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

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.