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

Figure 4-4: A high FGF4-concentration induces CDX2 + posterior gut tube. Flk1-LacZ cells differentiated for 5days in 100 ng/ml activin (Step 1) and 3 days in 25 ng/ml Wnt3a + 0, 10 or 100 ng/ml FGF4 (Step 2) were stainedfor FOXA2 and CDX2. 20× objective, scale bar indicates 50 µm.This resulted in numerous Sox2-LacZ + and SOX2 + cells as seen by β-Galactosidase stain andimmune-cytochemistry, respectively (Figures 4-2 and 4-3). Variation in the concentrations ofFGF4 and WNT3a did not seem to influence the numbers of Sox2-LacZ + or SOX2 + cells, aslong as both factors were present. We saw vast numbers of FOXA2 + cells throughout theconditions applied (Figure 4-3). FGF4 and WNT3a did not induce PDX1 + cells under anyconditions (Figure 4-3), but high concentrations of FGF4 in combination with WNT3a (5 or 25ng/ml) induced CDX2 + clusters in the culture (Figure 4-4). To be sure that we did not miss aweak expression of Pdx1 in the samples, we performed RT-PCR and saw only the housekeepinggene Glyceraldehyde 3-phosphate dehydrogenase (Gapdh) expressed (Figure 4-5).Thus, we concluded that FGF4 and WNT3a either alone or in combination were not enough todrive patterning of the DE into Pdx1-expressing posterior foregut cell types. We saw vastnumbers of Sox2-LacZ + and SOX2 + cells, indicative of an anterior DE-type. CDX2 wasinduced by 100 ng/ml FGF4, suggesting that this concentration is too high when aiming atPDX1-induction.52