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

alternative splicing of exons 8 and 9. The C-terminal half of the DIII domain is encoded by exon 8 to generate theFGFR(III)b isoforms while the C-terminal half of DIII is encoded by exon 9 to generate the FGFR(III)c isoforms.Modified from (Itoh and Ornitz 2004; Eswarakumar et al. 2005).HS, downstream signalling pathways and regulationHeparan sulfates (HSs) are proteoglycans consisting of repeated subunits of D-glucosamine anddisaccharides and are embedded in the extracellular matrix on the cell surface. Mutations in genesinvolved in the synthesis or modulation of HS results in developmental defects in mice, mostlikely due to impaired FGF-signalling (Bullock et al. 1998; Ornitz 2000; Kraushaar et al. 2010)).HS stabilizes FGFs to thermal denaturation, proteolysis and limit their diffusion and release tointerstitial spaces (Moscatelli 1987; Flaumenhaft et al. 1990). This leads to increased 1:1FGF:FGFR complex formation which then results in a transient receptor dimerization andsignalling from this 2:2 FGF:FGFR complex (Figure 1-6B; (Hsu et al. 1999; Plotnikov et al.1999; Pye and Gallagher 1999)).Intracellular phosphorylation of the kinase-domains leads to activation of intracellular signaltransduction pathways. Most commonly, the Ras/MAPK pathway is activated upon FGFsignalling,but also the PLCγ/Ca 2+ and PI3K/AKT pathways are activated (Bottcher and Niehrs2005). For activation of the Ras/MAPK pathway, FRS2 is recruited and forms a complexactivating Ras. Pathway activity ultimately leads to nuclear translocation of MAPK and activationof target genes, such as AP1, c-myc and ETS transcription factors (Wasylyk et al. 1998).Activation of PLCγ leads to intracellular release of Ca 2+ and activation of phosphokinase C (PKC;(Pawson 1995)). Finally, activation of PI3K through either direct interaction with the FGFR or bycomponents of the Ras/ MAPK pathway leads to activation of AKT (Carballada et al. 2001).FGF-signalling is regulated by members of the sprouty and sprouty-related EVH1 protein(SPRED) families by a feedback mechanism that regulates MAPK-signalling through receptortyrosine kinase binding (Hacohen et al. 1998; Lim et al. 2002). Furthermore, FGF-activity ismodified through a tight regulation of HS-synthesis and certain transmembrane regulators (SEFand FLRT), which interrupt FGF-FGFR complex formation or downstream signalling (Bottcherand Niehrs 2005). Synthetic small molecules such as SU5402 and PD173074 inhibit most FGFRsignallingwith some or little secondary effects, respectively (Mohammadi et al. 1997;Mohammadi et al. 1998).To date, the role of FGF-FGFR signalling in directed differentiation towards cell types of theendoderm germ layer has been investigated as a general activation or inhibition of FGF-FGFRsignals. The role of individual FGF- and FGFR family members in differentiation towards DEremains to be elucidated.18