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

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they were divided into three categories (Figure 2A; (Ornitz et al. 1996; Bottcher and Niehrs2005; Zhang et al. 2006)). FGF1, 2 and 9 activate a mixed population of both FGFRb- andFGFRc-isoforms, with a preference for the latter; FGF7 and 10 activate FGFRb-isoforms only;and FGF4, 5, 6, 8b, 8c, 8e and 16 activate one or more FGFRc-isoforms and/ or FGFR4(MacArthur et al. 1995; Ornitz et al. 1996; Olsen et al. 2006; Zhang et al. 2006; Mason 2007).FGFR4 is grouped with the FGFRc-type of receptors, based on the fact that it structurallyresembles this group of FGFRs (Vainikka et al. 1992). Importantly, most FGFs activatingFGFRc-isoforms also activate the FGFR4 (Mason 2007), making it also functionally anFGFRc-type. To evaluate the effect of the different FGFs in mesendodermal differentiation, wemonitored at the expression of PS and DE markers by means of reporter cell lines on days 3and 5.Accordingly, we induced mesoderm by 10 ng/ml BMP4 and added different FGFs to evaluatetheir effect on the PS marker T Gfp/ + cell line expression. FGF1, 2, 4, 6 and 9, binding a mixedpopulation of FGFRs or FGFRc-isoforms only, increased the number of T-GFP + cells on day 3by up to 20% compared to BMP4-treatment alone, i.e. 79 – 83 ± 6 – 10% and 69 ± 6%,respectively (mean % ± S.D., n=3; Figure 2B). FGF7 and 10 had no significant effect on T-GFP induction, nor did FGF8b, 8c, 8e or 16, but FGF5 slightly repressed T-induction (Figure2B). Looking at the same marker in a posterior streak/ mesoderm-inducing protocol, using 1ng/ml activin, we saw that FGF4 and 6 show a 31 – 42% increase in T-GFP induction on day 3(Figure 2C), while FGF5 and 10 show a smaller increase. FGF1, 2, 4, 6 and 9 induced numbersof T-GFP + cells by up to 34% on day 5. FGF7, 8b, 8c, 8e and 16 showed no effect on thenumbers of T-GFP + cells on either day 3 or 5. Thus, the largest effect was seen when addingFGFs binding FGFRc-isoforms or a mixed population of FGFRs, mediating an increase in T-GFP + cells in general and on day 5 in particular. The reason for this pronounced effect can bethrough either a delay in the response by some cells (in media containing BMP4 or activin only,it peaks on day 3), or maintaining the T-expression for a time period extending beyond day 3.Next, we looked at the effect of FGFs on anterior streak/ DE-induction by 30 ng/ml activin in amES cell line containing a GFP knock-in allele of the anterior streak marker Gsc, Gsc Gfp/ +(Tada et al. 2005). Addition of FGF1, 2, 4, 6, 8b and 9 increased the number of Gsc-GFP + cellsby 22 – 40% (Figure 2D). Activation of FGFRb-isoforms only, by FGF7 and 10, had no effectand nor did FGF5, 8c, 8e and 16. This finding was not due to the lack of receptors, as they werepresent in the cell population (Figure 1B). Looking at the DE marker Sox17, we saw up to a50% decrease of the Sox17-GFP Hi fraction, from 34 ± 4% to 17 ± 3% when adding FGFsactivating FGFRc-isoforms (Figure 2E). FGFs activating FGFRb-isoforms only, slightlyincreased the number of Sox17-GFP Hi cells or had no effect (FGF7 and FGF10, respectively).In summary, FGFs binding predominantly FGFR4/FGFRc-isoforms, i.e. FGF1, 2, 4, 6, 8b and9, promote differentiation towards a mesendoderm cell population expressing primitive andanterior streak markers.The mesendoderm population responds to FGFRc-isoform activation onlyNext we investigated whether the FGFs inhibiting DE formation would instead promote PS andmesoderm marker expression by analysing Sox17-GFP cells stained with antibodies against T,FLK1 and EpCAM. For analytical purposes, cell populations were divided into Sox17-GFP –/Loand Sox17-GFP Hi fractions. We limited the number of FGFs introduced in these experiments,focusing only on those showing the largest effect on PS and DE marker induction/ repression,namely FGF1, 2 and 9 (mixed FGFR-activation); FGF7 and 10 (FGFRb-activation only); andFGF4, 6 and 8b (FGFRc-activation only). As a control, we included differentiation by BMP4and saw that these cells expressed the most T in Sox17-GFP –/Lo cells as expected (Figures 3Aand B).In the Sox17-GFP Hi fraction, 12% of cells were T + on day 3 when treated with activin alone(Figure 3A). This number was increased by addition of FGF2, 4, 7 and 10, and decreased in thepresence of FGF6, 8b, 9 and especially FGF1. On day 5, numbers of T + cells were rather lowand an increase in the Sox17-GFP Hi /T + cell population was seen only when adding FGF2(Figure 3A). We also investigated the Sox17-GFP –/Lo fraction and saw that FGF7 and 10 had no64
influence on the numbers of Sox17-GFP –/Lo / T + cells on day 3 (Figure 3B), but the other FGFsall reduced cell numbers. On day 5, cell numbers were in general very low, showing a slightinduction of Sox17-GFP –/Lo / T + cells when adding FGF2, 4 or 6 (Figure 3B). These datademonstrate that the FGFs activating either a mixed population of FGFRs or FGFRc-isoformscan reduce the number of T + cells in the emerging DE and in the rest of the cell culture, whichhas been suggested to consist of cells of an anterior streak type, not yet determined to expressDE markers (Hansson et al. 2009).Tada and co-workers showed that the mesendoderm population can be distinguished by the PSmarker Gsc, and later the DE can be separated from the mesoderm by means of the DE markersSOX17, E-cadherin and FOXA2 and mesoderm markers FLK1 and platelet-derived growthfactor receptors (PDGFRs) α and β (Tada et al. 2005). Trying to define whether individualFGFs would switch the mesoderm vs. endoderm balance in the differentiating culture, weinvestigated markers able to distinguish between these cultures namely Epithelial cell adhesionmolecule (EpCAM) and FLK1. EpCAM is expressed in pluripotent mES cells and in the DEepithelium during embryonic development (Balzar et al. 1999; Sherwood et al. 2007) whereasFLK1 is expressed in all mesoderm cells leaving the embryonic posterior streak. Subsequently,FLK1 is observed in developing mesodermal cardiac crescent cells and in most extraembryonicmesoderm and at E8.5 it is expressed in splanchnic mesoderm and endothelial cellsof the dorsal aorta only (Ema et al. 2006). Focusing on day 5, we stained differentiated Sox17-GFP cells with antibodies for Flk1 and EpCAM. Looking at the three markers separately, wesaw a decrease in Sox17-GFP Hi cells in the presence of FGF1, 2, 4 and 6 in concordance withprevious data (Figures 3C and 2E). There were very few FLK1 + cells when treating withactivin, regardless of FGFs added, but a high induction in the BMP4-treated positive controland BSA control samples (Figure 3D). The induction of EpCAM is 87 ± 2% in the presence ofactivin (Figure 3E) and FGF6, 7, 8b and 9 modestly but significantly increase EpCAMexpression in the range of 88 ± 3% to 92 ± 3%. We see a high amount of cells that are FLK1 +or EpCAM + in the N2B27+BSA negative control medium. This condition gives rise to 50-75%Sox1-expressing neural progenitors at day 5 of culture ((Ying et al. 2003b); Peterslund et al.,unpublished data). The high amount of FLK1 + and EpCAM + cells in this condition is mostlikely due to random and/ or neural differentiation.In the Sox17-GFP Hi fraction, cells turned primarily into Sox17-GFP Hi / FLK1 – / EpCAM + cellsin the presence of activin, representative of definitive endoderm (Figure 3F; numbers shown are% of Sox17-GFP Hi cells). The addition of FGFs did not alter this picture, and also did notchange fates of the remaining cells, these being small fractions of Sox17 Hi / FLK1 + / EpCAM +and Sox17 Hi / FLK1 – / EpCAM – . The former are likely to represent cells in a transition phaseexpressing both EpCAM and FLK1 and were present mainly in the BSA control sample. Weonly found Sox17 Hi / FLK1 + / EpCAM – cells in the BMP4-condition, thus inducing what appearsto be a Sox17-expressing mesodermal cell type.We also looked at cell fates in the Sox17-GFP –/Lo fraction. Here, the vast majority of cells werestill Sox17 –/Lo / FLK1 – / EpCAM + (Figure 3G), and FGF1, 2, 8b and 9 had a positive effect onthis population, elevating numbers of this population by up to 10%.In summary, FGFs activating FGFRc-isoforms slightly decrease the number of Sox17-GFP + /T +and Sox17 –/Lo /T + cells, indicating random and/ or neural differentiation rather than PSformation at the expense of DE. In the Sox17-GFP + population, FGFs activating FGFRcisoformsincrease the number of FLK1 – /EpCAM + and decrease the number of FLK1 – /EpCAM –cells possibly indicating a pool of non-mesodermal cells at intermediary steps of differentiation.FGFRc-isotype activation induces cell growth during the first 3 days of cultureFGFs were originally discovered as having a mitogenic effect in fibroblast cells, and were laterfound to have adverse effects in embryonic development, including endoderm formation(Gospodarowicz and Moran 1975; Ornitz et al. 1996; Bottcher and Niehrs 2005). We analysedthe mitogenic effect of the FGFs in wt mES cells on days 3 and 5, and found that activintreatmentalone gave a 3,3 times increase in cell numbers by day 3 (from 2.000 cells/ cm 2 to6.600 cells/ cm 2 ; Figure 4A). All FGFs improved cell growth to varying degrees, FGF1, 2, 465
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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
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cell mass regenerates probably thro
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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 and 66: AbstractProgress in embryonic stem
Page 67 and 68: variants in their Ig-like domain II
Page 69: Cell count and proliferation assayC
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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