HE FORMATION OF ROXIMAL AND DISTAL …...2014/01/27 · Nostro et al., 2011). Analysing mechanisms that regulate gastrulation and endoderm formation in systems that initiate differentiation

THE FORMATION OF PROXIMAL AND DISTAL DEFINITIVE ENDODERM POPULATIONS IN CULTURE

REQUIRES P38 MAPK ACTIVITY

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

1Department of Zoology, University of Melbourne, Victoria, 3010, Australia 2The Menzies Research Institute Tasmania, University of Tasmania, Tasmania, 7000, Australia

* Authors have contributed equally to this work

Correspondence: Address: Dr. Joy Rathjen

Menzies Research Institute Tasmania,

17 Liverpool Street,

Hobart, TAS, 7000

Australia.

Email: [email protected];

Telephone: +61362262856

Fax: +61362267704

Running title: Endoderm formation and p38 MAPK

Author Disclosure: Charlotte Yap: Conception and design, Collection and/or assembly of data, Data analysis and interpretation,

Manuscript writing.

Hwee Ngee Goh: Conception and design, Collection and/or assembly of data, Data analysis and interpretation.

Mary Familari: Conception and design, Data analysis and interpretation.

Peter D. Rathjen: Conception and design, Financial support.

Joy Rathjen: Conception and design, Financial support, Data analysis and interpretation, Manuscript writing,

Final approval of manuscript.

Keywords: Embryonic Stem Cells; Endoderm; p38 MAP Kinase; Gastrulation; BMP4.

© 2014. Published by The Company of Biologists Ltd.Jo

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JCS Advance Online Article. Posted on 30 January 2014

SUMMARY

Endoderm formation in the mammal is a complex process with two lineages forming during the first weeks of

development, the primitive, or extraembryonic, endoderm that 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 definitive endoderm arises as two waves, which potentially reflect two distinct

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. Using EPL

cells and chemical inhibitors of p38 MAPK activity, roles for p38 MAPK in the formation of definitive

endoderm have been investigated. These approaches defined 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 demonstrated the formation of two distinct populations,

defined by gene expression and ontogeny, which were analogous to the proximal and distal definitive endoderm

populations of the embryo. Formation of proximal definitive endoderm required p38 MAPK activity and was

correlated with molecular gastrulation, defined by the expression of T. Distal definitive endoderm formation also

required p38 MAPK activity but could be formed when T expression was inhibited. Understanding lineage

complexity will be a prerequisite for the generation of endoderm derivatives for commercial and clinical use.

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3 Endoderm formation requires p38 MAPK activity

INTRODUCTION 5

Two distinct endoderm lineages arise during mammalian embryogenesis; 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 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,

mesendoderm, which arises in the primitive streak during gastrulation (Kinder et al., 2001; Lawson et al., 1991). 10

The ability of ES cells to self-renew indefinitely and to give rise to all embryonic and adult tissues in

response to appropriate signals in vitro and in vivo make them attractive tools for modelling the developmental

processes of gastrulation and definitive endoderm formation (Bradley et 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; Kinder et al., 2001; Kubo et al., 2004; Tada et al., 2005) and the 15

addition of Activin A has been shown to enrich the formation of endoderm during ES cell differentiation (Kubo

et al., 2004; Nostro et al., 2011). Furthermore, bipotent progenitors that differentiate into mesoderm and

definitive endoderm have been identified in culture; a Brachyury (T)-positive cell population (Kubo et al., 2004)

and a Goosecoid (Gsc), E-cadherin (ECD) and PDGFRα(αR) positive cell population that diverges to

Gsc+ECD+αR- and Gsc+ECD-αR+ populations (Tada et al., 2005). It is becoming clear, however, that definitive 20

endoderm formation, and subsequent differentiation, are complex processes, with induction strategies and

positional specification influencing outcomes (Gadue et al., 2009; Gadue et al., 2006; Jackson et al., 2010;

Nostro et al., 2011).

Analysing mechanisms that regulate gastrulation and endoderm formation in systems that initiate

differentiation from mouse ES cells is impacted by two confounding factors. Initially, ES cells differentiate to 25

form a later epiblast population, a population equivalent to the embryonic primitive ectoderm (Rathjen et al.,

2003), and primitive endoderm (Soudais et al., 1995). The primitive endoderm lineage is a potent source of

signals regulating pluripotent cell differentiation (Beddington and Robertson, 1998; Beddington and Robertson,

1999). Signals emanating from the primitive endoderm will have the potential to synergise or compete with

exogenous signals to influence differentiation outcome. The maturation of ES cells to later epiblast requires the 30

generation of endogenous signals within the differentiation system (Li et al., 2004). Primitive ectoderm

formation can be controlled in culture with the differentiation of ES cells to a second pluripotent cell population,

early primitive ectoderm-like (EPL) cells in response to MEDII, a medium conditioned by the human

hepatocarcinoma cell line, HepG2 (Chawengsaksophak et al., 2004; Lake et al., 2000; Rathjen et al., 1999;

Shimono and Behringer, 2003). EPL cells are similar to cells of the primitive ectoderm by morphology, gene 35

expression, cytokine response and differentiation potential (Harvey et al., 2010; Lake et al., 2000; Pelton et al.,

2002; Rathjen et al., 2002; Rathjen et al., 1999). ES cell differentiation in response to MEDII is not

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accompanied by the concomitant formation of primitive endoderm (Rathjen et al., 2002; Vassilieva et al., 2012).

Using EPL cells as the starting material for differentiation has resulted in a reliable model of gastrulation which

has been used to determine the role of growth factors, environmental manipulations and intracellular signalling 40

in cell differentiation and for identification of transient developmental intermediates (Harvey et al., 2010;

Hughes et al., 2009a; Hughes et al., 2009b; Zheng et al., 2010). Here, EPL cell differentiation will be used to

investigate the control of molecular gastrulation, focusing on the formation of definitive endoderm. Cells formed

as a result of molecular gastrulation will be referred to here as primitive streak intermediates; this term will cover

all cells expressing T, a marker of molecular gastrulation, and will include, but not be limited to, bipotent 45

mesendoderm.

p38 MAPK is a member of the mitogen-activated protein kinase (MAPK) family of kinases (Martin-Blanco,

2000; Nebreda and Porras, 2000) originally identified as involved in stress and inflammatory responses (Han et

al., 1994; Raingeaud et al., 1995; Rouse et al., 1994). Developmental roles for p38 MAPK activity have been

shown in the formation of cardiocytes (Aouadi et al., 2006), myocytes (Perdiguero et al., 2007), adipocytes 50

(Engelman et al., 1998), chondrocytes (Nakamura et al., 1999), erythroid cells (Nagata et al., 1998) and neurons

(Nebreda and Porras, 2000). Inhibiting p38 MAPK during ES cell differentiation promoted neurogenesis at the

expense of cardiogenesis and suggested 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 this study roles for p38 MAPK

activity in molecular gastrulation and definitive endoderm formation were identified. Inhibition of p38 MAPK 55

during EPL cell differentiation in response to serum reduced the expression of T, and promoted the formation of

neural lineages. In contrast, when cells were differentiated in response to BMP4, the inhibition of p38 MAPK

did not alter differentiation outcomes or the expression of differentiation markers. The analysis of the

differentiation outcomes from cells differentiated in Activin A, BMP4 or serum and the p38 MAPK inhibitor

showed that the formation of definitive endoderm from EPL cells was dependent on p38 MAPK activity. Further 60

characterisation suggested that EPL cell-derived definitive endoderm comprised two distinct populations,

representative of the proximal and distal definitive endoderm of the embryo, which formed in response to

alternate signalling environments and potentially from distinct progenitor populations.Jour

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RESULTS

BMP4 and serum induce the formation of primitive streak intermediates via distinct signalling pathways. 65

Primitive streak intermediates can be induced from EPL cells by BMP4 (Harvey et al., 2010; Zheng et

al., 2010) or serum (Hughes et al., 2009b). The requirement for p38 MAPK activity in primitive streak

intermediate formation in response to these inducers was investigated. Phosphorylated p38 MAPK (pp38

MAPK) and phosphorylated Heat shock protein 27 (pHsp27), a downstream target of p38 MAPK signalling,

were detected by western blot in EPL cells incubated in serum-free medium (SFM) with or without BMP4 or 70

serum (pp38 MAPK only) (Figure 1Ai, ii). No consistent increase in pp38 MAPK was seen in EPL cells after the

addition of serum. p38 MAPK activity was inhibited pharmacologically with SB203580 (4-(4´-fluorophenyl)-2-

(4´-methylsulfinylphenyl)-5-(4´-pyridyl)-imidazole; SB). This chemical inhibits p38α, p38β and p38β2

homologues by competing for ATP binding pockets (Cuenda et al., 1995). Levels of pp38 MAPK and pHsp27

were reduced in cells exposed to serum and SB when compared to cells exposed to serum (Figure 1Aii, S1A, B). 75

In the absence of serum or BMP4, EPL cells (as aggregates) formed almost exclusively neurons (Figure

1B; data not shown)(Zheng et al., 2010). The addition of BMP4, in BMP4-containing medium (BCM; Table S1),

or serum, in serum-containing medium (SCM; Table S1), during differentiation resulted in the formation of

cardiocytes and erthryocytes within the aggregates, as expected from previous analyses of differentiation

(Figures 1B) (Harvey et al., 2010; Zheng et al., 2010). The addition of SB to serum reduced the percentage of 80

aggregates that formed erthryocytes and increased the percentage of aggregates that formed neurons; cardiocyte

formation was unaffected (Figure 1B). In the presence of BMP4, however, there was no significant effect of the

SB inhibitor on the production of erythrocytes, neurons or cardiocytes. SB did not affect the ability of aggregates

to adhere to plasticware for scoring or the survival of mesoderm or ectoderm lineages scored in the assay (Table

S3). These data suggest a role for p38 MAPK in differentiation. 85

Previous analysis of EPL cell differentiation has suggested that the frequency of blood, cardiocyte and

neuron formation reflects the efficiency of primitive streak intermediate formation (Figure 1C, S1B) (Zheng et

al., 2010). Differentiating EPL cells were analysed for the expression of established markers for primitive streak

(T, Bmp4, Tgfb1, Wnt3 and 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 90

mesoderm (Mesp1, Hbb-b1, Nkx2-5 and Osr1) (Farace et al., 1984; Lints et al., 1993; Saga et al., 1996; So and

Danielian, 1999). Analysis was performed when gene expression was most reproducibly detected, day 2 for T,

Wnt3 and Fgf8 and day 4 for Bmp4 and Tgfb1. The expression of primitive streak and mesoderm marker genes

was reduced in EPL cells differentiated in SCM+SB compared to controls, consistent with the reduced formation

of mesoderm from these cells (Figures 1C, D, S2D). In contrast, only Bmp4 expression was decreased in EPL 95

cells differentiated in BCM+SB (Figure 1C). Lower expression of T, Wnt3 and Bmp4 was detected in cells

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differentiated in response to SCM compared with those differentiated in BCM (Figure S2E), suggesting that

differentiation in response to the inducers was not equivalent.

The significantly reduced expression of primitive streak intermediate markers and reduced erythrocyte

formation from EPL cells differentiated in serum when SB was added suggested a role for p38 MAPK in 100

primitive streak intermediate formation. Cardiocyte formation and the residual levels of erythrocyte formation in

a proportion of the aggregates differentiated in SCM+SB demonstrated the formation of a population of

primitive streak intermediate, albeit reduced, and a discord between the gene expression and differentiation data.

Most notably, the percentage of aggregates containing cardiocytes was unaffected by the inhibitor SB but the

expression of Nkx2-5, a cardiocyte marker, was undetectable, suggesting that SB did affect establishment of 105

cardiocytes. Differentiation assays score the presence, but not abundance, of a cell type in an aggregate.

Potentially, differentiation within aggregates cultured in serum and SB was reduced, delayed and asynchronous,

leading to a reduction in Nkx2-5 transcript levels at any given time point, and affecting the ability of the analysis

to detect expression.

Collectively, these data suggest that induction of primitive streak intermediates by serum, but not by 110

BMP4, requires p38 MAPK activity. The suppression of differentiation that resulted from the addition of the p38

MAPK inhibitor SB to serum-containing medium was specific to primitive streak intermediate formation.

Neurectoderm was formed, and the prevalence of the lineage was increased, in these conditions.

Formation of primitive streak intermediates in response to serum is reduced, but not abolished, by BMP4 115

inhibition.

BMP4 and serum potentially induce primitive streak intermediates by independent pathways.

Alternatively, serum may contain BMP activity at levels sufficient to induce primitive streak intermediates in

cells with p38 MAPK activity or 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). 120

Western blot analysis showed that the levels of phosphorylated Smad1/5/8 (pSmad1/5/8) were not increased in

EPL cells exposed to the serum used in this analysis (Figure 2A). In contrast, pSmad1/5/8 was markedly induced

in cells exposed to BMP4. These data indicate that our serum contained little or no BMP activity.

The possibility that BMP4 was induced in EPL cells as they differentiated in response to serum, and that

endogenously expressed BMP4 subsequently acted to induce the primitive streak intermediate, was investigated 125

by differentiating cells in BCM or SCM in the presence of the BMP inhibitor noggin. Noggin binds 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 and

exogenous BMP4. In BCM+noggin a higher percentage of aggregates contained neurons when compared to

controls, and effectively no aggregates contained cardiocytes and erythrocytes (Figure 2B). Consistent with this, 130

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the expression of primitive streak markers was decreased in cells differentiated in BCM+noggin compared to

BCM controls (Figure 2C, D). A significant decrease in the percentage of aggregates containing cardiocytes and

erythrocytes was also seen in aggregates cultured in SCM+noggin, although approximately 20% of aggregates

still formed mesoderm-derived lineages (Figure 2B). Similarly, this reduction in mesoderm formation was

reflected in a reduction in the expression of primitive streak intermediate markers in cells differentiating in 135

SCM+noggin when compared to SCM controls (Figure 2C, D). Mesoderm formation in cells differentiated in

SCM+noggin was reduced but not abolished (Figure 2B), suggesting that two pathways mediated the induction

of the primitive streak intermediate in these aggregates, a pathway dependent on endogenously generated BMP

activity and a second pathway independent of BMP signalling.

The formation of definitive endoderm in BMP4 and serum is impaired by the inhibition of p38 MAPK. 140

Mixl1, which has been implicated in definitive endoderm formation (Adams and Frank, 1980; Hart et al.,

2002), was expressed in cells differentiated in BMP4 and serum and was significantly decreased in both

conditions on the addition of the p38 MAPK inhibitor SB (Figures 3A) raising the possibility that p38 MAPK

was involved in definitive endoderm formation. The expression of additional endoderm markers Sox17, Ttr,

Gata4, Trh, and Eya2 (Gu et al., 2004a; Kanai-Azuma et al., 2002; Kinder et al., 2001; Lickert et al., 2002; 145

McKnight et al., 2007) in cells differentiated in BCM and SCM was examined. A novel endoderm marker,

serine protease inhibitor Kazal type 3 (Spink3), was included in the analysis. Previously, Spink3 has been shown

to be expressed in endoderm-derived populations, including cells of the gut and pancreas in E9.5 mouse embryos

(Wang et al., 2008). As shown here, Spink3 expression was detected in a band of definitive endoderm

immediately below the embryonic and extraembryonic boundary of gastrulating E7.5 embryos (Figure 3B). 150

Endoderm marker gene expression was detected in cells differentiated in BCM and SCM, but higher levels of

expression were generally detected in cells differentiated in SCM (Figure 3C). WISH detected endoderm on the

surface of aggregates differentiated in BCM and SCM, but more Ttr+ and Trh+ cells were seen on aggregates

differentiated in SCM when compared to those in BCM (Figure 3D). p38 MAPK inhibition led to a reduction of

most endoderm markers in EPL cells differentiated in SCM+SB but only a subset of markers (Spink3 and Ttr) in 155

EPL cells differentiated in BCM+SB (Figure 3C). The addition of SB resulted in similar expression levels of all

endoderm markers, with the exception of Gata4, regardless of the differentiation induction strategy used.

Sustained expression of Gata4 in BCM+SB may reflect expression of the gene in another lineage.

Some of the endoderm markers used here (Sox17 and Ttr) also mark visceral endoderm (Kanai-Azuma

et al., 2002; Kinder et al., 2001). Visceral endoderm can be distinguished from definitive endoderm and parietal 160

endoderm by the ability to endocytose HRP from the surrounding medium; internalised HRP can be detected

colourmetrically (Kanai-Azuma et al., 2002; Vassilieva et al., 2012). Embryoid bodies (EBs), which contain

visceral endoderm (Kubo et al., 2004; Vassilieva et al., 2012), developed areas of brown staining on their surface

(Figure 3E), indicating the presence of visceral endoderm. In contrast, EPL cells differentiated in SFM or SCM

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formed few cells capable of taking up HRP from the medium, demonstrating that little or no visceral endoderm 165

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 in the ATP binding site of p38 MAPKs (Young et al., 1997).

EPL cells differentiated in BCM or SCM supplemented with SB202190 showed reduced expression of endoderm

markers (Figure S3A). SB202190, however, impacted the expression of the primitive streak intermediate 170

markers in cells differentiated in SCM and BCM (Figure S3B), suggesting that, in comparison to SB203580, this

compound 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 in the endoderm outcomes from EPL cells differentiated in serum and BMP4. The 175

documentation of endoderm formation in response to BMP4 without the addition of Activin A is unprecedented

but perhaps not unexpected in light of reports that demonstrate formation of mesoderm and endoderm from a

common progenitor in culture (Kubo et al., 2004; Tada et al., 2005) and from the ability of BMP4 in conjunction

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 promotes the formation of a definitive endoderm 180

population that expresses a subset of markers.

The formation of endoderm in the embryo has been suggested to 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 encompassed within the primitive streak intermediate population. Mesendoderm

formation could underpin endoderm formation in serum and BMP4, with BMP4, or other inductive capabilities, 185

inducing a primitive streak intermediate with the properties of mesendoderm and a p38 MAPK-dependent

mechanism inducing an endoderm fate from this progenitor on further differentiation. Inhibiting BMP4 in

serum-containing medium with noggin did not affect the expression of the endoderm markers (Figure 4A),

suggesting that serum does not rely on endogenously-generated BMP4 to form mesendoderm.

In EPL cells differentiated in BCM+noggin marker gene expression and differentiation suggested that 190

formation of the primitive streak intermediate was significantly reduced (Figure 2C, D), the formation of

mesoderm effectively ablated (Figure 2B) and expression of endoderm markers Spink3, Ttr and Gata4 decreased

(Figure 4A), consistent with a two-step process reliant on the initial formation of a primitive streak intermediate

in response to BMP4. Trh and Eya2 expression, however, was increased to levels equivalent to those in cells

differentiated in SCM when compared to control (Figures 4A, S4A), suggesting the formation of endoderm in 195

BCM+noggin. The presence of endoderm on the surface of aggregates differentiated in BCM and BCM+noggin

was confirmed morphologically and by localisation of Trh expression (Figure 4B, C). Cell aggregates cultured

for an extended period expressed markers of endoderm derivatives including AFP and Ttr (gut/liver) and Fabp2

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(intestine) and Pdx1 (gut/pancreas) (Figure S4B). In EPL cells differentiated in BCM+noggin+SB the expression

of Trh and Eya2 was decreased by 10% and 40%, respectively when compared to cells differentiated in 200

BCM+noggin (Figure S4C), suggesting a requirement for endogenous p38 MAPK in Trh and Eya2 expression.

The co-regulation of Spink3, Ttr and Gata4 distinct from the co-regulation of Trh and Eya2 could arise

from the formation of two endoderm populations during EPL cell differentiation. Spink3, Ttr and Gata4

potentially mark endoderm that is produced in response to both BMP4 and serum but is reduced during

differentiation in BMP4-containing medium when noggin is present. A second population can be hypothesized 205

that expresses Trh and Eya2 and the formation of which is maintained in the absence of BMP4. In situ

hybridisation was used to confirm the generation of two, genetically distinct populations of endoderm during

differentiation (Figure 4D). Double-staining with probes against Spink3 and Trh detected differential staining on

the surface of the bodies that were either expressing Spink3 (blue) or Trh (magenta).

Bmp4 regulates the formation of Spink3-, Ttr- and Gata4- expressing endoderm through induction of the 210

primitive streak intermediate.

The induction of Spink3+, Ttr+ and Gata4+ endoderm in response to BMP4 is a two-step process

proceeding via a bipotent primitive streak intermediate, or mesendoderm; the likely role for BMP4 in this

process is indirect, by way of the induction of mesendoderm from EPL cells (Harvey et al., 2010). There exists

the possibility, however, that BMP4 has a role in the subsequent induction of endoderm from mesendoderm. 215

Activin A can induce primitive streak intermediates independently of BMP4 signalling from ES cells

and has been identified 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 differentiated in Activin A-containing media

(ACM) or ACM+noggin expressed markers of the primitive streak (Figure 5A). Expression of primitive streak

markers (except Bmp4) was reduced, and expression of the neural marker Sox1 increased, in cells differentiated 220

in ACM+noggin+SB (Figure 5A), suggesting that the ability of Activin A to induce primitive streak

intermediates required p38 MAPK. Western blot showed pp38 MAPK in cells that had been treated with ACM

(Figure 5B). The regulation of primitive streak intermediate formation in response to Activin A, therefore, is

distinct from primitive streak intermediate formation in response to BMP4 or serum.

In EPL cells differentiated in ACM, SFM+noggin or ACM+noggin, Sox17, Spink3 and Ttr were 225

expressed equivalently (Figure 5C). The expression of these genes in the absence of BMP4 signalling suggests

that BMP4 does not play additional roles in specification of endoderm from mesendoderm. Eya2 expression was

higher in EPL cells differentiated in ACM+noggin when compared to cells differentiated in ACM. The

expression of all endoderm markers in ACM+noggin suggests that Activin A, like serum, is able to induce both

proposed endoderm populations from EPL cells (Figure 4A). The increased expression of Eya2 suggests that the 230

formation of endoderm expressing these markers can be enhanced by BMP4 inhibition. The expression of Sox17,

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Spink3, Ttr and Eya2 (Figure 5C) was decreased in cells differentiated in ACM+noggin+SB, consistent with a

role for p38 MAPK activity in the Activin A-induced formation of endoderm.

The primitive streak intermediate is the progenitor for Spink3-, Ttr- and Gata4- expressing endoderm but not

necessarily for Trh- and Eya2- expressing endoderm. 235

The formation of Spink3-, Ttr- and Gata4-expressing endoderm appears to be dependent on prior

formation of primitive streak intermediates. Conversely, the expression of Trh and Eya2 in EPL cells

differentiated in BCM+noggin suggest the formation of an endoderm population does not rely on the prior

formation of this population. The formation of primitive streak intermediates from differentiating EPL cells can

be inhibited by DAPT (N-[N-(3,5-Difluorophenacetyl-L-alanyl)]-S-phenylglycine t-Butyl Ester), an antagonist 240

of γ-secretase (Hughes et al., 2009a). In cells formed from EPL cells differentiated in BCM+DAPT the

expression of Spink3 and Ttr was decreased, and Eya2 expression increased, when compared to cells formed in

BCM (Figure 5D). These data are consistent with formation of Trh+ and Eya2+ endoderm in the absence of

primitive streak intermediate formation, and a requirement for the initial formation of primitive streak

intermediates in the formation of Spink3+ and Ttr+ endoderm. Further differentiation of aggregates cultured in 245

BCM+DAPT showed the expression of later endoderm populations, consistent with the formation of an

endoderm progenitor (Figure S4B).

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DISCUSSION

p38 MAPK and the formation of primitive streak intermediates 250

The inhibition of p38 MAPK during EPL cell differentiation disrupts the formation of primitive streak

intermediates from EPL cells in response to Activin A or serum. Others have shown that p38 MAPK

inhibition during ES cell differentiation in serum promotes neurogenesis at the expense of cardiogenesis

(Barruet et al., 2011; Kodaira et al., 2006; Wu et al., 2010), and a requirement for p38 MAPK activity early

in cell differentiation (Barruet et al., 2011; Davidson and Morange, 2000; Duval et al., 2004; Wu et al., 255

2010). Inhibition of p38 MAPK activity did not affect the ability of BMP4 to induce primitive streak

intermediates or mesoderm derivatives, suggesting that this pathway is not essential for primitive streak

intermediate formation per se. The data presented here are consistent with a role for p38 MAPK in lineage

allocation or specification during differentiation, and specifically in the formation of primitive streak

intermediates, but only when molecular gastrulation is induced by serum or Activin A. Moreover, the inability of 260

cells cultured in SFM to form primitive streak intermediates, despite intracellular pp38 MAPK, suggests that p38

MAPK activity is not sufficient for differentiation but works in conjunction with other pathways.

In cells differentiated in serum, but not activin, one role for p38 MAPK appears to be up regulation of

Bmp4, which in turn initiates differentiation. The increased expression of Bmp4 in cells differentiated in serum,

and the significant reduction in expression in cells differentiated in BMP4-containing medium supplemented 265

with SB, is consistent with p38 MAPK acting upstream of Bmp4. Endogenously produced BMP4 acts in turn to

induce the primitive streak intermediate, an activity that is blocked by noggin. Noggin did not completely

suppress molecular gastrulation in response to serum, suggesting the presence of additional, potentially p38

MAPK dependent, pathways. In contrast, markers of molecular gastrulation were expressed robustly in cells

differentiated in Activin A-containing medium supplemented with noggin suggesting that Activin A activity was 270

independent of BMP. This is consistent with previous reports that Activin A does not induce 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 mesoderm specification. The

difficulty in resolving a role for p38 MAPK is most likely a consequence of the complexity of the molecular 275

mechanisms regulating gastrulation coupled with the use of ill-defined and poorly understood culture reagents.

The variability of outcomes elicited in cells cultured in p38 MAPK inhibitors (Barruet et al., 2011; Kodaira et

al., 2006; Wu et al., 2010), or from p38α-/- cells (Allen et al., 2000; Chakraborty et al., 2009; Guo et al., 2007;

Kodaira et al., 2006), could be attributed to the confounding use of serum in these experiments. Some sera have

been reported to contain exogenous BMP activity (Herrera and Inman, 2009; Kunath et al., 2007). BMP activity 280

within sera would be able to specify primitive streak intermediates and mesoderm lineages in the absence of p38

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MAPK activity. Our interpretation of the respective roles of serum and growth factors suggests that serum

variability is a contributing factor to variability in the analysis of molecular gastrulation, and the role of

p38MAPK specifically.

The ability of p38 MAPK inhibition to promote cardiocyte formation from human ES cells (Graichen 285

et al., 2008) contradicts the findings from mouse pluripotent cells reported here and by others (Barruet et al.,

2011; Kodaira et al., 2006; Wu et al., 2010). The differentiation of human ES cells was induced in cell

aggregates by a conditioned medium in which signalling activity was largely uncharacterised. Potentially,

increased cardiocyte formation resulted in an increase in the number of primitive streak intermediates, formed

in response to BMP or similar signalling within the conditioned medium, adopting a mesoderm fate. This 290

would occur when p38 MAPK was inhibited in these cells, preventing differentiation to the endoderm.

p38 MAPK comprises of α, β, γ and δ isoforms encoded separately. Of these, p38α and p38β are

expressed in the germ layers and primitive ectoderm respectively at gastrulation (Zohn et al., 2006). Ablation of

p38α resulted in placental defects and embryonic death mid-gestation (Adams et al., 2000; Mudgett et al., 2000).

Double mutant embryos, lacking p38α and p38β in embryonic tissues, gastrulate but fail mid-gestation with 295

diverse developmental defects (del Barco Barrantes et al., 2011). Mutations in the MKK3, 4 and 6, kinases that

have been shown to activate p38 MAPK, also survive beyond gastrulation (Lu et al., 1999; Tanaka et al., 2002;

Yang et al., 1997). p38IP (p38 MAPK interacting protein) deficient mice show a loss of p38 MAPK

phosphorylation in the primitive streak and a failure of cells without pp38 MAPK to migrate (Zohn et al., 2006).

This mutation did not, however, prevent primitive streak intermediate formation or mesoderm specification, but 300

raises the possibility that p38 MAPK has a role in the regulation of an epithelial to mesenchymal transition

during differentiation. The role for p38 MAPK is unlikely, therefore, to be essential for primitive streak

intermediate formation in vivo, but was revealed during in vitro differentiation in response to serum or Activin

A.

A novel role for p38 MAPK in the formation of definitive endoderm populations 305

Endoderm formation in the mammal is complex, with the formation of two endoderm lineages from the

pluripotent lineage during early development, primitive/visceral endoderm and definitive endoderm. Analysis of

EPL cell differentiation suggests that definitive endoderm may form as two distinct cell populations that can be

distinguished by gene expression and ontogeny. The formation of both lineages required p38 MAPK activity.

p38 MAPK inhibition during the differentiation of EPL cells in response to BMP4 resulted in the 310

reduced expression of Mixl1, Spink3 and Ttr , and when differentiated in response to serum reduced expression

of Mixl1, Sox17, Spink3, Ttr, Gata4, and Eya2, suggesting a previously unidentified role for p38 MAPK in

definitive endoderm formation. In the mouse, definitive endoderm formation is dependent on Nodal signalling

(Tremblay et al., 2000; Vincent et al., 2003), a TGFβ family member with similar signalling properties to

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Activin A (Conlon et al., 1994; Vincent et al., 2003). Similarly, ES cell differentiation induced by Activin A 315

results in the enrichment of definitive endoderm (Gadue et al., 2006; Kubo et al., 2004; Nostro et al., 2011).

Canonically, Nodal signalling is mediated by Smad2/3 and downstream effectors of Smads (Heldin et al., 1997;

Massaous and Hata, 1997). p38 MAPK has been shown to be activated by TGFβs and to mediate signalling in

response to these factors (Hanafusa et al., 1999; Hu et al., 2004; Yue and Mulder, 2000). Inhibition of p38

MAPK impaired the induction of definitive endoderm markers by Activin A, suggesting that Activin A 320

signalling was mediated, in part, by p38 MAPK. A role for p38MAPK has been shown in the positional

specification of the visceral endoderm in response to Nodal (Clements et al., 2011), and a similar requirement for

p38 MAPK in the induction of definitive endoderm formation in response to Nodal may exist.

The differential effects of the inhibitor SB on endoderm marker expression in aggregates differentiated

in serum or BMP4 infers that the specification of endoderm from pluripotent cells can occur via multiple 325

pathways and result in two populations. The expression of Spink3, Ttr, and Gata4 marked one of these

populations. Ttr has been previously used to mark visceral endoderm (Kinder et al., 2001) but the widespread

expression of Ttr in embryoid bodies derived from EPL cells, in which visceral endoderm is rarely formed

(Vassilieva et al., 2012), and the reliance of Ttr expression on molecular gastrulation by EPL cells, suggests that

Ttr can also be expressed in an endoderm formed during molecular gastrulation. The formation of a Spink3+, 330

Ttr+, and Gata4+ endoderm population from differentiating EPL cells correlated with the prior expression of

primitive streak markers. When primitive streak intermediate formation was inhibited, as happened when cells

were differentiated in BMP4-containing medium supplemented with noggin or DAPT, expression of Spink3, Ttr

and Gata4 was reduced. The second endoderm population was marked by the expression of Trh and Eya2. The

expression of these markers is maintained in cells differentiated in BMP4-containing medium supplemented with 335

noggin or DAPT, suggesting that Trh+ and Eya2+ endoderm can form in the absence of a T-expressing primitive

streak intermediate. The differential expression of Spink3, Ttr, and Gata4 between conditions that enriched or

suppressed the formation of the primitive streak intermediate, coupled with the persistence of Trh+ and Eya2+

endoderm when primitive streak intermediate formation was suppressed, is consistent with the formation of two

endoderm populations during EPL cell differentiation. 340

A model for the regulation of molecular gastrulation

Based on this analysis of primitive streak intermediate and endoderm formation from EPL cells we

propose a revised paradigm for molecular gastrulation (Figure 6). This model proposes that primitive streak

intermediates, which express T and other primitive streak markers, can be induced from EPL cells via multiple

pathways, including pathways dependent on BMP4 signalling, growth factors/cytokines, including Activin A 345

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 generate distinct primitive streak

intermediates distinguished by divergent differentiation potential, as has been shown for BMP4 and WNT

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(Tanaka et al., 2009). We propose that the primitive streak intermediate induced by BMP4 can differentiate to

form mesoderm and a population of endoderm expressing Spink3, Ttr and Gata4. This population is similar by 350

gene expression to the ring of endoderm marked by Spink3 in the proximal region of the embryo. Formation of

this endoderm population requires p38 MAPK activity in the primitive streak intermediate; activation of this

pathway is achieved through endogenous signalling in aggregates differentiated in BMP4. Alternatively, EPL

cells can differentiate into a Trh+ and Eya2+ endoderm that can be formed independently of the BMP4-induced

primitive streak intermediate; this population also requires p38 MAPK activity for formation. The gene 355

expression profile of this population mirrors the endoderm of the distal region of the embryo (Gu et al., 2004b;

McKnight et al., 2007).

The endoderm populations defined here are both are products of EPL cell differentiation and arise

during molecular gastrulation. We propose, therefore, that these populations are subpopulations of the definitive

endoderm and suggest the terminology proximal definitive endoderm and distal definitive endoderm to describe 360

them. Two waves of definitive endoderm, which populate the more proximal (lateral endoderm) and more distal

(medial endoderm) regions of the endoderm, have been proposed to occur during embryogenesis (Rouse et al.,

1994). These populations have been distinguished by their time of exit from the primitive streak, their direction

of migration across the egg cylinder, and their allocation to different regions of the gut tube in later development.

The populations defined from in vitro differentiation here potentially represent the populations identified by fate 365

mapping in vivo.

Our proposed model (Figure 6) addresses the role of mesendoderm in mammalian gastrulation,

propounding a population that satisfies the criteria of mesendoderm within the population of primitive streak

intermediates induced by BMP4 and which acts as a progenitor of the proximal definitive endoderm and

mesoderm. The model also describes distal definitive endoderm which can form independently of the BMP-370

signalling, suggesting that not all definitive endoderm formation proceeds via mesendoderm and raising the

possibility of an endoderm-specific progenitor. Definitive endoderm formation independent of a bipotent

progenitor has been previously suggested by fate mapping of the embryo (Lawson et al., 1991; Rouse et al.,

1994).

A goal of stem cell research is to generate, in sufficient quantity, functional cell types with commercial 375

and clinical applications. Many approaches for forming definitive endoderm and derivatives from ES cells have

been reported. These rely almost exclusively on the prior formation of primitive streak intermediates and, with

few exceptions (Mathew et al., 2012; Morrison et al., 2008), the positional identity of the endoderm population

formed has not been considered. What is clear from the literature is that the formation of later endoderm

populations is generally inefficient; this is potentially a consequence of the inefficient generation of the 380

appropriate progenitor at the onset of differentiation. Positional specification could restrict the developmental

potential of ES cell-derived definitive endoderm, and success may depend on enrichment for a specific

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endoderm population. Characterisation of the derivatives of proximal and distal definitive endoderm populations

in the embryo and in culture will allow differentiation protocols to be tailored to ensure enrichment of the

appropriate definitive endoderm for subsequent differentiation.385

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EXPERIMENTAL PROCEDURES

Cell culture

Mouse ES cells:

D3 ES cells (Doetschman et al., 1985) were used throughout. ES cell maintenance, EPL cell formation (as 390

aggregates), EB formation and conditioned media MEDII production 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 hours.

Differentiation assays

EPL cells were transferred to SCM (Table S1). The foetal calf serum (FCS; Life Technologies) used in these 395

experiments was chosen for the maintenance of pluripotency. Alternatively, EPL cells were transferred to SFM

(Table S1). SFM was supplemented with BMP4 (10 ng/mL; R&D Systems) (BCM; Table S1). Noggin (90

ng/mL; R&D Systems), SB (10 µM; Sigma) and/or 0.1% DMSO (Sigma), were added as described in the text,

and EPL cells 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 400

neurons (scored as long cell extensions emanating from the aggregated), from aggregates were determined as

described previously (Hughes et al., 2009a; Hughes et al., 2009b). Ideally a single aggregate was analysed / well

but in reality some wells contained more than one aggregate such that for each experimental condition ≥ 24

aggregates were analysed. 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. 405

Gene expression assays

EPL cells were transferred to SCM, BCM, ACM (SFM+Activin A (25 ng/mL); Table S1) and supplemented

with, noggin (90 ng/mL), SB (10 µM), DAPT (50 μM) 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/qPCR 410

Total cytoplasmic RNA was isolated using TRIzol® (Invitrogen). cDNA was synthesised as per the

manufacturer’s protocol (Promega). Primers (Table S2) were validated on differentiated ES cells (mouse) or

genomic DNA (human) and PCR products sequenced.

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RT-PCR: 25 µL reactions contained 1 ng/mL of forward and reverse primers, 1 x GoTaqH Green Master Mix

(Promega) and cDNA. Reactions were heated to 94°C for 2 minutes before cycles of 94°C for 30 s, 60°C for 30 s 415

and 72°C for 30 s, and finished with 5 minutes at 72°C, in an MJ Research thermocycler. PCR products were

visualised with a Molecular Imager® ChemiDoc™ XRS Imaging System (BioRad) with SYBR® Gold

(Invitrogen). Gene expression was quantified using Quantity One 1-D band analysis software (BioRad).

qPCR: Reactions, containing 1x Absolute blue QPCR SYBR Green Mix (Thermo Scientific), cDNA and 200

nM of forward and reverse primers, were performed on an MJ research thermocycler with a Chromo4 420

Continuous Fluorescence Detection system (MJ Research). Reactions were heated to 95°C for 15 minutes before

cycling at 95°C for 15 s, 60°C for 15 s and 72°C for 30 s. The raw data was analysed using the Q-Gene software

package (Muller et al., 2002; Simon, 2003).

Whole-mount in situ hybridization (WISH)

Embryos from time-mated Swiss mice and cell aggregates were fixed in 4% PFA and dehydrated in methanol. 425

WISH was performed as previously described (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). Hybridisation and post-hybridisation washes were performed at 65oC.

Embryos and aggregates were incubated overnight with anti-digoxigenin-AP Fab fragments (1:2000) (Roche) or 430

anti-fluorescein-AP Fab fragments (1:2000) (Roche), and developed with NBT/BCIP or INT/BCIP (Roche),

respectively, as per manufacturer’s instructions and photographed using an Olympus UC30 camera mounted on

a Motic SMX-143 stereomicroscope. Riboprobes were synthesized from pGEMT-easy vectors (Promega)

containing 300 bp Spink3, 460 bp Ttr or 408 bp Trh cDNA fragments, linearized with NcoI or Sal1 and

transcribed with SP6 (antisense) or T7 (sense) RNA polymerases. Aggregates were embedded in paraffin and 435

sectioned as required.

Western blotting

EPL cell aggregates were serum-starved for 2 hours 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 µM) 440

or LDN (350 nM) for 1 hour before addition of BMP4/FCS. Total proteins was analysed by Western blot.

Membranes were developed with ECL substrate (Amersham Pharmacia Biotech), scanned with a Molecular

Imager® ChemiDoc™ XRS Imaging System (BioRad) or Fujifilm LAS-3000 (Berthold Australia Pty Ltd) and

analysed by Quantity One™. Antibodies used: p38, pp38, pSmad1/5/8 (Cell Signalling Technologies), β-tubulin

I (Sigma), HRP-conjugated secondary antibody (Cell Signalling Technologies and DakoCytomation). 445

Horseradish peroxidase (HRP)-uptake assay

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HRP-uptake assay was performed as previously described (Kanai-Azuma et al., 2002; Vassilieva et al., 2012).

Statistical analysis

Experiments were analysed using unpaired one or two-tailed student’s t-test in Microsoft® Excel software.

Significance is denoted as follows: * p<0.05; ** p<0.01. Comparisons are made between outcomes in SB, 450

noggin and DAPT compared to equivalent base medium, with or without DMSO.

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ACKNOWLEDGEMENTS

The authors would like to thank members of the Rathjen laboratory for insightful discussions of the project. This 455

work was supported by the University of Melbourne and the Albert Shimmins Postgraduate Writing up Award.

CY and HNG were supported by Australian Postgraduate Awards, CY received additional support from the

Australian Stem Cell Centre.

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FIGURE LEGENDS

Figure 1. Inhibition of p38 MAPK signalling affects lineage choice from EPL cells.

(Ai) Pp38 MAPK and p38 MAPK in EPL cells incubated in SFM, SFM+serum (Serum), or SFM+BMP4

(BMP4) for 10, 30 and 60 minutes. n=3, β-tubulin was used as a loading control. The appearance of increased

pp38 MAPK with the addition of serum was variable. (Aii) pHsp27 and Hsp27 in serum starved EPL cells

transferred to SFM, SFM+serum+DMSO (Serum) and SFM+serum+SB for 15, 30 and 60 minutes. n=3, β-

tubulin was used as a loading control. (B) EPL cells differentiated in SFM+SB, BCM or SCM +/- SB or DMSO

were scored for the formation cardiocytes, erythrocytes and neurons. n=3; mean +/- SEM. Raw data for this

experiment can be found in Table S3. (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. Primitive streak markers were quantified on day 3 and have

been normalised to the expression of Gapdh (C). n=3; mean +/- SEM. Mesoderm and ectoderm markers were

analysed on day 4 (D). n=3, a representative result is shown. –R, no reverse transcriptase control; -D, no cDNA

control.

Figure 2. Inhibition of BMP signalling affects lineage choice in differentiating EPL cells.

(A) pSmad1/5/8 in EPL cells treated with SFM or SFM containing BMP4 or serum for 5, 10 and 30 minutes.

n=2; a representative result is shown. (B) EPL cell aggregates differentiated in BCM or SCM+/-noggin were

scored for the formation cardiocytes, erythrocytes and neurons. n=3; mean +/- SEM. Raw data for this

experiment can be found in Table S4. (C,D) Expression of primitive streak markers in EPL cells differentiated

for 2 (C) and 4 (D) days in treatments as for (B). Data has been normalised to β-actin transcript levels. n=3;

mean +/- SEM.

Figure 3. Inhibition of p38 MAPK signalling reduces endoderm marker gene expression in differentiating

EPL cells.

(A) Mixl1 expression in EPL cells differentiated in BCM or SCM, with or without SB or DMSO for 2 days. n=3.

Expression has been normalised to β-actin and expressed relative to expression in SCM. (B) WISH analysis of

Spink3 expression in E7.5 mouse embryos. Lateral views (i, iii); anterior view (ii). * marks the anterior

embryonic/extraembryonic boundary. Cross section of an E7.5 Spink3-stained mouse embryo (iv). (C) EPL cells

differentiated in BCM or SCM with or without SB for 4 days were analysed for the expression of endoderm

markers by qPCR. n=3-7; mean +/- SEM. Expression has been normalised to β-actin. (D) WISH analysis of EPL

cells differentiated in BCM and SCM for Ttr or Trh. (E) EPL cells differentiated in SCM and SFM were

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incubated with HRP protein. ES cell-derived aggregates (EBs) on day 7 were used as a positive control. Cells

that take up HRP stained brown after development (arrows).

Figure 4. Inhibition of BMP signalling affects endoderm formation in differentiating EPL cells.

(A) EPL cells differentiated in BCM or SCM with or without noggin or DMSO for 4 days were analysed by

qPCR for the expression of endoderm marker genes. Data has been normalised to β-actin transcript levels. n=3

or 4; mean +/-SEM. (B,C) EPL cells differentiated in BCM (B) and BCM+noggin (C) were sectioned and

stained with haemotoxylin and eosin for morphology (i) or for the expression of Trh (ii). Arrows indicate

squamous, endoderm-like cells on the surface of the aggregates. (D) EPL cells differentiated in SCM were

analysed by double WISH for the expression of Spink3 (blue, closed arrow) and Trh (magenta, open arrow).

Figure 5. The formation of primitive streak intermediates and endoderm in response to Activin A

signalling requires p38 MAPK activity.

(A) EPL cells differentiated in ACM, ACM+noggin with or without SB/DMSO or in SFM+noggin for 2 or 4

days were analysed by RT-PCR for the expression of primitive streak intermediate (day 2 and 4) or ectoderm

(day 4) markers. n=3, a representative result is shown. (B) Serum-starved aggregates were transferred to SFM or

SFM containing 25 ng/ml Activin A. Aggregates were collected 15, 30 and 60 minutes after transfer and

analysed by Western blot for phosphorylation of p38 MAPK. (C) EPL cells differentiated in ACM,

ACM+noggin with or without SB/ DMSO or in SFM+noggin for 4 days were analysed by qPCR for the

expression of endoderm markers. Data has been normalised to β-actin transcript levels. n=3; mean +/-SEM. (D)

Expression of endoderm markers by EPL cells differentiated in BCM and BCM+DAPT on day 4 of treatment.

Data has been normalised to β-actin transcript levels. n=3; mean +/-SEM.

Figure 6 Model of endoderm formation from EPL cells.

Formation of the proximal definitive endoderm is dependent on p38 MAPK activity and correlates with the prior

expression of primitive streak intermediate markers. The initial formation of the T-expressing primitive streak

intermediate can occur in response to a number of pathways including those regulated by BMP4, Activin A and

serum, and, we hypothesize, WNT, and likely results in a mixed population of progenitors (indicated by the

multiplicity of ovals). Formation of the distal definitive endoderm is dependent on p38 MAPK signalling but is

not correlated with the initial formation of the T-expressing primitive streak intermediate. PSI: primitive streak

intermediate; DE; definitive endoderm.

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EPL cellsDistal DE

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Tgfb1, Fgf8

Activity within serum(p38 MAPK dependent)

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Documents

HE FORMATION OF ROXIMAL AND DISTAL …...2014/01/27 · Nostro et al., 2011). Analysing mechanisms that regulate gastrulation and endoderm formation in systems that initiate differentiation