Developmental Biology 401 (2015) 17–24

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Developmental Biology
journal homepage: www.elsevier.com/locate/developmentalbiology

Epiblast morphogenesis before gastrulation
Guojun Sheng nLaboratory for Early Embryogenesis, RIKEN Center for Developmental Biology, Kobe 650-0047, Hyogo, Japan

art ic l e i nf o

a b s t r a c t

Article history:
Received 27 July 2014
Received in revised form
24 September 2014
Accepted 8 October 2014
Available online 19 October 2014

The epiblast is a single cell-layered epithelium which generates through gastrulation all tissues in anamniote embryo proper. Speciﬁcation of the epiblast as a cell lineage in early development is coupledwith that of the trophoblast and hypoblast, two lineages dedicated to forming extramebryonic tissues.
The complex relationship between molecular speciﬁcation and morphogenetic segregation of thesethree lineages is not well understood. In this review I will compare the ontogeny of epithelial epiblast indifferent amniote groups and emphasize the diversity in cell biological mechanisms employed by eachgroup to reach this conserved epithelial structure as the pre-requisite for gastrulation. The limitations ofassociating cell fate with cell shape and position will also be discussed. In most amniote groups, bipotential precursors for the epiblast and hypoblast, similar to the inner cell mass in the eutherianmammals, are not associated with an apolar, inside location in the blastocyst. Conversely, a blastocyst
cell with epithelial morphology and superﬁcial location is not indicative of its trophoblast fate. The polar
trophoblast is absent in all amniotes except for the eutherian mammals. In the avian, reptilian andeutherian groups, epithelialization of the epiblast occurs after its fate speciﬁcation and involves amesenchymal-to-epithelial transition (MET) process, whereas in the monotremes and marsupials, preepiblast cells adopt an epithelial morphology prior to their commitment to the epiblast fate. The
conservation of an epithelialized epiblast is viewed as an adaptation to evolutionary constraints placedon pre-gastrulation ectoderm in the ancestral amniote. The relationship between epiblast MET and
epiblast pluripontency will also be discussed. Whether such an MET/epithelialization process isadvantageous for the self-renewal and/or differentiation of human epiblast stem cells in vitro is unclear.
& 2014 Elsevier Inc. All rights reserved.

Keywords:
AmnioteEpiblast
Morphogenesis
Hypoblast
Trophoblast
Mesenchymal–epithelial transition

Introduction
The amniotes are a monophyletic clade of vertebrate animals consisting of three living groups: the mammals, reptiles and birds. The
reptiles and birds, together with the prototherian mammals (monotremes), share many early developmental features, whereas the early
embryogenesis in the metatherian (marsupial) and eutherian (placental) mammals are highly derived due to yolk reduction and anemphasis on placentation for nutritional supply. All amniote species,
however, have an extraembryonic tissue organization that is evolutionarily conserved (Ferner and Mess, 2011; Sheng and Foley, 2012) and its
genesis underlies key morphogenetic processes during their early
development. Future intraembryonic tissues originate from a part ofthe early amniote embryo called the epiblast, a single cell-layered epithelial sheet which is the starting material for gastrulation. Morphogenesis leading to the formation of this epithelial epiblast shows diverse
strategies used in different amniote groups to reach the earliest
phylogentically-conserved developmental stage. Understanding epiblastnCorresponding author. Fax: þ 81 78 306 3146.
E-mail address: sheng@cdb.riken.jp

http://dx.doi.org/10.1016/j.ydbio.2014.10.003
0012-1606/& 2014 Elsevier Inc. All rights reserved.

morphogenesis in model organisms serves as a basis for unraveling its
molecular regulation. Yet caution is needed when extrapolating species
speciﬁc features and mechanisms to other amniotes. In this review I
will provide a brief overview of major modes of epiblast formation in
the amniotes. Emphasis will be given to several features in the mousemodel which are not universally conserved, including the location of
the epiblast precursor cells, the association of trophoblast fate with theearliest epithelial structure in the blastocyst, and the relationshipbetween the polar trophoblast and the inner cell mass (ICM). I will
also underscore the difference between epiblast fate speciﬁcation andepiblast epithelialization (the process of collective polarization of
epiblast-fated cells to form an epithelium) (Nakaya and Sheng, 2013),
and emphasize the importance of the MET process accompanying theﬁnal step of epithelial epiblast formation in several amniote groups.
Epiblast formation in the mouse
Early development in the mouse has been discussed in detail inseveral recent reviews (Artus and Chazaud, 2014; Posfai et al., 2014;
Schrode et al., 2013; Stephenson et al., 2012). The following summaryis based on these reviews and on a recent paper describing the

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G. Sheng / Developmental Biology 401 (2015) 17–24

Fig. 1. Morphogenetic diversity in epiblast formation. Representative features of morphogenetic events leading to the formation of the epiblast in several amniote groups.
Red: epiblast; Yellow: hypoblast; Blue: trophoblast. (A) Mouse; (B) human; (C) rabbit; (D) Elephatulus; (E) Hemicentetes; (F) marsupials; (G) monotremes; (H) birds andreptiles. In ((D) and (E)) features after the segregation of the epiblast and hypoblast precursors are omitted.

epithelialization process of the epiblast (Bedzhov and ZernickaGoetz, 2014). After blastocyst formation, the ICM differentiates into
mixed populations of precursor cells for the epiblast and the primitive endoderm (referred to as hypoblast in this review) (Fig. 1A,left). The hypoblast precursors sort out and polarize ﬁrst to form anepithelialized sheet. The remaining apolar epiblast cells are stimulated to polarize by signals from the basement membrane of two
surrounding epithelia (the hypoblast and trophoblast) (Fig. 1A, middle), resulting in the formation of an epiblast rosette with its apicallumen at the center. The lumen expands and forms the rudimentary
proamniotic cavity. The epithelialized epiblast expands and fuseswith the epithelializing chorionic ectoderm precursors derived fromthe polar trophoblast, and as a consequence further expanding theproamniotic cavity (Fig. 1A, right). It is worth noting that the border
between the epiblast and trophoblast-derived chorionic ectoderm is

not the embryonic–extraembryonic boundary as often depicted,
because all of the ectoderm cells in the amnion, which is an extraembryonic structure, come from the epiblast. Furthermore, allmesoderm cells, including those in the amnion, chorion and chorioallantoic placenta, are also epiblast-derived.

Epiblast formation in the human
Based on limited morphological information (Niakan et al.,2012), early human development leading to the segregation of
epiblast and hypoblast precursor cells at late blastocyst and periimplantation stages is very similar to that described in the mouse(Fig. 1B, left and middle). However, molecular data suggest thathuman-speciﬁc variations to the mouse prototype may be more

G. Sheng / Developmental Biology 401 (2015) 17–24

prominent than previously expected (De Paepe et al., 2012; Kuijk
et al., 2012; Niakan and Eggan, 2012). Epithelialization of theepiblast in the human embryo takes place after implantation, andis inaccessible for molecular analysis. Morphological data suggested that the early phase of human epiblast epithelialization issimilar to the mouse in that polarization and epithelialization of
the epiblast precursor cells result in the formation of a rudimentary proamniotic cavity (Fig. 1B, right) (Luckett, 1975, 1978; Vögler,1987). Unlike in the mouse, however, this epithelialized epiblastball loses contact and does not fuse with the chorionic ectoderm.
The proamniotic cavity remains as the bona ﬁde apical lumen of
the epiblast sheet before and during gastrulation (Fig. 1B, right).
Radial symmetry-breaking in the human embryo thus does not
involve signals from the epiblast–chorionic ectoderm boundary as
known in the mouse embryo. The difference in the mode of
epiblast epithelialization and proamniotic cavity formation between these two species accounts for the “inverted” epiblast
topology in the mouse embryo and the more “ﬂat”, chick-likeone in the human embryo. As in the mouse, all amniotic ectoderm
and extraembryonic mesoderm cells in the human are derived
from the epithelialized epiblast.

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unilaminar epithelium with no ICM (Fig. 1D and E, left). These cells,
although polarized and located on the surface of the blastocyst, are
not restricted in their fate to the trophoblast lineage because both theepiblast and hypoblast precursors will come from this layer (Fig. 1D
and E, middle). In the elephant shrew, the mode of epiblast/hypoblast
formation from this unilaminar blastocyst is non-regionalized and theingression occurs throughout the unilaminar structure. In the tenrec,
the ingression takes place preferentially from one pole. As a cell
population, the unilaminar blastocyst in the elephant shrew andtenrec resembles the pluriblast (discussed below) in marsupialembryos in that these epithelial, surface-located cells are multipotential. Unlike in the marsupial case, however, the internalized
epiblast and hypoblast precursors in the elephant shrew and tenrec
aggregate and sort out, eventually reaching an embryonic organization similar to that in the mouse, with the epiblast cells surroundedby the trophoblast and hypoblast cells (Fig. 1D and E, right). These
epiblast cells will secondarily epithelialize in a manner akin to what is
known in the human (Van de Horst, 1949).

Epiblast formation in marsupials

In a large number of phylogenetically diverse eutherian species,
including the rabbit, pig, cow, sheep, cat and dog (Williams andBiggers, 1990), epiblast precursor cells polarize to form an epithelial
sheet in a manner very different from what is known in mouse andhuman embryos. In these animals, the overall morphologicalsequence of the initial ICM formation and epiblast/hypoblast precursor segregation takes place like in the mouse and human (Fig. 1C,
left and middle) (Degrelle et al., 2005; Oestrup et al., 2009; Vejlsted
et al., 2005). But during the process of epiblast epithelialization, theirpolar trophoblast, the Rauber’s layer, degenerates (Betteridge and
Flechon, 1988; Blomberg et al., 2008; Flechon et al., 2004; Vejlsted
et al., 2005; Williams and Biggers, 1990), and as a consequence theepiblast sheet is exposed directly to the external environment andfuses laterally with the mural trophoblast (Fig. 1C, right). The ﬁnalorganization of the epithelialized epiblast in these animals is
comparable to that in the birds/reptiles. Details of this fusion process
are poorly understood. For example, it is unclear how the disappearance/apoptosis of the polar trophoblast is coordinated with thepolarization of the epiblast cells to ensure the structural (epithelial)
integrity of the embryo, or whether the polar trophoblast makes any
cellular contribution to either the epiblast or the mural trophoblast.
Molecular mechanisms regulating early lineage segregation in theseanimals also seem to vary. The timing and lineage restriction in theexpression of NANOG, OCT4 and CDX2 in the cow were reported tobe remarkably different from that in the mouse (Berg et al., 2011;
Oestrup et al., 2009; Wolf et al., 2011). The above-mentioned
differences and the failure so far in deriving pluripotent stem cells
from these animals suggest that caution is needed in extrapolatingknowledge in mouse early development to all eutherian mammals(Berg et al., 2011; Gandolﬁ et al., 2012; Hall and Hyttel, 2014).

The marsupials consist of several hundred extant species andform a sister group to the eutherian mammals. Their early development has been studied in a dozen or so species. Post-fertilization
cleavage patterns vary, but in all of them a unilaminar, epithelial-likeblastocyst stage is reached, and all cells in this unilaminar structure appear indistinguishable (Selwood, 1992) (Fig. 1F, left). Like inthe elephant shrew and tenrec, these cells take up the morphologyof the trophoblast, but are not restricted fate-wise to this lineage. The
precursors for the epiblast and hypoblast, called the pluriblast in
marsupial embryos, are restricted in their position to one end of thisunilaminar structure. The inside-outside model for the segregation of
the ICM and trophoblast cell lineages is therefore not applicable tothe marsupial embryos. Nevertheless, it has been argued that acertain degree of ultrastructural/morphological heterogeneity can bediscerned among the epithelial blastocyst cells (Selwood, 1992). Thismay have a causal link to asymmetric cell divisions during earlycleavages (asymmetric inheritance of maternal determinants) and toa bias later on in their fate choices (trophoblast vs. pluriblast).
Molecular data from the opossum support this model (Morrison
et al., 2013), whereas data from the tammar wallaby argue against it(Frankenberg et al., 2013). By the late unilaminar and early bilaminarstages, heterogeneity in blastocyst cell morphology becomes more
prominent. The pluriblast cells now take up cuboidal or columnar
epithelial morphology and the hypoblast precursors delaminate fromthe pluriblast region of the blastocyst (Fig. 1F, middle). The internalized hypoblast cells spread and migrate to cover the under-surface
of both the epiblast (the remainder of the pluriblast after hypoblast
delamination) and the trophoblast (the non-delaminating part of theunilaminar blastocyst), thus generating a prototypic two-layered
structure with three well-deﬁned cell lineages (Fig. 1F, right). The
epiblast cells in marsupial embryos are considered to have polarized,
epithelial morphology from the very beginning, even prior to their
speciﬁcation as the epiblast precursor cells.

Epiblast formation in the elephant shrew and tenrec

Epiblast formation in monotremes

The furthest departure in eutherian early development from themouse prototype is seen in the elephant shrew (Elephatulus) andtenrec (Hemicentetes) (Goetz, 1939; Van de Horst, 1942, 1949;
Wimsatt, 1975). Both of them belong to the Afrotherian clade, theearliest offshoot in the eutherian tree (Romiguier et al., 2013). Early
embryogenesis in these two species passes through a hollow “blastocyst” stage at which all cells become part of a “trophoblast”-like

The monotremes (prototherians) have an oocyte organization
very similar to that of the reptiles and birds (Flynn and Hill, 1939).
They represent an early branch in mammalian evolution. Earlystage monotreme embryos are difﬁcult to obtain and our knowledge of their early development came primarily from three papers
published by Hill and colleagues (Flynn and Hill, 1939, 1947; Wilson
and Hill, 1908), the last of which investigated the developmental

Epiblast formation in the rabbit

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G. Sheng / Developmental Biology 401 (2015) 17–24

period leading to the formation of a bilaminar structure in Echidna
and the platypus and is summarized below. Up to the multilayered
blastoderm stage (Fig. 1G, left), monotreme early development
resembles that of the reptiles and birds (Bertocchini et al., 2013;
Flynn and Hill, 1947; Sheng, 2014). A reduction in the cell layer
number, likely through radial intercalation, results in the formationof a unilaminar, “blastocyst”-like structure (Flynn and Hill, 1947;
Selwood and Johnson, 2006) (Fig. 1G, middle). This “blastocyst” isincomplete since the epiboly movement (the process of blastoderm
cells migrating from the embryonic to the abembryonic side andeventually enclosing the entire yolk) at this point has brought theblastoderm edge cells only to the yolk equator. As the epiboly
continues and the entire yolk is being covered by a unilaminarepithelium, cells located in the central area of this unilaminarstructure, above the original germinal disc, are presumably speciﬁed as precursors for the epiblast and hypoblast, and the peripheral
cells become precursors for the trophoblast (Flynn and Hill, 1947;
Selwood and Johnson, 2006). It is unclear whether the central cells
are bi-potential for both the epiblast and hypoblast fates, as theequivalent of the eutherian ICM or the marsupial pluriblast, or are asalt-and-pepper mixture of pre-biased/pre-determined cell populations as suggested from morphological heterogeneity observed
among these cells (Flynn and Hill, 1947; Selwood and Johnson,2006). Formation of a bilaminar structure follows a similar process
as described in the marsupial embryo. The hypoblast precursor cells
delaminate from the central area of this unilamiar blastocyst andsubsequently spread underneath the epiblast and trophoblast(Fig. 1G, right). Selwood and Johnson (2006) had proposed that
segregation of the epiblast and hypoblast precursors in monotreme
embryos takes place even before the blastoderm achieves a unilaminar structure.

Epiblast formation in birds and reptiles
The sauropsids (birds and reptiles) are an important branch ofAmniota. Morphogenetic events leading to the separation of thetrophoblast, epiblast and hypoblast lineages in a eutherian
embryo are thought to have an evolutionary origin in theancestral mammal with an avian/reptilian-like developmental
program. The birds and reptiles are sometimes referred to as two
separate groups, but phylogenetic evidence strongly suggest thatthe avian clade is a part of the reptilian tree, with the crocodiles
being its closest living relatives (Chiappe, 2004; Chiari et al.,2012). Research on avian and reptilian early development has along history (Bertocchini et al., 2013; Sheng, 2014), but molecularinvestigation has been limited primarily to one avian species, thechicken (Gallus gallus). In a freshly-laid chicken egg, embryonicdevelopment has reached EGK-X (Eyal-Giladi and Kochav, 1976), astage at which three main cell lineages have already beendelineated: the epiblast, hypoblast and area opaca cells (Alevet al., 2013a; Lavial et al., 2007; Pernaute et al., 2010; Shin et al.,2011; Yatskievych et al., 1999) (Fig. 1H, right). These lineages arebroadly equivalent to the epiblast, hypoblast and trophoblast,respectively, in a mammal embryo. A notable difference is thatlower layer cells in the area opaca, located underneath thetrophoblast-like extraembryonic ectoderm and akin to the mammalian parietal primitive endoderm cells in their function, are
not derived from the hypoblast in the chick. Morphogenetic
events leading to the separation of these three lineages takeplace intrauterally and have not been studied at the molecularlevel. The model presented here (Fig. 1H, left and middle) is basedon our unpublished data on intrauterine chick development andon epiblast epithelialization and lineage segregation processes in
pre-gastrulation chick and zebra ﬁnch embryos. The ﬁrst half of
the intrauterine development in the chick embryo results in theformation of a multi-layered blastoderm (Fig. 1H, left), which
during the second half is gradually reduced to a two-layered
structure through shedding and radial intercalation (Fig. 1H,
middle, right). Molecular speciﬁcation of the three primarylineages accompanies this cellular rearrangement. Data fromzebra ﬁnch embryos suggest that lineage markers are alreadyexpressed in a salt-and-pepper manner at the beginning of thelayer reduction phase (Fig. 1H, left), with epiblast markers being
gradually restricted to the upper layers and hypoblast markers to
the low ones (Fig. 1H, middle), eventually leading to the twolayered epiblast–hypoblast organization with distinct molecularsignatures (Fig. 1H, right). This layer-reduction process is similar
to that described in monotreme embryos (Flynn and Hill, 1947;
Selwood and Johnson, 2006) and our molecular data appear to
support the pre-determination model proposed by Selwood andJohnson (2006). An important distinction between the layerreduction processes in the birds/reptiles on one hand and themonotremes on the other is that in the former the blastoderm
never reaches a unilaminar stage and juxtaposition of theepiblast and hypoblast precursors within a single epithelialized
layer is minimal.

A model for the amniote prototype
Based on the above discussion, two prototypic views of epiblast
epithelialization are presented in Fig. 2, one for the amniotes ingeneral and the other for the eutherian mammals in particular.
The ancestral amniote is considered to have an egg organizationand early embryonic development similar to what is known inextant sauropsids and prototherians (Fig. 2A). Early cleavages aremeroblastic, with cytokinesis lagging behind nuclear divisions.
This is followed by the cellularization process and by a phase of
rapid cell divisions, resulting in the formation of a multi-layered
blastoderm with dome-shaped and presumably totipotent “blastomeres” positioned on top of a giant yolk cell. After blastomere
layer reduction and the epiboly movement, the embryo reaches a
1- or 2-cell-layer thick “blastocyst” stage. Speciﬁcation of thetrophoblast, epiblast and hypoblast lineages takes place concomitant with this phase of cellular rearrangement. These two processes are presumed to be under separate molecular regulation. If
layer reduction (radial intercalation) advances more rapidly thanfate speciﬁcation, a unilaminar blastoderm, as seen in monotreme
embryos, is reached. In such a case, the central cells become
epiblast/hypoblast precursors either as a bi-potential cell population (similar to the pluriblast in marsupials) or a mixture of unipotential cells, and the peripheral ones become the trophoblast.
Hypoblast cells secondarily move out from this “pluriblast” layer,
resulting in the prototypic two-layered epiblast–hypoblast organization. If the layer reduction process lags behind the fatespeciﬁcation process, as seen in bird/reptile embryos, the epiblastand hypoblast cells are speciﬁed and sort out into two layers
before the embryo ever reaches a unilaminar stage. In bothscenarios, the relationship between the trophoblast and thepluriblast is that of peripheral vs. central, instead of outside vs.
inside (Johnson and Selwood, 1996). The epiblast as an epitheliumis therefore topographically continuous with the trophoblast
epithelium.

A model for the eutherian prototype
In ancestral eutherian mammals, the amniote prototype is
modiﬁed to accommodate two types of physiological changes: yolk
reduction and placentation. Early divisions in ancestral eutherians
are holoblastic and a unilaminar blastocyst stage is reached without

G. Sheng / Developmental Biology 401 (2015) 17–24

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Fig. 2. Models for the amniote and eutherian prototypes of epiblast epithelializaion. (A) The amniote prototype. (B) The eutherian prototype. Red: epiblast; Yellow:
hypoblast; Blue: trophoblast; tri-potential (T/E/H) or bi-potential (E/H) cells are also color-coded as indicated. In (A) represented by the reptilian, avian and monotremegroups, the mature oocyte is yolky and early cleavages meroblastic. Lineage segregation and blastomere layer reduction are hypothesized to be under separate regulation. If
layer reduction outpaces lineage speciﬁcation (as seen in monotreme embryos, bottom route), a unilaminar blastocyst stage is reached. This is followed by delamination ofhypoblast precursors. Otherwise, the bilaminar epiblast–hypoblast organization is reached without going through a unilaminar blastocyst stage (as seen in bird/reptile
embryos; top route). In (B) represented by all eutherian mammals, the oocyte contains little or no yolk. Cleavages are holoblastic. A unilaminar blastocyst stage, containing all(middle route) or partial (bottom route) tri-potential cells, is reached. Both the epiblast and hypoblast precursors delaminate from this unilaminar structure and aggregate toform the mixed epiblast and hypoblast cell mass, which after two rounds of epithelialization forms the epiblast and hypoblast epithelia. Pre-mature differentiation of thetrophoblast in some eutherians (top route) may have facilitated the formation of the ICM. An ICM also ultimately forms in the other two routes. Depending on theimplantation mode, the epithelialized epiblast may be covered by the polar trophectoderm (top right) or be in continuity with the mural trophectoderm (bottom right). The
dotted line represents the marsupial prototype, possibly by going through a bi-potential pluriblast stage.

undergoing the epiboly process (Fig. 2B), Marsupials represent an
evolutionary intermediate and the elephant shrew and tenrec
represent the early eutherian prototype. All cells of the unilaminar
blastocyst are epithelial and totipotent at this stage. Division into
the trophoblast and pluriblast areas may be decided stochasticallyor be inﬂuenced by asymmetric signals associated with implantation or inherent structural asymmetry. Early speciﬁcation of thetrophoblast lineage as seen in the mouse and human is likely aconsequence of early implantation in these species. If the embryonic side of the blastocyst is involved in the early implantation, alayer of polar trophoblast is evolved to cover the pluriblast (theICM) population. Although the origin of the polar trophoblast andits relationship to the pluriblast (the ICM) have not been clearly
resolved, in all eutherian species a conserved developmental stage
is eventually reached, with a well-differentiated trophoblast layer(including the mural and polar trophoblast) and an ICM consistingof mixed epiblast and hypoblast precursor populations (Fig. 2B). In
contrast, marsupial embryos never pass through this stage, suggesting that acquisition of the polar trophoblast fate was a eutherian invention, likely related to the mode of implantation in its

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G. Sheng / Developmental Biology 401 (2015) 17–24

ancestors. In many extant eutherian species in which the embryonicside is no longer actively involved in the establishment of fetal–
maternal contact, the polar trophoblast is secondarily lost, resulting
in an epithelialized epiblast which is topographically continuouswith the mural trophoblast as known in the sauropsids, monotremes and marsupials.

Epiblast formation as an evolutionary adaptation to functional
constraints
The epiblast as a cell lineage is deﬁned by its unique molecularsignature and its functional contribution to the three germ layers(Fig. 3A). The signiﬁcance of the epiblast as an epithelial structure isFig. 3. Relationship between form and function in epiblast morphogenesis. Form is the cell morphology and function is the cell lineage/fate. The epiblast lineage isdeﬁned by molecular criteria and the epithelial epiblast is deﬁned by cell biological criteria. Pre-epiblast indicates tri-potential or bi-potential precursor stage. (A) Lineage
segregation in the mouse embryo. After molecular speciﬁcation, the epiblast cells undergo morphological changes from mesenchymal to epithelial before imitating thegastrulation process. The hypothetical relationship between the epithelialization status and the type of pluripotent cells is also indicated. (B) Epiblast or pre-epiblast (tripotential or bi-potential precursor) cells in amniote embryos can exist in either mesenchymal or epithelial state. A fully epithelialized epiblast is the ﬁnal state in allamniotes. How to reach this ﬁnal state varies from species to species. For example, in the mouse embryo, the sequence is mesenchymal pre-epiblast/mesenchymal epiblast/epithelioid epiblast/fully epithelial epiblast. In stead, the sequence in marsupial embryos is epitheliod pre-epiblast/epithelioid epiblast/fully epithelial epiblast.

G. Sheng / Developmental Biology 401 (2015) 17–24

not clear. In vivo, all intraembryonic tissues are generated from theepiblast through gastrulation, the starting point of which is theunilaminar, fully-epithelialized epiblast. This requirement, however,is speciﬁc to the amniotes because in most amphibian and ﬁsh species the pre-gastrulation ectoderm is multi-cell layer thick and not as
well-epithelialized as the epiblast (Nakaya and Sheng, 2008). Germlayer differentiation per se, for example from the pluripotent amniote
ES or iPS cells, does not need to start from an epithelialized structure(Pieters and van Roy, 2014). The conservation of an epithelialized
epiblast in all extant amniotes suggests that their common ancestor
was under selection pressure to maintain the pre-gastrulation
ectoderm as a full epithelium to counteract mechanical stress uniqueto the amniote early development. Such a requirement for epithelial
integrity is likely associated with novel morphogenetic features in
the amniotes, including the need to generate extraembryonic tissues(e.g., the amnion and the chorion) and the shift from a circumblastoporal mode of gastrulation to a posterior-epiblast restricted one(Alev et al., 2013b; Bertocchini et al., 2013). These features necessitate
the subdivision of the ectoderm into embryonic and extraembryonicregions, the uncoupling of the gastrulation and epiboly processes,
and ﬁnally the full epithelialization of the epiblast.

Epiblast formation offers a unique window for capturing thepluripotent state of pre-gastrulation ectoderm
While many genes involved in pluripotency maintenance andreprogramming, e.g., NANOG (Scerbo et al., 2014) and class V POU
domain proteins (Frankenberg and Renfree, 2013), are molecularly
and functionally conserved and can trace their evolutionary origin to
at least the common ancestor of the osteichthyans (bony ﬁsh andtetropods), stable in vitro culture of pluripotent cells derived frompre-gastrula stage embryos or through reprogramming remains very
inefﬁcient in most species. Pre-gastrulation ectoderm cells by default
are pluripotent because they are capable of giving rise to all cell types
in the three germ layers after gastrulation. The pluripotency of thesepre-gastrulation ectoderm cells, however, is maintained only for abrief period of time during normal development. Capturing such anarrow developmental window in artiﬁcial culture conditions
requires proper understanding of how pre-gastrulation ectoderm
cells regulate the timing of lineage differentiation (e.g., as anevolutionarily conserved role of POU-V proteins (Tapia et al., 2012))
in coordination with other morphogenetic events that are notdirectly related to pluripotency maintenance (e.g., overlapping rolesof signaling pathways in regulating pluripotency and morphogenesis(Sokol, 2011)). The amniote epiblast from such a perspective can beviewed to go through divergent morphogenetic processes to reach aﬁnal cell biological status that is conserved among all amniotes, but
distinct from that in other vertebrate groups. Interestingly, epithelialization of the amniote epiblast may confer a certain advantage for
efﬁcient capture of such a transitory, pluripotent stage in vitro. It has
been shown that optimal reprogramming of somatic cells into
induced pluripotent cells requires a mesenchymal-to-epithelial transition process (Esteban et al., 2012), and that epithelial adhesion
protein E-cadherin mediates LIF-dependent self-renewal in mouse ES
cells (Bedzhov et al., 2013; del Valle et al., 2013; Pieters andvan Roy, 2014). Although mouse ES cells tend to form aggregates oftightly adhering cells without clear epithelial morphology, bothmouse epiblast-derived stem cells (EpiSCs) and human ESCs formcolonies reminiscent of an epithelialized epiblast. Recent transcriptional proﬁling data suggested that both mouse ESCs and mouse
EpiSCs capture transitory developmental status of the epiblast, albeit
at different stages of epiblast maturation (Boroviak et al., 2014)(Fig. 3A). These lines of evidence suggest that the process of epiblast
epithelialization in vivo may provide a unique cell biological window
for capturing pluripotent stages of pre-gastrulation ectoderm in vitro,

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with optimal “culturable” stages and culture conditions being variable in different amniote species due to variations in the molecularand cellular regulation of epiblast morphogenesis in vivo.

Epiblast formation in light of the evolutionary hourglass model
Using the analogy of the hourglass model, in which a pharyngula (a general term used to describe an embryonic stage after
the neurula, with conserved branchial arches and pharyngeal
pouches) is the phylotypic stage of all vertebrate embryos, one
may consider that amniote embryogenesis shares the earliest
phylotypic stage with two conserved features: the speciﬁcationof the three primary cell lineages (trophoblast-like extraembryonicectoderm, epiblast and hypoblast) and the epithelialization of the
pre-gastrulation epiblast. Development prior to the appearance of
these two features is not conserved and each group uses different
morphogenetic strategy to reach this conserved phylotypic stage(Fig. 3B). Commonly-held notions about how the three lineages
separate step-wise from each other and how these steps are
manifested morphologically should be considered in light of thismorphogenetic diversiﬁcation. With respect to mammalian early
development, three such notions, listed below, deserve reevaluation. (1) The ﬁrst step, the segregation between the trophoblast and the ICM (bi-potential precursors of the epiblast andhypoblast), is dictated by positional cues. Blastomeres locatedsuperﬁcially will become trophoblast cells and those locatedinternally will become ICM cells. (2) The trophoblast as anepithelium enclosing the blastocyst is the ﬁrst lineage to be
speciﬁed and after its speciﬁcation the trophoblast does not makeany contribution to the other two lineages. (3) From the earlyblastocyst stage the ICM is completely sealed off by polar trophoblast cells and the cellular and molecular distinction between
these two cell populations can be clearly made. These notions,obtained primarily from studies of the mouse embryo, cannot beapplied to the amniotes in general as we have discussed in thisreview. Neither can they be viewed as representing all mammals
or all eutherian mammals. Most importantly, an appreciation ofmorphogenetic diversity in epiblast formation may help us understand many subtle differences observed in mammalian earlylineage speciﬁcation which cannot be easily explained withcurrent prevailing models, including for instance the fact thattrophoblast cells in the cow maintain the ICM marker OCT4
expression up until the late blastocyst stage (Berg et al., 2011),
that human trophoblast cells express pluripotency markers andmaintain the capacity to differentiate into the ICM lineage (DePaepe et al., 2012), and that blastomeres located inside theblastocyst may initiate trophoblast differentiation in the absenceof maternal and zygotic E-cadherin (Stephenson et al., 2010).

Summary
The epiblast is one of the three primary lineages speciﬁed early
during amniote development. An epithelialized epiblast is evolutionarily conserved in the amniotes. It is the starting material for
gastrulation through which it gives rise to all intraembryonictissues. Morphogenetic processes leading to its formation vary
substantially in different amniote groups. Within the eutheriangroup, obvious departure from what is known in the mouse model
is noted. Subtler differences in epiblast morphogenesis and lineage
speciﬁcation between the mouse and the human may be understood more easily in light of such diversity. In reptiles, birds andeutherian mammals, epiblast cells are not organized as an epithelium when they are ﬁrst speciﬁed molecularly. An MET process has
to take place to reorganize these cells into a unilaminar structure

24

G. Sheng / Developmental Biology 401 (2015) 17–24

with all the essential features of a fully-formed epithelium (Fig. 3B).
Understanding the cell biology of this MET may ultimately help us
achieve optimal conditions to maintain pluripotent epiblast cells in
culture.
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