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From blastocyst to gastrula: gene
regulatory networks of embryonic stem
cells and early mouse embryogenesis
David-Emlyn Parfitt1,2,3,4,† and Michael M. Shen1,2,3,4
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1
Department of Medicine, 2Department of Genetics and Development, 3Department of Urology, and
Department of Systems Biology, Herbert Irving Comprehensive Cancer Center, Columbia University
College of Physicians and Surgeons, New York, NY 10032, USA
4
Review
Cite this article: Parfitt D-E, Shen MM. 2014
From blastocyst to gastrula: gene regulatory
networks of embryonic stem cells and early
mouse embryogenesis. Phil. Trans. R. Soc. B
369: 20130542.
http://dx.doi.org/10.1098/rstb.2013.0542
One contribution of 14 to a Theme Issue
‘From pluripotency to differentiation: laying
the foundations for the body pattern in the
mouse embryo’.
Subject Areas:
developmental biology, systems biology
Keywords:
gene regulatory network, mouse embryo,
stem cells, pluripotency, systems biology
Author for correspondence:
Michael M. Shen
e-mail: [email protected]
To date, many regulatory genes and signalling events coordinating mammalian development from blastocyst to gastrulation stages have been identified
by mutational analyses and reverse-genetic approaches, typically on a geneby-gene basis. More recent studies have applied bioinformatic approaches to
generate regulatory network models of gene interactions on a genome-wide
scale. Such models have provided insights into the gene networks regulating
pluripotency in embryonic and epiblast stem cells, as well as cell-lineage
determination in vivo. Here, we review how regulatory networks constructed
for different stem cell types relate to corresponding networks in vivo and
provide insights into understanding the molecular regulation of the blastocyst– gastrula transition.
1. Introduction
In the mammalian embryo, the transition from blastocyst to gastrula is a remarkably elaborate process involving a complex series of molecular and cellular events.
Many analyses of these developmental stages have typically been limited to
individual genes and/or pathways and their role in specific cellular and morphogenetic events. By contrast, recent studies have focused on dissecting the molecular
interactions that define the ‘regulatory logic’ of cells and tissues on a genome-wide
scale. The definition and validation of such context-specific regulatory networks
are likely to model experimental data more meaningfully than lists of differentially
expressed genes, and place regulatory genes in a larger framework that can provide insights into their function. Below, we highlight studies that shed light on
events taking place during the blastocyst–gastrula transition and have led to the
elucidation of gene regulatory networks (GRNs).
2. Properties of gene regulatory networks
†
Present address: Celmatix Inc., 1 Little West
12th St., New York, NY 10014, USA.
In general, cell types can be defined by expression of specific gene products,
including transcription factors (TFs), signalling molecules and regulatory
RNAs. GRN models describe predicted interactions between these gene products
as context-specific patterns of gene expression and activity. Thus, GRNs can be
considered as formal representations of the route from genomic information to
biological processes.
GRNs are typically visualized with ‘nodes’, representing genes, and ‘edges’
between nodes, representing molecular interactions. Establishment of GRNs for
a specific developmental process requires: (i) knowledge of the TFs and signalling
molecules involved in the process and (ii) inference of the interactions among
these components. For the first requirement, transcriptomic approaches such as
microarray and RNA sequencing analyses have been a common starting point.
These methods generate comprehensive gene expression profiles defining specific
cell populations or developmental time points. GRNs can then be inferred and
generated computationally from diverse experimental data typically provided
by functional perturbation experiments, which vary depending upon the
& 2014 The Author(s) Published by the Royal Society. All rights reserved.
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(a)
2
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FURIN
PACE4
proBMP4
BMP4
ExE
NODAL
CRIPTO
proNODAL
WNT3
epiblast
Phil. Trans. R. Soc. B 369: 20130542
(b)
Lefty
Nodal
VEGF3
Not
animal oral
ectoderm
Gsc
Veg1 lateral
ectoderm
Not
Wnt5A
Veg1 oral
ectoderm
Nk1
VEGF3
Veg input
Nk2.2
Wnt5A
VEGF3
Veg input
VEGFR
PMC
cell migration, spicule formation
Figure 1. GRNs for Nodal signalling in (a) mouse and (b) sea urchin. (a) A GRN that specifies mesendoderm containing a feedback loop between the NODAL
precursor ( proNODAL), NODAL, BMP4 and WNT3, as well as a CRIPTO-dependent feedback loop. Red indicates signals that promote mesendoderm formation,
green, mesoderm formation, and blue, ectoderm formation. (Adapted from [11].) (b) A GRN for Nodal signalling in sea urchin embryogenesis, illustrating its synergistic relationship with Not in ectoderm-specific gene expression. (Adapted from [12].)
experimental system. GRNs for chick and Xenopus embryos, for
example, have been constructed from expression data produced
after morpholino or siRNA silencing [1,2], while network construction in the mouse has benefited from targeted knock-out
lines. Such experiments reveal the impact of loss or gain of
function on downstream targets, which can be used for computational inference of gene interactions, often using probabilistic
graphical models and approaches based on information theory
and linear regression (reviewed in [3]). A key feature of GRNs
generated by such approaches is that they are scalable. Depending on the expression data provided, the resulting GRNs can
provide relatively simple models of tissue-specific interactions
or larger networks describing whole-genome processes. While
these models are typically generated from data that have been
experimentally acquired, it is important to emphasize that
the utility of network identification lies in the generation of
testable hypotheses about genetic relationships that direct and
facilitate subsequent experimental validation.
Although this review will focus on mouse development,
GRNs have provided the first truly global perspectives
of development and regulatory relationships in sea urchin, Drosophila and Caenorhabditis elegans, revealing conserved gene
regulatory mechanisms employed in distinct contexts [4–6].
In particular, the work of Davidson [7,8] has led to the characterization of an extensive regulatory network for sea urchin
development. This GRN represents a hierarchy of regulatory
steps that link components of embryonic development temporally and spatially, and has been used to define the roles of
‘high-ranking’ genes upstream of regulatory pathways, such
as those regulating mesendoderm formation [9].
Indeed, GRNs for development are generally hierarchical
and highly dynamic. Depending on biological context, they
can involve a single genetic input leading to the activation or
repression of a small number of effector genes, or progressive
interactions among multiple layers of regulatory genes, ultimately leading to the activation (or repression) of batteries of
‘differentiation’ genes. Indeed, as most genes probably have
limited pleiotropy [10], many operate within regulatory modules or subcircuits, examples of which include those regulated
by signalling molecules such as NODAL (figure 1) [12]. Such
structures result in high connectivity of particular regulatory
genes, called ‘hubs’, which often constitute candidates for
experimental validation of cis-regulatory relationships. The
dynamic features of development necessitate flexibility in
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Until recently, efforts to generate regulatory networks for
mammalian development in vivo have been relatively limited,
perhaps due to the small size and relative inaccessibility of
the embryo. These limitations have been at least partially overcome through the analysis of stem cells in culture, which have
served as paradigms for in vivo processes.
In particular, networks for the pluripotency and selfrenewal capacity of embryonic stem cells (ESCs), derived
from the inner cell mass (ICM) of the blastocyst, have been
widely studied [16,17]. Thus, gene targeting experiments
have established OCT4, NANOG and SOX2 as key TFs that
regulate pluripotency in vivo and in vitro [18 –20], while interactions among these TFs, their regulatory elements, and coregulated target genes have been proposed to constitute a
core transcriptional network for pluripotency [21 –24]. Similarly, networks have been constructed for epiblast stem cells
(EpiSCs) that are derived from the postimplantation epiblast
(Epi) [25,26]. Recent analyses have also included other factors
in the regulatory landscape of pluripotency. For example,
ESRRB, SALL4, TBX3, KLF4, KLF2 and REST have joined
the ranks of TFs constituting the ‘pluripotency network’
[21,27–31]. Moreover, non-coding RNAs such as miR-134,
miR-296 and miR-470 have been shown to directly regulate
Oct4, Nanog and Sox2 [32], while epigenetic modifiers such as
PRDM14 and WDR5 also display overlapping regulatory functions with the core pluripotency factors [33,34]. Although
understanding how these molecules are functionally integrated
represents a complex task, iterations of regulatory networks
have been generated on transcriptional [21,24,30,35] and posttranslational levels [36,37], while other studies have integrated
data from multiple regulatory levels [38,39].
Several features of these networks suggest how they might
operate to establish and/or maintain pluripotency. Firstly, and
perhaps unsurprisingly, they are enriched for genes involved
in regulation of the ICM or aspects of embryonic lineagespecific differentiation. Secondly, many genes are co-regulated
and are often downregulated during ESC differentiation,
suggesting their involvement in common cellular functions or
pathways. Thirdly, multiple interactions among genes within
these networks suggest that they affect a mutual function
and that a balance between these interactions is important for
4. Regulatory networks for pluripotency and
lineage commitment in the mouse embryo
During preimplantation mouse development, successive cleavages of the zygote lead to formation of the blastocyst,
containing the ICM and blastocyst cavity surrounded by the
trophectoderm (TE). Subsequently, the primitive endoderm
(PrE) becomes apparent at the surface of the ICM in contact
with the blastocyst cavity, with the remaining ICM forming
the Epi. After implantation into the maternal uterus, continued
cell divisions occur in concert with the morphogenetic changes
associated with gastrulation. While pluripotency is maintained
in cells of the Epi, it is gradually lost in descendants of the TE
and PrE, and eventually elsewhere during specification of the
primary germ layers.
(a) The gene regulatory network of the blastocyst
It has been long been established that Oct4, Nanog and Sox2
represent three genes that are critical to the maintenance of
pluripotency in vitro and to normal ICM development
in vivo [18 –20,59 –62]. SALL4 shares a similar role, as it
binds to the conserved regulatory region in the distal
3
Phil. Trans. R. Soc. B 369: 20130542
3. Using stem cells to model regulatory
networks for mammalian development
maintaining pluripotency. This view is consistent with
dosage-dependent effects for each of the core pluripotency factors [40–42], as well as significant intercellular differences in
their expression levels in ESCs and in vivo [43–46]. Moreover,
the broad range of genes present in most ESC regulatory networks implies their functional subdivision into sets of targets
regulated by different regulatory genes and/or complexes.
Thus, the control of target genes and signalling pathways in
the context of pluripotency is more likely to be combinatorial
than strictly hierarchical and represents a state of dynamic, as
opposed to constant, equilibrium so that ESCs are kept in an
undifferentiated state and retain the potential to undergo
multi-lineage differentiation.
Classically, pluripotency has been regarded as a ‘ground
state’ that is regulated by a TF network that inhibits differentiation, while the activation of one or more lineage-specifying
factors can trigger differentiation [47,48]. The interpretation
that the ground state is intrinsically stable was based on
observations that ESC pluripotency is maintained in culture
conditions that emulate the absence of ‘extrinsic instruction’
(figure 2a) [50,51]. However, this view seems to be at odds
with the idea that the ground state is actively maintained.
For example, the core pluripotency factors alone are tightly
auto-regulated and co-occupy the promoters of thousands
of targets [52].
As an alternative, a new ‘balance’ model of pluripotency
[49,53] proposes that pluripotency represents a state of transcriptional competition among regulatory factors that can
themselves act as lineage-specifiers in the proper context
(figure 2b) [40,54,55]. Thus, ESCs are poised at the cusp of
multiple opposing lineage commitment decisions, expressing
many TFs required to react to differentiation cues, such as
Brachyury, Hes1 and Eomes [56 –58]. Given these alternative
models for regulating the pluripotent state, it is important
to determine which GRNs more accurately reflect how pluripotency is maintained, altered and ultimately lost during
lineage-specific differentiation in stem cells in culture, as
well as in the embryo.
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GRNs, facilitated by gene redundancy and features such as
positive and negative feedback and feed-forward loops. Such
feedback loops may control transcriptional noise, as well as
the stability of transcriptional networks and linear dose–
response relationships [13]. These features are particularly
relevant in the context of signalling gradients that regulate
many aspects of early embryo development.
The elucidation of GRNs in mammalian species represents
a significant challenge, because the complexity of genome
organization and gene regulation tends to increase with the
number and diversity of cells. Despite these challenges, systems approaches should be valuable for understanding
mechanisms of cell fate determination in vertebrate systems.
For example, regulatory networks have been defined for
neural crest cell induction [14] and mesoderm specification
[15] in Xenopus, and similar networks are being constructed
for mammalian development.
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(a)
(b)
GSK3 inhibitor
4
Oct4
Sox2
Sall4
Nanog
Esrrb
Tbx3
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Wnt/b-catenin
signalling
Sall4
trophectoderm
MAPK inhibitor
endoderm
mesoderm
ectoderm
Nanog
Oct4
Klf4
lineage
commitment
Sox2
Esrrb
Tbx3
Figure 2. (a) The ‘classic’ model of pluripotency regulation proposes that a ground state is actively maintained by a core set of TFs in the absence of differentiation
signals, created by culture conditions involving inhibition of GSK3 and MAPK signalling. (b) The ‘balance’ model of pluripotency proposes that competition among
lineage-specifying TFs regulates pluripotency. Stimulatory (blue) and inhibitory (red) signals from each of the TFs confer multi-lineage differentiation potential, and
do not result in commitment to any particular lineage. (Adapted from [49].)
FGF signalling
c-Fos
Fgf4
Sox2
LIF
Esrrb
Jak/Stat3
Nanog
Oct4
IL-6
Sall4
Rex1
MAPK signalling
Zfp322a
Egr1a
Figure 3. A GRN for pluripotency in the blastocyst, constructed manually using recently published data from in vivo and in vitro studies [50,63 – 69]. The network
illustrates how LIF signalling components, MAPK signalling regulators and TFs such as Esrrb, Sall4 and c-Fos influence the dynamics of the core pluripotency network
in the blastocyst.
enhancer of Oct4 and activates transcription [63]; accordingly,
Sall4 null embryos display decreased Oct4, Sox2 and Nanog
expression [64]. Similarly, Stat3 regulates pluripotency in the
ICM via the OCT4–NANOG circuitry and is dependent on
leukaemia inhibitory factor (LIF) and interleukin (IL)-6 [65].
Such findings can be used to infer a simple network showing
how these factors regulate the core circuitry for pluripotency
(figure 3). Additional genes have been implicated in this network
by their ICM-specific expression and functional characterization
in ESCs. Thus, c-FOS is specifically expressed in the ICM in vivo
[66] and binds to the Nanog promoter in ESCs until its
expression is downregulated upon differentiation [67,68]. Similarly, Zfp322a and Egr1 are expressed specifically in the ICM
[43] and are thought to regulate pluripotency by suppressing
MAPK signalling [69].
However, the roles for several other genes in regulating pluripotency in vivo appear less clear. For example, Klf5 is expressed
ubiquitously in the preimplantation mouse embryo [70] and is
not only required for the normal transcription of Oct4, Sox2
and Nanog in the ICM, but also of Cdx2 in the TE [71,72].
Phil. Trans. R. Soc. B 369: 20130542
pluripotent
self-renewal
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(b) Regulatory networks governing the first cell
fate choices
Targeted deletion experiments in embryos and trophoblast
stem cells have together provided relevant data for inference
of a TE regulatory network that includes genes such as Cdx2,
Tead4, Eomes, Id2, Elf5, Tcfap2c, Esrrb, Sox2, Nanog and Oct4
(figure 4a) [20,73,91–95]). Reciprocal inhibition between Oct4,
Nanog and Cdx2 is perhaps the most central element of this network, as CDX2 and OCT4 form a functional repressor complex
in the early embryo [96], while Cdx2 is required for downregulation of Oct4 and Nanog in the TE, resulting in restriction of
their expression to the ICM [92]. Thus, TE and ICM specification is contingent upon regionalized alterations in the
balance of these proteins. One modulator of this balance is
the activity of the Hippo signalling pathway. Specifically, cell
polarity and positioning cues regulate the sub-cellular localization of LATS1/2, YAP, ANGIOMOTIN and NF2/MERLIN,
which in turn regulate YAP/TEAD4-mediated expression of
Cdx2, which becomes restricted to cells of the TE [94,97–100].
Other signalling events regulate maintenance of the
TE fate as the embryo transitions through peri- and early
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Phil. Trans. R. Soc. B 369: 20130542
in ICM cells than in ESCs [43]. While the high expression
levels of the miR-290 –295 cluster in both these different cell
types might suggest this cluster to be an exception, blastocyst
development is nonetheless normal in its absence [88]. Thus,
miRNAs might be functional yet dispensable in the establishment of pluripotency in vivo, a conclusion supported by other
recent experiments [89].
Indeed, these and other data [45] draw attention to fundamental differences between ESC and preimplantation biology.
Such differences may complicate comparisons between GRNs
for ESCs and the preimplantation embryo, in part perhaps
because embryo GRNs operate under strict time and spatial
constraints. Moreover, the de novo construction of preimplantation regulatory networks may be complicated by maternally
provided transcripts and proteins, which contribute to zygotic
genome activation and initial establishment of embryonic cell
lineages (reviewed in [90]). Moreover, the observation that
several pluripotency regulators appear to be dispensable for
ICM development in vivo might reflect functionally redundant
mechanisms within gene families or for genes within the same
regulatory subcircuit. Indeed, such redundancy may facilitate
the highly regulative nature of mammalian development, a
property not generally applicable to ESCs in culture. Furthermore, reciprocal signals between the ICM and TE may also
regulate their specification, and consequently networks constructed using whole blastocysts may miss features of networks
specific to these lineages.
Despite these limitations, our knowledge of the transcriptional architecture of the blastocyst has nonetheless been
extended by direct molecular analyses. Though correlative,
the quantitative transcriptional analysis of ICM cells as they
adopt properties of ESCs suggests that the regulatory network
of pluripotency in the ICM operates in parallel with the developmental programme driving early differentiation events, in
contrast to ESCs, where the developmental programme is
silenced in favour of unrestricted self-renewal [43]. These
findings raise the question of whether factors regulating pluripotent cells in the ICM and Epi also function during lineage
specification. This issue has been addressed in recent studies
of TE and PrE specification, as discussed below.
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Although Klf5 null embryos arrest and die at the blastocyst stage,
it is unclear whether this is due to defective establishment of TE
or ICM, or to a more general role for Klf5 in the cellular machinery of the early embryo. Conversely, the expression of other
pluripotency factors seems to be dispensable for early development. Notably, Esrrb null embryos survive until 10.5 days post
coitum (dpc) and die due to placental abnormalities [73]. Similarly, while Dppa4, Dppa5 and Utf1 are specifically expressed in
pluripotent cells in vivo and in culture, the impact of their absence
is only observed during late embryonic stages, though redundancy among these genes has not yet been investigated [74–76].
Given their common components, the GRNs modelling
ESC biology and preimplantation embryo development are
likely to be similar, at least to some extent. However, most
likely due to lack of accessibility of experimental material,
there have been few studies to date that have pursued the
de novo construction of regulatory networks for pre- and
postimplantation development in vivo. One example is the
description of two interlinked feedback loops regulated by
the NODAL precursor during mesendoderm specification
in the mouse (figure 1a) [11,77]. However, the utility of techniques such as single-cell RNA sequencing has made
quantitation of preimplantation transcriptomes more feasible.
For example, a comparative analysis of ESC and ICM transcriptomes revealed that a number of genes are positively correlated
with pluripotency in both contexts, including E-cadherin, Pim1,
Fzd9 and Dazl [43]. This study also identified altered gene
expression patterns during ICM outgrowth in culture, providing insights into differences between the gene networks
regulating ESCs and the ICM. For example, Nodal, Lin28 and
ERas are significantly upregulated during ICM outgrowth,
which is interesting as these genes are positive regulators of
self-renewal but are dispensable for pluripotency in ESCs,
suggesting specific roles in the embryo [78–80]. Notably,
genes involved in the development of TE, PrE and mesendoderm such as Cdx2, Gata6 and Tbx3 are downregulated during
ICM outgrowth in vitro [43].
Further efforts to define a regulatory network for the
blastocyst have analysed transcriptional profiles of preimplantation embryos in the context of reference protein
interaction networks [81], resulting in a network centred on
interactions between OCT4, SOX2, NANOG, SALL4 and
ILF2. The role of ILF2 during development is unclear, though
interestingly it is associated with active chromatin in numerous
embryonic tissues [82] and is known to interact with OCT4
and NANOG proteins in mouse ESCs [83]. Global transcriptome analysis of the embryo in the context of Oct4, Sall4 and
Nanog depletion have revealed that these factors act as combinatorial regulators of developmentally important genes at
preimplantation stages [84], including pluripotency factors
such as Klf5 and Klf2, the epigenetic modifiers Dnmt3a and
Dnmt3b, and the signalling molecules Fgf2, Fzd4 and Fzd7, as
well as miRNAs with enhancers occupied by OCT4, SALL4
or NANOG in ESCs [85,86]. These data suggest that a feedforward loop consisting of pluripotency factors, miRNAs,
Rbl2 and Dnmt3b regulates DNA methylation in the developing embryo and buffers fluctuations in the expression of
regulatory genes that might otherwise disrupt normal cell
fate specification [84].
Interestingly, however, a number of studies indicate
that miRNAs are largely dispensable for preimplantation
development (reviewed by Greve et al. [87]). Indeed, the
expression of several miRNA families is significantly lower
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Nanog
(a)
6
Tcfap2c
Gata3
Lats
Amot
Nff
Yap
Hippo
signalling
Tead4
Cdx2
Psx1
Eomes
Hand1
Elf5
Sox2
Esrrb
FGF
signalling
Gata6
Nanog
Fgf4
Fgfr2
ERK/MAPK
signalling
Nanog
Oct4
Sox7
Oct4
epiblast
primitive
endoderm
Sox17
Gata4
Pdgfra
Figure 4. GRNs for lineage commitment during preimplantation development. (a) A network of gene interactions involved in TE specification, centred on mutual
inhibition between Cdx2, Oct4 and Nanog. Hippo signalling components mediate positional cues in the regulation of Tead4 and Cdx2 expression, which in turn
regulate the expression of TE-specific genes such as Gata3, Psx1, Hand1, Eomes and Elf5. Concurrently, FGF signalling is thought to regulate a sub-network involving
Tcfap2c, Sox2 and Esrrb that inhibits Oct4 expression while stimulating Cdx2. (b) Interactions among genes involved in PrE specification. FGF4 signalling from the Epi
stimulates FGFR2-mediated ERK/MAPK signalling in cells of the nascent PrE, resulting in upregulation of Gata6 and Oct4, and inhibition of Nanog. Feedback loops
between Nanog, Gata6 and Fgfr2 enforce this dynamic, while Oct4 is thought to be upstream of PrE-specific genes such as Sox7, Sox17, Gata4 and Pdgfra.
postimplantation stages. In the ICM, Oct4 and Sox2 together
regulate the expression of Fibroblast growth factor 4 (Fgf4)
[101]), which in turn regulates cell proliferation via FGFR2mediated ERK/MAPK signalling. Indeed, targeted disruption
of ERK/MAPK signalling results in attenuated expression
of Cdx2 and Eomes, and peri-implantation lethality due to
impairment of trophoblast outgrowth [102]. In addition, Sox2
expression is essential for trophoblast maintenance in vivo
[20] and is likely to be a downstream target of FGF signalling,
forming a regulatory circuit with Esrrb and Tcfap2c that
promotes Cdx2 expression while inhibiting Oct4 [55].
Similarly, the network for PrE formation includes Oct4,
Nanog, Gata6 and Fgf4 (figure 4b). Fgf4 expression becomes
restricted to cells of the Epi by the time it becomes distinct
from the PrE [44,46]. FGF4 secretion by Epi cells regulates the
expression of Gata6, Gata4, Sox17 and Sox7 in the PrE, via
FGFR2, the GRB2–MAPK pathway and interactions with
GATA6 itself [103–105]. Accordingly, disruption of FGF4 signalling results in failure of PrE formation [106,107]. Indeed,
differential levels of FGF4 and FGFR2 between cells of the
ICM are essential for the dynamics of PrE/Epi formation
[46,107–109], leading to the mutually exclusive expression of
Nanog and Gata6 in the Epi and PrE, respectively [104]. In the
absence of Nanog, Epi formation is specifically impaired [19]
and Fgf4 expression is downregulated [110]. However, Sox17
and Gata4, which are markers of an established PrE identity,
are also absent in Nanog mutant embryos [61,110], suggesting
that Fgf4 expression is downstream of Nanog and is intrinsically
linked to Epi identity during specification of the PrE lineage.
Thus, Nanog is believed to have a non-cell-autonomous role
in PrE formation through this circuitry [61,110].
In some respects, Oct4 and Sox2 could play similar roles to
Nanog, as they are both required for Fgf4 expression [101]. However, unlike Sox2, Oct4 expression does not become restricted
to the Epi after implantation [20,111]. Recent studies have
demonstrated that Oct4 is required for the maintenance of
PrE-specific gene expression, which is disrupted in the absence
of zygotic OCT4 protein [59,60]. Notably, this requirement
for Oct4 cannot be rescued by exogenous FGF, demonstrating that Oct4 acts downstream of FGF signalling and plays a
cell-autonomous as well as non-cell-autonomous role in PrE
specification [59,60]. In addition, genome-wide expression
analysis of Oct4 mutant embryos demonstrates that Gata4 and
Sox17 expression are Oct4-dependent [59]. Interestingly, PrE
specification appears normal in the absence of either Gata4 or
Sox17 [112,113], perhaps reflecting functional redundancy
among PrE genes and their parallel regulation by Oct4.
Together, these studies demonstrate two central points.
First, they support the notion that the divergence of TE and
PrE involves reciprocal genetic changes within GRNs
shared, at least in part, with the ICM and Epi. These changes
ultimately derive from dynamic and reinforcing cellular interactions, in which cell polarity and positional cues appear to
trigger events separating TE from ICM, while cell division
orientation, migration and apoptosis drive the divergence
of PrE and Epi [114–116]. Secondly, these studies provide
evidence that pluripotency factors function in networks regulating lineage-specific differentiation in the embryo. Thus,
networks of genes containing at least some components
common to each developmental pathway drive pluripotency
and early lineage specification (figures 3 and 4). Notably,
switches in the function of these common components are
Phil. Trans. R. Soc. B 369: 20130542
(b)
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Oct4
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(c) Dynamics of the pluripotency network during
epiblast priming and lineage specification
7
Phil. Trans. R. Soc. B 369: 20130542
At the onset of gastrulation, the Epi undergoes regionalized
lineage specification, beginning with the specification of mesendoderm in the primitive streak posteriorly and embryonic
ectoderm in the anterior Epi. Although fate determination is
ultimately concomitant with a complete loss of pluripotency,
recent studies demonstrate that it is also contingent on dynamic
alterations of the regulatory network for pluripotency in the
Epi prior to its loss. However, analysis of these changes as
they occur has been hindered by embryo inaccessibility at
peri-implantation stages.
Consequently, the comparative study of ESCs and EpiSCs
has proved fruitful, as these stem cell types recapitulate many
features of the tissues from which they are derived. The
distinct signalling pathways that regulate pluripotency and
self-renewal in mouse ESCs and EpiSCs [117] are reflected
in non-identical TF binding and gene expression patterns
[118,119]. In particular, ESCs and EpiSCs define ‘naive’
and ‘primed’ states of pluripotency [120], which also exist in
vivo. While expression of the core pluripotency genes Oct4,
Sox2 and Nanog are associated with both naive and primed pluripotent states in the ICM and Epi [121,122], naive state markers
such as Rex1, Klf4, Klf2, Esrrb, Tbx3 and Stella are downregulated
after implantation in vivo [121,122]. Similarly, lineage specification markers such as Fgf5, Cer1 and T are expressed in the Epi
and in EpiSCs, but are absent in the ICM and ESCs [121,122].
Thus, it seems likely that similar batteries of genes are involved
in the transition between pluripotent states in vivo as in vitro.
Moreover, the primed pluripotent state could be relatively
stable throughout Epi development, because EpiSCs derived
from pre-gastrulation embryos of different stages display
similar global gene expression profiles [123].
Changes to the expression of core pluripotency genes
appear to have a significant role in the transition from a
naive to primed pluripotent state in vivo. Though its expression
is detected postimplantation at approximately 6.5 dpc, Nanog
expression is downregulated in the Epi during implantation
under direct repression by Tcf15 [124,125]. Nanog plays a central role in the regulation of naive pluripotency, directly
interacting with genes such as Esrrb, Sall4 and Tbx3 [126,127].
Reflecting this role, ESCs expressing Nanog at relatively low
levels are more prone to differentiation [128], while its overexpression in EpiSCs promotes their conversion to ESCs [62],
suggesting that downregulation of Nanog expression in the
Epi is important to establish or maintain the primed pluripotent state. A similar scenario for Oct4 seems plausible since
its expression in EpiSCs is typically lower than in ESCs or the
ICM [121]. EpiSCs expressing high levels of Oct4 in culture
display atypically high expression of naive markers such as
Esrrb, Klf4 and Rex1, and are able to contribute to blastocyst
chimaeras in vivo [121]. Therefore, the reduction in Oct4
expression that occurs upon implantation [124] could have a
significant impact on the nature of pluripotency in the Epi.
Studies conducted in vivo and in vitro have suggested
how other components could contribute to the regulatory
network of primed Epi. For example, regulatory models constructed from transcriptional signatures of EpiSCs in the
context of knock-down and overexpression experiments
have highlighted the pivotal roles of Zic2/Zic3 and Otx2 in
supporting the expression of Epi markers such as Fgf5,
while restricting Oct4 expression levels to those permissive
of the primed state [25]. Observations in the embryo support the role of Otx2 in this network [129,130]. However,
efforts to validate the role of Zic2/Zic3 in vivo have been complicated by their functional overlap and the lack of a Zic2 null
allele; interestingly, the most severe phenotype reported for
Zic3 mutant embryos corresponds to neural plate defects,
which could be linked to an earlier failure of Epi specification
[131]. In addition, Tcf7l1 (Tcf3) has been characterized as an
inhibitor of self-renewal and forms an auto-regulatory loop
with core pluripotency factors by co-regulation of several
targets [132]. In vivo and in vitro, Tcf3 expression is modulated
by canonical Wnt/b-catenin signalling as well as inhibition of
Gsk3 [133,134]. Thus, in the absence of Wnt signalling at periimplantation stages, Tcf3-mediated priming of the Epi can
occur through its connection with the core pluripotency circuitry [135]. Finally, the transcription factor TFE3 is thought
to promote a naive pluripotent state in ESCs through direct
transcriptional control of Esrrb, as well as the cis-regulatory
elements of Klf2 and Tbx3 [136]. In the postimplantation
embryo, Tfe3 becomes progressively restricted to the cytoplasm in the Epi, which is mediated by FOLLICULIN and
its binding partners FNIP1/FNIP2, thereby precluding the
transcriptional activity of TFE3 and promoting exit from the
naive state [136].
Subsequent to Epi priming, a web of regionalized signalling activities initiates lineage-specific differentiation [137].
These activities include canonical Wnt/b-catenin signalling,
which is essential for primitive streak formation and mesendoderm specification, while a downstream Tcf3-mediated circuit
leads to regionalized expression of several core pluripotency
factors [138,139]. Specifically, although it is expressed throughout the Epi at 6.5 dpc, Sox2 becomes anteriorly restricted at
7.5 dpc [20], while re-expression of Nanog at 5.5 dpc is specific
to the posterior Epi [124]. Lineage-specific gene expression is
associated with specific expression levels of core pluripotency
factors in differentiating ESCs, suggesting functional consequences for these dynamic changes [54,140]. For example,
high levels of Sox2 are associated with neuroectoderm specification, marked by Sox1, while mesendoderm specification is
associated with high levels of Oct4 and Nanog and expression
of Brachyury (T ). Similar associations are observed when
these lineages are induced by exogenous factors in culture
[54]. A network containing these genes, along with other
mesendoderm and neuroectoderm factors (FOXA2 and
BRN2, respectively) and the epigenetic regulators JARID2
and SUZ12 has been inferred from ESC ChIP-seq data
[54,85,141] and describes how expression dynamics of core
pluripotency factors may influence lineage commitment in
the gastrula (figure 5).
Such a pre-gastrulation Epi GRN may also explain how the
mutually exclusive expression of Sox2 and Nanog [20,124]
might initiate lineage-specific gene expression during emergence of the primary germ layers. Somewhat paradoxically,
Oct4 expression persists throughout the Epi until its elimination during somitogenesis, though this elimination begins in
the anterior Epi [124]. However, this observation is concordant
with the findings that Wnt/b-catenin-mediated primitive
rstb.royalsocietypublishing.org
mediated, at least in part, by cell-extrinsic signals that
ultimately trigger lineage specification. Further evidence
supporting this scenario has been provided by studies focusing on changes in the Epi during the transition towards
gastrulation, as described below.
Downloaded from http://rstb.royalsocietypublishing.org/ on June 15, 2017
8
SOX2
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OCT4
NANOG
JARID2
JMJD2
T
Brn2
Foxa2
neuroectoderm
mesoderm
endoderm
Sox1
Figure 5. A GRN for lineage commitment in the mouse gastrula. Interactions were inferred from published data [54,58,85,141] among the core pluripotency factors
OCT4, NANOG and SOX2, the epigenetic regulators JARID2, SUZ12 and JMJD2 and lineage-specifying factors Sox1 and Brn2 (for neuroectoderm) and Foxa2 and T (for
mesendoderm) and Eomes (for endoderm). The direction of the inferred interactions is not fully understood, but such a network suggests how localized changes in
the expression of the core pluripotency circuit might influence lineage commitment events. (Adapted from [54].)
streak formation is dependent on Nanog and that the association of Oct4 levels with specific lineages in differentiating
ESCs is weaker than that for Nanog [140]. Indeed, in the
embryo, high levels of Oct4 are required for the specification
of all three primary lineages [142].
Together, these results provide evidence that lineage specification at the onset of gastrulation involves changes to the core
pluripotency GRN, which in turn influences expression of
downstream lineage-specifying genes. Wnt/b-catenin signalling is undoubtedly central for these alterations, given the
role of this pathway during the onset of gastrulation. Indeed,
Wnt-mediated expression of pluripotency factors such as
Snail1 and Tcf3 is known to mediate mesendoderm specification [135,143]. Targets of other signalling pathways share
similar roles in regulating different states of pluripotency. For
example, in cooperation with Nodal signalling, and actively
induced by Nanog, Eomes directly regulates much of the transcriptional network for definitive endoderm specification in
the mouse [58]. Meanwhile, downstream of Nodal signalling,
Tbx3 regulates pluripotency in ESCs and expression of Eomes
and Brachyury upon differentiation towards mesendoderm
[144], though definitive confirmation of this relationship
in vivo has been limited to experiments in Xenopus embryos.
Similarly, Otx2 stabilizes the transition from a naive to primed
pluripotent state, antagonizing BMP4 signalling together with
its binding partner FGF2 [145], and is thought to function
in the same circuitry to regulate anterior neuroectoderm
differentiation in the embryo [145,146].
5. Conclusion and future perspectives
Based on recent studies, many of the genes that control pluripotency in the earliest stages of embryonic development also
regulate transitions through distinct pluripotent states, as
well as specific lineage decisions throughout pre- and early
postimplantation stages. Although not all such regulatory
genes are active at every stage, these observations suggest
that GRNs of the earliest events in mammalian development
are variations on a theme, involving different combinations of
the same components functioning under different constraints.
Central members of the core pluripotency circuitry, including
Oct4, Sox2 and Nanog, appear to be among the common components, while regionalized changes in the balance of the
circuit result in activation and repression of their respective
targets. Such changes are mediated in part by a gradual
extinction of their expression as the accessibility of their cisregulatory elements is decreased by nucleosome invasion
[124], which alters the effect of peripheral components such
as Tcf3 and Tbx3 on this circuitry, and initiates their switch
to roles as lineage-specifying genes. At present, it is unclear
how region-specific changes initiate this process, but alterations in cell contacts, signalling and morphogenetic events
are likely to be significant.
The clarification of these and related issues should be
greatly aided by the de novo construction of GRNs representing individual cell types at different stages of development.
In particular, such efforts could benefit from the application
of computational systems approaches that have not yet been
used to study the developing embryo. For example, although
many methods of co-expression analysis provide critical information about GRN architecture, they do not establish whether
regulatory interactions are direct or indirect. Approaches such
as Algorithm for the Reconstruction of Accurate Cellular Networks have been developed with this issue in mind, as it can
eliminate indirect transcriptional interactions from inferred
networks [147]. Furthermore, methods such as Modulator
Inference by Network Dynamics can detect three-way interactions between gene products (such as protein kinases),
downstream regulators and their targets [148].
Ideally, each predicted interaction within inferred GRNs
would be validated experimentally. Ultimately, such validation would require the identification of cis-regulatory
elements that underlie global gene expression dynamics. For
Phil. Trans. R. Soc. B 369: 20130542
SUZ12
Eomes
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In the future, GRNs should facilitate the integration of
upstream events and extrinsic signals with downstream
sub-networks of effector genes, in order to understand context-dependent function of these networks. Although such
efforts will present enormous computational challenges for
modelling the transition from blastocyst to early gastrulation
stages, these studies could ultimately lead to generation of
whole-organism GRNs for mammalian development.
Acknowledgements. We thank Bo Li and Hui Zhao for comments on the
manuscript.
from the NIH.
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