FGF-signalling in the differentiation of mouse ES cells towards ...
FGF-signalling in the differentiation of mouse ES cells towards ...
FGF-signalling in the differentiation of mouse ES cells towards ...
You also want an ePaper? Increase the reach of your titles
YUMPU automatically turns print PDFs into web optimized ePapers that Google loves.
294 M. Hansson et al. / Developmental Biology 330 (2009) 286–304dur<strong>in</strong>g <strong>the</strong> 5 day activ<strong>in</strong> treatment. To monitor <strong>the</strong> effectiveness <strong>of</strong>Dkk1 treatment <strong>in</strong> reduc<strong>in</strong>g canonical Wnt signal<strong>in</strong>g we firstgenerated an <strong>ES</strong> cell l<strong>in</strong>e stably transfected with a SuperTOP-Ceruleanreporter (SuTOP-CFP) and established its ability to report Wntsignal<strong>in</strong>g <strong>in</strong> E10.5 chimeric embryos after <strong>in</strong>jection <strong>in</strong>to E3.5blastocysts and implantation <strong>in</strong>to pseudo-pregnant females. Asshown <strong>in</strong> Fig. S5A, native Cerulean fluorescence can be observed <strong>in</strong>several sites known to harbor active Wnt signal<strong>in</strong>g at this stage,<strong>in</strong>clud<strong>in</strong>g <strong>the</strong> peripheral aspects <strong>of</strong> <strong>the</strong> otic vesicles (Maretto et al.,2003). When SuTOP-CFP <strong>cells</strong> were cultured <strong>in</strong> activ<strong>in</strong> we found thatCFP + colonies developed at day 3 even <strong>in</strong> <strong>the</strong> absence <strong>of</strong> exogenousWnt addition and that additional treatment with Dkk1 reduced orabolished reporter activity depend<strong>in</strong>g on <strong>the</strong> duration <strong>of</strong> treatment(Fig. S5B). Similarly, when SuTOP-CFP <strong>cells</strong> were assayed at day 5, wefound that Dkk1 treatment at days 3–4 or4–5 <strong>in</strong>hibited reporteractivity (Fig. S5C). Thus, treatment with Dkk1 for as little as 1 to 2 daysat <strong>the</strong> concentration used appeared quite effective <strong>in</strong> suppress<strong>in</strong>gSuTOP reporter activity.We <strong>the</strong>n analyzed <strong>the</strong> number <strong>of</strong> T-GFP + <strong>cells</strong> develop<strong>in</strong>g <strong>in</strong>response to activ<strong>in</strong> when Wnt signal<strong>in</strong>g was experimentally perturbed.T-GFP + cell numbers were augmented by Wnt3a with <strong>the</strong>largest effect reached when Wnt3a was added only at day 3.Treatment with Dkk1 on <strong>the</strong> o<strong>the</strong>r hand reduced <strong>the</strong> number <strong>of</strong> T-GFP + <strong>cells</strong> most effectively when <strong>in</strong>cluded <strong>in</strong> that last part <strong>of</strong> <strong>the</strong>three day period (Fig. 5A). Nei<strong>the</strong>r stimulat<strong>in</strong>g nor <strong>in</strong>hibit<strong>in</strong>g Wntsignal<strong>in</strong>g <strong>in</strong> Gsc Gfp/+ <strong>cells</strong>, by treatment with Wnt3a and Dkk1,respectively, resulted <strong>in</strong> a significant change <strong>in</strong> <strong>the</strong> number <strong>of</strong> activ<strong>in</strong><strong>in</strong>ducedGFP + <strong>cells</strong> irrespective <strong>of</strong> <strong>the</strong> treatment period (Fig. 5B).However, treatment <strong>of</strong> Sox17 Gfp/+ <strong>cells</strong> with Wnt3a prior toappearance <strong>of</strong> GFP + <strong>cells</strong> reduced <strong>the</strong> number <strong>of</strong> Sox17-GFP Hi <strong>cells</strong>,while later Wnt3a treatment had no effect. Conversely, treatment withDkk1 reduced <strong>the</strong> number <strong>of</strong> Sox17-GFP Hi <strong>cells</strong> only if present after <strong>the</strong>first appearance <strong>of</strong> <strong>the</strong>se (Fig. 5C).We next used siRNA mediated knock-down <strong>of</strong> β-caten<strong>in</strong> (encodedby Ctnnb1) to confirm that <strong>in</strong>hibition <strong>of</strong> canonical Wnt signal<strong>in</strong>g at <strong>the</strong>latter part <strong>of</strong> <strong>the</strong> 5 day activ<strong>in</strong> stimulation period could suppress <strong>the</strong>appearance <strong>of</strong> Sox17-GFP Hi <strong>cells</strong> and to fur<strong>the</strong>r test <strong>the</strong> Gsc Gfp/+ celll<strong>in</strong>e which appeared refractory to Wnt <strong>in</strong>hibition <strong>in</strong> previousexperiments. We transfected Gsc Gfp/+ and Sox17 Gfp/+ <strong>cells</strong> withcontrol and two different Ctnnb1 siRNAs at day 2 <strong>of</strong> <strong>differentiation</strong>and assayed β-caten<strong>in</strong> expression by western blott<strong>in</strong>g at days 3 and 5.In Gsc Gfp/+ <strong>cells</strong> we found a reduction <strong>of</strong> β-caten<strong>in</strong> expression at day 3which was normalized at day 5. siRNA treatment appeared moreeffective <strong>in</strong> Sox17 Gfp/+ <strong>cells</strong> with strong <strong>in</strong>hibition <strong>of</strong> β-caten<strong>in</strong>expression at day 3 which was only partly recovered by day 5(Fig. 6A). FACS analysis at day 5 showed a reduction <strong>in</strong> <strong>the</strong> number <strong>of</strong>Gsc-GFP + <strong>cells</strong> after β-caten<strong>in</strong> knock-down, although this onlyreached significance <strong>in</strong> Ctnnb1 siRNA2 treated samples (pb0.05,Fig. 6B). Fur<strong>the</strong>rmore, Q-RT-PCR analysis <strong>of</strong> Gsc Gfp/+ <strong>cells</strong> after β-caten<strong>in</strong> knock-down did not reveal significant changes <strong>in</strong> expression<strong>of</strong> Lhx1 and Chrd (Fig. S6).However, <strong>in</strong> agreement with <strong>the</strong> results obta<strong>in</strong>ed with Dkk1treatment, we found a prom<strong>in</strong>ent reduction <strong>in</strong> <strong>the</strong> number <strong>of</strong> Sox17-GFP Hi <strong>cells</strong> at day 5, <strong>in</strong> both siRNA1 and siRNA2 treated samples(pb0.05, Fig. 6B).Activ<strong>in</strong> dose-dependently <strong>in</strong>duces an anterior gene expression patternTo determ<strong>in</strong>e whe<strong>the</strong>r <strong>the</strong> DE formed <strong>in</strong> <strong>the</strong> presence <strong>of</strong> highconcentrations <strong>of</strong> activ<strong>in</strong> was anterior or posterior <strong>in</strong> character weanalyzed <strong>the</strong> expression <strong>of</strong> a number <strong>of</strong> markers display<strong>in</strong>g differentialexpression depend<strong>in</strong>g on <strong>the</strong> anterior–posterior position <strong>of</strong> <strong>the</strong> <strong>cells</strong>.RT-PCR analyses at day 5 showed that activ<strong>in</strong>, regardless <strong>of</strong>concentration, could <strong>in</strong>duce expression <strong>of</strong> genes associated withanterior cell fates, <strong>in</strong>clud<strong>in</strong>g Lefty1, Hex, (Mart<strong>in</strong>ez-Barbera et al.,2000) and Otx2, (Rh<strong>in</strong>n et al., 1998). However, robust expression <strong>of</strong>Fig. 6. Inhibition <strong>of</strong> canonical Wnt signal<strong>in</strong>g <strong>in</strong>hibits activ<strong>in</strong>-<strong>in</strong>duced Sox17-GFP Hi def<strong>in</strong>itive endoderm formation, but has little effect on Gsc expression. (A) Western blot analysis <strong>of</strong>β-caten<strong>in</strong> expression after siRNA mediated knock-down. Two different siRNAs target<strong>in</strong>g β-caten<strong>in</strong> (siRNA1 and siRNA2) or a scrambled control siRNA were <strong>in</strong>troduced to Gsc Gfp/+and Sox17 Gfp/+ cell l<strong>in</strong>es on day 2 and β-caten<strong>in</strong> levels assayed at day 3 and 5. An anti-β-act<strong>in</strong> western blot served as load<strong>in</strong>g control. (B) siRNA treated Gsc Gfp/+ and Sox17 Gfp/+ <strong>cells</strong>were analyzed for GFP expression by flow cytometry after 5 days <strong>of</strong> serum-free culture supplemented with 0, 3 or 100 ng/ml activ<strong>in</strong>. The mean % GFP + <strong>cells</strong> ±standard deviation <strong>of</strong>three <strong>in</strong>dependent experiments is presented. Untransfected and mock transfected controls are also shown.