Cancer Letters 341 (2013) 9–15

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Cancer Lettersjournal homepage: www.elsevier.com/locate/canlet

Mini-review

EMT in developmental morphogenesis
Yukiko Nakaya, Guojun Sheng ⇑Lab for Early Embryogenesis, RIKEN Center for Developmental Biology, 2-2-3 Minatojima-minamimachi, Chuo-Ku, Kobe, Hyogo 650-0047, Japana r t i c l e

i n f oKeywords:
Developmental EMT
Epithelium
Stratiﬁcation
Mesenchyme
AmnioteVertebrate
Mammals
Birds
Cancera b s t r a c t
Carcinomas, cancers of epithelial origin, constitute the majority of all cancers. Loss of epithelial characteristics is an early step in carcinoma progression. Malignant transformation and metastasis involve additional loss of cell-cycle control and gain of migratory behaviors. Understanding the relationships among
epithelial homeostasis, cell proliferation, and cell migration is therefore fundamental in understanding
cancer. Interestingly, these cellular events also occur frequently during animal development, but without
leading to tumor formation. Can we learn anything about carcinomas from developmental biology? In
this review, we focus on one aspect of carcinoma progression, the Epithelial–Mesenchymal Transition(EMT), and provide an overview of how the EMT is involved in normal amniote development. We discuss
12 developmental and morphogenetic processes that clearly involve the EMT. We conclude by emphasizing the diversity of EMT processes both in terms of their developmental context and of their cellular morphogenesis. We propose that there is comparable diversity in cancer microenvironment and molecularregulation of cancer EMTs.
Ó 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

2. Developmental EMTs with no cancer association

Epithelial–Mesenchymal Transition (EMT) and Mesenchymal–
Epithelial Transition (MET) are fundamental processes of cell shapechanges during animal development and disease progression (collectively referred to as EMT here, except when a distinction isneeded) [1]. Many excellent reviews have covered this subject inrecent years [2–6]. Its involvement in cancer underscores the needfor better molecular and cellular understanding of EMTs during
normal development. Developmental EMTs exhibit a wide rangeof phenotypic variations. For developmental models to be usefulin cancer studies, one needs to appreciate unique morphogenetic
parameters associated with each developmental EMT. In this review we survey developmental EMTs with an emphasis on theirmorphogenetic variables. The examples given are not meant tobe an exhaustive list, but to highlight the wide presence of EMTevents in development and the variations in their cellular organization. Many of them represent important but transitory morphogenetic events and do not have cancer counterparts. But manyoccur during organogenesis and have a direct link, in cellular origin
and intercellular organization, to organ-speciﬁc cancer EMTs in
adults. We will also provide a general discuss on epithelial structures in development and propose a conceptual framework to integrate diverse EMT phenomena in cell, developmental and cancer
biology.

2.1. Blastula formation⇑ Corresponding author. Tel.: +81 78 306 3132 (Lab); fax: +81 78 306 3146.
E-mail address: sheng@cdb.riken.jp (G. Sheng).
0304-3835/$ - see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.canlet.2013.02.037

The earliest MET during mammalian development takes placewhen blastomeres differentiate into three primitive cell lineages:
the trophoblast, epiblast and hypoblast [7]. This process actuallyconsists of three separate METs. The MET of the trophoblasts generates trophectoderm, the ﬁrst epithelial structure in a mammalianembryo (Fig. 1A, left). The second and third METs epithelialize thehypoblast and epiblast cells, respectively, from the inner cell mass
(Fig. 1A, right). Formation of these three cell lineages has often
been studied from the perspective of fate speciﬁcation and pluripotency maintenance, but of equal importance and involving distinct
molecular mechanisms is the cell biological regulation of these
MET processes [8–16]. Early mammalian development is heavily
inﬂuenced by maternal environment. The timing of blastomere
compaction, trophectoderm epithelialization, and epiblast andhypoblast epithelialization, relative to that of fate speciﬁcation,varies among mammalian species. Differences of these METs in primates and rodents may underlie difﬁculties in applying the advances in the ES cell ﬁeld, mostly derived from the mouse model,
to the humans [17,18].
2.2. Gastrulation and neural crest cell formation
Gastrulation and neural crest cell formation are two major
examples of developmental EMT. They have been reviewed extensively and are described here only brieﬂy. In gastrulation EMT,

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Fig. 1. Developmental EMTs. (A–E) Developmental EMTs with no direct cancer association. (A) Polarization and epithelialization of some blastomeres result in trophectoderm
formation (1st MET arrow). The inner cell mass is still mesenchymal at this stage. Epithelialization of the inner cell mass generates two additional epithelia (2nd MET arrow):
the epiblast and the hypoblast. (B) During gastrulation, epiblast cells undergo EMT to form mesoderm cells. (C) Neural crest cells leave the ectoderm during and after neuraltube closure. (D) Somitic mesoderm formation and differentiation involve several rounds of MET/EMT. PSM: presomitic mesoderm. (E) Formation of endocardium (theendothelial lining of the heart) involves MET. Endocardial cushion and cardiac valve formation involves EMT. (F–L): Developmental EMT with cancer association. (A)Anchoring chorionic villus; (B) Terminal end bud of the mammary gland; (C) Mesothelial cells; (D) Renal vesicle; (E) Liver bud; (F) Pancreatic bud; (G) Prostate gland.
Epithelial structures: yellow; mesenchymal structures: blue; basement membrane: red. EMT: yellow to blue arrow. MET: blue to yellow arrow. (For interpretation of thereferences to colour in this ﬁgure legend, the reader is referred to the web version of this article.)mesenchymal-shaped mesoderm cells are generated from theepithelial-shaped epiblast [19–23] (Fig. 1B). This is the earliest
developmental EMT. During the neural crest cell formation, precursor cells delaminate from border regions of neural and non-neural
ectoderm territories prior to and during neural tube closure andfrom dorsal neural tube after the closure [24–29] (Fig. 1C). Bothare stereotypic EMT events because mesoderm and neural crest
precursor cells are polarized epithelial cells with a basement membrane before EMT and start active migration after EMT. It is worth
pointing out that both EMTs exhibit variations in terms of species,
regions and developmental time, and should not be generalized.
There is no direct cancer association for gastrulation or neural crest
cell formation EMT. Postnatally, many cancers are of the mesoderm
origin, and neural crest cells are involved in melanoma andneuroblastoma.
2.3. Somitogenesis
Somites are derived from the paraxial mesoderm and begin as
mesenchymal-shaped presomitic mesoderm. Segmentation clock
regulates periodic budding of somites from its anterior tip [30].
Newly generated somite polarizes to form an epithelial ball, withan internal apical surface and external basement membrane(Fig. 1D, left). This is an MET process [31–33]. Each somite canbe broadly divided into two subdomains: the sclerotome anddermomyotome. Cells in the sclerotome subdomain, located ven-tromedially, lose their epithelial characteristics ﬁrst [34] (Fig. 1D,
middle). Somitocoel cells, located inside the epithelialized somite,remain mesenchymal and contribute to the sclerotome without
undergoing MET or EMT. Dermomyotome cells undergo complex
EMT and MET processes to generate the myotome (later on giving
rise to skeletal muscles) and an EMT process to generate the precursors of dorsal dermis [35–39] (Fig. 1D, right). Formation anddissolution of somites thus involve several rounds of extremely dynamic MET/EMTs.
2.4. Endocardium and endocardial cushion formation
Cardiac morphogenesis is important for embryonic survival. The
endocardium is the endothelial lining of the heart [40] and threesuccessive EMT/METs are involved in its formation and differentiation [41]. Cardiac mesodermal precursor cells undergo EMT during gastrulation, when these cells are speciﬁed as cardiac
progenitors. After subsequent bilateral migration within the lateralplate, mesenchymal-shaped endocardial progenitors separate frommyocardial progenitors before epithelialization of the latter. The
endocardial progenitors epithelialize to form endothelial plexus
and then tubes (Fig. 1E, left). This is an MET process. Early embryonic heart contains inner endocardial layer and outer myocardial
layer, separated by cardiac jelly, the extracellular matrix secreted
mainly by the myocardium. Endocardial cells delaminate basallyin the atrioventricular canal and outﬂow tract into this extracellu-

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lar matrix, contributing to the endocardial cushion in these areasand cardiac valves later in development [41] (Fig. 1E, right). Thisis an EMT process. In addition, similar to endocardial formation,
MET is involved in vaculogenesis in the entire embryo [42–44],
and further EMT is involved in hematopoietic stem cell formationfrom aortic endothelium [45].
3. Developmental EMTs with cancer association
3.1. Trophoblast invasion
The trophectoderm is a special type of primitive ectoderm andits origin in development precedes that of the three germ layers.
Trophectoderm cells (trophoblasts) mark the external boundaryof a mammalian embryo and mediate materno-fetal exchanges
during placental development. To achieve optimal contact withmaternal environment, trophoblasts proliferate, invade and branch
out within uterine endometrium. In most areas, the trophoblasts
form a cytotrophoblast inner layer and a syncytiotrophoblast outerlayer (Fig. 1F). The former is a stereotypic epithelial sheet with abasement membrane. The latter is a fusion product of the former
and covers its apical surface. EMT occurs in the anchoring villi
and some ﬂoating villi where cytotrophoblasts stratify and breachthe syncytiotrophoblast layer, forming extra-villous and interstitialcytotrophoblasts with mesenchymal characteristics [46–50](Fig. 1F). This EMT occurs apically and the inner-most cytotrophoblast layer remains epithelial and has an intact basement membrane. Associated cancer: Chorionic carcinoma.
3.2. Mammary gland
The mammary gland is an ectoderm-derived organ [51–54].
Thickening of mammary placode ectoderm is followed by local epithelial cell movement, leading to formation of mammary bud [55].
Cells within the mammary bud partially lose their epithelial characteristics, but re-epithelialize to form rudimentary mammary gland[56]. This early phase of mammary gland development thus involves partial EMT and MET processes. Onset of puberty initiates rapid ductal growth and morphogenesis, and pregnancy and lactation
cycles regulate the formation and involution of milk-producing
mammary lobular alveoli. Ductal elongation and bifurcation duringpuberty occur primarily at the terminal end buds (TEBs), the frontof stromal invasion for the ectoderm-derived mammary branches.
The TEB is a multi-layered epithelioid structure and is covered at
its leading edge by a broken basement membrane [51,57,58](Fig. 1G). Its luminal layer, and to some extent the basal layer, havepartial epithelial characteristics, but its inner layers have mesenchymal features [59]. The TEB also engages in collective cell migration [59,60]. Its formation therefore can be viewed as partial EMT.
Side branching also contributes to ductal growth and morphogenesis and may likewise involve similar partial EMT (Fig. 1G). Associated cancer: Breast cancer is the leading cancer in women.
3.3. Mesothelium
The mesothelium is a thin layer of epithelium lining the serosal(peritoneal, pleural and pericardial) cavities [61]. Derived from thelateral plate mesoderm during germ layer formation, the mesothelium is a genuine epithelium with tight junctions, a basement
membrane, and an apical surface facing the serosal cavities. Formation of the mesothelium thus involves both EMT and MET processes. After their formation, mesothelial cells can undergo
further EMTs (Fig. 1H) to generate coronary vascular smooth muscle cells and cardiomyocytes in the pericardial mesothelium [62–64], pulmonary vascular smooth muscle cells and lung mesen-

11

chyme in the pleural mesothelium [65] and gut vascular smoothmuscle cells in parietal peritoneal mesothelium [66]. EMT is also involved in generating sertoli cells from the mesothelium covering
the male gonad [67] and the granulosa cells from the mesotheliumof the ovary [68,69]. Furthermore, EMT has been implicated in the
repair of ovarian mesothelium after ovulation and in morphologicalresponses of the granulosa cells to follicle activation [68,70]. Associated cancer: Mesothelioma is the leading asbestos-related cancer.
Ovarian epithelial carcinoma makes up 90% of all ovarian cancer.
3.4. Kidney
Kidney is a mesoderm-derived organ. Adult kidney development begins when ureteric bud at posterior part of the nephricduct grows into surrounding metanephric mesenchyme [71–73].
The ureteric bud is an epithelial structure (formed at a much earlier stage of development through MET of the intermediate mesoderm) and remains so during its branching morphogenesis. The
metanephric mesenchyme cells organize themselves aroundbranching ureteric buds, coalesce into aggregates, and epithelialize
to form renal vesicles (Fig. 1I) [74,75]. Renal vesicles are surrounded by a basement membrane and have an apical lumen. Thisis a typical MET process [72,75–77]. Renal vesicles undergo further
tubular morphogenesis to give rise to renal tubules and non-vascular part of renal glomeruli [78]. Associated cancer: Renal cell carcinoma is the most common kidney cancer and is caused bymalignant transformation of renal tubule epithelial cells.
3.5. Liver
The liver starts off as liver bud, a ventral diverticulum of theforegut [79]. Hepatoblasts in the liver bud are initially of an epithelial morphology and are surrounded by a basement membrane. The
hepatoblasts proliferate and thicken to form a pseudostratiﬁed epithelium at the tip of the liver bud. This is followed by breaches inthe basement membrane, and hepatoblasts delaminate from the liver bud epithelium [80,81] (Fig. 1J, left). This is a typical EMT process. Mesenchymal hepatoblasts aggregate around the portal veinand differentiate into two types of epithelium cells: the biliary epithelial cells and hepatocytes (Fig. 1J, right). MET is involved in theformation of both. The biliary epithelium is a simple, stereotypic
epithelium with an apical lumen and a basal basement membrane[82]. The hepatocytes form a special epithelial structure [79]. It has
no basement membrane, but its basal side faces the sinusoid endothelial cells and its apical side faces the canalicular lumen. Tight
junctions separate the basolateral membrane from the apicalmembrane, from which bile acids and salts are secreted. Associatedcancer: Hepatocellular carcinoma and cholangiocarcinoma.
3.6. Pancreas
The pancreas is an endoderm-derived organ [83,84]. All of its
three main cell lineages, the exocrine, ductal and endocrine cells,
originate from the pancreatic bud epithelium. Pancreatic buds
are outgrowths of the foregut. Their formation involves local cellproliferation, lumenization and tubulogenesis [85]. During thisprocess, pancreatic bud cells are collectively bound by an intact
basement membrane. These cells transit from an initial foregut
epithelial state to a stratiﬁed epithelioid and then back to an epithelial one [85,86], involving partial EMT and MET. From this secondarily formed epithelium, pancreatic endocrine precursors
undergo a typical EMT process by breaking down the basement
membrane and leaving the epithelium [87–89] (Fig. 1K). Delaminated cells coalesce to form pancreatic islets that secret insulin,
glucagon and other pancreatic hormones into circulation. Endocrine islet cells are generally considered to be mesenchymal be-

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cause they do not have a typical epithelial organization and haveno lumen or basement membrane. But this notion may need revision. Each islet is enclosed by a sheet of basement membraneexternally, and internally-located islet cells rest on the basement
membrane of inﬁltrated vasculature [90–94]. They have asymmetrical membrane preference for glucose transporter localization [95]and insulin secretion [96]. Formation of pancreatic islets therefore
represents a special type of MET. Associated cancer: Pancreaticcancer is the fourth most common cancer and has among the lowest survival rate after diagnosis.
3.7. Prostate
The prostate gland is derived from androgen-regulated endodermal outgrowths of the urogenital sinus [97,98]. These outgrowths (prostate buds) are bound by the basement membrane[99], but cells in the buds form solid epithelioid mass with no lumen (Fig. 1L, left). Lumenization of prostate buds can therefore
be viewed as a partial MET process (Fig. 1L, right). Epithelialized
buds then undergo branching morphogenesis and cellular differentiation, making up the endoderm portion of the prostate gland. Partial reversion of this MET in adult may underlie prostatic
intraepithelial neoplasia, an early stage of prostate cancer[98,100]. Associated cancer: Prostate cancer tops new cancer cases
and is the second most deadly cancer in men.
4. What is an epithelium?
A stereotypic epithelium is generally considered to have the following characteristics: (1) It is a sheet of cells with a shared apicobasal polarity (e.g., polarities in membrane lipids and proteins, inintracellular molecular localization, in vesicular transport, and incytoskeletal and organellar organization); (2) There is no free passage of membrane lipids and proteins between the apical and basolateral compartments (e.g., through lipid rafts); (3) There is no freepassage of extracellular molecules between the apical and basolateral extracellular space (e.g., by forming tight junctions); (4) theirlateral membrane adheres to each other (e.g., through adherens
junctions); (5) Their basal membrane interacts with a specialized
extracellular matrix, the basement membrane (e.g., through integrins and dystroglycan) [2–4,6,20]. Cells organized this way are
considered to be fully epithelial (Fig. 2A). However, although thesecellular features are interconnected in their molecular regulation,
many epithelial-like structures do not have this full set of characteristics (Fig. 2A). This is especially true during embryonic development, when intercellular organizations are modiﬁed constantly toaccommodate for dynamic growth, morphogenesis and patternformation. We propose that the minimal criterion of an epithelial
structure is for its constituent cells to have a shared apico-basal
polarity. A group of cells without this minimal feature should becalled mesenchymal. If these mesenchymal cells are not migratory
and are constrained spatially, they can be viewed as having an epithelioid mesenchymal structure (Fig. 2A) (e.g., in luminal neoplasia
of many cancers and in the terminal end bud of growing mammary
gland). Transition from an epithelial structure (either full or partial
epithelium) to this epithelioid mesenchymal structure can therefore be considered as a partial EMT process. Molecularly, this basic
characteristic of an epithelium, the apico-basal polarity, isachieved through three polarity complexes: the PAR, the Crumbs
and the Scribble complexes [4]. The PAR complex contains PAR3,
PAR6 and aPKC, and sets the boundary between the apical andbasolateral membrane compartments. The Crumbs complex contains transmembrane protein Crumbs and cytoplasmic proteins
PLAS1 and PATJ, and controls the apical membrane formation.
The Scribble complex contains Scribble, LGL and DLG, andmaintains the basolateral membrane compartment. Mutations inmany of the apicobasal polarity complex genes have been associated with cancer EMTs [4] and known EMT inducers, such as Snailand ZEB1, can suppress transcription of adherens junction gene Ecadherin and cell polarity genes Crumbs3, Pals1 and Lgl2 [101–103].

Fig. 2. Partial EMT, epithelial stratiﬁcation and cancer EMT. (A) Between fully epithelial and fully mesenchymal structures, one can recognize partial epithelial and partial(epithelioid) mesenchymal structure. A partial EMT may mean one of the several stepwise transitions needed to convert a full epithelium to a full mesenchyme, or the modestchange from a partial epithelium to epithelioid mesenchyme by losing coordinated apico-basal polarity. (B) Stably-maintained stratiﬁed epithelium can be considered as anepithelial unit. Unstable stratiﬁcation causes temporary loss of epithelial characteristics. This happens both in development and during cancer progression. (C) A model for
how graded loss of epithelialness can lead to cancer. The stepwise nature of this transition is emphasized in both simple and complex EMTs. In cancer biology, this implies
that multiple independent molecular lesions are required for malignant transformation.

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5. Epithelial stratiﬁcation and EMT
In most cases, epithelial structures exist as a single-cell-layered,
two-dimensional sheet. Further morphogenetic modiﬁcations often result in forming complex, three-dimensional tissue architecture from such a two-dimensional sheet (e.g., lung, ureter).
Occasionally, single-layered epithelial structure can be organized
as a three-dimensional cell mass through special types of apico-basal polarization (e.g., among hepatocytes or pancreatic endocrine
cells) [79,91]. Epithelial structures can also be found as multi-layered, stratiﬁed cell sheets (e.g., mammary duct, skin epidermis).
Because all stratiﬁed epithelia originate in development as single-cell-layered epithelial structures, a stably-organized stratiﬁedepithelium can be viewed as one polarized epithelial unit. Maintenance of a stratiﬁed epithelium, however, requires delicate balanceof proliferation, mitotic spindle orientation, cell loss, and cross-talkbetween layers [104], and failure to do so often leads to cancer. In
many normal developmental scenarios (Fig. 2B), epithelial stratiﬁcation also leads to loss of epithelial characteristics among some or
all of its constituent cells. This can be viewed as partial EMT. Often
times, the non-epithelial cells originating from epithelial stratiﬁcation are spatially restricted and are not very motile. They canquickly revert to proper epithelial polarity and rarely result inmalignant growth. In cancer biology, such epithelial stratiﬁcationof normally single-cell-layered epithelia and the resulting partial
EMT in adult epithelial tissues are often signs of tumor initiation[105–107]. Most of these tumors are benign. Malignant transformation requires additional triggers for unregulated proliferationand the ability to breach the basement membrane (Fig. 2C).
6. Conclusions and outlook
EMT is a fundamental component of animal development, andas discussed in accompanying reviews, a hallmark of cancer progression. In this review, we have shown that there are many types
of epithelial organization and EMT in developmental morphogenesis. Not all epithelial structures undergo EMT (e.g., lung, colon), andnot all EMTs involve the conversion of fully epithelial cells to fully
mesenchymal ones. Recognizing partial epithelial structures andpartial EMTs may be the key to understanding both developmental
and cancer EMTs. Epithelial stratiﬁcation in development and cancer is often coupled with partial EMT. Diversity of developmental
EMTs in their cellular organization and surrounding tissue architecture strongly suggests that there is a similar level of diversity
in the molecular regulation of EMTs. Developmental EMT studies,with an emphasis on each’s uniqueness, may help us understandcancer EMTs. Conversely, emerging concepts in cancer and cancer-EMT research [108] offer fresh perspectives for developmental
EMT studies, and together these two research ﬁelds, development
and cancer, can beneﬁt from a win–win partnership.
Conﬂict of Interest
None
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