Development of Structures and Transport Functions in the
Mouse Placenta
Erica D. Watson and James C. Cross
Physiology 20:180-193, 2005. doi:10.1152/physiol.00001.2005
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PHYSIOLOGY 20: 180–193, 2005; doi:10.1152/physiol.00001.2005
Development of Structures and Transport
Functions in the Mouse Placenta
The placenta is essential for sustaining the growth of the fetus during gestation, and
Erica D. Watson
and James C. Cross
Department of Biochemistry and Molecular Biology,
Faculty of Medicine, University of Calgary,
Calgary, Alberta, Canada
jcross@ucalgary.ca
defects in its function result in fetal growth restriction or, if more severe, fetal death.
Several molecular pathways have been identified that are essential for development
of the placenta, and mouse mutants offer new insights into the cell biology of placental development and physiology of nutrient transport.
Placental Development in Mice and
Humans
Although the gross architecture of the human and
mouse placentas differ somewhat in their details,
their overall structures and the molecular mechanisms underlying placental development are
thought to be quite similar (72). As a result, the
180
mouse is increasingly used as a model for studying
the essential elements of placental development.
In mice, placental development begins in the blastocyst at embryonic day (E) 3.5 when the trophectoderm layer is set aside from the inner cell mass
(FIGURE 1) (15). At the time of implantation (E4.5),
the mural trophectoderm cells, which are those not
in contact with the inner cell mass, become trophoblast giant cells that are analogous to human
extravillous cytotrophoblast cells (72). These cells
stop dividing, yet they continue to replicate DNA
(endoreduplication) to become polyploid. In contrast, two diploid cell types emerge from the polar
trophectoderm, which are those cells immediately
adjacent to the inner cell mass: the extraembryonic ectoderm and the ectoplacental cone (72).
Subsequently, the extraembryonic ectoderm will
develop into the trophoblast cells of the chorion
layer and, later, the labyrinth. While developing,
the labyrinth is supported structurally by an ectoplacental cone-derived layer called the spongiotrophoblast. It forms a compact layer of cells sandwiched between the labyrinth and the outer giant
cell layer and corresponds to the column cytotrophoblast of the human placenta (72). During later
gestation, glycogen trophoblast cells begin to differentiate within the spongiotrophoblast layer, and
subsequently they diffusely invade the uterine wall
(2).
The vascular portion of the placenta is derived
from extraembryonic mesoderm (allantois) that
extends from the posterior end of the embryo at
E8.0 (14). At E8.5, the allantois and the chorion join
together in a process called chorioallantoic attachment. Soon thereafter, the chorion begins to fold to
form the villi, creating a space into which the fetal
blood vessels grow from the allantois (14). At this
time, the chorionic trophoblast cells begin to differentiate into two labyrinth cell types.
Multinucleated syncytiotrophoblast cells, formed
by the fusion of trophoblast cells, surround the
fetal endothelium of the capillaries (see FIGURE 3).
A mononuclear trophoblast cell type lines the
maternal blood sinuses. Together the trophoblast
and fetal vasculature generate extensively
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Survival and growth of the fetus are critically
dependent on the placenta. It forms the interface
between the maternal and fetal circulation, facilitating metabolic and gas exchange as well as fetal
waste disposal. In addition, the placenta produces
hormones that alter maternal physiology during
pregnancy and forms a barrier against the maternal
immune system (14). In humans and rodents, the
fully developed placenta is composed of three
major layers: the outer maternal layer, which
includes decidual cells of the uterus as well as the
maternal vasculature that brings blood to/from the
implantation site; a middle “junctional” region,
which attaches the fetal placenta to the uterus and
contains fetoplacental (trophoblast) cells that
invade the uterine wall and maternal vessels; and
an inner layer, composed of highly branched villi
that are designed for efficient nutrient exchange
(72). The villi are bathed by maternal blood and are
composed of outer epithelial layers that are derived
from the trophoblast cell lineage and an inner core
of stromal cells and blood vessels.
Many targeted mutations in mice exemplify how
single gene mutations can affect placental development or function (Tables 1, 2, and 3). A common
feature among these placental mutants is the
reduced ability to transport nutrients, which
results in fetal growth restriction or, under more
serious circumstances, embryonic death. The vast
majority of the placental phenotypes that have
been described to date result in defects in the
establishment or maturation of the placental villi,
which in mice comprise the so-called labyrinth
layer. Most of the defects are structural in nature,
although some of the mutants offer insights into
the regulation of nutrient transport.
REVIEWS
branched villi of the labyrinth (comparable with
human chorionic villi), which become larger and
more extensively branched until birth (E18.5–19.5)
(2). Maternal and fetal blood flows in a countercurrent manner within the labyrinth to maximize
nutrient transport (2). If the labyrinth is not appropriately vascularized with suitable patterning,
branching, and dilation, placental perfusion is
impaired, resulting in poor oxygen and nutrient
diffusion (63).
Chorioallantoic Attachment
E3.5
E8.0
Trophectoderm
Blastocoel
Inner cell
mass
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The first step in labyrinth development is chorioallantoic attachment, and defects in this process are
among the most common causes of midgestation
embryonic lethality (72). The allantois and chorion
trophoblast cells are derived in parallel from distinct cell populations. Originating from the epiblast, the allantois is composed of extraembryonic
mesoderm (16). Many genes are necessary for
proper development of the allantois (Table 1).
However, the bone morphogenetic protein (BMP)
signaling pathway appears to be particularly
important. Critical molecules have been knocked
out in mice, including Bmp2, -4, -5, and -7 (20, 86,
104) as well as Smad1, a downstream effector of
BMP signaling (43). The mutants display mesodermal differentiation defects contributing to abnormal allantoic development. Additionally, the allantois of a Foxf1-deficient mouse embryo is small and
shows a loss of BMP4 expression (52), suggesting
that this transcription factor is upstream of BMP.
The blood vessels in the allantois arise de novo due
to vasculogenesis, and this is not dependent on
attachment of the allantois to the chorion (16).
The majority of chorionic cells are derived from
the extraembryonic ectoderm, although they overlie a thin layer of chorionic mesothelium (72). Both
Err2/Err␤, a nuclear hormone receptor (49), and
fibroblast growth factor receptor 2 (Fgfr2) (99) are
expressed within chorion trophoblast cells and are
required for their maintenance. Proper formation
of the chorion and allantois are necessary for
E8.5
EPC
Trophoblast
giant cells
Chorionic ectoderm
Allantois
Chorionic
mesothelium
Ectoplacental
cone (EPC)
Extraembryonic
ectoderm
E9.5
Maternal decidua
Allantois
Epiblast
E14.5
Extravascular
trophoblast
giant cells
Maternal
spiral arteries
Umbilical
cord
Maternal
decidua
E10.5
Glycogen
trophoblast cells
Spongiotrophoblast
Trophoblast
giant cells
Labyrinth
Spongiotrophoblast
Villi
Blood
vessels
Maternal
blood sinus
Labyrinth
(placental villi)
FIGURE 1. Placental development of the mouse
The origins of the extraembryonic lineages begin at embryonic day
(E) 3.5 with the formation of the blastocyst. At E8.0, chorioallantoic
attachment occurs, followed by branching morphogenesis of the
labyrinth to form dense villi, within which nutrients are exchanged
(E8.5–10.5). The mature placenta (E14.5) consists of three layers: the
labyrinth, the spongiotrophoblast, and the maternal decidua.
PHYSIOLOGY • Volume 20 • June 2005 • www.physiologyonline.org
181
REVIEWS
attachment to occur. In addition, however, many
mutants exist in which the allantois and the chorion appear to have formed normally, yet chorioallantoic attachment fails to occur (Table 1). It is
known that attachment is dependent on the cell
adhesion molecule VCAM1 (25, 42), which is
expressed on the allantois, and its ligand ␣4-integrin (102), which is expressed by the chorionic
mesothelium. However, not all Vcam1- or ␣4-integrin-deficient mice fail in chorioallantoic attachment, suggesting that other redundant adhesion
mechanisms are involved. Indeed, other mutants
with defects in chorioallantoic attachment also display incomplete penetrance (Table 1). It will be
necessary to look more closely at these mutant placentas to determine if this seemingly random col-
lection of genes shares a common molecular pathway, allowing for a better understanding of the
attachment process. Importantly, in the event that
chorioallantoic attachment does occur in these
incompletely penetrant mutants, they will often
exhibit later defects in morphogenesis of the
labyrinth.
Initiation of Branching
Morphogenesis at the
Chorioallantoic Interface
At E9.0, immediately after chorioallantoic fusion
occurs, primary villi begin to develop, evenly
spaced across the chorionic surface (14), and blood
vessels soon fill in the villous folds (72). The process
Gene
Gene Product
Expression in Placenta
Bone morphogenetic protein
Mesodermal derivatives
Bmp4 chimera
Bone morphogenetic protein
Allantoic mesoderm; trophoblast
Bmp5/Bmp7
Bone morphogenetic proteins
Allantoic mesoderm
brachyury (T)
T-box transcription factor
Allantoic mesoderm
Cdx2 chimera
Homeobox transcription factor
Mesodermal derivatives; trophoblast
Edd
E3 ubiquitin ligase
Not known
Foxf1
Forkhead transcription factor
Allantoic mesoderm
Lim1(Lhx1)
Lim domain transcription factor
Mesodermal derivatives
Smad1
BMP signaling intermediate
Mesodermal derivatives
Allantoic Development
Bmp2
Chorionic Development
Err␤ (Esrrb)
Nuclear hormone receptor
Chorionic trophoblast
Fgfr2 null
Fibroblast growth factor receptor
Trophoblast derivatives
Chorioallantoic Attachment
␣4 integrin (Itga4)
Adhesion molecule (VCAM1receptor)
Chorionic mesothelium
CtBP1/CtBP2
COOH-terminal binding proteins
(downstream of WNT and BMP signaling)
Chorionic trophoblast
CyclinF
Cell cycle regulator; stem cell factor
E3-ubiquitin ligase complex
Trophoblast
Cyr61 (Cnn1)
ECM protein (integrin ligand)
Trophoblast; allantoic mesoderm
Dnmt1
DNA methyltransferase
Not known
Grb2 hypomorph
Adaptor protein (MAPK pathway)
Trophoblast derivatives
Lpp3
Lipid phosphate phosphatase
(inhibitor of Wnt signaling)
Chorionic trophoblast; allantoic
mesoderm/endoderm
Mrj
Cochaperone
Chorionic trophoblast
Rbp-J␬
Transcription factor
(Notch signaling pathway)
Mesodermal derivatives
Tcf1/Lef-1
Transcription factors (downstream of
Wnt signaling)
Not known
Vcam1
Adhesion molecule (␣4 integrin
ligand)
Allantoic mesoderm
Wnt7b
Secreted signaling molecule
Chorionic trophoblast
Zfp36L1
Zinc finger protein (RNA transcript
destabilizer)
Allantoic mesoderm; chorionic
trophoblast
*Incomplete penetrance of chorioallantoic attachment defect
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Table 1. Mouse mutants that affect chorioallantoic attachment
REVIEWS
is often described as “vascular invasion” of the
chorion, but this is misleading because the process
requires active participation of chorion trophoblast
and allantoic mesoderm. The branchpoints are
actively selected by clusters of chorion trophoblast
cells that express the Gcm1 gene (4). As each
branch elongates, Gcm1 expression remains at the
distal tip and continues to be expressed as long as
villi are branching. Gcm1 expression also initiates
the differentiation of chorionic trophoblast into
syncytiotrophoblast (4). Embryos deficient for
Gcm1 do not initiate chorioallantoic branching;
their chorion layer remains flat, trophoblast cells
do not differentiate, and the fetal vasculature
remains restricted to the allantois.
Gcm1 mRNA expression is first detected in the
chorion before chorioallantoic attachment, and
therefore branchpoint selection appears to be independent of allantoic attachment (4). However, the
phenotypes of several mouse mutants have suggested that the initiation of morphogenesis after
selection has occurred may require the interaction
of chorion trophoblast and allantois. For example,
the expression of Gcm1 mRNA is not maintained in
Mrj mutant mice in which chorioallantoic attachment fails to occur (31) and, in the absence of allantoic mesoderm, chorion trophoblast cells remain
undifferentiated (29). In addition, mutations in various genes within the Notch signaling pathway,
including Notch1/Notch4 (39), the Notch receptor
Delta-like 4 (17), and transcription factors
Hey1/Hey2 (19) and Rbpsuh (38), all appear to result
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Placental Phenotype of Mutant Mouse
Reference
Allantoic failure*
104
Allantoic failure
20
Unknown defect of chorioallantoic attachment*
86
Allantoic failure
70
Allantoic failure
11
Allantoic failure*
74
Allantoic failure; loss of Bmp4 expression
52
Allantoic failure
80
Allantoic failure*; downregulation of Vcam1
43
Failure of chorioallantoic attachment; trophoblast self-renewal defect
49
Failure of chorioallantoic attachment; trophoblast self-renewal defect*
99
Failure of chorioallantoic attachment*
102
Unknown defect of chorioallantoic attachment*
30
Unknown defect of chorioallantoic attachment*
90
Unknown defect of chorioallantoic attachment*
56
Unknown defect of chorioallantoic attachment
45
Unknown defect of chorioallantoic attachment*
75
Failure of chorioallantoic attachment; chorionic trophoblast defect
18
Failure of chorioallantoic attachment
31
Unknown defect of chorioallantoic attachment
62
Unknown defect of chorioallantoic attachment
22
Failure of chorioallantoic attachment*
25, 42
Failure of chorioallantoic attachment; downregulationof ␣4 integrin
65
Unknown defect of chorioallantoic attachment*
88
PHYSIOLOGY • Volume 20 • June 2005 • www.physiologyonline.org
183
REVIEWS
in early blocks to chorioallantoic branching.
Expression of these genes has only been reported
within the allantoic mesoderm/blood vessels, suggesting that the fetal vasculature may be important
for initiation of branching of the chorioallantoic
interface. There are several caveats with this
hypothesis, however. First, it is possible that these
mutant mice are simply developmentally delayed
or slowed and not arrested at the flat chorion stage,
as with Gcm1 mutants. To address this possibility,
later-stage placentas should be examined, as has
been done with Grb2 (75). Second, Hey1 mRNA has
also been detected within the trophoblast cells of
the ectoplacental cone at least at E8.5 (K. Dawson
and J. C. Cross, unpublished data), and therefore
expression of the Notch signaling components is
Table 2. Mouse mutants that affect branching morphogenesis of the labyrinth
Gene
Gene Product
Expression in Placenta
Transcription factor
Chorionic plate; distal tip of branches
in labyrinth
␣-adrenoreceptors
2a/2b/2c
␣4 integrin (Itga4)
␣v integrin (Itgav)
Arnt (Hif-1␤)
␤8 integrin
Adrenaline receptors (MAPK pathway)
Trophoblast giant cells,
spongiotrophoblast
Branching Initiation
Gcm1
Branching Morphogenesis
Chorionic trophoblast
Transmembrane adhesion molecule
Trophoblast, allantoic mesoderm
bHLH/PAS transcription factor
Labyrinth trophoblast
Transmembrane receptor (adhesion
molecule)
Trophoblast giant cells
Bmp2
Bone morphogenetic protein
Mesodermal derivatives
Bmp5/Bmp7
Bone morphogenetic proteins
Allantoic mesoderm
Bruce
E2/E3 ubiquitin ligase
Chorionic and labyrinth trophoblast,
endothelial cells
C-EBP␣/C-EBP␤
Transcription factors
Chorionic plate
Cited1 (Msg1)
Transcriptional cofactor
Labyrinth trophoblast and
spongiotrophoblast
Chm
Choroideremia (MAPK pathway)
Ubiquitous
c-Met
Met tyrosine kinase (HGF receptor)
Not known
CtBP2
COOH-terminal binding protein
(downstream of WNT and BMP signaling
pathways)
Labyrinth trophoblast and fetal
blood vessels
Cx26 (Gjb2)
Connexin, gap junction protein
Labyrinth trophoblast
Cx31 (Gjb3)
Connexin, gap junction protein
Trophoblast derivatives
Cx45
Connexin, gap junction protein
Allantoic mesoderm
CyclinF
Cell cycle regulator; stem cell factor
E3-ubiquitin ligase complex
Trophoblast
Dlx3
Homeodomain transcription factor
Trophoblast derivatives
Edd
E3-ubiquitin ligase
Not known
Erk2
Extracellular signal-related kinase 2
(MAPK signaling pathway)
Labyrinth trophoblast
Erk5
Extracellular signal-related kinase 5
(MAPK signaling pathway)
Not known
Fzd5
Wnt receptor
Labyrinth trophoblast
Fgfr2 null
Fibroblast growth factor receptor
Trophoblast derivatives
Fra1
AP transcription factor
Trophoblast giant cells, yolk sac
Gab1
Gab/Dos adaptor protein family (MAPK
signaling pathway)
Labyrinth trophoblast
Grb2 hypomorph
Adaptor protein (MAPK pathway)
Trophoblast derivatives
Hgf (Sf)
Hepatocyte growth factor/scatter factor
(through c-Met receptor)
Not known
Hsp90b (Hsp84)
Heat shock protein
Labyrinth trophoblast and allantoic
mesoderm
Igf2 (P0)
Insulin-like growth factor II
Labyrinth trophoblast
Continued on next page
184
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Adhesion molecule (VCAM1 receptor)
s
REVIEWS
not restricted to allantois. Third, human chorionic
villi develop before becoming vascularized (10),
implying that vascular interactions are not important for villous development, at least in humans.
Given these findings, it is clear that more work
needs to be done to address the signaling interactions between chorion trophoblast and allantois
during early stages of villous development.
Signaling and Morphogenesis of the
Labyrinth
A large number of genes have been identified that
are required for labyrinth development (Table 2).
However, for most of the genes, the specific cellular
phenotype is not clear based on the published
studies. Indeed, the most accurate description is
Placental Phenotype of Mutant Mouse
Reference
4
Small labyrinth; low Erk1 and Erk2 expression
67
Small labyrinth
102
Small labyrinth
7
Small labyrinth; labyrinth trophoblast defect; decreased VEGF expression
3, 37
Small labyrinth
106
Not known
104
Not known
86
Labyrinth normal size, decreased branching
48
Small labyrinth; limited branching potential
9
Small labyrinth, enlarged spongiotrophoblast
71
Small labyrinth
81
Small labyrinth
53
Small labyrinth
30
Small labyrinth, defect in glucose transport
21
Small labyrinth; trophoblast proliferation defect
68
Small labyrinth
40
Small labyrinth
90
Small labyrinth
58
Not known
74
Small labyrinth
27
Small labyrinth
85, 100
Small labyrinth
33
Small labyrinth; trophoblast self-renewal defect
99
Small labyrinth
78
Small labyrinth
34, 73
Small labyrinth
75
Small labyrinth, fewer trophoblast cells
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No labyrinth; block in branching morphogenesis
76, 93
Small labyrinth; trophoblast differentiation defect
94
Small labyrinth, diffusional surface area decreased
13
PHYSIOLOGY • Volume 20 • June 2005 • www.physiologyonline.org
185
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Table 2., continued
Gene Product
Expression in Placenta
Junb
AP-1 transcription factor
Trophoblast derivatives
Keratin8/Keratin19
Intermediate filaments (cytoskeleton)
Trophoblast derivatives
Laminin ␣5
Noncollagenous glycoprotein
Vascular endothelial cells
Lbp-1a
Grainyhead transcription factor
Ubiquitous
Lifr
Leukemia inhibitory factor receptor
Trophoblast and mesodermal
derivatives
Lkb-1
Ser/Thr kinase
Labyrinth
Lpp3 chimera
Lipid phosphate phosphatase (inhibitor
of Wnt signaling)
Trophoblast; allantoic endoderm
and mesoderm
Mek1 (Map2k1)
ERK/MAPK kinase
Labyrinth trophoblast
Mekk3 (Map3k3)
MAP kinase cascade
Not known
Muc1
Downstream effector of PPAR-␥ pathway
Trophoblast cells surrounding
maternal blood spaces
Ncx1
Na+/Ca2+ exchanger
Trophoblast derivatives
Nodal hypomorph
TGF-␤ family secreted signaling molecule
Spongiotrophoblast
Nte
Neuropathy target esterase
Chorion trophoblast, EPC
p38␣ MAPK (Map2k2)
MAPK
Labyrinth trophoblast
Pbp
PPAR-␥ coactivator
Not known
Pdgfb
Platelet-derived growth factor chain B
Trophoblast, mesodermal derivatives
Pkb␣ (Akt1)
Protein kinase B-␣ (PPAR-␥ pathway)
Labyrinth
Plk2 (Snk)
Polo-like kinase (cell cycle regulator)
Not known
Ppar␥
PPAR-␥ transcription factor
Labyrinth trophoblast, EPC derivatives
Prip (Rap250/Aib3)
PPAR-␥-interacting protein
Not known
Raf1
Kinase in MAPK pathway
Not known
Rb
Retinoblastoma tumor suppressor
Throughout placenta, strongest in
labyrinth
RockII
Kinase in Rho signaling
Labyrinth trophoblast and umbilical
blood vessels
Rxr␣/Rxr␤
Retinoid nuclear receptors (dimerize
with PPAR-␥)
EPC and derivatives
Smad1
BMP signaling intermediate
Mesodermal derivatives
Sos1
Ras-specific exchange factor (MAPK
pathway)
Labyrinth and spongiotrophoblast
Tfeb
bHLH –Zip transcription factor
Labyrinth trophoblast
UbcM4
Ubiquitin-conjugating enzyme
Ubiquitous
Vcam1
Adhesion molecule (␣4 integrin ligand)
Allantoic mesoderm
Vhlh
Tumor suppressor
Trophoblast
Wnt2
Secreted glycoprotein
Allantoic mesoderm, chorionic plate,
fetal blood vessels
Zfp36L1
Zinc finger protein (RNA transcript
destabilizer)
Allantoic mesoderm; chorionic
trophoblast
bHLH, basic helix-loop-helix domain; PAS, Per-Arnt-Sim domain; HGF, hepatocyte growth factor; EPC, ectoplacental
that the labyrinth is simply underdeveloped or
“small,” meaning that the chorioallantoic interface
remains underbranched and as a result there is a
relative reduction in the density of fetal blood vessels (FIGURE 2). Some mutants exhibit defects early
in labyrinth development such that their chorionic
plates remain compact with little branching and
little fetal blood vessel growth (8, 9, 23, 30, 64, 101).
Embryos in this case will die between E10.5 and
E12.5. Many other labyrinth phenotypes manifest
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PHYSIOLOGY • Volume 20 • June 2005 • www.physiologyonline.org
slightly later, with some evidence of chorioallantoic branching but with thick trilaminar trophoblast
layers and/or reduced vascularization (6, 13, 34, 71,
73, 96). The associated fetuses die either late in gestation or perinatally. The cause of lethality in all
cases is a result of insufficient metabolic exchange.
Despite the uncertainty about the specific underlying cellular defects, an important general conclusion to emerge from the study of small-labyrinth
mutants is that labyrinth development depends on
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Gene
co
ental
Placental Phenotype of Mutant Mouse
Reference
Small labyrinth
77
Small labyrinth; vascular lesions
89
Small labyrinth, adhesion between vascular endothelial cells and trophoblast lost
55
Small labyrinth
64
Small labyrinth, vascular lesions
96
Small labyrinth
105
Small labyrinth
18
Small labyrinth
23
Small labyrinth
101
Small labyrinth, vascular lesions
79
Small labyrinth
95
Small labyrinth, large spongiotrophoblast and giant cell layers
50
Small labyrinth
59
Small labyrinth
1, 60
Small labyrinth
107
Small labyrinth
61
Small labyrinth
103
Small labyrinth
51
Small labyrinth, defect in trophoblast differentiation
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es
REVIEWS
8
Small labyrinth
5, 41, 108
Small labyrinth
32
Excessive trophoblast proliferation, decreased vascularization, defect in
essential fatty acid transport
98
Small labyrinth, vascular lesions
92
Small labyrinth, defect in trophoblast proliferation
97
Not known
43
Small labyrinth, low ERK activity
69
Small labyrinth, decreased vascularization
87
Small labyrinth
26
Small labyrinth
25, 42
Small labyrinth, decreased vascularization
24
Small labyrinth, vascular lesions
57
Small labyrinth
88
cone; PPAR, peroxisome proliferator-activating receptor.
a number of intercellular signaling pathways.
Specific pathways that are critical include Fgf (99),
Egf (91), Notch (39), Lif (96), Pdgfb (61), and Wnt
(57). Likewise, a number of signaling adaptor proteins downstream of these signaling events are
implicated given the similarity of their mutant phenotypes, including Chm (80), CtBP2 (30), Erk2 (27),
Erk5 (85, 100), Gab1 (34, 73), Grb2 (75), Mek1 (23),
Mekk3 (101), p38␣ MAPK (1, 60), and Sos1 (69).
Based on restricted patterns of expression or
chimera experiments, it is apparent that these signaling pathways are largely required in the trophoblast cell compartment of the labyrinth (Refs. 27
and 67; reviewed in Refs. 72, 81, 85, 92, and 100).
In addition to the protein signaling systems,
nuclear receptors are also important for morphogenesis of the labyrinth. The retinoid X receptor
(RXR) proteins dimerize with a number of different
nuclear receptors, including retinoic acid receptors
(RARs) and the perioxisome proliferator-activating
PHYSIOLOGY • Volume 20 • June 2005 • www.physiologyonline.org
187
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Wild type
Spongiotrophoblast
Esx1 mutant
Rb mutant
“Small-labyrinth” mutant
Villi
Labyrinth
Blood
vessels
Umbilical
cord
Trophoblast
giant cells
FIGURE 2. Fetoplacental vascularization defects in various mutant placentas
receptor (PPAR). RXR-␣/RXR-␤ double-mutant
mice die at midgestation and show a smalllabyrinth phenotype (97). PPAR-␥ mutants show a
similar phenotype, implying that perhaps PPAR-␥
is the critical dimerization partner of the RXRs for
labyrinth development (8). In support of this
hypothesis, mutations in genes encoding the
PPAR-␥-associated
proteins
PKB-␣
(103),
PRIP/Rap250/AIB3 (5, 41, 108), and PBP (107), as
well as the transcriptional target gene Muc1 (79), all
have been implicated in labyrinth development.
Direct and Indirect Controls on
Vascularization of the Labyrinth
When morphogenesis of the labyrinth is diminished, one of the most obvious differences is that
the layer remains cell dense and there are fewer
maternal and fetal blood spaces. However, in the
vast majority of labyrinth mutants the differences
are likely to be secondary effects, and there are only
a few examples of mutants with primary vascular
defects.
The maternal blood spaces in the labyrinth
(termed sinusoids) often appear to be larger than
normal in mutants (56, 71), but this can be an indirect effect. The maternal sinusoids within the
labyrinth are lined and shaped by trophoblast cells
and normally diminish in size as gestation proceeds as a simple consequence of the increasing
density of trophoblast villous branching (2).
Therefore, whenever chorioallantoic branching is
reduced, the maternal blood spaces in the presumptive labyrinth layer will remain larger. The
more critical question would be whether the overall maternal blood volume in the presumptive
labyrinth is altered in a mutant. This can be difficult to assess accurately in histological sections,
however, because the blood will readily leak out
during tissue dissection unless the uterine blood
vessels are ligated before dissection and tissue fixation (2).
The focus only on fetal blood spaces within the
labyrinth can give investigators a false impression
about the nature of the primary defect in the
labyrinth. For many of the genes whose mutant
phenotypes were originally described as “vascular”
in nature, they are expressed exclusively within the
trophoblast and not the vasculature itself (Table 2).
More importantly, since fetal vessels can only grow
Table 3. Mouse mutants that affect vascularization of the labyrinth
188
Gene
Gene Product
Expression in Placenta
Cyr61 (Cnn1)
ECM protein (integrin ligand)
Trophoblast; allantoic mesoderm/
endothelium
Dll4
Delta-like 4 (Notch ligand)
Umbilical and vitelline arteries
Esx1
Homeobox transcription factor
Chorion, labyrinth trophoblast
Hey1/Hey2
bHLH transcription factors (Notch
signaling pathway)
Mesodermal derivatives
Notch1/Notch4
Transmembrane receptors
Vascular endothelial cells
Rbpsuh
Transcription factor (Notch signaling
pathway)
Not known
PHYSIOLOGY • Volume 20 • June 2005 • www.physiologyonline.org
Downloaded from physiologyonline.physiology.org on January 21, 2007
Within a wild-type placenta, the labyrinth trophoblast forms extensively branched villi, creating a path for the fetal
vasculature to grow. Esx1 mutants appear to undergo normal branching morphogenesis, yet the fetal vasculature
does not develop normally. In contrast, Rb-deficient labyrinths exhibit defects in trophoblast proliferation and differentiation and thus have reduced branching and extension of the trophoblast villi. Accordingly, these placentas
attempt to compensate for reduced nutrient transport by increasing their fetal capillary density. Most other mutants
with a “small-labyrinth” phenotype have reduced branching morphogenesis and villi formation and an apparent
reduction in vascularization, yet detailed analyses have not been done to determine the nature of the labyrinth defect
and/or if compensation occurs.
REVIEWS
tal vasculature (66). Accordingly, one would expect
that the majority of mutant placentas with defective labyrinth morphogenesis might also compensate for reduced nutrient and gas exchange. The Rb
mutant placenta is a well-documented example of
this phenomenon. Rb-deficient labyrinths have
abnormal architecture associated with fewer villi
due to an inappropriate proliferation of trophoblast cells and block to differentiation (98). The
villous surface area of Rb mutant labyrinth trophoblast is reduced to 62% of wild type. However,
fetal capillary density is only reduced to 88% and
essential fatty acid transport, as a measure of nutrient uptake capacity, is 86% of wild type (98).
Therefore, the villi that are able to form in Rb
mutants are relatively hypervascularized, and this
is apparently able to partially compensate at least
for fatty acid transport (FIGURE 2). Proper detailed
analyses of other small-labyrinth mutants may
reveal similar compensatory measures.
Tissue oxygenation is a critical regulator of vascular development (54). The expression of arylhydrocarbon receptor nuclear translocator (Arnt),
also known as hypoxia inducible factor-1␤ (Hif-1␤),
in the labyrinth suggests that tissue oxygenation
may be a normal regulator of placental growth (37,
72). Arnt heterodimerizes with Hif-1␣ to mediate
transcription of specific genes, including VEGF, in
response to oxygen deprivation (3). Particular
attention was given to the Arnt knockout mouse
when a defect within the labyrinth resulted (37). It
was, however, demonstrated by tetraploid chimera
experiments that Arnt function is actually required
within the trophoblast compartment and not the
vascular endothelium (3). As a result, the vascularization defect that was described is secondary to a
primary trophoblast defect. Consequently, the
Arnt-deficient mutants do not stand apart from the
other small-labyrinth mutants (Table 2 and FIGURE
2).
Downloaded from physiologyonline.physiology.org on January 21, 2007
into the core of villi within the labyrinth, all smalllabyrinth phenotypes with fewer villi would also be
described as having fewer overall fetal blood
spaces. The more accurate way to assess these
mutants is to compare vascular density with the
density of differentiated villi to determine if the
reduction in fetal blood vessel space is simply proportional to reduction in villi (98).
There are perhaps only a few examples of mouse
mutants that show a reduction in the vasculature of
the labyrinth that is not proportional to the extent
of villous development (Table 3). The extracellular
matrix protein Cyr61 (56) and the Notch-signaling
components Dll4 (17), Notch1/4 (39), Hey1/2 (19),
and Rbpsuh (38) are expressed in the vasculature
itself, and mutations in their genes result in a poorly vascularized allantois. The Esx1 gene, by contrast, encodes a homeobox transcription factor that
is expressed solely in trophoblast cells of the
labyrinth (46, 47). Placentas from Esx1 mutants
appear to undergo normal chorioallantoic branching morphogenesis but have obvious deficiencies
in fetal blood vessel growth into the labyrinth villi
(FIGURE 2) (46). This indicates that trophoblast
cells are actively involved in the vascularization of
the labyrinth and suggests that a possible transcriptional target of Esx1 is a signal from the trophoblast cells that induces or directs vascular morphogenesis. The trophoblast-derived factor(s) that
directly influence the development of fetal blood
vessels in the labyrinth remain elusive.
An important factor to bear in mind when studying a placental phenotype is that the overall size
and extent of vascularization of the placenta can
change in an apparent attempt to compensate for
primary defects. Esx1 mutant placentas are actually larger than their wild-type counterparts, perhaps
suggesting an attempt to compensate for the
reduced vascularization and nutrient transport
(46). Placentas from mothers who smoke throughout their pregnancy are disproportionately large
(66). Impaired oxygen transport caused by an
increase in carbon monoxide concentration
induces additional angiogenesis of the fetoplacen-
Placental Nutrient Transport
Nutrients are transferred across the placental barri-
Placental Phenotype of Mutant Mouse
Reference
Small labyrinth, downregulation of VEGF
56
Small labyrinth, reduced fetal vasculature
17
Vascularization defects; labyrinth trophoblast differentiation defects
46, 47
Small labyrinth; reduced fetal vasculature
19
No labyrinth; no fetal blood vessels
39
Small labyrinth, reduced fetal vasculature
38
PHYSIOLOGY • Volume 20 • June 2005 • www.physiologyonline.org
189
REVIEWS
Mononuclear
trophoblast cell
Maternal red
blood cell
Maternal
blood sinus
Fetal blood
vessel
Syncytiotrophoblast
bilayer
Fetal red
blood cell
Fetal vascular
endothelium
II
Fetal
nucleated
red blood cell
III
I
Fetal capillary
Maternal red
blood cell
III
II
I
Cx26
Fetal
capillary
Glucose transport
Mononuclear
trophoblast cells
layer I
Left: the trilaminar trophoblast layer consists of a bilayer of syncytiotrophoblast cells (layers II and III) that surround the fetal blood vessel endothelium and a mononuclear layer of trophoblast cells (layer I) that lines maternal blood sinusoids. Nutrients such as glucose must be transported through
four cell layers to get from the maternal blood space into fetal blood vessels. Cx26 and GLUT1 have been shown to aid in the transport of glucose.
Right: an electron micrograph of the trilaminar layer of labyrinth trophoblast
cells that separate the maternal and fetal blood spaces.
er via several mechanisms, including passive diffusion, facilitated diffusion, and active transport (83).
A significant amount of solute flux across the
mouse placenta is achieved by passive diffusion
(84). Therefore, in addition to the overall surface
area and permeability (83), diffusional distance is a
major factor influencing overall diffusional capacity of the placenta. In mice, a trilaminar layer of trophoblast cells separates the fetal capillary from the
maternal sinusoids: a bilayer of syncytiotrophoblast surrounds the fetal blood vessel endothelium and a layer of mononuclear cells lines the
maternal blood sinuses (2). Consequently the
nutrients, gases, and waste must diffuse or be
transported across four layers to get from one
blood compartment to the next (FIGURE 3).
Measuring the surface area and thickness of the
trophoblast layers in the labyrinth by stereological
analysis is a relatively easy way to assess the diffusional ability of mutant placentas (12, 84, 98).
Alkaline phosphatase is expressed by trophoblast
cells that line maternal blood sinusoids within the
labyrinth (2). Accordingly, quantifying this expression is a simple way of assessing diffusional surface
area present in the placenta (98).
190
Syncytiotrophoblast
layer II
FIGURE 3. Interaction between trilaminar trophoblast layer and
blood spaces within the labyrinth
Maternal
blood
sinus
Syncytiotrophoblast Mononuclear
trophoblast cells
bilayer
Syncytiotrophoblast
layer III
PHYSIOLOGY • Volume 20 • June 2005 • www.physiologyonline.org
Placental-specific knockout of the insulin-like
growth factor-II gene (Igf2) results in both a thickened diffusional barrier and a smaller overall surface area in the labyrinth (13, 84). Nutrient transport capacity can be assessed either directly by
measuring transport of radiolabeled nutrients (13,
36, 84) or by measuring the accumulated content of
nutrients such as essential fatty acids in the fetus
(98), as has been done with the Igf2 and Rb
mutants, respectively.
Few nutrient transporters have been studied in
the mouse placenta, and even fewer mutants have
been reported that show reduced nutrient transport. Placental-specific mutation of Igf2, in addition to producing anatomic changes, results in
altered system A amino acid transporter function
(13). Active calcium transport across the placenta is
reduced in parathyroid hormone/parathyroid hormone-related peptide receptor mutant mice (36).
The gap junction protein connexin 26 (Cx26) has
been shown to act in cooperation with the glucose
transporter GLUT1 in the facilitated diffusion of
glucose across the trophoblast layers of the
labyrinth (21) (FIGURE 3). Cx26 mutants die at
E11.0 and show a 60% decrease in glucose trans-
Downloaded from physiologyonline.physiology.org on January 21, 2007
Cx26
GLUT 1
GLUT3?
Fetal vascular
endothelium
REVIEWS
port (21). Although this suggests that Cx26 is
required for glucose transport, the reduction in glucose transport may also be due to the fact that the
surface area of the labyrinth is also probably
reduced (21). GLUT1 mutants show haploinsufficiency in that heterozygous embryos die at the
blastocyst stage due to increased apoptosis (28).
GLUT3 is also expressed in the placenta (44), yet no
knockout mouse model has been generated to test
its importance in placental glucose transfer.
Conclusions
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Work in the past 10 years has resulted in an explosion of information about the regulation of placental development and function, in particular that of
the villous placenta that is involved in nutrient
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