he formation of roximal and distal …...2014/01/27 · nostro et al., 2011). analysing mechanisms...
TRANSCRIPT
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
urna
l of C
ell S
cien
ceA
ccep
ted
man
uscr
ipt
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.
Jour
nal o
f Cel
l Sci
ence
Acc
epte
d m
anus
crip
t
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
Jour
nal o
f Cel
l Sci
ence
Acc
epte
d m
anus
crip
t
4 Endoderm formation requires p38 MAPK activity
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
nal o
f Cel
l Sci
ence
Acc
epte
d m
anus
crip
t
5 Endoderm formation requires p38 MAPK activity
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
Jour
nal o
f Cel
l Sci
ence
Acc
epte
d m
anus
crip
t
6 Endoderm formation requires p38 MAPK activity
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
Jour
nal o
f Cel
l Sci
ence
Acc
epte
d m
anus
crip
t
7 Endoderm formation requires p38 MAPK activity
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
Jour
nal o
f Cel
l Sci
ence
Acc
epte
d m
anus
crip
t
8 Endoderm formation requires p38 MAPK activity
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
Jour
nal o
f Cel
l Sci
ence
Acc
epte
d m
anus
crip
t
9 Endoderm formation requires p38 MAPK activity
(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,
Jour
nal o
f Cel
l Sci
ence
Acc
epte
d m
anus
crip
t
10 Endoderm formation requires p38 MAPK activity
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).
Jour
nal o
f Cel
l Sci
ence
Acc
epte
d m
anus
crip
t
11 Endoderm formation requires p38 MAPK activity
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
Jour
nal o
f Cel
l Sci
ence
Acc
epte
d m
anus
crip
t
12 Endoderm formation requires p38 MAPK activity
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
Jour
nal o
f Cel
l Sci
ence
Acc
epte
d m
anus
crip
t
13 Endoderm formation requires p38 MAPK activity
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
Jour
nal o
f Cel
l Sci
ence
Acc
epte
d m
anus
crip
t
14 Endoderm formation requires p38 MAPK activity
(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
Jour
nal o
f Cel
l Sci
ence
Acc
epte
d m
anus
crip
t
15 Endoderm formation requires p38 MAPK activity
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
Jour
nal o
f Cel
l Sci
ence
Acc
epte
d m
anus
crip
t
16 Endoderm formation requires p38 MAPK activity
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.
Jour
nal o
f Cel
l Sci
ence
Acc
epte
d m
anus
crip
t
17 Endoderm formation requires p38 MAPK activity
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
Jour
nal o
f Cel
l Sci
ence
Acc
epte
d m
anus
crip
t
18 Endoderm formation requires p38 MAPK activity
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.
Jour
nal o
f Cel
l Sci
ence
Acc
epte
d m
anus
crip
t
19 Endoderm formation requires p38 MAPK activity
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.
Jour
nal o
f Cel
l Sci
ence
Acc
epte
d m
anus
crip
t
20 Endoderm formation requires p38 MAPK activity
REFERENCES
Adams, E. and Frank, L. (1980). Metabolism of proline and the hydroxyprolines. Annu Rev Biochem 49, 1005-
61.
Adams, R. H., Porras, A., Alonso, G., Jones, M., Vintersten, K., Panelli, S., Valladares, A., Perez, L., Klein, R.
and Nebreda, A. R. (2000). Essential role of p38alpha MAP kinase in placental but not embryonic
cardiovascular development. Mol Cell 6, 109-16.
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, 859-70.
Aouadi, M., Bost, F., Caron, L., Laurent, K., Le Marchand Brustel, Y. and Binetruy, B. (2006). p38 mitogen-
activated protein kinase activity commits embryonic stem cells to either neurogenesis or cardiomyogenesis.
Stem Cells 24, 1399-406.
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). p38 mitogen activated protein kinase controls two successive-steps during
the early mesodermal commitment of embryonic stem cells. Stem Cells Dev 20, 1233-46.
Beddington, R. S. and Robertson, E. J. (1998). Anterior patterning in mouse. Trends Genet 14, 277-84.
Beddington, R. S. and Robertson, E. J. (1999). Axis development and early asymmetry in mammals. Cell 96,
195-209.
Bradley, A., Evans, M., Kaufman, M. H. and Robertson, E. (1984). Formation of germ-line chimaeras from
embryo-derived teratocarcinoma cell lines. Nature 309, 255-6.
Chakraborty, S., Kang, B., Huang, F. and Guo, Y. L. (2009). Mouse embryonic stem cells lacking p38alpha and
p38delta can differentiate to endothelial cells, smooth muscle cells, and epithelial cells. Differentiation 78, 143-
50.
Chawengsaksophak, K., de Graaff, W., Rossant, J., Deschamps, J. and Beck, F. (2004). Cdx2 is essential for axial
elongation in mouse development. Proc Natl Acad Sci U S A 101, 7641-5.
Clements, M., Pernaute, B., Vella, F. and Rodriguez, T. A. (2011). Crosstalk between Nodal/activin and MAPK
p38 signaling is essential for anterior-posterior axis specification. Curr Biol 21, 1289-95.
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 and maintenance of the primitive streak in the mouse.
Development 120, 1919-28.
Crossley, P. H. and Martin, G. R. (1995). The mouse Fgf8 gene encodes a family of polypeptides and is
expressed in regions that direct outgrowth and patterning in the developing embryo. Development 121, 439.
Cuenda, A., Rouse, J., Doza, Y., Meier, R., Cohen, P., Gallagher, T., Young, P. and Lee, J. (1995). SB 203580 is a
specific inhibitor of a MAP kinase homologue which is stimulated by cellular stresses and interleukin-1. FEBS
letters 364, 229-233.
Davidson, S. M. and Morange, M. (2000). Hsp25 and the p38 MAPK pathway are involved in differentiation of
cardiomyocytes. Dev Biol 218, 146-60.
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 and p38beta MAPKs during mouse development. Proc Natl Acad Sci
U S A 108, 12764-9.
Dickson, M. C., Martin, J. S., Cousins, F. M., Kulkarni, A. B., Karlsson, S. and Akhurst, R. J. (1995). Defective
haematopoiesis and vasculogenesis in transforming growth factor-beta 1 knock out mice. Development 121,
1845.
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., Malaise, M., Reinhardt, B., Kedinger, C. and Boeuf, H. (2004). A p38 inhibitor allows to dissociate
differentiation and apoptotic processes triggered upon LIF withdrawal in mouse embryonic stem cells. Cell
Death Differ 11, 331-41.
Jour
nal o
f Cel
l Sci
ence
Acc
epte
d m
anus
crip
t
21 Endoderm formation requires p38 MAPK activity
Engelman, J. A., Lisanti, M. P. and Scherer, P. E. (1998). Specific inhibitors of p38 mitogen-activated protein
kinase block 3T3-L1 adipogenesis. J Biol Chem 273, 32111-20.
Evans, M. J. and Kaufman, M. H. (1981). Establishment in culture of pluripotential cells from mouse embryos.
Nature 292, 154-6.
Farace, M. G., Brown, B., Raschella, G., Alexander, J., Gambari, R., Fantoni, A., Hardies, S., Hutchison, C. and
Edgell, M. (1984). The mouse beta h1 gene codes for the z chain of embryonic hemoglobin. Journal of
Biological Chemistry 259, 7123.
Gadue, P., Gouon-Evans, V., Cheng, X., Wandzioch, E., Zaret, K. S., Grompe, M., Streeter, P. R. and Keller, G.
M. (2009). Generation of monoclonal antibodies specific for cell surface molecules expressed on early mouse
endoderm. Stem Cells 27, 2103-13.
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 primitive streak formation using embryonic stem cells. Proc Natl Acad Sci
U S A 103, 16806-11.
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). Enhanced cardiomyogenesis of human embryonic stem cells by a small molecular
inhibitor of p38 MAPK. Differentiation 76, 357-70.
Gu, G., Wells, J. M., Dombkowski, D., Preffer, F., Aronow, B. and Melton, D. A. (2004a). Global expression
analysis of gene regulatory pathways during endocrine pancreatic development. Development 131, 165.
Gu, G., Wells, J. M., Dombkowski, D., Preffer, F., Aronow, B. and Melton, D. A. (2004b). Global expression
analysis of gene regulatory pathways during endocrine pancreatic development. Development 131, 165-79.
Guillemot, F., Lo, L.-C., Johnson, J. E., Auerbach, A., Anderson, D. J. and Joyner, A. L. (1993). Mammalian
achaete-scute homolog 1 is required for 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 mouse embryonic stem cells can
differentiate to endothelial cells, smooth muscle cells, and neurons. Dev Dyn 236, 3383-92.
Han, J., Lee, J. D., Bibbs, L. and Ulevitch, R. J. (1994). A MAP kinase targeted by endotoxin and hyperosmolarity
in mammalian cells. Science 265, 808.
Hanafusa, H., Ninomiya-Tsuji, J., Masuyama, N., Nishita, M., Fujisawa, J.-i., Shibuya, H., Matsumoto, K. and
Nishida, E. (1999). Involvement of the p38 Mitogen-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 axial mesendoderm morphogenesis and patterning in the murine embryo.
Development 129, 3597-608.
Harvey, N. T., Hughes, J. N., Lonic, A., Yap, C., Long, C., Rathjen, P. D. and Rathjen, J. (2010). Response to
BMP4 signalling during ES cell differentiation defines intermediates of the ectoderm lineage. J Cell Sci 123,
1796-804.
Heldin, C. H., Miyazono, K. and ten Dijke, P. (1997). TGF-signalling from cell membrane to nucleus through
SMAD proteins. Nature 390, 465-471.
Herrera, B. and Inman, G. J. (2009). A rapid and sensitive bioassay for the simultaneous measurement of
multiple bone morphogenetic proteins. Identification and quantification of BMP4, BMP6 and BMP9 in bovine
and human serum. BMC Cell Biol 10, 20.
Hu, M. C., Wasserman, D., Hartwig, S. and Rosenblum, N. D. (2004). p38MAPK acts in the BMP7-dependent
stimulatory pathway during epithelial cell morphogenesis and is regulated by Smad1. J Biol Chem 279, 12051-9.
Hughes, J. N., Dodge, N., Rathjen, P. D. and Rathjen, J. (2009a). A novel role for gamma-secretase in the
formation of primitive streak-like intermediates from ES cells in culture. Stem Cells 27, 2941-51.
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 mesoderm suppressing activity direct lineage choice from pluripotent
primitive ectoderm-like cells in culture. PLoS One 4, e5579.
Jour
nal o
f Cel
l Sci
ence
Acc
epte
d m
anus
crip
t
22 Endoderm formation requires p38 MAPK activity
Izumi, N., Era, T., Akimaru, H., Yasunaga, M. and Nishikawa, S. (2007). Dissecting the molecular hierarchy for
mesendoderm differentiation through a combination of embryonic stem cell culture and RNA interference.
Stem Cells 25, 1664-74.
Jackson, S. A., Schiesser, J., Stanley, E. G. and Elefanty, A. G. (2010). Differentiating embryonic stem cells pass
through 'temporal windows' that mark responsiveness 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 of definitive gut endoderm in Sox17-null mutant mice. Development 129,
2367-79.
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 a dynamic population of progenitor cells for the axial
mesoderm. Development 128, 3623-34.
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-like factor from bovine serum. Biochem Biophys Res Commun
345, 1224-31.
Kubo, A., Shinozaki, K., Shannon, J. M., Kouskoff, V., Kennedy, M., Woo, S., Fehling, H. J. and Keller, G.
(2004). Development of definitive endoderm from embryonic stem cells in culture. Development 131, 1651-62.
Kunath, T., Saba-El-Leil, M. K., Almousailleakh, M., Wray, J., Meloche, S. and Smith, A. (2007). FGF stimulation
of the Erk1/2 signalling cascade triggers transition of pluripotent embryonic stem cells from self-renewal to
lineage commitment. Development 134, 2895-902.
Lake, J., Rathjen, J., Remiszewski, J. and Rathjen, P. D. (2000). Reversible programming of pluripotent cell
differentiation. J Cell Sci 113 ( Pt 3), 555-66.
Lawson, K. A., Meneses, J. J. and Pedersen, R. A. (1991). Clonal analysis of epiblast fate during germ layer
formation in the mouse embryo. Development 113, 891-911.
Li, L., Arman, E., Ekblom, P., Edgar, D., Murray, P. and Lonai, P. (2004). Distinct GATA6- and laminin-
dependent mechanisms regulate endodermal and ectodermal embryonic stem cell fates. Development 131,
5277-86.
Lickert, H., Kutsch, S. and Kanzler, B. I. (2002). Formation of multiple hearts in mice following deletion of
[beta]-catenin in the embryonic endoderm. Developmental 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 cells and their myogenic descendants. Development 119, 419.
Liu, P., Wakamiya, M., Shea, M. J., Albrecht, U., Behringer, R. R. and Bradley, A. (1999). Requirement for
Wnt3 in vertebrate axis formation. Nature Genetics 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, 1845-57.
Martin-Blanco, E. (2000). p38 MAPK signalling cascades: ancient roles and new functions. Bioessays 22, 637-45.
Martin, G. R. (1981). Isolation of a pluripotent cell line from early mouse embryos cultured in medium
conditioned by teratocarcinoma stem cells. Proc Natl Acad Sci U S A 78, 7634-8.
Massaous, J. and Hata, A. (1997). TGF-[beta] signalling through the Smad pathway. Trends in cell biology 7,
187-192.
Mathew, S., Jaramillo, M., Zhang, X., Zhang, L. A., Soto-Gutierrez, A. and Banerjee, I. (2012). Analysis of
alternative signaling pathways of endoderm induction 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 of Thyrotropin releasing hormone in
the mouse definitive endoderm. Developmental Dynamics 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 ESCs reveals a role for FGF signaling. Cell Stem Cell 3, 402-15.
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 protein kinase in placental angiogenesis. Proc Natl Acad Sci U S A 97,
10454-9.
Jour
nal o
f Cel
l Sci
ence
Acc
epte
d m
anus
crip
t
23 Endoderm formation requires p38 MAPK activity
Muller, P. Y., Janovjak, H., Miserez, A. R. and Dobbie, Z. (2002). Short Technical Report Processing of Gene
Expression Data Generated by Quantitative Real-Time RT-PCR. Biotechniques 32.
Nagata, Y., Takahashi, N., Davis, R. J. and Todokoro, K. (1998). Activation of p38 MAP kinase and JNK but not
ERK is required for erythropoietin-induced erythroid differentiation. Blood 92, 1859-69.
Nakamura, K., Shirai, T., Morishita, S., Uchida, S., Saeki-Miura, K. and Makishima, F. (1999). p38 mitogen-
activated protein kinase functionally contributes to chondrogenesis induced by growth/differentiation factor-5
in ATDC5 cells. Exp Cell Res 250, 351-63.
Nebreda, A. R. and Porras, A. (2000). p38 MAP kinases: beyond the stress response. Trends Biochem Sci 25,
257-60.
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 TGFbeta family members and WNT regulates
patterning and pancreatic specification of human pluripotent stem cells. Development 138, 861-71.
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, 329-39.
Perdiguero, E., Ruiz-Bonilla, V., Gresh, L., Hui, L., Ballestar, E., Sousa-Victor, P., Baeza-Raja, B., Jardi, M.,
Bosch-Comas, A., Esteller, M. et al. (2007). Genetic analysis of p38 MAP kinases in myogenesis: fundamental
role of p38alpha in abrogating myoblast proliferation. EMBO J 26, 1245-56.
Pevny, L. H., Sockanathan, S., Placzek, M. and Lovell-Badge, R. (1998). A role for SOX1 in neural
determination. Development 125, 1967.
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). Directed differentiation of human embryonic stem cells into the
pancreatic endocrine lineage. Stem Cells Dev 16, 561-78.
Raingeaud, J., Gupta, S., Rogers, J. S., Dickens, M., Han, J., Ulevitch, R. J. and Davis, R. J. (1995). Pro-
inflammatory cytokines and environmental stress cause p38 mitogen-activated protein kinase activation by
dual phosphorylation on tyrosine and threonine. J Biol Chem 270, 7420-6.
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, 2649-61.
Rathjen, J., Lake, J. A., Bettess, M. D., Washington, J. M., Chapman, G. and Rathjen, 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 ( Pt 5), 601-12.
Rathjen, J. and Rathjen, P. D. (2003). Lineage specific differentiation of mouse ES cells: formation and
differentiation of early primitive ectoderm-like (EPL) cells. Methods Enzymol 365, 3-25.
Rathjen, J., Washington, J. M., Bettess, M. D. and Rathjen, P. D. (2003). Identification of a biological activity
that supports maintenance and proliferation of pluripotent cells from the primitive ectoderm of the mouse.
Biol Reprod 69, 1863-71.
Rouse, J., Cohen, P., Trigon, S., Morange, M., Alonso-Llamazares, A., Zamanillo, D., Hunt, T. and Nebreda, A.
R. (1994). A novel kinase cascade triggered by stress and heat shock that stimulates MAPKAP kinase-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 the nascent mesodermal cells during mouse gastrulation. Development
122, 2769.
Shimono, A. and Behringer, R. R. (2003). Angiomotin regulates visceral endoderm movements during mouse
embryogenesis. Curr Biol 13, 613-7.
Simon, P. (2003). Q-Gene: processing quantitative real-time RT–PCR data. Bioinformatics 19, 1439.
So, P. L. and Danielian, P. S. (1999). Cloning and expression analysis of a mouse gene related to Drosophila
odd-skipped. Mechanisms of Development 84, 157-160.
Jour
nal o
f Cel
l Sci
ence
Acc
epte
d m
anus
crip
t
24 Endoderm formation requires p38 MAPK activity
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). Targeted mutagenesis of the transcription factor GATA-4 gene in mouse
embryonic stem cells disrupts visceral endoderm differentiation in vitro. Development 121, 3877-88.
Tada, S., Era, T., Furusawa, C., Sakurai, H., Nishikawa, S., Kinoshita, M., Nakao, K. and Chiba, T. (2005).
Characterization of mesendoderm: a diverging point of the definitive endoderm and mesoderm in embryonic
stem cell differentiation culture. Development 132, 4363-74.
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, 126-41.
Tanaka, N., Kamanaka, M., Enslen, H., Dong, C., Wysk, M., Davis, R. J. and Flavell, R. A. (2002). Differential
involvement of p38 mitogen-activated protein kinase kinases MKK3 and MKK6 in T-cell apoptosis. EMBO Rep 3,
785-91.
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, 3079-90.
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 One 7, 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 graded Nodal signals. Genes Dev 17, 1646-62.
Wang, J., Chen, L., Ko, C. I., Zhang, L., Puga, A. and Xia, Y. (2012). Distinct signaling properties of mitogen-
activated protein kinase kinases 4 (MKK4) and 7 (MKK7) in embryonic stem cell (ESC) differentiation. J Biol
Chem 287, 2787-97.
Wang, J., Ohmuraya, M., Hirota, M., Baba, H., Zhao, G., Takeya, M., Araki, K. and Yamamura, K.-i. (2008).
Expression pattern of <i>serine protease inhibitor kazal type 3 (Spink3)</i> during mouse embryonic
development. Histochemistry and Cell Biology 130, 387-397.
Wilkinson, D. G., Bhatt, S. and Herrmann, B. G. (1990). Expression pattern of the mouse T gene and its role in
mesoderm formation.
Winnier, G., Blessing, M., Labosky, P. A. and Hogan, B. L. (1995). Bone morphogenetic protein-4 is required for
mesoderm formation and patterning in the mouse. Genes Dev 9, 2105-16.
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 kinase controls a switch between cardiomyocyte and neuronal
commitment of murine embryonic stem cells by activating myocyte enhancer factor 2C-dependent bone
morphogenetic protein 2 transcription. Stem Cells Dev 19, 1723-34.
Yang, D., Tournier, C., Wysk, M., Lu, H. T., Xu, J., Davis, R. J. and Flavell, R. A. (1997). Targeted disruption of
the MKK4 gene causes embryonic death, inhibition of c-Jun NH2-terminal kinase activation, and defects in AP-1
transcriptional activity. Proc Natl Acad Sci U S A 94, 3004-9.
Yue, J. and Mulder, K. M. (2000). Activation of the Mitogen-Activated Protein Kinase Pathway by Transforming
Growth Factor-ß. Methods Mol. Biol 142, 125-131.
Zheng, Z., de Iongh, R. U., Rathjen, P. D. and Rathjen, J. (2010). A requirement for FGF signalling in the
formation of primitive streak-like intermediates from primitive ectoderm in culture. PLoS One 5, e12555.
Zimmerman, L. B., De Jesus-Escobar, J. M. and Harland, R. M. (1996). The Spemann organizer signal noggin
binds and inactivates bone morphogenetic protein 4. Cell 86, 599-606.
Zohn, I. E., Li, Y., Skolnik, E. Y., Anderson, K. V., Han, J. and Niswander, L. (2006). p38 and a p38-interacting
protein are critical for downregulation of E-cadherin during mouse gastrulation. Cell 125, 957-69.
Jour
nal o
f Cel
l Sci
ence
Acc
epte
d m
anus
crip
t
25 Endoderm formation requires p38 MAPK activity
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
Jour
nal o
f Cel
l Sci
ence
Acc
epte
d m
anus
crip
t
26 Endoderm formation requires p38 MAPK activity
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.
Jour
nal o
f Cel
l Sci
ence
Acc
epte
d m
anus
crip
t
Jour
nal o
f Cel
l Sci
ence
Acc
epte
d m
anus
crip
t
Jour
nal o
f Cel
l Sci
ence
Acc
epte
d m
anus
crip
t
BCM/Ttr SCM/Ttr
i iviiiii
****
BCMDMSO SBSB DMSO
SCM
2.5
2.0
1.5
1.0
0.5
*M
ixl1
exp
ress
ion
rela
tive
to
SC
M
A
B
D E
Yap, Goh et al. Figure 3
**
SCM/TrhBCM/Trh
Sox17
Spink3 Ttr
Gata4 Trh
Eya210-3
10-2
10-1
100
101
102
***
**
***
*
**
**
*
Gen
e ex
pres
sion
nor
mal
ised
to G
AP
DH
BCMBCM+SBSCMSCM+SB
C
*
EBs SCM
SFM
Spink3
Jour
nal o
f Cel
l Sci
ence
Acc
epte
d m
anus
crip
t
B i
Yap, Goh et al. Figure 4
A
C
ii
i ii
D
Sox17
Spink3 Ttr
Gata4
Trh Eya2
100
10
1
0.01
0.001
0.1
Gen
e ex
pres
sion
nor
mal
ised
to β
-act
in
BCMBCM+NogginSCMSCM+Noggin
*
****
*** *
Jour
nal o
f Cel
l Sci
ence
Acc
epte
d m
anus
crip
t
Jour
nal o
f Cel
l Sci
ence
Acc
epte
d m
anus
crip
t
EPL cellsDistal DE
Trh, Eya2, Sox17
Proximal DESpink3, Ttr, Gata4,
Sox17Mesoderm
BMP4
pp38 MAPK
pp38 MAPK
Endoderm progenitor?
Yap, Goh et al. Figure 6
WNT3Activin A/Nodal
(p38 MAPK dependent)
Activin A/Nodal
Activin A/Nodal
pp38 MAPK?PSIT, Wnt3, Bmp4,
Tgfb1, Fgf8
Activity within serum(p38 MAPK dependent)
Jour
nal o
f Cel
l Sci
ence
Acc
epte
d m
anus
crip
t