Epiblast Development

Bernard AJ Roelen, Utrecht University, Utrecht, The Netherlands

© 2018 Elsevier Inc. All rights reserved.

Formation of the Mammalian Blastocyst

After fertilization of a mammalian oocyte, the formed totipotent zygote starts a series of cleavage divisions. During the first divisions
the blastomeres remain totipotent, but this potential gradually decreases upon further development with the timing of
developmental restriction dependent on the animal species. The first lineage segregation that occurs is the formation of outer
and inner cells around the morula stage. The outer cells form an epithelial sheet of trophectoderm that is important for
implantation into the uterus, the fetal maternal interaction and formation of the non-maternal components of the placenta.
Pump-like proteins within this epithelium facilitate the formation of a fluid filled cavity, called blastocoel, transforming the embryo
into a blastocyst. At this stage the embryo consists of two cell types, an outer trophectoderm layer and an inner cluster of cells
referred to as inner cell mass (ICM) facing the blastocoelic cavity. The POU transcription factor OCT3/4 is expressed at high levels
in the ICM and is critical for the formation of the pluripotent cell population in the early embryos, while the trophectoderm cells
express relatively high levels of the transcription factor CDX2. OCT3/4 and CDX2 not only function as transcription factors, but also
as repressors of each other’s gene transcription. It is the ICM that will give rise to the epiblast.

Second Lineage Segregation

After the first lineage segregation a second cell fate specification event occurs that segregates the ICM into epiblast, also called
primitive ectoderm, and hypoblast, also called primitive endoderm. In the mouse embryo the primitive endoderm after
implantation differentiates further to visceral endoderm. The epiblast consists of pluripotent cells that will form the fetus while
the primitive endoderm, an amniote specific cell type, although originating from the ICM will predominantly give rise to
extra-embryonic tissues such as the yolk sac that provides nutrients to the embryo. During gastrulation most of the visceral
endoderm disperses as the epiblast-derived definitive gut endoderm intercalates (Kwon et al., 2008). The primitive endoderm is
not only important for its nutritional role as yolk sac, but it also plays an essential role in the organization of the epiblast and
therefore the establishment of the body plan.
The transcription factors NANOG and GATA6 control the second lineage segregation of the ICM. Initially, from the 8-cell stage
onwards, both transcription factors are present in blastomeric nuclei. Soon after formation of the ICM however, NANOG and
GATA6 display a mutually exclusive “salt and pepper”-like expression pattern, with some ICM cells expressing predominantly
NANOG while other ICM cells predominantly express GATA6 (Chazaud et al., 2006). This distribution of NANOG and GATA6
appears to be stochastic in a position-independent manner. NANOG-expressing cells will sort out to become the pluripotent
epiblast, while GATA6 expressing cells will form an epithelial layer of primitive endoderm overlying the NANOG-positive epiblast
and facing the blastocoel. Indeed mice with a genetic deletion for Gata6 initially do form primitive endoderm but this fails to be
specified. After segregation the cells also become morphologically distinct. In mice, the dose of GATA6 in the early ICM regulates the
specification of primitive endoderm as demonstrated by a reduction of primitive endoderm cells in Gata6 heterozygous embryos
and defective primitive endoderm in null mutants of Gata6. Conversely, mouse embryos genetically deficient for Nanog fail to form
pluripotent epiblast cells and only produce primitive endoderm cells, besides TE (Mitsui et al., 2003). GATA6 and NANOG repress
each other’s transcription, and GATA6 seems to be working as an on/off switch since NANOG levels do not increase in the absence
of GATA6 (Schrode et al., 2014). After initial expression, the decision for exclusive Nanog or Gata6 expression is subsequently driven
by fibroblast growth factor (FGF) and mitogen-activated protein (MAP) kinase signaling. Pre-implantation mouse embryos treated
with an inhibitor of FGF receptor activity or an inhibitor of MAP kinase signaling blocked primitive endoderm formation, with all
ICM cells expressing NANOG. When embryos were on the other hand cultured in the presence of FGF4, all ICM cells became
GATA6-positive (Yamanaka et al., 2010). Exposure of Gata6 null mutants to exogenous FGF4 could not restore the specification
of primitive endoderm, indicating that GATA6 is required for primitive endoderm specification by FGF signaling (Schrode et al.,
2014).
In other mammals such as cattle and humans, NANOG and GATA6/GATA4 are initially also expressed in the precursors of
epiblast and primitive endoderm in a similar “salt-and-pepper” expression pattern, and later in the epiblast and primitive endoderm
respectively after segregation. The signals that regulate NANOG and GATA6 expression are different however. The formation of
human GATA6-positive primitive endoderm is for instance not dependent on MAP kinase signaling (Kuijk et al., 2012). In bovine
embryos, inhibition of MAP kinase signaling only partially inhibited primitive endoderm formation indicating that although FGF/
MAP kinase signaling inhibits NANOG expression, other signals directly stimulate GATA6 expression.
Exactly when the cells are irreversibly committed to either epiblast or primitive endoderm is not known. ICM cells of mouse
embryos exhibited plasticity in their developmental fate even after they had established exclusive NANOG or GATA6 expression
(Yamanaka et al., 2010). Isolated cells from the epiblast of day 4.5 post-coitum mouse embryos contribute to epiblast or derivatives
when injected into the blastocoelic cavity of a host blastocyst, and primitive endoderm cells will only give rise to extra-embryonic
endoderm, indicating that at this time epiblast and primitive endoderm are already committed to their lineages. Experiments with

Encyclopedia of Reproduction, 2nd edition, Volume 3 https://doi.org/10.1016/B978-0-12-801238-3.64480-X 341

342 Embryogenesis j Epiblast Development

bovine embryos revealed that cells are remarkably flexible and can change fate from becoming NANOG-expressing to GATA6-
expressing and vice versa at least until the late blastocyst stage, at day 8 of in vitro culture (Kuijk et al., 2012).

X-Chromosome Inactivation

Sex determination in mammals, with exception of that in monotremes, is determined by the sex chromosomes with homozygous
XX in females and heterozygous XY in males. The Y chromosome is much smaller and contains fewer genes that the X chromosome,
which could lead to an overexpression of X-linked genes in females. A solution for this phenomenon is a type of dosage
compensation by inactivation of one of the X chromosomes in female cells. Central in X-chromosome inactivation is the long
noncoding RNA Xist that is expressed from the X-chromosome that is to be silenced. Xist accumulates on the X-chromosome
from which it is expressed and recruits remodeling complexes that repress transcription from this chromosome.
In female cleavage stage mouse embryos, imprinted inactivation of specifically the paternal X-chromosome occurs. At the
blastocyst stage, X chromosome reactivation takes place such that in the inner cell mass both X-chromosomes are active in female
embryos. Upon formation of the epiblast, X-chromosome inactivation is reestablished but this time in a stochastic fashion in the
epiblast and derivatives thereof. In the mouse conceptus, imprinted paternal X chromosome inactivation is maintained in the
trophectoderm and primitive endoderm.
The pattern of X-chromosome inactivation in different mammalian species seems to vary in the cleavage stages, but in the
epiblast of most studied species random X-chromosome inactivation takes place (Dupont and Gribnau, 2013). Differences between
species are mostly prominent in the lineages that are not derived from the epiblast in particular the trophectoderm and primitive
endoderm. It has been suggested that in mice, rats and cattle the X chromosome inactivation is imprinted in the extraembryonic
lineages, while in primates, including human, rabbit and equine species, inactivation of the X chromosome in non-epiblast derived
tissues is also random (Dupont and Gribnau, 2013).

Organization of the Epiblast

Before the formation of the definitive fetal germ layers during gastrulation, the human embryo already has implanted into the
uterine tissue, making it less accessible for research, and therefore limited information is available regarding this species. In that
respect embryos from species such as cattle or horse are more accessible, but high breeding and housing costs together with limited
genetic and biochemical tools are disadvantages of these species. Most of the data available on epiblast differentiation therefore
have come from studies with mice. In addition, genetic manipulation of pluripotent embryonic stem cells combined with chimaera
formation has enabled the generation of gene knock-out and knock-in mice which has substantiated enormously to our current
understanding of gene function and pattern formation in the mouse.
The epiblast of mouse embryos forms a radially symmetric cup of epithelial cells, with the extraembryonic ectoderm as an
inverted cylinder on top and surrounded on the outside by a single-cell layered visceral endoderm, a derivative of the primitive
endoderm (Fig. 1A). The epiblast of most mammals including humans is formed as a flat epithelial sheet of cells, similar to the
epiblast of chicken embryos, with the primitive endoderm adjacent/underneath, hence the alternative name hypoblast (Fig. 1B).
Up until this stage the epiblast is still radially symmetric, but for induction of the definitive germ layers the establishment of ante-
rior–posterior polarity is essential.
The first sign of anterior–posterior polarity of the conceptus has been detected in the primitive/visceral endoderm of mouse
embryos. This axial information in the endoderm is subsequently translated to the epiblast. The endoderm furthest away from
the extraembryonic region, the distal visceral endoderm (DVE), derives from a few cells expressing both Gata6 and Lefty1 in the
late blastocyst. The position of these cells most likely indicates the future mid-anterior side of the conceptus (Takaoka and Hamada,
2012). These cells and progenitors move proximally towards the proximal anterior part of the egg cylinder, triggering global
movement of the visceral endoderm. Distal visceral endoderm cells will subsequently lose Lefty1 expression and migrate
laterally-distally away from the proximal anterior region. Simultaneously, prospective anterior visceral endoderm (AVE) cells are
newly induced at the distal part of the egg cylinder and migrate towards the anterior side. The function of the DVE is not well known,
while the role of AVE in patterning the epiblast has been well established.
The proximal epiblast starts to express proteins such as NODAL and WNT3a. Initially, NODAL is expressed throughout the entire
epiblast. The AVE cells on the other hand secrete the NODAL inhibitors LEFTY1 and CER1 and the Wnt inhibitor DKK1, and since
Wnt and NODAL are not repressed in the posterior part an anteroposterior axis is established in the epiblast (Perea-Gomez et al.,
2002). WNT3A and NODAL in the proximal-posterior part of the epiblast induce expression of the T-box transcription factor BRA-
CHYURY and delamination of epiblast cells through the primitive streak to form the definitive germ layers.

Non-Rodent Species

Compared to early cup-shaped embryos of mice and rats, embryos of other species, including the human, have a sheet-like
appearance, much more resembling a peri-gastrulating chick embryo. In the mouse embryo the part of the trophectoderm that
 Embryogenesis j Epiblast Development 343

Fig. 1 Schematic representation of lineage specification in developing mouse (A) and human (B) embryos. E refers to embryonic day; proximal
(pr)-distal (di and anterior (a)–posterior (p) axes are indicated. In the human embryo the anterior–posterior axis is perpendicular to the image.

is in contact with the inner cell mass, the polar trophectoderm, grows out to become the extra-embryonic ectoderm. In various
species however, including rabbits, ungulates, cats and dogs, the trophectoderm overlying the epiblast is referred to as Rauber’s layer
and will degenerate. After disappearance of the Rauber’s layer, the epiblast is directly exposed to the uterine milieu and connects
laterally with the remaining mural trophoblast to form a continuous sheet. In other animals that form a sheet-like epiblast, such
as primates, the polar trophectoderm establishes the connection with the uterine epithelium for implantation.
Most information from the epiblast of non-rodent species comes from ungulates such as the pig and cattle. At the
pre-implantation state the sheet-like epiblast of ungulates forms a pseudostratified epithelium with tight junctions, desmosomes
and a clear basement membrane separating it from the hypoblast (Hall et al., 2010). Ungulate embryos do not implant immediately
upon arrival in the uterus and hatching from the zona pellucida; instead their trophectoderm expands dramatically changing the
morphology of the conceptus from spherical to ovoid to tubular to filamentous (Fig. 2).
In embryos with a flat epiblast, extra-embryonic mesoderm cells have been identified that later contribute to chorion. The origin
of this type of mesoderm has been debated since mesenchymal cells have been identified relatively early in development. It has been
suggested that the extra-embryonic mesoderm cells delaminate from the early primitive streak, similar to what has been observed in
the mouse. On the other hand, data from monkey embryos suggest that the extra-embryonic mesoderm cells differentiate before
gastrulation from the reticulated primitive endoderm.

Gastrulation

The pluripotent epiblast is the source of most if not all fetal and adult lineages, both somatic and germline. In addition, extraem-
bryonic mesoderm is derived from the epiblast, as well as the ectodermal part of the amnion. Although these extra-embryonic
tissues do not contribute to the fetus, they are crucial for survival of the fetus by providing protection and nutrition. Gastrulation
344 Embryogenesis j Epiblast Development

(A) p (B)

te

m e

e
ve a

(C) (D)
te
e
ps

p
e a
te

Fig. 2 Microphotographs of (A) a sectioned cup-shaped E6.5 mouse embryo; (B) E10 porcine embryo, dorsal view; (C) E10 porcine embryo lateral
view (D) E14 horse embryo. a, anterior; e, epiblast; m, mesoderm; p, posterior; ps, primitive streak; te, trophectoderm; ve, visceral endoderm.

is a decisive moment in development as it transforms the epiblast from a single layer of similar cells into a multilayered embryo of
which the different layers have made unidirectional choices. The process of gastrulation commences with coordinated growth of the
epiblast and movement of epiblast cells towards the primitive streak followed by differentiation and ingression of mesendodermal
cells. The internalized cells subsequently converge to the midline and extend along the antero-posterior axis upon undergoing an
epithelial to mesenchymal transition and form the mesoderm and the definitive endoderm. The descendants of epiblast cells that do
not pass through the primitive streak will constitute the ectodermal lineages (Lawson et al., 1991; Fig. 2).
Proteins belonging to the fibroblast growth factor (FGF) family have been identified as key regulators of the epithelial to mesen-
chymal transition during gastrulation. Already before a primitive streak is visually present in the mouse embryo, Fgf8 is expressed in
the presumptive dorsal part of the proximal epiblast. Mouse embryos genetically deficient for Fgf8 besides a lack of FGF8 fail to
express FGF4. As a result, mesodermal cells are formed but they fail to migrate away from the streak and instead accumulate at
the proximal posterior side of the embryo. A similar clustering of cells at the posterior streak has been observed in embryos deficient
for FGF receptor 1 indicating that indeed FGF signaling is crucial for partitioning of the germ layers at the primitive streak. FGF
signaling induces the expression of the zinc finger transcription factor SNAIL. Cells that express high levels of SNAIL ingress through
the streak while cells that express high levels of the X-linked Sox3 gene maintain their position in the epiblast. SNAIL and SOX3
repress each other’s transcription, thereby ensuring proper subdivision of the epiblast into definitive germ layers (Acloque et al.,
2011). Cells of the epiblast have an epithelial character with apical-basal polarity and formation of a basal membrane. Ingression
of cells through the primitive streak requires that these break the basal membrane and undergo an epithelial to mesenchymal tran-
sition. The formed mesenchymal cells lose cell polarity and cell–cell adhesion but gain migratory and invasive properties. This tran-
sition is independent of the differentiation to mesoderm or endoderm, as it has been demonstrated that mouse embryos genetically
deficient of Snail still form mesoderm but the cells retain epithelial characteristics.
How do cells change from an epithelial sheet, the epiblast, to migratory mesenchymal cells? One important route for this
transition is loss of the adhesion protein E-cadherin, a protein that is important for maintenance of adhesion junctions and
epithelial tissue. By functioning as a transcriptional repressor, SNAIL can bind to the promoter of the gene coding for E-cadherin
leading to a downregulation of E-cadherin expression. The epiblast cells lose their epithelial character and gain the mesenchymal
phenotype required for gastrulation.

Fate Map

The developmental capacity of the rodent epiblast, after implantation and the formation of an egg cylinder and a pro-amniotic
cavity, has been studied by grafting epiblasts under the kidney capsule of adult animals and analysis of the resulting teratomas.
The explanted epiblast cells underwent an epithelial to mesenchymal transition as observed in cells migrating through the primitive
 Embryogenesis j Epiblast Development 345

streak and could give rise to descendants of all three germ layers. Experiments by which epiblast cells were meticulously labeled,
fragmented and subsequently grafted to other embryos indicated that although the cells are pluripotent, their progeny follows
a determined developmental fate based on their position in the epiblast. Labeling individual epiblast cells of pre-streak and early
primitive streak mouse embryos with horseradish peroxidase followed by in vitro culture revealed that epiblast cells differentiate
and migrate in a reproducible and coordinate way. This enabled the construction of a detailed fate map of presumptive embryonic
and extra-embryonic germ layers (Lawson et al., 1991). Cells of the lateral epiblast will primarily form embryonic mesoderm while
cells from the proximal region will predominantly form extra-embryonic mesoderm and primordial germ cells. Definitive
endoderm originates from a posterior region around the (future) streak.
Clonal analysis further revealed that the primordial germ cells also originate from but are not yet lineage restricted in the
epiblast. Cells in the proximal epiblast are induced by BMP signals from the extra-embryonic ectoderm to form precursors of
the primordial germ cells and allocation takes place during gastrulation in the mesoderm.
Clonal analysis also demonstrated that extensive cell mixing of epiblast cells occurred by morphogenetic movements and that
descendants of single cells can contribute to different germ layers (Lawson et al., 1991). The observation that precursor cells for
different lineages are present at specific regions of the embryo does not necessarily mean that these cells are irreversibly allocated
or restricted in their developmental capacity. Indeed heterotopic explant experiments with pre- and early streak embryos
demonstrated that it is the position of the cells, not the intrinsic characteristics of the cells that determines their fate (Tam and
Zhou, 1996).

Epiblast Cell Flexibility

It has been demonstrated that, at least in the mouse, epiblast cells can transmigrate into the (primitive) visceral endoderm layer
already before gastrulation. This is remarkable, since at this stage the epiblast and primitive endoderm are separated by a basement
membrane. Exactly how cell change fate and migrate though the basement membrane is unknown (Hiramatsu et al., 2013).
Whether epiblast transmigration to the visceral endoderm layer occurs specifically at the distal tip of the mouse egg cylinder remains
unclear. Alternatively, it has become clear that in the mouse, primitive endoderm cells are not all displaced by differentiating
epiblast cells that ingress through the primitive streak (Kwon et al., 2008). Rather than displacing and pushing the visceral endo-
derm cells proximally, the ingressing epiblast cells mix extensively with the visceral endoderm cells. As a result the gut tube, partic-
ularly the hindgut, is constituted by both visceral endoderm and epiblast-derived cells. Whether the flexibility of epiblast and
primitive endoderm is unique to mouse embryos remains to be established.

Human Epiblast

Understanding human epiblast development beyond the blastocyst stage has been hampered by the invasive implantation into the
uterus. Particularly the formation of the epiblast and the events associated with gastrulation has been a black box for the most part.
The development of in vitro implantation models has allowed the in vitro transition from pre/to postimplantation development,
although primitive streak formation and gastrulation has not been studied in these models due to ethical and legal restrictions
(Shahbazi et al., 2016). Indeed in vitro culture of human embryos beyond 14 days or the beginning of primitive streak formation
is not allowed in many countries. Questions that remain unresolved are the origin of the extraembryonic mesoderm, origin of the
primordial germ cells and the flexibility of epiblast cells to contribute to hypoblast cells prior to primitive streak formation.

Levels of Pluripotency

The epiblast cells of preimplantation embryos, in the mouse between days 3.5 and 4.5 post-coitum, are by definition pluripotent
since these cells can give rise to all somatic cell lineages and the germ cells. With the development of the embryo the pluripotency
levels gradually diminish, which can be observed for instance by the chromatin state of the cells and the gene expression profile.
Shortly after implantation, the pluripotency levels of mouse epiblast cells subside and after gastrulation and accompanying terminal
germ layer choice the cells have lost their pluripotent character. In developing embryos, the pluripotency state is very transient as the
function of the epiblast is to form a fetus. The pluripotency levels of pre- and post-implantation epiblast cells has been “captured”
by in vitro culture, and under the right conditions the cells can maintain their pluripotency levels as embryonic stem (ES) cells and
epiblast stem cells (EpiSCs) respectively. The pluripotency level of preimplantation epiblast and ES cells is considered to be unre-
stricted and is therefore referred to as “naïve” while that of post-implantation epiblast and EpiSCs are already “primed” for lineage
commitment. Interestingly, human ES cells share more characteristics with mouse EpiSCs and are therefore also considered to be at
a primed state of pluripotency. Indeed naïve ES cells form tightly adhering dome-shaped aggregates reminiscent of the blastocyst
stage epiblast, while EpiSCs form flat colonies similar to the epithelialized post-implantation epiblast. Naïve pluripotent cells are
characterized by high levels of OCT4, NANOG and KLF. Other indications of an open chromatin state and active transcription are
large regions of hypomethylated DNA, permissive H3K4 trimethylation and in female cells two active X-chromosomes. Conversely
primed epiblast cells, including EpiSCs, also express high levels of OCT4 and NANOG, but low levels of KLF4, and start to express
346 Embryogenesis j Epiblast Development

BRACHYURY, SOX17 and GATA4/6. In addition the EpiScs display higher DNA methylation levels, and repressive H3K27 trime-
thylation. In contrast to the naïve state, in female primed epiblast cells one of the X-chromosomes is inactivated. ES and EpiSCs have
revealed that alternate enhancer usage dictates the pluripotency character of these cells and it can be assumed that similar differences
in enhancer activity states are present in pre- and post-implantation epiblast cells.
During development, the naïve and primed pluripotency states of the epiblast are very transient. However, when the environ-
mental conditions are suboptimal for pregnancy, embryos can arrest in development before implantation, a state known as
diapause or delayed implantation. It is thought that in this diapause state the naïve pluripotent epiblast cells self-renew, very
much similar to the behavior of ES cells.

Conclusion

The epiblast is the pluripotent primary lineage that will form the definitive germ layers in a complex process of differentiation and
morphogenetic movements called gastrulation. After gastrulation the developmental capacity of the differentiating cells is restricted
to the residing germ layer. Most of what we know about epiblast development comes from mouse data. However, the mouse
epiblast is uniquely cup-shaped, opposed to the planar morphology of the epiblast of most mammalian species. Studies with
pluripotent stem cells and in vitro cultured embryos may in the future contribute to our knowledge of the epiblast.

Acknowledgments

Susana Chuva de Sousa Lopes is thanked for critically reading the manuscript. Apologies to colleagues whose work could not be mentioned or cited
due to space limitations.

References

Acloque, H., Ocana, O. H., Matheu, A., Rizzoti, K., Wise, C., et al. (2011). Reciprocal repression between Sox3 and snail transcription factors defines embryonic territories at
gastrulation. Developmental Cell, 21, 546–558.
Chazaud, C., Yamanaka, Y., Pawson, T., & Rossant, J. (2006). Early lineage segregation between epiblast and primitive endoderm in mouse blastocysts through the Grb2-MAPK
pathway. Developmental Cell, 10, 615–624.
Dupont, C., & Gribnau, J. (2013). Different flavors of X-chromosome inactivation in mammals. Current Opinion in Cell Biology, 25, 314–321.
Hall, V. J., Jacobsen, J. V., Rasmussen, M. A., & Hyttel, P. (2010). Ultrastructural and molecular distinctions between the porcine inner cell mass and epiblast reveal unique
pluripotent cell states. Developmental Dynamics, 239, 2911–2920.
Hiramatsu, R., Matsuoka, T., Kimura-Yoshida, C., Han, S. W., Mochida, K., et al. (2013). External mechanical cues trigger the establishment of the anterior-posterior axis in early
mouse embryos. Developmental Cell, 27, 131–144.
Kuijk, E. W., van Tol, L. T., Van de Velde, H., Wubbolts, R., Welling, M., et al. (2012). The roles of FGF and MAP kinase signaling in the segregation of the epiblast and hypoblast cell
lineages in bovine and human embryos. Development, 139, 871–882.
Kwon, G. S., Viotti, M., & Hadjantonakis, A. K. (2008). The endoderm of the mouse embryo arises by dynamic widespread intercalation of embryonic and extraembryonic lineages.
Developmental Cell, 15, 509–520.
Lawson, K. A., Meneses, J. J., & Pedersen, R. A. (1991). Clonal analysis of epiblast fate during germ layer formation in the mouse embryo. Development, 113, 891–911.
Mitsui, K., Tokuzawa, Y., Itoh, H., Segawa, K., Murakami, M., et al. (2003). The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells.
Cell, 113, 631–642.
Perea-Gomez, A., Vella, F. D., Shawlot, W., Oulad-Abdelghani, M., Chazaud, C., et al. (2002). Nodal antagonists in the anterior visceral endoderm prevent the formation of multiple
primitive streaks. Developmental Cell, 3, 745–756.
Schrode, N., Saiz, N., Di Talia, S., & Hadjantonakis, A. K. (2014). GATA6 levels modulate primitive endoderm cell fate choice and timing in the mouse blastocyst. Developmental Cell,
29, 454–467.
Shahbazi, M. N., Jedrusik, A., Vuoristo, S., Recher, G., Hupalowska, A., et al. (2016). Self-organization of the human embryo in the absence of maternal tissues. Nature Cell Biology,
18, 700–708.
Takaoka, K., & Hamada, H. (2012). Cell fate decisions and axis determination in the early mouse embryo. Development, 139, 3–14.
Tam, P. P., & Zhou, S. X. (1996). The allocation of epiblast cells to ectodermal and germ-line lineages is influenced by the position of the cells in the gastrulating mouse embryo.
Developmental Biology, 178, 124–132.
Yamanaka, Y., Lanner, F., & Rossant, J. (2010). FGF signal-dependent segregation of primitive endoderm and epiblast in the mouse blastocyst. Development, 137, 715–724.