DEVELOPMENTAL GENETICS 21:620 (1997)
REVIEW ARTICLE
Molecular Genetics of Implantation in the Mouse
JULIE L. RINKENBERGER,1* JAMES C. CROSS,2 AND ZENA WERB1
1Departmentof Anatomy, University of California, San Francisco
2Samuel Lunenfield Institute, Mount Sinai Hospital, Toronto, Ontario, Canada
ABSTRACT Embryo implantation is a complex
developmental process requiring precise coordination
between mother and offspring to ensure success. Implan-
tation failure is clinically relevant to in vitro fertilization
programs and to an understanding of diseases of
pregnancy like preeclampsia. Basic and clinical re-
search have identified a number of proteins involved in
peri-implantation development, but an understanding of
the implantation process and its cellular and molecular
components is just beginning. This review will focus on
the implantation and development of the murine em-
bryo and placenta. The significance of ectopic expres-
sion and targeted mutagenesis models to these pro-
cesses will be discussed. Dev. Genet. 21:620, 1997.
r 1997 Wiley-Liss, Inc.
Key words: Implantation; placentation; targeted muta-
genesis; mouse embryos
INTRODUCTION
Implantation is the process through which an embryo
attaches to and invades the maternal endometrium.
These connections provide access to the maternal circu-
lation that will sustain the embryo through the early
stages of gestation until the placenta has formed. The
peri-implantation period can be broken down into a
series of events, many of which take place simulta-
neously or overlap (Fig. 1). The first of these events is
the migration of the preimplantation blastocyst from
the oviduct into the uterus to its point of attachment to
the mother. For a review of preimplantation develop-
ment, see Kidder [1992], Cross et al. [1994], and Collins
and Fleming [1995]. At this time, the embryo hatches
from the zona pellucida. In the mouse, hatching is
achieved by a combination of rhythmic contractions of
the blastocyst and the production of a trypsin-like
protease, called strypsin, by the mural trophectoderm
that digests a hole in the zona pellucida [Perona and
Wassarman, 1986]. From gestational days 4.55, the
embryo attaches to the uterus (Fig. 1A). Then, in the
postattachment phase, from gestational days 4.58,
r 1997 WILEY-LISS, INC.
Reicherts membrane, the parietal endoderm, and the
embryonic trophoblast cells form a primitive diffusion
barrier to allow nutrients and gases from the maternal
blood vessels to nourish the embryo, without exposing it
to maternal blood. From gestation days 7.5 to 12, fetal
blood vessels begin to form and invade the yolk sac
placenta to provide nutrient and gas exchange until the
true chorioallantoic placenta forms at gestation day 9.
Failure to implant or establish a functional placenta
results in death and resorption of the embryo. Malforma-
tion of the placenta results in intrauterine growth
retardation and birth of runted embryos with poor
survival prospects. Thus, it is vital that a variety of
molecules including adhesion, signaling, transcription,
cell cycle, DNA repair, and replication proteins coordi-
nate fetal and maternal growth, differentiation, and
structure formation during this critical phase of devel-
opment. Ectopic expression and targeted mutagenesis
of a number of mouse genes are helping the molecular
genetic dissociation of implantation and placentation
(Table 1).
PREPARATION OF THE UTERUS
FOR EMBRYO IMPLANTATION
Contemporaneous with blastocyst hatching and mi-
gration into the uterus is the preparation of the uterus
for the apposition of the embryo to the uterine epithelia.
There is a defined implantation window in the mouse
during which the uterus will accept the embryo [Paria
et al., 1993]. This permissive time is determined by a
combination of progesterone [Huet-Hudson and Dey,
Contract grant sponsor: American Cancer Society; Contract grant
number: PF4228; Contract grant sponsor: National Institutes of
Health; Contract grant number: HD26732.
*Correspondence to: Julie Rinkenberger, Department of Anatomy,
LR208, University of California, 3rd and Parnassus Avenues, San
Francisco, CA 94143-0750.
Received for publication 7 April 1997; accepted 25 April 1997.

1990] and estrogen [Paria et al., 1993] signals that
prepare the uterus to accept embryo attachment. Fe-
male mice with a homozygous deletion of the estrogen
receptor fail to exhibit the hyperemia, cellular prolifera-
tion, and hypertrophy of the uterus characteristic of the
estrogen response and are sterile [Lubahn et al., 1993;
Course et al., 1995]. Estrogen directly or indirectly acts
on the embryo to facilitate implantation [Hou et al.,
1996]. If estrogen levels are kept low during the 24
hours before the embryo normally attaches, and if
progesterone is sustained at high levels, the mouse
blastocyst remains in diapause, a suspended state of
low metabolism that delays implantation for up to 30
days [Yoshinaga and Adams, 1966]. Further, embryos
that enter the uterus before it has been prepared by a
progesterone signal will not implant. Deletion of the
prolactin receptor results in implantation failure com-
bined with several maternal fertility defects including
abnormal estrous cycles and reduced levels of oocyte
maturation. Mutant females fail to undergo pseudopreg-
nancy in response to mating with vasectomized males,
indicating that prolactin signaling may be primarily
required to sustain luteal phase progesterone produc-
tion. In addition, fertilized embryos from homozygous
mutant females could not be rescued by transfer to
wild-type host mothers, suggesting that prolactin also
functions in preimplantation embryo development [Or-
mandy et al., 1997].
It is unclear how hormone signals prepare the uterus
for implantation of the embryo, but it is known that
they activate the expression of proteins thought to be
involved in increasing vascular permeability of the
uterus, both prior to and coincident with blastocyst
attachment. One such molecule, cyclo-oxygenase (COX-1),
is involved in the biosynthesis of prostaglandins from
arachidonic acid. COX-1 expression in the uterine
epithelium is upregulated by the combination of estro-
gen and progesterone before apposition of the embryo to
the uterine epithelium [Chakraborty et al., 1996]. How-
ever, COX-1 is not required for male or female fertility,
though its absence in homozygous to homozygous null
matings results in very low postnatal survival [Langen-
bach et al., 1995]. Expression of a second isoform,
COX-2, is not regulated by estrogen or progesterone in
the pregnant uterus, but its expression is required for
the formation of corpora lutea [Dinchuk et al., 1995;
Chakraborty et al., 1996]. Vascular endothelial growth
factor (VEGF), a protein that promotes angiogenesis
and increases vascular permeability, is also produced
within the peri-implantation uterus. However, it is not
clear that the preimplantation estrogen rise affects its
expression [Chakraborty et al., 1995]. Because null
mutants of VEGF and the VEGF receptors, flk-1 and
flt-1, die at mid-gestation [Fong et al., 1995; Shalaby et
al., 1995; Carmeliet et al., 1996; Ferrara et al., 1996], it
is impossible to assess any maternal requirement of
these factors for implantation.
IMPLANTATION IN THE MOUSE 7
Many cytokines are expressed in the uterus [for
review see Robertson et al. [1994], but only a few have
been shown to be required for embryonic implantation
and development. The clearest example is the require-
ment of maternally produced leukemia inhibitory fac-
tor (LIF) for embryo implantation. Homozygous LIF
mutants survive to adulthood, but females are sterile.
In the absence of maternally produced LIF, embryo
implantation fails, but embryos from null mothers will
implant and develop normally, if transferred to a wild-
type host mother [Stewart et al., 1992]. The fact that
LIF is produced in the uterus suggests the possibility
that it may allow LIF null embryos to develop normally.
Deletion of the low-affinity LIF receptor indicates that
LIF is indeed important for the postimplantation devel-
opment of the embryo [Ware et al., 1995; Li et al., 1995].
The cytokine interleukin-1 (IL-1) was also implicated
as required for blastocyst implantation in experiments
that injected IL-1 receptor antagonist into mice to block
the receptor [Simon et al., 1994]. However, the deletion
of the type I IL-1 receptor protein in mice contradicts
these data, because homozygous female mice are fertile
with a slight decrease in litter size resulting from
postnatal pup death [Abbondanzo et al., 1996].
EMBRYO ATTACHMENT TO THE UTERUS
Changes in estrogen and progesterone levels may
also regulate the expression of growth factors and
adhesion molecules or reduce the expression of inhibi-
tors of embryo attachment. Estrogen upregulates ex-
pression of the putative embryo growth and attachment
protein heparin-binding epidermal growth factor
(HB-EGF) in the uterine epithelium at sites of blasto-
cyst attachment, and in the uterine stroma in response
to estrogen plus progesterone in ovariectomized mice
[Wang et al., 1994]. HB-EGF and amphiregulin, both
members of the EGF family, function as growth or
differentiation factors and bind heparan sulfate in
addition to EGF. Amphiregulin expression increases in
response to progesterone and its expression in the
uterine epithelium peaks on day 4 of pregnancy at
blastocyst attachment sites [Das et al., 1995]. Expres-
sion of one potential inhibitor of embryo attachment,
mucin episialin (MUC1), declines at potential uterine
attachment sites in the mouse in response to the
progesterone decrease that occurs as estrogen levels
are rising [Braga and Gendler, 1993; Surveyor et al.,
1995]. However, its expression on uterine epithelia
peaks in humans at the time of implantation [Aplin and
Hey, 1995], indicating that it acts differently in mice
and humans, or that it is not directly involved in
embryo attachment. Direct demonstration of the role of
MUC1 in vivo is required to resolve this issue.
Embryo attachment occurs through the apposition of
the mural trophectoderm, the trophectoderm opposite
of the inner cell mass, to the uterine epithelium. The
molecules that mediate attachment have not been
8 RINKENBERGER ET AL.

Fig. 1.

completely elucidated, but a number of putative attach-
ment molecules have been identified by their expres-
sion on the uterine luminal epithelium or the embry-
onic mural trophectoderm during the peri-implantation
period. Included in this group are heparan sulfate
proteoglycan [Farach et al., 1987; Carson et al., 1993],
S-type lectins L14 and L30 [Poirier and Robertson,
1993], H type-I carbohydrates [Kimber et al., 1988;
Kimber and Lindenberg, 1990; Lindenberg et al., 1990;
White and Kimber, 1994], and the transmembrane form
of HB-EGF [Raab et al., 1996]. It is likely that multiple
receptor-ligand and carbohydrate interactions are re-
sponsible for the attachment of the blastocyst to the
uterine epithelium. In the mouse, deletion of the S-type
lectin L14, which is expressed on the trophectoderm of
hatching blastocysts, does not result in implantation
failure. S-type lectin L30, which has the same expres-
sion pattern as L14, may compensate for its loss
[Poirier and Robertson, 1993]. Another promising candi-
date, the transmembrane form of the HB-EGF protein,
was shown to confer blastocyst attachment ability to a
cell line in vitro. Luminal epithelial expression of
HB-EGF is proposed to enable blastocyst attachment
through heparan sulfate proteoglycans, the EGF recep-
tor, or both [Raab et al., 1996]. Targeted mutagenesis of
the EGF receptor results in different strain-specific
phenotypes. In one background, mutant embryos failed
soon after implantation, whereas in the other back-
grounds mouse embryos were viable to E12.5 or they
survived several days after birth [Miettinen et al., 1995;
Sibilia and Wagner, 1995; Threadgill et al., 1995]. The
strain specificity of the EGF receptor knockout pheno-
type is not understood, but deletion of HB-EGF may
help elucidate the role of the EGF receptor in embryo
adhesion to the uterus.
Recently, the adhesion molecule complex formed by
the proteins trophinin and tastin was cloned from a
human teratocarcinoma cell line. Characterization of
the complex by immunohistochemistry revealed its
Fig. 1. The peri-implantation period in the mouse. A: At E4.5,
embryos hatch from the zona pellucida and attach to the uterine
epithelium as shown by inset. B: Embryo at E5.5 with trophectoderm
invading through uterine epithelia into stromal tissue. Apoptosis of
uterine epithelia at the antimesometrial pole occurs but mesometrial
cells are intact. Primitive endoderm has produced visceral and pari-
etal endoderm. Parietal endoderm and Reicherts membrane line the
implantation chamber. Extraembryonic and embryonic ectoderm have
proliferated and extraembryonic ectoderm is starting to form the
ectoplacental cone. C: E7.5 gastrulating embryo is shown inside two
layers of decidual cells, with mesometrial cells being less abundant
resulting in an egg-shaped decidua. Primary and secondary trophecto-
derm-derived giant cells are shown, with secondary giant cells lining
the maternal blood spaces throughout the ectoplacental cone. The
ectoplacental cone contains secondary giant cells, maternal blood
sinuses, and extraembryonic ectoderm-derived cells. Mesoderm is
shown pushing up the extraembryonic ectoderm membrane to form
the chorion. Spaces between many tissue layers are shown for clarity,
but are smaller or nonexistent in vivo. D: Embryo is shown at E8.0
before turning to illustrate allantois growth toward the chorionic
IMPLANTATION IN THE MOUSE 9
expression on the human endometrial epithelium dur-
ing the implantation window and on the surface of
monkey blastocysts, as well as at the attachment sites
of a human teratocarcinoma cell line to an endometrial
adenocarcinoma cell line in vitro. Trophinin and tastin
also conferred attachment competence to Cos cells in
vitro [Fukuda et al., 1995]. Further demonstration of a
requirement for these molecules in implantation awaits
discovery of a homologous system in the mouse.
Several other proteins exhibit peri-implantation ex-
pression patterns or have been shown to facilitate
blastocyst outgrowth in vitro. For example, several of
the integrins, E-cadherin, laminin, fibronectin, tenas-
cin, and collagen IV, may facilitate early stages of
preimplantation development [Larue et al., 1994; Rieth-
macher et al., 1995], or may be required for cell
adhesion within the inner cell mass [Fassler and Meyer,
1995; Stephens et al., 1995], for the later steps of
trophoblast invasion of the uterine stroma [Damsky et
al., 1993; George et al., 1993; Sutherland et al., 1993;
Julian et al., 1994; Fassler et al., 1996], or for the
postimplantation development of the embryo [George et
al., 1993; Fassler et al., 1996]. However, none of these
molecules is required for the apposition of the embryo
to the uterus or its implantation. Two adhesion mol-
ecules that are critical for blastocyst development have
been identified by targeted mutagenesis. Both a-E-
catenin and its ligand E-cadherin are required for
blastocoel and trophectoderm formation, with mutants
degenerating before hatching at E4 [Larue et al., 1994;
Riethmacher et al., 1995; Torres et al., 1997]. Correct
integrin to receptor contacts are required for embryo
development after implantation begins. In the b1 inte-
grin knockout model, uterine attachment and invasion
by the trophectoderm begin, but the inner cell mass
dies without continuing to develop further [Fassler and
Meyer, 1995; Stephens et al., 1995]. The fibronectin
gene deletion mouse model has defects in mesoderm
development, neural tube formation, and in heart and
plate. Secondary giant cells in the primitive placenta are represented
as a single layer for clarity. Red dots in the chorionic plate indicate
maternal blood sinuses. Chorionic plate is derived from extraembry-
onic ectoderm-derived membranes fused to the mesoderm-derived
allantois. Embryonic blood islands are not depicted but would be on
the embryo side of the visceral endoderm. E: Embryo and placenta
from E13.5 are shown. Umbilical cord and inner layer of placenta are
fused for clarity. Fetal blood vessel growth has taken place at this time.
The trophoblast giant cell layer of the placenta is not shown. AL,
allantois; AM, amnion; AMD, antimesometrial decidua; AMP, antime-
sometrial pole; AUE, apoptotic uterine epithelium; BC, blastocoel
cavity; CP, chorionic plate; EE, embryonic ectoderm; EEE, extraembry-
onic ectoderm; EMB, embryo; EPC, ectoplacental cone; FBV, fetal
blood vessel; ICM, inner cell mass; LL, labyrinthine layer; MBS,
maternal blood sinus; MD, mesometrial decidua; MP, mesometrial
pole; MS, mesoderm; PE, primitive endoderm in (A) and parietal
endoderm in (B-E); PGC, primary giant cells; RM, Reicherts mem-
brane; SGC, secondary giant cells; SL, spongiotrophoblast layer; TE,
trophectoderm; UBC, umbilical cord vein; UC, uterine crypt; UE,
uterine epithelium; VE, visceral endoderm.
 TABLE 1. Gene Deletion Mouse Models That Result in Peri-Implantation Lethality

Gene Proposed function Cause of death Reference

Preimplantation failures
a-E-catenin Ligand of E-cadherin Fails to form blastocoel Torres et al., 1997
E-cadherin/uvomorulin Adhesion molecule Fails to form blastocoel Larue et al., 1994; Riethmacher et
al., 1995
Estrogen receptor Hormone receptor No estrogen response in uterus Lubahn et al., 1993
Hoxa-10 Transcription factor Weak vascular/decidual response Benson et al., 1996
by uterus to embryo
Prolactin receptor Hormone receptor Multiple reproductive abnormali- Ormandy et al., 1997
ties; defective preimplantation
development
Peri-implantation failures
APCmin Tumor suppressor Embryo failure Moser et al., 1995
b-catenin Ligand of E-cadherin No mesoderm, poor ectoderm devel- Haegel et al., 1995
opment
b1 integrin Adhesion molecule Embryo proliferation failure Fassler and Meyer, 1995; Stephens
et al., 1995
Brca-1 Tumor suppressor Embryo proliferation failure Hakem et al., 1996; Liu et al., 1996
Cyclin A2 Cell cycle factor Embryo proliferation failure Murphy et al., 1997
EGF receptor Cytokine receptor Background dependent: no implan- Threadgill et al., 1995; Sibilia and
tation; survives to E12.5; sur- Wagner, 1995; Miettinen et al.,
vives postnatally 1995
eed Transcriptional repressor Required for embryonic ectoderm Niswander et al., 1988, 1989; Schu
development and axial mesoderm macher et al., 1996
Evx-1 Transcription factor No extraembryonic or embryonic Spyropoulos and Capecchi, 1994
tissue
exed Required for development of extra- Niswander et al., 1988, 1989
embryonic ectoderm
FGF 4 Cytokine Inner cell mass failure Feldman et al., 1995
LIF Cytokine No implantation Stewart et al., 1992
Mcl-1 Anti-apoptotic factor No implantation Rinkenberger et al., unpublished
data
Mdm-2 Cell cycle factor Embryo proliferation failure Montes de Oca Luna et al., 1995;
Jones et al., 1995
msd No mesoderm is produced Holdener et al., 1994
Rad-51 DNA repair Blocked at two-cell stage or prolif- Tsuzuki et al., 1996; Lim and Hasty,
eration failure at E6 1996
Ref-1/AP endonuclease DNA repair; oxidation Embryo degeneration at E5.5 Xanthoudakis et al., 1996
Thioredoxin DNA synthesis; reduction Embryo gone by gastrulation Matsui et al., 1996
XRCC-1 DNA repair Embryonic tissue dies before gas- R.S. Tebbs and R. Pedersen, per-
trulation sonal communication
Placentation failures:
a4 integrin Ligand of VCAM-1 No allantois/Ectoplacental cone Yang et al., 1995
fusion
ERR2 Orphan hormone receptor Chorion failure V. Giguere, personal communica-
tion
Ets-2 Transcription factor Placenta failure R. Oshima, personal communica-
tion
Fibrinogen Coagulation factor Maternal death from hemorrhage Suh et al., 1995
at E10
Fibronectin ECM glycoprotein Failed embryonic and extraembry- George et al., 1993
onic vasculature development
Flk-1 VEGF receptor Blood island formation and vasculo- Shalaby et al., 1995
genesis failure
Flt-1 VEGF receptor Abnormal vasculogenesis and blood Fong et al., 1995
island formation
GATA-2 Transcription factor Embryo failure and reduced neo- Ma et al., 1997
vascularization of uterine
decidua
GATA-3 Transcription factor Embryo failure and reduced neo- Ma et al., 1997
vascularization of uterine
decidua
HGF/scatter factor Cytokine Lack of trophoblast giant cells in Uehara et al., 1995
labyrinthine layer of placenta
Hxt bHLH transcription factor Trophoblast failure J. Cross, unpublished data
Kuz ADAM/metalloproteinase No blood vessel development; dies D.J. Pan, personal communication
at E9.5
LCR-F1 Transcription factor No primitive streak or mesoderm Farmer et al., 1997
formed
Mash-2 Transcription factor Trophoblast failure Guillemot et al., 1994
N-cadherin Adhesion molecule Yolk sac vessel failure Radice et al., 1997
Thrombin receptor Coagulation factor receptor Half of embryos die at E9 exhib- Connolly et al., 1996
iting delayed placental develop-
ment
Thrombomodulin Anticoagulant factor Embryos die at E7.5 with maternal Healy et al., 1995
blood clots in blood spaces
Tissue factor Coagulation factor Embryos die between E8.5 and 10.5 Bugge et al., 1996
from hemorrhage from fetal ves-
sels
UBC-M4 Ubiquitin conjugating enzyme Labyrinthine trophoblast greatly Harbers et al., 1996
decreased in size
VCAM-1 Ligand for a4 integrin No allantois/Ectoplacental cone Gurtner et al., 1995; Kwee et al.,
fusion 1995
VEGF Angiogenesis factor Yolk sac vessel/blood island failure Carmeliet et al., 1996; Ferrara et
al., 1996

yolk sac development. Abnormal or retarded develop-
ment of embryonic and extraembryonic vasculatures
results in embryo degeneration after E8.5 [George et
al., 1993].
MATERNAL RESPONSE TO EMBRYO
ATTACHMENT
As mentioned earlier, just before attachment of the
embryo to the uterine epithelia, hormones induce
changes in the vascular permeability of the uterus.
Additionally, uterine stromal cells begin to proliferate
in anticipation of embryo attachment. The vascular
changes that occur in response to subsequent embryo
attachment can be visualized in the mouse by injection
of pontamine blue into the mother a few minutes before
sacrifice. Implantation sites are intensely labeled by
bands of dye. Hoxa-10 null females exhibit impaired
uterine vascular permeability and decidual responses
to the presence of embryos. These animals display
homeotic transformation of the proximal 25% of the
uterus to oviduct, which may limit their ability to
respond to and maintain pregnancy [Benson et al.,
1996].
The decidual response occurs in the immediate area
of the uterus surrounding an attached embryo by a
mechanism that is still poorly understood. Decidualiza-
tion involves the proliferation and differentiation of
uterine stromal cells, which results in the compression
of the uterine crypt around the embryo to form the
implantation chamber [Welsh, 1993]. The primary de-
cidual zone is the area in which stromal cells differenti-
ate to a pseudoepithelial phenotype and it forms around
the antimesometrial portion of the implantation cham-
ber at E5.0 to E5.5 in the mouse. The decidual cells in
this zone are large, multinucleate, and rich in endoplas-
mic reticulum with tight, desmosome-like and gap
junctions [Kleinfeld et al., 1976; Parr and Parr, 1986;
Tung et al., 1986]. The antimesometrial decidual cells
persist until the egg cylinder begins its expansion into
the antimesometrium, whereupon they begin to die
from the implantation chamber outwards and are con-
sumed by the trophectodermal giant cells, facilitating
their access to maternal blood vessels [Welsh and
Enders, 1985, 1987; Welsh, 1993]. Decidual cell remod-
eling appears to be regulated by a balance of metallopro-
teinases and their inhibitors [Alexander et al., 1996]
during the invasion of the trophoblast cells, as will be
discussed later.
Two days after the formation of the primary decidual
zone, the mesometrial decidua forms at the mesome-
trial pole of the embryo (Fig. 1C). Unlike the antimeso-
metrial decidual cells, the mesometrial decidual cells
are smaller and have a single nucleus. They are never
as tightly associated as the antimesometrial decidual
cells but do have extensive cellular processes connected
by gap and desmosome-like junctions [Welsh, 1993].
There are fewer trophoblast giant cells in the mesome-
trial region and it is likely that maternal blood vessel
IMPLANTATION IN THE MOUSE 11
growth contributes to the establishment of blood flow
through the mesometrial portion of the implantation
chamber [Welsh and Enders, 1991b]. The trophoblast
produces growth factors, like proliferin, that stimulate
uterine and possibly fetal neovascularization. Prolif-
erin production is regulated by the transcription factors
GATA-2 and GATA-3 in the trophoblast, and the de-
cidual tissue surrounding the null mutants of either
gene exhibits significantly reduced neovascularization,
compared to wild-type decidua within the same uterus
[Ma et al., 1997]. Both primary and mesometrial de-
cidual cells store large quantities of glycogen, leading to
speculation that one function of the decidua may be to
supply nutrition to the growing embryo until the forma-
tion of the placenta [Finn and Porter, 1975; Finn, 1977].
Another proposed function of decidualization is to
protect the mother from the invasive nature of the
embryo. Transplanting the mouse embryo outside of
the uterus results in extensive invasion and growth of
the embryo [Kirby, 1960, 1963a,b, 1965]. It is likely that
the decidua protects the uterus from inappropriate
invasion of the embryo through a physical barrier as
well as by the production of inhibitors of proteolytic
enzymes, like the tissue inhibitors of metalloprotein-
ases (TIMPs), that are produced by the trophectoderm
[Reponen et al., 1995; Alexander et al., 1996; Teesalu et
al., 1996]. The decidua may also protect the embryo,
which carries foreign paternal antigens, from the mater-
nal immune system by restricting passage of high
molecular weight substances like immunoglobulins,
microorganisms, and lymphocytes until the protective
layers of Reicherts membrane and the yolk sac have
formed [Parr and Parr, 1986].
Maintenance of the decidua throughout pregnancy
depends on the presence of progesterone. Formation of
the decidual reaction in mice and rats depends on
estrogen to drive the estrus cycle and then progesterone
and estrogen during the luteal phase of the cycle [Finn
and Porter, 1975]. Like the apoptosis of uterine epithe-
lial crypt cells, decidualization does not require the
presence of an embryo because it occurs in pseudopreg-
nancy [Finn and Porter, 1975], with expression of many
of the same genes in the deciduoma as in normal
decidua [Julian et al., 1994; Wang et al., 1994; Das et al.,
1995; Alexander et al., 1996; Brown et al., 1996].
Coincident with the apposition of the embryo to the
uterus, the nearby uterine epithelia begin to undergo
apoptosis. A series of studies using electron microscopy
first revealed the breakdown of the uterine epithelia
and the phagocytosis of these cells by the rapidly
proliferating and expanding cells of the mural trophec-
toderm in rats and mice [Enders and Schlafke, 1967;
El-Shershaby and Hinchliffe, 1975; Parr et al., 1987].
The trophectoderm appears to clear the dying cells from
the surface of the antimesometrial portion of the uter-
ine crypt to enable its invasion of the underlying
decidual tissue [Bevilacqua and Abrahamsohn, 1989;
for review, see Welsh, 1993]. Whether the trophecto-
12 RINKENBERGER ET AL.
derm is also providing some kind of a death signal to
trigger this breakdown is unclear. However, it seems
unlikely that such a signal is required because some
epithelial cell breakdown can occur in pseudopreg-
nancy in the absence of an embryo [El-Shershaby and
Hinchliffe, 1975; Welsh, 1993].
The breakdown of the uterine epithelia continues at
the antimesometrial pole of the implantation site, and
is followed a full gestational day later by the death of
the luminal epithelia at the mesometrial pole. It is
possible that some of the cell death observed in the
uterine epithelium at the mesometrial pole is not
apoptotic, but rather a type of necrosis [Welsh and
Enders, 1991a, 1993; Welsh, 1993]. At this time, the
maternal decidual cells form a network of spaces that
are filled with maternal blood. The trophectoderm-
derived giant cells of the embryo begin to make contacts
with these mesometrial maternal cells. Eventually,
trophoblasts will connect directly to the maternal endo-
thelium and express many endothelial markers, like
PECAM-1 [DeLisser et al., 1994].
GROWTH OF THE
POSTIMPLANTATION EMBRYO
During the time of its attachment to the uterine wall,
the embryo undergoes a tremendous burst of prolifera-
tion and a series of differentiative steps to form new
tissues. The parietal endoderm forms during the late
blastocyst stage at E4.5 and it expands into what
previously was the blastocoel cavity to begin to form the
yolk sac and Reicherts membrane. Formation of this
membrane begins with the mural trophectoderm on the
inside of the blastocoel cavity in the early blastocyst
and continues with the parietal endoderm cells as they
begin to migrate along the mural trophectoderm in the
peri-implantation embryo [Salamat et al., 1995].
Coincident with the expansion of the parietal endo-
derm there is substantial proliferation of the inner cell
mass to form the primitive ectoderm. The polar trophec-
toderm that overlays the primitive ectoderm prolifer-
ates and forms the ectoplacental cone and extraembry-
onic ectoderm. Eventually the extraembryonic ectoderm
will combine with mesoderm-derived tissue and fuse
with maternal blood vessels to form the placenta. The
primitive ectoderm will continue to grow down into the
yolk cavity to form the embryo which at this stage (E5.5
to E6.5) is called the egg cylinder [Rossant and Frels,
1981] (see Fig. 1B).
Targeted mutagenesis has identified proteins that
are critical for the development of the embryo during
this period of rapid proliferation and expansion. Not
surprisingly, this group includes DNA synthesis and
damage repair proteins as well as cell cycle factors. The
phenotype of these mutant embryos is very similar,
with early implantation of the embryo and formation of
a decidual reaction, followed by lack of proliferation or
expansion of embryonic size at E5.5, and its death and
resorption. Mutants of the DNA repair proteins Rad-51
[Lim and Hasty, 1996], Ref-1 [Xanthoudakis et al.,
1996], and XRCC-1 [R. Pedersen, personal communica-
tion], the DNA synthesis protein thioredoxin [Matsui et
al., 1996] as well as the cell cycle-associated proteins
cyclin A2 [Murphy et al., 1997] and Mdm-2 [Jones et al.,
1995; Montes de Oca Luna et al., 1995], and the tumor
suppressor Brca-1 [Hakem et al., 1996; Liu et al., 1996]
die from failure of embryo growth at E5.5E6.5. Inter-
estingly, in XRCC-1 and Rad-51 mutants, only embry-
onic cells, and not extraembryonic tissues, die initially.
In an elegant study using a series of monoclonal and
polyclonal antibodies, it has been shown that human
BRCA-1 and Rad-51 colocalize to chromosomes during
meiosis and that they may overlap in their expression
pattern on single-stranded chromosomes in the zygo-
tene period. They also coimmunoprecipitate in vitro,
suggesting that BRCA-1, like Rad-51, may function in
control of recombination and DNA repair [Scully et al.,
1997].
Implantation failure in the Ref-1/apurinic/apyrimi-
dinic endonuclease mutant may be an indirect result of
loss of the protein. Ref-1 is a bifunctional molecule that
has a role in DNA repair and reduces the oxidized form
of several transcription factors, resulting in their activa-
tion. It is thought that one of these proteins, rather
than Ref-1 directly, is crucial to embryo development
and is not activated in the Ref-1 mutant embryo,
although a target molecule has not been identified
[Xanthoudakis et al., 1996]. Another bifunctional pro-
tein, thioredoxin, has a role in both DNA synthesis and
the redox pathway. Mutant embryos lacking thiore-
doxin grow in culture quite well, suggesting that the
mutant phenotype is not so much an inhibition of DNA
synthesis, as it is a response to the intrauterine environ-
ment [Matsui et al., 1996]. However, it remains to be
determined which role of thioredoxin, DNA synthesis,
and/or reduction is required for correct implantation.
Both the cyclin A2 and Mdm-2 deletion mutants are
peri-implantation lethals, though neither may have a
direct role in the implantation process. The cell cycle
factor cyclin A2 is present early in development, and
the unexpected persistence of embryo growth to the
implantation phase is explained by the presence of
maternally derived protein [Murphy et al., 1997]. The
Mdm-2 knockouts, like the other mutants, stimulate a
decidual reaction, but by E5.5 there are few live cells
left in the implantation site. As Mdm-2 had been shown
to block the action of the p53 transcription factor, it was
thought that the phenotype of the mdm-2 mutant
animals may be a result of overactive p53. Interest-
ingly, the mdm-2 and p53 gene double knockout ani-
mals proved to be viable, suggesting that in early
development, Mdm-2 is required to inhibit the activity
of p53 in the embryo [Jones et al., 1995; Montes de Oca
Luna et al., 1995].
Another critical requirement of the implanting em-
bryo may be the presence of survival promoters, like the

Bcl-2 family member Mcl-1 [Kozopas et al., 1993]. In
the Mcl-1 knockout mouse model, homozygous mutant
embryos can be detected at E4.0, but they do not
implant. Unlike many of the other peri-implantation
lethals mentioned above, there is no homozygous mu-
tant embryo outgrowth or empty decidua visible from
E5.5 onward. Explanted preimplantation mutant em-
bryos fail to grow in culture at any time of isolation,
suggesting that they may be exquisitely sensitive to
culture conditions or require the maternal environment
to continue development to the blastocyst stage [J.
Rinkenberger and S.J Korsmeyer, unpublished data].
The Mcl-1 protein, like members of the Bcl-2 family,
shares a consensus homology region that has been
shown to function in the Bcl-XL protein as an ion
channel in vitro [Minn et al., 1997]. It is possible that all
of the members of this gene family exert either their
death-promoting or death-protecting function by con-
trol of ion or protein flow through the mitochondria and
other intracellular membranes. However, it has not
been demonstrated that any of the family members
function as a pore in vivo, and it is not known how this
function would regulate the cell death process.
FORMATION OF THE YOLK SAC PLACENTA
Rodents, like humans, form a hemochorial placenta
where the embryonic tissues actually invade beneath
the maternal epithelial cell layer and into the stromal
tissue to make direct contact with the maternal capil-
lary bed. Terminally differentiated embryonic tropho-
blast cells make direct contact with maternal blood,
and, in combination with Reicherts membrane and the
yolk sac in the early mouse embryo, allow gas and
nutrient exchange without exposing the embryo to the
maternal blood supply and immune system directly. By
day 8 of pregnancy in the mouse, antimesometrial
decidual cell breakdown has begun and trophectoderm
in contact with Reicherts membrane has formed a
network of pores on its surface. Reicherts membrane
consists of a basement membrane type of extracellular
matrix (ECM) initially produced by the mural trophec-
toderm and then by the parietal endoderm that sepa-
rates the embryo from the maternal blood cells that
flow through cavities around it as a result of the death
of antimesometrial decidual cells [Welsh and Enders,
1987; Bevilacqua and Abrahamsohn, 1988; Salamat et
al., 1993, 1995]. It is made up of a number of proteins
found in other kinds of basement membranes, such as
type IV collagen, laminin, entactin, fibronectin, and
heparan sulfate proteoglycan [Hogan et al., 1980, 1984;
Laurie et al., 1982a,b; Mazariegos et al., 1987; Inoue
and Leblond, 1988], but is thicker and more resilient
than many other types of ECM.
The clotting cascade must be carefully controlled
within the decidua, as the connection of maternal blood
IMPLANTATION IN THE MOUSE 13
vessels to the trophoblast-lined embryonic interface
forms during placentation, and for embryonic vasculo-
genesis in the yolk sac, placenta, and embryo proper.
Trophoblasts express endothelial markers and closely
associate with maternal blood vessels during this pro-
cess. Establishment of these connections requires a
delicate balance of both the fibrinogenic and fibrinolytic
mechanisms. Parietal endoderm cells produce an anti-
coagulation factor called thrombomodulin, which is
essential for the development of the embryo past E8.5
[Healy et al., 1995]. Thrombomodulin, in combination
with the heparan sulfate protein in Reicherts mem-
brane, probably prevents clotting of maternal blood
around the embryo at this stage of development. A lack
of tissue factor, which is a strong activator of the
coagulation cascade after tissue injury [Davie et al.,
1991], also results in embryonic lethality as a result of
hemorrhage of embryonic blood from both extraembry-
onic and embryonic vessels into the yolk and pericar-
dial sacs [Bugge et al., 1996]. Vessel integrity is compro-
mised between E8.5 and E9.5, a time when embryonic
and extraembryonic vasculatures are forming and fus-
ing, most likely as a result of failure to activate
thrombin generation or fibrin deposition at sites of
rupture.
Members of the clotting cascade are also required
during chorioallantoic placenta formation. Deletion of
the thrombin receptor results in loss of half of null
embryos at E910, with the other half surviving to
adulthood, although their fertility was not reported.
The dying embryos are phenotypically normal at E8.5,
but by E9.5 have no heartbeat and exhibit a delay in
placental development [Connolly et al., 1996]. Targeted
mutagenesis of fibrinogen demonstrates its require-
ment for the maternal response to trophoblast invasion,
as null embryos survive to term in heterozygous moth-
ers, but null mothers die at gestation day 10 from
massive hemorrhage into the uterus from the labyrin-
thine layer of the placenta [Suh et al., 1995].
At E8, embryo-derived blood islands are visible on
the inner wall of the yolk sac which is called the visceral
endoderm. In these hematopoietic colonies, the heman-
gioblasts give rise to both blood and blood vessels and
produce blood until the fetal liver and bone marrow
develop at later stages of gestation. VEGF and two of its
receptors, Flk-1 and Flt-1, are required by the embryo
for angiogenesis and blood island formation in the yolk
sac and vasculogenesis [Fong et al., 1995; Shalaby et al.,
1995; Carmeliet et al., 1996; Ferrara et al., 1996]. The
yolk sac begins to function as a primitive placenta to
promote gas and nutrient exchange between mother
and embryo at about day 9 of gestation. In the normal
embryo, the vessels of the yolk sac will continue to grow
until 1214 days of gestation, suggesting that it contin-
ues to function past the establishment of the placenta
[Muntener and Hsu, 1977].
14 RINKENBERGER ET AL.
INVASION OF THE DECIDUA
While the initial steps of implantation occur, the
trophectoderm cells, which have attached to the uterus,
endoreduplicate their DNA and become primary tropho-
blast giant cells. Giant cells are long-lived nonprolifer-
ating cells that produce proteinases and other mol-
ecules that facilitate the breakdown and invasion of the
decidua. They are actively involved in the phagocytosis
of dying maternal cells during invasion [Bevilacqua
and Abrahamsohn, 1988; Welsh, 1993]. Gelatinase
B/MMP9 [Brenner et al., 1989; Canete-Soler et al.,
1995; Harvey et al., 1995b; Alexander et al., 1996],
gelatinase A [Alexander et al., 1996], the urokinase-
type plasminogen activator (uPA) and its receptor
[Sappino et al., 1989; Harvey et al., 1995a; Teesalu et
al., 1996], and a number of other proteinases [Teesalu et
al., 1996] are expressed by the invading trophoblasts as
well as in the maternal decidua. The results of gene
deletion mouse models have indicated that, while pro-
teinase activity is crucial for implantation success,
there must be considerable functional redundancy.
Deletion of the low density lipoprotein receptor-related
protein that is expressed by trophoblast giant cells and
is involved in clearance of urokinase-type plasminogen
activator-plasminogen activator inhibitor 1 complexes
results in midgestation lethality well after implanta-
tion [Herz et al., 1992]. Surprisingly, deletion of most of
the plasminogen/plasmin system including tissue-type
plasminogen activator (tPA), uPA, plasminogen activa-
tor inhibitor-1 (PAI-1) [Carmeliet et al., 1994, 1995;
Carmeliet and Collen, 1995], uPA receptor [Dewerchin
et al., 1994; Bugge et al., 1995a], plasminogen [Ploplis et
al., 1995; Bugge et al., 1995b], or a2-macroglobulin
[Umans et al., 1995] does not affect viability of homozy-
gous knockout animals, which survive postnatally [Car-
meliet and Collen, 1995], although there is some de-
crease in the decidualization of the uterus in uPA
knockout pregnancies [Carmeliet et al., 1995]. While
deletion of the uPA and tPA proteinases separately does
not result in decreased fertility, double homozygotes are
less fertile in part because of their poor state of health
and excess fibrin deposition within the gonads [Carme-
liet et al., 1995]. It is not clear that the lack of uPA/tPA
really affects the implantation and placentation pro-
cess. A similar phenotype is also observed in the
plasminogen knockout females [Ploplis et al., 1995;
Bugge et al., 1995b]. Deletion of the matrix metallopro-
teinase gelatinase B/MMP9, implicated in the invasion
of maternal decidua, also does not affect the viability or
fertility of homozygous knockout animals. [Z. Werb,
unpublished data]. Together, these data suggest that
the proteinase network is complex, to ensure that
appropriate maternal and fetal connections are made
during implantation.
To counteract these proteinases, the decidua pro-
duces a number of inhibitors which may act to limit the
invasion of the embryo to the stromal layers nearest to
Fig. 2. Decidua and embryos from gestation day 6.5 are shown from
a TIMP-1 overexpressing transgenic mouse model and a control
nontransgenic mouse. TIMP-1 expression was driven by the b-actin
promoter and resulted in serum levels of TIMP-1 of 50 ng/ml. No
effects on fertility of TIMP-1 overexpressing mice were observed.
Decidua shown are from a homozygous TIMP-1 transgenic mouse and
a nontransgenic mouse, both on the CD-1 background.
it. In situ hybridization analysis and immunohistochem-
istry have identified overlapping expression patterns of
proteinases and their inhibitors in the early decidua
and embryo [Reponen et al., 1995; Alexander et al.,
1996]. Of the inhibitors examined, only TIMP-3 was
expressed in the decidua proximal to the embryo at
E7.5. Its expression pattern partially overlapped that of
gelatinase B, which was expressed in the primary
trophoblast giant cells, forming a boundary that may
limit their invasion of the decidua [Alexander et al.,
1996]. Levels of apoptosis in decidual cells proximal to
areas of gelatinase B expression were inversely propor-
tional to their level of TIMP-3 expression. However,
earlier TIMP-3 expression indicates it may have a role
in the implantation process, perhaps in decidual differ-
entiation. Consistent with a role for the matrix metallo-
proteinase system in regulating decidualization, injec-
tion of the synthetic metalloproteinase inhibitor MPI
reduced decidua formation [Alexander et al., 1996].
Additionally, overexpression of the metalloproteinase
inhibitor TIMP-1 in a transgenic mouse model slows
the development and remodeling of the decidual zone,
resulting in decidua with decreased length and smaller
size (Fig. 2). However, TIMP-1 transgenic animals are
fertile and do not exhibit higher levels of embryo
resorption suggesting that they form a functional,
albeit smaller decidua [Alexander et al., 1996]. It is
likely that the different gradients of expression of these
proteinases and their inhibitors act in concert with
other unknown factors to prevent embryo invasion
through the decidua and escape outside the uterus.
FORMATION OF THE PLACENTA
The definitive placenta is composed of cells of both
trophoblast (extraembryonic ectoderm of the chorion)

and mesoderm (allantois) origin. At E6.75, the primi-
tive ectoderm differentiates to form the three embry-
onic tissue layers: ectoderm, mesoderm, and endoderm.
This process is called gastrulation and it continues
until approximately E8.0 [Downs and Davies, 1993], at
which time primitive structures or domains of the
embryo that will become the head, somites, and neural
plate are visible [Lawson et al., 1987, 1991]. Highly
detailed analysis of gastrulation has identified nine
somewhat overlapping stages of structural and morpho-
logical change in the mouse embryo [Downs and Dav-
ies, 1993]. For the purposes of this review, gastrulation
results in the formation of mesoderm tissue that even-
tually forms the allantois. The allantois fuses to the
chorionic plate, resulting in the chorioallantoic part of
the placenta that occupies the labyrinthine layer (Fig.
1D). A number of targeted mutagenesis models present
defective formation of the mesoderm [for a more in
depth review, see Faust and Magnuson, 1993; Cross et
al., 1994; Copp, 1995; Farmer et al., 1997]. Mutation of
b-catenin, which has dual functions in connecting E-
cadherin to the cytoskeleton and also in transcriptional
regulation [Korinek et al., 1997; Morin et al., 1997;
Rubinfeld et al., 1997], results in failure to form the
mesoderm as well as defective extraembryonic ecto-
derm development visible at E7 [Haegel et al., 1995].
Three naturally occurring deletions within the albino
locus of the mouse genome also result in embryo failure
as a result of defective mesoderm production, with
abnormal morphology first visible at E7.5 (exed and
msd) and E8.5 (eed) [Holdener-Kenny et al., 1992]. The
exed homozygotes, which are resorbed by E8.5, have a
very small egg cylinder that lacks mesoderm, and
contains undifferentiated and dying extraembryonic
ectoderm. The msd mutant embryos fail to form meso-
derm by E7.5, but the egg cylinder continues to increase
in size and they exhibit an unusually large expansion of
the parietal endoderm [Holdener et al., 1994]. The eed
mutants develop amnion, chorion, exocoelom, ectopla-
cental cavity, and allantois, but they exhibit reduced
axial mesoderm formation resulting in no head process,
notochord, or somite formation [Holdener-Kenny et al.,
1992]. The eed gene has recently been cloned and is a
member of the Polycomb group of transcriptional repres-
sors [Schumacher et al., 1996]. Each of these deletions
likely affects a different part of the mesoderm induction
or production pathway, but all illustrate its require-
ment for both embryonic and placental structures.
The formation of the placenta requires the growth of
the allantois from the caudal end of the embryo in the
amniotic cavity through the exocoelomic cavity until it
fuses with the chorionic plate to form the chorioallan-
toic placenta (Fig. 1D). Deletion of the VCAM-1 mol-
ecule and its ligand, a4 integrin, has indicated that
they mediate allantois to chorion fusion. In their ab-
sence, embryos do not establish the placenta and die by
IMPLANTATION IN THE MOUSE 15
E12 [Gurtner et al., 1995; Kwee et al., 1995; Yang et al.,
1995]. A similar defect is observed in parthenogenote
embryos that have been aggregated to fertilized tetra-
ploid embryos [Spindle et al., 1996]. However, in this
case, some embryos may initiate allantois to chorion
fusion, but the labyrinthine layer of the placenta devel-
ops abnormally. None of the parthenogenotes from
these experiments survived past E12, indicating that
the failure of extraembryonic mesoderm to contribute
to correct placenta formation is one consequence of
paternal imprinting [Spindle et al., 1996].
By E10, four zones of the placenta can be visualized:
the chorionic plate formed by the membrane dividing
the exocoelomic cavity and the ectoplacental cavity
fused to the allantois, the labyrinthine layer composed
of embryonic trophoblast cell strands containing mater-
nal blood, the junctional zone spongiotrophoblast layer,
and the trophoblast giant cell zone that isolates the
embryo from the mother. Embryonic umbilical cord
vessel formation mediated by the allantoic mesoderm is
observed by E10 in the labyrinthine tissue and these
vessels contain nucleated fetal erythrocytes, indicating
that circulation through the placenta has begun [Mun-
tener and Hsu, 1977]. It is in the labyrinthine layer that
maternal and embryonic circulation will come closest to
each other and where gas and nutrient exchange will
take place. (Figure 1E shows an E13.514 embryo
attached to its placenta by the umbilical cord.) Labyrin-
thine layer development is abnormal in embryos lack-
ing hepatocyte growth factor/scatter factor, resulting in
embryonic lethality at E13 [Uehara et al., 1995]. A
labyrinthine layer defect is also observed in Wnt2
mutants, resulting in edema and pooling of maternal
blood as well as poor fetal capillary development and
fibrin deposition in the placenta. However, about 50% of
knockout animals survive to adulthood. Animals that
die are lost perinatally, suggesting that Wnt2 contrib-
utes to proper formation of the placenta, but that
mutant placenta can still support embryo development
to term [Monkley et al., 1996].
MOLECULAR DEFECTS IN HUMAN
PLACENTATION
Placental abnormalities are common in humans. For
example, preeclampsia affects as many as 710% of all
first pregnancies [Roberts et al., 1993]. It is diagnosed
after 20 weeks of gestation and is associated with
maternal hypertension and renal dysfunction as well as
with low birthweight or intrauterine death of the fetus.
One hallmark of the disease is the shallow invasion of
the spiral arterioles and maternal decidual stroma by
the placenta, resulting in poor maternal blood flow to
the fetus [Brown, 1995].
A number of investigators have begun to identify the
molecules involved in human trophoblast invasion of
the maternal stroma to assess their role in preeclamp-
16 RINKENBERGER ET AL.
sia. Many of the proteinases and adhesion molecules
identified thus far are also found in trophectoderm-
derived and uterine stroma cells during placenta forma-
tion in the mouse. Matrix metalloproteinases, including
gelatinase B and the plasminogen/plasmin system,
have been isolated from human placenta. At least one of
these molecules, gelatinase B, may be expressed pre-
dominantly in the zymogen form in media conditioned
by trophoblasts isolated from preeclamptic placentas
[Graham and McCrae, 1996]. Altered expression of two
fibrinolytic system inhibitors, PAI-1 and PAI-2, in pre-
eclamptic placenta and maternal plasma may explain
the increased fibrin deposition in the maternal kidney
and spiral arterioles [Gilabert et al., 1995]. Mice lack-
ing uPA and tPA, both targets of PAI-1, or plasminogen
do not have obvious placental abnormalities [Carmeliet
et al., 1994], which raises some doubts about the
importance of this observation. However, it is possible
that humans and mice differ in their sensitivity or
requirements for regulation of this system.
Similarities in the expression of integrins by the
trophoblast cells of E7.5 mouse ectoplacental cone
outgrowths and by differentiated human trophoblast
cells that have invaded the uterine wall have also been
demonstrated [Salamat et al., 1993; Sutherland et al.,
1993]. Included in this complex integrin repertoire is
the laminin and collagen receptor, a1b1. The switch in
the integrin repertoire from a5b1 to a1b1, which is
associated with human cytotrophoblast differentiation
to a more mature phenotype, is not observed in pre-
eclamptic cytotrophoblasts [Zhou et al., 1993; Lim et al.,
1994]. Recent work with explants of human trophoblast
cells cultured in vitro under hypoxic conditions has
shown that they are inhibited from proper differentia-
tion and do not switch to a1 integrin expression [Gen-
bacev et al., 1996], similar to what has been observed in
preeclamptic trophoblasts in vivo [Zhou et al., 1993]. In
the mouse, it is not known if integrin switching occurs
during placentation, but both subunits of a1b1 have
been deleted, with the a1 mutant mice surviving to
birth [Gardner et al., 1996]. The b1 integrin null mice
exhibit inner cell mass failure and die soon after
implantation [Fassler and Meyer, 1995; Stephens et al.,
1995]. The relevance of this observation to its involve-
ment in placentation is unclear, not only because of the
time of embryo failure, but also because b1 is a subunit
for many other integrin complexes found at this time of
development [Fassler et al., 1996]. Although the infor-
mation obtained from targeted mutagenesis models is
incomplete, it is likely that some species-specific differ-
ences in integrin usage during placentation exist be-
cause of differences in trophoblast-maternal interac-
tions between mouse and human [Damsky et al., 1993].
A newly described transgenic mouse model replicates
some of the hallmarks of preeclampsia including the
maternal hypertension, kidney damage, and placental
abnormalities observed in human patients. The af-
fected mice overexpress the human angiotensinogen
molecule, but only develop the preeclamptic phenotype
upon mating with transgenic males overexpressing
human plasma renin. Both renin and angiotensin II,
which is derived from angiotensinogen, are elevated in
the plasma of preeclamptic mice similar to what is
observed in pregnant women with preeclampsia. Thus,
in mice, the paternally derived renin produced by the
placenta in combination with the maternally overex-
pressed angiotensinogen results in preeclampsia [Taki-
moto et al., 1996]. It is likely that future investigation of
the normal placentation process in the mouse will
continue to provide clues to mechanisms of aberrant
placentation in humans.
A growing number of mouse models, both induced
and genetic, are helping to identify proteins that are
required to coordinate embryo and maternal interac-
tions during peri-implantation development. While an
understanding of the implantation process should di-
rectly impact infertility therapy and reproductive medi-
cine, this information may also provide clues about
similar mechanisms that may be involved in tumor
development and metastasis. Research has just started
to identify the molecules involved in the implantation
process. It is likely that as more are discovered, a
mechanistic rather than a descriptive understanding of
this most remarkable process will be obtained.
ACKNOWLEDGMENTS
We thank Elizabeth Hassell for the photograph in
Figure 2, and Margaret Flannery for comments on
Figure 1. J.C.C. is a scholar of the Medical Research
Council of Canada.
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