Develop. Growth Differ. (2008) 50, 755–766

doi: 10.1111/j.1440-169X.2008.01070.x

ReviewBlackwell Publishing Asia

Epithelial to mesenchymal transition during gastrulation:
An embryological view
Yukiko Nakaya and Guojun Sheng*
Laboratory for Early Embryogenesis, RIKEN Center for Developmental Biology, Kobe 650-0047, Japan

Gastrulation is a developmental process to generate the mesoderm and endoderm from the ectoderm, of which
the epithelial to mesenchymal transition (EMT) is generally considered to be a critical component. Due to increasing
evidence for the involvement of EMT in cancer biology, a renewed interest is seen in using in vivo models, such as
gastrulation, for studying molecular mechanisms underlying EMT. The intersection of EMT and gastrulation
research promises novel mechanistic insight, but also creates some confusion. Here we discuss, from anembryological perspective, the involvement of EMT in mesoderm formation during gastrulation in triploblastic
animals. Both gastrulation and EMT exhibit remarkable variations in different organisms, and no conserved rolefor EMT during gastrulation is evident. We propose that a ‘broken-down’ model, in which these two processes
are considered to be a collective sum of separately regulated steps, may provide a better framework for studying
molecular mechanisms of the EMT process in gastrulation, and in other developmental and pathologicalsettings.
Key words: Cell biology, epithelial to mesenchymal transition, gastrulation, mesoderm.

Introduction
During embryonic development, a single fertilized egg
eventually gives rise to an organism with hundreds ofdifferent cell types. The diverse cell types in complex
tissues and organs, however, can be loosely categorized
into two general types: those cells with a two-dimensionalorganization with their neighbors (epithelial) and thosewith a three-dimensional organization (mesenchymal).
The two-dimensional organization of an epithelium isrelative, of course, as it can fold into topologically complex structures, and epithelial cells often interact withother cells outside the epithelial sheet. Nevertheless,this general categorization has provided an importantframework for the description and understanding oftissue morphogeneses during animal development. Anymorphogenetic process can be viewed conceptually ascell organizational changes either within the epithelial
or mesenchymal state, or a transition between these*Author to whom all correspondence should be addressed.
Email: sheng@cdb.riken.jp
Received 07 August 2008; revised 04 September 2008;
accepted 04 September 2008.
© 2008 The Authors
Journal compilation © 2008 Japanese Society ofDevelopmental Biologists

two states (EMT for epithelial to mesenchymal transition
or MET for mesenchymal to epithelial transition).
The concept of EMT/MET, since first proposed four
decades ago in cell biological studies of chick embryos(Trelstad et al. 1966, 1967; Hay 1968), has been used
to describe diverse biological phenomena from trophectoderm formation in developing mammalian embryos(Collins & Fleming 1995; Morali et al. 2005; Eckert &Fleming 2008) to tumor metastasis and invasion(Savagner 2005; Yang & Weinberg 2008). Broad-scope
discussions of EMT/MET in development and diseasecan be found in several recent review articles (Hay
1995; Shook & Keller 2003; Thiery 2003; Huber et al.
2005; Savagner 2005; Zavadil & Bottinger 2005; Thiery& Sleeman 2006; Baum et al. 2008; Yang & Weinberg2008). This review will focus on EMT during gastrulation,
which is regarded as the earliest and most typical EMT
event during animal development.

Key concepts: epithelium, mesenchyme,
gastrulation and germ layers
Mesenchymal cells generally have more irregularmorphology and higher motility than epithelial cells. It
is, however, not easy to define these cells based on any
cell biological criterion. Instead, mesenchymal cells are756

Y. Nakaya and G. Shengoften regarded as cells that do not have an epithelial
morphology (Trelstad et al. 1967; Hay 1968). It is therefore
necessary to go through what are typically considered
to be the characteristics of an epithelium. A twodimensional epithelial structure is generally marked by
the presence of (i) an apico-basal polarity; (ii) a transepithelial barrier (tight junctions or septate junctions); (iii)
an epithelial specific cell–cell adhesion mediated byadherens junctions; and (iv) a basement membrane-likeextracellular matrix. These characteristics are not present
in all epithelial structures and none of them is uniquefor epithelial cells. When introducing the concept of
EMT/MET, Hay (1968) provided a much more generaldefinition for the epithelium as a tissue layer with a freesurface. This free surface is the side facing the exteriorof the embryo for the surface ectoderm, the gastrointestinal lumen for the gut endoderm, and a cavity formesoderm-derived epithelia such as somites, bloodvessels and nephritic tubules. This definition also has
its limitations. For instance, the stratification of an epithelium often results in epithelioid tissue organization
without a free surface, such as in deep layer ectoderm
cells in Xenopus embryos and in basal epithelial layerof skin epidermis. Therefore, the question about whatcriteria one should use to properly define an epithelial
cell remains to be one of the most debated in the EMT/MET field.
Gastrulation means the formation of the gut. Thisdevelopmental process gives rise to endoderm cells in
diploblasts (animals with two germ layers), and to mesoderm and endoderm cells in triploblasts (vast majorityof metazoans). In either diploblastic or triploblastic
animals, pre-gastrulation blastula cells (or epiblast cells inamniotes) are generally considered to have an epithelial
morphology. Endoderm formation in diploblasts cantake place either through an ingression/delamination
process, which would involve an EMT-like event, or
through an invagination/involution process, which does
not involve EMT (Byrum & Martindale 2004). In triploblasts, mesoderm formation often, but not always, as we
will discuss later, involves EMT-like morphological changes.
Endoderm formation in triploblasts, like in diploblasts,
may or may not involve EMT. In addition, gastrulation,a loose term describing the sum of morphogenetic
processes leading to the formation of three germ layers(ectoderm, mesoderm and endoderm), is also used toinclude morphogenetic processes within individual germlayers either prior to or after EMT. Our discussions will
not cover these topics. Instead, we will first discuss insome detail about how EMT is involved in mesoderm
formation in several experimental organisms, and thendiscuss recent findings on molecular regulations ofgastrulation EMT in mouse and chick embryos. We will
also provide some suggestions on how to view thegastrulation EMT from an evolutionary perspective, andon how the studies on gastrulation EMT in avian
embryos may provide useful insight for EMT studies ingeneral.

EMT in mesoderm formation during
gastrulation
Drosophila
In Drosophila, the invagination of mesoderm precursor
cells within the ventral blastoderm starts immediately
after the completion of cellularization (Leptin 2004) (Fig. 1A).
No basement membrane has been described so far
either for the cellularized blastdermal epithelium or for
the invaginating mesoderm precursors. Other epithelial
characteristics are present at the onset of invagination,
including markers for epithelial adherens junctions,septate junctions (functionally equivalent to tight junctions
as transepithelial barriers) and apico-basal polarity (Odaet al. 1998; Tepass et al. 2001; Pellikka et al. 2002;
Lecuit 2004; Kolsch et al. 2007). The invagination process
can be viewed as a topological rearrangement event of
an intact epithelium. Indeed, before mesoderm cells adopt
a mesenchymal morphology, all three epithelial characteristics are still present in invaginated mesoderm
precursors. The transition from epithelial-shaped mesoderm
precursors to mesenchymal-shaped mesoderm cells is
rapid, and appears to take place simultaneously for all
precursors (Leptin 2004). Localized expression ofadherens junction and septate junction markers, as wellas other non-junctional polarity markers, are lost in
dissociated mesoderm cells. The loss of DE-cadherin
is followed by the cytoplasmic export of DN-cadherin
mRNA, which has been present but restricted to the
nucleus prior to mesoderm precursor dissociation (Odaet al. 1998), marking a shift to the migratory behavior
of dissociated mesoderm cells. Molecules involved infibroblast growth factor (FGF) signaling, including FGF
receptor heartless, sugar modifying enzymes involved
in mediating FGF signaling, sugarless and sulfateless,and intracellular mediators dof, ras and pebble havebeen reported to control aspects of this EMT process(Beiman et al. 1996; Gisselbrecht et al. 1996; Shishido
et al. 1997; Michelson et al. 1998; Vincent et al. 1998;
Lin et al. 1999; Smallhorn et al. 2004).
Sea urchin
Mesoderm cells in sea urchins form in two phases(Fig. 1B). The primary mesenchymal cells, which give riseto the larval skeleton, ingress from the vegetal plate in theblastula stage embryo prior to the formation of archentenron (the primitive gut). The secondary mesenchymal© 2008 The Authors
Journal compilation © 2008 Japanese Society of Developmental Biologists

EMT in gastrulation

757

Fig. 1. Processes of mesoderm
formation in several experimental
triploblastic animals. (A) Dropshophila; (B) sea urchin; (C)zebrafish; (D) xenopus; (E) chicken;(F) mouse. Blue, mesoderm
precursors located in the ectoderm;yellow, mesoderm; green, endoderm;light brown, basement membrane.

cells form from the tip of the archenteron during thelatter half of the invagination process. The ingressionof primary mesenchymal cells is generally considered
as a typical EMT process. Prior to the ingression, thevegetal plate cells are of an epithelial morphology, withseptate junctions, adherens junctions, basement membrane and apico-basal polarity (Balinsky 1959; Wolpert& Mercer 1963; Gustafson & Wolpert 1967; Katow &Solursh 1979, 1980; Spiegel & Howard 1983; Andreuccetti et al. 1987; McClay et al. 2004; Itza & Mozingo

2005; Wessel & Katow 2005). Sea uchin blastula cells
have an additional feature in that apical membranes
establish prominent interactions with the hyaline layer,
an extracellular matrix covering the apical surface of
blastula epithelium (Fink & McClay 1985). The loss ofinteractions between the apical membrane processes
and the hyaline layer constitutes the earliest morphologicalsign of the initiation of EMT. Subsequently, ingressing
primary mesenchymal cells lose junctional interactions
with its neighbors and break through the basement© 2008 The Authors
Journal compilation © 2008 Japanese Society of Developmental Biologists

758

Y. Nakaya and G. Shengmembrane. The loss of junctional interaction is amultistep process. EM studies (Katow & Solursh 1980)
revealed that when the basal end has broken throughthe basement membrane, apical interactions withneighboring cells are still present. Ingression of primarymesenchymal cells takes place individually, and isaccompanied by extensive cell morphological changes
including apical constriction, although the ingression
and apical constriction have been postulated to involve
different mechanisms (Anstrom & Fleming 1994). Loose
basement membrane matrix covering the vegetal plate
cells shows discontinuity underneath ingressing cells,
and is resealed as a continuous layer after ingressionof all primary mesenchymal cells. The delamination of
secondary mesenchymal cells from the tip of invaginatingarchenteron is less characterized. Similar to Drosophila
mesoderm formation, archenteron invagination in sea
urchins can be viewed as a topological rearrangementof an intact epithelium, and invaginated endoderm cells
retain epithelial junctional interactions, polarity markers
and the basement membrane (Miller & McClay 1997;
McClay et al. 2004; Wessel & Katow 2005). The endoderm cell/basement membrane interaction is weaker
than that in the ectoderm (Hertzler & McClay 1999),
and adherens junctions are modulated to accommodate
the planar cell rearrangement within the archenteronepithelium (Ettensohn 1985; McClay et al. 2004).
Secondary mesenchymal precursor cells, located at the
tip of the invaginating archenteron, maintain apicalepithelial interactions with neighboring endoderm cells.
Half-way during the invagination process, these cells
lose the basement membrane and send out long basal
filopodia. These filopodia make contact with thebasement membrane of animal pole cells and the force
generated by this interaction is responsible for at least
the last third of archenteron invagination (Hardin 1988).
During this process, basal filopodia also make extensive
contacts with the extracellular matrix (instead of thebasement membrane) around the secondary mesenchyme cells. These cells eventually delaminate from thearchenteron and contribute to other mesoderm lineages,
such as muscle cells. The precise timing of apical junction
dissociation has not been investigated.
Zebrafish
Before the onset of epiboly, blastoderm cells in teleosts
can be separated into a surface enveloping layer anda deep enveloping layer (Fig. 1C). The former forms aprotective layer with squamous epithelial morphology(Betchaku & Trinkaus 1978; Keller & Trinkaus 1987;
Warga & Kimmel 1990), but does not give rise to either
mesoderm or endoderm cells. The deep enveloping
layer is the equivalent of the ectoderm/epiblast and will

give rise to the mesoderm and endoderm layers after
gastrulation. The deep enveloping layer is about four- to
five-cells-thick prior to gastrulation. During the involution
movement at the blastoderm margin, the deep enveloping layer flattens to two- to three-cells-thick due to radial
cell intercalation (Warga & Kimmel 1990). No epithelioid
structure is evident, however, either in the multi-cell-thickdeep enveloping layer or in the involuted mesendoderm(Montero et al. 2005), suggesting that gastrulation inzebrafish does not involve the EMT process in the
traditional sense. Instead, differential cell adhesion
mediated by cadherins and differential tensile force in cellsof different germ layers have been shown to contributeto germ layer segregation (Kane et al. 2005; Montero
et al. 2005; Shimizu et al. 2005; Krieg et al. 2008).
XenopusPrior to involution, the blastoderm in the marginal zone
is composed of two layers: a superficial layer and a
deep layer (Keller 1980; Keller & Shook 2004, 2008;
Shook & Keller 2008) (Fig. 1D). The superficial layer,
similar to the surface enveloping layer in zebrafish, serves
as a protective barrier for the developing embryo andcan be considered as a continuous epithelium with tightjunctions, adherens junctions and apico-basal polarity.
Although the superficial layer has a relatively smoothbasal surface facing the deep layer in sections and EM
analyses, no basement membrane has been reported
for it. Unlike the surface enveloping layer in zebrafish,
however, the superficial layer in Xenopus participates in
the involution and contributes to the endoderm. This
process can be viewed as not to involve EMT, for theepithelial sheet is somewhat retained in the involuted
endoderm. It is, however, unclear whether these cells
still retain tight junctions. Some of the involuted cells
located in the endoderm layer will later on contributeto the mesoderm through an EMT process. Underneaththe superficial layer, the deep layer cells undergo asimilar involution process in the marginal zone. Prior to
the involution, the deep layer cells intercalate radiallyand adopt, from multi-layered mesenchymal cells, anepithelioid morphology with a single-cell-thick layer anda thin basement membrane, but without tight junctions.
These cells involute and form the majority of themesoderm lineage. This can be viewed as an incomplete
EMT process. Many earlier involuting deep layer cells
contributing to the anterior mesendoderm, however,
never have the ‘opportunity’ to form this epithelioid
structure, and are instead pushed inside by the forcesthat initiate the blastopore formation (apical constrictionof bottle cells and vegetal endoderm rotation). Formationof these mesoderm cells therefore does not involve an
EMT process.

© 2008 The Authors
Journal compilation © 2008 Japanese Society of Developmental Biologists

EMT in gastrulation

Chick
Prior to primitive streak formation, the epiblast in chickembryos becomes epithelioid by late Stage HH1 (EyalGiladi Stage IX) (Bellairs et al. 1975, Eyal-Giladi &Kochav 1976; 1978; Kochav et al. 1980; Fabian & EyalGiladi 1981). The hypoblast cells form by a combinationof the segregation of initially multilayered blastoderm
disc before the formation of single cell-thick epiblastand the poly-ingression process during epithelioid
epiblast formation (Kochav et al. 1980; Fabian & EyalGiladi 1981). These hypoblast cells aggregate as islands
initially and, during primitive streak formation, spread out
and adopt the morphology of a loose mesenchymal
sheet (Stern 2004). As the primitive streak is being
formed, deep layer cells in the posterior marginal zone(including within the Koller’s sickle) contribute further tothe hypoblast and endoblast, whereas the superficial layer
will contribute to definitive endoderm and mesoderm
lineages (Bellairs 1986; Stern 1990; Bachvarova et al.
1998; Lawson & Schoenwolf 2001; Callebaut 2005;
Voiculescu et al. 2007). Formation of the primitive endoderm, the hypoblast and endoblast therefore can beconsidered not to involve the EMT process. Some of
the mesoderm cells are derived from the middle layerof the posterior marginal zone that has never adopted
an epithelial morphology (Bachvarova et al. 1998; Stern
2004; Callebaut 2005). Formation of these mesoderm
cells therefore also does not involve the EMT process.
Most of the mesoderm cells are formed by the convergence of epithelial-shaped epiblast cells towards theforming primitive streak, which subsequently undergo
EMT and ingress to adopt a mesenchymal morphology(Fig. 1E). These mesoderm precursor cells located in
the epiblast have all the characteristics of epithelial cells(Trelstad et al. 1967; Hay 1968; Nakaya et al. 2008).
Mouse
Trophectoderm is the first epithelial structure to form inmammalian embryos, with all associated epithelial characteristics (Vestweber et al. 1987; Collins & Fleming 1995;
Ohsugi et al. 1996, 1997; Fleming et al. 2000a and b;
Sheth et al. 2000; Flechon et al. 2003, 2007; MaddoxHyttel et al. 2003; Morali et al. 2005; Moriwaki et al.
2007; Eckert & Fleming 2008; Nishioka et al. 2008). The
inner cell mass cells, which are of mesenchymal morphology, soon give rise to two epithelial structures, theepiblast and the primitive endoderm (Fig. 1F). This is
generally considered as a primary epithelialization process
like the formation of trophectoderm, although in somepublished reports it is viewed as a secondary epithelialization process as some inner cell mass cells are derived
from polarized division of already epithelialized morula759

cells (Shook & Keller 2003). Some similarity can be drawn
between this and the stratification of already epithelialized blastoderm cells in Xenopus (Chalmers et al. 2003;
Ossipova et al. 2007) in that the ectoderm is separated
into two cell populations with different functions: anouter population maintains the epithelial morphology
and serves as a barrier, and an inner population serves
as cell source for three germ layers. The primitive endoderm differentiates into visceral endoderm and parietal
endoderm, with the former maintaining epithelial morphology and the latter becoming mesenchymal (Enders
et al. 1978; Hogan & Newman 1984; Verheijen & Defize
1999; Rivera-Perez et al. 2003; Perea-Gomez et al. 2007;
Gerbe et al. 2008). Similar to chick, the mesoderm anddefinitive endoderm cells in mammals are derived fromthe epithelial-shaped epiblast cells through the EMT
process (Tam & Beddington 1987, 1992; Lawson et al.
1991; Tam et al. 1993; Tam & Gad 2004).

Evolutionary considerations
The diverse mechanisms used for gastrulation and for
mesoderm formation, as exemplified above in several
model organisms and in many other triploblastic animals
investigated (Arendt & Nubler-Jung 1999; Stern 2004;
Solnica-Krezel 2005; Shook & Keller 2008), would tend
to suggest that there are few evolutionarily sharedmechanisms for the process of EMT in mesoderm
formation. Some generalized understanding, however,can be achieved by viewing it from the perspective ofphysical and physiological constraints imposed onembryonic development throughout evolution. The physicalconstraint is reflected in the effect of yolk content oncleavage, gastrulation and epiboly processes (Arendt &Nubler-Jung 1999; Shook & Keller 2008). The physiological constraint is reflected in the difference between
an embryo’s internal physiochemical properties andthose of the environment, including water, osmoticpressure and ion concentrations (Fesenko et al. 2000;
Fleming et al. 2000b; Kiener et al. 2008). The ectoderm,
prior to the generation of mesoderm and endoderm
layers, can be considered to have three main roles: (i)as a source of cells for the mesoderm and endoderm;(ii) as a protective layer between the developing embryoand the environment; and (iii) as a protective layer forthe nutritious substance of the embryo. The first is ofcourse generally regarded as the primary role of pregastrulation ectoderm. This role, however, is often
de-coupled with the second role. In such cases, there
may be no particular reason for the pre-gastrulation
ectoderm (as a cell source for the other two germ layers)
to adopt an epithelial morphology. The epithelializationof the ectoderm in post-gastrulation embryos can therefore be a secondary process, after the generation of© 2008 The Authors
Journal compilation © 2008 Japanese Society of Developmental Biologists

760

Y. Nakaya and G. Shengthe mesoderm and endoderm. Examples of this can befound in the separation of zebrafish ectoderm into thesuperficial enveloping layer and deep enveloping layer,in the stratification of Xenopus ectoderm into surface
layer and deep layer, and in some sense in the separation of trophectoderm and inner cell mass in mouseembryos. The de-coupling of the first and the third roles
is prominent in embryos with a significant amount ofyolk substance deposited in the fertilized egg. Whether
cellularized by holoblastic cleavages or syncytialized bymeroblastic cleavages, yolky cells are part of developing
embryos and will eventually become part of the endoderm. A large amount of yolk in many vertebrate
embryos therefore requires the de-coupling of the epibolylike process, as a means of internalizing these yolky cells,
and the formation of the mesoderm and endoderm fromthe ectoderm in the traditional sense of gastrulation.
Due to this de-coupling, mesoderm and endoderm
formation often takes place before gastrocoel formationand is shifted from a circumblastoporal process suchas in sea urchin, to dorsally favored as in Xenopus andZebrafish, and to dorsally restricted as in amniotes(Voiculescu et al. 2007; Shook & Keller 2008). Thesede-coupled roles of embryonic ectoderm suggest that
EMT, as a cellular morphogenetic process, is required
for mesoderm formation when and only when mesoderm
precursor cells are located in the ectoderm (and insome cases internalized mesendoderm) layer with anepithelial morphology, which does not have to be thecase as we have discussed above.

Genetic dissection of EMT in mesoderm
formation
Nevertheless, EMT was first proposed as a cellularmechanism for mesoderm formation in avian embryos(Trelstad et al. 1967; Hay 1968), and EMT/MET processes have since been used to describe a large variety
of other developmental processes, including neural crest
formation from the border of neural/non-neural ectoderm(Duband et al. 1995; Newgreen & McKeown 2005;
Sakai et al. 2005; Duband 2006; Sakai et al. 2006),
somitogenesis and sclerotome formation from epithelialized somite (Duband et al. 1987; Nakaya et al. 2004;
Hay 2005; Takahashi et al. 2005; Takahashi & Sato 2008)
and cardiac valve development from endocardium(Person et al. 2005; Runyan et al. 2005). This is not
surprising as all cells in an organism can in principle begrouped into either an epithelial or mesenchymal category.
Changes in tissue organization during normal andpathological development will often involve EMT or MET.
As in the case of mesoderm formation, EMT in otherdevelopmental processes often comes with evolutionary
variations. EMT as a descriptive term therefore would

be much less useful if there are no shared molecularmechanisms governing diverse EMT processes. Indeed,
uncovering and understanding these mechanisms has
been the focus of EMT-related research in the past
decade. With regard to EMT during gastrulation, genetic
and molecular studies have been primarily focused on
the mouse system.
As we have discussed earlier, the majority of mesoderm cells in amniotes (reptiles, birds and mammals)
form by a process of ingression during gastrulation. This
ingression is a typical EMT process. Pre-ingression
mesoderm precursor cells have all the characteristics
of epithelial cells, whereas post-ingression mesoderm cells
are mesenchymal. Genetic experiments in mice haverevealed that several extracellular signals, mediated bytransforming growth factor-β (TGF-β), Wnt and FGF
receptors, contribute to the EMT process in generating
mesoderm cells (Deng et al. 1994; Yamaguchi et al.
1994; Sun et al. 1999; Ciruna & Rossant 2001; Kemler
et al. 2004; Ben-Haim et al. 2006; Arnold et al. 2008),
although platelet-derived growth factor (PDGF), Notch,
Hedgehog and nuclear factor-κB (NF-κB) pathways
have also been implicated in other EMT processes, with
their roles in gastrulation EMT not carefully examinedyet (Huber et al. 2005; Baum et al. 2008; Van Den Akker
et al. 2008). Change of adherens junction types, from
E-cadherin mediated epithelial interactions to N-cadherin
mediated mesenchymal interactions, is generally considered as a critical step in this process (Hatta & Takeichi
1986; Cano et al. 2000; Zohn et al. 2006). FGF signaling
in the primitive streak has been shown to regulate gastrulation EMT in mouse (Yamaguchi et al. 1994; Sunet al. 1999; Zohn et al. 2006). FGF signals downregulate
E-cadherin expression to promote EMT during gastrulation by regulating snail gene expression (Ciruna &Rossant 2001). Snail, a zinc-finger transcription factor,
directly binds to E-boxes in the promoter region of Ecadherin gene and represses its transcription in cancer
cell lines (Batlle et al. 2000; Cano et al. 2000). Mouseembryos deficient in snail function exhibit abnormal
mesoderm cell morphology with apico-basal polarityand epithelial type adherence junctions, and E-cadherin
expression is retained in the mesoderm of Snail-/mutants (Carver et al. 2001). In a number of tumor celllines, it has been suggested that Wnt and receptor
tyrosine kinase mediated signals also promote Snailstabilization and its nuclear import, and subsequently
enhance EMT. The relationship between Snail andsignaling pathways other than FGF has yet to be clarified
at cellular levels in vivo. Furthermore, as a Snail independent pathway, it has been demonstrated that p38
mitogen-activated protein kinase (MAP kinase) and
p38-interacting protein and/or EPB4.1L5 (Band 4.1 superfamily) are required for the downregulation of E-cadherin© 2008 The Authors
Journal compilation © 2008 Japanese Society of Developmental Biologists

EMT in gastrulation

761

Fig. 2. Multi-step epithelial tomesenchymal transition (EMT)
process in mesoderm formation
during chicken gastrulation. In theprimitive streak where mesoderm
ingression takes place, precursor
cells first express mesoderm
markers (e.g. brachyury), then break
down the basement membrane.
These cells leave the epiblast after
disrupting the tight junctions andapical/basal polarity, followed by ashift from E-cadherin- to Ncadherin-based adherens junctions.

protein at the post-transcription level during EMT inmouse gastrulation (Zohn et al. 2006; Lee et al. 2007).
In addition to the mouse model, Snail gene family
members have been shown to play crucial roles in the
induction of EMT in other systems. For instance, Lvsnail, a member of the Snail family in sea urchin Lytechinus
variegatus, is required for the EMT process in primarymesenchyme formation by downregulating cadherin
expression and promoting cadherin endocytosis (Wuet al. 2007). Interestingly, Snail and cadherin regulated
EMT has also been proposed for mediating mesoderm
formation in zebrafish (Yamashita et al. 2004; Montero
et al. 2005; Krieg et al. 2008), an organism that does
not have a characteristic epithelial ectoderm as we havediscussed above. Overall, these studies have provided thegroundwork for investigating the molecular basis of EMT.

Basement membrane breakdown, a novel
aspect in regulating gastrulation EMT
It has been well recognized that the EMT process
involves the breakdown of the epithelial basement
membrane. In her earliest description of EMT, Hay(1968) considered basement membrane degradation
during mesoderm formation in chick embryos as one of
the most prominent features of EMT. Molecular mechanisms of basement membrane breakdown during
gastrulation EMT have not received careful investigation.
A recent report indicated that this step is also criticallyregulated (Levayer & Lecuit 2008; Nakaya et al. 2008)(Fig. 2). Breakdown of epithelial basement membrane,marked by fibronectin and laminin, was shown to be
the earliest event for gastrulation EMT in chick embryos,

taking place prior to the breakdown of tight junctions
and apico-basal polarity, whereas the shift in adherens
junction types was seen to take place after the ingression.
These observations indicate that EMT during gastrulation
may take place as a multi-step process, each of whichis controlled by a distinct set of signals. Supporting thismodel, it was shown that basally localized small GTPase
RhoA and its activator Net1, a RhoA GEF (Guanine
Nucleotide Exchange Factor), control the interaction
between the basement membrane and the basal
membrane of ectoderm cells. In ectoderm cells lateral
to the primitive streak, RhoA protein and activity aredetected both apically and basally, whereas in ectoderm
cells within the primitive streak, basal RhoA and Net1
(Net1 is detected only basally in lateral regions) are
downregulated, with apical RhoA still intact. Ectopic
expression of RhoA did not affect normal ectoderm lateral
to the streak, but resulted in the failure of basement
membrane breakdown in medial streak and in ingressed
mesoderm cells. Interestingly, many mesoderm precursor
cells overexpressing RhoA still manage to finish theingression process and move to the mesoderm layer,
indicating that other EMT related events, such as thedownregulation of tight junctions and the switch ofadherens junction types, are not obviously affected.
Furthermore, it was shown that the regulation of basement membrane breakdown mediated by RhoA takes
place in the context of dynamic cytoskeletal changes,
in particular the destabilization of basal microtubules,
during the process of ingression. These observations
further support the idea that EMT is a collective processcomposed of distinct cellular steps that are separately
regulated. It is so far still unclear however, what the© 2008 The Authors
Journal compilation © 2008 Japanese Society of Developmental Biologists

762

Y. Nakaya and G. Shengbiochemical mechanisms for the degradation of basement
membrane proteins are, and how this process is linked
to mesoderm fate specification marked by Brachyury
on the one hand, and to the later disruption of tightjunctions and apico-basal polarity, and the switch ofadherens junction types on the other.
Given the diversity of EMT, the sequence and timingof these cellular steps have been reported to vary in a
number of other EMT processes that have been investigated in some detail. During the formation of endocardial
cushion mesenchymal cells, it appears that the loss ofintercellular junctions takes place prior to the invasion
through lamina densa of endocardial epithelium (Person
et al. 2005). Sclerotome formation is another example
in which downregulation of adherens junctions takes
place before basement membrane disruption (Duband
et al. 1987). In the case of neural crest cell delamination,
while basement membrane breakdown is a necessary
step for cranial crest cells, at the trunk level, the delamination does not involve the basement membrane(Nichols 1981; Newgreen & Gibbins 1982; MartinsGreen & Erickson 1986). Moreover, no desmosomes orfunctional tight junctions are present in pre-delamination
crest cells (Erickson et al. 1987; Aaku-Saraste et al. 1996),
and breakdown of cadherin mediated adherens junctions
triggers immediate delamination (Newgreen & Gooday1985). A recent study with induced EMT in cultured
epithelial cells suggested that tight junction dissociation
occurs prior to adherens junction dissociation (Ozdamar
et al. 2005; Thiery & Huang 2005).
These differences highlight the complex nature ofcontext-dependent coordination of cellular components
in different EMT processes. It is thus not surprising that,in terms of signals regulating EMT, no clear consensus
has emerged as to whether a few ‘master regulatory’molecules control the entire EMT (i.e. one decision point
triggers a procession of cellular events), or an individual
cellular event takes distinct signaling cues. Observations
in gastrulation EMT favor the latter hypothesis, which isalso favored by recent in vitro studies on independent
regulations of tight junctions and adherens junctions(Hollande et al. 2003; Ozdamar et al. 2005). Nevertheless,signaling cues controlling individual cellular events likely
crosstalk, resulting in both the complexity for any given
EMT and the diversity of how EMT can be executed indifferent in vivo settings.

Future perspectives
In the four decades since EMT was first proposed, alarge number of descriptive and experimental studies
have been reported in literature, providing a rich resourceof both in vitro and in vivo data. Proper understandingof EMT will obviously require the integration of knowledge

from both fields. EMT studies using culture systems
remain to be an irreplaceable alternative for detailed
molecular analyses. However, one should not lose sightof the fact that EMTs in development and disease often
take place in a much more dynamic and complex
context. In addition, the relationship between EMT anddifferentiation is increasingly being appreciated in thestem cell research field. It has recently been reported
that the induction of EMT in human mammary epithelial
cells (HMLEs) by Twist, Snail or TGF-β1 leads to morphological change of these cells to a mesenchymal
shape with the upregulation of stem cell markers (Maniet al. 2008), pointing to a possible direct link between
EMT and pluripotency. This would not be unexpectedfrom the embryological perspective, as the first sign ofcellular differentiation during embryogenesis is often
manifested as the epithelialization of the ectoderm cells.
Furthermore, new molecular mechanisms, such as
through the investigation of microRNAs, are being
proposed for the regulation of EMT (Burk et al. 2008;
Cano & Nieto 2008; Gregory et al. 2008; Korpal et al.
2008; Park et al. 2008). By evaluating the expressionof 207 microRNAs in the many cancer cell lines, miR-200
family was identified as a general marker for E-cadherin
positive and vimentin negative cancer cells (Park et al.
2008). This and other recent reports also show thatmiR-200 family targets both ZEB1 and ZEB2 to inhibit
E-cadherin repression, and altering the levels of miR-200
in established cancer cell lines caused changes consistent
with either EMT or MET induction (Burk et al. 2008;
Cano & Nieto 2008; Gregory et al. 2008; Korpal et al.
2008; Park et al. 2008). Taken together, with a combination of novel tools and new perspectives, the EMT
research has entered an interesting time. This, in return,will undoubtedly contribute to the understanding of how
germ layers are formed during gastrulation, which
remains one of the most fascinating questions forembryologists.

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© 2008 The Authors
Journal compilation © 2008 Japanese Society of Developmental Biologists