The formation of proximal and distal definitive …...JournalofCellScience RESEARCH ARTICLE The formation of proximal and distal definitive endoderm populations in culture requires

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RESEARCH ARTICLE
The formation of proximal and distal definitiveendodermpopulations in culture requires p38 MAPK activity
Charlotte Yap1,*, Hwee Ngee Goh1,*, Mary Familari1, Peter DavidRathjen1,2 and Joy Rathjen1,2,`
ABSTRACT
Endoderm formation in the mammal is a complex process withtwo
lineages forming during the first weeks of development, theprimitive
(or extraembryonic) endoderm, which is specified in theblastocyst,
and the definitive endoderm that forms later, at gastrulation,as
one of the germ layers of the embryo proper. Fate mapping
evidence suggests that the definitive endoderm arises as two
waves, which potentially reflect two distinct cell populations.Early
primitive ectoderm-like (EPL) cell differentiation has beenused
successfully to identify and characterise mechanismsregulating
molecular gastrulation and lineage choice duringdifferentiation. The
roles of the p38 MAPK family in the formation of definitiveendoderm
were investigated using EPL cells and chemical inhibitors ofp38
MAPK activity. These approaches define a role for p38 MAPK
activity in the formation of the primitive streak and a secondrole in
the formation of the definitive endoderm. Characterisation ofthe
definitive endoderm populations formed from EPL cells
demonstrates the formation of two distinct populations, definedby
gene expression and ontogeny, that were analogous to theproximal
and distal definitive endoderm populations of the embryo.Formation
of the proximal definitive endoderm was found to require p38
MAPK activity and is correlated with molecular gastrulation,defined
by the expression of brachyury (T). Distal definitiveendoderm
formation also requires p38 MAPK activity but can form whenT
expression is inhibited. Understanding lineage complexity willbe a
prerequisite for the generation of endoderm derivatives for
commercial and clinical use.
KEY WORDS: Embryonic stem cells, Endoderm, p38 MAP kinase,
Gastrulation, BMP4
INTRODUCTIONTwo distinct endoderm lineages arise duringmammalian
embryogenesis – the primitive endoderm, a derivative of the
inner cell mass (ICM) of the blastocyst, which acts as a
progenitor for the extraembryonic visceral and parietal
endoderm, and the definitive endoderm, the progenitor of the
embryonic endoderm populations. In mouse, a proportion ofthe
definitive endoderm has been proposed to develop from a
bipotent progenitor, the mesendoderm, which arises in the
primitive streak during gastrulation (Kinder et al., 2001;Lawsonet al., 1991).
The ability of embryonic stem (ES) cells toself-renewindefinitely and to give rise to all embryonic and adulttissuesin response to the appropriate signals in vitro and in vivomakethem attractive tools for modeling the developmental processesof
gastrulation and formation of the definitive endoderm (Bradleyetal., 1984; Doetschman et al., 1985; Evans and Kaufman, 1981;Martin,1981). ES-cell-based approaches have been used to
identify and characterise endoderm formation (Izumi et al.,2007;Kubo et al., 2004; Tada et al., 2005), and the addition ofactivin Ahas been shown to increase the formation of endodermduring ES
cell differentiation (Kubo et al., 2004; Nostro et al.,2011).Furthermore, bipotent progenitors that differentiateintomesoderm and definitive endoderm have been identified in
culture – a brachyury (T)-positive cell population (Kubo etal.,2004) and a cell population that is positive for goosecoid(GSC),E-cadherin (ECD, also known as CDH1) and PDGFRathat divergesto Gsc+ECD+PDGFRa2 and Gsc+ECD2PDGFRa+
populations (Tada et al., 2005). It is becoming clear,however,that definitive endoderm formation and subsequentdifferentiationare complex processes, and outcomes are influencedby induction
strategies and positional specification (Gadue et al., 2009;Gadueet al., 2006; Jackson et al., 2010; Nostro et al., 2011).
Analysing the mechanisms that regulate gastrulation andendodermformation in systems that initiate differentiationfrom mouse EScells is impacted on by two confounding
factors. Initially, ES cells differentiate to form a laterpluripotentpopulation [a population that is equivalent to theembryonicprimitive ectoderm (Rathjen et al., 2003a)] andprimitiveendoderm (Soudais et al., 1995). The primitiveendoderm
lineage is a potent source of signals that regulatepluripotentcell differentiation (Beddington and Robertson, 1998;Beddingtonand Robertson, 1999). Signals emanating from theprimitive
endoderm have the potential to synergise or competewithexogenous signals to influence the differentiation outcome.Thematuration of ES cells to later pluripotent cell populations
requires the generation of endogenous signals withinthedifferentiation system (Li et al., 2004; Coucouvanis andMartin,1995). Primitive ectoderm formation can be controlled inculture
by the differentiation of ES cells to a second pluripotentcellpopulation, early primitive ectoderm-like (EPL) cells, inresponseto MEDII, a medium conditioned by the humanhepatocarcinomacell line HepG2 (Lake et al., 2000; Rathjen et al.,1999; Tan et al.,
2011; Washington et al., 2010). EPL cells are similar to cellsofthe primitive ectoderm. This has been shown using morphology,geneexpression, cytokine response and differentiation potential
(Lake et al., 2000; Rathjen et al., 2002; Tan et al.,2011;Washington et al., 2010). ES cell differentiation in responsetoMEDII is not accompanied by the concomitant formation of
primitive endoderm (Rathjen et al., 2002; Vassilieva et al.,2012).
1Department of Zoology, University of Melbourne, Victoria, 3010,Australia. 2TheMenzies Research Institute Tasmania, University ofTasmania, Tasmania, 7000,Australia.*These authors contributedequally to this work
`Author for correspondence ([emailprotected])
Received 2 May 2013; Accepted 4 January 2014
� 2014. Published by The Company of Biologists Ltd | Journal ofCell Science (2014) 127, 2204–2216 doi:10.1242/jcs.134502
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Using EPL cells as the starting material for differentiationhasresulted in a reliable model of gastrulation, which has beenused
to determine the role of growth factors,environmentalmanipulations and intracellular signalling in celldifferentiationand to allow the identification of transientdevelopmentalintermediates (Harvey et al., 2010; Hughes et al.,2009a;
Hughes et al., 2009b; Zheng et al., 2010). Here,thedifferentiation of EPL cells is used to investigate the controlofmolecular gastrulation, with a focus on the formation ofdefinitive
endoderm. Cells formed as a result of molecular gastrulationarereferred to here as primitive streak intermediates; this termcoversall cells expressing T, a marker of molecular gastrulation,and
includes, but is not limited to, bipotent mesendoderm.p38 MAPKis a member of the mitogen-activated protein
kinase (MAPK) family of kinases (Martı́n-Blanco, 2000;Nebreda
and Porras, 2000), and was originally identified throughitsinvolvement in stress and inflammatory responses (Han etal.,1994; Raingeaud et al., 1995; Rouse et al., 1994).Developmentalroles for p38 MAPK activity have been shown in theformation
of cardiocytes (Aouadi et al., 2006), myocytes (Perdigueroetal., 2007), adipocytes (Engelman et al., 1998),chondrocytes(Nakamura et al., 1999), erythroid cells (Nagata etal., 1998) and
neurons (Nebreda and Porras, 2000). Inhibiting p38 MAPKduring EScell differentiation promotes neurogenesis at theexpense ofcardiogenesis and suggests a role for p38 MAPK in
germ layer specification (Aouadi et al., 2006; Barruet et al.,2011;Wu et al., 2010) and cardiogenesis (Wang et al., 2012). Inthisstudy, roles for p38 MAPK activity in molecular gastrulationand
in definitive endoderm formation are identified. The inhibitionofp38 MAPK during EPL cell differentiation in response toserumreduced the expression of T and promoted the formation ofneurallineages. By contrast, when cells were differentiated inresponse
to BMP4 the inhibition of p38 MAPK did not alterdifferentiationoutcomes or the expression of differentiationmarkers. Theanalysis of the differentiation outcomes from cellsdifferentiated
in the presence of activin A, BMP4 or serum and the p38MAPKinhibitor showed that the formation of definitive endodermfromEPL cells was dependent on p38 MAPK activity. Further
characterisation suggested that EPL-cell-deriveddefinitiveendoderm comprised two distinct populations,representative ofthe proximal and distal definitive endoderm of theembryo, whichformed in response to alternative signallingenvironments and
potentially from distinct progenitor populations.
RESULTSBMP4 and serum induce the formation of primitivestreakintermediates through distinct signalling pathwaysPrimitivestreak intermediates can be induced from EPL cells by
BMP4 (Harvey et al., 2010; Zheng et al., 2010) or serum(Hugheset al., 2009b). The requirement for p38 MAPK activity intheformation of primitive streak intermediates in response tothese
inducers was investigated. Phosphorylated p38 MAPK (p-p38MAPK)and phosphorylated heat shock protein 27 (pHsp27), adownstreamtarget of p38 MAPK signalling, were detectedby western blot in EPLcells incubated in serum-free medium
(SFM) with or without BMP4 or serum (p-p38 MAPK only)(Fig.1Ai,Aii). No consistent increase in p-p38 MAPK was seenin EPL cellsafter the addition of serum. p38 MAPK activity was
inhibited pharmacologically with SB203580[4-(49-fluorophenyl)-2-(49-methylsulfinylphenyl)-5-(49-pyridyl)-imidazole;SB]. Thischemical inhibits p38a (MAPK14) and p38b(MAPK11)hom*ologues by competing for ATP-binding pockets (Cuenda
et al., 1995). The levels of p-p38 MAPK and pHsp27 werereducedin cells exposed to serum and SB as compared with cellsexposed to
serum alone (Fig. 1Aii; supplementary material Fig. S1A,B).Inthe absence of serum or BMP4, aggregates of EPL cells
almost exclusively formed neurons (Fig. 1B; data notshown)(Zheng et al., 2010). The addition of BMP4, inBMP4-containing
medium (BCM; supplementary material Table S1), or serum,inserum-containing medium (SCM; supplementary material TableS1),during differentiation resulted in the formation of cardiocytes
and erthryocytes within the aggregates, as expected frompreviousanalyses of differentiation (Fig. 1B) (Harvey et al.,2010;Zheng et al., 2010). The addition of SB to serum reducedthe
percentage of aggregates that formed erthryocytes andincreasedthe percentage of aggregates that formed neurons,whereascardiocyte formation was unaffected (Fig. 1B). In thepresence of
BMP4, however, there was no significant effect of theinhibitoron the production of erythrocytes, neurons or cardiocytes.SB didnot affect the ability of aggregates to adhere to plasticwareforscoring or the survival of mesoderm or ectoderm lineages
scored in the assay (supplementary material Table S3). Thesedatasuggest a role for p38 MAPK in differentiation.
Previous analysis of EPL cell differentiation has suggestedthat
the frequency of blood, cardiocyte and neuron formationreflectsthe efficiency of the formation of primitive streakintermediates(Fig. 1C; supplementary material Fig. S1B) (Zheng etal., 2010).
Differentiating EPL cells were analysed for the expressionofestablished markers of the primitive streak (T, Bmp4, Tgfb1,Wnt3and Fgf8) (Crossley and Martin, 1995; Dickson et al., 1995;
Liu et al., 1999; Wilkinson et al., 1990; Winnier et al.,1995),ectoderm (Sox1 and Ascl1) (Guillemot et al., 1993; Pevny etal.,1998) and mesoderm (Mesp1, Hbb-b1, Nkx2-5 and Osr1) (Faraceetal., 1984; Lints et al., 1993; Saga et al., 1996; So andDanielian,
1999). Analysis was performed when gene expression wasmostreproducibly detected (at day two for T, Wnt3 and Fgf8 and atdayfour for Bmp4 and Tgfb1). The expression of marker genes ofthe
primitive streak and mesoderm was reduced in EPL cells thatweredifferentiated in SCM+SB compared to the expression incontrols,consistent with the reduced formation of mesoderm
from these cells (Fig. 1C,D; supplementary material Fig. S2D).Bycontrast, only Bmp4 expression was decreased in EPL cellsthat weredifferentiated in BCM+SB (Fig. 1C). Lower expressionof T, Wnt3 andBmp4 was detected in cells that
were differentiated in response to SCM compared withthosedifferentiated in BCM (supplementary material Fig.S2E),suggesting that differentiation in response to these inducerswas
not equivalent.The significantly reduced expression of markersof the
primitive streak intermediates and reduced formation of
erythrocytes from EPL cells differentiated in serum when SBwasadded suggested that there is a role for p38 MAPK in theformationof the primitive streak intermediate. Cardiocyte
formation and the residual levels of erythrocyte formation inaproportion of the aggregates that were differentiated inSCM+SBdemonstrated the formation of a population ofprimitive streakintermediates, albeit a reduced one, and a
discord between the gene expression and differentiationdata.Most notably, the percentage of aggregatescontainingcardiocytes was unaffected by the inhibitor SB butthe
expression of Nkx2-5, a cardiocyte marker, wasundetectable,suggesting that SB did affect the establishment ofcardiocytes.Differentiation assays score the presence, but not theabundance,
of a cell type in an aggregate. Potentially, differentiationwithin
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aggregates cultured in serum and SB was reduced, delayedandasynchronous, leading to a reduction in Nkx2-5 transcriptlevelsat any given timepoint and affecting the ability of theanalysis to
detect expression.Collectively, these data suggest that theinduction of primitive-
streak intermediates by serum, but not by BMP4, requires
p38 MAPK activity. The suppression of differentiationthatresulted from the addition of the p38 MAPK inhibitor SBtoserum-containing medium was specific to the formation of the
primitive-streak intermediate. Neurectoderm was formed andtheprevalence of the lineage was increased in these conditions.
The formation of primitive streak intermediates in responsetoserum is reduced, but not abolished, by BMP4 inhibitionBMP4 andserum potentially induce primitive streakintermediates byindependent pathways. Alternatively, serum
might contain BMP activity at levels that are sufficient toinduceprimitive-streak intermediates in cells with p38 MAPKactivityor it might induce BMP expression and signalling duringcell
differentiation.Some sera have been shown to contain BMPactivity (Herrera
and Inman, 2009; Kodaira et al., 2006). Western blotanalysisshowed that the levels of phosphorylated Smad1, 5 and 8
(pSmad1/5/8) were not increased in EPL cells that were exposedtothe serum used in this analysis (Fig. 2A). By contrast, pSmad1/5/8was markedly induced in cells that were exposed to BMP4.
These data indicate that our serum contained little or noBMPactivity.
The possibility that BMP4 expression was induced in EPLcells
as they differentiated in response to serum, and thatendogenouslyexpressed BMP4 subsequently acted to induce theprimitivestreak intermediate, was investigated by differentiatingcells
See Also
16806 North Marie Village Pl, Conroe, TX 77306 - HAR.com
in BCM or SCM in the presence of the BMP inhibitor noggin.Nogginbinds to BMP4, BMP2 and, to a lesser extent,BMP7 proteins, andinhibits their interaction with receptors(Zimmerman et al., 1996).The inhibition of BMP4 signalling in
BCM will abrogate signalling from endogenous BMP activityandexogenous BMP4. In BCM+noggin a higher percentage ofa*ggregatescontained neurons when compared with controls,
and effectively no aggregates contained cardiocytesanderythrocytes (Fig. 2B). Consistent with this, the expressionofprimitive-streak markers was decreased in cells that were
differentiated in BCM+noggin compared with their expressioninBCM controls (Fig. 2C,D). A significant decrease in thepercentageof aggregates containing cardiocytes and erythrocyteswas also seenin aggregates that were cultured in SCM+noggin,
Fig. 1. Inhibition of p38 MAPK signalling affects lineage choicein EPL cells. (Ai) Western blot of p-p38 MAPK and p38 MAPK in EPLcells incubated inSFM, SFM+serum (Serum), or SFM+BMP4 (BMP4) for10, 30 or 60 min. b-tubulin was used as a loading control. Theappearance of increased levels of p-p38MAPK with the addition ofserum was variable. n53. (Aii) Western blot of pHsp27 and Hsp27 inserum-starved EPL cells that were transferred to SFM,SFM+serum andDMSO (Serum) or SFM+serum and SB for 15, 30 or 60 min. b-tubulinwas used as a loading control. n53. (B) EPL cells differentiatedinSFM+SB, or BCM or SCM with either SB or DMSO were scored for theformation of cardiocytes, erythrocytes and neurons. Raw data forthis experiment can befound in supplementary material Table S3.Results are means6s.e.m. n53. (C,D). Primitive streak (T, Bmp4,Tgfb1,Wnt3 and Fgf8), ectoderm (Sox1 and Ascl1)and mesoderm (Mesp1,Hbb-b1, Nkx2-5 and Osr1) markers were detected by RT-PCR in EPLcells differentiating in BCM or SCM with DMSO or SB.(C)Primitive-streak markers were quantified on day three and have beennormalised to the expression of Gapdh. Results are means6s.e.m.,n53.(D) Mesoderm and ectoderm markers were analysed on day four anda representative result is shown. n53. 2R, no reverse transcriptasecontrol; 2D, no cDNAcontrol. *P,0.05, #P,0.01.
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although ,20% of aggregates still formedmesoderm-derivedlineages (Fig. 2B). Similarly, this reduction inmesodermformation was reflected in a reduction in the expressionofmarkers of primitive streak intermediates in cells that
were differentiated in SCM+noggin when compared with
SCM controls (Fig. 2C,D). Mesoderm formation in cells thatweredifferentiated in SCM+noggin was reduced but not
abolished (Fig. 2B), suggesting that two pathways mediatetheinduction of the primitive streak intermediate in theseaggregates,a pathway that is dependent on endogenously generatedBMPactivity and a second pathway that is independent of BMP
signalling.
The formation of definitive endoderm in response to BMP4 orserumis impaired by the inhibition of p38 MAPKMixl1, which has beenimplicated in the formation of thedefinitive endoderm (Robb et al.,2000; Hart et al., 2002), was
found to be expressed in cells that were differentiated inBCMand SCM, and was significantly decreased in both conditionsonthe addition of the p38 MAPK inhibitor SB (Fig. 3A), raisingthe
possibility that p38 MAPK was involved in the formation ofthedefinitive endoderm. The expression of additionalendodermmarkers Sox17, Ttr, Gata4, Trh and Eya2 (Gu et al., 2004;Kanai-Azuma et al., 2002; Kwon et al., 2008; Lickert et al.,2002;
McKnight et al., 2007) in cells that were differentiated inBCMand SCM was examined. A novel endoderm marker, serineproteaseinhibitor Kazal type 3 (Spink3), was included in the
analysis. Previously, Spink3 has been shown to be expressedinendoderm-derived populations, including cells of the gutandpancreas in embryonic day (E)9.5 mouse embryos (Wang et al.,
2008). As shown here, Spink3 expression was detected in a bandofdefinitive endoderm that was located immediately below theembryonicand extraembryonic boundary of gastrulating E7.5
embryos (Fig. 3B). The expression of endoderm-marker geneswasdetected in cells that were differentiated in BCM or SCM buthigherlevels of expression were generally detected in cellsdifferentiatedin SCM (Fig. 3C). Whole-mount in situ
hybridisation (WISH) detected endoderm markers on thesurface ofa*ggregates that were differentiated in BCM or SCM,but more Ttr+ andTrh+ cells were seen on aggregates
differentiated in SCM when compared with those in BCM(Fig. 3D).Inhibition of p38 MAPK led to a reduction in theexpression of mostendoderm markers in EPL cells that were
differentiated in SCM, but only a subset of markers (Spink3andTtr) in EPL cells differentiated in BCM (Fig. 3C). The additionofSB resulted in similar expression levels of all endodermmarkers,with the exception of Gata4, regardless of thedifferentiation
induction strategy used. The sustained expression of Gata4 intheBCM+SB condition might reflect the expression of the geneinanother lineage.
Some of the endoderm markers used here (Sox17 and Ttr) alsomarkvisceral endoderm (Kanai-Azuma et al., 2002; Kwon et al.,2008).Visceral endoderm can be distinguished from definitive
endoderm and parietal endoderm by the ability toendocytosehorseradish peroxidase (HRP) from the surrounding medium–internalised HRP can be detected colorimetrically (Kanai-Azuma
et al., 2002; Vassilieva et al., 2012). Embryoid bodies(EBs),which contain visceral endoderm (Kubo et al., 2004;Vassilieva etal., 2012), developed areas of brown staining on theirsurface(Fig. 3E), indicating the presence of visceral endoderm.By
contrast, EPL cells that were differentiated in SFM or SCMformedfew cells that were capable of taking up HRP from themedium,demonstrating that little or no visceral endoderm was
formed in these conditions.The role of p38 MAPK in endodermformation was
investigated further using a second inhibitor, SB202190 (Lee
et al., 1994), which acts by binding the ATP-binding site ofp38
Fig. 2. Inhibition of BMP signalling affects lineage choiceindifferentiating EPL cells. (A) Western blot of pSmad1/5/8 in EPLcellstreated with SFM or SFM containing BMP4 or serum for 5, 10 or30 min. b-tubulin was used as a loading control. A representativeresult is shown.n52. (B) EPL cell aggregates differentiated in BCMor SCM with or withoutnoggin were scored for the formation ofcardiocytes, erythrocytes andneurons. The data represent themean6s.e.m. n53. Raw data for thisexperiment can be found insupplementary material Table S4. (C,D) Theexpression ofprimitive-streak markers in EPL cells differentiated for2 (C) and 4(D) days as in B. The data have been normalised tob-actintranscript levels. Results are means6s.e.m. n53. *P,0.05,#P,0.01.
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MAPKs (Young et al., 1997). EPL cells that were differentiatedinBCM or SCM supplemented with SB202190 showed reduced
expression of endoderm markers (supplementary material Fig.S3A).Treatment with SB202190, however, also reduced theexpression ofmarkers of the primitive streak intermediate in cells
that were differentiated in SCM or BCM (supplementarymaterialFig. S3B), suggesting that, in comparison to SB203580,thiscompound inhibited p38 MAPK and additional pathways that
were required for molecular gastrulation.These data areconsistent with a role for p38 MAPK activity in
the formation of definitive endoderm but suggest heterogeneityinthe endoderm outcomes from EPL cells that are differentiated
in serum and BMP4. The documentation of endoderm formationinresponse to BMP4 without the addition of activin A isunprecedentedbut perhaps not unexpected, given that mesoderm
and endoderm can form from a common progenitor in culture(Kuboet al., 2004; Tada et al., 2005) and that BMP4 can functioninconjunction with activin A or other growth factors to induce
definitive endoderm (Mathew et al., 2012; Phillips et al.,2007).
Inhibition of BMP signalling during differentiation promotestheformation of a definitive endoderm population thatexpresses asubset of markersThe formation of endoderm in the embryo has beensuggestedto proceed through the formation of a bipotent progenitorreferred
to as mesendoderm (Kinder et al., 2001; Lawson et al.,1991);mesendoderm arises in the primitive streak and isencompassedwithin the primitive-streak-intermediate population.Mesendoderm
formation could underpin endoderm formation in SCM andBCM, withBMP4, or other factors, inducing a primitive-streakintermediatewith the properties of mesendoderm and a p38-MAPK-dependentmechanism inducing an endoderm fate from this
progenitor on further differentiation. The inhibition ofBMP4signalling in SCM by noggin did not affect the expression oftheendoderm markers (Fig. 4A), suggesting that cells cultured inSCM
do not rely on endogenously generated BMP4 to formmesendoderm.In EPL cells differentiated in BCM+noggin, analysis ofthe
expression of marker genes and differentiation outcomesshowed
that formation of the primitive-streak intermediate was
Fig. 3. Inhibition of p38 MAPK signalling reducesthe expressionof endoderm marker genes indifferentiating EPL cells. (A) Mixl1expression in EPLcells differentiated in BCM or SCM, with orwithout SB orDMSO for 2 days. Expression has been normalised tob-actin and is shown relative to the expression in SCM.The datarepresent the mean6s.e.m. n53.(B) WISH analysis of Spink3expression in E7.5 mouseembryos. Panels i and iii show lateralviews, panel ii isan anterior view. The asterisk (*) marks theanteriorembryonic-extraembryonic boundary. Panel iv shows acrosssection of an E7.5 Spink3-stained mouse embryo.The embryo is ,500mm from anterior to posterior.(C) EPL cells differentiated in BCMor SCM with orwithout SB for 4 days were analysed for theexpressionof endoderm markers by qPCR. Expression wasnormalised tothe expression of the b-actin gene.Results are means6s.e.m. n53–7.(D) WISH analysisof Ttr or Trh expression in EPL cellsdifferentiated inBCM or SCM. Aggregates are ,250 mm in diameter.(E)EPL cells differentiated in SCM and SFM wereincubated with HRP. Dayseven EBs were used as apositive control. Cells that took up HRPstained brown(arrowheads). *P,0.05, **P,0.01.
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significantly reduced (Fig. 2C,D), the formation of mesoderm
was effectively ablated (Fig. 2B) and the expression oftheendoderm markers Spink3, Ttr and Gata4 decreased (Fig. 4A).Thisis consistent with a two-step process that is reliant on the
initial formation of a primitive-streak intermediate in responsetoBMP4. Trh and Eya2 expression, however, was increased tolevelsequivalent to those in cells differentiated in SCM whencomparedwith control (Fig. 4A; supplementary material Fig.
S4A), suggesting the formation of endoderm by cells culturedinBCM+noggin. The presence of endoderm on the surface of
aggregates differentiated in BCM or BCM+noggin wasconfirmedmorphologically and by localisation of Trh expression(Fig.4B,C). Cell aggregates that were cultured for an extendedperiodexpressed markers of endoderm derivatives including Afp
and Ttr (gut and liver), Fabp2 (intestine) and Pdx1 (gutandpancreas) (supplementary material Fig. S4B). In EPLcellsdifferentiated in BCM+noggin and SB, the expression of Trh
and Eya2 was decreased by 10% and 40%, respectively,whencompared with cells differentiated in BCM+noggin(supplementarymaterial Fig. S4C), suggesting a requirement
for endogenous p38 MAPK in Trh and Eya2 expression.Theco-regulation of Spink3, Ttr and Gata4 is distinct from
the co-regulation of Trh and Eya2, and this could arise from
the formation of two endoderm populations duringEPL-celldifferentiation. Spink3, Ttr and Gata4 potentially markendodermthat is produced in response to both BMP4 and serum butisreduced during differentiation in BCM when noggin is present.
A second population that expresses Trh and Eya2 canbehypothesised, the formation of which is maintained in theabsenceof BMP4. In situ hybridisation was used to confirm the
generation of two genetically distinct populations ofendodermduring differentiation (Fig. 4D). Double staining withprobesagainst Spink3 and Trh detected distinct populations of cellson
the surface of aggregates that expressed either Spink3 (blue)orTrh (magenta).
BMP4 regulates the formation of endoderm expressingSpink3, Ttrand Gata4 through induction of the primitivestreak intermediateTheinduction of Spink3+, Ttr+ and Gata4+ endoderm in response
to BMP4 is a two-step process proceeding through abipotentprimitive streak intermediate, or mesendoderm. The likelyrolefor BMP4 in this process is indirect, by way of theinduction
of mesendoderm from EPL cells (Harvey et al., 2010).Thepossibility exists, however, that BMP4 has a role inthesubsequent induction of endoderm from mesendoderm.
Activin A can induce primitive streak intermediatesindependentlyof BMP4 signalling from ES cells and has beenidentified as a potentinducer of endoderm lineages in culture(Izumi et al., 2007; Jacksonet al., 2010; Kubo et al., 2004; Tada
et al., 2005). As expected, EPL cells that were differentiatedinactivin-A-containing medium (ACM) or ACM+nogginexpressed markers ofthe primitive streak (Fig. 5A). In cells
differentiated in ACM+noggin+SB, the expression ofprimitivestreak markers (except Bmp4) was reduced and theexpression ofthe neural marker Sox1 was increased (Fig. 5A),suggesting that
the ability of activin A to induce primitive streakintermediatesrequired p38 MAPK. Western blot showed p-p38 MAPK incellsthat had been treated with ACM (Fig. 5B). The regulation ofthe
formation of primitive streak intermediates in response toactivinA, therefore, is distinct from that observed in response toBMP4or serum.
In EPL cells differentiated in ACM, SFM+noggin or
ACM+noggin, Sox17, Spink3 and Ttr were expressedequivalently(Fig. 5C). The expression of these genes in theabsence of BMP4signalling suggests that BMP4 does not play
additional roles in the specification of endodermfrommesendoderm. Eya2 expression was higher in EPLcellsdifferentiated in ACM+noggin when compared with cells
differentiated in ACM. The expression of all endodermmarkers
Fig. 4. Inhibition of BMP signalling affects endoderm formationindifferentiating EPL cells. (A) EPL cells that were differentiatedin BCM orSCM with or without noggin or DMSO for 4 days wereanalysed by qPCR forthe expression of endoderm-marker genes. Thedata have beennormalised to b-actin transcript levels. Results aremeans6s.e.m. n53 or 4.(B,C) EPL cells differentiated in BCM (B) andBCM+noggin (C) weresectioned and stained with haematoxylin andeosin for morphology (i) or forthe expression of Trh (ii).Arrowheads indicate squamous endoderm-likecells on the surface ofthe aggregates. Scale bars: 50 mm. (D) EPL cellsdifferentiated inSCM were analysed by double WISH for the expression ofSpink3 (blue,arrowhead) and Trh (magenta, arrow). *P,0.05, **P,0.01.Scale bar:100 mm.
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in cells that were cultured in ACM+noggin suggests thatactivin
A, like serum, is able to induce both of the proposedendodermpopulations from EPL cells (Fig. 4A). The increasedexpressionof Eya2 suggests that the formation of endoderm thatexpresses
these markers can be enhanced by BMP4 inhibition. Theexpressionof Sox17, Spink3, Ttr and Eya2 (Fig. 5C) wasdecreased in cellsdifferentiated in ACM+noggon+SB,
consistent with a role for p38 MAPK activity in theactivin-A-induced formation of endoderm.
The primitive streak intermediate is the progenitor forendodermthat expresses Spink3, Ttr and Gata4 but notnecessarily forendoderm that expresses Trh and Eya2The formation of endoderm thatexpresses Spink3, Ttr and Gata4
appears to be dependent on the prior formation of primitivestreakintermediates. By contrast, the expression of Trh and Eya2inEPL cells differentiated in BCM+noggin suggests
the formation of an endoderm that does not rely on thepriorformation of this population. The formation ofprimitive-streakintermediates from differentiating EPL cells can beinhibited by
DAPT{N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycinet-butylester}, an antagonist of c-secretase (Hughes et al., 2009a).Incells formed from EPL cells differentiated in BCM+DAPT,theexpression of Spink3 and Ttr was decreased, and Eya2expression
increased, when compared with cells formed in BCM (Fig. 5D).
These data are consistent with formation of Trh+ and Eya2+
endoderm in the absence of primitive-streakintermediateformation and with a requirement for the initialformation of
primitive-streak intermediates in the formation of Spink3+ andTtr+
endoderm. Further differentiation of aggregates culturedinBCM+DAPT showed the expression of markers of
later endoderm populations, consistent with the formation ofanendoderm progenitor (supplementary material Fig. S4B).
DISCUSSIONp38 MAPK and the formation ofprimitive-streakintermediatesThe inhibition of p38 MAPK duringEPL-cell differentiation
disrupts the formation of primitive-streak intermediates fromEPLcells in response to activin A or serum. Others have shownthatp38 MAPK inhibition during ES cell differentiation in serum
promotes neurogenesis at the expense of cardiogenesis (Aouadietal., 2006; Barruet et al., 2011; Wu et al., 2010), and that thereis arequirement for p38 MAPK activity early in celldifferentiation
(Barruet et al., 2011; Davidson and Morange, 2000; Duval etal.,2004; Wu et al., 2010). The inhibition of p38 MAPK activitydidnot affect the ability of BMP4 to induce primitivestreakintermediates or mesoderm derivatives, suggesting thatthis
Fig. 5. The formation of primitive-streak intermediates andendoderm in response to activin A signalling requires p38 MAPKactivity. (A) EPL cells thatwere differentiated in ACM, ACM+nogginwith or without SB or DMSO or in SFM+noggin for 2 or 4 days wereanalysed by RT-PCR for the expression of markersof primitive streakintermediates (day two and four) or ectoderm (day four). 2R, noreverse transcriptase control; 2D, no cDNA control. Arepresentative result isshown. n53. (B) Serum-starved aggregateswere transferred to SFM or SFM containing 25 ng/ml activin A.Aggregates were collected 15, 30 and 60 min aftertransfer and wereanalysed by western blot for the phosphorylation of p38 MAPK. (C)EPL cells differentiated in ACM, ACM+noggin with or without SB orDMSOor in SFM+noggin for 4 days were analysed by qPCR for theexpression of endoderm markers. The data have been normalised tob-actin transcript levels.Results are means6s.e.m. n53. (D) Theexpression of endoderm markers in EPL cells that weredifferentiated in BCM+DMSO or DAPT on day four oftreatment. Thedata have been normalised to b-actin transcript levels. Results aremeans6s.e.m. n53. *P,0.05, **P,0.01.
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pathway is not essential for the formation of primitivestreakintermediates per se. The data presented here are consistentwith
a role for p38 MAPK in lineage allocation or specificationduringdifferentiation, specifically in the formation of primitivestreakintermediates, but only when molecular gastrulation isinduced byserum or activin A. Moreover, the inability of cellscultured in
SFM to form primitive streak intermediates, despiteintracellularp-p38 MAPK, suggests that p38 MAPK activity is notsufficientfor differentiation but works in conjunction with otherpathways.
In cells differentiated in SCM, but not ACM, one role forp38MAPK appears to be the upregulation of Bmp4, which inturninitiates differentiation. The increased expression of Bmp4in
cells differentiated in SCM, and the significant reduction initsexpression in cells differentiated in BCM supplementedwith SB, isconsistent with p38 MAPK acting upstream of
Bmp4. Endogenously produced BMP4 acts in turn to inducetheprimitive streak intermediate, an activity that is blockedbynoggin. Noggin did not completely suppress moleculargastrulation inresponse to serum, suggesting the presence of
additional, potentially p38 MAPK dependent, pathways.Bycontrast, markers of molecular gastrulation wereexpressedrobustly in cells differentiated in activin-A-containingmedium
supplemented with noggin, suggesting that activin A activitywasindependent of BMP. This is consistent with previous reportsthatactivin A does not induce the expression of Bmp4 during EScell
differentiation (Jackson et al., 2010).The involvement of p38MAPK in lineage specification from
ES cells has proven difficult to demonstrate, withconflicting
reports on the need for p38 MAPK activity duringmesodermspecification. The difficulty in resolving a role for p38MAPK ismost likely a consequence of the complexity of themolecularmechanisms that regulate gastrulation coupled with the useof ill-
defined and poorly understood culture reagents. The variabilityofoutcomes elicited in cells cultured in the presence of p38MAPKinhibitors (Aouadi et al., 2006; Barruet et al., 2011; Wu etal.,
2010) or from Mapk142/2 cells (also known as p38a2/2cells)(Allen et al., 2000; Chakraborty et al., 2009; Guo et al.,2007;Aouadi et al., 2006) could be attributed to the confoundinguse of
serum in these experiments. Some sera have been reportedtocontain exogenous BMP activity (Herrera and Inman, 2009;Kodairaet al., 2006). BMP activity within sera would be able tospecifyprimitive streak intermediates and mesoderm lineages in
the absence of p38 MAPK activity. Our interpretation oftherespective roles of serum and growth factors suggests thatserumvariability is a contributing factor to variability in theanalysis of
molecular gastrulation, and in the analysis of the role ofp38MAPK specifically.
The ability of p38 MAPK inhibition to promote cardiocyte
formation from human ES cells (Graichen et al., 2008)contradictsthe findings from mouse pluripotent cells reported hereand by others(Barruet et al., 2011; Aouadi et al., 2006; Wu et al.,2010). The
differentiation of human ES cells was induced in cell aggregatesby aconditioned medium in which the signalling activity waslargelyuncharacterised. Potentially, increased cardiocyte formationresultedfrom an increase in the number of primitive streakintermediates
(formed in response to BMP or to similar signalling activitywithinthe conditioned medium) adopting a mesoderm fate. Thiswouldoccur when p38 MAPK was inhibited in these cells, preventingtheir
differentiation to the endoderm.p38 MAPK comprises a, b, c and disoforms that are encoded
separately. Of these, p38a and p38b are expressed in thegermlayers and primitive ectoderm, respectively, at gastrulation(Zohn
et al., 2006). The disruption of the gene encoding p38a resultedinplacental defects and embryonic death mid-gestation (Adams
et al., 2000; Mudgett et al., 2000). Double-mutantembryos,lacking p38a and p38b in embryonic tissues, gastrulate butfailmid-gestation with diverse developmental defects (delBarcoBarrantes et al., 2011). Embryos with mutations in theMKK3,
MKK4 and MKK6 kinases that have been shown to activate p38MAPKalso survive beyond gastrulation (Lu et al., 1999; Tanakaet al.,2002; Yang et al., 1997). Mice that are deficient in p38-
interacting protein (p38IP, also known as SUPT20H) show a lossofp38 MAPK phosphorylation in the primitive streak, and thecells thatlack p-p38 MAPK fail to migrate (Zohn et al., 2006).
This mutation did not, however, prevent the formationofprimitive-streak intermediates or mesoderm specification,butraises the possibility that p38 MAPK has a role in theregulation
of an epithelial-to-mesenchymal transition duringdifferentiation.It is unlikely, therefore, that p38 MAPK isessential for theformation of primitive streak intermediates invivo, but its role isrevealed during in vitro differentiation inresponse to serum or
activin A.
A novel role for p38 MAPK in the formation ofdefinitive-endoderm populationsEndoderm formation in the mammal iscomplex, with theformation of two endoderm lineages from thepluripotent
lineage during early development – primitive or visceralendodermand definitive endoderm. Analysis of EPL celldifferentiationsuggests that the definitive endoderm might form
as two distinct cell populations that can be distinguished bygeneexpression and ontogeny. The formation of both lineagesrequiredp38 MAPK activity.
p38 MAPK inhibition during the differentiation of EPL cellsin
response to BMP4 resulted in the reduced expression ofMixl1,Spink3 and Ttr. When p38 MAPK was inhibited in cellsthatdifferentiated in response to serum there was reducedexpression
of Mixl1, Sox17, Spink3, Ttr, Gata4 and Eya2. Theseresultssuggest a previously unidentified role for p38 MAPK intheformation of definitive endoderm. In the mouse, definitive
endoderm formation is dependent on signalling throughnodal(Tremblay et al., 2000; Vincent et al., 2003), a member oftheTGFb family with similar signalling properties to activinA(Conlon et al., 1994; Vincent et al., 2003). Similarly, EScell
differentiation induced by activin A results in the enrichmentofdefinitive endoderm (Gadue et al., 2006; Kubo et al., 2004;Nostroet al., 2011). Canonically, nodal signalling is mediated by
Smad2/3 and the downstream effectors of Smads (Heldin etal.,1997; Massaous and Hata, 1997). p38 MAPK has been shown tobeactivated by TGFbs and to mediate signalling in response tothesefactors (Hanafusa et al., 1999; Hu et al., 2004; Yue andMulder,2000). The inhibition of p38 MAPK impaired theinduction ofdefinitive endoderm markers by activin A,
suggesting that activin A signalling was mediated, in part,byp38 MAPK. A role for p38 MAPK has been shown in thepositionalspecification of the visceral endoderm in response tonodal(Clements et al., 2011) and a similar requirement may exist
for p38 MAPK in the induction of definitive endoderm formationinresponse to Nodal.
The differential effects of the inhibitor SB on the expressionof
endoderm markers in aggregates that were differentiated inSCM orBCM infers that the specification of endoderm frompluripotent cellscan occur through multiple pathways and results
in two populations. The expression of Spink3, Ttr and Gata4
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marked one of these populations. Ttr has been used previouslytomark visceral endoderm (Kwon et al, 2008), but the widespread
expression of Ttr in EBs derived from EPL cells, inwhichvisceral endoderm is rarely formed (Vassilieva et al., 2012),andthe reliance of Ttr expression on molecular gastrulation byEPLcells suggests that Ttr can also be expressed in an
endoderm formed during molecular gastrulation. The formationof aSpink3+, Ttr+ and Gata4+ endoderm population fromdifferentiatingEPL cells correlated with the prior expression of
primitive streak markers. When the formation of primitivestreakintermediates was inhibited, as happened when cellsweredifferentiated in BCM supplemented with noggin or DAPT, the
expression of Spink3, Ttr and Gata4 was reduced. Thesecondendoderm population was marked by the expression of TrhandEya2. The expression of these markers is maintained in cells
differentiated in BCM supplemented with noggin orDAPT,suggesting that Trh+ and Eya2+ endoderm can form in theabsenceof a T-expressing primitive-streak intermediate. Thedifferentialexpression of Spink3, Ttr and Gata4 between
conditions that enriched or suppressed the formation oftheprimitive-streak intermediate, coupled with the persistenceofTrh+ and Eya2+ endoderm when the formation of the primitive
streak intermediate was suppressed, is consistent withtheformation of two endoderm populations during EPLcelldifferentiation.
A model for the regulation of molecular gastrulationBased onthis analysis of the formation of the primitive-streak
intermediate and endoderm from EPL cells, we propose arevisedparadigm for molecular gastrulation (Fig. 6). Thismodelproposes that primitive streak intermediates, which expressTand other primitive streak markers, can be induced from EPL
cells through multiple pathways, including pathways dependentonBMP4 signalling, growth factors/cytokines, including activinA andthe active components of serum, which require p38 MAPK
signalling and, as has been reported by others, WNTsignalling(Tanaka et al., 2009). These pathways most likelygeneratedistinct primitive streak intermediates that aredistinguished by
divergent differentiation potential, as has been shown forBMP4and Wnt (Tanaka et al., 2009). We propose that the primitivestreak
intermediate induced by BMP4 can differentiate to formmesodermand a population of endoderm expressing Spink3, Ttr andGata4. Ingene expression, this population is similar to the ring ofendodermmarked by Spink3 in the proximal region of the embryo.Formation
of this endoderm population requires p38 MAPK activity intheprimitive streak intermediate; activation of this pathwayisachieved through endogenous signalling in aggregates that are
differentiated in the presence of BMP4. Alternatively, EPLcellscan differentiate into a Trh+ and Eya2+ endoderm that canbeformed independently of the BMP4-induced primitive streak
intermediate; this population also requires p38 MAPK activityforits formation. The gene expression profile of thispopulationmirrors the endoderm of the distal region of the embryo(Gu et al.,
2004; McKnight et al., 2007).The endoderm populations definedhere are both are products
of EPL cell differentiation and arise duringmoleculargastrulation. We propose, therefore, that thesepopulations are
subpopulations of the definitive endoderm and suggest thattheterms ‘proximal definitive endoderm’ and ‘distaldefinitiveendoderm’ are used to describe them. Two waves ofdefinitive
endoderm, which populate the more proximal (lateral endoderm)andmore distal (medial endoderm) regions of the endoderm, havebeenproposed to occur during embryogenesis (Tam, 2007).
These populations have been distinguished by their time ofexitfrom the primitive streak, their direction of migration acrosstheegg cylinder and their allocation to different regions of thegut
tube in later development. The populations defined from invitrodifferentiation here potentially represent thepopulationsidentified by fate mapping in vivo.
Our proposed model (Fig. 6) addresses the role of the
mesendoderm in mammalian gastrulation, suggesting apopulationthat (1) satisfies the criteria of mesendoderm withinthe populationof primitive streak intermediates induced by
BMP4 and (2) acts as a progenitor of the proximaldefinitiveendoderm and mesoderm. The model also describesdistaldefinitive endoderm that can form independently of theBMP
Fig. 6. A model of endoderm formation fromEPL cells. Formationof the proximal definitiveendoderm is dependent on p38 MAPKactivityand correlates with the prior expression ofmarkers of theprimitive-streak intermediate. Theinitial formation of theT-expressing primitive-streak intermediate can occur in response toanumber of pathways including those regulated byBMP4, activin A,serum and, we hypothesize,Wnt (WNT3), and likely results in amixedpopulation of progenitors (indicated by the use ofseveralovals). The formation of the distaldefinitive endoderm is dependenton p38 MAPKsignalling but is not correlated with theinitialformation of the T-expressing primitive-streakintermediate.PSI, primitive streak intermediate;DE, definitive endoderm.
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signalling, suggesting that not all definitive endodermformationproceeds through the mesendoderm and raising thepossibility
of an endoderm-specific progenitor. Definitive endodermformationindependent of a bipotent progenitor has beensuggested previouslyby fate mapping of the embryo (Lawsonet al., 1991; Rouse et al.,1994).
A goal of stem cell research is to generate, insufficientquantity, functional cell types with commercial andclinicalapplications. Many approaches have been reported forforming
definitive endoderm and derivatives from ES cells. Theserelyalmost exclusively on the prior formation of primitivestreakintermediates and, with few exceptions (Mathew et al.,2012;
Morrison et al., 2008), the positional identity of theendodermpopulation that is formed has not been considered. Whatisclear from the literature is that the formation of laterendoderm
populations is generally inefficient; this is potentiallyaconsequence of the inefficient generation of theappropriateprogenitor at the onset of differentiation. Positionalspecificationcould restrict the developmental potential ofES-cell-derived
definitive endoderm and success might depend on enrichment foraspecific endoderm population. Characterisation of the derivativesofproximal and distal definitive endoderm populations in the
embryo and in culture will allow differentiation protocols tobetailored to ensure the enrichment of the appropriatedefinitiveendoderm for subsequent differentiation.
MATERIALS AND METHODSCell cultureMouse ESD3 ES cells (Doetschmanet al., 1985) were used throughout. ES cell
maintenance, EPL cell formation (as aggregates), EB formationand the
production of the conditioned medium MEDII were performed asdescribed
previously (Rathjen and Rathjen, 2003). All treatments wereadministered
to EPL cells that had been maintained in 50% MEDII for 72 h.
Differentiation assaysEPL cells were transferred to SCM(supplementary material Table S1).
The fetal calf serum (FCS, Life Technologies) used in theseexperiments
was chosen for the maintenance of pluripotency. Alternatively,EPL cells
were transferred to SFM (supplementary material Table S1).SFM
was supplemented with BMP4 (10 ng/ml, R&D Systems) (BCM;
supplementary material Table S1). Noggin (90 ng/ml, R&DSystems),
SB (10 mM, Sigma) and/or 0.1% DMSO (Sigma) were added asdescribedin the text and EPL cells were cultured for 3 days withdaily medium
change. The formation of recognisable cell types [erythrocytes(scored as
the presence of red patches of cells), pulsing cardiocytes(scored as cell
movement) and neurons (scored as long cell extensions emanatingfrom the
aggregates)] from aggregates was determined as describedpreviously
(Hughes et al., 2009a; Hughes et al., 2009b). For eachexperimental
condition §24 aggregates were analysed. Ideally, a singleaggregate wasanalysed per well but in reality some wells containedmore than one
analysed aggregate. Alternatively, aggregates were treated for 4days in
suspension culture before they were mass-seeded in a 9.6 cm2dish and
maintained in SCM with regular medium change for a further 7days.
Gene expression assaysEPL cells were transferred to SCM, BCM orACM [SFM+activin A
(25 ng/ml); supplementary material Table S1] and weresupplemented
with noggin (90 ng/ml), SB (10 mM), DAPT (50 mM) and/or 0.1 or0.2%DMSO, as described in the text, and cultured for 4 days withdaily
medium change. Cells were collected after 2, 3 and 4 days oftreatment.
RT-PCR and quantitative PCRTotal cytoplasmic RNA was isolatedusing TRIzolH (Invitrogen). cDNAwas synthesised as per themanufacturer’s protocol (Promega). Primers
(supplementary material Table S2) were validated ondifferentiated ES
(mouse) or genomic DNA (human), and PCR products weresequenced.
For RT-PCR, 25 ml reactions contained 1 ng/ml of forward andreverseprimers, 16GoTaqH Green Master Mix (Promega) and cDNA.Reactionswere heated to 94 C̊ for 2 min before cycles of 94 C̊ for30 s, 60 C̊
for 30 s and 72 C̊ for 30 s, ending with 5 min at 72 C̊, in anMJ
Research thermocycler. PCR products were visualised with aMolecular
ImagerH ChemiDocTM XRS Imaging System (BioRad) with SYBRHGold(Invitrogen). Gene expression was quantified using Quantity One1-
D band analysis software (BioRad).
For quantitative PCR (qPCR), reactions containing 16AbsoluteblueQPCR SYBR Green Mix (Thermo Scientific), cDNA and 200 nM
of forward and reverse primers were performed on an MJresearch
thermocycler with a Chromo4 Continuous FluorescenceDetection
system (MJ Research). Reactions were heated to 95 C̊ for 15min
before cycling at 95 C̊ for 15 s, 60 C̊ for 15 s and 72 C̊ for30 s. The raw
data was analysed using the Q-Gene software package (Muller etal.,
2002; Simon, 2003).
Whole-mount in situ hybridisation (WISH)Embryos from time-matedSwiss mice and cell aggregates were fixed in
4% paraformaldehyde (PFA) and dehydrated in methanol. WISHwas
performed as described previously (Lake et al., 2000; Rosenand
Beddington, 1993) with modifications. Rehydrated embryos weretreated
with 6% H2O2. Probes were labelled using digoxigenin-11-dUTPor
fluorescein-12-UTP (Roche). The hybridisation andpost-hybridisation
washes were performed at 65 C̊.
Embryos and aggregates were incubated overnight with anti-
digoxigenin–AP Fab fragments (1:2000) (Roche) oranti-fluorescein–
AP Fab fragments (1:2000) (Roche) and were developed withNBT/
BCIP or INT/BCIP (Roche), respectively, as per themanufacturer’s
instructions. The samples were photographed using an OlympusUC30
camera mounted on a Motic SMX-143 stereomicroscope.Riboprobes
were synthesized from pGEMT-easy vectors (Promega)containing
300 bp of Spink3, 460 bp of Ttr or 408 bp of Trh cDNAfragments,
linearized with NcoI or SalI, and transcribed with SP6(antisense) or T7
(sense) RNA polymerases. Aggregates were embedded in paraffinand
sectioned as required.
Western blotEPL cell aggregates were serum starved for 2 h inSFM before BMP4
(10 ng/ml), 10% FCS or activin A (25 ng/ml) were added.Aggregates
were pretreated with noggin (90 ng/ml), 0.1 or 0.035% DMSO,SB
(10 mM) or LDN (350 nM) for 1 h before the addition of BMP4 orFCS.Total proteins was analysed by western blotting. Membraneswere
developed with ECL substrate (Amersham Pharmacia Biotech),scanned
with a Molecular ImagerH ChemiDocTM XRS Imaging System(BioRad)or Fujifilm LAS-3000 (Berthold Australia Pty Ltd) andanalysed by
Quantity OneTM. Primary antibodies were against p38, p-p38,pSmad1/5/
8 (Cell Signaling Technologies) and b-tubulin I (Sigma). Thesecondaryantibodies were HRP-conjugated (Cell SignalingTechnologies and
DakoCytomation).
HRP-uptake assayThe HRP-uptake assay was performed as describedpreviously (Kanai-
Azuma et al., 2002; Vassilieva et al., 2012).
Statistical analysisExperiments were analysed using unpairedone- or two-tailed Student’s t-
tests in MicrosoftH Excel software. The statistical significanceis denotedas follows: *P,0.05, **P,0.01. Comparisons are madebetweenoutcomes in SB, noggin and DAPT compared to the equivalentbase
medium, with or without DMSO.
AcknowledgementsThe authors would like to thank members of theRathjen laboratory for insightfuldiscussions of the project.
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Competing interestsThe authors declare no competinginterests.
Author contributionsC.Y.: conception and design, collectionand/or assembly of data, data analysisand interpretation,manuscript writing. H.N.G.: conception and design, collectionand/orassembly of data, data analysis and interpretation. M.F. conceptionanddesign, data analysis and interpretation. P.D.R.: conception anddesign. J.R.:conception and design, data analysis andinterpretation, manuscript writing, finalapproval ofmanuscript.
FundingThis work was supported by the University of Melbourneand the Albert ShimminsPostgraduate Writing up Award. C.Y. andH.N.G. were supported by AustralianPostgraduate Awards, C.Y.received additional support from the Australian StemCellCentre.
Supplementary materialSupplementary material available onlineathttp://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.134502/-/DC1
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The formation of proximal and distal definitive …...JournalofCellScience RESEARCH ARTICLE The formation of proximal and distal definitive endoderm populations in culture requires - [PDF Document] (2024)