# PPAR Signaling in Placental Development and Function

Authors: Yaacov Barak; Yoel Sadovsky; Tali Shalom-Barak
Journal: PPAR Research
 (2008)

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## Abstract

With the major attention to the pivotal roles of PPARs in diverse aspects of energy metabolism, the essential functions of PPARγ and PPARβ/δ in placental development came as a surprise and were often considered a nuisance en route to their genetic analysis. However, these findings provided an opportune entrée into placental biology. Genetic and pharmacological studies, primarily of knockout animal models and cell culture, uncovered networks of PPARγ and PPARδ, their heterodimeric RXR partners, associated transcriptional coactivators, and target genes, that regulate various aspects of placental development and function. These studies furnish both specific information about trophoblasts and the placenta and potential hints about the functions of PPARs in other tissues and cell types. They reveal that the remarkable versatility of PPARs extends beyond the orchestration of metabolism to the regulation of cellular differentiation, tissue development, and trophoblast-specific functions. This information and its implications are the subject of this review.


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## Body

## 1. Introduction

Mammalian
reproduction entails prolonged gestation, posing the challenge of securing the
thrift and long-term survival of the fetus in utero. The evolutionary answer to this challenge has been the
emergence of the placenta, whose roles are to facilitate efficient nutrient,
gas and waste exchange between the mother and fetus, while conferring immune
privilege on the embryo and secreting pregnancy hormones. The placental core
comprises a dense vascular array, where maternal and fetal circulations run in
close proximity, but are strictly separated by a trophoblast barrier that
specializes in essential bidirectional metabolite transport into and out of the
fetus. Placental dysfunction is associated with common disorders of pregnancy,
including spontaneous abortions, intrauterine growth restriction (IUGR), and
preeclampsia, all of which are commonly associated with compromised placental
vasculature [1–3]. In the mouse,
dozens of targeted gene mutations result in placental defects that underlie
stunted growth or midgestation lethality (reviewed in [4, 5]). Proof of direct
causative relationship between such defects and the lethal outcome comes from
the complete rescue of embryos by selective reconstitution of the trophoblast
in several knockout mouse strains [6–12].Among
the genes whose deficiency results in lethal placental defects are PPARγ
and PPARδ; the two are
closely related, yet functionally distinct members of the nuclear hormone
receptor superfamily of ligand-activated transcription factors. Obligate
heterodimers of PPARs and retinoid X receptors (RXRs) bind to PPAR-response
elements (PPREs) in the cis-regulatory regions of target genes and activate
transcription in response to small lipophilic ligands. While the identities of
endogenous PPAR ligands are still inconclusive, pharmaceutical development has
yielded several high-affinity synthetic agonists that are widely used in both
the clinic and the lab. Importantly, notwithstanding the primary focus of the
PPAR field on cellular and systemic metabolism, PPARs and their associated
regulators play at least equally essential roles in placental development and
function, as reviewed below.


## 1.1. Placental Development and Trophoblast Differentiation

The
deepest insights into the functions of PPARs in the placenta have been provided
by mouse genetic studies. This succinct overview and the accompanying Figure1
aim at providing the framework for these studies by summarizing placental
development in mice. One should bear in mind that while basic principles and
molecular regulation of placental development and function are similar across
mammals, morphological patterning and architecture of the placenta, and hence
terminology, vary considerably among species.Figure 1
Trophoblast lineages in the developing mouse placenta. Shown from left to right are a blastocyst (E3.5), an E6.5 embryo, and an E9.5 embryo. Respective trophoblast lineages are traced for clarity. Al: allantois; Ch: chrion; CP: chorionic plate; De: decidua; Emb: embryo; EPC: ectoplacental cone;1°GC: primary giant cells; 2°GC: secondary giant cells; ICM: inner cell mass; La: labyrinth; Sp: spongiotrophoblast; TE: trophectoderm. FGF4: fibroblast growth factor 4 secreted by the embryo to maintain the chorion. Blastocyst and E6.5 embryo picture courtesy of Drs. Mimi DeVries and Tom Gridley, respectively, The Jackson Laboratory.With
the exception of the percolating maternal blood, the placenta is exclusively an
embryonic tissue. The juxtaposed decidua is a maternal tissue formed from
endometrial lining of the uterus. The placenta is comprised of trophoblast
cells that originate from the trophectoderm layer of the blastocyst (Figure1).
Implantation of the embryo into the uterine wall triggers the expansion and initial
differentiation of trophectoderm cells to form both the chorion and, by process
of endoreduplication, primary giant cells. These giant cells facilitate uterine
invasion by the embryo. The chorion harbors trophoblast stem cells and, in the
mouse, gives rise to the ectoplacental cone (EPC). After initial expansion, the
EPC yields the spongiotrophoblast layer and secondary giant cells (Figure 1).
Giant cells separate the placenta from the maternal decidua and are responsible
both for maintaining the tight placenta-decidua interface and for executing
various endocrine functions, including secretion of steroid and prolactin
family pregnancy hormones. Spongiotrophoblasts perform (a) endocrine functions
by secreting pregnancy specific glycoproteins (PSGs) and prolactin-related
hormones, (b) metabolic functions, such as glycogen storage and production of
IGF2, and (c) presumed mechanical support functions. Syncytiotrophoblasts that
comprise the hemochorial trophoblast barrier between maternal and embryonic circulations
(the labyrinthine layer in mice; floating chorionic villi in humans) originate
directly from the chorion. In the mouse, vascularization of the placenta
initiates around E8.5, when the allantois, which harbors the future umbilical
blood vessels, attaches to the chorionic plate. Subsequently, the
chorioallantois invaginates into  the placenta and lays the vascular framework
of the labyrinth. Concomitantly, chorionic trophoblasts in the labyrinth differentiate three morphologically and functionally
distinct single cell layers that form a highly specialized epithelial barrier,
which  execute all bidirectional transport functions between the mother and the
fetus. Insights from mouse mutants demonstrate that  formations of
the labyrinthine trophoblast and
placental vascularization are highly concordant and involve extensive cellular
and molecular interactions between the allantoic endothelium and the
trophoblast [4]. The trophoblast is crucial for placental vascularization, as
evident from the complete correction of diverse placental vascular defects by
trophoblast-selective rescue [8–12]. In turn, multiple signaling factors
secreted by the embryonic endothelium, such as HGF, EGF, LIF, PDGFB, and WNT-2,
are essential for proper formation of the labyrinth [13–20].Cell culture
studies have facilitated the mechanistic understanding of molecular and
cellular processes involved in various aspects of trophoblast differentiation
and function. This area has been markedly advanced by the successful
establishment of protocols for procuring and manipulating trophoblast stem (TS)
cells from blastocysts or the EPC [21]. The stem cell status of TS cells can be
maintained by FGF4 and embryonic fibroblast-derived factors, possibly related
to TGFβ or activin [21, 22]. When FGF and conditioned media are withdrawn from the culture medium,
mimicking the growing distance between distal trophoblast layers and the
embryonic FGF4 source, TS cells differentiate spontaneously, primarily into giant cells and to some
extent also into spongiotrophoblast and multinucleated syncytial cells [21, 23]. Moreover, when reintroduced into
blastocysts, TS cells are able to differentiate into all trophoblast derivatives [21], demonstrating
their true stem cell nature.


## 2. PPARγ

In the absence of prior evidence that PPARγ is expressed
during early embryogenesis, the death of Pparg-null
embryos at the 10th day of gestation (E10.0) was initially surprising [12].
However, further inquiry revealed that Pparg is expressed abundantly in the placenta from E8.5 onward, and is not detected
in any other embryonic tissue until at least E13.5 (12). This expression
pattern provided circumstantial evidence that PPARγ may function in
the placenta, but the survival of tetraploid chimeras provided the definitive
proof that placental PPARγ deficiency was the cause of embryonic
lethality [12]. Tetraploid chimeras are generated by electrofusing 2-cell
embryos into single cells with tetraploid genomes. Such embryos resume development,
and their aggregation with diploid morulas or embryonic stem cells gives rise
to chimeras whose embryo derives exclusively from the diploid partner while
their placentas derive from the tetraploid partners [24]. When used to
reconstitute diploid Pparg-null
embryos with WT tetraploid
placentas, this procedure allowed survival of the mutant embryos until birth,
when they succumbed to unrelated defects that included severe cerebral and
intestinal hemorrhages [12]. The recent availability of epiblast-specific Cre
transgenes, which delete loxP-flanked (floxed) alleles efficiently in the
embryo but not extraembryonic tissue, has enabled to reprove this notion by
demonstrating that near-complete deficiency of Pparg in the embryo proper is not embryonic lethal [25, 26].


## 2.1. PPARγ and Trophoblast Differentiation

The complex histological and ultrastructural phenotype ofPparg-null placentas (Figure 2) provided insights into the
essential functions of PPARγ. Expression and
spatial distribution of prototypic trophoblast lineage markers are intact in
the mutant placentas, including the giant cell layer, the spongiotrophoblast,
the labyrinth, and the chorion [12]. However, labyrinthine trophoblast
precursors fail to terminally undergo differentiate, and instead, retain parenchymal morphology without
undergoing either compaction or syncytium formation [12]. The basement membrane
between the trophoblast and fetal endothelium is severely disrupted, loosening
the critical tight association between the two cell types [12]. This defect likely
hampers both the flow of metabolites from the trophoblast to the embryo and the
ability of embryonic vessels to use basement membrane tracks for extending and
branching into the labyrinth. Consequently, fetal vessels do not permeate the Pparg-null placenta and the labyrinthine
layer does not effectively form [12]. The trophoblast-lined maternal blood
pools are dilated and ruptured, leading to hemorrhages, fibrin deposition, and
overt phagocytosis of maternal erythrocytes by junctional zone trophoblasts [12].
Together, these observations indicate that while PPARγ
is dispensable for partition of trophoblasts to different lineages, it is
essential for terminal differentiation of labyrinthine syncytiotrophoblasts and
spongiotrophoblasts, and in turn for placental vascularization and integrity.
The further increase of Pparg levels
in the labyrinth during late gestation suggests that beyond its role in
establishing the vascular network of the placenta it may also play an important
role in its elaboration and maintenance [27].Schematic representation of thePparg-null phenotype. (a) WT placenta. Al: allantois; Ar: maternal artery; Ch: chorion; De: decidua; FV: fetal blood vessels; La: labyrinth; MBP: maternal blood pools; Sp: spongiotrophoblast; TGC: trophoblast giant cells. (b) Pparg-null placenta. Corresponding structures are as in (a). Differences of note are marked erythrophagocytosis by spongiotrophoblast cells (red speckles), absence of fetal vessels and breakdown of the maternal blood pools in the labyrinth, and thickening of the chorion. (c,d) Ultrastructural features of WT and Pparg-null hemochorial barriers (based on [12]). See legend in (c) for identity of major features. Differences include thickening of the three trophoblast layers, near elimination of lipid droplets in layer III, and loosening of the tight adherence between the trophoblast (green) and fetal endothelium (orange).
(a)(b)(c)(d)On
the opposite  pole of the PPARγ spectrum, feeding
pregnant mice a high dose of the PPARγ agonist
rosiglitazone (rosi) from mid to late gestation elicited severe thinning of the
spongiotrophoblast layer and substantial dilation of the maternal blood pools
in WT placentas [28]. Pparg+/- placentas were
protected from these effects, indicating that these are indeed the result of
excessive PPARγ activity. Reduced
expression of the trophoblast stem cell marker Eomes in rosi-treated WT placentas [28] suggested that excessive PPARγ
activity might cause these effects by accelerating stem cell differentiation,
concomitantly depleting the stem cell pool and destabilizing the balance
between differentiated
trophoblast cell types in the placenta. Warnings about embryonic toxicity in
rats in the inserts of two commonly prescribed PPARγ
agonists, Avandia (rosi) and Actos (pioglitazone), may reflect similar
phenomena. In contrast, short-term administration of acute doses of rosi to
pregnant rats during midgestation or chronic exposure of pregnant mice to
moderate doses of rosi was harmless [29, 30], as were anecdotal incidents in
which pregnant women were accidentally exposed to the drug [31, 32].The
functions of PPARγ in trophoblast
differentiation have been simulated in several in vitro systems. For example, stimulation of primary human term
trophoblasts by PPARγ agonists enhanced
their differentiation into multinucleated syncytiotrophoblasts, in agreement
with the critical role of PPARγ in syncytium
formation in the mouse labyrinth [33]. In TS cells, the association of PPARγ
with trophoblast differentiation is manifested in its dramatic induction during
transition from the undifferentiated to the differentiated state [34]. This
pattern demonstrates that PPARγ is integral to the
process of trophoblast differentiation and pinpoints TS cells as an ideal
platform for studying the placental functions of PPARγ.
On this front, we recently established Pparg-null
TS cell lines, whose analysis is currently underway [35].


## 2.2. PPARγ and Trophoblast Metabolism

The established roles of PPARγ in systemic and
cellular energy metabolism and the importance of trophoblast metabolism for
embryonic development raised the plausible hypothesis that PPARγ
might regulate metabolic functions of trophoblasts. This idea was strongly
supported by the near-complete absence of lipid droplets from the fetal
vessel-proximal trophoblast layer of Pparg-null placentas as opposed to their WT counterparts, in which these
droplets are abundant [12]. Moreover, PPARγ and RXR agonists
synergistically stimulate lipid uptake in both cultured trophoblasts in vitro and whole placentas in vivo [28, 36]. These processes are
associated with the upregulation of CD36, FABPpm, fatty acid transport proteins
1 and 4 (Fatp1, Fatp4), and the lipid
droplet proteins adipophilin, S3-12, and MLDP [28, 36]. Thus, PPARγ
is an important regulator of lipid dynamics in trophoblasts.Hypoxia of
trophoblasts due to hypoperfusion of the placental bed is a common complication
in human pregnancy. Interestingly, agonist-mediated stimulation of PPARγ protects
trophoblasts from an acute, but not a long-term apoptotic response to
hypoxia [37]. Potential mechanisms  underlying this protective effect include
PPARγ-dependent
differentiation of cytotrophoblasts to syncytiotrophoblasts, which are more
resistant to hypoxic death, or direct inhibition of apoptotic pathways by PPARγ.


## 2.3. Other PPARγ Functions in Trophoblasts

In  addition to the
role of PPARγ in trophoblast
differentiation and metabolism, it appears to contribute to specialized
functions of trophoblasts. One of these unique functions is invasion of the
endometrium. The strong coexpression of PPARγ and its obligatory RXRα partner in
extravillous cytotrophoblasts at the maternal-fetal interface of human embryos
suggested that PPARγ might regulate the invasive functions
of trophoblasts. The ability of PPARγ and RXR agonists to inhibit matrigel
invasion by both primary and transformed trophoblasts, and the enhancement of
invasion by PPARγ and RXR
antagonists, supported this hypothesis and implicated PPARγ as a negative regulator
of the process [38, 39]. This activity has been correlated to a 3-fold decrease
in the expression of pregnancy-associated plasma protein A (PAPP-A)—a protease essential for maturation of the
pro-invasive IGF2—and to a 3-fold induction of Interleukin-1β [40].Another critical
function of trophoblasts is the secretion of reproductive hormones, such as
placental lactogens (PL) and choriogonadotropin (hCG). Studies in primary human
trophoblasts showed that PPARγ and RXR agonists stimulate hCG and hPL
production, and that  PPARγ-RXRα heterodimers  directly activate hCGβvia a PPAR-response element (PPRE) in
its promoter [33, 38]. These findings suggest that PPARγ functions extend
to trophoblast-specific processes beyond cell differentiation, metabolism, and
motility.


## 2.4. Placental PPARγ Target Genes

PPARs are
transcription factors, and as such, their raison d’être is to regulate the
expression of target genes. Identification of these targets is therefore
fundamental for determining the biological functions of PPARs. Two primary
philosophies underlie target gene identification. The first is a candidate gene
approach, which involves hypothesis-driven testing of genes that make plausible
targets based either on their established regulation by PPARs in other tissues
or on their known relationship to PPAR-regulated processes; trophoblast targets
of PPARs found via this
approach are described throughout this review in relation to their biological
context. The second approach is discovery-based, and involves unbiased,
transcriptome-wide screening for target genes based on genetic, pharmacological,
and biochemical criteria. The strength of this strategy lies in its ability to
break ground and identify targets whose regulation by PPARs would not be
otherwise hypothesized.The identification ofMuc1 as
a PPARγ
target gene in trophoblasts by  subtraction of cDNA from WT versus
Pparg-null placentas has proven the power of the
latter approach to unearth unexpected targets [34]. Muc1 is very
tightly regulated by PPARγ,
and its expression is lost in both Pparg-null and
Rxra-null placentas and is upregulated by
PPARγ
agonists in both differentiated TS cells and
whole WT placentas [28, 34]. The Muc1 protein localizes to
apical labyrinthine trophoblasts surrounding maternal blood pools, analogous to
its luminal localization on simple secretory epithelia, such as those that abut
milk or salivary ducts [34]. This spatial pattern invokes unanticipated
anatomical and functional analogies between trophoblasts and prototypic luminal
epithelia, raising the provocative idea that some of the placental functions of
PPARγ are a carryover
from more ancient functions in classical epithelia. However, unlike Pparg, Muc1 is not essential for placental development and its deficiency
leads at worst to a mild dilation of the maternal blood pools in the labyrinth [34].
This benign phenotype indicates that other target genes must account for the
essential placental functions of PPARγ. Our ongoing microarray-based screens
start to uncover new PPARγ targets that may account for these
functions [35].In addition to
their prospect in illuminating PPAR functions, new target genes provide novel
templates for studying the details of native gene regulation by PPARs. Our
studies of theMuc1 promoter provide
an excellent example for the unique insights that such an approach can provide
over the study of synthetic promoters or isolated response elements. A proximal Muc1 promoter fragment responds
robustly and in an RXRα-dependent manner to PPARγ and rosi, yet
unlike most previously studied PPAR targets, let alone synthetic ones, is
entirely refractory to PPARα and PPARδ [34]. Detailed mutation analyses
reveal a weak PPRE in the proximal part of the Muc1 promoter that acts as a basal silencer, and whose derepression
by PPARγ is required for
robust and specific induction of Muc1 by an upstream, non-PPAR-binding enhancer [34]. This level of detail reveals
previously unappreciated layers of specificity and intricacy underlying the
regulation of real-life targets by PPARγ.


## 2.5. PPARγ and the Placenta-Heart Axis

Analysis ofPparg-null
embryos unexpectedly found accelerated cardiomyocyte differentiation and thinning of the
ventricular wall [12, 41]. This observation was intriguing because at that
developmental stage Pparg is
expressed nowhere but in the placenta. Consistent with this expression pattern,
complete reversal of the cardiac defects in Pparg-null
tetraploid chimeras confirmed that these anomalies are secondary to the
placental defects [12]. This result invoked a previously unappreciated
dependence of early heart development on placental integrity [12]. How placental Pparg deficiency underlies cardiac
malformation is currently unclear and could involve generalized nutritional,
vascular, or metabolic deficiencies, hypoxia, or a deficiency for
placenta-derived factors. However, similar cardiac defects are often observed
in association with placental anomalies (reviewed in [42]), and the
“placenta-heart axis” has been since reinforced in p38a-null embryos, which phenocopy the Pparg-null placental and cardiac defects and are similarly rescued
by tetraploid chimeras [11]. Therefore, myocardial failure is likely a general
attribute of placental insufficiency and not a specific consequence of PPARγ mutation.


## 3. PPARδ

As in the case of
PPARγ, the finding that Ppard-null embryos succumb to lethal placental defects was also
unexpected [43, 44]. The first Ppard-null
mouse strain reported was generated by truncating the gene a mere 60 amino
acids from its C-terminus (Ppard-ΔC60), leaving the
entire DNA-binding domain and most of the ligand-binding domain intact [45].
While this allele is likely a hypomorph, the authors reported significantly
smaller size and lower survival rates of the original F2 homozygotes for this
allele, which they have overcome by outbreeding and consecutive mating of the
survivors  [45]. In contrast, mice in
which PPARδ was inactivated by CRE/loxP-mediated truncation of the
N-terminal half of the DNA-binding domain and frame-shifting of the remaining
3’ part of Ppard mRNA exhibited
overwhelming embryonic lethality and placental defects, as detailed in Section
3.1 [43]. Nevertheless, a few homozygous-null mice survived gestation thanks to
a complex influence of genetics and maternal physiology (see Section 3.2). Two
other null configurations, one with lacZ insertion into the DNA-binding domain of PPARδ [46, 47] and
another that replaced the DNA-binding domain with PGK-neo [44], yielded
identical lethality and placental defects, confirming that PPARδ is indeed essential
for placental function.


## 3.1. PPARδ in Placental Development and Integrity

Lethality and
sub-Mendelian ratios ofPpard-null
embryos are observed from E9.5–10.5 onward. Rare null embryos surviving beyond
that stage typically exhibit severe flooding of maternal blood into the
placental and embryionic space and are significantly smaller than their WT and heterozygous siblings and the few
that survive to birth are markedly runt [43, 44]. Still, none dies after birth
and all thrive and become generally healthy and fertile adults, despite
remaining slightly smaller than their Ppard sufficient counterparts [43]. The combination of strictly prenatal mortality,
growth restriction, and abundant expression of Ppard in the placenta points to critical defects in extraembryonic
tissue.From as early as E8.5 onward,Ppard-null embryos and placentas are significantly smaller than
their littermates [43, 44]. All placental compartments are smaller, including
the labyrinth, the spongiotrophoblast, and the giant cell layer. The latter is
severely thinner and discontinuous, with cells that do not attain the maximal
size typical of WT
giant cells (43,
44). This compromise in giant cell size and continuity likely underlies the
observed loosening of the normally tight placenta-decidua interface and the
inability to retrieve Ppard-null specimens
from E9.5 onward without substantial detachment of placentas from the deciduas [43].
In contrast, while the labyrinth is smaller, its vascular structure is fully
elaborated, clearly distinguishing the Ppard-null
from the Pparg-null placental phenotype
[43]. These features are summarized schematically in Figure 3.Schematic representation of thePpard-null phenotype. (a) WT placenta (similar to Figure 2(a)). (b) Ppard-null placenta. Hr: hemorrhage; for all other abbreviations see the legend for Figure 2. Notable differences include smaller and discontinuous giant cells, reduced size of the entire placenta and loosening of its attachment to the decidua, and sporadic severe hemorrhages at various locations in or around the placenta.
(a)(b)Consistent with
the implicated role of PPARδ in giant cell differentiationin
vivo, studies of the trophoblast cell line Rcho-1 have unequivocally
demonstrated that PPARδ is crucial for giant cell
differentiation in vitro [44].
Agonist-mediated stimulation of PPARδ dramatically
accelerated differentiation of Rcho-1 cells into giant cells, whereas
siRNA-mediated knockdown of PPARδ severely inhibited the process. PPARδ was necessary and
sufficient for suppression of Id-2, which inhibits giant cell differentiation,
and for upregulation of I-mfa, which promotes giant cell differentiation by
antagonizing the bHLH transcription factor Mash-2. Interestingly, in
trophoblasts, just like in keratinocytes, PPARδ upregulates the
expression of two key nodes in the PI3 kinase (PI3K)/Akt signaling pathway: PDK1
and ILK. These, in turn, activate Akt by phosphorylating two residues: Thr308
and Ser473. Activation of this pathway is critical for the ability of PPARδ to accelerate
giant cell differentiation, and a synthetic PI3K inhibitor completely reversed upregulation of PL-1, downregulation of Id-2, and giant cell formation.
However, additional pathways are at play downstream of PPARδ, as evident in
the insensitivity to PI3K inhibition of PPARδ-dependent I-mfa
activation.


## 3.2. Genetic and Maternal Modifications of  the Ppard-Null Phenotype

Surprisingly, allPpard deficient alleles exhibit highly variable penetrance
of both the placental phenotype and lethality itself. Our early studies of Ppard-null mice encountered a clear
maternal effect on the fate of Ppard-null
embryos. These studies were carried out on either a pure 129/SvJae 129 background or a segregating F2, F3,
and F4-C57BL/6J [B6]: 129 background, in which the vast
majority of homozygous null embryos die during gestation [43]. However, 2–5% of 129-Ppard-null mice and 10–15% of B6: 129-Ppard-null
mice survived to parturition. These rare survival events were not randomly
distributed. First, litters with multiple null pups (up to 4 in one litter)
were frequently observed [43, 47]. Second, all survival cases occurred in
first-time pregnancies, none recurring in the same breeding pair. Third,
survival was not heritable in these cases, that is, null mice were fully fertile, but never gave birth to Ppard-null progeny when crossed with Ppard+/- or Ppard-/- mates. This
substantial deviation from random distribution suggested that survival on these
genetic backgrounds is modified primarily by maternal conditions rather than
genetics. A hypothetical example of such conditions is slow immune attack of
first-time mothers on embryos with breached immune privilege.Notwithstanding
maternal effects, thePpard-null
phenotype is also clearly subject to genetic modification. Peters et al.
alluded to poor survival of the initial batch of homozygous Ppard-ΔC60
mice and the complete resolution of this problem by an additional backcross of
F1 mice with inbred C57BL/6N mates, which yielded normal Mendelian distribution
of the progeny starting at F3 [45]. Similarly, Nadra et al. reported very low
survival rates of outbred B6:129-Ppard-null mice, which was eventually
overcome by intercrossing rare surviving mutants [44]. Our work in progress
sheds further light on the effects of genetic modifiers on the Ppard-null phenotype. First, repetitive
backcrosses onto B6 completely
obliterates survival of mutants beyond E9.5, indicating that 129-specific alleles allow mutants to
survive 1-2 days longer than B6 alleles
and are more permissive towards the survival of Ppard-null embryos to term [47]. Second, when B6:Ppard+/- mice are backcrossed onto an FVB/NJ (FVB) background, intercrosses of the heterozygous F1 generation
result in survival of ∼15% of the expected Ppard-null
progeny [47]. On this background, survival of F2 FVB:B6-Ppard-null mice is
evenly distributed and not limited to first time pregnancies. Thus, FVB alleles are permissive for survival
of Ppard-null embryos, yet in a
substantially different way than the 129 or B6:129 backgrounds. Third, survival
of FVB:B6 Ppard-null embryos is
heritable, and multigenerational intercrosses of F2-FVB:B6-Ppard-null parent
pairs and their progeny led to the establishment of a semistable stock of
viable Ppard-null mice [47]. This
stock has reached a reproductive plateau by F4, and now consistently yields
survival of approximately 50% of the Ppard-null
progeny. Further inspection reveals that all progeny survive to E10.0, when
approximately half of the litter develops abnormal histological features at the
placenta-decidua interface and succumbs to transplacental infiltration of
maternal blood and fatal hemorrhaging and necrosis. In contrast, the placentas
of viable Ppard-null embryos from
this stock are broadly normal. At present, it is not clear whether this sharp
partition represents a stochastically incomplete penetrance or rather a
discrete genetic or epigenetic modifier that is inherited by only 50% of the
progeny.In
conclusion, placental PPARδ regulates essential
processes, which are highly interactive with the genetic and maternal
environments. Further studies of the Ppard-null
phenotype, its response to experimentally defined maternal variables, and
identification of genes that modify its nature and outcomes should yield new
insights into the biology of both PPARδ
and the placenta.


## 4. Transcriptional Partners of PPARs

The ability of
PPARs to bind DNA and activate transcription depends strictly on
heterodimerization with retinoid-X receptors (RXRs) [48]. In addition, diverse transcriptional coactivator proteins are
indispensable for transcriptional activation by PPAR-RXR heterodimers. These
interdependencies imply that both RXRs and relevant coactivators should be
essential for placental functions of PPARs and their deficiencies should yield
comparable phenotypes.


## 4.1. RXRs

RXRα
is the major RXR isoform in the placenta [49], and its deficiency is therefore
expected to recapitulate lethal placental defects of Pparg-null and Ppard-null
embryos. Indeed, Rxra-null placentas
exhibit multiple defects, some of which are similar to defects in Pparg-null placentas, including the
following: (a) incomplete compaction of labyrinthine trophoblasts, (b)
disruption of the basement membrane and the tight contact between labyrinthine
trophoblasts and infiltrating fetal endothelium, (c) a marked reduction in
lipid droplet content of labyrinthine trophoblasts, and (d) maternal hematomas
at the junctional zone [50]. Other defects, such as partial disorganization of
the labyrinthine zone, invasion of spongiotrophoblast cells into the labyrinth,
and reduced number of glycogen cells, are not an obvious extrapolation of
either the Pparg-null or the Ppard-null phenotype.Still,Rxra-null embryos die between E12.5
and E16.5 [51, 52], and the aforementioned placental anomalies are observed
later than the lethal endpoints of either PPAR deficiency. Therefore, these
defects can represent at best an incomplete knockdown of PPARγ
and δ  activities. This milder phenotype is
apparently rooted in functional redundancy with RXRβ,
as evident in the markedly accelerated and exacerbated Rxra/Rxrb double null
phenotype [53]. Rxra/b double null
embryos die at E9.5 while exhibiting a combination of failed placental
vascularization, which is a hallmark of Pparg deficiency, and severe placenta-decidua detachment, as in Ppard-null embryos. This phenotype suggests that although RXRα
is the primary PPAR partner in the placenta, RXRβ
provides a redundant, albeit incomplete backup for PPAR function in the
placenta.The
most conspicuous phenotype ofRxra-null
embryos is severe thinning and incomplete septation of the cardiac ventricles,
which is the likely cause of their death [51, 52]. This phenotype is non-cardiomyocyte-autonomous
[54] and has been successfully recapitulated by ablation of retinoic acid
signaling in the epicardium [55]. Consequently, its relationship to the
placental defects has never been investigated. Nevertheless, the proven dependence
of myocardial hypoplasia on placental defects in Pparg-null embryos raises the need to examine whether at least some
aspects of the cardiac Rxra-null
phenotype can be traced back to placental defects.


## 4.2. CoActivators

Among the large
array of cofactors that mediate transactivation functions of PPAR-RXR
heterodimers, two stand out in the context of placental functions:
PBP/DRIP205/TRAP220 (official gene name:Pparbp)
and PRIP/AIB3/RAP250 (official name: Ncoa6).
Three teams knocked out Pparbp and
found that homozygous null embryos die at E11.5 concomitant with growth
restriction and myocardial hypoplasia [56–58]. One team
described placental defects that included poor compaction of labyrinthine
trophoblasts, reduced vascularization, and phagocytosis of maternal
erythrocytes, recapitulating multiple histological and ultrastructural features
of Pparg-null placentas [56]. These
observations suggested that PPARBP coactivates essential developmental targets
of PPARγ-RXRα/β heterodimers in
the placenta, and the later lethality of these mutants suggested partial
redundancy with other coactivators. A second team saw no overt morphological
defects in Pparbp-null placentas, but
found that tetraploid chimeras postponed lethality of the mutants from E11.5 to
E13.5, proving that the homozygous-null embryos nevertheless die due to
placental defects [57]. Interestingly, tetraploid chimeras did not rescue the
cardiac defects of Pparbp-null mice,
demonstrating that these defects evolve irrespective of the placental problems,
unlike in the case of Pparg deficiency.Three teams of
investigators generated and analyzed differentNcoa6-null mouse strains that exhibited different grades of
phenotypic severity [59–61]. One team
targeted Ncoa6 by deleting exons 4
through 7 [59]. Homozygous-null embryos died around E10.0, preceded by
substantial growth restriction, severe myocardial thinning, and a series of
placental defects that closely resembled those of Pparg-null placentas. These included (a) failed vascularization of
the labyrinth, (b) poor compaction of syncytiotrophoblasts, (c) dilation and
rupture of the maternal blood pools, and (d) erythrophagocytosis in the
junctional zone. An additional placental phenotype not shared with Pparg-null placentas was thickening of
the giant cell layer alongside thinning of the spongiotrophoblast and the
labyrinthine zones [59]. These overall similarities indicated that Ncoa6 is critical for the essential
transcriptional functions of PPARγ and perhaps additional transcription
factors in the placenta and that Ncoa6 deficiency is not compensated for by genetic redundancy. The other two teams
interrupted the gene downstream of exon 6, and reported undetectable levels of Ncoa6 gene products, but a significantly
milder phenotype [60, 61], which suggested that both configurations are
functional hypomorphs. Homozygous-targeted embryos for these alleles died
around E13.5 and exhibited myocardial hypoplasia and placental defects that
included a thin spongiotrophoblast layer, ectopic spongiotrophoblasts within
the labyrinth, reduced vascularization of the labyrinth, and stasis and
necrosis in the junctional zone [60, 61]. Interestingly, these features are
highly reminiscent of the Rxra-null
phenotype, suggesting that they indeed reflect incomplete loss of Pparg function.While the
phenotypes ofNcoa6 and Pparbp-null mice pinpoint the two as
essential coactivators of PPARγ-RXRα/β transcription complexes in the
developing placenta, this is by no means the complete inventory of cofactors
that are crucial for placental functions of PPARs. First, no cofactor knockout
has so far yielded a Ppard-null-like
phenotype. Second, possible roles of cofactors that have not yielded clear
placental phenotypes cannot be ruled out. For example, mice deficient for
either CBP or p300 die during early gestation [62–64], and because
extraembryonic tissues were not carefully examined in these mutants, placental
defects are still a strong possibility. Another complication is presented by
families of homologous cofactors with a high potential for functional
redundancies, such as the p160 coactivators SRC-1, TIF2, and ACTR/SRC-3 or the
PGC-1 family, that is,  PGC-1α, PGC-1β, and PRC. While single deficiencies
for any of these cofactors are not embryonic lethal, therefore precluding
serious placental defects, one should keep in mind that compensation by
remaining family members may well be at play.


## 5. Conclusions and Prospects

As detailed in this review, PPARγ and PPARδ
play nonredundant roles in placental development and physiology. PPARγ
is a key regulator of trophoblast differentiation and metabolism, PPARδ
is essential for giant cell function and placental integrity, and their coreceptors
RXRα and β   are instrumental for the execution of these
functions. At least two transcriptional coactivators, PPARBP and NCOA6, are
critical for essential functions of PPARγ in the placenta, as
deduced from the Pparg-null-like
phenotype of their deficiencies, and additional cofactors are likely crucial
for those of PPARδ.Still,
the network of signals upstream, alongside, and downstream of PPARγ
and PPARδ is far from
elucidated. Several PPAR targets have been identified in trophoblasts,
providing initial mechanistic insights into PPAR function in the placenta. However,
the discovery of as many new target genes will be indispensable for fully
deciphering these functions. Another important effort should be to determine
the various regulators that control or modify PPAR expression and activity in
trophoblasts. These include, but are not limited to upstream transcriptional
regulators, molecules that control the stability of PPAR gene products, posttranslational
modifications that alter the functions of PPARs, RXRs, or their cofactors, and
the production and dissemination of endogenous ligands. Many of these processes
may constitute key regulatory nodes in placental physiology. In addition,
PPAR-specific features, such as the identity of genes that modify the outcomes
of PPARδ deficiency, would
provide invaluable insights.Finally,
identifying compelling similarities between thePpar-null placental phenotypes and published descriptions of
targeted genes with previously unknown connections presents a complementary
approach for identifying critical nodes in placental PPAR signaling. Such a
strategy has been widely successful in identifying a plethora of epistatic
relationships in lower eukaryotes such as yeast, nematodes, and flies, and more
recently in identifying novel SHH signaling components in mice [65]. Because
placental defects are among the earliest roadblocks in the development of many
gene-targeted embryos, such opportunities abound. For example, the published
analyses of single and compound keratin 8 (mK8), mK18, and mK19 knockouts reveal remarkable similarities to the Ppard-null placental phenotype [66–69]. Similarly,
the placental and cardiac phenotypes of αV- and β8-integrins,
p38α, JunB, and Fra1 knockouts are strikingly similar to those of Pparg-null embryos [9–11, 70, 71].
Integrating studies of these genes and their corresponding pathways into the
functional studies of PPARs and their regulators, associated factors, and
transcriptional targets should provide further insights into the mode by which PPAR
signaling networks regulate placental development.



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*Source: 142082-2007-11-21.xml*