How to make a primordial germ cell

© 2014. Published by The Company of Biologists Ltd | Development (2014) 141, 245-252 doi:10.1242/dev.098269
How to make a primordial germ cell
Primordial germ cells (PGCs) are the precursors of sperm and eggs,
which generate a new organism that is capable of creating endless
new generations through germ cells. PGCs are specified during early
mammalian postimplantation development, and are uniquely
programmed for transmission of genetic and epigenetic information
to subsequent generations. In this Primer, we summarise the
establishment of the fundamental principles of PGC specification
during early development and discuss how it is now possible to make
mouse PGCs from pluripotent embryonic stem cells, and indeed
somatic cells if they are first rendered pluripotent in culture.
KEY WORDS: Epigenetic programming, Primordial germ cells,
Specification, Transcription factors
Primordial germ cells are highly specialised cells that are precursors
of gametes, which, following meiosis, develop as haploid sperm and
eggs that generate a new organism upon fertilisation. They transmit
genetic and epigenetic information between generations and ensure
the survival of a species. Although germ cells are set aside during
early development in almost all animals, the mechanism of germ cell
specification is not conserved among animals. Typically, specification
of germ cells occurs either through the inheritance of preformed germ
plasm (Weissmann, 1885), or is induced among equipotent cells by
instructive signals. For example, germ cell specification in Xenopus
and C. elegans occurs via the inheritance of germ plasm, whereas, in
axolotls, germ cell specification occurs in animal cap cells in response
to signals (Johnson et al., 2011; Niewkoop, 1979). In principle, both
these mechanisms ensure suppression of somatic fate while promoting
the onset of the germ cell programme. In mice, primordial germ cells
(PGCs) originate from the early postimplantation epiblast cells, which
also give rise to all somatic cells in response to signals from the
extraembryonic tissues.
Mammalian sperm and eggs make an equal genetic contribution
to a new organism, but their epigenetic contributions are unique and
complementary. Both a male and a female genome are necessary for
development to term because of the parent of origin-dependent
‘imprinting’ of the genome in the germ line (McGrath and Solter,
1984; Surani et al., 1984). Investigations of mammalian germ cells
provide a unique insight into how epigenetic information with
respect to ‘imprints’ is first erased and then re-initiated, and becomes
Wellcome Trust, Cancer Research UK, Gurdon Institute, University of Cambridge,
Cambridge CB2 1QN, UK. 2Department of Physiology, Development and
Neuroscience, University of Cambridge, Cambridge CB2 3EG, UK. 3Wellcome
Trust-Medical Research Council, Stem Cell Institute, University of Cambridge,
University of Cambridge, Cambridge CB2 3DY, UK. 4Department of Biochemistry
and Molecular Biology, University of Iceland, Vatnsmyrarvegi 16, 101 Reykjavík,
*This article is dedicated to the memory of Sheila Carrie Barton, whose
exceptional skills and knowledge of mouse embryology made it possible to
identify the key regulators of primordial germ cells.
Author for correspondence ([email protected])
heritable from the germ line into adulthood. Imprinting results in the
parent of origin-dependent monoallelic expression of imprinted
genes during development. Faulty imprints can lead to
developmental, physiological and behavioural anomalies in mice,
and result in diseases in humans (reviewed by Grossniklaus et al.,
2013). The process of robust erasure and resetting of the epigenome
in early PGCs also ensures that aberrant epigenetic information is
not transmitted to the offspring. Nonetheless, there are recent reports
suggesting that environmental factors can induce epigenetic changes
that can be transmitted through the germline to subsequent
generations with detrimental consequences (reviewed by Tomizawa
and Sasaki, 2012). Studies on the germ cell lineage might test the
validity of these claims or provide the mechanistic basis for them.
The resetting of the germline epigenome is also crucial for the
establishment of the totipotent state following fertilisation. The
underlying mechanisms involved may be applicable to the
experimental reprogramming of the epigenome and to
the manipulation of cell fates in vitro, and potentially to the
reprogramming of the endogenous cells of diseased tissues. This has
important implications for regenerative medicine and human diseases,
including germ cell tumours. Germ cells also provide opportunities
with which to uncover the mechanisms of chromatin modifications,
as well as of DNA methylation/demethylation. How the factors
involved induce significant epigenetic changes might be useful in
other contexts during normal and aberrant development, and could
lead to the development of novel therapeutic agents.
More directly, investigation of germ cells could lead to advances
in reproductive medicine. For example, defective mitochondria
transmitted by oocytes have major health implications; how they
accumulate and are transmitted thereafter is of particular interest, as
are the other causes of infertility. Advances in germ cell biology and
genome editing, together with the ability to generate germ cells from
pluripotent stem cells might lead to advances in assisted
reproduction in some mammals.
Major advances over the past decade have been the identification
of key regulators of PGC specification (reviewed by Leitch et al.,
2013a), how the genetic network functions during PGC specification
and during the onset of a unique epigenetic programme
(Magnúsdóttir et al., 2013), and the establishment of an epigenetic
ground state and the initiation of the imprinting cycle (Hackett et al.,
2012; Hayashi et al., 2007). Here, we focus on the mechanism of
PGC specification in mice, which is currently the best-characterised
germ cell lineage among mammals. Its characterisation has led to
the development of experimental systems that can be used to
generate PGC-like cells from pluripotent stem cells, and potentially
from any somatic cell via induced pluripotent stem cells (iPSCs),
which in turn can give rise to viable gametes in vivo that are capable
of generating a live organism (Hayashi and Surani, 2009; Hayashi
et al., 2011; Hayashi et al., 2012). We are currently able to generate
early germ cells and rudimentary oocyte-like structures in vitro
(Hayashi and Surani, 2009), but further advances might lead to the
ability to induce meiosis and advanced development of gametes in
Erna Magnúsdóttir1,2,3,4,‡ and M. Azim Surani1,2,3,*
Development (2014) doi:10.1242/dev.098269
The instructive nature of PGC specification in mice
The classical work on clonal analysis by Lawson and Hage showed
that the progenitors of PGCs are located in the most proximal region
of the postimplantation epiblast, close to the extraembryonic tissues
(Fig. 1). These PGC precursors are detected here at embryonic day
(E) 6.0-6.5, and eventually generate a founding population of ~40
PGCs at ~E7.25 (Chiquoine, 1954; Ginsburg et al., 1990; Lawson
and Hage, 1994).
The key instructive signals for PGC specification are the bone
morphogenetic proteins (BMP2, BMP4 and BMP8B), which
originate from the extraembryonic tissues and act though SMAD1
and SMAD5 (Hayashi et al., 2002; Lawson et al., 1999; Tam and
Snow, 1981; Ying and Zhao, 2001; Ying et al., 2000). The response
of epiblast cells to PGC specification is BMP dose dependent in vivo
(Lawson et al., 1999), as the number of PGCs decreases in
proportion to the loss of BMP2 and BMP4 alleles. In vitro, BMP4
is sufficient for PGC induction, whereas WNT3 is required to induce
competence for PGC fate in epiblast cells and enable their
appropriate response to BMP signalling towards PGC specification
(Ohinata et al., 2009). In principle, any postimplantation epiblast cell
at this time of development can potentially acquire PGC fate under
appropriate conditions, although this response is normally restricted
in vivo by diverse signalling gradients, not only of the inductive
BMP signals but also gradients of inhibitory signals from the distal
and anterior visceral endoderm, such as cerberus 1 (CER1),
dickkopf 1 (DKK1) and LEFTY1 (Ohinata et al., 2009; Tam and
Zhou, 1996) (Fig. 1).
Intrinsic regulators of mouse PGC fate
The genetic basis for PGC specification has emerged only over the
past decade following attempts to discover the key regulators of
PGC fate by single cell analysis. Among the first genes to be
identified was developmental pluripotency-associated 3 [Dppa3
(previously Stella)] as the definitive marker of founder PGCs
(Saitou et al., 2002; Sato et al., 2002), followed by Prdm1, which
encodes BLIMP1, a PR domain zinc-finger protein that is a key
regulator of PGC specification (Chang et al., 2002; Hayashi et al.,
2007; Ohinata et al., 2005; Vincent et al., 2005). Expression of
BLIMP1 in a few randomly distributed most proximal epiblast cells
at ~E6.25 marks the onset of PGC specification, which fits with the
origin of PGCs by clonal analysis (Lawson and Hage, 1994).
BLIMP1 expression demarcates germ cells from the neighbouring
somatic cells through the repression of the incipient mesodermal
programme (Hayashi et al., 2007; Kurimoto et al., 2008; Ohinata et
al., 2005). A mutation in BLIMP1 resulted in a small cluster of
aberrant PGC-like cells at ~E8.5 that were more like the
neighbouring somatic cells due to a failure to repress somatic gene
expression and to induce PGC-specific genes (Ohinata et al., 2005).
A comparison between wild-type and mutant BLIMP1 PGC-like
cells was subsequently pivotal for the identification of Prdm14 (a
In vivo
In vitro
2i + LIF
Day 2
Epiblast-like cells
Activin A +
~Day 2-4
Fig. 1. Specification of primordial germ cells. Primordial germ cells (PGCs) originate from the postimplantation epiblast cells in vivo during embryonic days
(E) ~6.5-7.5 (in vivo column) in response to BMP4 from the extra-embryonic ectoderm (ExE), with BMP2 from the visceral endoderm (VE) (left). They bind to
receptors that phosphorylate SMAD1 and SMAD5, which dimerise with SMAD4, translocate to the nucleus and induce the key transcriptional regulators of
PGCs. These events are recapitulated in vitro (in vitro column) with naïve pluripotent embryonic stem cells (ESCs), which acquire a primed competent state on
day 2 when cultured in activin/FGF2. PGCs can be induced in these cells either by cytokines or directly by BLIMP1, PRDM14 and AP2γ. 2i, culture media for
the propagation of naive embryonic stem cells; AP2γ, a helix-loop-helix transcription factor; BLIMP1 (PRDM1), a transcription factor with a PR and ZNF
domain; BMP, bone morphogenetic protein; EGF, epidermal growth factor; FGF, fibroblast growth factor; ICM, inner cell mass; LIF, leukaemia inhibitory factor;
PRDM14, transcription factor with a PR and ZNF domain; SCF, stem cell factor.
PGC-like cells
member of the Prdm family) (Grabole et al., 2013; Kurimoto et al.,
2008; Yamaji et al., 2008). A mutation in Prdm14 led to the
formation of aberrant PGCs that were lost by ~E11.5. The cells
exhibited a defective epigenetic programme, as judged by the failure
of the characteristic global erasure of H3K9me2 histone
modification at ~E8.5, partly because the Ehmt1-Ehmt2-mediated
H3K9 methylasae activity was not repressed. The cells also failed
to show genome-wide induction of the polycomb enzyme EZH2mediated H3K27me3. This suggests that PRDM14 is important at
least for the epigenetic programme in early germ cells (Hajkova et
al., 2008; Hajkova et al., 2010; Yamaji et al., 2008; Seki et al.,
2007). Furthermore, BLIMP1 expression is sustained by PRDM14,
which might explain the induction of somatic genes in mutant cells
(Grabole et al., 2013; Magnúsdóttir et al., 2012; Magnúsdóttir et al.,
2013). PRDM14 also induces Dppa3 and Sox2 at ~E8.5. Thus,
neither PGC-specific gene expression nor the re-initiation of
pluripotency gene expression occurred in Prdm14-null cells
(Grabole et al., 2013; Yamaji et al., 2008).
Finally, Tcfap2c, which encodes AP2γ (a direct target of
BLIMP1), is also crucial for PGC specification (Kurimoto et al.,
2008; Magnúsdóttir et al., 2013; Weber et al., 2010) because a
mutation in this gene caused very early loss of PGCs. Although
these cells remain to be fully characterised, it is possible that AP2γ
might also be involved in the repression of somatic genes, including
early mesodermal marker Hoxb1 (Weber et al., 2010). Thus,
BLIMP1, PRDM14 and AP2γ together constitute a mutually
interdependent transcriptional network for PGC specification
(Magnúsdóttir et al., 2013; Nakaki et al., 2013).
PGCs versus soma: the underlying pluripotency of the
unipotent germ cell lineage
Evidence suggests that at the onset of PGC specification, the
postimplantation epiblast cells are destined for somatic fate, with an
increase in DNA methylation and H3K9me2 histone modification,
including the onset of X inactivation (Hackett et al., 2013);
pluripotency genes, such as Nanog, Rex1 and Dppa3 are also
repressed. BLIMP1 arrests this trend towards somatic fate at the
onset of PGC specification, and initiates a reversion of some of these
properties, including initiation of X-reactivation and re-expression
of key pluripotency genes, such as Sox2 and Nanog.
Comprehensive re-expression of pluripotency genes is unique to
cells that adopt PGC fate, which is evident to only a limited extent
in somatic lineages as in neural progenitors where Sox2 promotes
cell identity (Graham et al., 2003). The expression of pluripotency
genes in PGCs is likely permissive for the initiation of an epigenetic
programme, such as DNA demethylation (Hackett et al., 2013;
Leitch et al., 2013b; Seisenberger et al., 2012). Despite the similarity
to the inner cell mass (ICM) and embryonic stem cells (ESCs) with
respect to the expression of pluripotency genes, PGCs are unique
because they exhibit the imprinting cycle and have the potential for
meiosis (reviewed by Surani et al., 2007). Genes such as Nanog are
apparently necessary for the maintenance of early germ cells
because mutant cells undergo apoptosis during migration (Chambers
et al., 2007; Yamaguchi et al., 2009). OCT4 might have a role in
establishing competence for PGC fate and thereafter, perhaps by
priming of the appropriate enhancers (Kehler et al., 2004; Soufi et
al., 2012). Expression of OCT4 in PGCs depends on the activation
of its distal enhancer (Bao et al., 2009; Chen et al., 2008; Yeom et
al., 1996), reflecting the key attributes of underlying pluripotency in
PGCs, although they develop into only gametes, sperm and eggs.
Early PGCs can, however, undergo dedifferentiation into
pluripotent embryonic germ cells (EGCs) in response to fibroblast
Development (2014) doi:10.1242/dev.098269
growth factor (FGF)/leukaemia inhibitory factor (LIF) signalling in
culture (Matsui et al., 1992). EGCs resemble ESCs, probably owing
to the ‘erasure’ of the epigenetic memory of their origin from the
postimplantation epiblast. Dedifferentiation of PGCs to EGCs
entails rapid repression of BLIMP1 (Durcova-Hills et al., 2008;
Leitch et al., 2010), suggesting that BLIMP1 might have a role in
lineage restriction in PGCs; its repression is essential for the reexpression of Myc and Klf4, which are repressed in PGCs but are
essential for the self-renewal of EGCs and ESCs (Durcova-Hills et
al., 2008).
The tripartite transcriptional network for PGC specification
The tripartite network of BLIMP1, AP2γ and PRDM14 is important
not only for the initiation of PGC specification, but also for the
unfolding epigenetic programme that ensues. To identify direct
targets of BLIMP1 and AP2γ, the embryonal carcinoma cell line
P19 (P19EC) was used as a surrogate system because PGCs are
limited in number and difficult to culture and manipulate. P19EC
cells are appropriate because they originate from postimplantation
epiblast cells, the precursors of PGCs (McBurney, 1993; Spivakov
and Fisher, 2007). Notably, expression of BLIMP1, PRDM14 and
AP2γ in P19EC cells, both individually and in different
combinations, elicits expression of several PGC genes and induces
repression of somatic genes (Magnúsdóttir et al., 2013).
Bioinformatics analysis has provided a wealth of information on
the potential targets of the tripartite transcriptional network. Single
cell transcriptome data of normal and mutant PGCs, combined with
the information on the targets of BLIMP1 and AP2γ in P19EC, as
well as of PRDM14 in ESCs (Ma et al., 2011), has revealed that the
germ cell programme is executed by a high degree of cooperative
activity between the three factors. This enables initiation of several
programmes in early PGCs, including the repression of somatic
genes. For example, the migratory programme, as well as expression
of PGC-specific genes, is executed mainly by PRDM14 and AP2γ,
whereas PRDM14 and BLIMP1 collaborate in both the induction
and repression of epigenetic modifiers.
The majority of the AP2γ-bound genes were also bound by
BLIMP1 and PRDM14 (Fig. 2), to facilitate changes in gene
expression (Fig. 3). AP2γ is enriched both on distal elements as well
as on promoters, unlike BLIMP1, which is predominantly bound to
promoters, whereas PRDM14 binds mainly to distal regulatory
regions (Ma et al., 2011). It is possible that PRDM14 and BLIMP1
make the initial engagement with genomic binding sites to initiate
transcriptional changes, which are then ‘locked in’ by AP2γ
(Fig. 2C). BLIMP1 also binds to cell cycle and multiple
transcriptional regulators, including all four Hox gene loci (HoxA,
HoxB, HoxC and HoxD), possibly to ‘protect’ PGCs with their
underlying pluripotency from responding to various extrinsic signals
as they migrate to the gonads.
Activation of the PGC programme
A pivotal role of BLIMP1 is to induce Tcfap2c expression directly
(Kurimoto et al., 2008; Magnúsdóttir et al., 2013; Weber et al.,
2010). How BLIMP1 can both repress and induce different targets
is unknown, but it might recruit transcriptional co-repressors and coactivators under some specific conditions (Ancelin et al., 2006; Ren
et al., 1999; Su et al., 2009; Yu et al., 2000). Notably, in zebrafish,
BLIMP1 directly activates Tfap2a (encoding AP2α), which is
crucial for the specification of neural crest cells (Powell et al., 2013).
The induction of AP2γ by BLIMP1 is crucial for the combinatorial
role of PRDM14 and AP2γ in the induction of PGC-specific genes
such as Dnd1, as well as Nanos3 (Fig. 3B). They also bind to the
Development (2014) doi:10.1242/dev.098269
Tag density
Distal element
Germ cell
Fig. 2. Differential occupancy and
combinatorial roles of BLIMP1,
PRDM14 and AP2γ. (A) BLIMP1 mainly
occupies promoters (left), whereas
PRDM14 occupies the distal regulatory
elements (right). AP2γ binds both
promoters and distal elements (centre).
The sequence tag densities (y-axis)
relative to distance from transcriptional
start sites (TSS) (x-axis) are shown.
(B) The overlap between genes bound
by the three factors in the whole
genome; the majority of the genes bound
by AP2γ are bound by either BLIMP1 or
PRDM14. (C) Gene expression changes
are mediated by the initial engagement
of both BLIMP1 and PRDM14 together to
repress somatic genes or by PRDM14
alone to activate germ cell genes. The
induction of AP2γ by BLIMP1
strengthens the network via the cobinding of AP2γ with BLIMP1 and
PRDM14, and ‘locks in’ the initial
engagement. AP2γ, a helix-loop-helix
transcription factor; BLIMP1 (PRDM1), a
transcription factor with a PR and ZNF
domain; PRDM14, a transcription factor
with a PR and ZNF domain.
‘Locking in’ of
transcriptional state
distal enhancer of Pou5f1 (previously Oct4), to maintain its
expression in PGCs. Overall, PRDM14 probably contributes to
epigenetic reprogramming in PGCs, as shown by its potential for
reprogramming of epiblast stem cells (EpiSCs) to naive ESCs
(Gillich et al., 2012). PRDM14 alone and in collaboration with
AP2γ regulates a multitude of genes involved in cell-cell adhesion
and migration (Magnúsdóttir et al., 2013; Ma et al., 2011).
The genetic network for PGC specification initiates the
epigenetic programme
The epigenetic programme induced primarily by BLIMP1 and
PRDM14 leads to global DNA demethylation towards an epigenetic
ground state in early PGCs (Hackett et al., 2013; Hajkova et al.,
2008; Hajkova et al., 2010; Ohno et al., 2013; Seisenberger et al.,
2012) (Fig. 4). For example, the repression of Uhrf1 (Bostick et al.,
2007) and Dnmt3b by BLIMP1 and PRDM14 promotes DNA
replication-coupled DNA demethylation in PGCs. Additionally,
TET1 and TET2, which are bound by PRDM14, catalyse
hydroxylation of 5-methyl-cytosine, which provides an additional
parallel redundant mechanism for DNA demethylation in PGCs
(Hackett et al., 2013; Ma et al., 2011; Ohno et al., 2013;
Seisenberger et al., 2012). There is potentially a role for a base
excision repair mechanism (Hajkova et al., 2008) that could
contribute to the erasure of imprints. Although PRDM14 contributes
to DNA hypomethylation in naïve pluripotent ESCs, imprints are
retained in these cells (Leitch et al., 2013b).
The transcriptional network for PGC specification also regulates
histone modifications, as PRDM14 induces H3K9 histone
Germ cell
demethylases Kdm3a and Kdm4b. This induction, together with the
repression of Ehmt1, ensures rapid and global loss of H3K9me2, and
could account for the re-expression of pluripotency genes and
promote DNA demethylation (Liu et al., 2013; Rothbart et al.,
2012). The repression of Kdm6b demethylase by PRDM14 and
BLIMP1 contributes to the global increase in H3K27me, which is
also seen in the inner cell mass of blastocysts. Furthermore,
PRDM14 induces Hdac6, which, unlike its orthologues, does not
seem to participate in transcriptional regulation, but promotes cell
mobility, an important aspect of PGC biology (Li et al., 2013). The
re-activation of the inactive X-chromosome in PGCs might be
facilitated by PRDM14 binding to intron 1 of Xist and to Rnf12
(Rlim – Mouse Genome Informatics), an activator of Xist (Barakat
et al., 2011; Ma et al., 2011; Shin et al., 2010).
Generating PGCs in vitro: recapitulating development
The increasing knowledge of PGC specification in vivo has led to
attempts to mimic the process in vitro (Hayashi and Surani, 2009;
Hayashi et al., 2011; Magnúsdóttir et al., 2013; Matsui et al., 1992;
Nakaki et al., 2013; Ohinata et al., 2009; Tam and Zhou, 1996).
Initial attempts were made with the whole postimplantation epiblast,
with or without the visceral endoderm (VE) still attached, as well as
with or without the extraembryonic ectoderm (ExE), which was
cultured in the presence of cytokines (Lawson et al., 1999). These
studies were then comprehensively validated using Prdm1, Prdm14
and Dppa3 promoters as reporters (Ohinata et al., 2009), which
established that most, if not all, epiblast cells are competent for PGC
fate in the presence of BMP4. The efficiency increased with stem
Development (2014) doi:10.1242/dev.098269
cell ge
Ge Dppa3 Dnd1
ti c
E pi
ti c r e g u l a t o r
Fig. 3. The transcriptional network for
primordial germ cell specification. (A) The
interdependent network for primordial germ cell
(PGC) specification consisting of BLIMP1,
PRDM14 and AP2γ is depicted at the centre. The
network performs crucial functions by the binding
of a combination of factors represented in the
individual segments that control expression of key
genes shown within them. Blue and yellow
segments indicate repressed and induced genes,
respectively. (B) Genome browser views, adapted
from Magnúsdóttir et al. (Magnúsdóttir et al.,
2013) of ChIP-seq peaks depicting examples of
combinatorial binding of BLIMP1, AP2γ and
PRDM14. Although Pou5f1, which encodes
OCT4, is not differentially expressed between
PGCs and their precursors, and is therefore not
shown in A, the enhancers used for its expression
switch from a proximal one in the epiblast to that
of a distal one in PGCs, which is also the case in
the inner cell mass (ICM) and in embryonic stem
cells. AP2γ, a helix-loop-helix transcription factor;
BLIMP1 (PRDM1), a transcription factor with a
PR and ZNF domain; PRDM14, a transcription
factor with a PR and ZNF domain.
P r o li f e r a
ti o n
cell factor (SCF), epidermal growth factor (EGF), LIF and BMP8B,
partly due to increased cell viability in vitro because some factors,
such as LIF, are not implicated in PGC specification in vivo. The
efficiency increased when the VE and ExE were removed. This
response to cytokines for PGC specification was predictably
abrogated in the absence of SMAD1 and SMAD5, eliminating the
possibility of indirect effects of BMP signalling molecules in PGC
induction. The in vitro generated PGCs could develop into viable
gametes in vivo that were able to give rise to viable offspring
(Ohinata et al., 2009).
Importantly PGC-like cells could also be generated starting with
naïve pluripotent ESCs that were first cultured for 2 days in basic
FGF (bFGF) and activin A, followed by other cytokines, including
BMP4, BMP8B, SCF, EGF and LIF, to obtain PGC-like cells
(Hayashi et al., 2011). BMP4 is probably the most important
cytokine for the induction of PGCs, whereas other factors, such as
LIF and SCF, aid the survival of PGCs, which currently can be
maintained for only 5-6 days in vitro. The initial culture of ESCs in
bFGF and activin A signalling molecules drives them towards a
postimplantation epiblast character (Guo et al., 2009). These
epiblast-like cells (EpiLCs) form embryoid bodies when cultured as
cell aggregates on non-adherent surfaces. The embryoid bodies
respond to BMP4 and other cytokines and start to develop as PGClike cells from day 2 onwards, resulting in 40-60% of cells
Development (2014) doi:10.1242/dev.098269
Facilitators of DNA methylation
H3K9 methylation
DNMT1 targeting/
methylation maintenance
De novo DNA methylation
De novo DNA methylation
H3K27 de-methylase
Antagonists of DNA methylation
H3K9 de-methylase
H3K9 de-methylase
Transcriptional co-represssors
Histone deacetylase
Histone deacetylase
Uhrf1 status
Not expressed
Fig. 4. Regulation of epigenetic modifiers by BLIMP1 and PRDM14.
(A) BLIMP1 and PRDM14 repress the facilitators, and induce the antagonists
of DNA methylation and other histone modifiers. (B) The combinatorial role of
BLIMP1 and PRDM14 in the repression of Uhrf1, a DNMT1 accessory
protein (also known as NP95), promotes global DNA demethylation in PGCs.
BLIMP1 occupies the main promoter of Uhrf1, whereas PRDM14 occupies
an alternative downstream promoter. Uhrf1 is expressed in embryonic stem
cells despite the presence of PRDM14, which indicates that PRDM14 is not
sufficient for Uhrf1 repression.
becoming PGCs after about 6 days (Fig. 1). These in vitro-generated
PGCs can develop into viable sperm and oocytes (Hayashi et al.,
2011; Hayashi et al., 2012). Although this in vitro method is
appropriate for studying PGC specification, these cells do not
exhibit imprint erasure or enter meiosis in vitro, whereas they can
undergo gametogenesis in vivo when introduced back into the
gonads of fully grown animals (Hayashi et al., 2011; Hayashi et al.,
The PGC in vitro culture system has allowed the interrogation of
several key regulators of PGC specification. In recent studies, two
independent groups forced the expression of BLIMP1, AP2γ and
PRDM14, and found that these proteins could directly induce PGClike fate in vitro in the absence of cytokines (Magnúsdóttir et al.,
2013; Nakaki et al., 2013). This is consistent with the role of these
proteins in vivo (summarised in Fig. 3). Importantly, the somatic
programme was not upregulated in the absence of cytokines during
the direct induction of the PGC-like cells (Magnúsdóttir et al., 2013;
Nakaki et al., 2013). Although PRDM14 alone was also able to
induce PGC-like cells at a low frequency in vitro (Nakaki et al.,
2013), this apparently occurred concomitant with the induction of
endogenous Prdm1 and Tfap2c (the gene encoding AP2γ). This does
not rule out the possibility that all three factors are necessary for
PGC fate. The absence of somatic gene upregulation during the
direct induction of PGC-like fate does not obviate the need for
BLIMP1 in PGC specification in vitro, in part because it directly
induces AP2γ, as well as inducing the epigenetic programme
together with PRDM14. Thus, the role of BLIMP1 extends beyond
the repression somatic genes per se. For example, BLIMP1 and
PRDM14 together are essential for the repression of Uhrf1, which
is crucial for DNA demethylation in PGCs and for the re-expression
of pluripotency genes (Magnúsdóttir et al., 2013) (Fig. 4B).
Furthermore, BLIMP1 represses Myc, which permits exit from
pluripotency and development of the restricted but dynamic nature
of the early germ cell lineage (Lin et al., 2012).
Investigations from over a decade of research have led to major
advances in mouse germ cell biology, which will provide the basis
for studies on germ cells of other mammalian species, including
humans. The identification of the key regulators of PGC
specification, BLIMP1, PRDM14 and AP2γ, which together
constitute the tripartite genetic network for specification PGCs,
represents an important advance. There is also a greater and precise
understanding of their combinatorial roles in the repression of genes
of the somatic programme, induction of the germ cell genes and in
the initiation of epigenetic modifications in early germ cells.
BLIMP1 is expressed first and it is the key regulator of PGC
specification, not least because it induces AP2γ, and together with
PRDM14, they control all the major aspects of PGC specification
and the key attributes of early germ cells, such as the epigenetic
programme (Magnúsdóttir et al., 2013; Nakaki et al., 2013; Ohinata
et al., 2005; Weber et al., 2010; Yamaji et al., 2008). The germline
also represents an elegant system combining an underlying
pluripotency and lineage restriction that is critically balanced by the
co-expression of the appropriate transcriptional regulators.
The major advances in mammalian germ cell biology have led
to the exploration of PGC specification from pluripotent stem
cells. These PGC-like cells can progress towards the formation of
rudimentary oocyte-like structures (Hayashi and Surani, 2009).
Starting with naïve ESCs, such PGC-like cells are formed,
although they do not exhibit some key properties such as the
erasure of imprints or meiosis, but can undergo gametogenesis
under appropriate conditions in vivo to form viable gametes that
are capable of generating a live organism (Hayashi et al., 2011;
Hayashi et al., 2012). Notably, the three key regulators of PGC
specification can directly induce PGC-like fate in vitro in the
absence of cytokines (Magnúsdóttir et al., 2013; Nakaki et al.,
2013), although such direct induction of PGC-like cells occurs
only from the appropriately primed epiblast cells but not from the
naïve ESCs. This suggests the importance of appropriate priming
of cells for PGC fate. Indeed, loss of competence also occurs in
the pluripotent EpiSC, which is due to the increase in DNA
methylation and includes the promoters of PGC genes (Bao et al.,
2009; Gillich et al., 2012). These in vitro models might be useful
for investigating the molecular prerequisites of competence for
PGC-like fate and its counterpart, the somatic fate. It is also
possible to induce PGC-like fate in iPSCs derived from somatic
cells. This might make it possible to generate viable gametes from
somatic cells, perhaps by direct transdifferentiation of somatic
cells into germ cells with the three key regulators of PGCs.
Further studies will show how some mutations, both naturally
occurring and introduced, influence the development and properties
of the germ cell lineage. This may allow investigation into the longterm consequences of transgenerational inheritance of genetic and
epigenetic information. The causes of some forms of infertility
might also be uncovered, as well as the factors implicated in germ
cell tumours such that amelioration of these diseases may one day
be possible.
Finally, the comprehensive epigenetic programming, including
global DNA demethylation, observed in early germ cells provides a
unique opportunity with which to gain knowledge of key enzymes
and epigenetic modifiers that promote the epigenetic ground state
(Hackett et al., 2013; Hajkova et al., 2008; Hajkova et al., 2010; Ng
et al., 2013). These mechanisms might be applicable more widely
for the experimental manipulation of the epigenome and cell fates
of normal and diseased tissues, through the erasure and re-initiation
of novel epigenetic information.
This article is dedicated to the memory of Sheila Carrie Barton, whose exceptional
skills and knowledge of mouse embryology made it possible to identify the key
regulators of primordial germ cells.
Competing interests
The authors declare no competing financial interests.
This work was supported by grants from the Wellcome Trust and Human Frontiers
Science Program to M.A.S., and by the European Commision/Marie Curie
Programme to E.M.
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