunath forthe T-GFP, Gsc-GFP, Sox17-GFP, FGF4 +/– and FGF4 –/– cell lines, respectively. We thankSøren Refsgaard Lindskog for excellent technical assistance and Mads Daugaard for criticallyreading of the manuscript.Figure legendsFigure 1: Screen for FGFR-isotypes during DE differentiation in sorted fractions of Sox17-GFP cellsThe expression of each FGFR-isoform was analysed by qPCR in both sorted and unsortedfractions of Sox17-GFP cells, differentiated by our DE protocol (30 ng/ml activin for 5 days).A) A histogram showing sorting gates in GFP – , GFP Lo and GFP Hi fractions. B) The absoluteexpression of each FGFR-isoform was standardised to the house-keeping gene TATA-bindingprotein (Tbp). Sox17 Hi fractions are shown only at day 4 and 5, when they appeared in theculture. The fraction of Sox17-GFP – cells was to low for RNA-extraction. We did not obtainfunctional primers for FGFR3b. The relative mean expression ± S.E.M. of 3 independentexperiments is shown, using a Student’s paired, two-tailed t-test for the statistical analysis: * =P < 0,05; ** = P < 0,01 compared to the ESC condition for each fraction (Sox17-GFP +fractions were compared to the unsorted ESC sample).Figure 2: Activation of FGFRc-isoforms boosts mesendoderm but inhibits DE markerexpressionUsing GFP-reporter cell lines T-GFP, Gsc-GFP and Sox17-GFP, we differentiated cells for 3(T-GFP cell line only) and 5 days in BMP4- or activin-containing media, adding differentFGFs. Cells were analysed by a FACS. A) Table of FGF – FGFR binding of selected FGFs69