Transcriptional control of pluripotency: decisions in early

development
Brett Vaughan Johnson
1
, Joy Rathjen
2
and Peter David Rathjen
2
The pathways controlling the maintenance and loss of
pluripotency in cells of the early embryo regulate the formation
of the tissues that will support development. Several
transcription factors have been identied as being integral to
the establishment and/or maintenance of pluripotency,
coordinately regulating the expression of genes within
pluripotent cells and acting as gene targets of these same
processes. Recent advances in understanding the
transcriptional regulation of these factors have revealed
differences in the transcriptional complexes present within sub-
populations of the pluripotent lineage and in the mechanisms
regulating the loss of pluripotency on differentiation.
Addresses
1
School of Molecular and Biomedical Science and the Australian Stem
Cell Centre, University of Adelaide, Adelaide 5005, Australia
2
School of Molecular and Biomedical Science, the Australian Stem Cell
Centre and the ARC SRC for the Molecular Genetics of Development,
University of Adelaide, Adelaide 5005, Australia
Corresponding author: Rathjen, Peter D (peter.rathjen@adelaide.edu.au)
Current Opinion in Genetics & Development 2006, 16:447454
This review comes from a themed issue on
Differentiation and gene regulation
Edited by Vincenzo Pirrotta and Maarten van Lohuizen
Available online 17th August 2006
0959-437X/$ see front matter
# 2006 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.gde.2006.08.012
Introduction
Pluripotency describes the developmental competence of
cells that display a specialised differentiation capability
the ability to give rise, through differentiation, to all
cell types of the embryo and adult. Within the embryo
(Figure 1), the pluripotent lineage is rst apparent at one
pole of the expanding blastocyst as a small population of
massed cells, the inner cell mass (ICM), which arises at
the time that the extraembryonic trophectoderm lineage
forms from outer cells of the early embryo. Cells of the
ICM give rise to two daughter populations: the extraem-
bryonic endoderm, fated to form extraembryonic tissues;
and the primitive ectoderm, or epiblast. Pluripotency is
preserved only in cells of the primitive ectoderm, a
temporally distinct population that can be discriminated
from the ICM by its morphology, its repertoire of
expressed genes, and by virtue of having a differentiation
potential that excludes the extraembryonic endoderm[1].
Pluripotency is lost as somatic cell populations are
formed by differentiation of the primitive ectoderm at
gastrulation, and it is subsequently retained only in
those cells allocated to the germline. Faithful progression
of embryogenesis therefore requires establishment
and maintenance of pluripotency in two discrete popula-
tions, the ICM and the primitive ectoderm, and tempo-
rally and spatially regulated loss of pluripotency in the
trophectoderm, extraembryonic endoderm and somatic
cells. Our emerging knowledge of the molecular and
cellular basis for pluripotency has been obtained from
genetic dissection of regulatory pathways in vivo,
coupled with genetic and biochemical analysis of plur-
ipotent embryonic stem (ES) cell lines derived from
the ICM.
In this review, we discuss our understanding of the
essential genes involved in regulating pluripotency and
assess how their transcription is controlled during the
maintenance and loss of pluripotency, both within the
mammalian embryo and in vitro.
The transcription factors Oct4, Nanog and
Sox2 are required for pluripotency in the
ICM lineage
The POU homeodomain transcription factor Oct4 (also
known as Pou5f1) is expressed in all pluripotent cells of
the mammal and is downregulated upon formation of
extraembryonic and somatic lineages (Figure 1). Oct4
/
blastocysts arise, but cells within the ICM lack pluripo-
tency and form only the trophectoderm, resulting in peri-
implantation developmental arrest. In vitro culture of
isolated inner cells from these embryos suggests an abso-
lute requirement for Oct4 in establishment of the plur-
ipotent lineage [2]. Oct4 is not sufcient for the
maintenance of pluripotency, because forced expression
fails to render mouse ES cells independent of extracel-
lular factors that activate gp130 signalling [3]. Genomic
targets bound by Oct4 in mouse ES cells were predomi-
nantly but not exclusively downregulated upon cell dif-
ferentiation [4

], consistent with evidence identifying

Oct4 as a transcriptional activator and repressor in differ-
ent promoter contexts [5,6

].
The variant homeodomain transcription factor Nanog,
shown to activate transcription through multiple protein
domains [7], is expressed throughout the pluripotent cells
of the ICM but is downregulated in extraembryonic
lineages and in pluripotent cells of the peri-implantation
embryo [8]. Forced expression of Nanog, unlike that of
Oct4, is sufcient to maintain the pluripotent state in
www.sciencedirect.com Current Opinion in Genetics & Development 2006, 16:447454
mouse ES cells in the absence of LIF (leukemia inhibi-
tory factor) [8,9], and in human ES cells results in cells
that can be propagated in the absence of feeder cells and
associated signalling [10

]. Nanog
/
embryos fail to
maintain the pluripotent lineage and arrest at peri-
implantation; the ICM from Nanog
/
embryos and
Nanog-depleted ES cells differentiate into parietal endo-
derm-like cells in culture. The nding that Nanog
/
ICM is capable of forming extraembryonic endoderm,
but not trophectoderm, supports the hypothesis that
Nanog is involved in the maintenance of pluripotency
downstream of Oct4-mediated suppression of trophecto-
derm formation and acts to prevent differentiation to
extraembryonic endoderm [8,9,11,12

,13

]. The loss of
Nanog expression in the peri-implantation embryo coin-
cides with downregulation of ICM markers such as Zfp42
(formerly known as Rex1) and Crtr1 (CP2-related transcrip-
tional repressor 1) [14], loss of developmental competence
for extraembryonic endoderm differentiation, and forma-
tion of the primitive ectoderm. Differential requirements
for Oct4 and Nanog between the ICM and the primitive
ectoderm suggest distinct regulatory pathways for enfor-
cement of pluripotency (Figure 1).
Nanog is subsequently upregulated in the posterior pri-
mitive ectoderm from 6.5 days post-coitum (dpc) [8,15].
Whether this expression is associated with pluripotency
in this cell type has not been determined, although it is
worthy of note that overexpression of NANOG in human
ES cells resulted in a primitive ectoderm-like phenotype,
with associated upregulation of FGF5 ( broblast growth
factor 5) and downregulation of REX1 [10

].
The Sox2 (SRY-related HMG box) gene is expressed in
the ICM, early primitive ectoderm, anterior primitive
ectoderm, germ cells and multipotent extraembryonic
ectoderm cells [16,17]. Sox2
/
embryos arrest at a similar
time to Oct4
/
and Nanog
/
embryos, and blastocyst-
like structures are formed, but developmental arrest,
characterised by a lack of primitive ectoderm, occurs
around the time of implantation. Sox2
/
blastocysts
are refractory to ES cell isolation, an observation that
has been interpreted as evidence for a role for Sox2 in the
maintenance of pluripotency in the ICM, although an
earlier role in establishment of the lineage might be
masked by persistence of maternal protein [16].
STAT3 (signal transducer and activator of transcription
3) is activated by gp130-mediated signalling in mouse ES
cells and is required for maintenance of pluripotency in
these cells [18]. Stat3
/
embryos fail around 6.5 dpc,
following establishment of the pluripotent cell lineage,
andit is unclear if lethality is associatedwithfailure of the
pluripotent cells or if it occurs because STAT3 is
required in the visceral endoderm [19]. In contrast to
mouse ES cells, human ES cells do not require gp130
signalling and STAT3 activation for their maintenance
[20,21]. In vivo and in vitro data are therefore inconsistent
with a broad physiological relevance of STAT3 in
pluripotency.
448 Differentiation and gene regulation
Figure 1
The changing repertoire of transcriptional regulators in the pluripotent lineage. Diagrammatic representation of the developmental origin and
organisation of early embryonic lineages, including pluripotent cell types: inner cell mass (ICM) and primitive ectoderm (blue), and non-pluripotent
derivatives trophectoderm (purple), extraembryonic endoderm (green) and somatic cell types (red). The repertoire of transcriptional regulators of
pluripotency (Oct4, Nanog and Sox2) expressed in different pluripotent cell populations is indicated (boxed) at the early blastocyst and 6.5 dpc stages.
Transcription factors implicated in establishment of the trophectoderm (Cdx2), extraembryonic endoderm (Gata6) and somatic (GCNF) lineages are
indicated. LRH-1, which is required for maintenance of Oct4 expression in the epiblast, is also shown (orange). Accumulated data support a model
whereby the repertoire of regulatory proteins defines cell fate and, in the correct combinations, maintains pluripotency. Oct4 is required for the
establishment and maintenance of pluripotency in the ICM, where it acts to prevent formation of trophoblast cells. Nanog and Sox2 are also required
for maintenance of pluripotency in the ICM. Nanog is required to prevent formation of extraembryonic ectoderm at the ICM stage; however, it appears
that Oct4 and Sox2 are sufficient to maintain a pluripotent state in primitive ectoderm in the absence of Nanog.
Current Opinion in Genetics & Development 2006, 16:447454 www.sciencedirect.com
Establishment and maintenance of
pluripotency building the transcription
factor networks
Transcriptional regulation of Oct4, Nanog and Sox2 is
crucial for establishment and maintenance of the plur-
ipotent lineage and provides insights into the regulatory
networks that control pluripotency. Two broad enhancer
regions contribute to Oct4 expression in the early mouse
embryo (Figure 2a). The distal enhancer region is
required for normal levels of Oct4 expression in the
ICM and ES cells [22,23,24

], whereas the proximal

enhancer is required for normal expression of Oct4 in
the primitive ectoderm but is not required for expression
in the ICM[22,23]. The conserved region CR4 (Figure 2),
located within the distal enhancer, is required for Oct4
expression in mouse ES and human embryonal carcinoma
cells and binds both Oct4 and Sox2 [4

,24

,25]. Interac-
tion between these proteins on DNA can result in syner-
gistic regulation of transcription from the Fgf4 enhancer
[26,27] and, on the UTF1 (undifferentiated embryonic cell
Transcriptional control of pluripotency: decisions in early development Johnson, Rathjen and Rathjen 449
Figure 2
Regulation of Oct4 transcription in the pluripotent lineage. (a) Diagrammatic representation of the Oct4 promoter and factors implicated in
regulation of Oct4 expression in the mouse: the distal enhancer (required for ICM expression) and proximal enhancer (required for primitive ectoderm
expression) in conjunction with the proximal promoter are shown. Regions of strong sequence conservation between mouse and human are
represented (conserved regions, CR1CR4) as is the hormone response element (HRE/DR0). Binding sites for Sox2, Oct4, Nanog, LRH-1 and
GCNF, identified by chromatin immunoprecipitation, are also indicated. Specific sites identified as occupied prior to ES cell differentiation are denoted
1A, 1B, 2A and 2B. (b) The occupancy of sites in the Oct4 regulatory region at discrete developmental stages is shown. The occupancy of the
LRH-1 sites in primitive ectoderm is based on a requirement for LRH-1 to maintain Oct4 expression at this stage. The sites of embryonic Oct4
expression are shown in blue. Sox2 is represented by a brown oval, Oct4 a yellow oval, Nanog a green rectangle, LRH-1 an orange rectangle,
and GCNF by paired grey ovals. Abbreviations: EP, epiblast (also known as primitive ectoderm); EX, extraembryonic ectoderm; ICM, inner cell mass;
Me, methylation of DNA; PE, primitive endoderm; TP, trophectoderm; VE, visceral endoderm.
www.sciencedirect.com Current Opinion in Genetics & Development 2006, 16:447454
transcription factor 1) enhancer, correlates with mainte-
nance of the pluripotent state [28

]. RNAi silencing of
Oct4 or Sox2 reduced transcription from a CR4-containing
reporter, supporting a role for these proteins in positive
regulation of Oct4 expression [4

,24

]. Nanog binding has

been demonstrated in both the proximal enhancer and
the distal enhancer in ES cells. Overexpression or RNAi
silencing of Nanog results in upregulation and downregu-
lation of Oct4, respectively, consistent with positive
regulation of Oct4 by Nanog in the mouse. The main-
tenance of Oct4 and pluripotency in ICM and ES cells
thus appears to require cross-regulation between key
transcriptional regulators of pluripotency: the mainte-
nance of Oct4 transcriptional activity in the inner cells
of Oct4
/
blastocysts, as shown by reporter gene expres-
sion, presumably occurs through the activity of other
pluripotency regulators such as Sox2 and Nanog [2].
The LRH-1 (liver receptor homologue 1; also known as
Nr5a2 [nuclear receptor subfamily 5, group A, member 2])
orphan nuclear receptor has been shown to bind within the
proximal enhancer and the proximal promoter, and an
LRH-1 transcript expressed during embryonic develop-
ment has been identied in ES cells [29

,30]. Oct4 expres-

sion was maintained in LRH-1
/
ES cells, and within the
ICM of LRH-1
/
embryos, consistent with the proposed
requirement for distal enhancer but not proximal enhancer
in these cells [29

]. However, LRH-1
/
embryos lacked
Oct4 expression in the primitive ectoderm, consistent with
a requirement for LRH-1 in proximal enhancer function
and Oct4 expression in this tissue. Similarly, during differ-
entiation, LRH-1
/
ES cells downregulate Oct4 more
rapidly than do wild type cells, consistent with failure to
maintain Oct4 expression in a transient primitive ectoderm
differentiationintermediate [29

]. The distinct regulatory

mechanisms responsible for Oct4 expression in the ICM,
ES cells and primitive ectoderm (Figure 2b) might be a
consequence of alternate signalling environments. LRH-1
transactivation has been shown to be mediated by phos-
phorylation through the ERK (extracellular signal-regu-
lated kinase) pathway in HeLa cells [31]. It will be
interesting to determine if a similar signalling pathway is
involved in the regulation of LRH-1 activity in pluripotent
cells.
Nanog promoter activity has been shown to require Oct4
expression in ES cells [4

,32

]. A 178 bp region, contain-

ing a highly conserved Oct4Sox2 consensus binding site,
is required for pluripotent-specic Nanog expression in
mouse and human ES cells [12

], and mutation of the

Oct4 and Sox2 consensus sites in this element disrupted
pluripotent cell-specic promoter function [12

]. Chro-
matin immunoprecipitation has demonstrated the bind-
ing of both Oct4 and Sox2 to the Nanog promoter region in
mouse and human ES cells [32

]. Similarly, siRNAknock-
down of Sox2 in mouse ES cells results in loss of Nanog
promoter activity [32

].
Like Oct4, Nanog expression in pluripotent cells
is cross-regulated by transcription factors implicated
in pluripotency. However, the more dynamic regulation
of Nanog throughout early development, particularly
the downregulation of Nanog expression in the Oct4-
and Sox2-expressing cells of the late blastocyst
and early egg cylinder embryo [8,15], suggests that
alternative, as yet uncharacterised, mechanisms contri-
bute to the regulation of Nanog in pluripotent cells in
vivo. It is of note that using gel shift analysis of the
Oct4Sox2 binding element from the Nanog promoter
with R1 ES cell extract suggested preferential associa-
tion of Oct4 with an unidentied protein, termed PSBP
(pluripotent cell-specic Sox element-binding protein)
[12

].
The Sox2 locus is bound by both Oct4 and Nanog in the
mouse and human and represents one of the few known
conserved targets of these two transcription factors [4

].
Two Sox2 regulatory regions, SRR1 and SRR2, are impli-
cated in ES cell-specic expression of Sox2. SRR2 contains
a composite Oct4Sox2 element, which is required for
SRR2 enhancer activity [33]. Oct4 binding to SRR1 has
also been demonstrated [34]. Reduction or extinction of
Oct4 levels results in loss of function of the SRR2 enhancer
and a resulting reduction in expression [33]. RNAi deple-
tion of Nanog in ES cells reduces Sox2 expression, suggest-
ing positive regulation of Sox2 by Nanog in the mouse [4

].
The existence of Sox2 as both a regulator and a target of
Oct4 and Nanog is consistent with the existence of a
transcriptional network that establishes and maintains
pluripotency.
Loss of pluripotency dismantling the
pluripotent networks
Timing and regulation of loss of pluripotency is crucial
for the orderly establishment of the embryo and the
elimination of potentially teratogenic cells. The variant
homeodomain proteinCdx2 () is preferentially expressed
in and localised to the nucleus of the lagging blastomere
at the two-cell stage and in the descendants of this cell,
which establish the trophectodermal lineage [35

]. The
trophectoderm fails to form in embryos in which Cdx2 is
depleted by siRNA in the lagging blastomere [35

], and
Cdx2
/
embryos show defects in trophectoderm integ-
rity. Neither Oct4 nor Nanog expression is downregulated
in the outer cells of Cdx2
/
blastocysts, suggesting a role
for Cdx2 in the downregulation of these genes during
segregation of the ICM and trophectoderm cell lineages
[36]. Consistent with this, Cdx2 has been shown to
interact with Oct4 on the Oct4Sox2 binding site
of the Oct4 distal enhancer, thereby repressing transcrip-
tion [6

]. Co-expression of Oct4 and Cdx2 in the trophec-

toderm lineage from the 2-cell stage, preceding
differentiation to trophectoderm at 3.5 dpc, suggests
a requirement for additional regulation of this develop-
mental event.
450 Differentiation and gene regulation
Current Opinion in Genetics & Development 2006, 16:447454 www.sciencedirect.com
Extraembryonic endoderm arises from cells of the ICM
shortly after blastocyst formation, and this process is
negatively regulated by Nanog [13

,37

]. In ES cells,
forced expression of Gata4 (GATA-binding protein 4)
or Gata6 results in extraembryonic endoderm formation,
suggesting that Nanog acts to suppress Gata6 transcrip-
tion [38]. However, the transcriptional networks that
regulate Gata6 and other extraembryonic endoderm
genes, and which suppress pluripotency genes such as
Nanog, are unknown. Activation of the Ras pathway in ES
cells also results in upregulation of extraembryonic endo-
derm markers [39] and appears to downregulate Nanog
expression. However, a requirement for Ras pathway
activation for extraembryonic endoderm formation in vivo
has not been demonstrated, and H-ras
/
, N-ras
/
and
K-ras
/
mice all develop normally past the epiblast
stage [4043].
Forced expression of Oct4 in somatic tissues after gas-
trulation results in embryonic lethality around 13.5 dpc
[44]. Repression of Oct4 transcription associated with
establishment of the somatic lineages is dependent on
binding of the orphan nuclear receptor GCNF (germ cell
nuclear factor; also known as Nr6a1) [45

], because
GCNF
/
embryos and ES cells fail to downregulate
Oct4 appropriately at gastrulation and during retinoic
acid-induced differentiation, respectively [46,47]. GCNF
binds to a site within the Oct4 proximal promoter [45

]
that overlaps a binding site for the Oct4 regulator LRH-1
[29

], suggesting that repression requires displacement

of LRH-1. Following transcriptional downregulation by
GCNF, a series of hierarchical epigenetic events,
mediated by G9a and Dnmt3a/3b, is required for stable
maintenance of Oct4 silencing [48]. The Nanog locus also
contains several consensus-binding sites for GCNF,
GCNF binding has been detected at the Nanog locus
during retinoic acid-induced differentiation, and GCNF
/

embryos show inappropriate expression of Nanog at

gastrulation [45

], suggesting a similar mechanism of

transcriptional repression.
The transcriptional cross-regulation of Oct4, Nanog and
Sox2 means that alterations in one regulator will affect the
expression of others within the cell, providing a mechan-
ism for collapsing pluripotency and eliminating poten-
tially teratogenic cells from the embryo. The sensitivity
of this network to even minor perturbations is illustrated
by experiments in ES cells that suggest that precise levels
of Oct4 are required for pluripotency, with differentiation
resulting from both increased and decreased Oct4 expres-
sion. A reduction in Oct4 levels, either by approximately
50% using genetic manipulation or by approaching 90%
using siRNA, leads to the formation of trophectoderm
and, in many approaches, primitive endoderm from
mouse and human ES cells [3,11,49

]. Increasing levels
of Oct4 by less than two-fold results in the formation of
primitive endoderm and mesoderm [3,5,49

]. It has been
suggested from these approaches that the ratio of Oct4 to
other factors within the cell is crucial for the maintenance
of pluripotency, and disturbance of the ratio results in
differentiation. Oct4 DNA binding activity is not
required for differentiation when overexpressed, suggest-
ing that Oct4 overexpression results in sequestration of
other regulators within the cell [5].
Conclusions
Cross-regulation of Oct4, Sox2 and Nanog by their gene
products reveals a regulatory network in the ICM and ES
cells that is required for pluripotency. Loss of pluripo-
tency is induced by the activation of pathways, such as
those associated with Cdx2 and GCNF function, which
perturb the levels of pluripotent regulators and impose
alternate cellular fates. The role of this network in the
regulation of other genes in pluripotent cells is indicated
by the analysis of genomic regions that bind Oct4: chro-
matin immunoprecipitation and DNA microarray (ChIP-
chip) analysis of OCT4-bound DNAfromhuman ES cells
revealed 623 bound promoter regions, over 353 of which
are also bound by SOX2 and NANOG [50

]. However, a
comparison of the genomic regions bound by Nanog and
Oct4 between mouse and human in ES cells revealed an
unexpectedly low number of common targets between
species [4

]. The common regulation of Sox2 in both

systems provides condence that equivalent regulatory
pathways exist and function in pluripotent cells of human
and mouse.
Remarkably little is known about links connecting the
extracellular environment and the regulation of pluripo-
tency at the level of transcription extracellular factors
and intracellular signalling pathways that directly regulate
the activity of Oct4, Sox2 and Nanog are poorly under-
stood. The analysis of mouse ES cells, which have a non-
physiological reliance on gp130 signalling and STAT3
activation for the maintenance of pluripotency [51], has
shed little light on the extracellular molecules required
for pluripotency maintenance in vivo. Maintenance of
pluripotency in human ES cells requires signalling from
members of the FGF and TGFb (transforming growth
factor b) families of growth factors [52] and might prove to
be a more physiological model for understanding the
role of extracellular signalling and intracellular signal
transduction in pluripotency in the ICM. Deciphering
these pathways holds promise for generating improved
methodologies for maintenance and proliferation of
pluripotent human ES cells in vitro.
A complete understanding of pluripotency requires that
our analysis be extended to the primitive ectoderm,
which contains a distinct repertoire of pluripotency reg-
ulators activated by alternate signalling pathways. Inves-
tigation of pluripotency regulation in the primitive
ectoderm is hampered in vivo by the peri-implantation
lethality associated with the loss of several pluripotency
Transcriptional control of pluripotency: decisions in early development Johnson, Rathjen and Rathjen 451
www.sciencedirect.com Current Opinion in Genetics & Development 2006, 16:447454
regulators and the difculty of manipulating the embryo
during implantation. Early primitive ectoderm-like
cells are formed from ES cells and share many features
with the embryonic primitive ectoderm, including gene
expression patterns and developmental potential [14,53
55]. These cells might prove valuable as a surrogate for
genetic and biochemical analysis of pluripotency in pri-
mitive ectoderm, equivalent to the important role played
by ES cells in deciphering pluripotency in the ICM.
Comparison of the differing signalling networks that
regulate pluripotency in these lineages is expected to
provide insight into the core requirements for enforce-
ment of the pluripotent state.
Update
A recent study demonstrates that the ICM at E3.5 is
composed of cells that express either Nanog or Gata6 and
that are effectively already restricted to an epiblast or
primitive endoderm lineage, respectively, at this early
stage [56

]. Similar restricted gene expression within a

population, which pre-empts later developmental events,
was observed for specication of trophectoderm cell fate
by Cdx2 expression in one cell of the two-cell embryo
[35

].
Arecent study reported that regions of H3K4 methylation
within overlapping regions of extended H3K27 trimethy-
lation are characteristically associated with active and
inactive chromatin states, respectively, in ES cells
[57

]. Occurring frequently at Oct4 and Nanog target

genes, these regions are hypothesized to correspond to
developmentally regulated genes that are silenced in ES
cells. Lee et al. [58

] identied more than 200 human

genes that were bound by the Polycomb repressive com-
plex 2 (PRC2) subunit SUZ12, display H3K27 trimethy-
lation and are transcriptionally repressed in human ES
cells. A signicant subset of these genes was occupied by
OCT4, SOX2 and NANOG. Consistent results were
observed in the mouse [59

]. Disruption of H3K27
methylation resulted in de-repression of most target
genes examined. These results suggested that ES cell
pluripotency requires repression by Polycomb com-
plexes. A similar epigenetic prole of repressed genes
was reported by Azuara et al. [60

], and H3K27 methyla-

tion deciency also resulted in premature expression of
repressed genes. These data suggest that specic genes
are primed for expression in pluripotent cells and that
opposing epigenetic marks are involved in this process
that occurs at many genes that are also targets of Nanog,
Oct4 and Sox2.
Acknowledgements
The authors would like to thank members of the Rathjen laboratory, past
and present, for discussions and contributions that have shaped the thinking
behind this review. Research within the Rathjen laboratory is funded by the
Australian Stem Cell Centre and the ARC Special Research Centre for the
Molecular Genetics of Development. Because of space limitations and the
breadth of this review the work of many authors who have made valuable
contributions to this eld has not been cited.
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454 Differentiation and gene regulation
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