Download Ectodermal progenitors derived from epiblast

doi:10.1093/jmcb/mjv030
Published online May 19, 2015
Journal of Molecular Cell Biology (2015), 7(5), 455 – 465 | 455
Article
Ectodermal progenitors derived from epiblast stem
cells by inhibition of Nodal signaling
Lingyu Li1,4,† , Lu Song1,†, Chang Liu1, Jun Chen1, Guangdun Peng1, Ran Wang1, Pingyu Liu1,
Ke Tang2, Janet Rossant3, and Naihe Jing1, *
1
State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences,
Shanghai 200031, China
2
Institute of Life Science, Nanchang University, Nanchang 330031, Jiangxi, China
3
Program in Developmental and Stem Cell Biology, Hospital for Sick Children Research Institute, Toronto, ON M5G 1X8, Canada
4
Present address: Department of Developmental Biology, Stanford University School of Medicine, Stanford, CA 94305, USA
†
These authors contributed equally to this work.
* Correspondence to: Naihe Jing, E-mail: [email protected]
The ectoderm has the capability to generate epidermis and neuroectoderm and plays imperative roles during the early embryonic development. Our recent study uncovered a region with ectodermal progenitor potential in mouse embryo at embryonic day 7.0 and
revealed that Nodal inhibition is essential for its formation. Here, we demonstrate that through brief inhibition of Nodal signaling
in vitro, mouse embryonic stem cell (ESC)-derived epiblast stem cells (ESD-EpiSCs) could be committed to transient ectodermal
progenitor populations, which possess the ability to give rise to neural or epidermal ectoderm in the absence or presence of BMP4,
respectively. Mechanistic studies reveal that BMP4 recruits distinct transcriptional targets in ESD-EpiSCs and ectoderm-like cells.
Furthermore, FGF– Erk signaling may also be alleviated during the generation of ectoderm-like cells. Thus, our data suggest that instructive interactions among several extracellular signals participate in the commitment of ectoderm from ESD-EpiSCs, which shed
new light on the understanding of the formation of ectoderm during the gastrulation in early mouse embryo development.
Keywords: EpiSCs, ectoderm, BMP4, Nodal, FGF
Introduction
Three germ layers, ectoderm, mesoderm, and endoderm, are
generated in the early mouse embryo during gastrulation. As gastrulation initiates, the pluripotent epiblast cells migrate through
the primitive streak to form mesendoderm, which later gives rise
to mesoderm and definitive endoderm. The layer of cells that do
not ingress into the primitive streak is referred as ectoderm (Tam
and Behringer, 1997). By the end of gastrulation, the ectoderm was
restricted to the epidermal and the neural lineages (Carey et al.,
1995). Fate map studies in mouse embryo have revealed that the
anterior/proximal part of the ectodermal layer at embryonic day
7.5 (E7.5) is usually differentiated into epidermal lineage, while
other parts of the anterior ectodermal layer give rise to progenitors
of the central nervous system (Tam, 1989; Tam and Quinlan, 1996).
Single cell lineage tracing experiment demonstrates that neural
ectoderm and surface ectoderm progenitors are localized in the anterior midline ectoderm at the late gastrulation stage (Cajal et al.,
2012). However, whether ectoderm progenitor populations exist
Received October 31, 2014. Revised January 22, 2015. Accepted January 27, 2015.
# The Author (2015). Published by Oxford University Press on behalf of Journal of
Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved.
in mouse embryos has not been fully elucidated. We recently
showed that an anterior/proximal region of the ectodermal layer
in E7.0 mouse embryo contains transient bi-potential ectodermal
progenitor populations, which can be efficiently differentiated
into either epidermis or neural tissue depending on BMP signaling
(Li et al., 2013).
Several studies show that there may be intermediate ectodermal
progenitor cells, such as neuro-ectodermal progenitors or epidermal
progenitors, existing during the differentiation of the pluripotent
stem cells (Kawasaki et al., 2000; Aberdam et al., 2007b; Harvey
et al., 2010). During mouse embryonic stem cell (ESC) neural differentiation, when ESCs were treated with BMP4 and co-cultured with
PA6 stromal cells, the expression of the epidermal marker E-cadherin
but not mesodermal markers is significantly increased (Kawasaki
et al., 2000). When cultured on fixed feeders in serum-free condition, mouse ESCs could be specified to either neural or epidermal
fate depending on the absence or presence of BMP4, respectively
(Aberdam et al., 2007b). Furthermore, a transient ectoderm population is identified during the neural fate commitment of ESC in HepG2
cell-conditioned medium (Harvey et al., 2010). However, the mechanism involved in ectodermal progenitor formation during the
process of pluripotent stem cell differentiation is not clear.
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Epiblast stem cells (EpiSCs) are pluripotent stem cells, which are
derived from the epiblast of postimplantation mouse embryos from
E5.5 to E7.5 (Brons et al., 2007; Tesar et al., 2007; Kojima et al.,
2014). Recently, EpiSC cell lines are established from mouse ESCs
and named ESC-derived epiblast stem cells (ESD-EpiSCs), which
share similar characteristics as EpiSCs (Zhang et al., 2010). In
EpiSCs, Activin/Nodal and bFGF signaling pathways are required
to maintain the undifferentiated state (Greber et al., 2010). Upon
EpiSCs differentiation, Activin/Nodal signaling is necessary to
induce mesendoderm differentiation (Vallier et al., 2009a).
Inhibition of Activin/Nodal signaling by SB431542 (Inman et al.,
2002; Laping et al., 2002) impairs mesendoderm lineage commitment and promotes neural induction (Patani et al., 2009; Vallier
et al., 2009b; Chng et al., 2010). So far, little is known about
whether and how ectodermal progenitor populations could be
derived from EpiSCs.
In this study, we conducted differentiation in chemically defined
medium (CDM), and observed that ESD-EpiSCs treated with a Nodal
inhibitor for a short period could be committed to ectodermal progenitors, which have the potential to generate either neural or epidermal cells. In addition, BMP4 plays distinct roles in EpiSCs and
ectodermal progenitors. The inhibition of FGF signaling also promotes ectodermal differentiation of ESD-EpiSCs, and both Nodal
and FGF signaling pathways participate in the commitment from
ESD-EpiSCs to ectodermal progenitors.
Results
Nodal inhibition promotes the specification from ESD-EpiSCs
to ectoderm lineage
In order to investigate the developmental potential of ESDEpiSCs by Nodal inhibition, ESD-EpiSCs were differentiated under
four conditions, CDM only (Control), CDM with BMP4 (BMP4), CDM
with SB431542 (SB43), and CDM with BMP4 plus SB43 (BMP4/
SB43), for 3 days. Intriguingly, cells cultured under different conditions showed distinct morphologies (Figure 1A). A homogenous
cell population with stratified epidermis-like morphology was
observed in BMP4/SB43 treatment (Figure 1Ad). In addition, realtime quantitative PCR (Q-PCR) showed that the expression of pluripotent markers Oct4 and Fgf5 was sharply reduced in CDM medium
with SB43 (Figure 1Ba). In the absence of Nodal inhibition, BMP4
mainly induced the expression of mesendodermal markers, such
as T, Flk1, Gata4, Gata6, and Sox17 (Figure 1Bb). In the absence
of BMP4, SB43 promoted the expression of neural markers Sox1,
Sox2, Pax6, and MAP2 (Figure 1Bc). The combination of BMP4
and SB43 treatment resulted in high expression of epidermal
markers K8, K18, K5, K14, and K15 (Figure 1Bd) (Moll et al.,
1982; Kirfel et al., 2003; Aberdam et al., 2007a; Troy et al., 2011).
Consistent with the Q-PCR results, immunostaining assay
revealed similar expression profiles of lineage markers assessed
in differentiated ESD-EpiSCs (Figure 1C and D). Compared with the
control, the expression of MAP2 was higher in SB43-treated cells, indicating that the neural differentiation was enhanced (Figure 1Cg
and D). In the presence of BMP4, 40% of cells were positive for
T, a mesendoderm marker (Figure 1Cb and D), and only 10%
were positive for K18, an epidermal marker (Figure 1Cj and D).
Interestingly, 82% of ESD-EpiSCs treated with both SB43 and
BMP4 for 3 days were positive for K18 (Figure 1Cl and D). Taken together, these data suggest that Nodal inhibition probably cooperates with BMP signals to modulate the lineage commitment
from ESD-EpiSCs to either neural or epidermal fate, and repression
of Nodal signaling by SB43 in ESD-EpiSCs might induce an ectodermal progenitor, which could give rise to either epidermal or neural
cells.
To further confirm the existence of an ectodermal progenitor stage
during ESD-EpiSC differentiation, we characterized the lineage
progression of ESD-EpiSCs differentiation for 3 days under four conditions (Control, BMP4, SB43, BMP4/SB43). Q-PCR analyses revealed
that after 24 h of culture, the cell population exhibited features reminiscent of the ectodermal progenitor populations (Supplementary
Figure S1). On Day 1 (D1), in SB43-treated cells (SB43 or BMP4/
SB43), the expression of the pluripotent marker Oct4 was decreased,
and the expression of epiblast markers Fgf5 and Nodal was also
sharply reduced to the basal level (Supplementary Figure S1A).
However, neural markers Sox1, Pax6, and MAP2, as well as epidermal
markers K8, K18, and DNp63 (Aberdam et al., 2007b), could not be
detected in these cells on D1. But after D1, the expression of neural
and epidermal markers was increased gradually (Supplementary
Figure S1B and C). SB43 treatment also suppressed the expression
of mesendoderm markers T, Mixl1, and Gata4 to the ground level
on D1 (Supplementary Figure S1D). These results suggest that cells
treated with Nodal inhibition for 24 h have progressed through the
pluripotent state to a transient intermediate ectodermal progenitor
status, but not been committed to either neural or epidermal fate.
Derivation of the ectoderm lineage by brief inhibition of Nodal
signaling
To investigate the temporal-window of the Nodal inhibition to
derive the transient ectodermal progenitor populations, ESDEpiSCs were cultured in CDM with SB43 for 12 h, 18 h, 24 h, and
30 h, respectively (Figure 2A). After the removal of SB43, cells
were grown in CDM without or with BMP4 till Day 3. Then, total
RNAs were collected and analyzed by Q-PCR assays. The result
showed that in the absence of BMP4, the expression of neural
markers Sox1, Pax6, and MAP2 is dramatically increased in
ESD-EpiSCs treated with SB43 for 18 h or 24 h (Figure 2B). In contrast, in the presence of BMP4, the expression of epidermal
markers K8, K18, DNp63, K5, and K14 (Figure 2C), but not mesendodermal markers Flk1, Sox17, Gata6, Cdx2, Hand1, and Eomes
(Figure 2D), was induced in ESD-EpiSCs treated with SB43 for 18 h
or 24 h. When cells were treated first with SB43 for a shorter
period (12 h) and with BMP4 thereafter, high expression levels of
mesendoderm markers were observed (Figure 2D), suggesting that
the mesendoderm differentiation potential was still reserved in
these cells. Meanwhile, prolonged inhibition of Nodal signaling
(30 h) prior to the BMP induction failed to induce the expression of
epidermal markers to the similar levels as cells cultured with both
BMP4 and SB43 (Figure 2C), suggesting that probably cells treated
by SB43 for 30 h have differentiated beyond the ectodermal progenitor stage. These data demonstrate that an intermediate ectodermal
progenitor population is induced by SB43 treatment for 18 h or 24 h.
Ectodermal progenitors derived from epiblast stem cells
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Figure 1 Nodal inhibition promotes neural and epidermal differentiation in ESD-EpiSCs. (A) The morphology of cells differentiated in four kinds of
medium: CDM without any other factor (Control), CDM with BMP4 (BMP4), CDM with SB43 (SB43), and CDM with BMP4 and SB43 (BMP4/SB43).
BMP4 concentration: 10 ng/ml; SB43 concentration: 10 mM. Scale bar, 200 mm. (B) Q-PCR analysis of marker gene expression in cells differentiated under the four medium conditions. (C) Immunostaining for T, MAP2, and K18 in cells differentiated under the four medium conditions.
Scale bar, 200 mm. (D) Percentage of T+, MAP2+, and K18+ cells after differentiation under the four medium conditions.
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Figure 2 ESD-EpiSCs treated with SB43 for 18 h are in a transient stage similar to the ectodermal progenitor stage. (A) Schematic of the timewindow experiment. Red line indicates the addition of SB43 (10 mM). Blue line indicates the addition of BMP4 (10 ng/ml). Dotted black line indicates control medium without any factor or inhibitor. (B) The expression of neural markers in each group of cells. (C) The expression of epidermal
marker genes in each group of cells. (D) The expression of mesendodermal markers in each group. The red rectangle is used to highlight Q-PCR data
in the 18– 24 h window. (E) Western blot analysis of p-Smad2 and Smad2 in ESD-EpiSCs treated with SB43 (SB) for 3 h, 6 h, 12 h, and 18 h.
Nodal signal is transducted by extracellular ligand binding to the
receptor, then recruits and phosphorylates cytoplasmic effectors
Smad2/3 (Schier, 2003). In order to investigate whether SB43
treatment interfered Nodal signaling cascade, western blot was
conducted to assess the expression of phosphorylated Smad2
(p-Smad2) during the ESD-EpiSC differentiation. Compared with
the control, the expression of p-Smad2 was reduced gradually
with SB43 treatment at 3 h, 6 h, and 12 h, and was almost undetectable at 18 h (Figure 2E). Interestingly, the lowest Nodal activity
indicated by undetectable p-Smad2 level and the induction of the
transient ectodermal progenitor occurred co-incidentally around
18 h after SB43 treatment, suggesting that cells treated with
SB43 for 18 h or 24 h are bi-potency to differentiate to either
neural or epidermal fate.
To validate that the ectoderm specification by Nodal inhibition
was not SB43 specific, A-83-01, another ALK4/5/7 receptors
inhibitor, was used to substitute SB43 (Tojo et al., 2005).
Consistent with the observations in SB43-treated cells, the
Ectodermal progenitors derived from epiblast stem cells
expression of the neural marker MAP2 or the epidermal marker K18
was clearly detected in A-83-01-treated cells without or with BMP4,
respectively (Supplementary Figure S2A). Interestingly, it is the
cells treated with A-83-01 for 15 h or 18 h that display the dual
potency for neural or epidermal fate (Supplementary Figure S2B).
In addition, an epiblast stem cell line generated from early mouse
embryo could also generate the intermediate ectodermal progenitor after SB43 treatment (data not shown). Thus, an ectodermal
progenitor population, named ectoderm-like cells (EctlCs), could
be derived from EpiSCs by transient inhibition of Nodal signaling.
BMP4 activates divergent transcriptional networks in EpiSCs
and EctlCs
Given that EctlCs were different from EpiSCs, their properties were
characterized by whole genome microarrays. The RNA samples were
collected from ESD-EpiSCs, EctlCs, and neuronal and epidermal cells
differentiated from EctlCs, and were analyzed on Agilent Whole
Mouse Genome Oligo 4X44K Microarrays. The global transcriptome
of EctlCs was different from ESD-EpiSCs (Figure 3A), which indicates
that EctlCs have already exited from the pluripotent epiblast state.
Meanwhile, hierarchical clustering analysis also showed the low
similarity in gene expression profiles between EctlCs and differentiated ectodermal cells, including NPCs (neural progenitor cells)
and epidermis (Figure 3A). Based on microarray data, we chose a
few genes that are specifically and highly expressed in EctlCs, and
verified their expression pattern by Q-PCR. Both microarray results
and Q-PCR data revealed that the expression of Eras, Sez6, Stmn3,
and Stmn4 genes was high in EctlCs, compared with ESCs, EpiSCs,
ESD-EpiSCs, NPCs, and epidermis (Figure 3Ba). Furthermore, we isolated 38 and 37 cells from ESD-EpiSC and EctlC population, respectively, for single cell Q-PCR assays. The single cell Q-PCR results
showed that Eras was exclusively highly expressed in EctlCs, while
Stmn3 had a little noise expression in ESD-EpiSCs (Supplementary
Figure S3Ab, e). Moreover, Fgf5 and Nodal were preferentially expressed in ESD-EpiSCs (Supplementary Figure S3Aa, d), whereas
the expression of Sox1, a NPC marker, and K18, an epidermis marker,
was barely detected in either EctlCs or ESD-EpiSCs (Supplementary
Figure S3Ac, f). Next, we assessed the in vivo expression pattern of
Eras and Stmn3 in early mouse embryo by whole-mount in situ hybridization. The Eras was highly and specifically expressed in vivo
at the anterior part of the mouse epiblast at E7.0 (Figure 3Bb), corresponding to the ectoderm stage (Li et al., 2013). However, Stmn3
was expressed in the whole epiblast and extraembryonic ectoderm,
and had no significant region specificity (Supplementary Figure
S3B). These data indicate that compared with Stmn3, Eras is a better
marker for EctlCs. Together, these results suggest that EctlCs could
represent a transient ectoderm population in mouse embryo, and
the differentiation potency is associated with BMP activity.
EpiSCs is derived from egg cylinder epiblast at E5.5, and EctlCs
may be corresponding to the ectoderm stage at E7.0. BMP4
induces mesendodermal differentiation at the late epiblast stage,
but promotes epidermal induction at the ectoderm stage (Li
et al., 2013). In order to investigate the distinct mechanisms of
BMP4 functions at different stages, the activation of BMP signaling
in ESD-EpiSCs and EctlCs was assessed. First, these two types of
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cells were treated with BMP4 for 1 h, 6 h, and 24 h, respectively.
Then, phosphorylated Smad1/5/8 (p-Smad1/5/8), an active
BMP downstream effector, was analyzed by western blot. In both
ESD-EpiSCs and EctlCs, the phosphorylation of Smad1/5/8 was
clearly induced by BMP4 at 1 h and 6 h, but was diminished at
24 h (Figure 3C), indicating that BMP signaling cascade in
ESD-EpiSCs was the same as that in EctlCs.
To distinguish possible mechanisms of BMP4 actions in different
cells, RNAs were prepared from ESD-EpiSCs and EctlCs treated with
BMP4 for 3 h, and were analyzed by microarray assays. The results
showed that 371 and 726 genes were upregulated (≥2-fold) in
ESD-EpiSCs and EctlCs, respectively (Figure 3D). Detailed analysis
demonstrated that only 87 genes were upregulated in both cells,
suggesting that BMP4 regulates distinct groups of downstream
genes in different cells. Gene Ontology term enrichment assays
revealed that the upregulated genes in ESD-EpiSCs were mostly
involved in the mesendoderm differentiation, whereas the upregulated genes in EctlCs were highly associated with the epidermal fate
commitment (Figure 3E).
To further validate microarray data, Q-PCR assay was conducted.
Notably, Mesp1, Flk1, Hoxd1, and Hoxb9 genes, which play crucial
roles in mouse embryo gastrulation and mesoderm differentiation
(Saga et al., 1999; Fehling et al., 2003), were upregulated only in
ESD-EpiSCs after BMP4 treatment (Figure 3F). Krt6b (K6b), Krt13
(K13), Krt83 (K83), and Ovol2 genes, which are essential for epidermis development (Wells et al., 2009), were specifically activated in
EctlCs (Figure 3F). Therefore, in different cells, BMP signaling may
cooperate with distinct cofactors to program the unique cell fate
commitment, such as mesendoderm fate from ESD-EpiSCs and epidermal fate from EctlCs.
Inhibition of FGF – Erk signaling promotes ectodermal
specification
Both Nodal/Activin and bFGF signalings are required for
ESD-EpiSCs maintenance (Brons et al., 2007; Tesar et al., 2007;
Kojima et al., 2014), and transient Nodal inhibition promotes
EctlCs derivation (Figure 1). Then, we asked whether the inhibition
of FGF– Erk signaling could also generate similar effect. ESD-EpiSCs
were treated under four conditions, Control, BMP4, FGF– Erk signaling inhibitor PD173074 (PD17) (Trudel et al., 2004), and BMP4 plus
PD17, for 3 days. Similar to SB43 treatment, distinct cell morphologies were detected, especially that BMP4/PD17-treated cells displayed epidermal morphology (Figure 4A). The gene expression
pattern was analyzed by Q-PCR. Consistently, the expression of
neural markers Sox1, Pax6, and MAP2 was sharply increased in
ESD-EpiSCs treated with PD17 (Figure 4Ba). The expression of epidermal markers K5, K8, K14, K18, and DNp63 was dramatically
enhanced in cells treated with both BMP4 and PD17 (Figure 4Bb).
The expression of mesendodermal markers Flk1, Sox17, and
Gata6 was only induced by BMP4 (Figure 4Bc). Western blot analysis revealed that phosphorylated Erk (p-Erk) was reduced
within 1 h after PD17 treatment (Figure 4C), indicating that FGF–
Erk signaling activity was inhibited. The results show that, similar
to Nodal inhibition, EctlCs could also be derived from ESD-EpiSCs
through the inhibition of FGF– Erk activity.
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Figure 3 Downstream transcriptional networks of BMP4 are different between ESD-EpiSCs and EctlCs. (A) Microarray gene expression heat-map of
ESD-EpiSCs, EctlCs, NPCs, and epidermis. Heat-map colors (red, upregulation; green, downregulation) indicate gene expression in units of standard deviation from the mean across all samples. (B) Genes upregulated only in EctlCs, including Eras, Sez6, Stmn3, and Stmn4 (a). Eras expression
pattern in E7.0 mouse embryo (b). (C) Western blot analysis of p-Smad1/5/8 and Smad1 in ESD-EpiSCs and EctlCs that were treated with BMP4 for
1 h, 6 h, and 24 h. (D) ESD-EpiSCs and EctlCs were treated with BMP4 for 3 h. The same types of cells treated with 4 mM HCl-1‰ BSA (BMP4 solution buffer) were used as control. Comparison of upregulated genes in response to BMP4 is performed between ESD-EpiSCs and EctlCs. (E) GO
analysis of biological processes of the overlap genes in microarray analysis described in D. (F) Gene expression levels in ESD-EpiSCs and EctlCs
without or with BMP treatment for 3 h.
To investigate the possible crosstalk between Nodal/Activin and
FGF signaling pathways during the ectoderm formation, p-Erk
levels were examined in ESD-EpiSCs treated with Nodal inhibitor
SB43 in the absence or presence of BMP4. Indeed, p-Erk level
was reduced in ESD-EpiSC treated with SB43 for 6 h (Figure 5Aa),
suggesting that FGF pathway is modulated by SB43. In contrast,
inhibition of FGF signaling by PD17 had no effects on p-Smad2
levels at 6 h (Figure 5Ab). To further confirm this pathway regulation, we asked whether FGF signaling could interfere Nodal
inhibition-induced ectodermal specification from ESD-EpiSCs. As
expected, the expression of neural markers Sox1, Pax6, and
MAP2 was reduced in ESD-EpiSCs treated with both bFGF and
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Figure 4 Inhibition of FGF – Erk signaling promotes ectoderm commitment in ESD-EpiSCs. (A) The morphology of cells differentiated in four kinds of
medium: CDM without any other factor (Control), CDM with BMP4 (BMP4), CDM with PD173074 (PD17), and CDM with BMP4 and PD173074
(BMP4/PD17). BMP4: 10 ng/ml; PD173074: 50 ng/ml. Scale bar, 500 mm. (B) Marker gene expression in cells differentiated under the four
medium conditions for 3 days. (C) Western blot analysis of phospho-Erk (p-Erk) and Erk in ESD-EpiSCs that were cultured with control medium
(C), BMP4 (B), PD173074 (PD), BMP4 and PD173074 (B/PD) for 1 h, 6 h, and 24 h. BMP4: 10 ng/ml; PD173074: 50 ng/ml.
SB43 (SB43 compared with SB43+bFGF in Figure 5Ba). The expression of epidermal markers K14, K15, and K18 was also decreased in
BMP4/SB43/bFGF-treated cells, compared with BMP4/SB43treated cells (Figure 5Bb). The expression of mesendodermal
markers T, Flk1, and Gata6 was not promoted in BMP4/bFGFtreated cells, compared with BMP4-treated cells (Figure 5Bc). On
the other hand, the expression of neural markers Sox1, Pax6, and
MAP2 was not altered with the addition of Activin (PD17 compared
with PD17+Activin in Figure 5Bd), neither the expression of epidermis markers K14, K15, and K18 (BMP4+PD17 compared with
BMP4+PD17+Activin in Figure 5Be). The expression of mesendodermal markers T, Flk1, and Gata6 was not significantly altered
after BMP4 and Activin treatment (BMP4 compared with
BMP4+Activin in Figure 5Bf). These results suggest that both
Nodal signaling and FGF signaling should be alleviated during ectoderm formation, and Nodal inhibition promotes the ectoderm formation partially through interfering FGF– Erk signaling.
Discussion
It has been known for decades that in vertebrates, ectoderm,
mesoderm, and endoderm, three basic germ layers are generated
during gastrulation. However, until recently, we demonstrate that
the bi-potential ectodermal progenitors are located in the
anterior/proximal part of the ectodermal layer in E7.0 mouse
embryo in vivo (Li et al., 2013). In this study, we characterized an
ectodermal intermediate progenitor population during EpiSCs differentiation by inhibiting Nodal or FGF signaling in vitro. BMP signaling promotes the specification of EpiSCs to mesendoderm
fate, while it programs the commitment of ectodermal progenitors
to epidermal lineage (Figure 6).
Nodal signaling plays an essential role to ensure the appropriate
embryo patterning in vivo, and anterior visceral endoderm (AVE)
generates Nodal antagonists Lefty1 and Cerberus-1 (Cer1) to
prevent the enlarged primitive streaks (Perea-Gomez et al.,
2002). The balance between Nodal activity and its antagonists is
crucial for the anterior – posterior (A – P) patterning of the epiblast
(Thomas and Beddington, 1996; Kimura et al., 2000). In mouse
EpiSCs, Nodal signaling is imperative in mesendodermal differentiation and pluripotency maintenance (Vallier et al., 2009a). We
found that repression of Nodal signaling by SB43 in ESD-EpiSCs
could induce an ectodermal progenitor population, which has
dual potency to generate epidermal or neural cells in a BMP4dependent manner (Figure 1). Furthermore, the transient ectoderm
progenitors could be derived by brief inhibition of Nodal signaling
with SB43 or A-83-01 for 15– 24 h during ESD-EpiSCs differentiation (Figure 2 and Supplementary Figure S2). Clearly, the
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Figure 5 The relationship between Nodal signaling and FGF–Erk signaling during ectoderm commitment from ESD-EpiSCs. (A) Western blot analysis
of phospho-Erk (p-Erk) and Erk (a), phospho-Smad2 (p-Smad2) and Smad2 (b) in ESD-EpiSCs that were cultured with control medium (C), BMP4 (B),
SB43 (SB), BMP4 and SB43 (B/SB), PD173074 (PD), BMP4 and PD173074 (B/PD) for 1 h, 6 h, and 24 h. BMP4: 10 ng/ml; SB43: 10 mM; PD173074:
50 ng/ml. (B) (a–c) Marker gene expression patterns of cells cultured in eight kinds of medium for 3 days: CDM without any other factor (Ctrl), CDM
with BMP4 (BMP4), CDM with SB43 (SB43), CDM with BMP4 and SB43 (BMP4/SB43), and the above four treatments with bFGF. bFGF: 12 ng/ml. (d–f)
Marker gene expression pattern of cells cultured in eight kinds of medium for 3 days: CDM without any other factor (Ctrl), CDM with BMP4 (BMP4),
CDM with PD173074 (PD), CDM with BMP4 and PD173074 (BMP4/PD17), and the above four treatments with Activin. Activin: 20 ng/ml. The values
are represented as mean + SD. **P , 0.01. N.S., non-significant difference between the two groups (P . 0.05).
ectoderm progenitor could be induced from the pluripotent EpiSCs
in vitro.
FGF signaling is important for early embryonic development and
the self-renewal of EpiSCs (Ciruna and Rossant, 2001). In mouse
embryo, Fgf4 and Fgf8, which are expressed in the primitive streak
(Niswander and Martin, 1992; Kinder et al., 1999), induce mesoderm
formation and regulate gastrulation movement. Treatment with both
BMP4 and PD17 increases the proportion of K8/K18-positive
Ectodermal progenitors derived from epiblast stem cells
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Figure 6 The model for ectoderm formation during mouse embryonic development and stem cell differentiation. With low level of Nodal signalling
in the anterior ectoderm, ectodermal progenitor populations are formed. The formation of ectodermal cells can be mimicked by stem cell differentiation process in vitro. ESD-EpiSCs lose their ability to differentiate into mesendodermal cells by inhibition of Nodal signalling. Meanwhile, they
are induced to an ectoderm-like stage. The cells at this stage develop into neurons in the absence of BMP4 and give rise to epidermis in the presence of BMP4.
populations during ESC differentiation (Stavridis et al., 2010), indicating that epidermis development is enhanced by these two
factors. Treatment with PD17 could promote ESD-EpiSCs to
become ectodermal lineages (Figure 4), suggesting that probably
FGF–Erk signaling should be attenuated for ectoderm commitment.
Interestingly, p-Erk level was reduced after blocking Nodal signaling
by SB43 (Figure 5). However, p-Smad2 level was hardly affected by a
FGF signaling inhibitor PD17 (Figure 5). Altogether, FGF signal repression might be one of the downstream processes caused by Nodal inhibition during the ectoderm formation.
Moreover, our microarray and Q-PCR data revealed that Eras,
Sez6, Stmn3, and Stmn4 genes were specifically and highly
expressed in EctlCs derived from pluripotent stem cells in vitro
(Figure 3). Furthermore, single cell Q-PCR results showed that
Eras is a specific marker for EctlCs (Supplementary Figure S3A).
As expected, in situ hybridization assay confirmed that Eras is exclusively expressed in the anterior part of epiblast in E7.0 mouse
embryo in vivo (Figure 3). Thus, the genes identified in the study
could be used as potential ectoderm markers, labeling the transient ectodermal progenitors.
BMP4 signal is imperative for the cell fate commitment, and it
induces mesoderm development in EpiSCs and promotes epidermal
specification in ectoderm. Nevertheless, so far, how to distinguish
downstream targets associated with BMP4 to participate in the specification of different lineages is still largely unclear. Our microarray
data revealed that K6b, K13, K83, and Ovol2 are possible BMP4
effectors to induce epidermis differentiation (Figure 3); meanwhile,
Mesp1, Flk1, Hoxd1, and Hoxb9 genes are BMP downstream mediators to promote mesoderm development. These results indicate that
the same signaling pathway could activate distinct downstream
effectors to program the commitment of different cell fate, depending on various states of the cells, as well as surrounding environments. The cell fate commitment in vivo or in vitro is achieved by
the instructive interactions among multiple extracellular signaling
pathways, as well as the extrinsic and intrinsic regulators. The findings in the study shed new light on the understanding of not only the
formation of ectoderm, but also the generation of all three basic
germ layer, including ectoderm, mesoderm, and endoderm.
Materials and methods
ESD-EpiSCs culture and monolayer differentiation
ESD-EpiSCs were cultured in chemically defined medium (CDM)
supplemented with 20 ng/ml Activin A (R&D systems) and 12 ng/ml
bFGF (Invitrogen) (CDM/AF). Cells were passaged every 3 days
through dissociation with 2 mg/ml collagenase IV (Invitrogen),
plated onto cell culture dishes that were pre-coated with FBS for
24 h at 378C, and then washed twice in PBS before use. CDM contains
50% IMDM (Gibco) and 50% F12 NUT-MIX (Gibco), supplemented
with 7 mg/ml insulin (Roche), 15 mg/ml transferrin (Roche), 450 mM
monothioglycerol (Sigma), and 5 mg/ml BSA (Roche).
For monolayer differentiation, ESD-EpiSCs were cultured in
CDM/AF for 1 day, washed twice in CDM, and then differentiated in
CDM containing different factors or inhibitors, including 10 ng/ml
BMP4 (R&D systems), 10 mM SB431542 (Sigma), 1 mM A-83-01
(Tocris Bioscience), 20 ng/ml Activin, 12 ng/ml bFGF (Pufei), or
50 ng/ml PD173074, for 3 days. To detect the definitive ectoderm
stage, ESD-EpiSCs colonies were fragmented into small pieces after
treatment with 2 mg/ml collagenase IV (Invitrogen). The clumps
were cultured in CDM supplemented with SB43 or A-83-01 for
12– 30 h. After SB43 treatment, cells were washed twice to remove
residual inhibitors, and cultured in CDM supplemented with or
without BMP4 for 2 days.
RNA preparation and Q-PCR analysis
Total RNA was extracted from cells using Trizol reagent (Pufei),
according to the manufacturer’s instructions. Reverse transcription
was performed with 1.0 mg of total RNA using SuperScript III
reverse transcriptase (Invitrogen). Quantitative PCR (Q-PCR) was
464
| Li et al.
performed in accordance with the manufacturer’s instructions,
using Opticon Monitor (Eppendorf). Single cell Q-PCR was performed as described previously (Picelli et al., 2014). Each sample
was analyzed in triplicate, with the Ct values averaged and then normalized to GAPDH control. The primers are listed in Supplementary
Table S1.
Western blot
Immunocytochemistry was performed as described previously
(Sheng et al., 2010). The following antibodies were used: antiphosphorylated Smad (pSmad) 1/5/8 (1:2000; Cell Signaling
Technology), anti-Smad1 (1:2000; Cell Signaling Technology),
anti-phosphorylated Erk1/2 (1:1000; Cell Signaling Technology),
anti-Erk1/2 (1:3000; Santa Cruz).
Immunofluorescence analysis
Immunocytochemistry was performed as described previously
(Gao et al., 2001). The following primary antibodies were used:
mouse monoclonal anti-Cytokeratin 18 (Krt18/K18) (1:150, Abcam),
mouse monoclonal anti-Mtap2/MAP2 (1:200, Sigma), goat polyclonal
anti-T (1:200, R&D systems). Primary antibodies were detected with
Fluorescein isothiocyanate (FITC)- and Cy3-conjugated secondary
antibodies (1:500; Jackson Immunoresearch).
Whole-mount in situ hybridization
Whole-mount in situ hybridizations were performed as described
previously (Zhu et al., 2014). The probes of Eras and Stmn3 were
PCR-amplified from mouse cDNA. The PCR primers are listed in
Supplementary Table S1.
Microarray analysis
The experiments were described in previous study (Zhang et al.,
2010). Agilent Whole Mouse Genome Oligo 4X44K Microarrays (onecolor platform) were used to analyze the gene profile of ESD-EpiSCs,
ectoderm-like cells (EctlCs), ectoderm-like cells differentiated in CDM
for 2 days (NPCs), and ectoderm-like cells differentiated in CDM plus
BMP4 for 2 days (Epidermis). In order to explore the BMP4 transcriptional networks, ESD-EpiSCs were treated with 4 mM HCl-1‰ BSA
(BMP4 solution buffer used as control) or BMP4 for 3 h, and EctlCs
were treated with 4 mM HCl-1‰ BSA (control) or BMP4 for 3 h.
Statistics
Each experiment was performed at least for three times, and similar
results were obtained. The data are presented as mean + SD.
Student’s t-test was used to compare the effects of all treatments.
Statistically significant differences are indicated as *P , 0.05 and
**P , 0.01.
Supplementary material
Supplementary material is available at Journal of Molecular Cell
Biology online.
Acknowledgements
We thank Jodi Garner at ES Cell Facility, Developmental and Stem
Cell Biology, Sickkids, Toronto for providing tissue culture system.
Funding
This work was supported in part by the Strategic Priority Research
Program of the Chinese Academy of Sciences (XDA01010201), the
National Key Basic Research and Development Program of China
(2014CB964804, 2015CB964500), and the National Natural
Science Foundation of China (91219303, 31430058).
Conflict of interest: none declared.
References
Aberdam, D., Gambaro, K., Medawar, A., et al. (2007a). Embryonic stem cells as a
cellular model for neuroectodermal commitment and skin formation.
C. R. Biol. 330, 479– 484.
Aberdam, D., Gambaro, K., Rostagno, P., et al. (2007b). Key role of p63 in
BMP-4-induced epidermal commitment of embryonic stem cells. Cell Cycle
6, 291 – 294.
Brons, I.G., Smithers, L.E., Trotter, M.W., et al. (2007). Derivation of pluripotent
epiblast stem cells from mammalian embryos. Nature 448, 191 – 195.
Cajal, M., Lawson, K.A., Hill, B., et al. (2012). Clonal and molecular analysis of the
prospective anterior neural boundary in the mouse embryo. Development
139, 423– 436.
Carey, F.J., Linney, E.A., and Pedersen, R.A. (1995). Allocation of epiblast cells to
germ layer derivatives during mouse gastrulation as studied with a retroviral
vector. Dev. Genet. 17, 29 –37.
Chng, Z., Teo, A., Pedersen, R.A., et al. (2010). SIP1 mediates cell-fate decisions
between neuroectoderm and mesendoderm in human pluripotent stem cells.
Cell Stem Cell 6, 59 – 70.
Ciruna, B., and Rossant, J. (2001). FGF signaling regulates mesoderm cell fate
specification and morphogenetic movement at the primitive streak. Dev.
Cell 1, 37 –49.
Fehling, H.J., Lacaud, G., Kubo, A., et al. (2003). Tracking mesoderm induction
and its specification to the hemangioblast during embryonic stem cell differentiation. Development 130, 4217 –4227.
Gao, X., Bian, W., Yang, J., et al. (2001). A role of N-cadherin in neuronal differentiation of embryonic carcinoma P19 cells. Biochem. Biophys. Res. Commun.
284, 1098 – 1103.
Greber, B., Wu, G., Bernemann, C., et al. (2010). Conserved and divergent roles of
FGF signaling in mouse epiblast stem cells and human embryonic stem cells.
Cell Stem Cell 6, 215 –226.
Harvey, N.T., Hughes, J.N., Lonic, A., et al. (2010). Response to BMP4 signalling
during ES cell differentiation defines intermediates of the ectoderm lineage.
J. Cell Sci. 123, 1796 – 1804.
Inman, G.J., Nicolas, F.J., Callahan, J.F., et al. (2002). SB-431542 is a potent and
specific inhibitor of transforming growth factor-b superfamily type I activin
receptor-like kinase (ALK) receptors ALK4, ALK5, and ALK7. Mol. Pharmacol.
62, 65 – 74.
Kawasaki, H., Mizuseki, K., Nishikawa, S., et al. (2000). Induction of midbrain
dopaminergic neurons from ES cells by stromal cell-derived inducing activity.
Neuron 28, 31 – 40.
Kimura, C., Yoshinaga, K., Tian, E., et al. (2000). Visceral endoderm mediates
forebrain development by suppressing posteriorizing signals. Dev. Biol.
225, 304 – 321.
Kinder, S.J., Tsang, T.E., Quinlan, G.A., et al. (1999). The orderly allocation
of mesodermal cells to the extraembryonic structures and the anteroposterior axis during gastrulation of the mouse embryo. Development 126,
4691 – 4701.
Kirfel, J., Magin, T.M., and Reichelt, J. (2003). Keratins: a structural scaffold with
emerging functions. Cell. Mol. Life Sci. 60, 56– 71.
Kojima, Y., Kaufman-Francis, K., Studdert, J.B., et al. (2014). The transcriptional
and functional properties of mouse epiblast stem cells resemble the anterior
primitive streak. Cell Stem Cell 14, 107 – 120.
Laping, N.J., Grygielko, E., Mathur, A., et al. (2002). Inhibition of transforming
growth factor (TGF)-b1-induced extracellular matrix with a novel inhibitor of
the TGF-b type I receptor kinase activity: SB-431542. Mol. Pharmacol. 62,
58 – 64.
Ectodermal progenitors derived from epiblast stem cells
Li, L., Liu, C., Biechele, S., et al. (2013). Location of transient ectodermal progenitor potential in mouse development. Development 140, 4533 –4543.
Moll, R., Franke, W.W., Schiller, D.L., et al. (1982). The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells.
Cell 31, 11 – 24.
Niswander, L., and Martin, G.R. (1992). Fgf-4 expression during gastrulation,
myogenesis, limb and tooth development in the mouse. Development 114,
755 – 768.
Patani, R., Compston, A., Puddifoot, C.A., et al. (2009). Activin/Nodal inhibition
alone accelerates highly efficient neural conversion from human embryonic
stem cells and imposes a caudal positional identity. PLoS One 4, e7327.
Perea-Gomez, A., Vella, F.D., Shawlot, W., et al. (2002). Nodal antagonists in the
anterior visceral endoderm prevent the formation of multiple primitive
streaks. Dev. Cell 3, 745 – 756.
Picelli, S., Faridani, O.R., Bjorklund, A.K., et al. (2014). Full-length RNA-seq from
single cells using Smart-seq2. Nat. Protoc. 9, 171 –181.
Saga, Y., Miyagawa-Tomita, S., Takagi, A., et al. (1999). MesP1 is expressed in
the heart precursor cells and required for the formation of a single heart
tube. Development 126, 3437 – 3447.
Schier, A.F. (2003). Nodal signaling in vertebrate development. Annu. Rev. Cell
Dev. Biol. 19, 589 – 621.
Sheng, N., Xie, Z., Wang, C., et al. (2010). Retinoic acid regulates bone morphogenic protein signal duration by promoting the degradation of phosphorylated Smad1. Proc. Natl Acad. Sci. USA 107, 18886 – 18891.
Stavridis, M.P., Collins, B.J., and Storey, K.G. (2010). Retinoic acid orchestrates
fibroblast growth factor signalling to drive embryonic stem cell differentiation.
Development 137, 881 –890.
Tam, P.P. (1989). Regionalisation of the mouse embryonic ectoderm: allocation of
prospective ectodermal tissues during gastrulation. Development 107, 55–67.
Tam, P.P., and Behringer, R.R. (1997). Mouse gastrulation: the formation of a
mammalian body plan. Mech. Dev. 68, 3– 25.
| 465
Tam, P.P., and Quinlan, G.A. (1996). Mapping vertebrate embryos. Curr. Biol. 6,
104 –106.
Tesar, P.J., Chenoweth, J.G., Brook, F.A., et al. (2007). New cell lines from mouse
epiblast share defining features with human embryonic stem cells. Nature
448, 196 –199.
Thomas, P., and Beddington, R. (1996). Anterior primitive endoderm may be responsible for patterning the anterior neural plate in the mouse embryo. Curr.
Biol. 6, 1487 –1496.
Tojo, M., Hamashima, Y., Hanyu, A., et al. (2005). The ALK-5 inhibitor A-83-01
inhibits Smad signaling and epithelial-to-mesenchymal transition by transforming growth factor-b. Cancer Sci. 96, 791 – 800.
Troy, T.C., Arabzadeh, A., and Turksen, K. (2011). Re-assessing K15 as an epidermal stem cell marker. Stem Cell Rev. 7, 927 – 934.
Trudel, S., Ely, S., Farooqi, Y., et al. (2004). Inhibition of fibroblast growth factor
receptor 3 induces differentiation and apoptosis in t(4;14) myeloma. Blood
103, 3521 – 3528.
Vallier, L., Mendjan, S., Brown, S., et al. (2009a). Activin/Nodal signalling maintains pluripotency by controlling Nanog expression. Development 136,
1339 –1349.
Vallier, L., Touboul, T., Chng, Z., et al. (2009b). Early cell fate decisions of human
embryonic stem cells and mouse epiblast stem cells are controlled by the
same signalling pathways. PLoS One 4, e6082.
Wells, J., Lee, B., Cai, A.Q., et al. (2009). Ovol2 suppresses cell cycling and terminal differentiation of keratinocytes by directly repressing c-Myc and Notch1.
J. Biol. Chem. 284, 29125 –29135.
Zhang, K., Li, L., Huang, C., et al. (2010). Distinct functions of BMP4 during different stages of mouse ES cell neural commitment. Development 137,
2095 –2105.
Zhu, Q., Song, L., Peng, G., et al. (2014). The transcription factor Pou3f1 promotes neural fate commitment via activation of neural lineage genes and inhibition of external signaling pathways. ELife 3, e02224.