Pluripotency Factors in Embryonic
Stem Cells Regulate Differentiation
into Germ Layers
Matt Thomson,1,2,3Siyuan John Liu,1,6Ling-Nan Zou,1,3Zack Smith,4Alexander Meissner,3,4
and Sharad Ramanathan1,2,3,5,6,*
1FAS Center for Systems Biology
3Harvard Stem Cell Institute
4Department of Stem Cell and Regenerative Biology
5School of Engineering and Applied Sciences
6Department of Molecular and Cellular Biology
Harvard University, Cambridge, MA 02138, USA
Cell fate decisions are fundamental for development,
but we do not know how transcriptional networks
reorganize during the transition from a pluripotent
to a differentiated cell state. Here, we asked how
mouse embryonic stem cells (ESCs) leave the plurip-
otent state and choose between germ layer fates. By
analyzing the dynamics of the transcriptional circuit
that maintains pluripotency, we found that Oct4
and Sox2, proteins that maintain ESC identity, also
orchestrate germ layer fate selection. Oct4 sup-
presses neural ectodermal differentiation and pro-
motes mesendodermal differentiation; Sox2 inhibits
mesendodermal differentiation and promotes neural
ectodermal differentiation. Differentiation signals
continuously and asymmetrically modulate Oct4 and
Sox2 protein levels, altering their binding pattern in
the genome, and leading to cell fate choice. The
same factors that maintain pluripotency thus also
integrate external signals and control lineage selec-
tion. Our study provides a framework for under-
standing how complex transcription factor networks
control cell fate decisions in progenitor cells.
Howprogenitor cells decide their fate isa question that underlies
all of developmental biology but is poorly understood. While
complex regulatory networks are known to maintain cells in
distinct cell fates (Davidson et al., 2002; Novershtern et al.,
signals and reorganize these networks to allow fate transitions.
Mouse embryonic stem cells (ESCs) provide a model system
for studying cell fate choice (Nishikawa et al., 2007; Niwa,
2010). The cells integrate signals in their environment and
choose whether to remain pluripotent or to differentiate into
progenitors of the mesendoderm (ME) or neural ectoderm (NE)
(Figure 1A) (Greber et al., 2010; Nishikawa et al., 2007; Niwa,
2007, 2010; Niwa et al., 2000; Tesar et al., 2007; Yamaguchi
et al., 1999; Ying et al., 2003b). A complex circuit of transcription
factors and epigenetic regulators (including Oct4, Sox2, Nanog,
Klf4, Klf5, Tbx3; Jarid2, Suz12) holds the ESC in a pluripotent
state (Figure 1B) by repressing genes required for ME and NE
differentiation (Ema et al., 2008; Han et al., 2010; Jiang et al.,
2008; Pasini et al., 2010; Peng et al., 2009; Schuettengruber
and Cavalli, 2009; Silva and Smith, 2008). High-throughput
experiments have provided a complex but static picture of the
pluripotency circuit (Chen et al., 2008; Lu et al., 2009; Marson
et al., 2008; Wang et al., 2006) (a part of which is shown in Fig-
ure 1B), but we do not know how an ESC leaves the pluripotent
state and selects between the ME and NE cell fate.
Since pluripotency circuit expression is sufficient to block all
differentiation (Kim et al., 2008; Mitsui et al., 2003; Takahashi and
Yamanaka, 2006), the circuit must be reorganized during germ
layer differentiation so that cells can release the gene expression
program associated with either ME or NE lineage. Here,
we study the dynamic regulation of pluripotency circuit compo-
nents during in vitro differentiation to gain insight into the regula-
tory mechanisms underlying cell fate selection in this system.
By analyzing circuit dynamics during lineage selection, we are
able to disentangle the complex network (Figure 1B) and focus
on key factors that both regulate pluripotency and control
germlayer differentiation. Whilemostpluripotency circuitfactors
are downregulated or variably expressed during differentiation,
Oct4 and Sox2 are not. Oct4 is upregulated in cells choosing
the ME fate but repressed in cells choosing the NE fate.
Conversely, Sox2 protein level is upregulated in cells choosing
the NE and repressed in those choosing the ME fate. Oct4 and
Sox2 protein levels provide continuous temporal markers of
the cell’s progression toward lineage selection before lineage
specific markers are activated. The lineage specific regulation
Cell 145, 875–889, June 10, 2011 ª2011 Elsevier Inc. 875
Fold Change (ES Cell/Mesendoderm)
Fold Change (ES Cell/Neural Ectoderm)
A binds to the promoter of B (ChIP Seq Data)
A B :
DAPI Brachyury Sox1
ME class embryonic stem cell gene
NE class embryonic stem cell gene
Neural Ectoderm (NE)
Figure 1. ESCs, Defined by Correlated Expression of Pluripotency Factors, Select between NE and ME Fate In Vitro
(A) ESCs lose pluripotency and differentiate into ME progenitor cells to express Brachyury in response to Wnt3a or Wnt agonist, CHIR. They differentiate into NE
progenitors to express Sox1 in response to FGF signals or retinoic acid.
(B) Diagram of interactions between the pluripotency factors (yellow), key epigenetic regulators (gray), and regulators of the ME (red) and NE (green) lineage
inferred from ChIP-seq data in the literature.
(C) Plot of fold change in expression levels (obtained from microarray data in Shen et al. ) of genes expressed in ESCs compared to their expression in ME
progenitors (x axis) versus the same fold change comparison in NE progenitors (y axis). Pluripotency factors and key epigenetic regulators are indicated with
black dots, ME class genes in red, and NE class genes in green.
(D) Fluorescence images of Sox1-GFP ESCs exposed to 3 mM CHIR and stained with DAPI and immunostained for Brachyury show cytoplasmic GFP expression
(green, NE marker) or nuclear Brachyury expression (red, ME marker). Scale bars represent 32 mm.
(E) Scatter plot of Sox1 levels versus Brachyury levels in differentiated cells. Each point represents Brachyury and Sox1-GFP signal in a single cell.
(F) Scatter plot of protein levels in single cells for Oct4, Sox2, and Nanog derived from immunofluorescence measurements for n > 1000 ESCs growing in LIF and
BMP. Protein levels measured in units normalized to the population mean. Figure S1F shows population distributions of Oct4 and Sox2 protein levels.
(G) Pearson correlation coefficients for pairs of pluripotency factors measured using immunofluorescence and FACS.
(H) Fluorescence images of ESC nuclei stained simultaneously for DAPI, Sox2, Oct4, and Nanog. Scale bars represent 32 mm.
(I) Fluorescence images of ESCs stained for DAPI, Nanog and Tbx3. White arrow points to punctate Tbx3 expression (red) in a nucleus with low Nanog (green).
Intensity of the delocalized nuclear Tbx3 and Nanog have a correlation of R = 0.65. Scale bars represent 32 mm.
See also Figure S1.
876 Cell 145, 875–889, June 10, 2011 ª2011 Elsevier Inc.
of Oct4 and Sox2 is necessary for germ layer fate choice and
alters their binding pattern in the genome. The intimate involve-
ment of these key nodes of the pluripotency circuit in initiating
differentiation enables the cell to integrate signals and choose
between different fates.
Gene Expression Patterns Define Three Classes
of ES Factors
Weidentified transcription factorsand DNAbinding proteins that
are expressed in ESCs and studied their regulation in ME and
NE progenitor cells (Figure 1C) using published microarray
data (Shen et al., 2008). We found that many genes (diagonal
points in Figure 1C) present in the ESC are downregulated in
both ME and NE cells, including Klf4, a pluripotency circuit
factor, and Rex1, a commonly used ESC marker (Masui et al.,
2008) (see the Extended Experimental Procedures available
online). However, several genes were present in the ESC and
differentially present in either ME or NE progenitors and fell
near either axis in Figure 1C.
We identified a class of genes that was expressed in ESC and
ME progenitors but specifically downregulated in NE progenitor
cells (Figure 1C, red points). This ME class of genes contained
the pluripotency genes Oct4, Nanog, Klf5, Tbx3, and Klf9.
Complementarily, a class of genes, we call the NE class, was
expressedinESCs andinNEprogenitor cellsbutdownregulated
in ME progenitor cells (Figure 1C, green points). This class con-
Dnmt3a, and Zfp532. We classified genes as belonging to the
ME or NE class based upon their distance from the diagonal in
Figure 1C (a table with distances and p values is in Figure S1C;
see the Extended Experimental Procedures for details). Surpris-
ingly, many of the core pluripotency circuit genes, including
Nanog, Oct4, Sox2, Klf5, and Tbx3, had signatures of lineage
specific regulation, suggesting a deeper connection between
pluripotency maintenance and lineage choice.
We then developed an experimental system for studying the
expression of the ME and NE class genes during lineage choice
in single cells.
ESCs Differentiate into Germ Layer Progenitors In Vitro
We reconstituted the NE versus ME cell fate decision in vitro and
established an experimental system in which we could differen-
tiate a cell population into either the ME or NE lineage by adding
Wnt or retinoic acid.
We maintained ESCs in the pluripotent state using LIF and
BMP in defined conditions without serum or feeder cells using
methods described previously (Ying et al., 2003a; Ying et al.,
2008) (Extended Experimental Procedures). Cells removed
from pluripotency promoting conditions did not immediately
respond to NE or ME inducing signals, and this effect has been
reported in the literature (Jackson et al., 2010). However, after
48 hr in N2B27, a defined medium that lacks differentiation
signals (Extended Experimental Procedures), cells became
competent to respond to signals.
After 48 hr in N2B27, cells responded to Wnt3a or CHIR, a
Wnt agonist, by activating the core mesendodermal regulators
Brachyury and Foxa2 (Figure 1D and Figure S1D), with more
than 70% of cells expressing Brachyury within 36 hr of signal
addition at high levels of signal (e.g., 3 mM CHIR). After 48 hr in
N2B27, retinoic acid (RA) or FGF drove NE differentiation and
triggered the activation of NE markers, Sox1, Brn2, and Nestin
(Figure 1D and Figure S1E). Consistent with the published litera-
ture, 70% of cells activated Sox1 within 36 hr of signal addition
(Abranches et al., 2009; Ying et al., 2003b). In this way, Wnt or
retinoic acid signals drove high-efficiency differentiation of
ESCs to the ME or NE cell fate, respectively.
Even in the presence of Wnt3a or CHIR, a population of cells
activated Sox1, the NE regulator, and not Brachyury, the ME
regulator. This is presumably due to the paracrine FGF signaling
between the cells leading some cells to adopt the NE fate (Ying
et al., 2003b). Using a previously validated Sox1-GFP reporter
cell line (Ying et al., 2003b) and Brachyury immunofluorescence,
we could detect Sox1 and Brachyury activation in the same cell
population (Ying et al., 2003b). In cell populations treated with
Wnt3a or CHIR, Sox1-GFP expression and Brachyury staining
were mutually exclusive (Figures 1D and 1E). There are no points
on the diagonal of the scatter plot in Figure 1E, illustrating the
absence of simultaneous high Sox1 and Brachyury expression
in single cells. The ‘‘L’’ shape of the scatter plot shows that cells
make a discrete decision to activate either Sox1 or Brachyury
Our ability to induce and detect Sox1 positive and Brachyury-
positive cells under identical conditions provided a defined
experimental system for studying the regulation of the pluripo-
tency circuit factors during ME versus NE lineage selection.
We validated the differentiation protocol with a variety of cell
Nanog Downregulation Is Necessary
for Lineage Selection
We used immunofluorescence to measure the levels of the
proteins identified in Figure 1C in single cells as we took the cells
through the differentiation protocol described in the previous
section. We performed immunofluorescence staining under
be quantitatively compared between different cell populations
(Sachs et al., 2005).
Consistent with the dense network of positive regulatory inter-
actions that have been inferred between pluripotency factors
(Chew et al., 2005; Ivanova et al., 2006; Masui et al., 2007), in
populations of cells growing under pluripotency promoting
conditions, pluripotency circuit protein levels were strongly
correlated across the cell population (Figures 1F–1I).
Cells responded to differentiation signals only 48 hr after the
withdrawal of pluripotency-promoting conditions (Figure 2A).
After 48 hr in N2B27, microarrays showed that 87% of genes
changed by less than 2-fold, while 9% (including many pluripo-
tency factors) were downregulated by more than 2-fold, and
4% were induced by more than 2-fold after 48 hr, as shown by
the histogram in Figure 2B. Nanog, Oct4, Sox2, Klf4, Klf5, and
Tbx3 messenger RNA (mRNA) levels were 5%, 74%, 30%,
14%, 19%, and 12% of their levels in ESCs (Figure 2B). A small
et al., 2009). Dnmt3b is a de novo DNA methyltransferase that
Cell 145, 875–889, June 10, 2011 ª2011 Elsevier Inc. 877
acts with Dnmt3a to methylate the promoters of the pluripotency
circuit factors including Nanog during germ layer differentiation
(Li et al., 2007).
Consistent with the microarray data, the protein levels of Oct4,
Sox2, Nanog, Klf4, Klf5, Tbx3, and Klf9 fell during the transition,
and 2D and Figure S2A). In Figure 2C, the mean levels of Oct4
and Sox2 in more than 99% of the cells are below the mean
expression levels of these proteins in pluripotency supporting
conditions. Cell images (Figure 2C) show that Tbx3 and Klf4
protein levels are severely downregulated upon withdrawal of
pluripotency sustaining conditions (compare Figure S2A).
Nanog (Fold Change)
Sox2 (Fold Change)
Oct4 (Fold Change)
Mean level in
48 hours after withdrawal
48 hours after withdrawal
48 hours after
02468 10 12 14
48 hours after
0.06 0.25 0416
(48 hr / ES)
48 hr / ES ± SD
0.0470 ± 0.0045
0.0598 ± 0.0176
0.1222 ± 0.0094
0.1435 ± 0.0032
0.1853 ± 0.0544
0.2254 ± 0.0466
0.2990 ± 0.0102
0.7447 ± 0.0590
12.6108 ± 0.3100
13.5833 ± 1.1557
Number of genes
Nanog (Fold Change)
Figure 2. Downregulation of ESC-Specific Factors Is Necessary for Differentiation
(A) Schematic of ESC differentiation protocol. 48 hr after the withdrawal of pluripotency promoting conditions (LIF and BMP), cells are exposed to differentiation
signals. Cells then respond to ME inducing signals (Wnt3a or CHIR) by activating Brachyury and to NE inducing signals (retinoic acid or endogenous FGF) by
activation of Sox1.
(B) Histogram of fold change of mRNA expression levels in the cell population 48 hr afterwithdrawal of LIF and BMP as measured by microarray. The table shows
fold changes for a set of pluripotency factors as well as for Dnmt3b and Fgf5.
(C) The scatter plot (left) of Oct4 versus Sox2 expression in n > 1000 single cells 48 hr after withdrawal of pluripotency conditions. The red dot indicates mean
expressionlevel of these proteins in pluripotency conditions.Fluorescent images (right) of cells immunostained for DAPI, Tbx3 (above) and Klf4 (below) 48hrafter
withdrawal of pluripotency conditions. Scale bars represent 32 mm.
(D) Scatter plot of Nanog versus Dnmt3a levels in single cells under pluripotency supporting conditions (black) and 48 hr after the withdrawal of pluripotency
represent 32 mm.
(E) Plot of the fold changes of mean Nanog protein levels (>20,000 cells) in cell populations fixed every 6 hr after withdrawal of LIF and BMP. Error bars indicate ±
standard deviation (SD) in Nanog protein levels in the cell population. Solid line shows Nanog decay fit to an exponential with a half-life of 7.5 hr.
(F) Fluorescence images of cells in pluripotency conditions, LIF and BMP, stained for DAPI, Nanog, and Brachyury after CHIR addition for 24 hr and siRNA
knockdown of Nanog. siRNA construct was added to cells for 24 hr prior to CHIR exposure. Scale bars represent 32 mm.
See also Figure S2.
878 Cell 145, 875–889, June 10, 2011 ª2011 Elsevier Inc.
Thus, while in ESCs Nanog is expressed at high levels and
Dnmt3a at low levels (black scatter plot in Figure 2D), after 48 hr,
the cells show a complementary pattern of expression with high
levels of Dnmt3a and low levels of Nanog (red scatter plot in
Figure 2D; images in Figure 2D), consistent with an antagonistic
regulatory relationship between these factors (Li et al., 2007).
Next, we asked whether a fall in Nanog levels might drive the
ESC into the responsive state. We found that the mean Nanog
protein level in the cell population fell exponentially in time
(half-life 7.5 hr) by 100 fold after 48 hr in N2B27 (Figure 2E), sug-
gesting that Nanog downregulation might be an early and causal
event for driving the ESC into the responsive state.
Consistently, short interfering RNA (siRNA)-mediated knock-
down of Nanog in ESCs growing in pluripotency promoting
conditions led to a correlated decrease in Oct4 and Sox2 protein
levels (Figure S2). Further, in these conditions, CHIR drove
Brachyury activation only in cells that had lost Nanog (Figure 2F).
In the presence of Nanog, activation of the Wnt pathway with
CHIR or other pathway agonists is known to promote pluripo-
tency (Sato et al., 2004; Ying et al., 2008). Thus, Nanog knock
down was necessary and sufficient for Brachyury activation by
Oct4 and Sox2 Are Differentially Regulated
during Fate Choice
The correlated expression pattern of the pluripotency factors in
ESCs (Figure 1F) contrasts sharply with the lineage-specific
expression pattern that we observed in our microarray analysis
(Figure 1C). We asked whether this change in correlation pattern
is reflected in protein levels of pluripotency factors in individual
cells during differentiation.
cells were not committed to a lineage and could be driven to
either cell fate by signal addition (Figure 2A). We added differen-
tiation signals to cells 48 hr after withdrawal of pluripotency
conditions. Upon addition of signal (3 mM CHIR, Figures 1D
and 1E), some cells adopted the ME (Brachyury expression)
and others the NE (Sox1 expression) lineage.
We studied the regulation of genes in the ME class (Oct4,
Rbpj, and Zfp532) defined above to ask how the pluripotency
circuit is regulated during lineage selection. In addition to the
ME and NE class, we studied the pluripotency factor, Klf4, and
the key epigenetic regulator, Jarid2, which controls the targeting
of repressive epigenetic modifications to the genome in pluripo-
tent and differentiating ESCs (Peng et al., 2009).
Of the genes identified from the microarray analysis as
belonging to the ME or NE class, Tbx3, Klf4, Klf9, and Rbpj
proteins were absent or present at very low levels in cells
entering both the NE and ME lineages and were not reactivated
during differentiation (Figure 2C and Figure S3). This suggests
that these factors play a role in pluripotency maintenance but
not in lineage choice. In contrast, Oct4, Sox2, Nanog, Jarid2,
Klf5, Foxp1, and Dnmt3a proteins were present in the nucleus
of differentiating cells (Figure 3), and we classified these factors
into groups based on their expression pattern.
tive (ME) cells but absent in Sox1-positive (NE) cells. Consis-
in single cells correlated positively (Figure 3A, red scatter plot).
The scatter plots of Oct4 versus Sox1 levels showed that cells
with Sox1 have very low levels of Oct4 (Figure 3A, green scatter
plot). Klf5 had a similar pattern of protein expression to Oct4.
However, rather than being absent in Sox1-positive cells, Klf5
was confined to a subcellular compartment distinct from the
nucleus (Figure 3B).
The second group of proteins included only Sox2, which was
expressed in a complementary pattern to Oct4 and Klf5 (Fig-
ure 3C). In cells undergoing ME differentiation, Sox2 and
Brachyury were mutually exclusive (Figure 3C, red scatter
plot). In contrast, Sox2 and Sox1 (NE marker), expression corre-
lated strongly during NE lineage induction (Figure 3C, green
As the final class of factors, Foxp1, Nanog, Dnmt3a, and
Jarid2 were present variably in cells that had activated either
Sox1 or Brachyury (Figures 3D–3G). For example, scatter plots
of Dnmt3a protein levels quantify this observation and illustrate
that the distribution of Dnmt3a protein is similar in both in Sox1
and Brachyury expressing cells (Figure 3F).
We quantified the data in the scatter plots by measuring the
probability of observing a given protein conditioned on the pres-
ence of each lineage marker (Figure 3H and the Extended Exper-
imental Procedures). On this plot, variably expressed proteins
like Dnmt3a lie on the diagonal and are present with high proba-
bility in both Sox1 and Brachyury-expressing cells. Lineage
specific proteins like Oct4 and Sox2 fall on the extreme off-diag-
onal of the plot and are present with high probability in either ME
or NE progenitor cells but not both, consistent with the images in
Figures 3A and 3C (see the Extended Experimental Procedures).
To determine whether the above patterns were established
prior to lineage choice, we used FACS and live-cell imaging to
study the dynamics of protein regulation in response to signal.
We did not study Klf5 further because Klf5 knockout ESC lines
can differentiate into all three germ layers (Ema et al., 2008).
Differential Modulation of Oct4 and Sox2 Levels
Precedes Cell Fate Selection
We asked how the relative levels of Oct4 and Sox2 protein
change in time during ME and NE differentiation. We added
CHIR or RA to cell populations to induce ME or NE differentiation
as described (Experimental Procedures), and determined the
levels of both factors in individual cells (n > 40,000) using FACS.
After CHIR addition, the meanlevelof Sox2 proteinfellby72%
after 8hrand by 77%at 12hr (Figure 4A). The mean Oct4 protein
level, on the other hand, fell by 5% over the first 12 hr. At 12 hr
after CHIR addition, 42% of cells had activated Oct4 to levels
greater than the mean level in the cell population prior to signal
addition, while only 1.7% of cells had Sox2 protein levels
exceeding the Sox2 mean in the initial cell population. After
12 hr, the fold change in relative Oct4 and Sox2 protein levels
protein in the cell population. The activation of ME regulator
Brachyury occurred between 12 and 15 hr after CHIR addition.
By 24 hr, Oct4 levels were upregulated and Sox2 repressed in
cells that had activated Brachyury (Figure 4A) as shown in the
Cell 145, 875–889, June 10, 2011 ª2011 Elsevier Inc. 879