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

More documents

Recommendations

Info

and 9 being the most effective ones (up to 20.400 cells/ cm 2 or a 3 times increase compared tothe activin-treated cells). By means of 5-ethynyl-2’-deoxyuridine (EdU)-incorporation (Salicand Mitchison 2008) we could quantify the proliferation of cells (see Figure S1 for gating andcontrols). We found that the absolute number of proliferating cells was higher at day 3 than day5, by an approximate 4 time increase for the activin-treated cells and a 4-7 times increase forthe conditions supplemented with FGFs. At day 3, the relative number of proliferating cells didnot differ much from the activin-treated sample (Figure 4A) except for a small reduction whenadding FGF1 or not adding any growth factors at all, i.e. the BSA control.On day 5, there was app. 80.000 cells/cm 2 in samples treated with activin alone. This numberincreased 1,6 times to app. 130.000 cells/ cm 2 when adding FGF2 (Figure 4B). This indicatesthat the effect of FGFs on cell growth is decreasing over time probably because more cells aredifferentiated at day 5. At this stage, especially FGF1 and 2 positively affect cell numbers.These have been shown also in other cell systems to have the largest mitogenic effect (Ornitz etal. 1996; Zhang et al. 2006). By EdU-incorporation, we saw a 50% higher proliferation rate inthe BSA control than with cells treated with activin, showing 25% and 15% total proliferation,respectively (Figure 4B). The FGFs most frequently reduced proliferation; FGF1, 2, 4 and 6 by40 – 50% and FGF8b, 9 and 10 showing moderate reductions. These data indicate that the maineffect seen by FGF1, 2, 4, 6 and 9 on proliferation occurs prior to day 3, and that most of thecells in these cultures have left the proliferative state by day 5.FGF4 is dispensable for the formation of endodermOf the FGFs tested, FGF4 and 6 binding FGFRc-isotypes only, increase BMP4 and activininducedexpression of T and Gsc the most. FGF4 is important during gastrulation where it isresponsible for the cell movements through the PS (Bottcher and Niehrs 2005). We wanted toinvestigate whether an FGF4 –/– cell line (Wilder et al. 1997) would be able to i) differentiateinto the endoderm lineage; and ii) promote differentiation of DE cell types, in that the latterwould not be inhibited by the FGFRc isoform-activating FGF4. When cells were maintained aspluripotent cells, the FGF4 –/– cell line showed a different cell morphology than the E14 andFGF4 +/– cell lines. Cells grew in small, very dense clusters indicative of pluripotent cells(Figure 5A) and growth rates were slower, confirming the mitogenic effect of FGF4. Cellsstained positive for the pluripotency marker OCT4 and negative for the endoderm markerSox17, similar to the wt and heterozygote cell lines.We subjected the wt E14, FGF4 +/– and FGF4 –/– cell lines to our DE induction-protocol.Through antibody staining of SOX17, E-cadherin (Ecad) and FOXA2 we identified cells of anendoderm origin. Foxa2 is expressed during embryonic development in the anterior primitivestreak, the newly formed definitive endoderm and is maintained throughout most matureendoderm-derived tissues (Kaestner et al. 1994; Weinstein et al. 1994). FGF4 +/– cells behavedmuch like E14 wt cells, showing vast numbers of OCT4 – /SOX17 + and SOX17 + /FOXA2 + /Ecadherin+ cells by day 5 (Figure 5B). In each cell line, differentiation was not 100% and smallclusters of tightly connected, undifferentiated OCT4 + cells persisted in the culture. Remarkably,FGF4 –/– cells readily differentiated along the endoderm lineage, showing mainly OCT4 –/SOX17 + cells and only a few more OCT4 + cells than the wt and heterozygote cell lines (Figure5A). When administrating FGF4 protein ectopically, the number of OCT4 + cells was reduced towt levels. There were comparable numbers of SOX17 + /FOXA2 + /E-cadherin + in the knock-outcell line and wt or heterozygote cell lines, and these did not change by the addition of FGF4 tothe medium (Figure 5B).Thus, we conclude that FGF4 is dispensable for differentiation of mES cells along theendoderm lineage and for cells to leave the pluripotent state when the differentiation protocolapplied includes activin.Definitive endoderm is formed in the absence of FGF4Looking for expression of Sox7 and Thermostable direct hemolysin gene (Tdh), markers ofvisceral endoderm (VE; (Sherwood et al. 2007)), we wanted to see whether the endodermformed was definitive or visceral. Staining for endoderm and VE markers in the66
undifferentiated cells, we found that E-cadherin was abundantly expressed but not SOX17 orSOX7 expression (Figure 6A). When cells had undergone differentiation for five days, E14 andFGF4 +/– cell lines were SOX17 + /E-cadherin + /SOX7 – indicative of a DE identity (Figure 6B).FGF4 –/– cells mainly showed the same expression pattern but also showed small clusters ofSOX17 + /E-cadherin + /SOX7 + cells indicating formation of VE in these areas (Figures 6B andS2). When adding ectopic FGF4 to the growth medium, hardly any SOX7 + cells were seen inthe culture (Figure 6B).By qPCR analyses, we confirmed the DE phenotype of all three cell lines but did not seeevidence of a VE sub-population in the FGF4 –/– cell culture. We saw a large induction of Sox17and especially Cxcr4 transcription upon DE-induction (Figure 6C) indicative of the formationof DE rather than VE. The VE marker Sox7 showed similar levels of expression in thepluripotent and differentiated states for all three cell lines and the absolute amount oftranscription was very low, i.e. similar to Sox17-expression levels in mES cells. Tdh wasexpressed at intermediary levels in mES cells but was down-regulated upon DE-induction.Interestingly, the FGF4 +/– cell line showed a somewhat elevated expression of Sox17 and Cxcr4both in the pluripotent and differentiated states whereas the FGF4 –/– cell line expressed thesegenes at levels comparable to wt cells. This suggests that FGF4 acts as a morphogen and that anintermediary expression level most efficiently induces DE-formation whereas high or lowlevels in the wt and FGF4 –/– cell lines, respectively, fail to do so. In summary, we conclude thatFGF4-signalling is dispensable for induction of DE in FGF4 –/– mES cells and that anintermediary FGF4-koncentration may be beneficial to DE formation.DiscussionDuring embryonic development, epithelial tissues express FGFRb-isoforms whilemesenchymal tissues express mainly FGFRc-isoforms (Ornitz and Itoh 2001). FGFsspecifically activating FGFRb-isoforms (i.e. FGF7 and 10) are mainly expressed in themesenchyme and FGFs activating FGFRc-isoforms (i.e. FGF4, 8 and 9) are mainly expressedin the epithelium, resulting in specificity during reciprocal epithelial-mesenchymal signalling indeveloping organs such as the lung, cecum, salivary glands and pancreas (Stark et al. 1991;Orr-Urtreger et al. 1993; Colvin et al. 2001; Ornitz and Itoh 2001; Elghazi et al. 2002; Manfroidet al. 2007). Data from mouse embryos obtained in our group confirm findings by others that inthe developing pancreas FGFR2b and 4 are expressed in the epithelium, whereas FGFR1c and2c are expressed in the mesenchyme (Kathrine Beck Sylvestersen, personal communication;(Stark et al. 1991; Orr-Urtreger et al. 1993; Elghazi et al. 2002)). In the present report we showhow FGFR2b and 4 are up-regulated in the Sox17-GFP Hi or DE-fraction during DE formationin mES cells. FGFR1c, 2c and 3c were up-regulated in the Sox17-GFP Lo fraction alone or inboth fractions. The reason for this unexpected high expression of FGFRc-isoforms in both theSox17-GFP Lo and Sox17-GFP Hi fractions may be that the Sox17-GFP Lo fraction contains alarge pool of cells not yet committed to an epithelial fate or cells that are undifferentiated. Thefraction is characterised by high numbers of non-mesodermal Sox17-GFP –/Lo / FLK1 – /EpCAM + expressing cells. These cells may still undergo differentiation and maturation andthus have the potential to later become epithelial endoderm or they may contain mesenchymalcells to some degree. The Sox17-GFP Lo expressing cells may have a function in the culturesimilar to that of the mesenchyme in pancreatic development, i.e. signalling to direct cell fate ofDE or foregut progenitors. Accordingly, preliminary data in our lab show that if Sox17-GFPcells are sorted after 5 days of DE formation, then re-plated and cultured under conditionsinducing pancreatic progenitors (Hansson et al. 2009), the Sox17-GFP Hi fraction will looseGFP expression and fail to turn on foregut markers such as SOX2 and PDX1 (Maria Winzi,personal communication). On the contrary, cells of the Sox17-GFP Lo fraction will turn on GFPexpression and differentiate into SOX2 + , NKX6.1 + and PDX1 + positive cells similar to theunsorted culture. This suggests that signals from the Sox17-GFP Lo to the Sox17-GFP Hi cellsinclude FGFs and is crucial for their propagation and ability to differentiate further.67
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 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: influence on the numbers of Sox17-G
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 ...

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?