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 ...
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M. Hansson et al. / Developmental Biology 330 (2009) 286–304291expression <strong>of</strong> <strong>the</strong> PS marker Mixl1 (Pearce and Evans, 1999) was<strong>in</strong>duced by activ<strong>in</strong>, <strong>the</strong> number <strong>of</strong> Mixl1-GFP + <strong>cells</strong> was <strong>in</strong>dependent<strong>of</strong> <strong>the</strong> activ<strong>in</strong> concentration (Fig. 1C).In vivo, BMP4 is a ventraliz<strong>in</strong>g agent, act<strong>in</strong>g dur<strong>in</strong>g gastrulation to<strong>in</strong>duce T and repress Gsc expression (Fa<strong>in</strong>sod et al., 1994; Jones et al.,1996; Ste<strong>in</strong>beisser et al., 1995). We found a strong but transient<strong>in</strong>duction <strong>of</strong> T expression <strong>in</strong> response to BMP4 (Fig. 1A). Peak<strong>in</strong>g at day3, we observed 48±15% T-GFP + <strong>cells</strong>, which was significantly higherthan detected <strong>in</strong> vehicle-treated <strong>cells</strong> (pb0.05). The <strong>in</strong>duction <strong>of</strong> Texpression by BMP4 was observed regardless <strong>of</strong> <strong>the</strong> presence orabsence <strong>of</strong> activ<strong>in</strong>. However, <strong>the</strong> activ<strong>in</strong>-mediated <strong>in</strong>duction <strong>of</strong> Gsc-GFP + <strong>cells</strong> at day 5 was strongly <strong>in</strong>hibited by BMP4 (pb0.05),irrespective <strong>of</strong> <strong>the</strong> activ<strong>in</strong> concentration (Fig. 1B). Cultures conta<strong>in</strong><strong>in</strong>gBMP4 never conta<strong>in</strong>ed more than 10±6% Gsc-GFP + <strong>cells</strong>, which iscomparable to vehicle-treated <strong>cells</strong>. BMP4 also stimulated Mixl1expression peak<strong>in</strong>g at day 3 with 26±12% Mixl1-GFP + <strong>cells</strong> butfur<strong>the</strong>r addition <strong>of</strong> activ<strong>in</strong> did not affect <strong>the</strong> number <strong>of</strong> Mixl1-GFP +<strong>cells</strong> (Fig. 1C).In Xenopus, Wnt molecules have both organizer-<strong>in</strong>duc<strong>in</strong>g andposterioriz<strong>in</strong>g activities (Niehrs, 2004) and <strong>in</strong> mice Wnt3 is requiredfor proper axis formation and <strong>in</strong>duction <strong>of</strong> <strong>the</strong> primitive streak(Barrow et al., 2007; Liu et al., 1999). We <strong>the</strong>refore tested <strong>the</strong> ability<strong>of</strong> Wnt3a by itself or <strong>in</strong> comb<strong>in</strong>ation with different doses <strong>of</strong> activ<strong>in</strong> to<strong>in</strong>duce PS markers. 5 ng/ml Wnt3a <strong>in</strong>duced 23±4% T-GFP + <strong>cells</strong> (datanot shown), whereas 100 ng/ml <strong>in</strong>duced 30±9% T-GFP + <strong>cells</strong> at day 3,significantly higher than <strong>the</strong> control <strong>cells</strong> (Fig. 1A; pb0.05). Activ<strong>in</strong> didnot have a significant effect on Wnt3a-<strong>in</strong>duced T expression althoughwe observed a tendency to reduced numbers <strong>of</strong> T-GFP + <strong>cells</strong> with <strong>the</strong>highest doses <strong>of</strong> activ<strong>in</strong>. The prom<strong>in</strong>ent <strong>in</strong>duction <strong>of</strong> T expression seenwith both BMP4 and Wnt3a was confirmed by immun<strong>of</strong>luorescence atday 3 (Fig. S2A). Wnt3a also <strong>in</strong>duced Mixl1 expression. Culturesconta<strong>in</strong><strong>in</strong>g 100 ng/ml Wnt3a conta<strong>in</strong>ed 11±4% Mixl1-GFP + <strong>cells</strong> atday 4. Notably, <strong>the</strong> comb<strong>in</strong>ation <strong>of</strong> 100 ng/ml activ<strong>in</strong> and 100 ng/mlWnt3a <strong>in</strong>duced 22±3% Mixl1-GFP + <strong>cells</strong> at day 4, approximatelytwice that achieved by ei<strong>the</strong>r factor alone (Fig. 1C; pb0.05). Whenexam<strong>in</strong><strong>in</strong>g co-expression <strong>of</strong> Tand GFP at day 3 by immun<strong>of</strong>luorescencewe found that most, if not all, Mixl1 express<strong>in</strong>g <strong>cells</strong> also expressed T,while <strong>the</strong> converse was not <strong>the</strong> case (Fig. S2B). While Wnt3astimulated T and Mixl1 expression, it did not affect <strong>the</strong> number <strong>of</strong>Gsc-GFP + <strong>cells</strong> (Figs. 1B and S2). The <strong>in</strong>duction <strong>of</strong> Gsc expression byactiv<strong>in</strong> <strong>in</strong> <strong>the</strong> presence or absence <strong>of</strong> Wnt3a and its <strong>in</strong>hibition by BMP4was confirmed by immun<strong>of</strong>luorescence at day 5 (Fig. S2C). Notably, <strong>the</strong>few T-express<strong>in</strong>g <strong>cells</strong> present <strong>in</strong> activ<strong>in</strong>-treated Gsc Gfp/+ <strong>cells</strong> after5 days did not express GFP. Consider<strong>in</strong>g that T is found not only <strong>in</strong> <strong>the</strong>PS, but also <strong>in</strong> <strong>the</strong> emerg<strong>in</strong>g mesoderm at <strong>the</strong> late gastrula stage(Inman and Downs, 2006), this may <strong>in</strong>dicate that mesoderm is als<strong>of</strong>ormed <strong>in</strong> activ<strong>in</strong>-treated cultures. Collectively, <strong>the</strong> different <strong>ES</strong> l<strong>in</strong>es allresponded similarly to growth factor treatment (Fig. S2). Overall, ourresults <strong>in</strong>dicate an anterioriz<strong>in</strong>g role <strong>of</strong> activ<strong>in</strong> dur<strong>in</strong>g <strong>ES</strong> cell<strong>differentiation</strong> that can be modulated by <strong>the</strong> posterioriz<strong>in</strong>g factorsBMP4 and Wnt3a, consistent with <strong>the</strong> roles <strong>of</strong> <strong>the</strong>se factors before anddur<strong>in</strong>g gastrulation (reviewed <strong>in</strong> Tam and Loebel, 2007).BMP4 <strong>in</strong>duces mesoderm and blocks activ<strong>in</strong>-mediated <strong>in</strong>duction <strong>of</strong>def<strong>in</strong>itive endodermTo establish if our cultures conta<strong>in</strong>ed embryonic or extraembryoniccell types we first exam<strong>in</strong>ed expression <strong>of</strong> CXCR4 (chemok<strong>in</strong>e (C-X-Cmotif) receptor-4), which is expressed <strong>in</strong> embryonic but not <strong>in</strong>extraembryonic tissues (McGrath et al., 1999; Sherwood et al., 2007).Us<strong>in</strong>g FACS analysis we compared surface expression <strong>of</strong> CXCR4 on <strong>cells</strong>isolated from dissociated E11 <strong>mouse</strong> embryo heads with that <strong>of</strong>differentiated <strong>ES</strong> cell progeny. Two dist<strong>in</strong>ct CXCR4-expression populationscould be detected among <strong>cells</strong> from <strong>mouse</strong> embryos, a CXCR4 lo andaCXCR4 hi population (Fig. S3A). When <strong>ES</strong> cell progeny from ei<strong>the</strong>rvehicle or activ<strong>in</strong>-treated cultures was analyzed it was clear that <strong>the</strong> vastFig. 3. Activ<strong>in</strong>-<strong>in</strong>duced expression <strong>of</strong> <strong>the</strong> anterior primitive streak marker Gsc is <strong>in</strong>hibited by BMP4 but not by Dkk1. Gsc Gfp/+ <strong>ES</strong> <strong>cells</strong> were grown <strong>in</strong> serum-free mediumsupplemented with one or more <strong>of</strong> <strong>the</strong> follow<strong>in</strong>g growth factors or <strong>in</strong>hibitor; 10 ng/ml BMP4, 100 ng/ml Wnt3a, 320 ng/ml Dkk1, 3 or 100 ng/ml activ<strong>in</strong> as <strong>in</strong>dicated. (A)Triple-label immun<strong>of</strong>luorescence was performed to analyze <strong>the</strong> co-expression <strong>of</strong> E-cad (Cdh1), Gsc (GFP), and Sox17 <strong>in</strong> <strong>cells</strong> grown for 5 days under <strong>the</strong> <strong>in</strong>dicated conditions.Note Cdh1 + GFP − Sox17 − <strong>cells</strong> (white arrows), Cdh1 − GFP + Sox17 − <strong>cells</strong> (red arrows), Cdh1 + GFP − Sox17 + <strong>cells</strong> (white arrowheads), and Cdh1 + GFP + Sox17 + <strong>cells</strong> (with yellownuclei, red arrowheads) (B) Triple-label immun<strong>of</strong>luorescence was also performed to analyze co-expression <strong>of</strong> Cdh1, Gsc (GFP), and Oct4 on day 5. Note Cdh1 + GFP − Oct4 + <strong>cells</strong>(white arrows), Cdh1 − GFP + Oct4 − <strong>cells</strong> (red arrows), and Cdh1 + GFP + Oct4 − <strong>cells</strong> (red arrowheads).