the formation of proximal and distal definitive …...journalofcellscience research article the...

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RESEARCH ARTICLE

The formation of proximal and distal definitive endodermpopulations in culture requires p38 MAPK activity

Charlotte Yap1,*, Hwee Ngee Goh1,*, Mary Familari1, Peter David Rathjen1,2 and Joy Rathjen1,2,`

ABSTRACT

Endoderm formation in the mammal is a complex process with two

lineages forming during the first weeks of development, the primitive

(or extraembryonic) endoderm, which is specified in the blastocyst,

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 been used

successfully to identify and characterise mechanisms regulating

molecular gastrulation and lineage choice during differentiation. The

roles of the p38 MAPK family in the formation of definitive endoderm

were investigated using EPL cells and chemical inhibitors of p38

MAPK activity. These approaches define a role for p38 MAPK

activity in the formation of the primitive streak and a second role in

the formation of the definitive endoderm. Characterisation of the

definitive endoderm populations formed from EPL cells

demonstrates the formation of two distinct populations, defined by

gene expression and ontogeny, that were analogous to the proximal

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 definitive endoderm

formation also requires p38 MAPK activity but can form when T

expression is inhibited. Understanding lineage complexity will be 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 during mammalian

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 of the

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 to self-renewindefinitely and to give rise to all embryonic and adult tissuesin response to the appropriate signals in vitro and in vivo makethem attractive tools for modeling the developmental processes of

gastrulation and formation of the definitive endoderm (Bradleyet al., 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 of activin Ahas been shown to increase the formation of endoderm during ES

cell differentiation (Kubo et al., 2004; Nostro et al., 2011).Furthermore, bipotent progenitors that differentiate intomesoderm and definitive endoderm have been identified in

culture – a brachyury (T)-positive cell population (Kubo et al.,2004) and a cell population that is positive for goosecoid (GSC),E-cadherin (ECD, also known as CDH1) and PDGFRathat diverges to Gsc+ECD+PDGFRa2 and Gsc+ECD2PDGFRa+

populations (Tada et al., 2005). It is becoming clear, however,that definitive endoderm formation and subsequent differentiationare complex processes, and outcomes are influenced by 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 andendoderm formation in systems that initiate differentiationfrom mouse ES cells is impacted on by two confounding

factors. Initially, ES cells differentiate to form a later pluripotentpopulation [a population that is equivalent to the embryonicprimitive ectoderm (Rathjen et al., 2003a)] and primitiveendoderm (Soudais et al., 1995). The primitive endoderm

lineage is a potent source of signals that regulate pluripotentcell differentiation (Beddington and Robertson, 1998; Beddingtonand Robertson, 1999). Signals emanating from the primitive

endoderm have the potential to synergise or compete withexogenous signals to influence the differentiation outcome. Thematuration of ES cells to later pluripotent cell populations

requires the generation of endogenous signals within thedifferentiation system (Li et al., 2004; Coucouvanis and Martin,1995). Primitive ectoderm formation can be controlled in culture

by the differentiation of ES cells to a second pluripotent cellpopulation, early primitive ectoderm-like (EPL) cells, in responseto MEDII, a medium conditioned by the human hepatocarcinomacell line HepG2 (Lake et al., 2000; Rathjen et al., 1999; Tan et al.,

2011; Washington et al., 2010). EPL cells are similar to cells ofthe primitive ectoderm. This has been shown using morphology,gene expression, 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 response toMEDII 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 of Tasmania, Tasmania, 7000,Australia.*These authors contributed equally to this work

`Author for correspondence ([email protected])

Received 2 May 2013; Accepted 4 January 2014

� 2014. Published by The Company of Biologists Ltd | Journal of Cell Science (2014) 127, 2204–2216 doi:10.1242/jcs.134502

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Using EPL cells as the starting material for differentiation hasresulted in a reliable model of gastrulation, which has been used

to determine the role of growth factors, environmentalmanipulations and intracellular signalling in cell differentiationand to allow the identification of transient developmentalintermediates (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 control ofmolecular gastrulation, with a focus on the formation of definitive

endoderm. Cells formed as a result of molecular gastrulation arereferred to here as primitive streak intermediates; this term coversall cells expressing T, a marker of molecular gastrulation, and

includes, but is not limited to, bipotent mesendoderm.p38 MAPK is a member of the mitogen-activated protein

kinase (MAPK) family of kinases (Martı́n-Blanco, 2000; Nebreda

and Porras, 2000), and was originally identified through itsinvolvement in stress and inflammatory responses (Han et al.,1994; Raingeaud et al., 1995; Rouse et al., 1994). Developmentalroles for p38 MAPK activity have been shown in the formation

of cardiocytes (Aouadi et al., 2006), myocytes (Perdigueroet al., 2007), adipocytes (Engelman et al., 1998), chondrocytes(Nakamura et al., 1999), erythroid cells (Nagata et al., 1998) and

neurons (Nebreda and Porras, 2000). Inhibiting p38 MAPKduring ES cell differentiation promotes neurogenesis at theexpense of cardiogenesis 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). In thisstudy, roles for p38 MAPK activity in molecular gastrulation and

in definitive endoderm formation are identified. The inhibition ofp38 MAPK during EPL cell differentiation in response to serumreduced the expression of T and promoted the formation of neurallineages. By contrast, when cells were differentiated in response

to BMP4 the inhibition of p38 MAPK did not alter differentiationoutcomes or the expression of differentiation markers. Theanalysis of the differentiation outcomes from cells differentiated

in the presence of activin A, BMP4 or serum and the p38 MAPKinhibitor showed that the formation of definitive endoderm fromEPL cells was dependent on p38 MAPK activity. Further

characterisation suggested that EPL-cell-derived definitiveendoderm comprised two distinct populations, representative ofthe proximal and distal definitive endoderm of the embryo, whichformed in response to alternative signalling environments and

potentially from distinct progenitor populations.

RESULTSBMP4 and serum induce the formation of primitive streakintermediates through distinct signalling pathwaysPrimitive streak 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 in theformation of primitive streak intermediates in response to these

inducers was investigated. Phosphorylated p38 MAPK (p-p38MAPK) and phosphorylated heat shock protein 27 (pHsp27), adownstream target of p38 MAPK signalling, were detectedby western blot in EPL cells 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 cells after 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)homologues by competing for ATP-binding pockets (Cuenda

et al., 1995). The levels of p-p38 MAPK and pHsp27 were reducedin cells exposed to serum and SB as compared with cells exposed to

serum alone (Fig. 1Aii; supplementary material Fig. S1A,B).In the absence of serum or BMP4, aggregates of EPL cells

almost exclusively formed neurons (Fig. 1B; data not shown)(Zheng et al., 2010). The addition of BMP4, in BMP4-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 from previousanalyses of differentiation (Fig. 1B) (Harvey et al., 2010;Zheng et al., 2010). The addition of SB to serum reduced the

percentage of aggregates that formed erthryocytes and increasedthe percentage of aggregates that formed neurons, whereascardiocyte formation was unaffected (Fig. 1B). In the presence of

BMP4, however, there was no significant effect of the inhibitoron the production of erythrocytes, neurons or cardiocytes. SB didnot affect the ability of aggregates to adhere to plasticware forscoring or the survival of mesoderm or ectoderm lineages

scored in the assay (supplementary material Table S3). These datasuggest a role for p38 MAPK in differentiation.

Previous analysis of EPL cell differentiation has suggested that

the frequency of blood, cardiocyte and neuron formation reflectsthe efficiency of the formation of primitive streak intermediates(Fig. 1C; supplementary material Fig. S1B) (Zheng et al., 2010).

Differentiating EPL cells were analysed for the expression ofestablished 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 et al.,1998) and mesoderm (Mesp1, Hbb-b1, Nkx2-5 and Osr1) (Faraceet al., 1984; Lints et al., 1993; Saga et al., 1996; So and Danielian,

1999). Analysis was performed when gene expression was mostreproducibly detected (at day two for T, Wnt3 and Fgf8 and at dayfour for Bmp4 and Tgfb1). The expression of marker genes of the

primitive streak and mesoderm was reduced in EPL cells thatwere differentiated 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).By contrast, only Bmp4 expression was decreased in EPL cellsthat were differentiated in BCM+SB (Fig. 1C). Lower expressionof T, Wnt3 and Bmp4 was detected in cells that

were differentiated in response to SCM compared with thosedifferentiated in BCM (supplementary material Fig. S2E),suggesting that differentiation in response to these inducers was

not equivalent.The significantly reduced expression of markers of the

primitive streak intermediates and reduced formation of

erythrocytes from EPL cells differentiated in serum when SBwas added suggested that there is a role for p38 MAPK in theformation of the primitive streak intermediate. Cardiocyte

formation and the residual levels of erythrocyte formation in aproportion of the aggregates that were differentiated inSCM+SB demonstrated the formation of a population ofprimitive streak intermediates, albeit a reduced one, and a

discord between the gene expression and differentiation data.Most notably, the percentage of aggregates containingcardiocytes was unaffected by the inhibitor SB but the

expression of Nkx2-5, a cardiocyte marker, was undetectable,suggesting that SB did affect the establishment of cardiocytes.Differentiation assays score the presence, but not the abundance,

of a cell type in an aggregate. Potentially, differentiation within

RESEARCH ARTICLE Journal of Cell Science (2014) 127, 2204–2216 doi:10.1242/jcs.134502

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aggregates cultured in serum and SB was reduced, delayed andasynchronous, leading to a reduction in Nkx2-5 transcript levelsat any given timepoint and affecting the ability of the analysis to

detect expression.Collectively, these data suggest that the induction of primitive-

streak intermediates by serum, but not by BMP4, requires

p38 MAPK activity. The suppression of differentiation thatresulted from the addition of the p38 MAPK inhibitor SB toserum-containing medium was specific to the formation of the

primitive-streak intermediate. Neurectoderm was formed and theprevalence of the lineage was increased in these conditions.

The formation of primitive streak intermediates in responseto serum is reduced, but not abolished, by BMP4 inhibitionBMP4 and serum potentially induce primitive streakintermediates by independent pathways. Alternatively, serum

might contain BMP activity at levels that are sufficient to induceprimitive-streak intermediates in cells with p38 MAPK activityor it might induce BMP expression and signalling during cell

differentiation.Some sera have been shown to contain BMP activity (Herrera

and Inman, 2009; Kodaira et al., 2006). Western blot analysisshowed that the levels of phosphorylated Smad1, 5 and 8

(pSmad1/5/8) were not increased in EPL cells that were exposedto the serum used in this analysis (Fig. 2A). By contrast, pSmad1/5/8 was markedly induced in cells that were exposed to BMP4.

These data indicate that our serum contained little or no BMPactivity.

The possibility that BMP4 expression was induced in EPL cells

as they differentiated in response to serum, and that endogenouslyexpressed BMP4 subsequently acted to induce the primitivestreak intermediate, was investigated by differentiating cells

in BCM or SCM in the presence of the BMP inhibitor noggin.Noggin binds to BMP4, BMP2 and, to a lesser extent,BMP7 proteins, and inhibits their interaction with receptors(Zimmerman et al., 1996). The inhibition of BMP4 signalling in

BCM will abrogate signalling from endogenous BMP activity andexogenous BMP4. In BCM+noggin a higher percentage ofaggregates contained neurons when compared with controls,

and effectively no aggregates contained cardiocytes anderythrocytes (Fig. 2B). Consistent with this, the expression ofprimitive-streak markers was decreased in cells that were

differentiated in BCM+noggin compared with their expressionin BCM controls (Fig. 2C,D). A significant decrease in thepercentage of aggregates containing cardiocytes and erythrocyteswas also seen in aggregates that were cultured in SCM+noggin,

Fig. 1. Inhibition of p38 MAPK signalling affects lineage choice in EPL cells. (Ai) Western blot of p-p38 MAPK and p38 MAPK in EPL cells incubated inSFM, SFM+serum (Serum), or SFM+BMP4 (BMP4) for 10, 30 or 60 min. b-tubulin was used as a loading control. The appearance of increased levels of p-p38MAPK with the addition of serum was variable. n53. (Aii) Western blot of pHsp27 and Hsp27 in serum-starved EPL cells that were transferred to SFM,SFM+serum and DMSO (Serum) or SFM+serum and SB for 15, 30 or 60 min. b-tubulin was used as a loading control. n53. (B) EPL cells differentiated inSFM+SB, or BCM or SCM with either SB or DMSO were scored for the formation of cardiocytes, erythrocytes and neurons. Raw data for this 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 EPL cells differentiating in BCM or SCM with DMSO or SB.(C) Primitive-streak markers were quantified on day three and have been normalised to the expression of Gapdh. Results are means6s.e.m., n53.(D) Mesoderm and ectoderm markers were analysed on day four and a representative result is shown. n53. 2R, no reverse transcriptase control; 2D, no cDNAcontrol. *P,0.05, #P,0.01.


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although ,20% of aggregates still formed mesoderm-derivedlineages (Fig. 2B). Similarly, this reduction in mesodermformation was reflected in a reduction in the expression ofmarkers of primitive streak intermediates in cells that

were differentiated in SCM+noggin when compared with

SCM controls (Fig. 2C,D). Mesoderm formation in cells thatwere differentiated in SCM+noggin was reduced but not

abolished (Fig. 2B), suggesting that two pathways mediate theinduction of the primitive streak intermediate in these aggregates,a pathway that is dependent on endogenously generated BMPactivity and a second pathway that is independent of BMP

signalling.

The formation of definitive endoderm in response to BMP4 orserum is impaired by the inhibition of p38 MAPKMixl1, which has been implicated in the formation of thedefinitive endoderm (Robb et al., 2000; Hart et al., 2002), was

found to be expressed in cells that were differentiated in BCMand SCM, and was significantly decreased in both conditions onthe addition of the p38 MAPK inhibitor SB (Fig. 3A), raising the

possibility that p38 MAPK was involved in the formation of thedefinitive endoderm. The expression of additional endodermmarkers 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 in BCMand SCM was examined. A novel endoderm marker, serineprotease inhibitor Kazal type 3 (Spink3), was included in the

analysis. Previously, Spink3 has been shown to be expressed inendoderm-derived populations, including cells of the gut andpancreas in embryonic day (E)9.5 mouse embryos (Wang et al.,

2008). As shown here, Spink3 expression was detected in a bandof definitive endoderm that was located immediately below theembryonic and extraembryonic boundary of gastrulating E7.5

embryos (Fig. 3B). The expression of endoderm-marker geneswas detected in cells that were differentiated in BCM or SCM buthigher levels of expression were generally detected in cellsdifferentiated in SCM (Fig. 3C). Whole-mount in situ

hybridisation (WISH) detected endoderm markers on thesurface of aggregates that were differentiated in BCM or SCM,but more Ttr+ and Trh+ 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 most endoderm markers in EPL cells that were

differentiated in SCM, but only a subset of markers (Spink3 andTtr) in EPL cells differentiated in BCM (Fig. 3C). The addition ofSB resulted in similar expression levels of all endoderm markers,with the exception of Gata4, regardless of the differentiation

induction strategy used. The sustained expression of Gata4 in theBCM+SB condition might reflect the expression of the gene inanother lineage.

Some of the endoderm markers used here (Sox17 and Ttr) alsomark visceral endoderm (Kanai-Azuma et al., 2002; Kwon et al.,2008). Visceral endoderm can be distinguished from definitive

endoderm and parietal endoderm by the ability to endocytosehorseradish 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 their surface(Fig. 3E), indicating the presence of visceral endoderm. By

contrast, EPL cells that were differentiated in SFM or SCMformed few 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 endoderm formation was

investigated further using a second inhibitor, SB202190 (Lee

et al., 1994), which acts by binding the ATP-binding site of p38

Fig. 2. Inhibition of BMP signalling affects lineage choice indifferentiating EPL cells. (A) Western blot of pSmad1/5/8 in EPL cellstreated with SFM or SFM containing BMP4 or serum for 5, 10 or 30 min. b-tubulin was used as a loading control. A representative result is shown.n52. (B) EPL cell aggregates differentiated in BCM or SCM with or withoutnoggin were scored for the formation of cardiocytes, erythrocytes andneurons. The data represent the mean6s.e.m. n53. Raw data for thisexperiment can be found in supplementary material Table S4. (C,D) Theexpression of primitive-streak markers in EPL cells differentiated for2 (C) and 4 (D) days as in B. The data have been normalised to b-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 differentiatedin BCM or SCM supplemented with SB202190 showed reduced

expression of endoderm markers (supplementary material Fig.S3A). Treatment with SB202190, however, also reduced theexpression of markers of the primitive streak intermediate in cells

that were differentiated in SCM or BCM (supplementary materialFig. S3B), suggesting that, in comparison to SB203580, thiscompound inhibited p38 MAPK and additional pathways that

were required for molecular gastrulation.These data are consistent with a role for p38 MAPK activity in

the formation of definitive endoderm but suggest heterogeneity inthe endoderm outcomes from EPL cells that are differentiated

in serum and BMP4. The documentation of endoderm formationin response to BMP4 without the addition of activin A isunprecedented but perhaps not unexpected, given that mesoderm

and endoderm can form from a common progenitor in culture(Kubo et al., 2004; Tada et al., 2005) and that BMP4 can function inconjunction 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 promotesthe formation of a definitive endoderm population thatexpresses a subset of markersThe formation of endoderm in the embryo has been suggestedto proceed through the formation of a bipotent progenitor referred

to as mesendoderm (Kinder et al., 2001; Lawson et al., 1991);mesendoderm arises in the primitive streak and is encompassedwithin the primitive-streak-intermediate population. Mesendoderm

formation could underpin endoderm formation in SCM andBCM, with BMP4, or other factors, inducing a primitive-streakintermediate with the properties of mesendoderm and a p38-MAPK-dependent mechanism inducing an endoderm fate from this

progenitor on further differentiation. The inhibition of BMP4signalling in SCM by noggin did not affect the expression of theendoderm markers (Fig. 4A), suggesting that cells cultured in SCM

do not rely on endogenously generated BMP4 to form mesendoderm.In EPL cells differentiated in BCM+noggin, analysis of the

expression of marker genes and differentiation outcomes showed

that formation of the primitive-streak intermediate was

Fig. 3. Inhibition of p38 MAPK signalling reducesthe expression of endoderm marker genes indifferentiating EPL cells. (A) Mixl1 expression in EPLcells differentiated in BCM or SCM, with or without SB orDMSO for 2 days. Expression has been normalised to b-actin and is shown relative to the expression in SCM.The data represent the mean6s.e.m. n53.(B) WISH analysis of Spink3 expression in E7.5 mouseembryos. Panels i and iii show lateral views, panel ii isan anterior view. The asterisk (*) marks the anteriorembryonic-extraembryonic boundary. Panel iv shows across section of an E7.5 Spink3-stained mouse embryo.The embryo is ,500 mm from anterior to posterior.(C) EPL cells differentiated in BCM or SCM with orwithout SB for 4 days were analysed for the expressionof endoderm markers by qPCR. Expression wasnormalised to the expression of the b-actin gene.Results are means6s.e.m. n53–7. (D) WISH analysisof Ttr or Trh expression in EPL cells differentiated inBCM or SCM. Aggregates are ,250 mm in diameter.(E) EPL cells differentiated in SCM and SFM wereincubated with HRP. Day seven EBs were used as apositive control. Cells that took up HRP stained 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 of theendoderm markers Spink3, Ttr and Gata4 decreased (Fig. 4A).This is consistent with a two-step process that is reliant on the

initial formation of a primitive-streak intermediate in response toBMP4. Trh and Eya2 expression, however, was increased tolevels equivalent to those in cells differentiated in SCM whencompared with control (Fig. 4A; supplementary material Fig.

S4A), suggesting the formation of endoderm by cells cultured inBCM+noggin. The presence of endoderm on the surface of

aggregates differentiated in BCM or BCM+noggin was confirmedmorphologically and by localisation of Trh expression(Fig. 4B,C). Cell aggregates that were cultured for an extendedperiod expressed markers of endoderm derivatives including Afp

and Ttr (gut and liver), Fabp2 (intestine) and Pdx1 (gut andpancreas) (supplementary material Fig. S4B). In EPL cellsdifferentiated in BCM+noggin and SB, the expression of Trh

and Eya2 was decreased by 10% and 40%, respectively,when compared with cells differentiated in BCM+noggin(supplementary material Fig. S4C), suggesting a requirement

for endogenous p38 MAPK in Trh and Eya2 expression.The co-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 during EPL-celldifferentiation. Spink3, Ttr and Gata4 potentially mark endodermthat is produced in response to both BMP4 and serum but isreduced during differentiation in BCM when noggin is present.

A second population that expresses Trh and Eya2 can behypothesised, the formation of which is maintained in theabsence of BMP4. In situ hybridisation was used to confirm the

generation of two genetically distinct populations of endodermduring differentiation (Fig. 4D). Double staining with probesagainst Spink3 and Trh detected distinct populations of cells on

the surface of aggregates that expressed either Spink3 (blue) orTrh (magenta).

BMP4 regulates the formation of endoderm expressingSpink3, Ttr and Gata4 through induction of the primitivestreak intermediateThe induction of Spink3+, Ttr+ and Gata4+ endoderm in response

to BMP4 is a two-step process proceeding through a bipotentprimitive streak intermediate, or mesendoderm. The likely rolefor BMP4 in this process is indirect, by way of the induction

of mesendoderm from EPL cells (Harvey et al., 2010). Thepossibility exists, however, that BMP4 has a role in thesubsequent induction of endoderm from mesendoderm.

Activin A can induce primitive streak intermediatesindependently of BMP4 signalling from ES cells and has beenidentified as a potent inducer of endoderm lineages in culture(Izumi et al., 2007; Jackson et al., 2010; Kubo et al., 2004; Tada

et al., 2005). As expected, EPL cells that were differentiatedin activin-A-containing medium (ACM) or ACM+nogginexpressed markers of the primitive streak (Fig. 5A). In cells

differentiated in ACM+noggin+SB, the expression of primitivestreak markers (except Bmp4) was reduced and the expression ofthe neural marker Sox1 was increased (Fig. 5A), suggesting that

the ability of activin A to induce primitive streak intermediatesrequired p38 MAPK. Western blot showed p-p38 MAPK in cellsthat had been treated with ACM (Fig. 5B). The regulation of the

formation of primitive streak intermediates in response to activinA, therefore, is distinct from that observed in response to BMP4or 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 BMP4 signalling suggests that BMP4 does not play

additional roles in the specification of endoderm frommesendoderm. Eya2 expression was higher in EPL cellsdifferentiated in ACM+noggin when compared with cells

differentiated in ACM. The expression of all endoderm markers

Fig. 4. Inhibition of BMP signalling affects endoderm formation indifferentiating EPL cells. (A) EPL cells that were differentiated in BCM orSCM with or without noggin or DMSO for 4 days were analysed by qPCR forthe expression of endoderm-marker genes. The data have beennormalised to b-actin transcript levels. Results are means6s.e.m. n53 or 4.(B,C) EPL cells differentiated in BCM (B) and BCM+noggin (C) weresectioned and stained with haematoxylin and eosin for morphology (i) or forthe expression of Trh (ii). Arrowheads indicate squamous endoderm-likecells on the surface of the aggregates. Scale bars: 50 mm. (D) EPL cellsdifferentiated in SCM 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 that activin

A, like serum, is able to induce both of the proposed endodermpopulations from EPL cells (Fig. 4A). The increased expressionof Eya2 suggests that the formation of endoderm that expresses

these markers can be enhanced by BMP4 inhibition. Theexpression of Sox17, Spink3, Ttr and Eya2 (Fig. 5C) wasdecreased in cells differentiated in ACM+noggon+SB,

consistent with a role for p38 MAPK activity in the activin-A-induced formation of endoderm.

The primitive streak intermediate is the progenitor forendoderm that expresses Spink3, Ttr and Gata4 but notnecessarily for endoderm that expresses Trh and Eya2The formation of endoderm that expresses Spink3, Ttr and Gata4

appears to be dependent on the prior formation of primitive streakintermediates. By contrast, the expression of Trh and Eya2in EPL cells differentiated in BCM+noggin suggests

the formation of an endoderm that does not rely on the priorformation of this population. The formation of primitive-streakintermediates from differentiating EPL cells can be inhibited by

DAPT {N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycinet-butyl ester}, an antagonist of c-secretase (Hughes et al., 2009a).In cells formed from EPL cells differentiated in BCM+DAPT, theexpression of Spink3 and Ttr was decreased, and Eya2 expression

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-streak intermediateformation and with a requirement for the initial formation of

primitive-streak intermediates in the formation of Spink3+ and Ttr+

endoderm. Further differentiation of aggregates cultured inBCM+DAPT showed the expression of markers of

later endoderm populations, consistent with the formation of anendoderm progenitor (supplementary material Fig. S4B).

DISCUSSIONp38 MAPK and the formation of primitive-streakintermediatesThe inhibition of p38 MAPK during EPL-cell differentiation

disrupts the formation of primitive-streak intermediates from EPLcells in response to activin A or serum. Others have shown thatp38 MAPK inhibition during ES cell differentiation in serum

promotes neurogenesis at the expense of cardiogenesis (Aouadi etal., 2006; Barruet et al., 2011; Wu et al., 2010), and that there is arequirement for p38 MAPK activity early in cell differentiation

(Barruet et al., 2011; Davidson and Morange, 2000; Duval et al.,2004; Wu et al., 2010). The inhibition of p38 MAPK activity didnot affect the ability of BMP4 to induce primitive streakintermediates or mesoderm derivatives, suggesting that this

Fig. 5. The formation of primitive-streak intermediates and endoderm in response to activin A signalling requires p38 MAPK activity. (A) EPL cells thatwere differentiated in ACM, ACM+noggin with or without SB or DMSO or in SFM+noggin for 2 or 4 days were analysed by RT-PCR for the expression of markersof primitive streak intermediates (day two and four) or ectoderm (day four). 2R, no reverse transcriptase control; 2D, no cDNA control. A representative result isshown. n53. (B) Serum-starved aggregates were transferred to SFM or SFM containing 25 ng/ml activin A. Aggregates were collected 15, 30 and 60 min aftertransfer and were analysed by western blot for the phosphorylation of p38 MAPK. (C) EPL cells differentiated in ACM, ACM+noggin with or without SB or DMSOor in SFM+noggin for 4 days were analysed by qPCR for the expression of endoderm markers. The data have been normalised to b-actin transcript levels.Results are means6s.e.m. n53. (D) The expression of endoderm markers in EPL cells that were differentiated in BCM+DMSO or DAPT on day four oftreatment. The data have been normalised to b-actin transcript levels. Results are means6s.e.m. n53. *P,0.05, **P,0.01.


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pathway is not essential for the formation of primitive streakintermediates per se. The data presented here are consistent with

a role for p38 MAPK in lineage allocation or specification duringdifferentiation, specifically in the formation of primitive streakintermediates, but only when molecular gastrulation is induced byserum or activin A. Moreover, the inability of cells cultured in

SFM to form primitive streak intermediates, despite intracellularp-p38 MAPK, suggests that p38 MAPK activity is not sufficientfor differentiation but works in conjunction with other pathways.

In cells differentiated in SCM, but not ACM, one role for p38MAPK appears to be the upregulation of Bmp4, which in turninitiates differentiation. The increased expression of Bmp4 in

cells differentiated in SCM, and the significant reduction inits expression in cells differentiated in BCM supplementedwith SB, is consistent with p38 MAPK acting upstream of

Bmp4. Endogenously produced BMP4 acts in turn to inducethe primitive streak intermediate, an activity that is blockedby noggin. Noggin did not completely suppress moleculargastrulation in response to serum, suggesting the presence of

additional, potentially p38 MAPK dependent, pathways. Bycontrast, markers of molecular gastrulation were expressedrobustly in cells differentiated in activin-A-containing medium

supplemented with noggin, suggesting that activin A activity wasindependent of BMP. This is consistent with previous reports thatactivin A does not induce the expression of Bmp4 during ES cell

differentiation (Jackson et al., 2010).The involvement of p38 MAPK in lineage specification from

ES cells has proven difficult to demonstrate, with conflicting

reports on the need for p38 MAPK activity during mesodermspecification. The difficulty in resolving a role for p38 MAPK ismost likely a consequence of the complexity of the molecularmechanisms that regulate gastrulation coupled with the use of ill-

defined and poorly understood culture reagents. The variability ofoutcomes elicited in cells cultured in the presence of p38 MAPKinhibitors (Aouadi et al., 2006; Barruet et al., 2011; Wu et al.,

2010) or from Mapk142/2 cells (also known as p38a2/2 cells)(Allen et al., 2000; Chakraborty et al., 2009; Guo et al., 2007;Aouadi et al., 2006) could be attributed to the confounding use of

serum in these experiments. Some sera have been reported tocontain exogenous BMP activity (Herrera and Inman, 2009;Kodaira et al., 2006). BMP activity within sera would be able tospecify primitive streak intermediates and mesoderm lineages in

the absence of p38 MAPK activity. Our interpretation of therespective roles of serum and growth factors suggests that serumvariability is a contributing factor to variability in the analysis of

molecular gastrulation, and in the analysis of the role of p38MAPK 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 here and by others(Barruet et al., 2011; Aouadi et al., 2006; Wu et al., 2010). The

differentiation of human ES cells was induced in cell aggregates by aconditioned medium in which the signalling activity was largelyuncharacterised. Potentially, increased cardiocyte formation resultedfrom an increase in the number of primitive streak intermediates

(formed in response to BMP or to similar signalling activity withinthe conditioned medium) adopting a mesoderm fate. This wouldoccur when p38 MAPK was inhibited in these cells, preventing their

differentiation to the endoderm.p38 MAPK comprises a, b, c and d isoforms that are encoded

separately. Of these, p38a and p38b are expressed in the germlayers and primitive ectoderm, respectively, at gastrulation (Zohn

et al., 2006). The disruption of the gene encoding p38a resulted inplacental defects and embryonic death mid-gestation (Adams

et al., 2000; Mudgett et al., 2000). Double-mutant embryos,lacking p38a and p38b in embryonic tissues, gastrulate but failmid-gestation with diverse developmental defects (del BarcoBarrantes et al., 2011). Embryos with mutations in the MKK3,

MKK4 and MKK6 kinases that have been shown to activate p38MAPK also 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 lossof p38 MAPK phosphorylation in the primitive streak, and thecells that lack p-p38 MAPK fail to migrate (Zohn et al., 2006).

This mutation did not, however, prevent the formation ofprimitive-streak intermediates or mesoderm specification, butraises the possibility that p38 MAPK has a role in the regulation

of an epithelial-to-mesenchymal transition during differentiation.It is unlikely, therefore, that p38 MAPK is essential for theformation of primitive streak intermediates in vivo, but its role isrevealed during in vitro differentiation in response to serum or

activin A.

A novel role for p38 MAPK in the formation of definitive-endoderm populationsEndoderm formation in the mammal is complex, with theformation of two endoderm lineages from the pluripotent

lineage during early development – primitive or visceralendoderm and definitive endoderm. Analysis of EPL celldifferentiation suggests that the definitive endoderm might form

as two distinct cell populations that can be distinguished by geneexpression and ontogeny. The formation of both lineages requiredp38 MAPK activity.

p38 MAPK inhibition during the differentiation of EPL cells in

response to BMP4 resulted in the reduced expression of Mixl1,Spink3 and Ttr. When p38 MAPK was inhibited in cells thatdifferentiated in response to serum there was reduced expression

of Mixl1, Sox17, Spink3, Ttr, Gata4 and Eya2. These resultssuggest a previously unidentified role for p38 MAPK in theformation of definitive endoderm. In the mouse, definitive

endoderm formation is dependent on signalling through nodal(Tremblay et al., 2000; Vincent et al., 2003), a member of theTGFb family with similar signalling properties to activin A(Conlon et al., 1994; Vincent et al., 2003). Similarly, ES cell

differentiation induced by activin A results in the enrichment ofdefinitive endoderm (Gadue et al., 2006; Kubo et al., 2004;Nostro et al., 2011). Canonically, nodal signalling is mediated by

Smad2/3 and the downstream effectors of Smads (Heldin et al.,1997; Massaous and Hata, 1997). p38 MAPK has been shown tobe activated by TGFbs and to mediate signalling in response tothese factors (Hanafusa et al., 1999; Hu et al., 2004; Yue andMulder, 2000). The inhibition of p38 MAPK impaired theinduction of definitive 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 thepositional specification 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 formationin response to Nodal.

The differential effects of the inhibitor SB on the expression of

endoderm markers in aggregates that were differentiated inSCM or BCM infers that the specification of endoderm frompluripotent cells can 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 previously tomark visceral endoderm (Kwon et al, 2008), but the widespread

expression of Ttr in EBs derived from EPL cells, in whichvisceral endoderm is rarely formed (Vassilieva et al., 2012), andthe reliance of Ttr expression on molecular gastrulation byEPL cells suggests that Ttr can also be expressed in an

endoderm formed during molecular gastrulation. The formationof a Spink3+, Ttr+ and Gata4+ endoderm population fromdifferentiating EPL cells correlated with the prior expression of

primitive streak markers. When the formation of primitive streakintermediates was inhibited, as happened when cells weredifferentiated in BCM supplemented with noggin or DAPT, the

expression of Spink3, Ttr and Gata4 was reduced. The secondendoderm population was marked by the expression of Trh andEya2. The expression of these markers is maintained in cells

differentiated in BCM supplemented with noggin or DAPT,suggesting that Trh+ and Eya2+ endoderm can form in theabsence of a T-expressing primitive-streak intermediate. Thedifferential expression of Spink3, Ttr and Gata4 between

conditions that enriched or suppressed the formation of theprimitive-streak intermediate, coupled with the persistence ofTrh+ and Eya2+ endoderm when the formation of the primitive

streak intermediate was suppressed, is consistent with theformation of two endoderm populations during EPL celldifferentiation.

A model for the regulation of molecular gastrulationBased on this analysis of the formation of the primitive-streak

intermediate and endoderm from EPL cells, we propose a revisedparadigm for molecular gastrulation (Fig. 6). This modelproposes that primitive streak intermediates, which express Tand other primitive streak markers, can be induced from EPL

cells through multiple pathways, including pathways dependenton BMP4 signalling, growth factors/cytokines, including activinA and the active components of serum, which require p38 MAPK

signalling and, as has been reported by others, WNT signalling(Tanaka et al., 2009). These pathways most likely generatedistinct primitive streak intermediates that are distinguished by

divergent differentiation potential, as has been shown for BMP4and Wnt (Tanaka et al., 2009). We propose that the primitive streak

intermediate induced by BMP4 can differentiate to form mesodermand a population of endoderm expressing Spink3, Ttr and Gata4. Ingene expression, this population is similar to the ring of endodermmarked by Spink3 in the proximal region of the embryo. Formation

of this endoderm population requires p38 MAPK activity inthe primitive streak intermediate; activation of this pathway isachieved through endogenous signalling in aggregates that are

differentiated in the presence of BMP4. Alternatively, EPL cellscan differentiate into a Trh+ and Eya2+ endoderm that can beformed independently of the BMP4-induced primitive streak

intermediate; this population also requires p38 MAPK activityfor its formation. The gene expression profile of this populationmirrors the endoderm of the distal region of the embryo (Gu et al.,

2004; McKnight et al., 2007).The endoderm populations defined here are both are products

of EPL cell differentiation and arise during moleculargastrulation. We propose, therefore, that these populations are

subpopulations of the definitive endoderm and suggest thatthe terms ‘proximal definitive endoderm’ and ‘distal definitiveendoderm’ are used to describe them. Two waves of definitive

endoderm, which populate the more proximal (lateral endoderm)and more distal (medial endoderm) regions of the endoderm, havebeen proposed to occur during embryogenesis (Tam, 2007).

These populations have been distinguished by their time of exitfrom the primitive streak, their direction of migration across theegg cylinder and their allocation to different regions of the gut

tube in later development. The populations defined from invitro differentiation here potentially represent the populationsidentified by fate mapping in vivo.

Our proposed model (Fig. 6) addresses the role of the

mesendoderm in mammalian gastrulation, suggesting apopulation that (1) satisfies the criteria of mesendoderm withinthe population of primitive streak intermediates induced by

BMP4 and (2) acts as a progenitor of the proximal definitiveendoderm and mesoderm. The model also describes distaldefinitive endoderm that can form independently of the BMP

Fig. 6. A model of endoderm formation fromEPL cells. Formation of the proximal definitiveendoderm is dependent on p38 MAPK activityand correlates with the prior expression ofmarkers of the primitive-streak intermediate. Theinitial formation of the T-expressing primitive-streak intermediate can occur in response to anumber of pathways including those regulated byBMP4, activin A, serum and, we hypothesize,Wnt (WNT3), and likely results in a mixedpopulation of progenitors (indicated by the use ofseveral ovals). The formation of the distaldefinitive endoderm is dependent on p38 MAPKsignalling but is not correlated with the initialformation of the T-expressing primitive-streakintermediate. PSI, primitive streak intermediate;DE, definitive endoderm.


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signalling, suggesting that not all definitive endoderm formationproceeds through the mesendoderm and raising the possibility

of an endoderm-specific progenitor. Definitive endodermformation independent of a bipotent progenitor has beensuggested previously by fate mapping of the embryo (Lawsonet al., 1991; Rouse et al., 1994).

A goal of stem cell research is to generate, in sufficientquantity, functional cell types with commercial and clinicalapplications. Many approaches have been reported for forming

definitive endoderm and derivatives from ES cells. These relyalmost exclusively on the prior formation of primitive streakintermediates and, with few exceptions (Mathew et al., 2012;

Morrison et al., 2008), the positional identity of the endodermpopulation that is formed has not been considered. What isclear from the literature is that the formation of later endoderm

populations is generally inefficient; this is potentially aconsequence of the inefficient generation of the appropriateprogenitor at the onset of differentiation. Positional specificationcould restrict the developmental potential of ES-cell-derived

definitive endoderm and success might depend on enrichment fora specific endoderm population. Characterisation of the derivativesof proximal and distal definitive endoderm populations in the

embryo and in culture will allow differentiation protocols to betailored to ensure the enrichment of the appropriate definitiveendoderm for subsequent differentiation.

MATERIALS AND METHODSCell cultureMouse ESD3 ES cells (Doetschman et al., 1985) were used throughout. ES cell

maintenance, EPL cell formation (as aggregates), EB formation and the

production of the conditioned medium MEDII were performed as described

previously (Rathjen and Rathjen, 2003). All treatments were administered

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 these experiments

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&D Systems),

SB (10 mM, Sigma) and/or 0.1% DMSO (Sigma) were added as describedin the text and EPL cells were cultured for 3 days with daily 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 emanating from the

aggregates)] from aggregates was determined as described previously

(Hughes et al., 2009a; Hughes et al., 2009b). For each experimental

condition §24 aggregates were analysed. Ideally, a single aggregate wasanalysed per well but in reality some wells contained more than one

analysed aggregate. Alternatively, aggregates were treated for 4 days in

suspension culture before they were mass-seeded in a 9.6 cm2 dish and

maintained in SCM with regular medium change for a further 7 days.

Gene expression assaysEPL cells were transferred to SCM, BCM or ACM [SFM+activin A

(25 ng/ml); supplementary material Table S1] and were supplemented

with noggin (90 ng/ml), SB (10 mM), DAPT (50 mM) and/or 0.1 or 0.2%DMSO, as described in the text, and cultured for 4 days with daily

medium change. Cells were collected after 2, 3 and 4 days of treatment.

RT-PCR and quantitative PCRTotal cytoplasmic RNA was isolated using TRIzolH (Invitrogen). cDNAwas synthesised as per the manufacturer’s protocol (Promega). Primers

(supplementary material Table S2) were validated on differentiated ES

(mouse) or genomic DNA (human), and PCR products were sequenced.

For RT-PCR, 25 ml reactions contained 1 ng/ml of forward and reverseprimers, 16GoTaqH Green Master Mix (Promega) and cDNA. Reactionswere heated to 94 C̊ for 2 min before cycles of 94 C̊ for 30 s, 60 C̊

for 30 s and 72 C̊ for 30 s, ending with 5 min at 72 C̊, in an MJ

Research thermocycler. PCR products were visualised with a Molecular

ImagerH ChemiDocTM XRS Imaging System (BioRad) with SYBRHGold (Invitrogen). Gene expression was quantified using Quantity One 1-

D band analysis software (BioRad).

For quantitative PCR (qPCR), reactions containing 16Absolute blueQPCR SYBR Green Mix (Thermo Scientific), cDNA and 200 nM

of forward and reverse primers were performed on an MJ research

thermocycler with a Chromo4 Continuous Fluorescence Detection

system (MJ Research). Reactions were heated to 95 C̊ for 15 min

before cycling at 95 C̊ for 15 s, 60 C̊ for 15 s and 72 C̊ for 30 s. The raw

data was analysed using the Q-Gene software package (Muller et al.,

2002; Simon, 2003).

Whole-mount in situ hybridisation (WISH)Embryos from time-mated Swiss mice and cell aggregates were fixed in

4% paraformaldehyde (PFA) and dehydrated in methanol. WISH was

performed as described previously (Lake et al., 2000; Rosen and

Beddington, 1993) with modifications. Rehydrated embryos were treated

with 6% H2O2. Probes were labelled using digoxigenin-11-dUTP or

fluorescein-12-UTP (Roche). The hybridisation and post-hybridisation

washes were performed at 65 C̊.

Embryos and aggregates were incubated overnight with anti-

digoxigenin–AP Fab fragments (1:2000) (Roche) or anti-fluorescein–

AP Fab fragments (1:2000) (Roche) and were developed with NBT/

BCIP or INT/BCIP (Roche), respectively, as per the manufacturer’s

instructions. The samples were photographed using an Olympus UC30

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 cDNA fragments,

linearized with NcoI or SalI, and transcribed with SP6 (antisense) or T7

(sense) RNA polymerases. Aggregates were embedded in paraffin and

sectioned as required.

Western blotEPL cell aggregates were serum starved for 2 h in SFM 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 or FCS.Total proteins was analysed by western blotting. Membranes were

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) and analysed by

Quantity OneTM. Primary antibodies were against p38, p-p38, pSmad1/5/

8 (Cell Signaling Technologies) and b-tubulin I (Sigma). The secondaryantibodies were HRP-conjugated (Cell Signaling Technologies and

DakoCytomation).

HRP-uptake assayThe HRP-uptake assay was performed as described previously (Kanai-

Azuma et al., 2002; Vassilieva et al., 2012).

Statistical analysisExperiments were analysed using unpaired one- or two-tailed Student’s t-

tests in MicrosoftH Excel software. The statistical significance is denotedas follows: *P,0.05, **P,0.01. Comparisons are made betweenoutcomes in SB, noggin and DAPT compared to the equivalent base

medium, with or without DMSO.

AcknowledgementsThe authors would like to thank members of the Rathjen laboratory for insightfuldiscussions of the project.


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Competing interestsThe authors declare no competing interests.

Author contributionsC.Y.: conception and design, collection and/or assembly of data, data analysisand interpretation, manuscript writing. H.N.G.: conception and design, collectionand/or assembly of data, data analysis and interpretation. M.F. conception anddesign, data analysis and interpretation. P.D.R.: conception and design. J.R.:conception and design, data analysis and interpretation, manuscript writing, finalapproval of manuscript.

FundingThis work was supported by the University of Melbourne and the Albert ShimminsPostgraduate Writing up Award. C.Y. and H.N.G. were supported by AustralianPostgraduate Awards, C.Y. received additional support from the Australian StemCell Centre.

Supplementary materialSupplementary material available online athttp://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.134502/-/DC1

ReferencesAdams, R. H., Porras, A., Alonso, G., Jones, M., Vintersten, K., Panelli, S.,Valladares, A., Perez, L., Klein, R. and Nebreda, A. R. (2000). Essential roleof p38alpha MAP kinase in placental but not embryonic cardiovasculardevelopment. Mol. Cell 6, 109-116.

Allen, M., Svensson, L., Roach, M., Hambor, J., McNeish, J. and Gabel, C. A.(2000). Deficiency of the stress kinase p38alpha results in embryonic lethality:characterization of the kinase dependence of stress responses of enzyme-deficient embryonic stem cells. J. Exp. Med. 191, 859-870.

Aouadi, M., Bost, F., Caron, L., Laurent, K., Le Marchand Brustel, Y. andBinétruy, B. (2006). p38 mitogen-activated protein kinase activity commitsembryonic stem cells to either neurogenesis or cardiomyogenesis. Stem Cells24, 1399-1406.

Barruet, E., Hadadeh, O., Peiretti, F., Renault, V. M., Hadjal, Y., Bernot, D.,Tournaire, R., Negre, D., Juhan-Vague, I., Alessi, M. C. et al. (2011). p38mitogen activated protein kinase controls two successive-steps during the earlymesodermal commitment of embryonic stem cells. Stem Cells Dev. 20, 1233-1246.

Beddington, R. S. and Robertson, E. J. (1998). Anterior patterning in mouse.Trends Genet. 14, 277-284.

Beddington, R. S. and Robertson, E. J. (1999). Axis development and earlyasymmetry in mammals. Cell 96, 195-209.

Bradley, A., Evans, M., Kaufman, M. H. and Robertson, E. (1984). Formation ofgerm-line chimaeras from embryo-derived teratocarcinoma cell lines. Nature309, 255-256.

Chakraborty, S., Kang, B., Huang, F. and Guo, Y. L. (2009). Mouse embryonicstem cells lacking p38alpha and p38delta can differentiate to endothelial cells,smooth muscle cells, and epithelial cells. Differentiation 78, 143-150.

Clements, M., Pernaute, B., Vella, F. and Rodriguez, T. A. (2011). Crosstalkbetween Nodal/activin and MAPK p38 signalling is essential for anterior-posterior axis specification. Curr. Biol. 21, 1289-1295.

Conlon, F. L., Lyons, K. M., Takaesu, N., Barth, K. S., Kispert, A., Herrmann, B.and Robertson, E. J. (1994). A primary requirement for nodal in the formation andmaintenance of the primitive streak in the mouse. Development 120, 1919-1928.

Coucouvanis, E. and Martin, G. R. (1995). Signals for death and survival: a two-step mechanism for cavitation in the vertebrate embryo. Cell 83, 279-287.

Crossley, P. H. and Martin, G. R. (1995). The mouse Fgf8 gene encodes a familyof polypeptides and is expressed in regions that direct outgrowth and patterningin the developing embryo. Development 121, 439-451.

Cuenda, A., Rouse, J., Doza, Y. N., Meier, R., Cohen, P., Gallagher, T. F.,Young, P. R. and Lee, J. C. (1995). SB 203580 is a specific inhibitor of a MAPkinase homologue which is stimulated by cellular stresses and interleukin-1.FEBS Lett. 364, 229-233.

Davidson, S. M. and Morange, M. (2000). Hsp25 and the p38 MAPK pathway areinvolved in differentiation of cardiomyocytes. Dev. Biol. 218, 146-160.

del Barco Barrantes, I., Coya, J. M., Maina, F., Arthur, J. S. and Nebreda, A. R.(2011). Genetic analysis of specific and redundant roles for p38alpha andp38beta MAPKs during mouse development. Proc. Natl. Acad. Sci. USA 108,12764-12769.

Dickson, M. C., Martin, J. S., Cousins, F. M., Kulkarni, A. B., Karlsson, S. andAkhurst, R. J. (1995). Defective haematopoiesis and vasculogenesis intransforming growth factor-beta 1 knock out mice. Development 121, 1845-1854.

Doetschman, T. C., Eistetter, H., Katz, M., Schmidt, W. and Kemler, R. (1985).The in vitro development of blastocyst-derived embryonic stem cell lines:formation of visceral yolk sac, blood islands and myocardium. J. Embryol. Exp.Morphol. 87, 27-45.

Duval, D., Malaisé, M., Reinhardt, B., Kedinger, C. and Boeuf, H. (2004). A p38inhibitor allows to dissociate differentiation and apoptotic processes triggered uponLIF withdrawal in mouse embryonic stem cells. Cell Death Differ. 11, 331-341.

Engelman, J. A., Lisanti, M. P. and Scherer, P. E. (1998). Specific inhibitors ofp38 mitogen-activated protein kinase block 3T3-L1 adipogenesis. J. Biol. Chem.273, 32111-32120.

Evans, M. J. and Kaufman, M. H. (1981). Establishment in culture of pluripotentialcells from mouse embryos. Nature 292, 154-156.

Farace, M. G., Brown, B. A., Raschellà, G., Alexander, J., Gambari, R.,Fantoni, A., Hardies, S. C., Hutchison, C. A., 3rd and Edgell, M. H. (1984).The mouse beta h1 gene codes for the z chain of embryonic hemoglobin. J. Biol.Chem. 259, 7123-7128.

Gadue, P., Huber, T. L., Paddison, P. J. and Keller, G. M. (2006). Wnt and TGF-beta signaling are required for the induction of an in vitro model of primitivestreak formation using embryonic stem cells. Proc. Natl. Acad. Sci. USA 103,16806-16811.

Gadue, P., Gouon-Evans, V., Cheng, X., Wandzioch, E., Zaret, K. S., Grompe,M., Streeter, P. R. and Keller, G. M. (2009). Generation of monoclonalantibodies specific for cell surface molecules expressed on early mouseendoderm. Stem Cells 27, 2103-2113.

Graichen, R., Xu, X., Braam, S. R., Balakrishnan, T., Norfiza, S., Sieh, S., Soo,S. Y., Tham, S. C., Mummery, C., Colman, A. et al. (2008). Enhancedcardiomyogenesis of human embryonic stem cells by a small molecular inhibitorof p38 MAPK. Differentiation 76, 357-370.

Gu, G., Wells, J. M., Dombkowski, D., Preffer, F., Aronow, B. and Melton, D. A.(2004). Global expression analysis of gene regulatory pathways duringendocrine pancreatic development. Development 131, 165-179.

Guillemot, F., Lo, L.-C., Johnson, J. E., Auerbach, A., Anderson, D. J.and Joyner, A. L. (1993). Mammalian achaete-scute homolog 1 is requiredfor the early development of olfactory and autonomic neurons. Cell 75, 463-476.

Guo, Y. L., Ye, J. and Huang, F. (2007). p38alpha MAP kinase-deficient mouseembryonic stem cells can differentiate to endothelial cells, smooth muscle cells,and neurons. Dev. Dyn. 236, 3383-3392.

Han, J., Lee, J. D., Bibbs, L. and Ulevitch, R. J. (1994). A MAP kinase targetedby endotoxin and hyperosmolarity in mammalian cells. Science 265, 808-811.

Hanafusa, H., Ninomiya-Tsuji, J., Masuyama, N., Nishita, M., Fujisawa, J.,Shibuya, H., Matsumoto, K. and Nishida, E. (1999). Involvement of the p38mitogen-activated protein kinase pathway in transforming growth factor-beta-induced gene expression. J. Biol. Chem. 274, 27161-27167.

Hart, A. H., Hartley, L., Sourris, K., Stadler, E. S., Li, R., Stanley, E. G., Tam,P. P., Elefanty, A. G. and Robb, L. (2002). Mixl1 is required for axialmesendoderm morphogenesis and patterning in the murine embryo.Development 129, 3597-3608.

Harvey, N. T., Hughes, J. N., Lonic, A., Yap, C., Long, C., Rathjen, P. D. andRathjen, J. (2010). Response to BMP4 signalling during ES cell differentiationdefines intermediates of the ectoderm lineage. J. Cell Sci. 123, 1796-1804.

Heldin, C. H., Miyazono, K. and ten Dijke, P. (1997). TGF-beta signalling fromcell membrane to nucleus through SMAD proteins. Nature 390, 465-471.

Herrera, B. and Inman, G. J. (2009). A rapid and sensitive bioassay for thesimultaneous measurement of multiple bone morphogenetic proteins.Identification and quantification of BMP4, BMP6 and BMP9 in bovine andhuman serum. BMC Cell Biol. 10, 20.

Hu, M. C., Wasserman, D., Hartwig, S. and Rosenblum, N. D. (2004). p38MAPKacts in the BMP7-dependent stimulatory pathway during epithelial cellmorphogenesis and is regulated by Smad1. J. Biol. Chem. 279, 12051-12059.

Hughes, J. N., Dodge, N., Rathjen, P. D. and Rathjen, J. (2009a). A novel role forgamma-secretase in the formation of primitive streak-like intermediates from EScells in culture. Stem Cells 27, 2941-2951.

Hughes, J. N., Washington, J. M., Zheng, Z., Lau, X. K., Yap, C., Rathjen, P. D.and Rathjen, J. (2009b). Manipulation of cell:cell contacts and mesodermsuppressing activity direct lineage choice from pluripotent primitive ectoderm-like cells in culture. PLoS ONE 4, e5579.

Izumi, N., Era, T., Akimaru, H., Yasunaga, M. and Nishikawa, S. (2007).Dissecting the molecular hierarchy for mesendoderm differentiation through acombination of embryonic stem cell culture and RNA interference. Stem Cells25, 1664-1674.

Jackson, S. A., Schiesser, J., Stanley, E. G. and Elefanty, A. G. (2010).Differentiating embryonic stem cells pass through ‘temporal windows’ that markresponsiveness to exogenous and paracrine mesendoderm inducing signals.PLoS ONE 5, e10706.

Kanai-Azuma, M., Kanai, Y., Gad, J. M., Tajima, Y., Taya, C., Kurohmaru, M.,Sanai, Y., Yonekawa, H., Yazaki, K., Tam, P. P. et al. (2002). Depletion ofdefinitive gut endoderm in Sox17-null mutant mice.Development 129, 2367-2379.

Kinder, S. J., Tsang, T. E., Wakamiya, M., Sasaki, H., Behringer, R. R., Nagy, A.and Tam, P. P. (2001). The organizer of the mouse gastrula is composed of adynamic population of progenitor cells for the axial mesoderm. Development128, 3623-3634.

Kodaira, K., Imada, M., Goto, M., Tomoyasu, A., Fukuda, T., Kamijo, R., Suda, T.,Higashio, K. and Katagiri, T. (2006). Purification and identification of a BMP-likefactor from bovine serum. Biochem. Biophys. Res. Commun. 345, 1224-1231.

Kubo, A., Shinozaki, K., Shannon, J. M., Kouskoff, V., Kennedy, M., Woo, S.,Fehling, H. J. and Keller, G. (2004). Development of definitive endoderm fromembryonic stem cells in culture. Development 131, 1651-1662.

Kwon, G. S., Viotti, M. and Hadjantonakis, A. K. (2008). The endoderm of themouse embryo arises by dynamic widespread intercalation of embryonic andextraembryonic lineages. Dev. Cell 15, 509-520.


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http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.134502/-/DC1http://dx.doi.org/10.1084/jem.191.5.859http://dx.doi.org/10.1084/jem.191.5.859http://dx.doi.org/10.1084/jem.191.5.859http://dx.doi.org/10.1084/jem.191.5.859http://dx.doi.org/10.1634/stemcells.2005-0398http://dx.doi.org/10.1634/stemcells.2005-0398http://dx.doi.org/10.1634/stemcells.2005-0398http://dx.doi.org/10.1634/stemcells.2005-0398http://dx.doi.org/10.1089/scd.2010.0213http://dx.doi.org/10.1089/scd.2010.0213http://dx.doi.org/10.1089/scd.2010.0213http://dx.doi.org/10.1089/scd.2010.0213http://dx.doi.org/10.1089/scd.2010.0213http://dx.doi.org/10.1016/S0168-9525(98)01499-1http://dx.doi.org/10.1016/S0168-9525(98)01499-1http://dx.doi.org/10.1016/S0092-8674(00)80560-7http://dx.doi.org/10.1016/S0092-8674(00)80560-7http://dx.doi.org/10.1038/309255a0http://dx.doi.org/10.1038/309255a0http://dx.doi.org/10.1038/309255a0http://dx.doi.org/10.1016/j.diff.2009.05.006http://dx.doi.org/10.1016/j.diff.2009.05.006http://dx.doi.org/10.1016/j.diff.2009.05.006http://dx.doi.org/10.1016/j.cub.2011.06.048http://dx.doi.org/10.1016/j.cub.2011.06.048http://dx.doi.org/10.1016/j.cub.2011.06.048http://dx.doi: 10.1016/0092-95)90169-http://dx.doi: 10.1016/0092-95)90169-http://dx.doi.org/10.1016/0014-5793(95)00357-Fhttp://dx.doi.org/10.1016/0014-5793(95)00357-Fhttp://dx.doi.org/10.1016/0014-5793(95)00357-Fhttp://dx.doi.org/10.1016/0014-5793(95)00357-Fhttp://dx.doi.org/10.1006/dbio.1999.9596http://dx.doi.org/10.1006/dbio.1999.9596http://dx.doi.org/10.1073/pnas.1015013108http://dx.doi.org/10.1073/pnas.1015013108http://dx.doi.org/10.1073/pnas.1015013108http://dx.doi.org/10.1073/pnas.1015013108http://dx.doi.org/10.1038/sj.cdd.4401337http://dx.doi.org/10.1038/sj.cdd.4401337http://dx.doi.org/10.1038/sj.cdd.4401337http://dx.doi.org/10.1038/sj.cdd.4401337http://dx.doi.org/10.1074/jbc.273.48.32111http://dx.doi.org/10.1074/jbc.273.48.32111http://dx.doi.org/10.1074/jbc.273.48.32111http://dx.doi.org/10.1038/292154a0http://dx.doi.org/10.1038/292154a0http://dx.doi.org/10.1073/pnas.0603916103http://dx.doi.org/10.1073/pnas.0603916103http://dx.doi.org/10.1073/pnas.0603916103http://dx.doi.org/10.1073/pnas.0603916103http://dx.doi.org/10.1002/stem.147http://dx.doi.org/10.1002/stem.147http://dx.doi.org/10.1002/stem.147http://dx.doi.org/10.1002/stem.147http://dx.doi.org/10.1111/j.1432-0436.2007.00236.xhttp://dx.doi.org/10.1111/j.1432-0436.2007.00236.xhttp://dx.doi.org/10.1111/j.1432-0436.2007.00236.xhttp://dx.doi.org/10.1111/j.1432-0436.2007.00236.xhttp://dx.doi.org/10.1242/dev.00921http://dx.doi.org/10.1242/dev.00921http://dx.doi.org/10.1242/dev.00921http://dx.doi.org/10.1016/0092-8674(93)90381-Yhttp://dx.doi.org/10.1016/0092-8674(93)90381-Yhttp://dx.doi.org/10.1016/0092-8674(93)90381-Yhttp://dx.doi.org/10.1016/0092-8674(93)90381-Yhttp://dx.doi.org/10.1002/dvdy.21374http://dx.doi.org/10.1002/dvdy.21374http://dx.doi.org/10.1002/dvdy.21374http://dx.doi.org/10.1126/science.7914033http://dx.doi.org/10.1126/science.7914033http://dx.doi.org/10.1074/jbc.274.38.27161http://dx.doi.org/10.1074/jbc.274.38.27161http://dx.doi.org/10.1074/jbc.274.38.27161http://dx.doi.org/10.1074/jbc.274.38.27161http://dx.doi.org/10.1242/jcs.047530http://dx.doi.org/10.1242/jcs.047530http://dx.doi.org/10.1242/jcs.047530http://dx.doi.org/10.1038/37284http://dx.doi.org/10.1038/37284http://dx.doi.org/10.1186/1471-2121-10-20http://dx.doi.org/10.1186/1471-2121-10-20http://dx.doi.org/10.1186/1471-2121-10-20http://dx.doi.org/10.1186/1471-2121-10-20http://dx.doi.org/10.1074/jbc.M310526200http://dx.doi.org/10.1074/jbc.M310526200http://dx.doi.org/10.1074/jbc.M310526200http://dx.doi.org/10.1074/jbc.M310526200http://dx.doi.org/10.1371/journal.pone.0005579http://dx.doi.org/10.1371/journal.pone.0005579http://dx.doi.org/10.1371/journal.pone.0005579http://dx.doi.org/10.1371/journal.pone.0005579http://dx.doi.org/10.1634/stemcells.2006-0681http://dx.doi.org/10.1634/stemcells.2006-0681http://dx.doi.org/10.1634/stemcells.2006-0681http://dx.doi.org/10.1634/stemcells.2006-0681http://dx.doi.org/10.1371/journal.pone.0010706http://dx.doi.org/10.1371/journal.pone.0010706http://dx.doi.org/10.1371/journal.pone.0010706http://dx.doi.org/10.1371/journal.pone.0010706http://dx.doi.org/10.1016/j.bbrc.2006.05.045http://dx.doi.org/10.1016/j.bbrc.2006.05.045http://dx.doi.org/10.1016/j.bbrc.2006.05.045http://dx.doi.org/10.1242/dev.01044http://dx.doi.org/10.1242/dev.01044http://dx.doi.org/10.1242/dev.01044http://dx.doi: 10.1016/j.devcel.2008.07.017.http://dx.doi: 10.1016/j.devcel.2008.07.017.http://dx.doi: 10.1016/j.devcel.2008.07.017.

Jour

nal o

f Cel

l Sci

ence

Lake, J., Rathjen, J., Remiszewski, J. and Rathjen, P. D. (2000). Reversibleprogramming of pluripotent cell differentiation. J. Cell Sci. 113, 555-566.

Lawson, K. A., Meneses, J. J. and Pedersen, R. A. (1991). Clonal analysis ofepiblast fate during germ layer formation in the mouse embryo. Development113, 891-911.

Lee, J. C., Laydon, J. T., McDonnell, P. C., Gallagher, T. F., Kumar, S., Green,D., McNulty, D., Blumenthal, M. J., Heys, J. R., Landvatter, S. W. et al.(1994). A protein kinase involved in the regulation of inflammatory cytokinebiosynthesis. Nature 372, 739-746.

Li, L., Arman, E., Ekblom, P., Edgar, D., Murray, P. and Lonai, P. (2004). DistinctGATA6- and laminin-dependent mechanisms regulate endodermal andectodermal embryonic stem cell fates. Development 131, 5277-5286.

Lickert, H., Kutsch, S., Kanzler, B., Tamai, Y., Taketo, M. M. and Kemler, R.(2002). Formation of multiple hearts in mice following deletion of beta-catenin inthe embryonic endoderm. Dev. Cell 3, 171-181.

Lints, T. J., Parsons, L. M., Hartley, L., Lyons, I. and Harvey, R. P. (1993). Nkx-2.5: a novel murine homeobox gene expressed in early heart progenitor cellsand their myogenic descendants. Development 119, 419-431.

Liu, P., Wakamiya, M., Shea, M. J., Albrecht, U., Behringer, R. R. and Bradley,A. (1999). Requirement for Wnt3 in vertebrate axis formation. Nat. Genet. 22,361-365.

Lu, H. T., Yang, D. D., Wysk, M., Gatti, E., Mellman, I., Davis, R. J. and Flavell,R. A. (1999). Defective IL-12 production in mitogen-activated protein (MAP)kinase kinase 3 (Mkk3)-deficient mice. EMBO J. 18, 1845-1857.

Martin, G. R. (1981). Isolation of a pluripotent cell line from early mouse embryoscultured in medium conditioned by teratocarcinoma stem cells. Proc. Natl. Acad.Sci. USA 78, 7634-7638.

Martı́n-Blanco, E. (2000). p38 MAPK signalling cascades: ancient roles and newfunctions. Bioessays 22, 637-645.

Massaous, J. and Hata, A. (1997). TGF-beta signalling through the Smadpathway. Trends Cell Biol. 7, 187-192.

Mathew, S., Jaramillo, M., Zhang, X., Zhang, L. A., Soto-Gutiérrez, A. andBanerjee, I. (2012). Analysis of alternative signaling pathways of endoderminduction of human embryonic stem cells identifies context specific differences.BMC Syst. Biol. 6, 154.

McKnight, K. D., Hou, J. and Hoodless, P. A. (2007). Dynamic expression ofthyrotropin-releasing hormone in the mouse definitive endoderm. Dev. Dyn.236, 2909-2917.

Morrison, G. M., Oikonomopoulou, I., Migueles, R. P., Soneji, S., Livigni, A.,Enver, T. and Brickman, J. M. (2008). Anterior definitive endoderm from ESCsreveals a role for FGF signaling. Cell Stem Cell 3, 402-415.

Mudgett, J. S., Ding, J., Guh-Siesel, L., Chartrain, N. A., Yang, L., Gopal, S.and Shen, M. M. (2000). Essential role for p38alpha mitogen-activated proteinkinase in placental angiogenesis. Proc. Natl. Acad. Sci. USA 97, 10454-10459.

Muller, P. Y., Janovjak, H., Miserez, A. R. and Dobbie, Z. (2002). Short technicalreport processing of gene expression data generated by quantitative real-timeRT-PCR. Biotechniques 32, 1372-1374.

Nagata, Y., Takahashi, N., Davis, R. J. and Todokoro, K. (1998). Activation ofp38 MAP kinase and JNK but not ERK is required for erythropoietin-inducederythroid differentiation. Blood 92, 1859-1869.

Nakamura, K., Shirai, T., Morishita, S., Uchida, S., Saeki-Miura, K. andMakishima, F. (1999). p38 mitogen-activated protein kinase functionallycontributes to chondrogenesis induced by growth/differentiation factor-5 inATDC5 cells. Exp. Cell Res. 250, 351-363.

Nebreda, A. R. and Porras, A. (2000). p38 MAP kinases: beyond the stressresponse. Trends Biochem. Sci. 25, 257-260.

Nostro, M. C., Sarangi, F., Ogawa, S., Holtzinger, A., Corneo, B., Li, X.,Micallef, S. J., Park, I. H., Basford, C., Wheeler, M. B. et al. (2011). Stage-specific signaling through TGFb family members and WNT regulates patterningand pancreatic specification of human pluripotent stem cells. Development 138,861-871.

Pelton, T. A., Sharma, S., Schulz, T. C., Rathjen, J. and Rathjen, P. D. (2002).Transient pluripotent cell populations during primitive ectoderm formation:correlation of in vivo and in vitro pluripotent cell development. J. Cell Sci. 115,329-339.

Perdiguero, E., Ruiz-Bonilla, V., Gresh, L., Hui, L., Ballestar, E., Sousa-Victor,P., Baeza-Raja, B., Jardı́, M., Bosch-Comas, A., Esteller, M. et al. (2007).Genetic analysis of p38 MAP kinases in myogenesis: fundamental role ofp38alpha in abrogating myoblast proliferation. EMBO J. 26, 1245-1256.

Pevny, L. H., Sockanathan, S., Placzek, M. and Lovell-Badge, R. (1998). A rolefor SOX1 in neural determination. Development 125, 1967-1978.

Phillips, B. W., Hentze, H., Rust, W. L., Chen, Q. P., Chipperfield, H., Tan, E. K.,Abraham, S., Sadasivam, A., Soong, P. L., Wang, S. T. et al. (2007). Directeddifferentiation of human embryonic stem cells into the pancreatic endocrinelineage. Stem Cells Dev. 16, 561-578.

Raingeaud, J., Gupta, S., Rogers, J. S., Dickens, M., Han, J., Ulevitch, R. J.and Davis, R. J. (1995). Pro-inflammatory cytokines and environmental stresscause p38 mitogen-activated protein kinase activation by dual phosphorylationon tyrosine and threonine. J. Biol. Chem. 270, 7420-7426.

Rathjen, J. and Rathjen, P. D. (2003). Lineage specific differentiation of mouseES cells: formation and differentiation of early primitive ectoderm-like (EPL)cells. Methods Enzymol. 365, 1-25.

Rathjen, J., Lake, J. A., Bettess, M. D., Washington, J. M., Chapman, G. andRathjen, P. D. (1999). Formation of a primitive ectoderm like cell population,

EPL cells, from ES cells in response to biologically derived factors. J. Cell Sci.112, 601-612.

Rathjen, J., Haines, B. P., Hudson, K. M., Nesci, A., Dunn, S. and Rathjen, P. D.(2002). Directed differentiation of pluripotent cells to neural lineages:homogeneous formation and differentiation of a neurectoderm population.Development 129, 2649-2661.

Rathjen, J., Washington, J. M., Bettess, M. D. and Rathjen, P. D. (2003).Identification of a biological activity that supports maintenance and proliferationof pluripotent cells from the primitive ectoderm of the mouse. Biol. Reprod. 69,1863-1871.

Robb, L., Hartley, L., Begley, C. G., Brodnicki, T. C., Copeland, N. G., Gilbert,D. J., Jenkins, N. A. and Elefanty, A. G. (2000). Cloning, expression analysis,and chromosomal localization of murine and human homologues of a Xenopusmix gene. Dev. Dyn. 219, 497-504.

Rosen, B. and Beddington, R. S. (1993). Whole-mount in situ hybridization in themouse embryo: gene expression in three dimensions. Trends Genet. 9, 162-167.

Rouse, J., Cohen, P., Trigon, S., Morange, M., Alonso-Llamazares, A.,Zamanillo, D., Hunt, T. and Nebreda, A. R. (1994). A novel kinasecascade triggered by stress and heat shock that stimulates MAPKAPkinase-2 and phosphorylation of the small heat shock proteins. Cell 78, 1027-1037.

Saga, Y., Hata, N., Kobayashi, S., Magnuson, T., Seldin, M. F. and Taketo,M. M. (1996). MesP1: a novel basic helix-loop-helix protein expressed in thenascent mesodermal cells during mouse gastrulation. Development 122, 2769-2778.

Simon, P. (2003). Q-Gene: processing quantitative real-time RT-PCR data.Bioinformatics 19, 1439-1440.

So, P. L. and Danielian, P. S. (1999). Cloning and expression analysis of a mousegene related to Drosophila odd-skipped. Mech. Dev. 84, 157-160.

Soudais, C., Bielinska, M., Heikinheimo, M., MacArthur, C. A., Narita, N.,Saffitz, J. E., Simon, M. C., Leiden, J. M. and Wilson, D. B. (1995). Targetedmutagenesis of the transcription factor GATA-4 gene in mouse embryonic stemcells disrupts visceral endoderm differentiation in vitro. Development 121, 3877-3888.

Tada, S., Era, T., Furusawa, C., Sakurai, H., Nishikawa, S., Kinoshita,M., Nakao, K., Chiba, T. and Nishikawa, S. (2005). Characterization ofmesendoderm: a diverging point of the definitive endoderm and mesodermin embryonic stem cell differentiation culture. Development 132, 4363-4374.

Tam, P. P. L., Khoo, P. L., Lewis, S. L., Bildsoe, H., Wong, N., Tsang, T. E., Gad,J. M. and Robb, L. (2007). Sequential allocation and global pattern ofmovement of the definitive endoderm in the mouse embryo during gastrulation.Development 134, 251-260.

Tan, B. S., Lonic, A., Morris, M. B., Rathjen, P. D. and Rathjen, J. (2011). Theamino acid transporter SNAT2 mediates L-proline-induced differentiation of EScells. Am. J. Physiol. Cell Physiol. 300, C1270-1279.

Tanaka, N., Kamanaka, M., Enslen, H., Dong, C., Wysk, M., Davis, R. J.and Flavell, R. A. (2002). Differential involvement of p38 mitogen-activatedprotein kinase kinases MKK3 and MKK6 in T-cell apoptosis. EMBO Rep. 3, 785-791.

Tanaka, M., Jokubaitis, V., Wood, C., Wang, Y., Brouard, N., Pera, M., Hearn,M., Simmons, P. and Nakayama, N. (2009). BMP inhibition stimulates WNT-dependent generation of chondrogenic mesoderm from embryonic stem cells.Stem Cell Res. 3, 126-141.

Tremblay, K. D., Hoodless, P. A., Bikoff, E. K. and Robertson, E. J. (2000).Formation of the definitive endoderm in mouse is a Smad2-dependent process.Development 127, 3079-3090.

Vassilieva, S., Goh, H. N., Lau, K. X., Hughes, J. N., Familari, M., Rathjen, P. D.and Rathjen, J. (2012). A system to enrich for primitive streak-derivatives,definitive endoderm and mesoderm, from pluripotent cells in culture. PLoS ONE7, e38645.

Vincent, S. D., Dunn, N. R., Hayashi, S., Norris, D. P. and Robertson, E. J.(2003). Cell fate decisions within the mouse organizer are governed by gradedNodal signals. Genes Dev. 17, 1646-1662.

Wang, J., Ohmuraya, M., Hirota, M., Baba, H., Zhao, G., Takeya, M., Araki, K.and Yamamura, K. (2008). Expression pattern of serine protease inhibitor kazaltype 3 (Spink3) during mouse embryonic development. Histochem. Cell Biol.130, 387-397.

Wang, J., Chen, L., Ko, C. I., Zhang, L., Puga, A. and Xia, Y. (2012). Distinctsignaling properties of mitogen-activated protein kinase kinases 4 (MKK4) and 7(MKK7) in embryonic stem cell (ESC) differentiation. J. Biol. Chem. 287, 2787-2797.

Washington, J. M., Rathjen, J., Felquer, F., Lonic, A., Bettess, M. D., Hamra,N., Semendric, L., Tan, B. S., Lake, J. A., Keough, R. A. et al. (2010). L-Proline induces differentiation of ES cells: a novel role for an amino acid in theregulation of pluripotent cells in culture. Am J Physiol Cell Physiol 298, C982-992.

Wilkinson, D. G., Bhatt, S. and Herrmann, B. G. (1990). Expression pattern ofthe mouse T gene and its role in mesoderm formation. Nature 343, 657-659.

Winnier, G., Blessing, M., Labosky, P. A. and Hogan, B. L. (1995). Bonemorphogenetic protein-4 is required for mesoderm formation and patterning inthe mouse. Genes Dev. 9, 2105-2116.

Wu, J., Kubota, J., Hirayama, J., Nagai, Y., Nishina, S., Yokoi, T., Asaoka, Y.,Seo, J., Shimizu, N., Kajiho, H. et al. (2010). p38 Mitogen-activated protein


2215

http://dx.doi:10.1038/372739a0http://dx.doi:10.1038/372739a0http://dx.doi:10.1038/372739a0http://dx.doi:10.1038/372739a0http://dx.doi.org/10.1242/dev.01415http://dx.doi.org/10.1242/dev.01415http://dx.doi.org/10.1242/dev.01415http://dx.doi.org/10.1016/S1534-5807(02)00206-Xhttp://dx.doi.org/10.1016/S1534-5807(02)00206-Xhttp://dx.doi.org/10.1016/S1534-5807(02)00206-Xhttp://dx.doi.org/10.1038/11932http://dx.doi.org/10.1038/11932http://dx.doi.org/10.1038/11932http://dx.doi.org/10.1093/emboj/18.7.1845http://dx.doi.org/10.1093/emboj/18.7.1845http://dx.doi.org/10.1093/emboj/18.7.1845http://dx.doi.org/10.1073/pnas.78.12.7634http://dx.doi.org/10.1073/pnas.78.12.7634http://dx.doi.org/10.1073/pnas.78.12.7634http://dx.doi.org/10.1002/1521-1878(200007)22:73.0.CO;2-Ehttp://dx.doi.org/10.1002/1521-1878(200007)22:73.0.CO;2-Ehttp://dx.doi.org/10.1016/S0962-8924(97)01036-2http://dx.doi.org/10.1016/S0962-8924(97)01036-2http://dx.doi.org/10.1186/1752-0509-6-154http://dx.doi.org/10.1186/1752-0509-6-154http://dx.doi.org/10.1186/1752-0509-6-154http://dx.doi.org/10.1186/1752-0509-6-154http://dx.doi.org/10.1002/dvdy.21313http://dx.doi.org/10.1002/dvdy.21313http://dx.doi.org/10.1002/dvdy.21313http://dx.doi.org/10.1016/j.stem.2008.07.021http://dx.doi.org/10.1016/j.stem.2008.07.021http://dx.doi.org/10.1016/j.stem.2008.07.021http://dx.doi.org/10.1073/pnas.180316397http://dx.doi.org/10.1073/pnas.180316397http://dx.doi.org/10.1073/pnas.180316397http://dx.doi.org/10.1073/pnas.180316397http://dx.doi.org/10.1006/excr.1999.4535http://dx.doi.org/10.1006/excr.1999.4535http://dx.doi.org/10.1006/excr.1999.4535http://dx.doi.org/10.1006/excr.1999.4535http://dx.doi.org/10.1016/S0968-0004(00)01595-4http://dx.doi.org/10.1016/S0968-0004(00)01595-4http://dx.doi.org/10.1242/dev.055236http://dx.doi.org/10.1242/dev.055236http://dx.doi.org/10.1242/dev.055236http://dx.doi.org/10.1242/dev.055236http://dx.doi.org/10.1242/dev.055236http://dx.doi.org/10.1038/sj.emboj.7601587http://dx.doi.org/10.1038/sj.emboj.7601587http://dx.doi.org/10.1038/sj.emboj.7601587http://dx.doi.org/10.1038/sj.emboj.7601587http://dx.doi.org/10.1089/scd.2007.0029http://dx.doi.org/10.1089/scd.2007.0029http://dx.doi.org/10.1089/scd.2007.0029http://dx.doi.org/10.1089/scd.2007.0029http://dx.doi.org/10.1074/jbc.270.13.7420http://dx.doi.org/10.1074/jbc.270.13.7420http://dx.doi.org/10.1074/jbc.270.13.7420http://dx.doi.org/10.1074/jbc.270.13.7420http://dx.doi.org/10.1016/S0076-6879(03)65001-9http://dx.doi.org/10.1016/S0076-6879(03)65001-9http://dx.doi.org/10.1016/S0076-6879(03)65001-9http://dx.doi.org/10.1095/biolreprod.103.017384http://dx.doi.org/10.1095/biolreprod.103.017384http://dx.doi.org/10.1095/biolreprod.103.017384http://dx.doi.org/10.1095/biolreprod.103.017384http://dx.doi: 10.1002/1097-2000)9999:99993.0.CO;2-. Epub 2000 Oct 16.http://dx.doi: 10.1002/1097-2000)9999:99993.0.CO;2-. Epub 2000 Oct 16.http://dx.doi: 10.1002/1097-2000)9999:99993.0.CO;2-. Epub 2000 Oct 16.http://dx.doi: 10.1002/1097-2000)9999:99993.0.CO;2-. Epub 2000 Oct 16.http://dx.doi.org/10.1016/0092-8674(94)90277-1http://dx.doi.org/10.1016/0092-8674(94)90277-1http://dx.doi.org/10.1016/0092-8674(94)90277-1http://dx.doi.org/10.1016/0092-8674(94)90277-1http://dx.doi.org/10.1016/0092-8674(94)90277-1http://dx.doi.org/10.1093/bioinformatics/btg157http://dx.doi.org/10.1093/bioinformatics/btg157http://dx.doi.org/10.1016/S0925-4773(99)00058-1http://dx.doi.org/10.1016/S0925-4773(99)00058-1http://dx.doi.org/10.1242/dev.02005http://dx.doi.org/10.1242/dev.02005http://dx.doi.org/10.1242/dev.02005http://dx.doi.org/10.1242/dev.02005http://dx.doi.org/10.1242/dev.02005http://dx.doi: 10.1242/dev.02724 Epub 2006 Dec 6.http://dx.doi: 10.1242/dev.02724 Epub 2006 Dec 6.http://dx.doi: 10.1242/dev.02724 Epub 2006 Dec 6.http://dx.doi: 10.1242/dev.02724 Epub 2006 Dec 6.http://dx.doi: 10.1152/ajpcell.00235.2010. Epub 2011 Feb 23.http://dx.doi: 10.1152/ajpcell.00235.2010. Epub 2011 Feb 23.http://dx.doi: 10.1152/ajpcell.00235.2010. Epub 2011 Feb 23.http://dx.doi.org/10.1093/embo-reports/kvf153http://dx.doi.org/10.1093/embo-reports/kvf153http://dx.doi.org/10.1093/embo-reports/kvf153http://dx.doi.org/10.1093/embo-reports/kvf153http://dx.doi.org

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