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)
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
(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
scatter plot in Figure 4A, where cells adopting the ME fate
showed high Oct4 and low Sox2 expression.
After RA addition, Oct4 and Sox2 protein levels diverged but
on a longer time scale than after CHIR addition (Figure 4B). At
14 hr after RA addition, the mean level of Sox2 protein had fallen
were 100% of their initial level while Oct4 had fallen by 64%. At
the same time point, 37% of cells had Sox2 levels greater
than the mean level in the cell population prior to signal addition,
while less than 1% of cells had Oct4 levels exceeding the mean
in the initial cell population. At 18 hr, the relative fold change in
Oct4 and Sox2 differed by more than the sum of the standard
Figure 3. Key ESC Transcription Factors Are Differentially Expressed in ME and NE Cells
(A–G) Cells were differentiated in conditions (see the Experimental Procedures) where some cells adopted the ME fate (Brachyury positive) and others adopted
the NE fate (Sox1-GFP positive). Images of a field of cells immunostained 36 hr after signal addition for ME marker Brachyury, NE marker Sox1, DAPI, and
aspecificfactor(left)areshown.Scatterplots(right)areoftheexpressionlevel ofthatfactor againstBrachyuryinn>300Brachyury-positive cells(red points)and
against Sox1, n > 300 Sox1-positive cells (green points) for Oct4 (A), Klf5 (B), Sox2 (C), Foxp1 (D), Nanog (E), Dnmt3a (F), and Jarid2 (G). All scale bars represent
(H) The data from (A)–(G) are summarized in a conditional probability plot. Each point in the plot represents the probability of finding the expression of the specific
protein in ME progenitors (y axis) versus NE progenitors (x axis).
See also Figure S3.
880 Cell 145, 875–889, June 10, 2011 ª2011 Elsevier Inc.
510 15 20
Oct4-mCitrine (Fold Change)
0510 15 20
Oct4-mCitrine (Fold Change)
Oct4-mCitrine (Fold Change)
Neural ectodermal Differentiation
Pluripotent CellsPluripotent Cells
Neural ectodermal (NE) Differentiation
Neural ectodermal (NE) Differentiation
1.25 hrs5.55 hrs10.25 hrs14.55 hrs19.25 hrs
Mesendodermal (ME) Differentiation
Mesendodermal (ME) Differentiation
Sox2 (Fold Change)
Oct4 (Fold Change)
Sox2 (Fold Change)
Oct4 (Fold Change)
Mesendodermal Differentiation Neural ectodermal Differentiation
Relative Fold ChangeRelative Fold Change
Figure 4. Oct4 and Sox2 Protein Levels Diverge Continuously during Lineage Selection
(A) Left: Plot of mean ± SD of Oct4 (purple squares) and Sox2 (blue circles) protein levels in n > 40,000 cells at 0, 8, and 12 hr after CHIR addition, measured
simultaneously in single cells via immunofluorescence and FACS. Protein levels were normalized to the mean protein level in the cell population prior to signal
addition. Right: Scatter plot of Oct4 and Sox2 protein levels 24 hr after signal addition in single ME progenitor cells (?2400 cells), with levels normalized to the
mean level of these proteins in pluripotent cells.
(B) Plot of mean ± SD of Oct4 (purple squares) and Sox2 (blue circles) levels in populations of n > 40,000 cells after RA addition.
(C) Scatter plot of Oct4 versus Sox2 protein levels in n > 2000 cells 24 hr after RA addition. Protein levels were normalized to the mean protein level in the cell
population prior to signal addition.
(D) Confocal microscopy images of cells undergoing ME differentiation stained for DAPI, Oct4, Sox2, and Brachyury.
(E) Single-cell trajectories of Oct4-mCitrine levels obtained via time lapse microscopy for ESCs growing under pluripotency-promoting conditions.
(F) Single-cell trajectories of Oct4-mCitrine in cells differentiating into the neural ectodermal lineage. Conditions supporting pluripotency were removed for 48 hr,
and then retinoic acid (500 nM) was added at t = 0 to induce differentiation.
(G) Temporal trajectories of Oct4-mCitrine in n > 100 cells differentiating into NE (mean in solid line, standard deviation as a light green background) and
ME progenitors (mean in solid line, standard deviation as a light red background) obtained by fluorescence time-lapse microscopy of the Oct4-mCitrine cell line
(t = 0 corresponds to time of signal addition, Figure 2A).
(H–J) Fluorescence images from a time lapse imaging experiment following a field of Oct4-mCitrine cells under pluripotency promoting conditions (H), during
retinoic acid driven NE differentiation (I), and (during CHIR driven ME differentiation (J). Scale bars represent 32 mm.
See also Figure S4.
Cell 145, 875–889, June 10, 2011 ª2011 Elsevier Inc. 881
deviations of either protein in the cell population. The NE maker
Sox1 was activated between 17 and 19 hr after RA addition. By
24 hr, Sox2 was activated and Oct4 repressed in Sox1-express-
ing cells as seen in Figure 4C.
Thus, after the addition of differentiation signals, Oct4 and
Sox2 protein levels diverged in time, and the correlated pat-
tern of Oct4 and Sox2 expression of the ESC (Figure 1F)
broke continuously into either a high Oct4 and low Sox2 or
a low Oct4 and high Sox2 pattern during differentiation (Figures
To validate our FACS results, we measured Oct4 expression
dynamics in single live cells during ME and NE differentiation.
We created and validated (Experimental Procedures and Fig-
ure S4) (Tesar et al., 2007) an Oct4-mCitrine fusion reporter
cell line (Experimental Procedures and Figure S4). We used
time-lapse epifluorescence microscopy to track Oct4-mCitrine
expression in hundreds of single cells both under pluripotency-
promoting conditions and during differentiation. Under pluripo-
tency-promoting conditions, Oct4 levels in single cells varied
during normal rounds of cell division (Figures 4E and 4H). After
48 hr in N2B27, Oct4-mCitrine levels in single cells decreased
to 60% ± 12% of ESC levels.
NE differentiation with retinoic acid drove a rapid downregula-
tion of Oct4-mCitrine level that was detectable in single cells
vidual cells decreased their Oct4 level to half of its initial value by
16 hr after RA addition (Figure 4F). At the population level, Oct4
level decreased linearly in time (Figure 4G).
ME differentiation drove a complementary response. Cells
responded to CHIR by upregulating Oct4 (Figures 4J and 4G).
In the cell population, Oct4 level increased linearly in time within
6 hr of CHIR addition (Figure 4G).
In this way, cells differentiating into the ME or NE lineage
showed a detectable response in Oct4 protein levels within
6 hr of signal addition, a time that is on average 10 hr before
the earliest detectable lineage specific marker expression.
Further, the Oct4 temporal trajectories in cells differentiated
with CHIR diverged from those differentiated with RA within
6 hr (Figure 4G). These results show that the differential modula-
tion of Oct4 occurs prior to the expression of ME or NE lineage-
We next determined the functional roles of Oct4 and Sox2 in
lineage choice. The observed Oct4 and Sox2 protein expression
patterns (Figures 3A, 3C, 4A, and 4C) suggested that Oct4
might specifically inhibit the NE lineage while promoting ME
differentiation and that that Sox2 might specifically inhibit ME
differentiation while promoting NE lineage choice. Our analysis
of published ChIP-seq data (Chen et al., 2008) provided further
evidence for the asymmetric role for Oct4 and Sox2 in regulating
ME and NE differentiation (Extended Experimental Procedures
and Figure S5).
Oct4 and Sox2 Bind DNA in Lineage-Specific Patterns
relative levels are modulated during differentiation, we used
ChIP-qPCR to probe the binding of Oct4 and Sox2 along five
genomic regions (Oct4, Sox2, Nanog, Brachyury, and Sox1) in
ESCs and during ME and NE differentiation. We could not obtain
reproducible ChIP-qPCR results along the Sox1 locus in ESCs,
ME, or NE progenitors (see the Experimental Procedures).
We measured the spatial distribution of binding enrichment
along each genomic locus using tiled qPCR primers on ChIP
samples for each factor Oct4 and Sox2, in the three lineages:
ES, ME and NE. Experiments were performed on threebiological
replicates, and in three technical replicates for each primer pair.
targeted to and around the region of interest.
We validated our experimental system by comparing the
results of our Oct4 and Sox2 ChIP-qPCR experiments on
ESCs to published ChIP-seq data (Chen et al., 2008; Marson
et al., 2008). We found that Oct4 (Figures 5B, 5E, 5I, and 5L,
blue bars) and Sox2 (Figures 5C, 5F, 5J, and 5M, blue bars)
binding were enriched at locations previously detected in
ChIP-seq experiments (Figures 5A, 5D, 5H, 5K, blue hash marks)
near the genomic loci of Nanog (Figures 5A–5C, blue bars), Sox2
(Figures 5D–5F, blue bars), Oct4 (Figures 5H–5J, blue bars), and
Brachyury (Figures 5K–5M, blue bars). The correspondence
between the peaks in our ChIP-qPCR measurement and peak
calls in published ChIP-seq data suggested that the ChIP-
qPCR accurately reflects Oct4 and Sox2 binding.
then are differentially regulated during ME and NE lineage selec-
NE differentiation, Oct4 and Sox2 enrichment decreased at the
Nanog promoter (Figures 5B and 5C, red bars for ME and green
bars for NE lineage) and also at the ?3.5 kb ES-specific regula-
tory region of Sox2 (Figures 5E and 5F, red and green bars).
However, a class of regulatory sites became differentially
occupied by Oct4 or Sox2 during lineage choice. In ME progen-
itor cells, Oct4 is induced and Sox2 downregulated (Figure 4). In
these cells, Oct4 binding was enriched at the +3.7 kb regulatory
region of the Sox2 locus (red bars in Figure 5E; Figure 5G,
p = 0.0014), while Sox2 enrichment was uniformly depleted
across the entire Sox2 locus in ME cells (Figure 5F, red bars).
sion in neural progenitor cells (Miyagi et al., 2006; Sikorska et al.,
2008; Tomioka et al., 2002).
In NE progenitor cells, Sox2 is induced while Oct4 is downre-
gulated (Figure 4). In these cells, Sox2 binding was enriched in
the +3.7 kb NE regulatory region of its own enhancer, consistent
with the role of this region in controlling Sox2 expression in NE
cells (Figure 5F, green bar at +3700 bp from Sox2 TSS, p =
0.0019). Sox2 enrichment also increased at the ?4.3 kB region
of the Brachyury promoter (Figure 5M, green bar at ?4250 bp
from Brachyury TSS; Figure 5N, green line, p = 0.0171) where
Sox2 binding was enriched in all three NE biological replicates.
In ME cells, Sox2 enrichment at this locus was detected in
only one of three biological replicates (Figure 5M, red bar
at ?4250 bp from Brachyury TSS; Figure 5N, red line), likely
due to contaminating subpopulations of NE progenitors, and
was not statistically significant (p = 0.37).
In this way, while many ESC-specific binding events are
depleted of Oct4 and Sox2 during differentiation, a fraction of
regions become differentially occupied by either Oct4 or Sox2.
As Oct4 protein levels increase relative to Sox2, Oct4 binding
increases in regions of the genome associated with NE
882 Cell 145, 875–889, June 10, 2011 ª2011 Elsevier Inc.
Distance from Oct4 TSS (bps)
-1kb +1kb-2kb-3kb-4kb -5kb-6kb
-5280-4582-3514-2228-1012 -398 1460
Distance from Nanog TSS (bps)
-5280-4582-3514-2228-1012 -398 1460
Distance from Nanog TSS (bps)
Distance from Oct4 TSS (bps)
-5606 -4578 -3526 -2264 -1010 -500
Distance from Sox2 TSS (bps)
-5606 -4578 -3526 -2264 -1010
Distance from Sox2 TSS (bps)
Distance from Brachyury TSS (bps)
Distance from Brachyury TSS (bps)
Oct4 ChIP in ME cells
Oct4 LocusBrachyury Locus
Nanog LocusSox2 Locus
Distance from Brachyury TSS (bps)
Sox2 ChIP in ME cells
Sox2 ChIP in NE cells
Oct4 ChIPOct4 ChIP
Distance from Sox2 TSS (bps)
Oct4 ChIPOct4 ChIP
Figure 5. Oct4 and Sox2 Bind Asymmetrically in NE and ME Regulatory Regions during Differentiation
(A–N)ChIP-qPCRassaysforOct4 andSox2binding atNanog(A–C),Sox2(D–G),Oct4 (H–J),andBrachyury(K–N)lociinESCs(blue bars),ME cells(red bars),and
NE cells (green bars). Each genomic region is depicted with qPCR primer coordinates (orange hashes) and previously reported Oct4 and Sox2 ESC-specific
binding sites (blue hashes; hash heights are proportional to published ChIP-seq peak heights). Mean fold enrichment is normalized to input, and error bars
represent ± standard error of the mean (SEM) of technical triplicates. The x axis represents positions relative to the transcriptional start site.
(G) ME lineage-specific binding of Oct4 at the Sox2 +3700 bp neural enhancer probed with multiple primer pairs. Each point and error bars represent mean
enrichment values ± SEM for three biological replicates (p = 0.0014).
(N) Lineage-specific binding of Sox2 at the Brachyury ?4250 bp region was probed with multiple primer pairs. Points show mean enrichment values for Sox2
in the NE lineage (green circles) and ME lineage (red circles) over three biological replicates.
Error bars (black for NE lineage, gray ME lineage) represent ± SEM of biological replicates. NE peak at ?4250 bp has p = 0.0171, while ME enrichment has
p = 0.37. See also Figure S5.
Cell 145, 875–889, June 10, 2011 ª2011 Elsevier Inc. 883
differentiation. As Sox2 protein levels increase, Sox2 binding
increases in the Brachyury promoter. Thus, these two regulatory
regions become enriched for either Oct4 or Sox2 during lineage
choice. These results together with our imaging data suggest
that Oct4 and Sox2 might perform lineage specific repression
during differentiation. In such a model, Oct4 would repress the
NE lineage and Sox2 the ME lineage.
Oct4 or Sox2 Perturbation Modulates Cell Fate
To test the functional roles of Oct4 and Sox2 in lineage selection,
we interfered with the levels of these proteins by transfecting
differentiating cells with overexpression plasmids and siRNA
constructs (Figure S5). In these experiments, we drove differen-
tiation into NE or ME progenitor cells at high efficiency by adding
the induction signals retinoic acid or CHIR to ESCs after 48 hr in
N2B27 (see ‘‘ESCs Differentiate into Germ Layer Progenitors
In Vitro’’). We performed the transfection experiments without
selection markers so that cells transfected with a given plasmid
or siRNA occurred next to untransfected cells, and in a single
field of view we could examine perturbed and unperturbed cells
We confirmed that Oct4 inhibits NE differentiation by trans-
fecting cells with a constitutively expressing Oct4 plasmid while
inducing NE differentiation with retinoic acid. Cells with high
Oct4 did not express Sox1, and cells expressing Sox1 had low
Oct4 levels (Figure 6A and 6B), as illustrated by the ‘‘L’’ shape
of the scatter plot (Figure 6A). In contrast, Oct4 overexpression
during mesendodermal differentiation did not block Brachyury
expression (Figure 6C).
We confirmed that Sox2 inhibits ME differentiation by driving
constitutive expression of Sox2 during mesendodermal differen-
tiation driven by CHIR. Cells with high Sox2 expression did not
express Brachyury (Figures 6D and 6E), again highlighted by
Sox2 (Fold Change
Oct4 (Fold Change
Oct4 over-expression, NE differentiation
Oct4 over-expression, ME differentiation
Sox2 over-expression, ME differentiation
Sox2 over-expression, Oct4 siRNA, ME differentiation
Figure 6. Oct4 Specifically Represses the NE Lineage and Sox2 the ME Lineage
(A) Scatter plot of Oct4 versus Sox1 in 2500 cells from a population in which constitutive Oct4 plasmid has been transfected and neural differentiation inducing
signal retinoic acid (1 mM) was added. Oct4 levels are measured as fold change over levels in ESCs under pluripotency conditions.
(B) Images of cells from (A) costained for Oct4, Sox2, Sox1, and DAPI.
(C) Images of cells transfected with a constitutive Oct4 plasmid for 24 hr prior to CHIR-induced ME differentiation costained for Oct4, Brachyury, and DAPI.
(D) Scatter plot of Brachyury versus Sox2 in over 5000 cells transfected with a Sox2 plasmid 24 hr prior to the induction of mesendodermal differentiation with
200 ng/ml Wnt3a (identical results obtained with CHIR, data not shown) costained for Sox2, Brachyury, and DAPI.
(E) Images of cells from (D).
(F) Images of cells transfected with siRNA against Oct4 and a constitutive Sox2 plasmid costained for Oct4, Sox2, Brachyury, and DAPI.
(G) Images of ESCs transfected with a Brn2 plasmid and stained for Brn2 and Oct4 under pluripotency-promoting conditions.
See also Figure S5.
884 Cell 145, 875–889, June 10, 2011 ª2011 Elsevier Inc.
the ‘‘L’’-shaped scatter plot (Figure 6D). Further, cells in which
Sox2 was driven constitutively and Oct4 expression was
abrogated using siRNA did not express Brachyury (Figure 6F),
showing that ME inhibition by Sox2 was independent of Oct4.
Furthermore, Brachyury expression was confined to cells that
retained Oct4 expression suggesting that Oct4 is necessary for
Further, overexpressing Sox2 while abrogating Oct4 expres-
sion under conditions supporting pluripotency led to a fraction
of cells activating the neural ectodermal regulator Sox1 (Fig-
ure S5), suggesting that Sox2 drives NE differentiation.
As another test of relevance,we asked whether forced expres-
sion of terminal lineage markers induces the observed pattern of
identity that is induced in NE cells in our experimental system at
a similar time as Sox1 (Figure S1). Forced Brn2 expression can
convert fibroblasts directly into neurons (Vierbuchen et al.,
2010). We found that constitutive expression of Brn2 in ESCs
regulated Oct4 in these cells, as shown by images in Figure 6G.
These experiments show that Oct4 specifically represses
Sox1 and the NE lineage, and Sox2 specifically represses
Brachyuryand themesendodermal lineage.Thus,the differential
activation of these genes critically regulates cell fate choice.
Differential Regulation of Oct4 and Sox2 Enables
Together, the data presented above lead us to a model of pluri-
potency circuit regulation during differentiation and lineage
choice. In the pluripotent state, positive regulatory interactions
between Oct4, Sox2, Nanog, Klf4, Klf5, and Tbx3 maintain these
proteins at high and correlated levels, preventing differentiation.
We verified positive regulatory interactions between Oct4, Sox2,
and Nanog using siRNA (Figures S5 and S6). Upon withdrawal of
pluripotency promoting factors, LIF and BMP, or with the direct
abrogation of Nanog expression with siRNA, the levels of pluri-
potency factors fall. This fall allows the differentiation signals,
Wnt and retinoic acid or FGF, to differentially regulate elements
of the circuit. The differential regulation competes with the
intrinsic positive interactions between the pluripotency factors.
The ME inducing signals drive Sox2 levels down and Oct4 levels
up, while the NE inducing signals drive Oct4 down and Sox2 up.
Because Oct4 specifically inhibits the NE lineage and Sox2
specifically inhibits the ME lineage, the cells make a lineage
We modeled the competition between the intrinsic positive
regulation of the pluripotency factors and the differential regula-
tion by signals. Model analysis (Extended Experimental Proce-
dures, Figures 7B–7F, and Figures S6C and S6D) revealed that
this competition allows the pluripotency circuit to perform signal
integration, so that one differentiation signal can change the
cell’s interpretation of the other. Specifically, the model pre-
dicted that exposure of the cell to Wnt and retinoic acid simulta-
neously can jam the pluripotency circuit in a high Oct4 and high
Sox2 state, leading to the expression of Nanog (Figure 7F) and
thus preventing the activation of either Brachyury or Sox1.
To test the prediction of our model, we added combinations of
RA and CHIR to differentiating ESCs and quantified the fraction
of cells that activated the germ layer markers Sox1-GFP or
Brachyury by microscopy. Experimentally, we found that RA
changes the cell’s interpretation of the CHIR signal. We added
treated with CHIR between 3–5 mM and 250 nM retinoic acid, we
did not detect either Sox1-GFP or Brachyury staining by micros-
copy (Figure 7G), while cells treated with these concentrations of
CHIR alone would have activated Brachyury at 70% efficiency.
To ascertain the fate of these cells, we stained the same cells
for Oct4, Sox2, and Nanog, and we found that many cells con-
tained levels of these proteins similar to levels observed under
pluripotency promoting conditions (Figure 7H). This implies that
the cells do not enter either the ME or NE fate within the duration
of the experiment. This could be because of delayed lineage
choice or because the cells have entered a distinct state from
which they cannot reach the ME and NE fate. A detailed analysis
of the gene expression pattern, epigenetic state, and potency of
these cells is the subject of future work and will give additional
insight into the state of these jammed cells.
In this work, we asked how ESCs leave the pluripotent state and
select between the ME and NE cell fate. As a cell transitions from
ESC to germ layer progenitor, several hundred genes are down-
regulated. By reconstituting the lineage branch in vitro, we could
classify DNA binding proteins based on their expression pattern
duringlineage selection.Wefoundthatvery fewproteins thatare
present inESCs areregulated in alineage specific fashion during
differentiation providing a substantial simplification of the
complex, underlying transcriptional circuit. By using the under-
lying lineage branch as a guide, we can identify small, crucial
sets of factors that control lineage choice within the complex
regulatory circuits described by genomic methods.
Through this approach, we found that pluripotency mainte-
nance and lineage choice are intricately linked. The pluripotency
circuit is known to act as a unit that strongly represses lineage
specific gene expression in ESCs (Figure 1B). However, rather
than being a monolithic entity, the pluripotency circuit compo-
nents have lineage specific roles, so that the same proteins
can also be used for lineage selection. While the intact pluripo-
tency circuit inhibits all germ layer differentiation, Oct4 specifi-
cally represses only the neural ectodermal fate, while Sox2
specifically represses only the mesendodermal fate. Together,
Oct4 and Sox2 repress differentiation into either germ layer
fate. When these two proteins are differentially regulated leading
to high Oct4 and low Sox2 levels, or low Oct4 and high Sox2
levels, either the mesendodermal fate or the neural ectodermal
of the same proteins that together repress differentiation, a cell
can select a germ layer fate.
Terminal markers of lineage choice, like Sox1 and Brachyury,
have been identified in many progenitor cell populations.
However, such terminal markers only report on the outcome of
to cell fate choice. Oct4 and Sox2 protein levels provide a way to
track the state of the cell continuously from ESC to NE or ME
progenitor cell. By studying proteins that are present and
Cell 145, 875–889, June 10, 2011 ª2011 Elsevier Inc. 885
Neural ectodermal Signal
Neural ectodermal and
Fraction of Cells
Wnt agonist CHIR
Fraction of cells expressing Sox1
Fraction of cells expressing brachyury
Concentration of Wnt agonist alone for
70% of brachyury expression
0.25µM retinoic acid
Neural ectodermal Signal
Oct4 (A.U.)Oct4 (A.U.)
Oct4 (A.U.)Oct4 (A.U.)
Figure 7. The Architecture of the Pluripotency Circuit Enables Signal Integration and Lineage Choice
(A) Model for the coupling between the pluripotency circuit, differentiation signals, and cell fates based upon data from Figures 3, 4 and 5. The asymmetric
inhibition of Oct4 and Sox2 by differentiation signals competes with positive feedback between circuit elements (circular arrow), Oct4, Sox2, Nanog, Klf5, Klf4,
(B–E) Plots of the Oct4 and Sox2 phase space showing the stable fixed points in thepresence of differentcombinations ofdifferentiation signalsas defined by the
mathematical model (Extended Experimental Procedures). In each panel, arrows depict the magnitude and direction of the time rate of change of Oct4 and Sox2
concentration in Oct4 and Sox2 space. Conditions are in the absence of differentiation signals (B), in the presence of NE inducing signals (C), in the presence of
ME inducing signals (D), and in the presence of inducers of both lineages (E).
(F) Plot of steady state Nanog level for combinations of ME- and NE-inducing signals. Nanog is near zero in black regions but is present at ESC levels in yellow
(G) A titration of the Wnt agonist CHIR in cells also treated with 250 nM retinoic acid. Green dots show fraction of cells activating Sox1 and red dots indicate
fraction of cells activating Brachyury.
(H) Images of Sox1-GFP cells treated with 5 mM CHIR and 250 nM retinoic acid immunostained for Brachyury, Oct4, Sox2, and Nanog.
See also Figure S6.
886 Cell 145, 875–889, June 10, 2011 ª2011 Elsevier Inc.
modulated continuously during lineage selection, we gain
access to the detailed dynamics of the process. For example,
sorting cells by their Oct4 or Sox2 levels during the cell fate tran-
sition would enable examination of epigenetic regulation and
transcription factor binding patterns as a function of the cell’s
differentiation status. Having continuous markers of lineage
array of cellular processes and their impact on a cell’s develop-
In many systems, a progenitor cell’s response to differentia-
tion signals depends on the cell’s history, its context, and the
presence of other signals in the environment. For example, in
the developing limb bud, FGF and Wnt signals together retain
progenitor cells in a multipotent state but individually promote
differentiation and cell lineage specification (Hu et al., 2009;
ten Berge et al., 2008a). Our study demonstrates that a
context-dependent interpretation of the signals can arise in
part from the architecture of the transcriptional circuit that
governs the multipotent state. External signals differentially acti-
vate Oct4 and Sox2, so that conflicting signals alter the cell’s
interpretation of one another through their combined influence
on the pluripotency circuit dynamics as seen in Figure 7.
Core circuits of transcription factors also operate in neural
progenitor cells (Briscoe et al., 2000) and hematopoietic progen-
itor cells (Cantor and Orkin, 2001). These circuits might also
play a role in signal integration and cell fate choice. While each
type of progenitor cell might use a different combination of tran-
scription factors to make lineage choices, circuits that make
information from external signals and select a fate (Anderson,
2001). Our study provides a general approach for isolating deci-
sion-making circuits and for relating broad scale circuit structure
to the dynamic behavior of single cells.
ESC Culture and Differentiation Assays
Cells were propagated in feeder-free, serum-free N2B27 media supplemented
with LIF and BMP as described previously (Ying and Smith, 2003; Ying
et al., 2008). ESC media was supplemented with the FGF receptor inhibitor
PD173074 (Sigma, StemGent) at 100 nM to suppress background differentia-
tion. For differentiation toward the neural ectoderm or mesendoderm line-
ages, ESCs were plated into N2B27 for 48 hr, followed by addition of Wnt3a
(200 ng/ml) or CHIR99021 (3 mM) for ME differentiation (ten Berge et al.,
2008b) or RA (500 nM) for NE differentiation (Ying et al., 2003b). For differenti-
ation experiments, cells were immunostained 36 hr after addition of the differ-
Immunofluorescence and FACS
Immunofluorescence and flow cytometry were performed via standard tech-
niques (Extended Experimental Procedures).
Live-Cell Fluorescence Microscopy
mentally enclosed Zeiss Axiovision.
Analysis of Published Microarray and ChIP-Seq Data
were identified from GEO GSE12982 (Shen et al., 2008). Published ChIP-seq
data (Chen et al., 2008; Marson et al., 2008) were analyzed with custom
code in Mathematica (Wolfram) (Extended Experimental Procedures).
Gene Expression Microarray for ES versus 48 hr state
Microarrayswere conducted in triplicates on Illumina Mouse-Ref8BeadChips,
according to the manufacturer’s protocol. Quantile normalization was used in
Overexpression and siRNA Knockdown
Overexpression experiments used CAG promoter-driven Oct4 and Sox2 plas-
mids (Mitsui et al., 2003). siRNA constructs were validated in previous studies
(Hu et al., 2009). Plasmid and/or siRNA transfections were performed 24 hr
before addition of differentiation signals.
Generation of Oct4-mCitrine Transgenic ESC Line
Oct4-mCitrine fusion construct was generated with Red/ET Recombination
(Gene Bridges). The construct consisted of the distal and proximal enhancers
of Oct4, the Oct4 open reading frame, the linker, mCitrine cDNA, the native 30
untranslated region of Oct4, and the loxP-PGK-gb2-neo-loxP selection
cassette (Gene Bridges).
Chromatin Immunoprecipitation and qPCR
Mikkelsen et al. (2007). qPCR was performed on an ABI 7900HT with primers
from ChIP-qPCR tiling arrays (SABiosciences) and custom primer sets
(Operon) (Table S1). Fold enrichment was calculated relative to input.
Mathematical Modeling of Pluripotency Circuit
Simulation of mathematical models was performed using custom written code
in MATLAB (MathWorks) (Extended Experimental Procedures).
(http://www.ncbi.nlm.nih.gov/geo/) under the accession number GSE29005.
Supplemental Information includes Extended Experimental Procedures, six
figures, and one table and can be found with this article online at doi:
We thank Doug Melton, Sean Eddy, Areez Mody, and Alexander Schier for
scientific discussions and Rene Maehr, Masa Yamagata, and Dawen Cai for
discussions and technical assistance. We thank Manfred Baetscher and the
Harvard Genome Modification Facility for assistance with the cell line genera-
tion. We thank Bodo Stern, Nicole Francis, and in particular Sean Eddy and
three anonymous referees for extensive comments on the manuscript. We
Life Science Center (A.M.) for support. Microarray hybridization and measure-
ments were performed by the Molecular Genetics Core Facility at Children’s
Hospital Boston supported by NIH-P50-NS40828, and NIH-P30-HD18655.
M.T. and S.R. conceived the project. M.T. established the experimental
system and performed the immunofluorescence and perturbation experi-
ments. S.J.L. and M.T. built the cell lines and performed microarray experi-
ments. L.N.Z. performed the FACS experiments and established techniques
for long term microscopy of ESCs on glass. M.T. and S.J.L. performed the
time-lapse microscopy experiments. Z.S. and A.M. did the ChIP for Oct4
and Sox2. S.J.L., M.T., and S.R. performed the tiling qPCR experiments.
M.T., S.R., and S.J.L. performed the data and mathematical analyses and
wrote the manuscript.
Received: October 15, 2010
Revised: March 3, 2011
Accepted: May 16, 2011
Published: June 9, 2011
Cell 145, 875–889, June 10, 2011 ª2011 Elsevier Inc. 887
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