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

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FGFR(IIIc)-activation induces mesendoderm but isdispensable for definitive endoderm formation in mouseembryonic stem cellsJanny Marie L. Peterslund and Palle Serup*Department of Stem Cell Biology, Hagedorn Research Institute, Niels Steensens Vej 6, DK-2820 Gentofte, Denmark.Running title: FGF(R)s and mesendoderm differentiation in mESCsKeywords: mouse embryonic stem cell, fibroblast growth factor (FGF), FGF receptors,mesendoderm, definitive endoderm, FGF4 –/– cell line*Corresponding author:Dr. Palle SerupHagedorn Research InstituteNiels Steensens Vej 6DK-2820 GentofteDenmarkE-mail: pas@hagedorn.dkPhone: +45 44439822Fax: +45 4443800058
AbstractProgress in embryonic stem (ES) cell research over the last decade has outlined a possible cellbased intervention strategy in diabetes based on the hierarchical differentiation of embryonicstem cells into insulin producing beta cells. The definitive endoderm (DE) cell type constitutesan essential milestone in this differentiation pathway and fibroblast growth factor (FGF)-signaling drives the formation of DE in the mouse ES cell model system. More specifically, ithas been shown that FGF4 is crucial for cells to leave the pluripotent state and differentiate toectoderm and mesoderm cell lineages. The FGF system counts several receptor isoforms with apossible functional redundant role in conducting the differentiation signal. Here, we investigatethe spatio-temporal dynamics of FGF receptor (FGFR) distribution in the forming DE and findthat FGFR(III)c-isoforms are highly represented in the whole culture, whereas FGFR2(III)band FGFR4 are found in the DE fraction, specifically. The FGFR(III)c isoform-activating FGFsinduce mesendoderm markers T and Gsc, but reduce the DE marker Sox17 whereas FGFsactivating FGF(III)b-isoforms have no effect on either cell type. Notably, FGFR(III)c isoformactivatingFGFs exhibit strong mitogenic effects on ES cells early in the differentiation periodwhere FGFs activating FGFR(III)b-isoforms have only a moderate mitogenic potentialconfined to the late differentiation period. Interestingly, when applying our DE inductionprotocol onto an FGF4 –/– cell line, we find that cells readily differentiate into endoderm cellswithout ectopic administration of FGF4. By antibody staining and qPCR analyses for definitiveand visceral endoderm markers, we show that this endoderm is definitive rather than visceral.We conclude that FGFR(III)c-isoform activation selectively drives the differentiation of mEScells towards mesendoderm and that FGF4 is dispensable for the final differentiation step intoDE.59
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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: 5. Paper IIFGFR(IIIc)-activation in
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 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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