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

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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
variants in their Ig-like domain III, the FGFR(III)b or FGFR(III)c isoforms (hereafter FGFRbor FGFRc, respectively; (Ornitz and Itoh 2001; Itoh and Ornitz 2004)). FGFs are involved inmany functions such as germ layer formation, limb development, cell proliferation and cellmigration in the developing embryo (Ornitz and Itoh 2001). In early mouse development, FGFsignallingis necessary for the migration of epiblast cells through the PS (Ciruna et al. 1997;Guo and Li 2007). The loss of FGF4 is lethal at E4-5, due to the inability of epiblast cells toundergo epithelial-to-mesenchymal transition and migrate through the PS (Feldman et al.1995). FGFR1 –/– mice also die at gastrulation and both FGF4 and FGFR1 are expressed in theICM and PS (Deng et al. 1994; Yamaguchi et al. 1994). FGF4 is expressed in pluripotent mEScells and has been shown to be necessary for differentiation into ectoderm and mesodermlineages, suggesting a crucial role of FGF4 in the initiation of differentiation (Kunath et al.2007). However, Wilder et al. showed that FGF4 –/– cells could differentiate in vitro, albeit at alow frequency, and gave rise to tumours consisting of a wide range of differentiated cell typesin vivo (Wilder et al. 1997).In this study, we expand on our previous finding that active FGF-signalling is necessary for DEformation (Hansson et al. 2009), and investigate the effects of different FGFR-isoforms onmesendoderm and DE differentiation. We first analyse the expression patterns of FGFRisoformsin the forming DE and find that FGFRc-isoforms are up-regulated in the bulk culturewhereas FGFR2b and 4 are up-regulated specifically in the DE-fraction. By means of reportercell lines and antibody staining, we find that FGFs activating primarily FGFRc-isoforms inducePS and mesendoderm markers T and Gsc but reduce the DE marker Sox17. FGFs activatingFGFRb-isoforms have no effect on either cell type. The FGFRc isoform-activating FGFs showthe highest mitogenic effects early in the differentiation period, and suggestively speeds updifferentiation, as proliferation rates in the presence of these FGFs are reduced later in theculture period. Remarkably, an ES cell line carrying a knockout for one such FGFRc isoformactivatingFGF, FGF4 –/– was able to differentiate to endoderm cells at levels comparable to wtand FGF4 +/– situations. The absence of FGF4 gave rise to DE differentiation, and although afew cells stained positive for the VE marker Sry-related HMG box 7 (SOX7), qPCR analysesconfirmed the DE fate of the culture. Thus, we conclude that FGFRc-isoforms specify themesendoderm but not DE cell population and that FGF4-signalling is dispensable for inductionof DE cells.Materials and MethodsCell culture and differentiation of mESCsWe used the following mouse ES cell lines: E14 (Hooper et al. 1987), T-GFP (Fehling et al.2003), Gsc-GFP (Tada et al. 2005), Sox17-GFP (Kim et al. 2007), FGF4 +/– and FGF4 –/–(Wilder et al. 1997). Cells were grown as previously described (Ying et al. 2003a; Hansson etal. 2009) on cell culture plastic ware (Nunc) coated with 0,1% gelatine (Sigma), using 0,05%Trypsin-EDTA (Invitrogen) for dissociation of cells during passage. Trypsin was inactivated byN2B27 medium: KO-DMEM supplemented with N2, B27, 0.1 mM non-essential amino acids,2 mM L-glutamine, Penicillin/Streptomycin (all from Invitrogen), 0.1 mM 2-mercaptoethanol(Sigma-Aldrich). Cells were grown for at least 3 passages before onset of differentiation.For differentiation purposes, cells were dissociated into single cells and seeded at 2.000 cells/cm 2 in N2B27 medium containing one or more of the following growth factors: BMP4 (10ng/ml), activinA (1 or 30 ng/ml; both from R&D Systems), FGF1 (100 ng/ml; ChemiconInternational), FGF2 (100 ng/ml; Invitrogen), FGF4, FGF5, FGF6, FGF7, FGF8b, FGF8c,FGF8e, FGF9, FGF10, FGF16 (5 or 100 ng/ml; all from R&D Systems). Media containingFGFs were supplemented with 10 µg/ml heparan sulfate (Sigma-Aldrich).Flow cytometryFor GFP-analysis of reporter cell lines, live cells were dissociated into single cells by 0,05%Trypsin-EDTA (Invitrogen) and analysed by FACS Calibur flow cytometer (BD Biosciences).61
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PhD thesisCand.scient. Janny Marie
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ResuméSukkersyge er en sygdom der
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Table of contents1
Page 12 and 13:
ICMinner cell massIdInhibitor of di
Page 14 and 15:
cell mass regenerates probably thro
Page 16 and 17: Figure 1-1: Early embryo developmen
Page 18 and 19: Figure 1-3: Regional expression of
Page 20 and 21: The pluripotent stateThe pluripoten
Page 25 and 26: There are four membrane-bound FGFRs
Page 27: 2. AimsThe aim of this study was to
Page 30 and 31: Developmental Biology 330 (2009) 28
Page 32 and 33: 288 M. Hansson et al. / Development
Page 50 and 51: Figure S2Figure S2: A subpopulation
Page 52 and 53: Figure S4Figure S4: Expression of T
Page 54 and 55: Figure S6Figure S6: qRT-PCR analyse
Page 56 and 57: epithelium; Cdx2, expressed posteri
Page 58 and 59: Figure 4-4: A high FGF4-concentrati
Page 60 and 61: Figure 4-6: A 3-step protocol does
Page 63 and 64: 5. Paper IIFGFR(IIIc)-activation in
Page 65: AbstractProgress in embryonic stem
Page 69 and 70: Cell count and proliferation assayC
Page 71 and 72: influence on the numbers of Sox17-G
Page 73 and 74: undifferentiated cells, we found th
Page 75 and 76: FGF4, 5, FGF8b and FGFR1, are expre
Page 77 and 78: with EdU-stain (blue sample); and w
Page 79 and 80: Olsen, S.K., J.Y. Li, C. Bromleigh,
Page 81 and 82: FiguresFigure 1: Screen for FGFR-is
Page 83 and 84: Figure 3: Activation of FGFRb or FG
Page 87 and 88: Opposite, Figure 6: In the absence
Page 89 and 90: 6. General discussionEndoderm diffe
Page 91 and 92: Overall, the multitude of FGF-signa
Page 93 and 94: transplantation is the spread of an
Page 96: AcknowledgementsThe work presented
Page 99 and 100: Chambers, I., D. Colby, M. Robertso
Page 101 and 102: Hawkins, V.J. Wroblewski, D.S. Li,
Page 103 and 104: Nishikawa, S.I., S. Nishikawa, M. H
Page 105 and 106: Tanimizu, N., H. Saito, K. Mostov,
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