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

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292 M. Hansson et al. / Developmental Biology 330 (2009) 286–304majority of the cells were CXCR4 hi (Fig. S3A), indicating that most cells, inboth vehicles and activin-treated cultures, are embryonic rather thanextraembryonic in nature. Furthermore, significant levels of Sox7 (SRYboxcontaining gene 7) transcripts, which is exclusively expressed in theextraembryonic part of the endoderm in the gastrula stage embryo(Kanai-Azuma et al., 2002), could only be detected in Wnt3a-treatedcultures but not in vehicle, BMP4-, or activin-treated cultures (Fig. S3B).Having established the embryonic nature of the ES cell progeny inour cultures we next examined the expression of a number of germlayer specific markers. We initially analyzed expression of thetranscription factor gene Foxa2 and the epithelial marker E-cadherin(E-cad; Cdh1), both of which are expressed in developing endoderm(Ang et al., 1993; Sasaki and Hogan, 1993). Immunofluorescentstaining of cells grown in 3 or 100 ng/ml activin showed that thesecultures contained many Foxa2 + Cdh1 + cells compared to vehicletreatedsamples (Fig. 2). Notably, addition of BMP4 (10 ng/ml) but notWnt3a (100 ng/ml) was able to drastically reduce the number ofFoxa2 + Cdh1 + cells induced by activin (Fig. 2). Analysis of VEGFreceptor-2 (Kdr or Flk1) expression using Flk1-LacZ ES cells revealedthat both BMP4 (with or without 100 ng/ml activin) and Wnt3a werecapable of inducing Flk1-expressing cells (Fig. 2), indicative ofmesoderm formation (Ema et al., 2006).Wnt signaling augments the development of Sox17-expressing definitiveendoderm induced by activinBased on the analysis of Foxa2 and Cdh1 expression it was not clearif the concentration of activin used influenced subsequent differentiationtowards DE. Furthermore, analyses of Foxa2 and Cdh1 expressioncannot distinguish between DE from different A–P positions. Wetherefore examined the number of Gsc Gfp/+ cells that co-expressedGFP, Cdh1, and Sox17 by ICC as an indicator of anterior DE (ADE), inresponse to varying doses of activin with or without additional BMP4,Wnt3a, or Dkk1 treatment. Notably, we found that 100 ng/ml activinresulted in higher numbers of Cdh1 + GFP + Sox17 + triple positive cellsthan seen with 3 ng/ml activin (Fig. 3A, compare panels b and c),supporting that efficient formation of ADE depends on the activinconcentration (Yasunaga et al., 2005). Treatment with 3 ng/ml activinresulted in many Cdh1 + cells but the majority of these were not coexpressingGFP or Sox17 and most likely represent undifferentiated EScells (see below). Most of the GFP + cells generated in response to 3 ng/ml activin were Cdh1 − Sox17 − , suggesting that they may representmesoderm (Fig. 3A, panel b). Similarly, after treatment with Wnt3aalone (100 ng/ml) most GFP + cells were Cdh1 − Sox17 − (Fig. 3A, paneld). We tested if Wnt signaling was required for the development ofFig. 4. The requirement for canonical Wnt signaling during activin-induced Sox17 expression is more pronounced in aggregate culture than in adherent culture, and Dkk1 inhibitsnodal-induced Sox17 expression more than activin-induced Sox17 expression. (A) Nodal/activin and Wnt signaling interactions were analyzed in Mixl1 Gfp/+ and Gsc Gfp/+ cells atdays 2–6 of differentiation using flow cytometry. (B) Gsc Gfp/+ cells cultured in the presence of Dkk1 prior to and during activin induction were analyzed by flow cytometry. (C) Gsc Gfp/+cells were induced to form embryoid bodies in the presence of the indicated growth factors and analyzed for GFP expression by flow cytometry after 5 days of culture. (D) Sox17 Gfp/+cells cultured for 5 days were analyzed for GFP expression by flow cytometry. The mean % GFP + cells ±S.E.M. of three independent experiments is presented. (E) Sox17 Gfp/+ cellscultured for 2–7 days in the presence of activin (30 or 100 ng/ml) were analyzed for GFP expression by flow cytometry at the indicated time points. (F) Sox17 Gfp/+ cells were induced toform embryoid bodies in the presence of the indicated growth factors and analyzed for GFP expression by flow cytometry after 5 days of culture. The mean % GFP + cells ±standarddeviation of three independent experiments is presented for all flow cytometric analyses unless otherwise noted.
M. Hansson et al. / Developmental Biology 330 (2009) 286–304293Cdh1 + GFP + Sox17 + triple positive cells in response to activin andfound that such cells were still generated efficiently in response to100 ng/ml activin if Dkk1 was included (Fig. 3A, panel f). In contrast,addition of BMP4 completely prevented the development of such cells(Fig. 3A, panel g). We often found clusters of Cdh1 + GFP − Sox17 − cellsin cultures treated with different combinations of activin and Wnt3a(Fig. 3A, panels b–e). Immunocytochemistry revealed that manyCdh1 + Gsc − cells were Oct4 + and thus likely represent undifferentiatedES cells (Fig. 3B). Attempts to quantify the relative number ofGsc-GFP + Cdh1 + and Gsc-GFP − Cdh1 + cells by FACS under the variousconditions failed due to problems with achieving reliable FACS datausing the Chd1 monoclonal antibodies available.Although it was not evident from the above experiments with theGsc Gfp/+ cells that canonical Wnt signaling influenced the expressionof PS markers or the formation of DE in response to activin we wantedto examine closer if Wnt activity was required for expression of otherPS markers and DE formation in our ES cell cultures since gastrulationand thereby also endoderm formation requires Wnt3 activity in vivo(Barrow et al., 2007; Liu et al., 1999) and because we detectedexpression of Wnt3 and Wnt3a in our differentiating ES cell cultures(Fig. S4). To test this notion, we first cultured Mixl1 Gfp/+ cells withactivin or Wnt3a in the presence or absence of 320 ng/ml Dkk1 or1 μM of the ALK4/5/7-specific inhibitor SB431542, respectively. Theconcentration of the inhibitors was titrated by using a Wnt- or activinresponsiveluciferase assay (data not shown), choosing the concentrationthat blocked the response to exogenous Wnt3a or activin,respectively, without causing non-specific toxicity in ES cells. Whenanalyzing the number of Mixl1-GFP + cells after 4 days of activintreatment (30 ng/ml) we found that these were significantly reducedwhen Dkk1 was included (26±3% vs. 4±3%; pb0.05; Fig. 4A).Similarly, the number of Mixl1-GFP + cells induced by 100 ng/mlWnt3a was reduced from 11±4% on day 4 to 3±4% by simultaneousSB431542 treatment (Fig. 4A). Thus, activin and Wnt3a act cooperativelyto induce Mixl1 expression and both signaling pathways arerequired for Mixl1 expression in ES cell progeny. Although Wnt3a onlyinduced low numbers of Gsc-expressing cells, these were dependenton endogenous nodal/activin signaling as SB431542 significantlyinhibited the development of Gsc-GFP + cells in response to Wnt3a(Fig. 4A). In contrast, FACS analyses confirmed that activin-inducedGsc expression was not inhibited by Dkk1 treatment. Cultures ofGsc Gfp/+ cells contained 17±4% Gsc-GFP + cells at day 5 when grownin 30 ng/ml activin and 23±12% when cultured in the presence ofactivin and Dkk1 (Fig. 4A) consistent with the above mentionedimmunofluorescent analysis that demonstrated that Gsc-GFP + Sox17 +Cdh1 + triple positive cells were efficiently generated in response toactivin treatment, regardless of the presence of Dkk1. UndifferentiatedES cells have a low level of active canonical Wnt signaling despite thelack of exogenously added Wnt factors (Sato et al., 2004). To test if thislow level of Wnt signaling has any influence on the later activation ofGsc expression we cultured undifferentiated Gsc Gfp/+ cells for threepassages in media containing Dkk1 before differentiation was inducedby removing LIF and BMP4 and adding activin and Dkk1 for 5 days. Wefound that inclusion of Dkk1 prior to differentiation did not preventactivin from efficiently inducing Gsc expression (Fig. 4B). Furthermore,Dkk1 failed to prevent activin from inducing Gsc-GFP + cells inaggregate culture (Fig. 4C).To obtain quantitative data on the number of DE cells after growthfactor treatment we subjected a Sox17 Gfp/+ reporter line (Kim et al.,2007) to our differentiation protocol. At mid-streak stage Sox17expression marks the definitive endoderm forming adjacent to theanterior end of the PS. Simultaneous with the movement of DE to theanterior region of the gastrula, the Sox17 expression domain expandsto include the endoderm underlying the neural plate of the early tailbud-stageembryo (Kanai-Azuma et al., 2002). Thus, Sox17 is an earlymarker that similar to genes such as Hex, Foxa2, and Cer1, areexpressed simultaneously in the anterior visceral endoderm and theDE in the embryonic part of the gastrulating embryo. Sox17 Gfp/+ cellsexpress a low level of GFP (Sox17-GFP Lo ) when kept undifferentiatedin the presence of LIF and BMP4 (not shown). Differentiation underneural promoting conditions (Ying et al., 2003a) results in thedevelopment of a Sox17-GFP − population and some remainingSox17-GFP Lo cells while treatment with activin for 5 days inducesSox17-GFP Hi and Sox17-GFP − populations in addition to a remainingSox17 Lo population (Fig. S1). Increasing concentrations of activinresulted in development of progressively more Sox17-GFP Hi cells withthe highest numbers reached with 30 and 100 ng/ml of activin (Fig.4D). We observed an increase in Sox17-GFP Hi cells over time, peakingat day 5, followed by a modest decrease at days 6 and 7 (Fig. 4E). Theinduction of Sox17-GFP Hi cells was at least partly dependent on Wntsignaling as treatment with Dkk1 reduced the number of GFP Hi cellsby ∼50% at the highest activin concentration (Fig. 4D). Notably, thenumber of Sox17-GFP Hi cells induced by 1 μg/ml nodal appeared morestrongly reduced in response to Dkk1 treatment than did a similarnumber of Sox17-GFP Hi cells induced by 30 and 100 ng/ml of activin(Fig. 4D). Furthermore, when differentiation was performed inaggregate culture, which may rely more on endogenous signaling(Sachlos and Auguste, 2008; ten Berge et al., 2008), the number ofactivin-induced Sox17-GFP Hi cells were strongly reduced by Dkk1treatment (Fig. 4F). As expected, the addition of BMP4 preventedactivin-induced formation of Sox17-GFP Hi cells (pb0.001), whileaddition of Wnt3a resulted in a marginal, but significant (pb0.05)increase in the development of Sox17-GFP Hi cells (Fig. 4D).To further define the time at which canonical Wnt signaling wasrequired for the formation of T-, Gsc- and Sox17-GFP Hi cells, wecultured the cells with activin for three (T Gfp/+ )orfive (Gsc Gfp/+ andSox17 Gfp/+ ) days and added either Wnt3a or Dkk1 for shorter periodsFig. 5. Wnt signaling is required during late stages of activin-induced definitiveendoderm formation. ES cells were cultured in serum-free medium with 100 ng/mlactivin and supplemented with 100 ng/ml Wnt3a or 320 ng/ml Dkk1 for a variablenumber of days. T Gfp/+ cells (A) were cultured for 3 days and Gsc Gfp/+ (B) and Sox17 Gfp/+(C) cells were cultured for 5 days before being analyzed for GFP expression by flowcytometry. The mean % GFP + cells ±standard deviation of three independentexperiments is presented.
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 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
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Opposite, Figure 6: In the absence
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6. General discussionEndoderm diffe
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Overall, the multitude of FGF-signa
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transplantation is the spread of an
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AcknowledgementsThe work presented
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Chambers, I., D. Colby, M. Robertso
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Hawkins, V.J. Wroblewski, D.S. Li,
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Nishikawa, S.I., S. Nishikawa, M. H
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Tanimizu, N., H. Saito, K. Mostov,
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