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

IntroductionBased on knowledge obtained in developmental biology, mouse embryonic stem (mES) cellscan be directed towards differentiation into specific germ layers and more mature tissues. Sucha differentiation into glucose-responsive β cell-like insulin-secreting cells, serves in theory as acure for type I diabetes mellitus (McCall et al. 2010). For this purpose, the first step is togenerate definitive endoderm (DE) with the potential to further differentiate into cellsresembling the primitive gut tube (reviewed by (Van Hoof et al. 2009)). Understanding the roleof each component used in this directed differentiation is crucial for obtaining the optimalprogenitor cell population in each step.In the late blastocyst stage of the developing mouse embryo (E4.5), the inner cell mass (ICM) isdivided into the epiblast and the primitive, later visceral endoderm. The visceral endoderm(VE) is involved in the asymmetric anterior-posterior patterning of the epiblast, resulting in theonset of gastrulation (reviewed by (Rossant 2004)). In the gastrulating mouse embryo, epiblastcells migrate through the primitive streak (PS), and in this process become determined towardseither mesoderm or DE germ layers (Lawson et al. 1991; Tam et al. 1993; Carey et al. 1995).The transforming growth factor-β family member nodal, an activator of SMAD2/3 signalling, isthe main initiator of epiblast patterning and PS formation (Conlon et al. 1994; Waldrip et al.1998). At high levels, nodal induces anterior PS structures and DE and at low doses it inducesmore posterior streak fates (Ben-Haim et al. 2006). At the posterior-most end of the PS, bonemorphogenetic protein 4 (BMP4) is produced by the extra-embryonic ectoderm and establishesa signal gradient. BMP4 is critical for formation of the PS and induces mesoderm formation(reviewed by (Gadue et al. 2005)). It is currently accepted that cells in the mesoderm andendoderm tissues arise from a common progenitor cell population, the mesendoderm (Lawsonet al. 1991; Kinder et al. 2001). The PS marker Brachyury (T) is expressed in the nascent andmigrating mesoderm of the primitive streak during gastrulation in the mouse embryo (Kispertand Herrmann 1994). Goosecoid (Gsc) is located in the progressing primitive streak at E6.5,and later localizes to the anterior streak, from which the DE arises (Blum et al. 1992). It isinduced by high concentrations of activin in animal cap explants from Xenopus and in mEScells, it is used as a marker for the mesendoderm cell population (Kubo et al. 2004; Gadue et al.2006). Sry-related HMG box gene 17 (Sox17) is an early marker specifically expressed in thedefinitive endoderm of the gastrula, and later expands to the endoderm underlying the neuralplate of the early-bud-stage embryo (Kanai-Azuma et al. 2002). Sox17 is also expressed in theextra-embryonic, but not in the embryonic visceral endoderm.In mES cell cultures, cells take on a mesendoderm-type fate before being committed to eitherthe mesoderm or DE lineages (Tada et al. 2005). ActivinA (activin hereafter) is used as asurrogate for nodal as they both activate SMAD2/3 signalling by binding to the ALK4 receptor,thus functioning in the same manner (Schier 2003). In the mesendoderm population, highconcentrations of nodal/ activin-signalling induce anterior streak and DE cells while BMP4 orlow concentrations of nodal/ activin induce posterior streak or mesoderm (Kubo et al. 2004;Willems and Leyns 2008; Hansson et al. 2009). The PS genes T, Mix-like 1 (Mixl1) and Gsc areexpressed in this population in response to increasing concentrations of activin. High activinlevelsfurther induce the DE markers Sox17, E-cadherin and Forkhead box A2 (Foxa2). BMP4induces T, Mixl1 and the mesodermal marker Fetal like kinase 1 (Flk1; VEGFR2/ Kdr; (Gadueet al. 2005)). During mES cell differentiation, T-expressing cells give rise to endoderm andmesoderm derivatives (Kubo et al. 2004) and we have previously shown that a T-GFP reportercell line (T Gfp/ + ; (Fehling et al. 2003)) can be activated by BMP4 and a low concentration ofactivin (Hansson et al. 2009).For the differentiation of mesendoderm and DE to occur properly in mES cells, fibroblastgrowth factor (FGF)-signalling is required (Funa et al. 2008; Morrison et al. 2008; Willems andLeyns 2008; Hansson et al. 2009). The FGF family of proteins consists of 22 members namedFGF1-23 (FGF15 is the mouse ortholog of human FGF19). They activate one or more of fourreceptor tyrosine kinases, the FGF receptors (FGFRs)1-4. FGFRs1-3 have two secreted splice60