SS Literature Review 2012-2013
Syncytin genes and the evolutionary
invention of the mammalian placenta
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i Contents
Abstract…………………………………………………………………………………..………1
Introduction………………………………………………………………………………...……1
Endogenous retroviruses……………………….……………………………………………….4
Retroviral envelope proteins……………………………………………………………………5
Placental structure and diversity………………………………………………….……………7
Retroviral env genes in the human placenta………......……………………..………………10
ERV3…………………………………………………………………………………………10
Syncytin-1…………………………………………………………..……………………..…12
Syncytin-2……………………………………………………………………………………15
Syncytins in Mice………………………………………………………………………………17
Identification……………………………………………………...…………………………17
Functional Analysis…………………………………………………………………………18
Syncytins in other Mammals…………………………..………………………………………20
Beyond Syncytins………………………………………………………………………………22
Syncytin-like genes…………………..………………………………………………………23
Other retroelements in placental evolution………...………...……………………………24
Conclusions and outlook…………………………….…………………………………………25
References………………………………………………………………………………………27
ii Abstract
There has been a longstanding evolutionary interplay between retroviruses and the placenta. The
placenta provides retroviruses access to germline cells, allowing them to become endogenous
retroviruses (ERVs), transmitted vertically between the host generations. In turn, ERVs have
provided the host with a wealth of novel genetic material that has been utilized in placental
development and evolution. The most well characterized example of this phenomenon is the
domestication of retroviral envelope genes, named syncytins, whose fusogenic capacity appears to
have
allowed
for
the
invention
of
a
unique
tissue,
the
syncytiotrophoblast.
The
syncytiotrophoblast is associated with more invasive placental types and acts as the barrier
between the foetal and maternal tissues during gestation. Syncytin genes have been captured on
at least six independent occasions in diverse mammalian lineages, including primates, rodents,
rabbits and carnivores. Syncytin-like genes have also been identified in guinea pigs and sheep.
There is much evidence to suggest that syncytins play an essential role in placentation and
syncytiotrophoblast formation. Indeed, it is hypothesised that syncytins or syncytin-like gene
capture may have mediated the transition from a primitive ‘yolk-sac’ placenta to the diverse
range of complex placental types exhibited by eutherian mammals today.
Keywords: Syncytin genes, Placentation, Retroviral Capture, Convergent Evolution, Eutherian
Evolution.
1 Introduction
It can be argued that the invention of the placenta has been one of the most pivotal innovations in
mammalian evolution. Without the ability to bear live young, mammals could never have colonized the
vast range of aquatic and terrestrial habitats they occupy today. The evolution of the placenta allowed
for the protection of offspring from the environment for an extended amount of time. It also gave the
mother the ability to provide prolonged nourishment and oxygen to the embryo, which is necessary for
more complex foetal development. It is these innovations that have made placental mammals one of the
most successful vertebrate groups on earth today.
The placenta is a transient and autonomous organ, and can be defined as an apposition of foetal
to maternal tissue. It has two primary functions, firstly to facilitate and maximise metabolic exchanges
between the mother and foetus, and secondly to protect and mask the foetus from the mother’s immune
system. These two goals are frequently antagonistic, owing to the difficulty of simultaneously
nourishing and camouflaging a foreign, invasive structure (Wooding and Burton, 2008). These
competing constraints, along with the fact that genes involved in both immune processes (Hughes,
2002) and reproduction evolve rapidly (Crespi, 2010), have led to the placenta being one of the most
diverse and mutable organs to evolve.
The complexity of placental evolution is reflected in the myriad of diverse structures, shapes,
physiological adaptions and cellular compositions of placentas found today, particularly within
placental mammals, with different clades and species developing different solutions to the conflicting
pressures and to their own unique situations. Indeed, structural diversity in other mammalian organs,
such as the lung or the eye, appears remarkably uniform when compared to the extensive range of
placenta types (Wooding and Burton, 2008). The exact processes by which this startling variety has
developed remain an evolutionary mystery.
The chorioallantoic placenta is the defining characteristic of the mammalian clade Placentalia,
which encompasses all extant eutherian mammals (Carter and Mess, 2007). It is placentation in this
dominant group of mammals, which includes our own species, that this review will focus on. However,
this is not to say that placentas do not occur in other types of mammals or vertebrates. The amniote
structure of foetal membranes has undergone modification on multiple independent occasions to allow
for placentation in a number of vertebrate species. This modification typically involves the fusing of
the chorionic membrane with yolk sac or allantois to allow the foetal blood vessels access to the
extraembryonic maternal environment (Carter and Enders, 2004).
Indeed, ‘yolk-sac’ placentas are common in marsupials, squamate reptiles and even sharks
(Renfree, 2010; Blackburn and Flemming, 2009; Jones and Hamlett, 2004). In this type of placentation,
2 while the majority of nourishment supplied to the foetus is derived from the yolk sac, some nutrients
diffuse through the uterine wall via a choriovitelline placenta. In eutherian mammals the placenta has
evolved to fulfil a more central role, providing virtually all nourishment to the developing foetus via
the heavily vascularised chorioallantoic membrane. Intriguingly, chorioallantoic placentas are not
limited to eutherians, but in rare cases have also separately evolved in some lizards and marsupials to a
level of complexity equal to that of eutherians (Blackburn and Flemming, 2011; Freyer et al, 2003).
It is clear that placentation, as a solution to nourishing unborn young, has appeared
independently or semi-independently on more than one occasion (Wildman, 2011). Even in the
eutherian clade it can be difficult to untangle truly homologous features of placentation from cases of
parallel evolution. This limits to some degree the usefulness of anatomical and physiological data in
establishing evolutionary relationships between the large diversity of mammalian placental types
(Elliot and Crespi, 2009).
A more recent approach to reconstructing placental evolution in eutherians has been to examine
the genes expressed during placental development in different species (Knox and Baker, 2008).
Interestingly, many of the genes required for placentation are also implicated in the development of
other organs. These genes tend to be conserved throughout eutherian mammals and other vertebrate
clades. This suggests that the initial invention of the placenta was not accompanied by the emergence
of an entirely novel set of genes, but rather by the co-opting of existing genes to perform the same or a
similar role to that performed in other organs (Cross et al, 2003; Knox and Baker, 2008). In modern
eutherians, these genes typically are involved in the early stages of placentation, such as stimulating the
accelerated growth of the extraembryonic membranes, which are the precursors to the placenta. For
example, fibroblast growth factor (FGF), which is involved the growth and regeneration of a wide
variety of tissues, serves a vital role in trophoblast proliferation in the early embryo (Xu et al, 1998).
Nevertheless, there does exist a subset of genes whose expression is limited to the placenta.
Intriguingly, the majority of these ‘placenta-specific’ genes seem to be restricted to different species or
families rather than being conserved throughout all eutherian mammals. Furthermore, a substantial
number of the genes studied to date, appear to be involved in important features of placentation (Rawn
and Cross, 2008). Overall, this implies an evolutionary scenario whereby a rudimentary placenta was
first formed by recruiting ancient genes involved in growth, metabolic and signalling pathways.
Following the development of this primitive placenta, gene duplication, retroviral capture and
alternative promoter use all contributed to a bank of novel genetic material used by the fast diversifying
eutherian clade to develop a large range of structural and physiological reproductive strategies (Knox
and Baker, 2008). In some cases different clades have arrived at the same strategy independently
(Wildman, 2011). In this way, placental evolution can be compared to the invention of the eye where
3 similar genetic elements were repeatedly co-opted by different phyla for convergent evolution
(Kozmik, 2008).
There is considerable focus on the study of placenta-specific genes today in the hope that this
will contribute to the understanding of the evolutionary puzzle that is placentation. The majority of
these genes have emerged through the process of gene duplication (Rawn and Cross, 2008). Gene
duplication is a powerful process for generating novel genetic material that can fuel evolution (Ohno,
1970). Lineage-specific gene duplication has occurred multiple times in placental evolution. These
genes are usually involved in the later stages of placental development. Some have evolved to fulfil the
needs of species-specific pregnancy physiology, while others have more fundamental roles in
placentation and have unrelated functional counterparts in other species (Knox and Baker, 2008).
For example, the prolactin (Prl) locus has undergone extensive expansion in the rodent lineage,
resulting in a Prl gene family encoding a range of uterus and placenta-specific cytokines and hormones
implicated in regulation of adaptions to pregnancy. Interestingly multiple copies of the Prl locus are
also found in the cow genome, showing high levels of placental expression. However, these genes are
not orthologous with the genes of the rat Prl family, and seem to have arisen from an independent
expansion of the Prl locus in ruminants (Soares, 2004). In humans there has been no expansion of the
Prl locus. There has, however, been duplication of the related growth hormone (GH) locus. These GHlike genes may be fulfilling similar roles in human gestation to the prolactin family in other species
(Alam et al, 2006).
However, in addition to gene duplication, there has been another key process that has allowed
eutherian mammals to obtain the novel gene functions needed for placentation. The capture of
retroviral genes has played a pivotal role in the evolution of the mammalian placenta (Mi et al, 2000;
Dupressoir et al, 2005; Heidmann et al 2009; Cornelis et al, 2012; Haig, 2012). Although retroviruses
have long been observed in the placenta, the full significance of their presence there has only recently
been appreciated (Harris, 1998). Indeed, the placenta appears to be a ‘hotspot’ for retroviral
domestication, where retroviral genes have been co-opted by the host to serve functional roles,
including the exploitation of maternal tissues for the benefit of the foetus. These retroviral captures
have occurred independently multiple times in a range of eutherian lineages (Heidmann et al 2009;
Cornelis et al, 2012).
The most striking example of this phenomenon, and the subject of this review, is the capture of
retroviral envelope genes, named ‘syncytins’, for the purpose of enabling trophoblast fusion and
possibly maternal immunosuppression. Trophoblast fusion allows for the emergence of a more invasive
placental type that is thought to promote better metabolic exchange between the foetus and its mother
(Elliot and Crespi, 2008). It appears that syncytins have been captured on at least six separate occasions
4 in different eutherian lineages and co-opted for the development of a more invasive placenta type (Mi
et al, 2000; Dupressoir et al, 2005; Heidmann et al 2009; Cornelis et al, 2012).
This review will focus on the role these syncytins have served in placental function and
evolution. The following sections will contain an account of the relationship between retroviruses and
their vertebrate hosts, as well as a specific description of retroviral envelope proteins and their presence
in the placenta. Following this it will be necessary to review the specifics of placental development.
The remaining sections are devoted to syncytins and syncytin-like genes, their functional roles in
humans, mice and other mammals, and their evolutionary implications.
5 Endogenous retroviruses
Throughout their evolution, vertebrates have been continually subjected to retroviral infection. The
remnants of these ancient infections have been found in the genomes of virtually every vertebrate class
studied (Herniou et al, 1998). Retroviruses possess the unique ability to integrate their DNA into the
host genome. This allows the viral DNA, known as a provirus, to be replicated as part of the host
genome without damage to the cell. In a minority of cases, if the cell infected happens to be a germ
cell, it is possible for the provirus to colonise the germline. Retroviruses that enter the germline will be
transmitted vertically from generation to generation of the host organism in a Mendelian fashion. This
type of retrovirus is known as an endogenous retrovirus or ERV (Stoye, 2012).
The true extent of ERV coverage in vertebrate genomes was only realised with the advent of
whole genome sequencing. ERV sequences were found to comprise a substantial portion of mammalian
genomes (8% and 10% in mouse and human respectively) (Lander et al, 2001; Waterston et al, 2002).
This large-scale distribution of ERVs throughout vertebrate genomes is the result of numerous
independent germline infections and subsequent amplification of provirus copy number via reinfection
or retrotransposition. As a result there now exist large multigene families of retroviral origin in
vertebrate genomes, although single copy families also exist. By analysing the distribution of the
various families of ERVs it can be shown that they entered the vertebrate lineage at different points in
evolutionary history (Gifford and Tristem, 2003).
The large majority of these retroviral elements have accumulated deletions and mutations over
time, which has rendered them non-functional. As a consequence, it is only ERVs that have recently
integrated into the germline that still retain the ability to both express their viral proteins and remain
infectious. Examples of modern ERVs, still undergoing the process of endogenization, include enJSRV
in sheep (Varela et al, 2009) and KoRV in koalas (Tarlinton et al, 2006). Once incorporated into the
host genome, ERVs are exposed to the same selective pressures as any other piece of chromosomal
DNA. The ability of an ERV to transpose or to create infectious particles is typically detrimental to the
host organism and consequently is lost through purifying selection (McAllister and Werren, 1997).
ERVs that survive this selective screening through the loss of their infectious properties are then
subject to further mutational decay, simply due to a lack of selective pressure on the viral sequence.
Some of these neutral ERV insertions go on to hitchhike their way to fixation in the host population
(Belshaw et al, 2004). Effective host defences for inactivating transposable elements contribute to the
number of neutral ERV sequences in the genome thus increasing the amount of ERV insertions that
progress to fixation (Jern and Coffin, 2008).
6 However, ERV insertions can also reach fixation in a host population by way of a selective
sweep. There are a number of rare cases where certain endogenous retroviral genes have been
continually expressed in the host organism over millions of years without succumbing to deleterious
mutation (Varela et al, 2009). These cases are of intense interest to researchers as they are indicative of
positive selection being exerted on the viral protein by the host organism. This suggests that the host
organism has recruited retroviral proteins to perform specific biological functions. In particular, the
retroviral envelope protein appears to be a good candidate for recruitment by the host organism, as has
been the case in placentation.
7 Retroviral envelope proteins
The retroviral genome is comprised of three transcriptional units, gag, pol and env, flanked on either
end by long terminal repeats (LTRs). The env gene codes for a glycoprotein, which, together with
lipids derived from the host cell plasma membrane, forms the viral envelope. This glycoprotein
mediates cellular receptor binding and membrane fusion. It is thus essential for viral entry into and exit
from the host cell (Stoye, 2012).
The envelope glycoprotein is composed of two subunits, a surface subunit (SU) and a
transmembrane subunit (TM). Cleavage of the original env polyprotein into these two separate subunits
is essential for fusogenic activity (Hunter, 1997). The two subunits are assembled on the host cell
surface to be attached to the virion during budding (see Fig. 1). The SU includes a receptor-binding
domain, which will recognise specific surface proteins present on host cell membranes. Binding of the
SU to a receptor activates a fusion peptide located at the n-terminus of the TM. This drives the fusion
of the virion membranous envelope with the membrane of the target cell.
Furthermore, retroviruses can also drive cell-cell membrane fusion, which aids viral spread
(Poste et al., 1978). This occurs when envelope
A
proteins expressed on the surface of host cells
syncytia (see Fig 1). It is this fusogenic property
that is utilized by the syncytin proteins in
B
bind to the receptors of neighbouring cells. This
triggers membrane fusion and results in the
formation of large multinucelated cells called
trophoblast fusion. The TM also contains a highly
conserved hydrophilic 17-amino acid sequence
named the immunosuppressive domain, which can
suppress T and B cell function
(Good et al,
1990). This domain permits the uninhibited spread
of viral infection and has also been implicated in
the development of neoplasms produced by
oncogenic retroviruses (Oostendorp et al, 1993).
There are a substantial number of intact
ERV env genes that have been conserved in the
human genome (de Parseval, 2003). This suggests
8 Fig. 1. Structure and function of the retroviral envelope protein. (A) Schematic representation of the envelope glycoprotein. The transmembrane subunit (TM) contains a fusion peptide, immunosuppressive domain (ISD) and transmembrane anchoring domain. The surface subunit (SU) contains a receptor binding domain (RBD) and signal peptide (right). After cleavage, TM and SU remain in association (left). (B) Envelope protein mediates membrane fusion. Binding of SU to cell surface receptor can enable viral entry (left) or cell fusion (right). Figures adapted from Cornelis et al, 2012, a nd Dupressoir et al, 2012. that the expression of the retroviral envelope protein may, with appropriate host regulation, be
advantageous. One of the main ways these proteins can confer a beneficial effect to their host cell is by
preventing subsequent infection from related exogenous retroviruses by cell surface receptor
interference. This is the case with the murine fv-4 ERV envelope gene, which confers resistance to
Friend murine leukaemia virus by blocking its cell surface receptor (Gardner, 1991).
Receptor blocking has also been observed sheep, where endogenous JSRV inhibits the entry of
exogenous JSRV into the cell (Palmarini et al, 2004). However, there are also some cases where
envelope proteins have been co-opted by host organisms to perform completely novel biological roles
(Jern and Coffin, 2008). The most striking example of this phenomenon and the subject of this review
is the utilization of envelope genes in placentation.
There are several ERV families in humans and other eutherians that have placenta-specific
expression, some of which contain intact env genes (Blond et al, 2000; Dupressoir et al, 2005, Dunlap
et al, 2006). This implies that reinfection, mediated by the envelope protein, has been an important
proliferation mechanism within these ERV families (Belshaw et al, 2004). The placenta gives direct
access to foetal germline cells via the mother’s somatic cells and provides an effective site for viral
transmission from mother to offspring and vice versa. Expression of infectious ERVs in the trophoblast
could have permitted entry of the ERVs’ progenitors into the germline. Sustained placental expression
of these ERVs could have then allowed for repeated germline colonisations during early retroviral
endogenisation (Haig, 2012).
Extraordinarily, it seems that while ERV sequences were exploiting placentas for their own
benefit, placentas began to exploit these ERVs for their own uses. Indeed, parallels can be made
between the parasitic nature of the foetus towards its mother and of a retrovirus towards its host.
Retroviral envelope genes that allow for successful invasion of host cells appear to have been co-opted
by mammalian placentas for more effective infiltration of the uterine wall (Dupressoir et al, 2011;
Cornelis et al, 2012). Trophoblast cell fusion and suppression of the maternal immune system are two
important processes in invasive placental development (Wooding and Burton, 2008). As described,
fusogenicity and immunosuppression are also two key properties of the retroviral envelope protein.
Indeed, envelope proteins have been implicated in the formation and function of the multinucleated
syncytiotrophoblast, a characteristic feature of many invasive placental types. However, in order to
properly comprehend how retroviral envelope proteins can mediate placentation a short review of
placental structure and development is necessary.
9 Placenta structure and diversity
Placentation typically initiates upon the implantation of the blastocyst into the uterine wall (see Fig. 2).
The trophoblast, derived from the outer cells of the blastocyst, is the first cell line to differentiate
during embryogenesis and is the forerunner of the placenta. The blastocyst also contains an inner cell
mass composed of larger cells, which go on to form the embryoblast. The embryoblast will give rise to
the embryo, the umbilical cord and the inner lining membrane of the amniotic cavity, the amnion. In
invasive placental types, the embryoblast also contributes to the formation of the connective tissue and
capillary blood vessels at the core of the placental chorionic villi (Wooding and Burton, 2008).
The trophoblast forms the outer layer of the placenta and thus is the direct interface between
maternal and foetal tissues. As previously alluded to, there is wide diversity of placental structures
between and within mammalian groups. Much of this diversity can be attributed to the degree of
invasion of the uterine wall by the trophoblast and the differences in structure of the resulting interface
(Carter and Enders, 2004). Placenta types can be roughly divided into four main groups based on the
degree of invasiveness (see Fig. 3A). These are not, however, predictable on the basis of taxonomy.
Indeed, there is much speculation as to what type of placenta the last common ancestor of placental
mammals possessed (Wildman, 2012).
In the epitheliochorial placenta there exists a simple apposition of a trophoblast monolayer to
Fig. 2. Human blastocyst invasion and syncytiotrophoblast formation. (A) Implantation of the blastocyst. The blastocyst reaches the uterine cavity and orientates the inner cell mass (blue) towards maternal epithelium. Adhesive trophoblast cells (pink) then appose to epithelial lining. The trophoblast cells form thin folds, which invade and digest epithelial cells and the basement membrane. Cytotrophoblast cells (CTB) begin to fuse to form the highly invasive syncytiotrophoblast, which penetrates the maternal endometrium. Foetal villi form and maternal blood supply is accessed. (B) Structure of invasive foetal villi. Continuous syncytiotrophoblast outer layer forms direct barrier between the maternal blood and foetal tissue. This outer layer surrounds free floating villi that exist in maternal blood spaces. An underlying layer of villous cytotrophoblasts m aintains the syncytiotrophoblast via cell fusion. Foetal villi are also anchored in the uterine tissue. Extra-­‐
villous cytotrophoblasts exist at base of anchored villi. These cells are invasive and migrate into uterine decidua, penetrating maternal arteries. Figures adapted from Bischof and Irminger-­‐Finger, 2004, and Dupressoir et al, 2012. 10 the maternal epithelium, with no uterine invasion. The remaining three types of placentation:
synepitheliochorial, endotheliochorial and haemochorial, show increasing levels of invasiveness
defined by the number of uterine cell layers between the trophoblast and the maternal blood, illustrated
in Fig. 3A. The vast majority of placentas in these three groupings also show varying levels of
trophoblast fusion to form multinucleated syncytium (Wooding and Burton, 2008). This suggests that
syncytium formation is an important process in the invasion of maternal tissues, although the exact
mechanisms of invasion remain to be deciphered.
In most types of endotheliochorial and haemochorial placentas, mononuclear cytotrophoblasts,
after a period of rapid proliferation, undergo differentiation and fusion to form a continuous
multinucleated syncytiotrophoblast outer layer, which covers the underlying cytotrophoblasts. While
the gross architecture of the syncytiotrophoblast varies from species to species, its fundamental
structural and functional properties remain fairly uniform. It is an invasive structure that penetrates the
uterine wall and produces enzymes and hormones necessary to progress placentation. It forms the
barrier between maternal and foetal tissue and after initial invasion the syncytiotrophoblast goes on to
mediate metabolic exchange between the mother and foetus, as well as modulating the maternal
immune system. Moreover, the continuity of the syncytial layer is thought to provide better protection
from potentially harmful maternal cells and molecules than a simple cellular layer. In addition to this,
as a result of fusion, the syncytiotrophoblast possesses an excess of plasma membrane in proportion to
cytoplasm, which enables the formation of microvilli (Bischof and Irminger-Finger, 2005). These
microvilli, along with the chorionic villi, allow for increased surface area between the foetal and
maternal tissues. Indeed, more invasive placental types can allow for more efficient metabolic
exchange between the mother and foetus (Elliot and Crespi, 2008).
Syncytiotrophoblast structure and function has been best characterized in humans and other
primates, who possess a haemochorial placenta. In humans, after the formation of syncytiotrophoblast,
vascular spaces occur in the syncytial layer allowing the rapidly proliferating layer of cytotrophoblasts
to grow through the syncytiotrophoblast and form primary chorionic villi, which are in turn covered by
a syncytiotrophoblast layer (see Fig. 2B). These finger-like projections are then able to invade the
uterine wall, growing through the progressive endometrial cell layers to reach the maternal blood
vessels. The chorionic villi continue to develop forming a vascularised core of mesenchymal cells
surrounded by the inner layer of cytotrophoblasts and the outer syncytiotrophoblast layer. They can
either exist embedded in the uterine wall or floating in the intervillous maternal blood spaces (Gude et
al, 2004) (See Fig. 2B).
11 A B Fig. 3. Placental diversity and
distribution. A) Four types of
placenta based on extent of
trophoblast invasion.
Epitheliochorial placenta shows no
invasive phenotype.
Mononucleated trophoblast cells
adhere to maternal epithelium.
Synepitheliochorial type shows
some invasive properties.
Trophoblast cells fuse with each
other and with maternal epithelial
cells to form syncytial plaques.
Endotheliochorial and
haemochorial placenta are highly
invasive and penetrate uterine
endometrium. Majority of these
placental types exhibit
syncytiotrophoblast (SyT)
formation. In endotheliochorial
placentas the SyT adheres to outer
wall of maternal blood vessels. In
haemochorial placenta the SyT is
bathed in maternal blood.
B) Distribution of placental types
throughout eutherian clades.
epitheliochorial-blue,
synepitheliochorial-green,
endotheliochorial-red,
haemochorial-black.
Figures adapted from Wooding
and Burton, 2008, and
Dupressoir et al, 2012.
The syncytiotrophoblast cannot undergo nuclear division and may only expand through fusion
with
cytotrophoblast
cells.
Thus,
throughout
pregnancy,
as
the
placenta
develops,
the
syncytiotrophoblast layer is replenished and regenerated by fusion with the lower layer of proliferating
cytotrophoblasts. This supply of cytotrophoblastic cell components is needed to counteract the high
turnover rate of the syncytiotrophoblast, which is thought to continually release apoptotic bodies,
known as syncytial knots, into the maternal blood (Burton and Jones, 2009). It is self-apparent that
these fusion processes require tight regulation. Indeed, too much fusion would lead to a depleted
regenerative pool of cytotrophoblastic cells, whereas too little fusion could cause breakdown or defects
in the syncytiotrophoblast.
Haemochorial and endotheliochorial placental types possessing a syncytiotrophoblast are found
in a range of species from three of the four eutherian superorders, namely Xenarthra, Laurasiatheria
and Euarchontoglires (See Fig. 3B). While endotheliochorial and haemochorial placental types are
observed in Afrotheria, their structure is quite distinct from that observed in other orders and the
presence of syncytiotrophoblast tissue in this clade has not yet been established (Carter and Enders,
2004). Using molecular phylogenetic analysis, a number of studies have inferred that a haemochorial or
endotheliochorial placenta that was present in the most recent common ancestor (MRCA) of all
placental mammals. These studies also suggest that this primitive placenta possessed a
syncytiotrophoblast and indicate that less invasive placental types are, in fact, a derived state (Wildman
et al, 2006; Carter and Mess, 2007; Elliot and Crespi, 2009). However, it is possible that the MRCA
had a placental type unlike any seen today (Wildman, 2011).
12 In the past 15 years, studies have implicated retroviral envelope proteins as key effectors in
syncytiotrophoblast formation and function (Dupressoir et al, 2009). Furthermore, env genes involved
in syncytiotrophoblast formation in different species appear to have been captured independently by
different mammalian lineages, in a striking case of convergent evolution (Cornelis et al, 2012). If the
MRCA of placental mammals did indeed possess a syncytiotrophoblast, it may also have relied on
domesticated retroviral genes. The study of these env genes has the potential to reveal much about
placental evolution and diversity among eutherian mammals. The earliest identification of retroviral
env genes with a putative role in trophoblast fusion was in humans, which will be the subject of the
next section of this review.
13 Retroviral env genes in the human placenta
It has long been recognised that retroviruses have a particular affinity for the placenta. Throughout the
1970’s retroviral particles had been observed in the placentas of cats, mice, guinea pigs, humans and
other primates via the use of electron microscopy (Daniel and Chilton, 1978). Further research in the
1980’s led to the immunolocalisation of human endogenous retrovirus (HERV) envelope protein to the
syncytiotrophoblast (Suni et al, 1984). Today it is known that no less than 18 retroviral env genes in the
human genome contain open reading frames (ORFs) and of these, at least four are expressed at
significant levels in trophoblast tissues (de Parseval et al, 2003; Blaise et al, 2005). Three of these
genes have been well-characterized, namely the ERV3 env gene, syncytin-1 and syncytin-2. Much
research has been undertaken in an attempt to understand the relevance of their expression in the
placenta, with emphasis on the functional properties they demonstrate in vitro and the expression
patterns they display in situ.
The two retroviral env genes that have received the most attention are syncytin-1 and syncytin2, as they have both exhibited potent fusogenic capacity and have been implicated in
syncytiotrophoblast formation (Mi et al, 2000; Blaise et al, 2003). However, there are a number of
other innate properties retroviral envelope proteins possess, which could facilitate placental
development, the first and foremost of these being immunosuppression (Mangeney et al, 2007). The
following sections will attempt to review what is known at present about the functional roles of the
various envelope proteins expressed in the human placenta.
ERV3
The first placenta-specific ERV possessing a coding env gene to be identified in humans was ERV3.
ERV3 is a single-copy, full-length HERV sequence and was identified through the use of hybridization
probes from known primate proviruses (O’Connell et al, 1984). Sequence analysis of ERV3 revealed
an ORF for the viral env gene (Cohen et al, 1985). Expression of ERV3 was shown to be restricted
primarily to the placenta, with low levels of expression in the testis, and was found to be specifically
upregulated in cytotrophoblast cells undergoing fusion (Boyd et al, 1993). ERV3 entered into the
Catarrhini lineage (apes and old world monkeys) approximately 20 million years ago (Shih et al, 1991).
The ERV3 ORF has been shown to be conserved in at least five old world monkey and great ape
species, suggesting it is being subjected to purifying selection (Herve et al, 2004).
The identification of a premature stop codon in the ERV3 env gene that is homozygous in 1%
of the Caucasian population (de Parseval and Heidmann, 1998) and the total absence of the ERV3 open
reading frame in gorillas (Herve et al, 2004) led to the conclusion that, despite its tissue-specific
14 expression and high level of conservation, the ERV3 env gene is not essential for reproductive success
or survival. It is, however, probable that ERV3 has a beneficial function, just not a vital one.
Alternatively, the ERV3 protein may previously have had a beneficial role, but is now redundant due to
functional replacement by another captured env gene.
It has been hypothesized that the ERV3 envelope protein could play a role in cytotrophoblast
differentiation, syncytiotrophoblast formation or maternal immunosuppression (Larsson et al, 1994;
Venables et al, 1995, Boyd et al, 1993). It has also been suggested that the protein could be involved in
protecting the foetus from viral infection, as had been observed previously with other conserved
envelope proteins (Good et al, 1990). However, at present there is no direct evidence to support any of
these possible functions.
The ERV3 envelope protein lacks the hydrophobic transmembrane domain, which eliminates a
possible fusogenic role for the protein (O’Connell et al, 1984). Instead, it has been suggested the
protein might be involved in modulation of the maternal immune system, given the similarity of its
immunosuppressive domain to putative immunosuppressive sequences (Boyd et al, 1993). Indeed, the
protein’s immunosuppressive domain is one of the most highly conserved sequences in ERV3 among
primate species (Herve et al, 2004). Recent support of ERV3s possible role in immunosuppression
comes from in vivo studies, which demonstrated ERV3’s ability to inhibit the rejection of allogeneic
tumour cells by the mouse immune system (Mangeney et al, 2007).
Early studies observed that ERV3 expression was absent in trophoblastic tumour,
choriocarcinoma (Kato et al, 1990). Choriocarcinoma is a fast-growing cancer of the early,
undifferentiated trophoblast cells. Transfection of BeWo choriocarcinoma cell lines with ERV3 env
was found to cause decreased cell proliferation and increased cell differentiation, accompanied by a
slight increase in cell fusion (Lin et al, 2000). Decrease in cell proliferation is thought to be a necessary
first step in cytotrophoblast morphological differentiation and consequently cytotrophoblast fusion
(Morrish et al, 1998). It is possible the ERV3 could facilitate in syncytiotrophoblast formation by
assisting in the regulation of cytotrophoblast growth and differentiation. The exact mechanisms by
which ERV3 inhibits cell proliferation are unknown.
Recent work on LTR methylation in placenta-specific ERVs has given insight into their spatial
and temporal expression (Gimenez et al, 2009). Methylation of retroviral LTRs, which contain
regulatory elements and the retroviral promoter sequence, allows for the silencing of retroviral genes.
Methylation of repeated sequences appears to have evolved as a host defence against the potentially
deleterious effects of retroelements (Yoder et al, 1997). It also may allow the host to regulate the
expression patterns of viral genes (Reiss et al, 2007). Interestingly, ERV3 in placental tissue appears to
be heavily methylated during the first trimester, when most cytotrophoblast proliferation takes place.
Subsequent hypomethylation of ERV3 in the second trimester may contribute to the decrease in
15 cytotrophoblast proliferation in the later stages of gestation (Gimenez et al, 2009). However,
conflicting evidence has recently been published, suggesting ERV3 mRNA levels are higher in early
gestation than in the later stages (Holder et al, 2012).
Although there has been much focus on the ERV3 envelope protein in earlier studies, the
discovery of the fusogenic syncytin genes has led to ERV3 being left relatively neglected. More in
depth analysis of ERV3’s expression patterns and functional properties is needed to allow for better
characterization of its possible role in placentation.
Syncytin-1
Over a decade after the discovery of ERV3, a new multicopy family of retroviruses was identified,
named HERV-W (Blond et al, 1999). HERV-W sequences were isolated from a placenta cDNA
library. Furthermore, HERV-W cDNA fragments containing a complete ORF coding for a retroviral
envelope protein were recovered (Blond et al, 1999). This env gene mapped to a HERV-W sequence on
the long arm of chromosome seven, named ERVWE1 (Mi et al, 2000; Blond et al, 2000). As with the
ERV3 sequence, this HERV-W env gene, subsequently dubbed syncytin-1, exhibited high levels of
expression in the placenta, more specifically in trophoblast cells, with low levels of expression also
seen in the testis (Blond et al, 1999, Mi et al, 2000).
Unlike ERV3 however, the syncytin-1 protein possessed all the required elements necessary for
membrane fusion, including the transmembrane domain missing from the ERV3 protein. Cell fusion
assays in vitro uniformly demonstrated syncytin-1’s fusogenic properties. Syncytin-1 expression in a
range of cell types, including primate, pig and human cell lines, can trigger the formation of large
multinucleated syncytia (see Fig. 4) (Mi et al, 2000, Blond et al, 2000). Syncytin-1 expression can
drive both homotypic and heterotypic cell fusion. Interestingly, the genetic background of the cell
Fig. 4. Syncytin-1 induced COS cell fusion. A) COS cells (fibroblast-like cell line) transfected with vector containing syncytin-1 in reverse
orientation for expression. No cell fusion observable. B) COS cells transfected with vector containing syncytin-1 gene in the sense orientation.
Large multinucleated syncytia can be seen. Adapted from Mi et al, 2000.
16 expressing syncytin-1 does not influence fusogenic activity, indicative of a highly fusogenic envelope
glycoprotein (Blond et al, 2000). It was postulated that this novel retroviral envelope protein could be
responsible for cytotrophoblast cell fusion.
In vitro analysis of primary cytotrophoblast cultures, which differentiate spontaneously to form
syncytiotrophoblasts, showed syncytin-1 expression to be collinear with cytotrophoblast differentiation
and fusion, as well as with the expression of human chorionic gonadotropin (hCG) (Frendo et al, 2003).
Previous studies have shown hCG activates different pathways involved in cytotrophoblast
differentiation (Cronier et al, 1994). It was also demonstrated that HERV-W antisense oligonucleotides
could inhibit syncytiotrophoblast formation in vitro and decrease levels of hCG secretion fivefold
(Frendo et al, 2003).
Syncytin-1 expression can be stimulated in vitro by cyclic AMP (cAMP) (Mi et al, 2000,
Frendo et al, 2003). Interesting, a rise in cAMP levels is needed for the synthesis of multiple
trophoblast specific hormones and proteins (Keryer et al, 1998). Syncytin-1 expression has also been
shown to be under the regulation of the placenta-specific transcription factor Glial-Cell Missing 1
(GCM1, also known as GCMa), whose pattern of expression coincides with that of syncytin-1 (Yu et
al, 2002). GCM1 has been shown to be essential for syncytiotrophoblast formation in the mouse
placenta (Anson-Cartwright et al, 2000). It has been postulated that a cAMP mediated kinase signalling
pathway controls GMC1 expression, which in turn regulates syncytin-1 expression. There is, as of yet,
no physiological explanation for how syncytin-1 expression may be stimulated. Corticotropin-releasing
hormone (CRH) has been proposed as a possible inducer (Tolosa et al, 2012). CRH is produced by the
placenta and is capable of increasing levels of cAMP, which could in turn increase syncytin-1
expression.
Syncytin-1 was found to interact with the type D mammalian retrovirus receptor (RDR/ASCT2) by the use of fusion-assays with a range of receptor-blocked cells (Blond et al, 2000). Interestingly,
syncytin-1 is highly fusogenic in vitro in comparison to related exogenous retroviral envelope proteins,
which share the same receptor, again indicative of a highly fusogenic protein. It has been suggested that
this could be due to the longer cytoplasmic tail of syncytin-1 relative to that of type D exogenous
retroviruses, which may contain a determinant that facilitates fusogenicity (Blond et al, 2000). Overall
these findings indicated that syncytin-1 was serving an important role in cytotrophoblast fusion.
Comparative genome analysis has revealed that the progenitor of the HERV-W family entered
the ancestors of higher primates approximately 25 million years ago. Furthermore, the ORF of
syncytin-1 showed remarkable conservation between species (Mallet et al, 2004). Orthologous loci
isolated from gibbon, chimpanzee, orang-utan and gorilla, all demonstrated the same fusogenic
capacity in vitro. In addition to this, surveys carried out of genetic diversity in human populations
showed syncytin-1 to have an extremely low rate of polymorphism (Mallet et al, 2004). All
17 polymorphic variants detected have subsequently been shown to be capable of trophoblast fusion (de
Parseval et al, 2005). Conservation of this fusogenic function throughout humans and great apes is a
strong indicator of an important functional role for the syncytin-1 protein in cytotrophoblast
differentiation and fusion. The exact nature of this fusogenic role however has remained elusive.
Expression of syncytin-1 was originally thought to be limited to the syncytiotrophoblast (Mi et
al, 2000), however further in situ studies using immunohistochemical techniques and RT-PCR
demonstrated that, while syncytin-1 levels were highest in the syncytiotrophoblast, syncytin-1
expression was present in all other trophoblast lineages, including villious and extravillious
cytotrophoblasts (Malassine et al, 2005; Muir et al, 2006). These studies also reported that the syncytin1 receptor RDR was expressed in extravillious cytotrophoblasts. The co-expression of syncytin-1 and
its receptor in trophoblast cells that do not undergo fusion indicates that, while syncytin-1 may be
required for cytotrophoblast fusion, it is not alone sufficient.
Syncytin-1 is expressed in the placenta throughout gestation, with mRNA levels higher during
the first trimester than at term (Holder et al, 2012). These findings corresponded with earlier studies
that showed higher levels of methylation on the LTRs of the ERVWE1 locus in later stages of gestation
(Gimenez et al, 2009). A reduction in syncytin-1 expression at term could inhibit cytotrophoblast
fusion, decreasing maintenance of the syncytiotrophoblast.
Despite ongoing research, there is still no consensus as to the expression pattern for the
syncytin-1 receptor RDR/ASCT-2. Immunolocalisation studies have delivered different results
depending on the type of antibodies involved (Malassine et al, 2005). Thus, at this time, owing to the
widespread expression and uncharacterized receptor location of syncytin-1, the exact nature of its
involvement in cytotrophoblast fusion cannot be inferred.
In addition to its fusogenic function, syncytin-1 has also been shown to exhibit other properties
that could possibly be involved in placenta morphogenesis. Syncytin-1 has been reported to exert an
anti-apoptotic effect (Knerr et al, 2007), and to induce cell proliferation (Strick et al, 2007).
Additionally, it is hypothesised that syncytin-1 may also play a role in immunosuppression (Blond et
al, 2000; Tolosa et al, 2012). Experiments in mouse models found recombinant syncytin-1 to be
ineffective at suppressing the host immune response to tumour growth (Mangeney et al, 2007).
However, more recent work has found that syncytin-1 does indeed have an immunosuppressive effect.
It was demonstrated that syncytin-1 could inhibit the production of the Th1 (A type of T helper cell)
cytokines IFN-γ, TNF-α and CXCL10 in vitro (Tolosa et al, 2012). This latter study also found that
increasing syncytin-1 expression in vitro, did not lead to increased levels of syncytin-1 protein in the
cytotrophoblast cells, but in exosomes secreted by the cells. Based on these findings a mechanism has
been proposed whereby CRH stimulation of syncytin-1 expression leads to the rapid packaging and
18 secretion of syncytin-1 in these exosomes. This could bring syncytin-1 in direct contact with target
cells of the mother’s immune system, allowing it to modulate their cytokine activity.
Remarkably, a recent study has implicated syncytin-1 as an effector of osteoclast fusion (Søe et
al, 2011). Together with cytotrophoblasts and myotubes, osteoclasts are among the few cell types that
can undergo fusion (Zambonin-Zallone et al, 1984). The idea that syncytins could have been exapted
for functional roles outside the placenta is a novel one and the mechanism by which this could have
occurred is as of yet unknown.
At present, more research is needed to elucidate the exact temporal and spatial expression
patterns of syncytin-1 and its receptor. Furthermore, it has been suggested that syncytin-1 may have an
alternative cell surface receptor and this warrants further investigation (Marin et al, 2003). With more
insight into the localisation of syncytin-1 and its receptors, a clearer picture will emerge of the precise
processes in which its fusogenic and other putative properties are being utilized.
Syncytin-2
The publishing of the complete human genome sequence in 2001 (Lander et al, 2001), allowed for
extensive, systematic searches for all retroviral
env genes with ORFs present in the human
genome (de Parseval et al, 2003; Villesen et al,
2004). This led to the identification of sixteen
intact env genes, three of which were found to
be expressed at high levels in the placenta by
use of qRT-PCR (de Parseval et al, 2003). Two
of these genes were the previously identified
ERV3 and syncytin-1. However, the third was a
novel env gene of the HERV-FRD family.
Fusion assays were performed on the sixteen
coding env genes and of these only two could
induce syncytia formation, namely syncytin-1
and
the
HERV-FRD
gene,
which
was
subsequently named syncytin-2 (Blaise et al,
2003).
Comparative genome analysis revealed
syncytin-2 to be older than syncytin-1, being
conserved in the genome of all New and Old
Fig 5. In situ hybridization of syncytin-2 and MFSD2 in term placenta
and model of cell fusion. A) Syncytin-2 antisense riboprobe reveal the
protein to be localising to the membranes of sparse cytotrophoblasts in
contact with the syncytiotrophoblast (top). Syncytiotrophoblast membrane
is labelled for MFSD2 using same techniques (bottom). B) Proposed
mechanism for “in-fusion” of cytotrophoblasts into the syncytiotrophoblast.
Syncytin-2 expressed on the surface of villous cytotrophoblasts binds to
MFSD2 receptor on syncytiotrophoblast plasma membrane, inducing
fusion of cytotrophoblast into the syncytial layer. Cytotrophoblasts are
unable to fuse with each other, due to lack of MFSD2 expression. Adapted
from Esnault et al, 2008.
19 World monkeys, with an estimated age of insertion of 40 million years ago. The corresponding
syncytin-2 envelope genes from various simians were cloned and shown to be both highly conserved
and capable of syncytia formation (Blaise et al, 2004). Surveys of human genetic polymorphism also
revealed extremely low levels of diversity at the syncytin-2 locus (de Parseval et al, 2005). Thus it
appears, as is the case with syncytin-1, that syncytin-2 is being exposed to high levels of purifying
selection, indicative of a important physiological role.
After its identification, fusion assays were carried out on syncytin-2 in a variety of cell lines
(Blaise et al, 2003). Syncytin-2 was found to be an extremely fusogenic envelope protein capable of
inducing fusion in a large variety of cell types, including the BeWo choriocarcinoma cell line.
Furthermore, syncytin-2 triggers cell fusion in a different set of cell lines to syncytin-1, indicating
different receptor usage. Later research identified a syncytin-2 binding receptor, MFSD2, with
placenta-specific expression (Esnault et al, 2008).
In contrast to the widespread expression of syncytin-1 in all trophoblast cell lines, syncytin-2
expression was shown to be restricted to the villous cytotrophoblast cells (Malassine et al, 2007).
Furthermore, in situ immunostaining demonstrated that syncytin-2 was more frequently expressed in
cytotrophoblast cells that were in direct contact with the syncytiotrophoblast. Within these cells,
syncytin-2 appeared to be localising to the sections of the plasma membranes that comprised the
interface with the syncytiotrophoblast (see Fig 5A). Remarkably, MFSD2 receptor expression is
confined to the syncytiotrophoblast (Fig 5B) (Esnault et al, 2008). It has been proposed that the highly
restricted expression of syncytin-1 and MFSD2 allows for fusion of villous cytotrophoblasts into the
syncytiotrophoblast, while preventing fusion between the cytotrophoblasts themselves. This
mechanism may promote syncytiotrophoblast maintenance throughout gestation, without depleting the
supply of villous cytotrophoblasts (Esnault et al, 2008).
Interestingly, both syncytin-2 and its receptor are regulated by the placenta-specific
transcription factor GCM1 (Liang et al, 2010), as is the case with syncytin 1. This indicates GMC1 as
being a critical factor in regulating cytotrophoblast differentiation and fusion.
In order to establish the relative contributions of syncytin-1 and syncytin-2 in cytotrophoblast
fusion, siRNA knockout studies of syncytin-1 and syncytin-2 in primary trophoblast cell lines were
performed (Vargas et al, 2009). Interestingly, while cell fusion was reduced in both cases, siRNA
against syncytin-2 has the more detrimental phenotype. This implies that syncytin-2 may play a more
essential part in cytotrophoblast fusion than syncytin-1. Experiments were also carried out on BeWo
choriocarcinoma cell lines, which were incubated with agents that induce cytotrophoblast cell fusion.
Incubation coincided with an increase of both syncytin-1 and syncytin-2 mRNA transcripts, however
syncytin-2 expression experienced a more drastic increment. Furthermore syncytin-2 was poorly
expressed in non-induced cells relative to syncytin-1. These findings all point to a central role for
20 syncytin-2 in cytotrophoblast cell fusion, with a possible subsidiary role for syncytin-1 (Vargas et al,
2009).
Syncytin-2 has also demonstrated definite immunosuppressive properties. This was manifested
by its ability to mask allogeneic tumour cells from the mouse immune system (Mangeney, 2007). In
addition to this, the immunosuppressive domain of syncytin-2 exhibits 100% amino-acid conservation
in all primates sequenced (Blaise et al, 2004). Thus it is possible that syncytin-2 carries out two
complementary functions, syncytiotrophoblast formation, and protection of the invading foetal villi
from the maternal immune system.
Unravelling the precise functional roles of the two syncytins in placental morphogenesis poses a
complex problem. Syncytin-1 and syncytin-2 have no orthologous counterparts outside the primate
lineage. Thus in vivo knockout studies are at present an impossibility. In vitro functional assays and in
situ expression analysis, while important, can only continue to provide indirect evidence as to their
putative roles. More in depth analysis of human and primate polymorphism at the syncytin loci and its
possible correlation to disease may contribute to our understanding of syncytin function. Several
studies have correlated reduced syncytin expression with severity of preeclampsia and pregnancyinduced hypertension (Vargas et al, 2011; Kudaka et al, 2008). The discovery of non-orthologous
syncytin genes in mice has given researchers an invaluable comparative model for syncytin function. It
is these mouse syncytins that will be discussed in the next section.
21 Syncytins in Mice
Identification
As previously stated, research on the physiological properties of syncytin-1 and syncytin-2 was
somewhat restricted due to lack of a model organism. Furthermore, the absence of syncytin-1 and
syncytin-2 orthologs in other mammals with invasive placental types called into question their
functional and evolutionary importance. Although it was suggested that different families of ERVs
could perform the same function in other mammals, it was thought unlikely that the function in
question would be an essential one (Stoye, 2000). However, the discovery of two fusogenic envelope
proteins in mice in challenged this idea by irrefutably illustrating the essential role that syncytin genes
can play placentation.
The search for a model organism led to the large-scale screening of the mouse genome for intact
retroviral env genes (Dupressoir et al, 2005). This screen identified two single copy ERV sequences,
both unrelated to each other or to any known murine ERV families. The env genes of these ERV
sequences both contained complete ORFs and their expression was found to be placenta-specific via
qRT-PCR. The env genes, subsequently named syncytin-A and syncytin-B, were shown to have highly
conserved orthologs in all examined members of the Muridae family and their entry into the rodent
lineage was dated at approximately 20 million years ago for both genes (Dupressoir et al, 2005).
In vitro experiments showed the mouse syncytins to be extremely fusogenic when expressed in
transfected cells. Moreover, syncytin-A and syncytin-B, caused fusion in different sets of cell types,
signifying different receptor usage, as is the case with the human syncytins. In addition to this,
syncytin-B was found to be under the control of GCM1, the same placenta-specific transcription factor
that regulates the expression of the human syncytins. This suggests that human and mouse syncytins
are similarly regulated, further supporting the use of the mouse as model organism for syncytin
function in placental development (Dupressoir et al, 2005).
Remarkably however, the mouse syncytins were shown to be entirely distinct from those of
humans. Their entry dates into their respective lineages occurred well after the speciation of rodents
and primates. Furthermore, they are not syntenic and phylogenetic analysis places them into distinct
retroviral families (Dupressoir et al, 2005). These significant findings implied that retroviral gene
domestication had occurred separately at least twice in eutherians resulting in convergent placental
evolution. In addition to this the mouse syncytins provided a platform to ascertain the exact functional
role of placental syncytins in vivo.
22 Functional Analysis
Primates and rodents both possess haemochorial placentas (see Fig 3A). In this type of placentation
there is direct contact between the syncytiotrophoblast and the maternal blood. Structural differences in
haemochorial placentation have been observed throughout various mammalian species, with muroids
having two syncytiotrophoblast layers, while non-muroid rodents, simians and lagomorphs possess
only one (Wooding and Burton, 2008). In addition to this, rodents possess labyrinthine interdigitation,
in which foetal and maternal villi exist together in a mesh, with more minimal invasion of the uterine
wall by foetal villi. In contrast, primates have evolved villous interdigitation, which as previously
described, involves heavy invasion of the maternal tissue by foetal villi (Carter and Mess, 2007). It has
been hypothesised that some of these structural disparities could be accounted for by the differing
properties of the syncytin genes that have been captured by the different lineages (Cornelis et al, 2012).
Despite the structural differences between human and mouse syncytiotrophoblast, there are
enough similarities between the two to allow for functional comparison (Georgiades et al, 2002). The
two syncytiotrophoblast layers of the murine placenta (ST-I and ST-II) are tightly adherent, with gap
junctions between the layers allowing for cytoplasmic exchange and communication. Thus they can be
thought of as one functional structure. ST-II is in contact with the foetal epithelium and ST-I with the
maternal blood (See Fig. 6). As in humans, the mouse syncytial layers are formed through the fusion of
mononuclear cytotrophoblasts (Georgiades et al, 2002).
In vitro analysis of syncytin-A demonstrated its ability to mediate cytotrophoblast fusion.
Fig. 6. Mouse placental labyrinth
structure and knockout
phenotypes. A) Schematic
representation of the murine
placenta. The labyrinth zone is the
direct interface between maternal
and foetal tissues. It comprises of
maternal and foetal blood channels
enclosed by trophoblast-derived
cells.
B) Interaction between trophoblast
cells and blood spaces in the
labyrinth. A syncytiotrophoblast
bilayer surrounds the foetal blood
vessel endothelium. Mononuclear
trophoblast cells line the maternal
blood sinusoids.
C) Knockout studies show both
mouse syncytins are needs for
normal syncytiotrophoblast bilayer
formation. Syncytin-A knockout
mice show defects in the ST-I layer
(middle), while syncytin-B
knockout results in an unfused STII layer (right), relative to the
control (left).
Adapted from Watson and Cross,
2005, and Dupressoir et al, 2012
23 Withdrawal of fibroblast growth factor 4 (FGF4) from primary mouse trophoblast cell cultures induces
cytotrophoblast differentiation and fusion, accompanied by a collinear increase in syncytin-A mRNA
transcripts and protein. Furthermore, inhibition of syncytin-A through the use of antisense
oligonucleotides, results in a drastic decrease in cell fusion (Gong et al, 2007). Syncytin-B was not
examined in this study, though previous work had demonstrated its fusogenic capacity (Dupressoir et
al, 2005).
In addition to its fusogenic properties, syncytin-B has also exhibited immunosuppressive ability
in the mouse tumour model (Mangeney et al, 2007). In contrast, syncytin-A transfection did not prevent
tumour rejection by the mouse immune system. However, this does not preclude an
immunosuppressive function for syncytin-A. Human syncytin-1 also gave a negative result in this
study, but was subsequently shown to inhibit T cell function (Tolosa et al, 2012).
The syncytin-A and syncytin-B genes were shown to exhibit similar patterns of expression, with
near identical levels of transcript accumulation during placentation. However, syncytin-A is localized
to the ST-I layer while, syncytin-B along with GCM1 are expressed in ST-II (Simmons et al, 2008).
Prior to syncytiotrophoblast formation these genes are expressed in distinct layers in precursor
trophoblast cells. As previously mentioned, GCM1 has been demonstrated to be directly involved in
syncytin-B expression (Dupressoir et al, 2005). GCM1 has also been implicated in syncytin-A
expression, although the evidence is somewhat more oblique. GCM1 binding motifs have been located
on the syncytin-A ORF (Asp et al, 2007). Overexpression of GCM1 in transfected cell lines did not
increase levels of syncytin-A transcripts. In addition to this, elevation of cAMP levels, thought to
increase GCM1 activity, did not increase syncytin-A expression. However, a combination of both
treatments saw a significant increment in syncytin-A mRNA transcripts (Asp et al, 2007). Furthermore,
syncytin-A expression is down-regulated in GCM1-deficient murine chorionic tissue (Schubert et al,
2008). It will be important for future research to establish the exact role of GCM1 in syncytin-A
expression, in order to better our understanding of the comparative aspects of human and mouse
syncytins.
The most significant findings regarding the mouse syncytins have come from in vivo knockout
studies, which have given great insight into the central role of syncytins in both mouse and human
placentation. Remarkably, it was found that the homozygous syncytin-A null phenotype was lethal,
with mouse embryos invariably dying in utero halfway through gestation (Dupressoir et al, 2009). This
corresponds to the period in which the placental labyrinth develops, containing the two
syncytiotrophoblast layers. On examination, it was found that the ST-I layer showed defects in
syncytial formation and indications of apoptosis, leaving it unable to interact sufficiently with ST-II for
adequate metabolic exchange (see Fig. 6C). Furthermore, there existed an excess of mononuclear
trophoblast cells, which were seen to be disrupting labyrinth vascularization. In comparison the ST-II
24 layer appeared to develop normally, which agrees with the expression patterns of the mouse syncytins.
These findings demonstrated syncytin-A to be crucial for the fusion of progenitor trophoblast cells to
form the ST-I syncytiotrophoblast layer, a vital step in murine placentation.
In contrast, the loss of the syncytin-B gene in homozygous null mutants resulted in viable
embryos, which presented only limited late-onset growth retardation and a decrease in neonate numbers
(Dupressoir et al, 2011). The mutant placenta showed defects in syncytial formation in the ST-II layer,
which resulted in the disruption of labyrinth architecture (see Fig. 6C). Interestingly, double knockout
mice, possessing both syncytin-A and syncytin-B homozygous deletions, showed earlier embryonic
death than syncytin-A single knockout mice. This suggests that, while the physiological consequences
of syncytin-B knockout are only evident in late gestation, ST-II layer formation is important in early
placentation. These findings established two distinct, non-redundant roles for the mouse syncytins,
each of which appeared to be involved in the formation of a different syncytiotrophoblast layer, and
both required for healthy embryonic development. It could be postulated that the capture of two distinct
syncytin genes led to the development of two distinct syncytiotrophoblast layers (Dupressoir et al,
2011). It has yet to be determined whether the human syncytins play equally distinct roles in
placentation or if there is some level of redundancy between them.
In any event, this was the first demonstration that syncytins could indeed play an essential role
in mammalian placental morphogenesis. Moreover, the independent capture of placental syncytins in
the muridae lineage had huge evolutionary implications. Could retroviral domestication in placental
evolution be a recurring phenomenon? This possibility raised the further question as to whether
fusogenic retroviral envelope proteins existed in other species with invasive, syncytial placental types.
This led to genomewide searches in a range of eutherian mammals from a variety of clades for intact
retroviral env genes. Remarkably, syncytin and syncytin-like genes were identified in multiple diverse
lineages, including ovines, leporids, caviids and carnivorans (Dunlap et al, 2006; Heidmann et al, 2009;
Vernochet et al, 2011; Cornelis et al, 2012).
25 Syncytins in other mammals
The fifth syncytin gene to be discovered belonged to the Leporidae lineage (Rabbits, Hares). Leporidae
are members of the Lagomorpha order, all of whom possess haemochorial placentas. This novel
syncytin, subsequently named syncytin-Ory1, was identified during an in silico search of the fully
sequenced rabbit genome (Heidmann et al, 2009). Further experiments, demonstrated its placentaspecific expression and fusogenic properties, the hallmarks of a bona fide syncytin gene. More
specifically its expression was localized to the placenta junctional zone, where invading syncytial tissue
makes contact with the uterine wall. Interestingly, syncytin-ory1 was found to share the same receptor
as human syncytin-1, namely the type D mammalian retrovirus receptor (RDR/ASCT-2). However,
syncytin-ory1 was found to be unrelated to the four previously described syncytins, belonging to a
distinct retroviral lineage. Comparative genome analysis found syncytin-Ory1 to be conserved in
diverse species of the Leporidae family but absent in Ochotonidae (pikas), indicating its entry into the
lagomorph lineage approximately 15 million years ago (Heidmann et al, 2011).
Thus far, the three lineages in which syncytin genes had been described, lagomorphs, murids
and primates, were all part of the Euarchontoglire superorder of eutherian mammals. Euarchontoglires
diverged from a second eutherian superorder, the Laurasiatheria, approximately 100 million years ago.
The Laurasiatheria include an extensive range of species in several orders, including Perissodactyla,
Cetartiodactyla, Insectivora, Chiroptera, Pholidota and Carnivora, and exhibit a wide variety of
placental types (Carter and Enders, 2004).
In order to establish whether syncytin domestication was a general process, which transcended
ancient evolutionary divergences, and to investigate whether syncytins were present in
nonhaemochorial type placentas, members of the Carnivora order were investigated for placental env
genes. Carnivorans possess an endotheliochorial placenta in which the syncytiotrophoblast is apposed
to the uterine blood vessels rather than coming into direct contact with the maternal blood as is the case
with the haemochorial placenta (see Fig. 3A).
Name
Time of Insertion
Conservation
Expression Site
Receptor
Syncytin-1
>25 My
hominoids
all trophoblast cell types
ASCT-2
Syncytin-2
>40 My
simians
villous cytotrophoblasts
MFSD2A
Syncytin-A
>20 My
muroids
syncytiotrophoblast layer-I
unknown
Syncytin-B
>20 My
muroids
syncytiotrophoblast layer-II
unknown
Syncytin-Ory1
>12 My
leproids
syncytiotrophoblast
ASCT-2
Syncytin-Car1
>65 My
carnivorans
syncytiotrophoblast
unknown
Table 1. Known mammalian syncytin genes.
26 A positive result was obtained when in silico searches of the cat and dog genomes identified an
env gene with all the recognised characteristics of a syncytin, including placenta-specific expression
(more specifically at the level of the invading foetal villi), fusogenic properties and exposure to high
levels of purifying selection, with full conservation of its coding status throughout 26 representatives of
the Carnivora (Cornelis et al, 2012). The env gene, dubbed syncytin-Car1, was found to be distinct
from the previously described syncytins and signifies an independent retroviral capture by the common
ancestor of today’s carnivorans. Pholidota (pangolins) are the closest outgroup to Carnivora and
possess a non-invasive epitheliochorial placenta. Interestingly, syncytin-Car1 was found to be absent
from the two different pangolin species investigated. Thus it was concluded that syncytin-Car1 entered
the Carnivora order sometime before its radiation approximately 60 million years ago. This makes
syncytin-Car1 the oldest syncytin discovered to date. It also extends the presence of syncytins outside
of both the Euarchontoglire clade and the haemochorial placenta type.
These findings unambiguously demonstrated that syncytin gene capture is not a rare occurrence
in eutherian mammals, but instead is a widespread process, which has occurred independently in
diverse lineages multiple times. Moreover, evidence suggests that syncytins serve a major role in
cytotrophoblast fusion, an important process in invasive placentation (Frendo et al, 2003; Dupressoir et
al, 2009). Thus we have a striking example of convergent
evolution mediated by retroviral capture. Furthermore, it is
tempting to hypothesise that the large diversity of invasive
placenta structures (Wooding and Burton, 2008) is the
consequence of innate differences between the independently
captured syncytin genes, which belong to a wide variety of
retroviral families. It will be of great interest to investigate
the presence of syncytin genes in other mammalian lineages,
with a particular emphasis on the remaining two eutherian
superorders, Xenarthra (sloths, armadillos) and Afrotheria
(elephants, hyraxes).
There is also a need to characterize the precise
functional roles, fusogenic and otherwise, that the various
mammalian syncytins play in placentation. Although much
work has been done on the mouse and human syncytins, their
carnivoran and lagomorph counterparts are relatively
uncharacterized. Studies of this kind will contribute to our
understanding of how the various syncytins have influenced
placental evolution in these lineages.
27 Fig. 7. Eutherian phylogeny with syncytin integration
events indicated. Syncytin captures have occurred
independently on at least six separate occasions, five
times in the Euarchontoglire order and once in
Laurasiatheria.
Adapted from Cornelis et al, 2012
Beyond Syncytins
It must be emphasised that the involvement of retroviral capture in placental evolution is not only
limited to the fusogenic syncytins. In addition to the syncytin genes, there are an abundance of other
retroelements, derived from both ERVs and retrotransposons, which have been coopted for placental
development. Other env genes have been identified, which have not fulfilled all the criteria used to
define syncytin genes, such as fusogenicity or long-term evolutionary conservation. However, these
syncytin-like genes can still contribute to placental development, as described below. Also implicated
in placental evolution are regulatory retroelements, which can drive placenta specific expression of
cellular genes.
Syncytin-like genes
As mentioned previously, there are other env genes in the human genome, aside from the syncytins,
with placenta-specific expression, such as the ERV3 env gene. Although this protein is not vital for
successful human placentation, there is evidence that it may perform a beneficial function (Herve et al,
2004). In addition to ERV3, other coding env genes in humans have been identified that show high
levels of placental expression (Blaise et al 2005). However, their envelope proteins have all been found
to be non-fusogenic and their possible functional role has yet to be investigated. Outside of humans,
‘syncytin-like’ proteins have been identified in both sheep and guinea pigs.
Sheep have a synepitheliochorial placenta, a placental type found only in ruminants. It is
comparable to the epitheliochorial placenta, but in synepitheliochorial placentas the uterine wall is
modified by invasion of binucelate trophoblast cells (BNCs). These BNCs fuse with uterine epithelial
cells to form multinucleated syncytial plaques (see Fig. 3A) (Carter and Enders, 2004).
The Jaagsiekte sheep retrovirus (JSRV) is at present in the process of endogenization. The
sheep genome contains approximately 20 ERVs that are closely related to the exogenous JSRV
(Palmarini et al, 2004). Interestingly, the env genes of multiple endogenous JSRVs (enJSRVs) are
expressed at high levels in uterine epithelium as well as in the BNCs, their mononuclear progenitors
and the syncytial plaques (Dunlap et al, 2005). Loss-of function experiments in vitro demonstrated that
enJSRV envelope proteins play a vital role in trophoblast differentiation and growth. Inhibition of
enJSRV protein production in utero, on both sides of the maternal-foetal interface, resulted in
trophoblast abnormalities and abortion (Dunlap et al, 2006). However, the exact mechanisms by which
these envelope proteins stimulate trophoblast growth and differentiation have yet to be elucidated.
Endogenous JSRVs can interfere with the replication cycle of exogenous JSRVs (Palmarini et
al, 2004). The protection of their hosts from subsequent infection may have allowed some enJSRVs to
28 become fixed in host populations. After fixation, their expression in placental and uterine tissues could
have contributed to reproductive efficiency. Given the apparently crucial role of enJSRV envelope
proteins in trophoblast implantation, it is possible that some host mechanisms, which originally
governed this process, were lost through redundancy (Dunlap et al, 2006).
The hunt for syncytin genes in the different mammalian lineages led to the identification of a
single copy ERV sequence in the guinea pig genome with an intact env gene (Vernochet et al, 2011).
Guinea pigs are members of the Caviomorpha lineage, a South American branch of the Hystricognathi
suborder of rodents, who possess haemochorial placentas. The env gene, dubbed env-Cav1, was found
to have a conserved coding status throughout the Caviomorpha clade, consistent with a time of entry
more than 30 million years ago. Although this gene showed placenta-specific expression at the level of
the invasive trophoblasts, no fusogenic activity could be demonstrated in vitro. Although env-Cav1
may not be directly involved in cytotrophoblast cell fusion, its level of conservation suggests that this
envelope protein has an important functional role, and it may be involved in other elements of invasion
or immunosuppression.
Overall, these findings reinforced the idea that retroviral env genes have been co-opted for
placental morphogenesis multiple times in eutherian evolution. Furthermore, they imply that envelope
proteins can contribute to placentation, not just through the stimulation of cell-cell fusion, but in
multiple and diverse ways.
Other retroelements in placental evolution
In addition to the creation and exploitation of novel genetic material, evolution is also driven by
changes in gene regulatory patterns, via mechanisms such as alternative promoter usage, alternative
splicing and epigenetic regulation (Sverdlov, 2005). Indeed, changes in transcriptional level and pattern
can have a substantial impact on species diversity (Wray, 2007).
Most ERVs in the genome exist as solo long terminal repeats (LTRS), after recombination
between their flanking LTRs led to the deletion of all intermediate retroviral material. A significant
fraction of these solo LTRs have retained their regulatory sequences and can act as alternative
promoters for nearby cellular genes (van de Lagemaat et al, 2003). While in many cases these
alternative promoters show only slight changes in expression pattern from native promoters, there are
cases where ERV LTR usage can result in completely novel patterns of gene expression. Moreover, the
majority of these cases seem to be limited to the placenta (Cohen et al, 2009).
In many mammals, high levels of ERV transcripts are found in germline cells, testis, and the
placenta. As stated previously, expression of ERVs in reproductive tissues may have allowed for the
colonization of the germline by their progenitors (Belshaw et al, 2004). Thus, it may have been that
29 LTRs with placenta-specific transcription factor binding sites were preferentially selected. Another
possibility is that ERV LTRs are subject to less DNA methylation in reproductive tissues. Indeed, the
placenta does exhibit an atypical pattern of global hypomethylation, which allows retroviral promoter
sequences to remain active (Rawn and Cross, 2008).
Methylation of LTRs allows for the silencing of genes under their regulation. Indeed, the LTR
sequences of placenta-specific ERVs with intact env genes, such as the syncytins, show
hypermethylation in most tissues, with hypomethylation in the placenta (Gimenez et al, 2009).
However, it has not been established whether methylation is the sole mechanism for LTR placentaspecific transcriptional regulation (Reiss et al, 2007).
LTR regulatory sequences are used for the placenta-specific expression of a number of cellular
genes. One of the first of these to be characterized was Cyp19, which encodes the enzyme aromatase,
responsible for the conversion of androgens to estrogens, which regulate placental growth (Simpson et
al, 1994). Cyp19 has evolved multiple promoters for expression in different tissues, including a
placenta-specifc promoter in humans derived from an LTR. Interestingly, bovine and ovine Cyp19
have each independently evolved a placenta-specific promoter, without the help of retroviral insertion,
and show different patterns of placental expression (Vanselow et al, 2004).
In addition to Cyp19, there are at least five other genes showing placenta-specific expression in
humans as a result of retroviral insertion (Rawn and Cross, 2008). Interestingly, these five genes are all
restricted to the simian lineage and may have been involved in the evolution of the particularly invasive
primate placenta with its characteristic villous interdigitation (Cohen and Bischof, 2007). For example,
one of these genes, leptin, stimulates trophoblast cell proliferation (Magarinos et al, 2007), while
another, INSL4, increases invasiveness and mobility of certain cancer cells (Brandt et al, 2005).
In several cases, including Cyp19, orthologs of these genes have placenta-specific expression in
other mammals, although not as a result of LTR regulation. This suggests that these genes have
conserved placental function, while their placental-specific expression is the consequence of
convergent evolution (Rawn and Cross, 2008). Further studies need to be done, in an attempt to
characterize placenta-specific genes in other mammals, which are under the regulation of LTR
promoters.
Retrotranspon-derived genes have also been implicated in placental development. Two genes
from the suchi-ichi class of Ty3/gypsy retrotransposons, Peg10 and Rtl1, have been shown to be vital
for placental development and appear to be conserved throughout eutherian mammals, although their
exact functions have yet to be established (Ono et al, 2006; Sekita et al, 2008).
30 Conclusions and Outlook
There is a unique relationship between endogenous retroviruses and the placenta, which is evidenced in
the longstanding evolutionary interplay between the two. Placental expression of ERVs has most likely
evolved to facilitate the entry of their progenitors into host germline cells (Belshaw et al, 2004). This
subsequently led to the continuous domestication of placenta-specific retroviral genes and elements by
the host to serve adaptive functions in placentation. Consequently, retroviral co-option has had a
substantial impact on placental evolution and may have contributed, not only to the striking diversity of
placenta types seen in eutherians today, but to the very invention of the placenta itself.
There are a variety of ways in which endogenous retroviral sequences, both coding and
regulatory, can contribute to placental morphogenesis. Retroviral promoters can be exapted to serve as
alternative promoters, driving the placenta-specific expression of cellular genes (Cohen et al, 2009).
Moreover, retroviral genes have also been co-opted to serve novel functions in placentation. In
particular, the retroviral env gene, which codes for the viral envelope glycoprotein, demonstrates a
number of functional properties that make it an excellent candidate for co-option by the host, including
fusogenicity, immunosuppression, host protection and stimulation of cell proliferation (Varela et al,
2009). The best-documented examples of this phenomenon are the syncytin genes. The defining
characteristic of syncytins, which sets them apart from other placenta-specific env genes, is their ability
to induce cell-cell fusion (Blond et al, 2000; Blaise et al 2005). So far six syncytin genes have been
identified, two in primates, two in murids, one in lagomorphs and one in carnivorans, all of which were
independently captured in their respective lineages (Heidmann et al, 2009; Cornelis et al 2012).
These syncytin genes may have, owing to their fusogenic properties, allowed for the
evolutionary invention of a novel tissue, the syncytiotrophoblast. The syncytiotrophoblast plays an
essential role in the development of most invasive placental types and is found in a diverse range of
eutherian mammals (Carter and Enders, 2004). In order to determine how critical syncytins are for
cytotrophoblast fusion, a search for their presence in other mammalian clades possessing invasive
syncytiotrophoblast tissues is of prime importance. It will also be of particular interest if there presence
can be demonstrated in the two more ancient mammalian superorders, Xenarthra and Afrotheria, the
latter of which, although possessing a wide range of invasive placental types, has not shown evidence
of syncytial tissue formation. Of some interest, syncytium formation also takes place in the
chorioallantoic placenta of the marsupial bandicoot family (Padykula and Taylor, 1976). This could
also plausibly be the result of retroviral capture and warrants investigation.
To date there has been no study done on retroviral envelope proteins in species with noninvasive epitheliochorial placentas. This placental type shows no evidence of syncytiotrophoblast
formation, hence it must be asked whether syncytin or syncytin-like proteins are present in these
31 species and if so what possible role they serve. Syncytins and other envelope proteins also have potent
immunosuppressive properties, which may turn out to be just a critical in placental development as
their fusogenic ones.
Indeed, modulation of the maternal immune system is vital in all placental types, not just the
invasive subset (Wooding and Burton, 2008). It is possible that the capture of a retroviral envelope
gene with immunosuppressive properties enabled the emergence of a primitive placenta in oviparous
vertebrates, by facilitating the grafting of foetal tissues to the uterine wall.
If syncytin genes are present in all invasive placental types, this will have considerable
evolutionary implications. The most recent common ancestor of all placental mammals is believed to
have had an invasive placental type containing syncytiotrophoblast tissue, with epitheliochorial
placentation in eutherians thought to be a derived state (Carter and Mess, 2007; Elliot and Crespi,
2009). If this hypothesis is correct it is probable that this placental ancestor was also relying on the
fusogenic properties conferred to it by retroviral envelope proteins.
Thus it is possible to envision an evolutionary scenario, whereby a primitive placenta existed in
an early mammalian ancestor, which was already relying on the immunosuppressive properties of
envelope proteins. Some of these envelope proteins may also have been exhibiting fusogenic
properties, which, by allowing deeper invasion of the uterine wall, could have contributed to
reproductive efficiency. This invasive phenotype would have been selected for, leading to the
development of an endotheliochorial or haemochorial placenta, which was present in the MRCA of
placental mammals. Indeed, it could have been the success of this invasive placental type that triggered
the rapid expansion of early placental mammals. As the mammalian lineages diversified the founding
env genes could have been replaced by the successive and independent capture and co-option of new
retroviral env genes, each of which conferred their hosts with a selective advantage. If this is the case,
the syncytins present today may only be the most recent in a series. Moreover, env genes such as
ERV3, could possibly be defunct syncytin genes in the process of being made redundant.
There have been a number of studies published on the intrinsic properties and functional
characteristics of the human and mouse syncytins, in order to establish their exact role in placentation.
Studies of this kind must also be performed on the lagomorph and carnivoran syncytins. This will allow
comparative analysis of the different types of invasive placenta with respect to the different properties
of their domesticated syncytins. Indeed, it could be hypothesised that the extensive range of placental
types that exist today could in part be due to diversity among the stochastically captured syncytin genes
in different lineages. Variable factors, such as level of fusogenic capacity, availability of the
appropriate cell surface receptor, regulation of temporal and spatial expression and immunosuppressive
ability, exist between syncytin proteins, which could result in drastically different structures of the
maternal foetal interface.
32 In conclusion, it is apparent that retroviral capture and domestication has played a central role in
placental evolution. Both the discovery of new syncytins and syncytin-like genes, and the functional
characterization of those already identified will be critical in our understanding of their role in
placentation and mammalian evolution.
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