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

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used in this paper. Modified from (Ornitz et al. 1996; Olsen et al. 2006; Zhang et al. 2006;Mason 2007). B-C) Cells were differentiated in 10 ng/ml BMP4 (= mesoderm-induction) or 1ng/ml activin (= posterior streak/ mesoderm-induction) w/wo FGFs, and expression of T-GFPwas measured at days 3 and 5. D) Gsc-GFP cells were differentiated in 30 ng/ml activin w/woFGFs, and expression of GFP was measured at day 5. E) Sox17-GFP cells were differentiatedin 30 ng/ml activin w/wo FGFs, and expression of GFP was measured at day 5. The meanexpression ± S.E.M. of 3 independent experiments is shown, using a Student’s t-test for thestatistical analysis: * = P < 0,05; ** = P < 0,01 compared to the BMP4 or activin conditions.Figure 3: Activation of FGFRb or FGFRc-isoforms differentially affects the expression ofPS, DE and mesoderm markersSox17-GFP cells were differentiated in media containing BMP4 or activin w/wo FGFs for 3-5days before harvest. Cells were stained for markers of PS, DE or mesoderm and analysed usinga FACS. A & B) Cells were stained for T and analysed on days 3 and 5. Shown here are therelative number of T + cells in the Sox17-GFP Hi fraction A), or the Sox17-GFP –/Lo fraction B).The mean expression ± S.E.M. of 2 independent experiments are shown, no statistical analysisperformed. C-G) Cells were stained for FLK1 and EpCAM and analysed in either singlechannel for C) Sox17-GFP, D) FLK1, E) EpCAM; or in multichannel for the Sox17-GFP Hifraction F), or Sox17-GFP –/Lo fraction G) dividing data into four fractions: FLK1 – /EpCAM + ;FLK1 + /EpCAM + ; FLK1 – /EpCAM – ; FLK1 + /EpCAM – . The mean expression ± S.E.M. of 3independent experiments is shown, using a Student’s paired, two-tailed t-test for the statisticalanalysis: * = P < 0,05; ** = P < 0,01 compared to the activin conditions.Figure 4: FGFs activating FGFRc-isoforms affect early cell growth and proliferationA wt mES cell line (E14) was grown in media containing activin w/wo FGFs and harvested foranalysis of total cell number and proliferation on days 3 &5. A count of total number of cellsand relative proliferation of cells is shown for day 3 A) and day 5 B). The mean expression ±S.E.M. of 3 independent experiments is shown, using a Student’s t-test for the statisticalanalysis: * = P < 0,05; ** = P < 0,01 compared to the activin conditions.Figure 5: FGF4-signalling is dispensable for endoderm differentiationE14, FGF4 +/– and FGF4 –/– cells were stained for markers of pluripotency and endoderm. A)Undifferentiated cells were stained for OCT4 and SOX17 and the nuclear stain DAPI. B) Cellswere differentiated for 5 days in 30 ng/ml activin w/wo 5 ng/ml FGF4 and stained for OCT4and endoderm markers SOX17, FOXA2, E-cadherin (Ecad) and DAPI. Representative imagesare shown for each condition. Scale bar: 100 µm.Figure 6: In the absence of FGF4, DE rather than VE is formedAntibody stain and qPCR-analyses of DE and VE markers in E14, FGF4 +/– and FGF4 –/– cells.A) Undifferentiated cells were stained for SOX17, E-cadherin, SOX7 and the nuclear stainDAPI. B) Cells were differentiated for 5 days in 30 ng/ml activin w/wo 5 ng/ml FGF4 andstained for the same markers. Representative images are shown for each condition. Scale bar:100 µm. C) qPCR data showing the relative expression levels of Sox17, Cxcr4, Sox7 and TdhmRNA present in undifferentiated and differentiated cells of each cell line compared to E14ESCs, all standardized to the house-keeping gene Tbp. The mean expression ± S.E.M. of 3independent experiments is shown (n=2 for FGF4 –/– cells treated w/ activin + FGF4), using aRatio t-test for the statistical analysis: # = P < 0,05; ## = P < 0,01 compared to the E14 mES cellcondition.SUPPLEMENTARY DATAFigure S1: Negative controls of EdU-incorporation and sort gateA histogram showing the distribution of pluripotent E14 mES cells with/without EdUincorporationand without EdU-stain (red and black samples); without EdU-incorporation and70
with EdU-stain (blue sample); and w/ EdU-incorporation and with EdU-stain (stained withAlexa-488, i.e. positive control; green sample).Figure S2: A few areas of SOX7 + cells in the FGF4 –/– cell line after DE-inductionCells were differentiated for 5 days in 30 ng/ml activin w/wo 5 ng/ml FGF4 and stained forSOX17, E-cadherin, SOX7 and the nuclear stain DAPI. The yellow frame indicates area blownup and shown to the right (red signal boosted). Representative images are shown for eachcondition, except for FGF4 –/– cells in activin alone which shows a SOX7 + area in the culture.White scale bar: 100 µm; yellow scale bar: 50 µm.ReferencesBalzar, M., M.J. Winter, C.J. de Boer, and S.V. Litvinov. 1999. The biology of the 17-1A antigen (Ep-CAM). J Mol Med 77: 699-712.Ben-Haim, N., C. Lu, M. Guzman-Ayala, L. Pescatore, D. Mesnard, M. Bischofberger, F. Naef, E.J.Robertson, and D.B. Constam. 2006. The nodal precursor acting via activin receptors inducesmesoderm by maintaining a source of its convertases and BMP4. Dev Cell 11: 313-23.Blum, M., S.J. Gaunt, K.W. Cho, H. Steinbeisser, B. Blumberg, D. Bittner, and E.M. De Robertis. 1992.Gastrulation in the mouse: the role of the homeobox gene goosecoid. Cell 69: 1097-106.Bottcher, R.T. and C. Niehrs. 2005. Fibroblast growth factor signaling during early vertebratedevelopment. Endocr Rev 26: 63-77.Carey, F.J., E.A. Linney, and R.A. Pedersen. 1995. Allocation of epiblast cells to germ layer derivativesduring mouse gastrulation as studied with a retroviral vector. Dev Genet 17: 29-37.Chang, T.C., Y.C. Chen, M.H. Yang, C.H. Chen, E.W. Hsing, B.S. Ko, J.Y. Liou, and K.K. Wu. 2010.Rho kinases regulate the renewal and neural differentiation of embryonic stem cells in a cellplating density-dependent manner. PLoS One 5: e9187.Ciruna, B.G., L. Schwartz, K. Harpal, T.P. Yamaguchi, and J. Rossant. 1997. Chimeric analysis offibroblast growth factor receptor-1 (Fgfr1) function: a role for FGFR1 in morphogeneticmovement through the primitive streak. Development 124: 2829-41.Colvin, J.S., A.C. White, S.J. Pratt, and D.M. Ornitz. 2001. Lung hypoplasia and neonatal death in Fgf9-null mice identify this gene as an essential regulator of lung mesenchyme. Development 128:2095-106.Conlon, F.L., K.M. Lyons, N. Takaesu, K.S. Barth, A. Kispert, B. Herrmann, and E.J. Robertson. 1994.A primary requirement for nodal in the formation and maintenance of the primitive streak in themouse. Development 120: 1919-28.Deng, C.X., A. Wynshaw-Boris, M.M. Shen, C. Daugherty, D.M. Ornitz, and P. Leder. 1994. MurineFGFR-1 is required for early postimplantation growth and axial organization. Genes Dev 8:3045-57.Elghazi, L., C. Cras-Meneur, P. Czernichow, and R. Scharfmann. 2002. Role for FGFR2IIIb-mediatedsignals in controlling pancreatic endocrine progenitor cell proliferation. Proc Natl Acad Sci U SA 99: 3884-9.Ema, M., S. Takahashi, and J. Rossant. 2006. Deletion of the selection cassette, but not cis-actingelements, in targeted Flk1-lacZ allele reveals Flk1 expression in multipotent mesodermalprogenitors. Blood 107: 111-7.Fehling, H.J., G. Lacaud, A. Kubo, M. Kennedy, S. Robertson, G. Keller, and V. Kouskoff. 2003.Tracking mesoderm induction and its specification to the hemangioblast during embryonic stemcell differentiation. Development 130: 4217-27.Feldman, B., W. Poueymirou, V.E. Papaioannou, T.M. DeChiara, and M. Goldfarb. 1995. Requirementof FGF-4 for postimplantation mouse development. Science 267: 246-9.Funa, N.S., J. Saldeen, B. Akerblom, and M. Welsh. 2008. Interdependent fibroblast growth factor andactivin A signaling promotes the expression of endodermal genes in differentiating mouseembryonic stem cells expressing Src Homology 2-domain inactive Shb. Differentiation 76: 443-53.Gadue, P., T.L. Huber, M.C. Nostro, S. Kattman, and G.M. Keller. 2005. Germ layer induction fromembryonic stem cells. Exp Hematol 33: 955-64.71
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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
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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
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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 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: FGF4, 5, FGF8b and FGFR1, are expre
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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