300 M. Hansson et al. / Developmental Biology 330 (2009) 286–304Fig. 12. <strong>ES</strong> cell-derived endoderm acquire foregut cell fates. (A–J′ and P–Z′) OS25 (Sox2β geo/+ )or(K–O) Pdx1 LacZ/+ <strong>cells</strong> were cultured for 5 days <strong>in</strong> 100 ng/ml activ<strong>in</strong> andsubsequently treated with 5 ng/ml Wnt3a, 10 ng/ml <strong>FGF</strong>4 and/or 0,1 μM RA for 3 days as <strong>in</strong>dicated. The co-expression <strong>of</strong> Foxa2 and Sox2 (foregut), Pdx1 (midgut) or Cdx2 (h<strong>in</strong>dgut)was analyzed by immun<strong>of</strong>luorescence. The expression <strong>of</strong> Sox2 (A–E) and Pdx1 (K–O) was confirmed by analyz<strong>in</strong>g β-galactosidase activity us<strong>in</strong>g X-gal sta<strong>in</strong><strong>in</strong>g.Fur<strong>the</strong>r analysis <strong>of</strong> BMP4- (and Wnt3a-) treated <strong>cells</strong> revealed <strong>the</strong>presence <strong>of</strong> Flk1 + <strong>cells</strong> demonstrat<strong>in</strong>g that mesodermal <strong>differentiation</strong>had occurred. Interest<strong>in</strong>gly, Smith et al. recently reported thatBMP4 treatment <strong>of</strong> m<strong>ES</strong> <strong>cells</strong>, under conditions nearly identical toours, resulted <strong>in</strong> <strong>cells</strong> <strong>of</strong> unknown identity that were unlikely to bemesodermal based on presence <strong>of</strong> E-cad immunoreactivity (Kunath etal., 2007). While we occasionally detect a fa<strong>in</strong>t plasma membranesta<strong>in</strong><strong>in</strong>g <strong>in</strong> BMP4-treated <strong>cells</strong> (see for example Fig. 3), <strong>the</strong> <strong>in</strong>tensity <strong>of</strong><strong>the</strong> sta<strong>in</strong><strong>in</strong>g is strongly reduced compared to <strong>the</strong> signal seen <strong>in</strong> activ<strong>in</strong>treated<strong>cells</strong>, and most <strong>cells</strong> appear E-cad − <strong>in</strong> our experiments.Fur<strong>the</strong>rmore, <strong>the</strong> presence <strong>of</strong> Flk1 express<strong>in</strong>g <strong>cells</strong> would appear toconclusively demonstrate mesodermal <strong>differentiation</strong>. This is alsoconsistent with a number <strong>of</strong> studies where embryoid bodies arestimulated with BMP4 (Czyz and Wobus, 2001; F<strong>in</strong>ley et al., 1999;Johansson and Wiles, 1995; Lengerke et al., 2008; Ng et al., 2005;Willems and Leyns, 2008).Our exam<strong>in</strong>ation <strong>of</strong> <strong>the</strong> role <strong>of</strong> Wnt signal<strong>in</strong>g dur<strong>in</strong>g mesendoderm<strong>differentiation</strong> seems to <strong>in</strong>dicate a major difference between <strong>differentiation</strong><strong>in</strong> embryoid bodies and <strong>in</strong> adherent monoculture. Apparently,<strong>the</strong> extent to which Wnt signal<strong>in</strong>g is required to <strong>in</strong>duce anteriorPS formation is different <strong>in</strong> <strong>the</strong> two systems. Gadue et al. found that100 ng/ml Wnt could <strong>in</strong>duce formation <strong>of</strong> Foxa2-CD4 + T-GFP +anterior PS-like <strong>cells</strong>, and that this was dependent on endogenousALK4/5/7 signal<strong>in</strong>g. Moreover, <strong>the</strong> ability <strong>of</strong> activ<strong>in</strong> to <strong>in</strong>duce <strong>the</strong>same fate was dependent on Wnt signal<strong>in</strong>g s<strong>in</strong>ce Dkk1 could prevent<strong>the</strong> effect <strong>of</strong> activ<strong>in</strong> (Gadue et al., 2006). At first glance <strong>the</strong>se f<strong>in</strong>d<strong>in</strong>gsmay appear to contrast with our results, namely that <strong>the</strong> sameconcentration <strong>of</strong> Wnt3a could not <strong>in</strong>duce significant numbers <strong>of</strong> Gsc-GFP + <strong>in</strong> adherent culture and that Dkk1 could not prevent <strong>in</strong>duction<strong>of</strong> Gsc-GFP + <strong>cells</strong> by activ<strong>in</strong>. However, <strong>the</strong> Gsc-GFP + <strong>cells</strong> <strong>in</strong> this workdo not express Brachyury and very likely represent a mixed populationand are thus difficult to compare to <strong>the</strong> Foxa2/T double positive <strong>cells</strong>
M. Hansson et al. / Developmental Biology 330 (2009) 286–304301studied by Gadue et al. Fur<strong>the</strong>rmore, although siRNA mediated knockdown<strong>of</strong> β-caten<strong>in</strong> significantly reduced <strong>the</strong> number <strong>of</strong> Gsc-GFP + <strong>cells</strong>develop<strong>in</strong>g <strong>in</strong> response to activ<strong>in</strong> <strong>the</strong> effect never exceeded 50%. Incontrast, <strong>the</strong> formation <strong>of</strong> Sox17-GFP Hi DE <strong>cells</strong> was more sensitive to<strong>in</strong>hibition <strong>of</strong> canonical Wnt signal<strong>in</strong>g, but only when signal<strong>in</strong>g was<strong>in</strong>hibited at a relatively late stage <strong>of</strong> development, suggest<strong>in</strong>g that <strong>the</strong>requirement for Wnt signal<strong>in</strong>g is not dur<strong>in</strong>g <strong>the</strong> formation <strong>of</strong> PS <strong>cells</strong>but may ra<strong>the</strong>r represent a Wnt-mediated l<strong>in</strong>eage choice by acommon mesendodermal precursor or alternatively, a requirementfor Wnt signal<strong>in</strong>g <strong>in</strong> <strong>the</strong> ma<strong>in</strong>tenance <strong>of</strong> Sox17-express<strong>in</strong>g DE <strong>cells</strong>.Indeed, our observation that Dkk1 only reduces <strong>the</strong> number <strong>of</strong> Sox17 Hi<strong>cells</strong> if present at day 4, i.e. after Sox17 expression is <strong>in</strong>itiated, isconsistent with a requirement for Wnt signal<strong>in</strong>g <strong>in</strong> <strong>the</strong> ma<strong>in</strong>tenance <strong>of</strong>DE. Also, our observations are consistent with <strong>the</strong> requirement for β-caten<strong>in</strong> function <strong>in</strong> DE to acquire or ma<strong>in</strong>ta<strong>in</strong> DE fate (Lickert et al.,2002) as well as <strong>the</strong> observation <strong>of</strong> synergy between Sox17 and β-caten<strong>in</strong> <strong>in</strong> <strong>the</strong> activation <strong>of</strong> DE gene expression (S<strong>in</strong>ner et al., 2004).We also speculate that <strong>the</strong> proposed action <strong>of</strong> Wnt3 dur<strong>in</strong>ggastrulation might <strong>of</strong>fer at least a partial explanation for <strong>the</strong> difference<strong>in</strong> sensitivity <strong>towards</strong> Dkk1 treatment observed between activ<strong>in</strong> andnodal as well as between adherent and aggregate culture. Wnt3 isrequired <strong>in</strong> vivo for nodal to <strong>in</strong>itiate its auto-stimulatory cascade. In<strong>the</strong> absence <strong>of</strong> Wnt3, nodal expression is <strong>in</strong>itiated but <strong>the</strong>n regressesra<strong>the</strong>r than expands (Barrow et al., 2007; Liu et al., 1999). Wnt3 isthought to <strong>in</strong>duce expression <strong>of</strong> Cfc1 which encodes <strong>the</strong> obligatenodal co-receptor Cripto (Morkel et al., 2003). It was recently reportedthat <strong>the</strong> three dimensional environment <strong>in</strong> embryoid bodies preventexogenously added growth factors from diffus<strong>in</strong>g more than a limiteddistance <strong>in</strong>to <strong>the</strong> embryoid body (Sachlos and Auguste, 2008) possiblymak<strong>in</strong>g <strong>the</strong>se more reliant on endogenous relay signal<strong>in</strong>g via nodalwhich subsequently would require Wnt activity to <strong>in</strong>duce Criptoexpression. This notion is supported by our observation that nodal<strong>in</strong>ducedSox17-GFP Hi <strong>cells</strong> appear more sensitive to Dkk1-mediatedsuppression than activ<strong>in</strong> <strong>in</strong>duced Sox17-GFP Hi <strong>cells</strong>, and by ourobservation that activ<strong>in</strong>-<strong>in</strong>duced Sox17-GFP Hi <strong>cells</strong> are more sensitiveto Dkk1 treatment <strong>in</strong> aggregate culture compared to adherent culture.The apparent absence <strong>of</strong> such an autocr<strong>in</strong>e nodal/Wnt signal<strong>in</strong>g loop<strong>in</strong> adherent culture is not surpris<strong>in</strong>g given <strong>the</strong> remarkable <strong>in</strong>stability<strong>of</strong> nodal when secreted by cultured <strong>cells</strong> (Le Good et al., 2005). Nodalmay simply be degraded before it can reach o<strong>the</strong>r <strong>cells</strong> <strong>in</strong> <strong>the</strong> dishwhereas <strong>in</strong> <strong>the</strong> conf<strong>in</strong>ed environment <strong>of</strong> an embryoid body it may wellsignal to nearby <strong>cells</strong> and such signal<strong>in</strong>g might propagate <strong>in</strong> a relayfashion. Thus, Wnt3a treatment would not be sufficient to <strong>in</strong>duce thisloop <strong>in</strong> adherent culture. Conversely, s<strong>in</strong>ce all <strong>the</strong> <strong>cells</strong> <strong>in</strong> adherentculture are exposed evenly to exogenous activ<strong>in</strong> <strong>the</strong>re may be norequirement for Wnt signal<strong>in</strong>g to facilitate ALK4 signal<strong>in</strong>g. Never<strong>the</strong>less,Gsc-GFP + <strong>cells</strong> appear largely refractory to <strong>the</strong> <strong>in</strong>hibitoryeffects <strong>of</strong> Dkk1 treatment. S<strong>in</strong>ce Wnt <strong>in</strong>duced expression <strong>of</strong> Xgsc ismediated by two homeodoma<strong>in</strong> prote<strong>in</strong>s (Siamois and Tw<strong>in</strong> whichhave no apparent homologs <strong>in</strong> <strong>the</strong> <strong>mouse</strong>) b<strong>in</strong>d<strong>in</strong>g to <strong>the</strong> PE-element,this f<strong>in</strong>d<strong>in</strong>g raises <strong>the</strong> question <strong>of</strong> whe<strong>the</strong>r <strong>the</strong> conserved Wntresponsiveelement <strong>in</strong> <strong>the</strong> <strong>mouse</strong> Gsc promoter (Watabe et al., 1995)isfunctional <strong>in</strong> all contexts. It is possible that Gsc is not always <strong>in</strong>ducedby Wnt as Gsc expression is reduced ra<strong>the</strong>r than <strong>in</strong>creased <strong>in</strong> E8.5Dkk1 mutant embryos (Lewis et al., 2008). Lastly, we cannot ruleout a more trivial explanation related to <strong>the</strong> particular Gsc-GFP celll<strong>in</strong>e, which may potentially harbor a defect <strong>in</strong> its responsiveness toDkk1-mediated Wnt <strong>in</strong>hibition. It is possible that more efficientknock-down <strong>of</strong> β-caten<strong>in</strong> than we achieved might fur<strong>the</strong>r reduce oreven prevent <strong>the</strong> formation <strong>of</strong> Gsc-GFP + <strong>cells</strong>.In contrast, we do observe a requirement for Wnt signal<strong>in</strong>g for<strong>in</strong>duction <strong>of</strong> Mixl1 expression. Our experiments revealed <strong>the</strong> simultaneousrequirement <strong>of</strong> nodal/activ<strong>in</strong> and Wnt signal<strong>in</strong>g to <strong>in</strong>duceexpression <strong>of</strong> Mixl1 <strong>in</strong> <strong>ES</strong> cell progeny. Stimulation <strong>of</strong> one pathwaywhile <strong>in</strong>hibit<strong>in</strong>g <strong>the</strong> o<strong>the</strong>r reduced Mixl1 <strong>in</strong>duction, which corroboratesprevious f<strong>in</strong>d<strong>in</strong>gs (Gadue et al., 2006). The Mixl1 promoter haspreviously been shown to be nodal/activ<strong>in</strong>-responsive via <strong>the</strong>presence <strong>of</strong> Foxh1 and Smad b<strong>in</strong>d<strong>in</strong>g sites <strong>in</strong> <strong>the</strong> promoter (Hart etal., 2005) and <strong>in</strong>spection <strong>of</strong> <strong>the</strong> published sequence reveals <strong>the</strong>presence <strong>of</strong> consensus TCF/LEF b<strong>in</strong>d<strong>in</strong>g sites <strong>in</strong> <strong>the</strong> promoter as well.In vivo, Wnt/β-caten<strong>in</strong> signal<strong>in</strong>g is upstream <strong>of</strong> two dist<strong>in</strong>ct geneexpression programs act<strong>in</strong>g dur<strong>in</strong>g anteroposterior axis and mesodermformation, respectively (Morkel et al., 2003). We suspect thatwe observe a requirement for Wnt signal<strong>in</strong>g <strong>in</strong> mesoderm formationbut did not pursue this aspect fur<strong>the</strong>r.The two <strong>differentiation</strong> systems, adherent and aggregate culture,also differ strik<strong>in</strong>gly <strong>in</strong> <strong>the</strong> requirement for <strong>FGF</strong> signal<strong>in</strong>g. In agreementwith our results a recent study us<strong>in</strong>g embryoid body formation foundthat <strong>FGF</strong> signal<strong>in</strong>g was not required for BMP4-<strong>in</strong>duced T expression(Willems and Leyns, 2008), which is unlike <strong>the</strong> situation <strong>in</strong> adherentculture where BMP4-<strong>in</strong>duced T expression is completely prevented bySU5402. Moreover, consistent with our results Kunath et al., also us<strong>in</strong>gadherent culture, recently reported that <strong>FGF</strong>4 deficiency or treatmentwith PD173074 prevented <strong>the</strong> switch from BMP4-mediated support <strong>of</strong>pluripotency to BMP4-<strong>in</strong>duced <strong>differentiation</strong> (Kunath et al., 2007).The requirement for <strong>FGF</strong> signal<strong>in</strong>g <strong>in</strong> <strong>in</strong>duction <strong>of</strong> mesodermal geneexpression is also consistent with <strong>the</strong> <strong>in</strong> vivo requirement for <strong>FGF</strong>signal<strong>in</strong>g <strong>in</strong> Xenopus and zebrafish mesodermal <strong>in</strong>duction (Cornell etal., 1995; Mathieu et al., 2004). In zebrafish, <strong>FGF</strong> signal<strong>in</strong>g is requireddownstream <strong>of</strong> Nodal for <strong>in</strong>duction <strong>of</strong> One-eyed-p<strong>in</strong>head, <strong>the</strong> zebrafishhomolog <strong>of</strong> Cripto (Mathieu et al., 2004). However, as discussedabove activ<strong>in</strong> does not require Cripto to activate its receptors, thusactiv<strong>in</strong> <strong>in</strong>duced Sox17 expression should not necessarily be <strong>in</strong>hibitedby <strong>FGF</strong>R <strong>in</strong>hibitors. This is <strong>in</strong>deed what we f<strong>in</strong>d when <strong>in</strong>hibit<strong>in</strong>g <strong>FGF</strong>signal<strong>in</strong>g prior to appearance <strong>of</strong> Sox17-GFP Hi <strong>cells</strong>, <strong>the</strong> time where onewould expect to f<strong>in</strong>d a requirement for Cripto function if Nodal was <strong>the</strong><strong>in</strong>ducer. It might be illum<strong>in</strong>at<strong>in</strong>g to determ<strong>in</strong>e if Nodal <strong>in</strong>duced Sox17expression would be sensitive to <strong>the</strong>se <strong>in</strong>hibitors. Never<strong>the</strong>less, we doobserve that Sox17 expression depends on <strong>FGF</strong> signal<strong>in</strong>g after <strong>the</strong><strong>in</strong>itial appearance <strong>of</strong> Sox17-GFP Hi <strong>cells</strong> at <strong>the</strong> same time where weobserve a dependency for Wnt signal<strong>in</strong>g. It thus appears thatma<strong>in</strong>tenance <strong>of</strong> Sox17 expression and/or propagation <strong>of</strong> Sox17express<strong>in</strong>g <strong>cells</strong> depend on both <strong>FGF</strong> and Wnt signal<strong>in</strong>g. Consistentwith our results, Brickman et al. also observed an absolute requirementfor <strong>FGF</strong> signal<strong>in</strong>g at days 3–7 for <strong>the</strong> formation <strong>of</strong> Hex + CXCR4 + ADE<strong>cells</strong> when differentiat<strong>in</strong>g <strong>ES</strong> <strong>cells</strong> <strong>in</strong> monolayer culture under def<strong>in</strong>edconditions (Morrison et al., 2008).In some cases we noticed what appeared to be conflict<strong>in</strong>g resultswhen <strong>in</strong>hibit<strong>in</strong>g <strong>FGF</strong> and <strong>FGF</strong>R signal<strong>in</strong>g by soluble <strong>FGF</strong> receptors andSU5402, respectively. It is possible that this is a reflection <strong>of</strong> <strong>FGF</strong><strong>in</strong>dependent <strong>FGF</strong>R activation. <strong>FGF</strong> receptors are known to formcomplexes with N-cadher<strong>in</strong> and N CAM <strong>in</strong> neurons and <strong>in</strong> pancreaticβ-<strong>cells</strong>, result<strong>in</strong>g <strong>in</strong> ligand <strong>in</strong>dependent receptor activation (Cavallaroet al., 2001; Saffell et al., 1997; Williams et al., 1994), and this wouldnot be expected to be sensitive to <strong>the</strong> addition <strong>of</strong> soluble <strong>FGF</strong>receptors. More trivial explanations are also possible. We cannot ruleout that SU5402 may have unknown non-<strong>FGF</strong>R-mediated effects or becerta<strong>in</strong> that our soluble <strong>FGF</strong>R preparations are capable <strong>of</strong> <strong>in</strong>hibit<strong>in</strong>g all<strong>FGF</strong> family members. Additional experiments with dom<strong>in</strong>ant negativereceptors and cell l<strong>in</strong>es mutated <strong>in</strong> genes cod<strong>in</strong>g for <strong>FGF</strong> signal<strong>in</strong>gcomponents will likely shed more light on <strong>the</strong>se questions.The expression <strong>of</strong> anterior markers such as Otx2 and Cer1 <strong>in</strong> Sox17-GFP Hi <strong>cells</strong> suggest that <strong>the</strong>se could be anterior def<strong>in</strong>itive endoderm.This notion is supported by <strong>the</strong> selective presence <strong>of</strong> Pyy transcripts <strong>in</strong>this population, but <strong>the</strong> lack <strong>of</strong> good ADE specific markers prevents usfrom categorically mak<strong>in</strong>g this conclusion. However, <strong>in</strong> vivo ADEforms under conditions <strong>of</strong> high nodal signal<strong>in</strong>g (Ben-Haim et al.,2006; Lu and Robertson, 2004; V<strong>in</strong>cent et al., 2003) which we believewe mimic with culture conditions conta<strong>in</strong><strong>in</strong>g 30–100 ng/ml activ<strong>in</strong> or1 μg/ml nodal. Thus, it is likely that <strong>the</strong> Sox17-GFP Hi <strong>cells</strong> formed <strong>in</strong>our cultures represent ADE. However, it is also likely that mesoderm isformed to some extent <strong>in</strong> cultures treated with high doses <strong>of</strong> activ<strong>in</strong> or
- 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
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- 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
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- 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
- 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
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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,