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

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The definitive problem of endodermWhen initiating differentiation into endoderm there is a great concern as to whether it hasbecome visceral or definitive endoderm. These cell types share many markers (Sox17, Foxa2,E-cadherin), and it is therefore necessary to test the obtained cell population for the divergingmarkers to make a solid conclusion. In pratice this has proven less important, as induction ofDE is based on addition of activin, in which visceral endoderm (VE) does not form (Yasunagaet al. 2005). However, it is still necessary to show more than the shared markers to drawconvincing conclusions.In the developing embryo, the forming DE cells have traditionally been believed to replace theouter layer of VE, thus forming a homogeneous cell layer. A recent study performing lineagetracingof VE cells in the mouse embryo suggests that DE cells insert into the VE cellpopulation (Kwon et al. 2008). The VE cells are diluted by the faster proliferating DE cells, butVE cells are found in the gut tube lining as late as the 16-18 somite stages. This indicates thatthe VE does indeed contribute to the embryo proper and suggests that it may not be detrimentalto have a small population of VE cells in the differentiating culture after all.FGFR isoform-specific activation in differentiationFGFs in evolution: why are there so many in Mammalia?The temporal and spatial expression of FGFs and FGFRs determine signalling at any giventime. The large number of FGFs and FGFRs may seem excessive since a simpler system wouldbe more ‘cost-efficient’ to the cell. FGFs are found only in multicellular organisms (metazoa),ranging from the nematode C. elegans to mouse and human (Itoh and Ornitz 2004). In C.elegans there are two FGFs and one FGFR and in Drosophila melanogaster there are threeFGFs (Ornitz and Itoh 2001; Itoh and Ornitz 2004). The reason behind the large family of FGFsfound in mouse and human lies in two phases of expansion during evolution. The firstexpansion occurred by genome duplication before chordate evolution. The second expansionoccurred during early vertebrate evolution by two large-scale gene-duplications, possibly of thewhole genome each time (Itoh and Ornitz 2004). This resulted in the 22 FGFs found in miceand humans today. FGFRs co-evolved with the FGFs and they have since evolved splicevariants,which add to the complexity of ligand-receptor specificity and downstream signalling(Itoh and Ornitz 2004). The temporal and spatial expression of FGFs and FGFRs determinesignalling at any given time, and an additional layer of complexity is added by the evolution ofHSs, which are also differentially expressed (Allen and Rapraeger 2003).The many different functions of FGFs during development and adult homeostasis is probablythe basis for the abundance of FGFs and FGFRs found in mammals, as specific regulation oftheir function is important. Most null mutants show different phenotypes, arguing for distinctfunctions of each FGF during development. These specific functions are tissue- and time pointdependent:some FGFs are expressed at the same time in certain tissues, but have distinctfunctions in other tissues, as is the case for the FGF8 family, comprising FGF8, 17 and 18.These are co-expressed in the midbrain-hindbrain junction, but FGF8 and FGF18 showdifferential expression and effects in limb and bone development, respectively (Xu et al. 2000;Liu et al. 2002). This overlapping expression of FGFs belonging to the same sub-family, andtheir shared FGFR-binding properties, argue for a functional redundancy among FGFs (Itohand Ornitz 2004). Redundancy between sub-families is also seen, for example by FGF3 and 8in the induction of otic placode and forebrain development in zebrafish (Liu et al. 2003; Walsheand Mason 2003). In embryonic development, the specific activation of FGFRs is not onlydependent on the concentration of FGFs secreted, but also on their diffusion through theextracellular matrix where HS retains them. The effective dose may therefore be differentamong cells, and could explain e.g. the differential expression of epiblast and primitiveendoderm markers in the ICM, which was recently shown to be FGF concentration-dependent(Yamanaka et al. 2010).84
Overall, the multitude of FGF-signalling is huge and has to be interpreted in the spatio-temporalcontext in which it is investigated. In in vitro ES cell differentiation, however, one can makeuse of redundant functions of the different FGFs as the effect upon differentiation is onlydependent on the expression of FGFRs and not necessarily on the endogenous range of FGFsexpressed. Still, the endogenously expressed FGFs must be taken into account when aiming forthe optimal protocol to ensure no conflicting signalling is taking place in the cells.FGFR-isoforms in DE formation and patterningWe have shown that FGFs activating FGFRc-isoforms increase the numbers of cells expressingmesendoderm markers but reduce the numbers of Sox17-GFP + DE cells. On the other hand,FGFs activating only FGFRb-isoforms have no effect on these cells, most likely due to theirlack of expression. This argues for a beneficial effect of FGFRc-activation during early stageDE-induction, namely in the generation of a mesendodermal population of cells, but aninhibitory effect of these same FGFs during the later DE-specification. Also, the concentrationsof FGFs used in this study may prove to be important. DE-formation is dependent on FGFRsignalling,as inhibition by small molecules inhibits Sox17-GFP + cells. Likewise, highconcentrations of FGFs have an inhibitory effect on this same population, arguing for anoptimal intermediate concentration. Such an intermediate concentration is possibly even belowthe endogenous FGF concentration, as the FGF4 +/– cell line seems to differentiate into DE at ahigher success that the wt or the FGF4 –/– cell line, even when the latter is supplemented with alow concentration of FGF4.Meanwhile, experiments in ‘Step 2’ of the Pdx1-inducing protocol (Chapter 4) show thataddition of FGFs increases the number of Pdx1-GFP + cells, possibly through their mitogeniceffect. Furthermore, FGF7 and 10 function even better than FGF4 in induction of Pdx1-GFP +cells (Nina Engberg and Claude Rescan, unpublished data), arguing that FGFRb-isoforms arepresent and have an additional role to the mitogenic alone. These DE cells are epithelial and assuch probably require activation of FGFRb-isoforms during DE patterning and/ ororganogenesis. This would make sense from a developmental point of view as the developmentof epithelial components in many organs depends on FGF10 for epithelio-mesenchymalinteractions (Min et al. 1998; Ohuchi et al. 2000). Also, FGFRb-isoforms are expressed inepithelial tissues during development (Kathrine Beck Sylvestersen, unpublished data; (Ornitzand Itoh 2001)).Overall, addition of FGFs activating FGFRc-isoforms during mesendoderm formation followedby absence of FGFs during DE-formation and finally FGFRb isoform-activation duringinduction of posterior foregut, i.e. Pdx1-expressing cells, could prove the most beneficial.Successful DE formation in an FGF4 null cell lineAt a low cell density, we see that the FGF4 –/– cell line readily differentiates into DE, showingindependence of FGF4-signalling in induction of differentiation. This was surprising, as aprevious study showed dependence for FGF4 in the induction of ectoderm and mesodermdifferentiation, thus suggesting that FGF4-signalling is needed for cells to leave the pluripotentstate altogether (Kunath et al. 2007). This inhibition of differentiation could be reverted byaddition of FGF4, but not by FGF5, which is expressed early during differentiation andactivates FGFR1c as does FGF4 (Haub and Goldfarb 1991). However, FGF4 additionallyactivates FGFR2c, 3c and 4 and a redundancy by FGF6 or 8(b) may prove significant as thesebind FGFR1c, 2c, 4 and FGFR2c, 3c, 4, respectively, similar to FGF4 (Ornitz et al. 1996;Zhang et al. 2006; Mason 2007).From in vitro studies of mammalian cell cultures, it is known that there is a positive correlationbetween cell density and cellular response, measured by receptor phosphorylation or geneexpression (Polk et al. 1995; Bedrin et al. 1997; Batt and Roberts 1998; Mukhopadhyay et al.1998). On the other hand, an inverse correlation between cell density and gene expression hasbeen shown in other systems (Li and Goldstein 1996; Singh et al. 1996; Posern et al. 1998). InES cell cultures little is known about cell density and gene expression responses. It was shownthat cells grown at high densities have a pool of β-catenin located at the cell surface, where it is85
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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
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Figure 1-3: Regional expression of
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The pluripotent stateThe pluripoten
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There are four membrane-bound FGFRs
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2. AimsThe aim of this study was to
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Developmental Biology 330 (2009) 28
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288 M. Hansson et al. / Development
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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: 6. General discussionEndoderm diffe
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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