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

More documents

Recommendations

Info

alternative splicing of exons 8 and 9. The C-terminal half of the DIII domain is encoded by exon 8 to generate theFGFR(III)b isoforms while the C-terminal half of DIII is encoded by exon 9 to generate the FGFR(III)c isoforms.Modified from (Itoh and Ornitz 2004; Eswarakumar et al. 2005).HS, downstream signalling pathways and regulationHeparan sulfates (HSs) are proteoglycans consisting of repeated subunits of D-glucosamine anddisaccharides and are embedded in the extracellular matrix on the cell surface. Mutations in genesinvolved in the synthesis or modulation of HS results in developmental defects in mice, mostlikely due to impaired FGF-signalling (Bullock et al. 1998; Ornitz 2000; Kraushaar et al. 2010)).HS stabilizes FGFs to thermal denaturation, proteolysis and limit their diffusion and release tointerstitial spaces (Moscatelli 1987; Flaumenhaft et al. 1990). This leads to increased 1:1FGF:FGFR complex formation which then results in a transient receptor dimerization andsignalling from this 2:2 FGF:FGFR complex (Figure 1-6B; (Hsu et al. 1999; Plotnikov et al.1999; Pye and Gallagher 1999)).Intracellular phosphorylation of the kinase-domains leads to activation of intracellular signaltransduction pathways. Most commonly, the Ras/MAPK pathway is activated upon FGFsignalling,but also the PLCγ/Ca 2+ and PI3K/AKT pathways are activated (Bottcher and Niehrs2005). For activation of the Ras/MAPK pathway, FRS2 is recruited and forms a complexactivating Ras. Pathway activity ultimately leads to nuclear translocation of MAPK and activationof target genes, such as AP1, c-myc and ETS transcription factors (Wasylyk et al. 1998).Activation of PLCγ leads to intracellular release of Ca 2+ and activation of phosphokinase C (PKC;(Pawson 1995)). Finally, activation of PI3K through either direct interaction with the FGFR or bycomponents of the Ras/ MAPK pathway leads to activation of AKT (Carballada et al. 2001).FGF-signalling is regulated by members of the sprouty and sprouty-related EVH1 protein(SPRED) families by a feedback mechanism that regulates MAPK-signalling through receptortyrosine kinase binding (Hacohen et al. 1998; Lim et al. 2002). Furthermore, FGF-activity ismodified through a tight regulation of HS-synthesis and certain transmembrane regulators (SEFand FLRT), which interrupt FGF-FGFR complex formation or downstream signalling (Bottcherand Niehrs 2005). Synthetic small molecules such as SU5402 and PD173074 inhibit most FGFRsignallingwith some or little secondary effects, respectively (Mohammadi et al. 1997;Mohammadi et al. 1998).To date, the role of FGF-FGFR signalling in directed differentiation towards cell types of theendoderm germ layer has been investigated as a general activation or inhibition of FGF-FGFRsignals. The role of individual FGF- and FGFR family members in differentiation towards DEremains to be elucidated.18
2. AimsThe aim of this study was to investigate the role of FGF-signalling in directed differentiation ofmouse ES cells towards i) mesendoderm formation, and ii) definitive endoderm formation andpatterning.We applied a mono-layer, serum-free culture system in which differentiation of mouse ES cellstowards mesendoderm was achieved by addition of BMP4 and a range of activin-concentrationsto the culture medium. Formation of definitive endoderm was reached using high concentrationsof activin and further patterning hereof was done using various growth factors and inhibitors. Weinvestigated the influence of FGFs on this system by addition of a range of FGFs, small moleculeFGF-inhibitors and soluble FGFRs. We analysed the resulting cell populations by using greenfluorescent protein (GFP)-linked reporter cell lines and knockout cell lines, immunecytochemistry,RT-PCR and qPCR.19
Page 1: PhD thesisCand.scient. Janny Marie
Page 5: ResuméSukkersyge er en sygdom der
Page 9: 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: There are four membrane-bound FGFRs
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 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,
show all

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

Create successful ePaper yourself

Delete template?

Save as template?