Aberrant Growth and Pattern Formation in Peromyscus Hybrid

BIOLOGY OF REPRODUCTION 83, 988–996 (2010)
Published online before print 11 August 2010.
DOI 10.1095/biolreprod.110.085654
Aberrant Growth and Pattern Formation in Peromyscus Hybrid
Placental Development1
Amanda R. Duselis and Paul B. Vrana2
Department of Biological Chemistry, School of Medicine, University of California Irvine, Irvine, California
Crosses between the North American deer mouse species
Peromyscus maniculatus (BW) and P. polionotus (PO) produce
dramatic asymmetric developmental effects. BW females mated
to PO males (female bw 3 male po) produce viable growthretarded offspring. In contrast, PO females mated to BW males
(female PO 3 male BW) produce overgrown but dysmorphic
conceptuses. Most female PO 3 male BW offspring are dead by
midgestation; those surviving to later time points display
numerous defects reminiscent of several diseases. The hybrid
effects are particularly pronounced in the placenta. Here we
examine placental morphological defects via histology and in
situ hybridization as well as the relationship between growth
and mortality in the female PO 3 male BW cross. These assays
indicate altered hybrid fetal:placental ratios by the equivalent of
mouse (Mus) Embryonic Day (E) 13 and disorganization and
labyrinth defects in female PO 3 male BW placentas and confirm
earlier suggestions of a severely reduced junctional zone in the
female bw 3 male po hybrids. Further, we show that both
cellular proliferation and death are abnormal in the hybrids
through BrdU incorporation and TUNEL assays, respectively.
Together the data indicate that the origin of the effects is prior to
the equivalent of Mus E10. Finally, as the majority of these assays
had not previously been performed on Peromyscus, these studies
provide comparative data on wild-type placentation.
apoptosis, developmental biology, placenta, trophoblast
INTRODUCTION
The placenta and associated extraembryonic tissues perform
multiple functions during mammalian prenatal development [1,
2]. In addition to mediating maternal-fetal nutrient/waste
transfer and immune relationships, these tissues are major
endocrine organs and contribute to early pattern formation and
hematopoiesis [3]. Thus, perturbations of placental function
may result in fetal and/or maternal health consequences [4–6].
However, there are few mammalian models for studying the
potential interactions of natural genetic variants to produce
developmental dysplasias. For example, the genomes of
commonly used rodent strains do not represent naturally
occurring allelic combinations and have been affected by
inbreeding [7, 8]. Similarly, other domesticated mammals are
1
Supported by NSF #MCB-0517754 and NIH grant #P40 RR014279.
Correspondence and current address: Paul B. Vrana, Peromyscus
Genetic Stock Center, Department of Biological Sciences, University of
South Carolina, Columbia, SC 29208. FAX: 803 576 5780;
e-mail: pbvrana@gmail.com or vranap@mailbox.sc.edu
2
Received: 29 April 2010.
First decision: 29 May 2010.
Accepted: 28 July 2010.
Ó 2010 by the Society for the Study of Reproduction, Inc.
eISSN: 1529-7268 http://www.biolreprod.org
ISSN: 0006-3363
988
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known to have been altered by human selection (e.g., dogs,
cows) [9].
Deer mice (Peromyscus) offer such an opportunity. These
native North American cricetid rodents are .25 million years
diverged from both laboratory mice (Mus) and Rats (Rattus)
and not capable of interbreeding with them. Crosses between
two species, P. maniculatus (stock ¼ BW) and P. polionotus
(stock ¼ PO), produce asymmetric effects on growth and
development. Both parental stock strains were derived from
single wild populations [10].
While PO and BW animals have identical term fetal and
placental weights, BW females crossed with PO males produce
growth-retarded offspring [11, 12]. These animals remain
smaller than either parental species throughout life (denoted as
female bw 3 male po to indicate the effect). Litter sizes of the
female bw 3 male po hybrid cross are slightly smaller than
those of either parental strain, indicating some prenatal lethality
[13]. However, the growth-retarded survivors do not exhibit a
noticeably shortened life span (PO, BW live to 4 þ yr), are
fertile, and the sexes are equally represented.
In contrast, PO females mated to BW males (female PO 3
male BW) produce overgrown and severely dysmorphic
conceptuses. Roughly half of all female PO 3 male BW
breedings end in complete death of the litter by the time
gestation is two-thirds complete [14, 15]. Female PO 3 male
BW hybrids that survive to late gestation display numerous
developmental defects, many reminiscent of human syndromes
[14]. Rare female PO 3 male BW litters that reach parturition
typically result in maternal death because of an inability to pass
the hybrid offspring through the birth canal [13].
The placenta is particularly affected in both crosses [13,
16, 17]. The female bw 3 male po placentas average half the
weight of those from the parental strains, while those of the
female PO 3 male BW cross weigh about three times that of
the parental strains. An additional portion (;10%) of female
PO 3 male BW conceptuses lack any obvious embryonic
structures [14]. These conceptuses typically resemble placental tissue and thus may be analogous to hydatidiform
moles.
The hybrid placentas (particularly female PO 3 male BW)
have been shown to misexpress extracellular matrix (ECM)related loci, loci-encoding cell-cycle regulators, and loci
subject to genomic imprinting [18–20]. Numerous imprinted
gene products have been documented as playing significant
roles in mammalian placental growth and development [21].
Similar misexpression or loss of imprinting (LOI) of imprinted
genes has been linked to the molar phenotype [22, 23].
However, neither the relationship of the altered gene
expression to the hybrid phenotypes nor the nature of the
placental defects have yet been elucidated.
To understand the basis of these defects, we therefore
assessed hybrid placental patterning, growth dynamics, and
their potential association with female PO 3 male BW hybrid
mortality. We also assessed parental strain and hybrid placental
growth, morphology, and gene expression over the time
ABSTRACT
HYBRID PLACENTAL GROWTH AND PATTERN FORMATION
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TABLE 1. Peromyscus placental weights (g) at selected ages.a
Group/ageb
E10
E13
E16
PO/BW
bw 3 po
PO 3 BW
0.075 6 0.013
0.046 6 0.007
0.083 6 0.006
0.101 6 0.014
0.041 6 0.004
0.278 6 0.140
0.180 6 0.020
0.091 6 0.007
0.458 6 1.00
a
b
Mean weights are shown, 6 1 SD.
All ages are Mus equivalent.
MATERIALS AND METHODS
Animals
We purchased PO and BW animals from the Peromyscus Genetic Stock
Center (http://stkctr.biol.sc.edu). Animals were housed and bred under
approved institutional animal care protocols of the University of California,
Irvine, or the University of South Carolina and in accordance with the Guide
for the Care and Use of Laboratory Animals. Animals were housed with food
and water ad libitum on a 16:8-h light:dark cycle. We bred PO females with
BW males and BW females with PO males to obtain reciprocal hybrids. Males
and females were placed in the same cage with a separation cage top to allow
the estrous cycle of the female to be induced by the presence of a male without
contact. After 3 days, a normal cage top was used so that females and males
could mate at will. Vaginal smears were performed two times a day (morning
and evening) and visually assessed for sperm. We designated Day 0 of
development when sperm was found in vagina of cycling females. Placentas
(and associated fetuses) were dissected at each time point listed and for each
grouping (PO and BW, female bw 3 male po, and female PO 3 male BW).
Placentas were then weighed (wet), and the average for each group and time
point was determined.
Histological Analysis
Placentas were fixed in 4% paraformaldehyde. Fixed samples were
dehydrated through ascending concentrations of ethanol and then sent to
Histoserv Inc. (www.histoservinc.com) for sectioning and hematoxylin and
eosin staining. The laminin (catalog #ab7463) antibody was purchased from
Abcam (www.abcam.com) and used according to the manufacturers’ protocol.
In Situ Hybridization
Detection of messenger RNA by in situ hybridization in tissue sections was
performed by standard methods [25]. Peromyscus sequences (;500 base-pair
FIG. 1. Average placental:fetal weight ratios from E10 to E16. Genotype
is indicated in key. With the exception of the parental strains (PO/BW) and
female bw 3 male po at E14, all three classes differed significantly from
each other after E12 as judged by the Student t-test; (P , 0.001) as
implemented in Microsoft Excel. All ages are Mus equivalent.
[bp] cDNA fragment from exons 1–2 of the H19 gene, ;400-bp fragment
spanning exons 2–3 of Cdkn1c, and a full-length Esx1 cDNA) were cloned into
the Topo TA dual promoter cloning vector (Invitrogen). Sp6- and T7-derived
RNA probes were generated from linearized plasmids in the presence of
digoxygenin to generate both sense and anti-sense hybridization probes. Tpbpa
in situ hybridization was performed with a full-length Mus cDNA probe. The
Tpbpa and Esx1 procedures were performed by Histoserv, Inc. For both
histology and in situ hybridizations, at least three sections from three
individuals per time point and subgrouping were examined. Sense-strand
probes for each gene were also hybridized; none exhibited staining.
Gene Expression
Quantitative real-time PCR for Esx1 was performed as previously
described. The Peromyscus Rpl32 gene was used as a control. Reactions were
done in triplicate in 96-well plates and run on an ABI 7900 real-time PCR
instrument. Quantitative analysis was conducted according to Critical Factors
for Successful Real-Time PCR from Qiagen located at www.Qiagen.com. A
minimum of three samples were used for each subgrouping (PO, BW, female
PO 3 male BW and female bw 3 male po) in the analysis.
Proliferation and Apoptosis Assays
Females with litters at the equivalent of Mus E10, E13, and E16 were
injected with BrdU at 1.5 ml/100 g and euthanized 1 h later. Sections were
stained for apoptosis and amount of proliferation with the In situ Cell Death
Detection Kit, AP (catalog #11684809910) and 5-bromo-2 0 -deoxy-uridine
Labeling Detection Kit II (catalog #1299964) assay kits from Roche Applied
Science. The number of BrdU-incorporated and TUNEL-positive cells were
counted under 303 magnification. The numbers presented were derived by
averaging at least three sections from four individuals per time group from each
subgrouping (the two parental strains—denoted as PO/BW, female bw 3 male
po, and female PO 3 male BW).
RESULTS
Placental Growth Versus Hybrid Survival
The placental weights of the parental strains and reciprocal
hybrids at three time points (equivalent to Mus ages E10, 13,
and 16) are summarized in Table 1. The reduction in female bw
3 male po placental weight is apparent at the earliest stage
examined and throughout the remainder of gestation (i.e.,
including times later than E16).
Surprisingly, placentas of the female PO 3 male BW
conceptuses we examined at E10 do not vary significantly in
weight from those of the parental stock animals. However, we
did observe developmental defects in the E10 female PO 3
male BW conceptuses. By E13, however, the average female
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corresponding to Mus Embryonic Day (E) 10–16. Peromyscus
development differs from that of Mus in that it is approximately
4 days longer prior to implantation. That is, a Peromyscus
embryo E14 is the equivalent of a Mus E10 embryo. However,
we refer to Mus equivalent developmental time points to avoid
confusion.
Peromyscus placentas are discoidal and similar in morphology to those of other muroid rodents [24]. As in other muroids,
Peromyscus placentation results in three distinct layers: 1) the
labyrinth, where maternal and fetal blood supplies meet; 2) the
junctional zone (or spongiotrophoblast), which gives rise to
invasive and endocrine cells; and 3) the maternal decidua—that
part of the endometrium that has been remodeled by invasive
zygotic cells and serves as the attachment point. The
morphology of the junctional zone/labyrinth boundary and
maternal interface exhibit some differences from those of house
mice (Mus) and other genera. Studies to date indicate that the
female bw 3 male po placental phenotype is due to a reduction
of the junctional zone [16, 17]. The structural changes
occurring in female PO 3 male BW placentation are less
clear, however, as are the dynamics of the abnormal
proliferation. Here we investigate these changes through
histology and gene expression of loci known to exhibit hybrid
misexpression and/or trophoblast lineage markers. In addition,
we assess BrdU incorporation and TUNEL assays to pursue
these questions.
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DUSELIS AND VRANA
PO 3 male BW placental weight is greater than that of mature
PO/BW embryos (;E15 or later).
Our studies indicate that the majority of female PO 3 male
BW conceptuses are dead by E16. While many of these deaths
occur prior to E10, overall female PO 3 male BW mortality
continues to increase from E10 to E16. We had previously
observed that greater placental weight was correlated with the
degree of LOI in backcross (PO females 3 F1 males) that
mimics the female PO 3 male BW cross [26]. Similarly, the
degree of LOI in mixed-background animals is associated with
prenatal mortality. We therefore considered whether increased
placental growth might be generally associated with the female
PO 3 male BW mortality.
To test this hypothesis, we plotted placental weights of
timed pregnancies vs. the percentage of female PO 3 male BW
conceptuses alive in litters at that age (Supplemental Fig. S1
available at www.biolreprod.org). These data provided little
evidence of a specific association at any particular age. A
possible exception is seen from E12 to E13; the percentage of
live conceptuses observed at E12 was only ;50%, while only
;30% of those observed at E13 were alive. However,
examination of timed pregnancies indicates that the cumulative
total of live conceptuses by this point is well below 50%
(meaning that there may be a selection bias such that
conceptuses that survive to E13 are atypical). This observation,
combined with the phenotypic heterogeneity of the female PO
3 male BW conceptuses, makes determining such associations
difficult.
We also assessed fetal:placental weight ratios as a measure
of placental efficiency [27]. As expected, the average value of
this ratio increases steadily over the second half of gestation
in parental strain (PO or BW) animals (Fig. 1); it reaches 10
the day prior to parturition. While the trajectory of the female
bw 3 male po ratio is more erratic, it reaches slightly greater
values than that of the parental strains. This increased ratio
reflects the observation that female bw 3 male po placental
weight is more affected than fetal weight. In contrast, the
maximum average female PO 3 male BW ratio is approximately six. The lowered ratio in female PO 3 male BW
conceptuses may reflect both placental defects and cell
lineage shifts toward extraembryonic fates (as suggested by
the molar conceptuses).
Female PO 3 Male BW Hybrid Placental Hemorrhaging
One cause of the female PO 3 male BW placental
inefficiency is suggested by the frequent occurrence of
macroscopic blood pools (i.e., indicating intraplacental hemorrhaging). We used hematoxylin and eosin staining to assess
the nature and ontogeny of this major defect in placental
pattern formation (Fig. 2). The female PO 3 male BW blood
pools we have observed are more often located at the placental
edges. However, as shown in Figure 2, large pools occupying a
central sinus occur at least as early as E10 (note that the degree
of hemorrhaging is heterogeneous). This suggests that a
possible underlying cause of embryonic death may be failure
of maternal-fetal nutrient transfer due to blood vessel instability
in the labyrinth. Moreover, our previous research suggested a
deficiency of some ECM-related proteins in female PO 3 male
BW placentas [20].
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FIG. 2. Hematoxylin and eosin-stained histological sections illustrating the hemorrhaging and disorganization observed in the female PO 3 male BW
placentas. Genotype is indicated at top and age (Mus equivalent) at left. Original magnification 31; bars ¼ 1 mm.
HYBRID PLACENTAL GROWTH AND PATTERN FORMATION
991
FIG. 3. Representative examples of laminin immunohistochemistry to assess labyrinth vasculature. Genotype is indicated at
top and age at left. Columns represent
groups of placentas by genotype: PO/BW,
female PO 3 male BW and female bw 3
male po, respectively. A–C ¼ E10 placentas, D–F ¼ E13 placentas and G–I ¼ E16
placentas (all ages are Mus equivalent).
Double arrowheads represent embryonic
blood vessels; single arrowheads indicate
compromised maternal blood sinuses. Such
tears were never observed in PO/BW or
female bw 3 male po sections. All pictures
were taken of the labyrinth at 340 magnification.
Labyrinth and Junctional Zone Markers
The product of the X-chromosome-linked Esx1 gene is a
homeodomain transcription factor critical for proper morphogenesis of vascularization within the labyrinth layer [29, 30].
Ablation of mouse Esx1 expression results in enlarged
placentas with little demarcation between the labyrinth and
junctional zone. The Esx1 mutant placentas also exhibit
abnormal capillary dilation and branching. In addition to these
similarities between the knockout and female PO 3 male BW
placentas, Esx1 is tightly linked to a Peromyscus locus mapped
on the basis of association with the placental hypertrophy [26,
31]. Thus, variation in this gene may contribute to aspects of
the female PO 3 male BW dysgenesis.
Our previous study indicated Esx1 overexpression in female
bw 3 male po placentas—possibly reflecting the relatively high
proportion of labyrinthine cells in this hybrid cross. We
reassessed Esx1 expression using the same quantitative PCR
assay at E10, E13, and E16 (Fig. 4A). The temporally
expanded data reveals that the female bw 3 male po Esx1
overexpression is transitory; at E13, female bw 3 male po
expression drops significantly below that of PO/BW. While the
average female PO 3 male BW expression is lower than that of
the parental strains at E10 and E16, the values substantially
overlap. Therefore, while it is unlikely that reduced Esx1
expression levels contribute to the female PO 3 male BW
defects, it is possible that spatial misexpression plays a role.
To investigate this possibility, we performed in situ
hybridization with a Peromyscus Esx1 probe on placental
sections. As expected, the highest proportion of expressing
cells is seen at E10, with few positive cells at E16 in the
parental strains or either hybrid type. As in Mus, E10
expression appeared highest in the developing labyrinth. At
E13 and E16, however, expressing cells are more dispersed
throughout the placenta (Fig. 4B). Esx1 staining in the E10
female bw 3 male po hybrid placentas reflects the apparent
reduction of junctional zone trophoblast. Similarly, female PO
3 male BW E10 Esx1 expression is more dispersed,
presumably reflecting that spatial disorganization has already
occurred.
We also performed in situ hybridization experiments with
probes from two additional loci whose primary placental
expression is within the labyrinthine layer. Cdkn1c and H19 are
linked imprinted loci that we have previously shown to exhibit
female PO 3 male BW hybrid misexpression [18–20]. While
both are normally expressed from the maternal allele, H19
exhibits increased (biallelic) female PO 3 male BW expression,
while placental Cdkn1c expression is reduced. The potential
effects of increased H19 expression are unclear [32]. However,
the Cdkn1c product is a negative regulator of growth, and its
downregulation is associated with hyperproliferation in normal
development and diseases, including hydatidiform moles [33].
Figure 5 illustrates that Cdkn1c stains both the Peromyscus
junctional zone and the labyrinth at E10. Thereafter, expression
is confined largely to the labyrinth, trophoblast giant cells
(TGCs), and probable immature TGCs. In contrast, H19 is
found in the labyrinth and mature TGCs at all ages. The H19
TGC expression illustrates a similar migration of these cells in
Peromyscus as in other rodent species: at E10, the majority of
TGCs are seen between the labyrinth and spongiotrophoblast
layer but by E16 are within the decidua and junctional zone.
However, the staining patterns of these two genes suggest a
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To further investigate this possibility, we stained placental
sections with a laminin antibody. These proteins are key
components of the basal lamina and therefore useful for
enhancing the clarity of placental blood vessels [28].
Examination of the stained sections suggests a reduction in
female PO 3 male BW embryonic blood vessels (i.e., relative
to PO/BW; Fig. 3). Moreover, the walls of the maternal blood
sinuses within the female PO 3 male BW labyrinth typically
appear thinner and often compromised. These defects are
consistent with the previously documented reduction in female
PO 3 male BW placental collagen expression as well as similar
hemorrhaging in the associated embryos [11, 20].
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DUSELIS AND VRANA
FIG. 4. Quantitative PCR (A) and in situ
hybridization assays (B) of placental Esx1
expression. In A, genotype is indicated at
bottom. Bars indicate standard error. Female
bw 3 male po expression was significantly
different (P , 0.001) than the other two
classes at both E10 and E13 (Mus equivalent). In B, genotype is indicated at top and
age at left. Note the expanded area of Esx1
expression at E10 in both hybrid types
relative to the parental strains (PO/BW).
However, the expressing cells are more
dispersed in the female PO 3 male BW
hybrids. Arrows indicate areas of relatively
intense staining. Original magnification
310; bars ¼ 100 lm.
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lack of demarcation between the two zones in female PO 3
male BW and are consistent with an early reduction in the
female bw 3 male po junctional zone/spongiotrophoblast.
We used the well-established marker Tpbpa (i.e., 4311) to
further assess possible aberrant junctional zone patterning in
the hybrids (Fig. 6). In Mus, this gene is expressed in all
junctional zone cell types except TGCs [34]. Surprisingly, E10
Tpbpa staining was most intense in female bw 3 male po
placental sections. However, by E13 the female bw 3 male po
staining region was considerably narrower than that of the
parental strains and nearly absent at E16. This result suggests
an initial surfeit of female bw 3 male po spongiotrophoblast
progenitors that fail to multiply and/or survive at a normal rate.
In contrast, female PO 3 male BW Tpbpa staining was less
cohesive than that of the parental strains at all three ages: E10
expression is scattered and found on the edges of the areas
stained by H19 and Cdkn1c. By E13, parental strain Tpbpa
staining has expanded considerably, while female PO 3 male
BW is even more scattered. This suggests that very early
female PO 3 male BW placental patterning is at least slightly
perturbed, and the disorganization is magnified as development
proceeds.
Proliferation and Cell Death
The observed aberrant growth and pattern formation in the
hybrid placentas suggests possible imbalances between proliferation and apoptosis. As we previously found genes encoding
cell-cycle regulators to be affected in the hybrid placentas [20],
we investigated whether gross differences in cellular proliferation and death were apparent in the hybrid placentas. We
utilized BrdU incorporation to measure proliferation and
TUNEL staining, which identifies cells with DNA fragmentation typically resulting from apoptosis.
In Mus, placental growth shows increasing proliferation and
organization of the placenta until approximately E14.5, when it
is considered mature [35]. Reciprocally, regionalized apoptosis
in the maternal decidua and fetal glycogen cells start at mid- to
late gestation. Figure 7 indicates that these patterns are very
similar in wild-type Peromyscus: the number of BrdU-positive
cells declines past E10, becoming virtually absent by E16.
Correspondingly, the number of PO/BW TUNEL-positive
placental cells increased over this period.
Both the female PO 3 male BW and the female bw 3 male
po placentas exhibited shifted proliferation/apoptosis curves.
Surprisingly, both hybrid types had higher numbers of BrdU-
HYBRID PLACENTAL GROWTH AND PATTERN FORMATION
993
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FIG. 5. Representative results of placental in situ hybridization experiments with Cdkn1c and H19 probes. Columns represent groups of placentas (PO/
BW, female PO 3 male BW and female bw 3 male po, respectively). A–H ¼ E10 placentas, I–P ¼ E13 placentas, Q–X ¼ E16 placentas (all ages are Mus
equivalent). Rows alternate between the two genes as indicated in the leftmost panels. Single arrow ¼ mature TGCs, double arrow ¼ probable immature
TGCs. Brackets in B and D outline spongiotrophoblast. Column 1 pictures were taken at 31 magnification; column 2–4 pictures were taken at 315
magnification. Note that the PO/BW 310 magnification picture at E10 (B) appears rotated, as it is a close-up of the rightmost section of A.
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DUSELIS AND VRANA
FIG. 6. Representative results of Tpbpa in
situ placental hybridization. Genotypes and
ages indicated at the top and left of figure,
respectively (all ages are Mus equivalent).
The first column pictures were taken at 31
magnification, while all others were taken
at 310 magnification. Note that the 310
pictures are rotated 90 degrees clockwise
relative to the 31 pictures.
after E12. This period of placental weight increase is
immediately preceded by a rise in fetal mortality. Among the
potential causes of mortality in this time frame are the observed
labyrinth defects; these include unstable maternal blood spaces
(i.e., presumably due to the thinner walls) and a decrease in the
number of fetal capillaries. These defects would be expected to
inhibit maternal-fetal nutrient and waste exchange.
DISCUSSION
The placenta is a transient organ whose proper function is
nevertheless critical for both maternal and fetal health [6].
Placental hypoplasia is associated with intrauterine and other
growth retardation [2, 4] syndromes, while hyperplasia is
associated with fetal overgrowth syndromes such as BeckwithWiedemann [38] as well as gestational diabetes [39] and
various forms of gestational trophoblast disease [40]. Peromyscus offer the opportunity for assessing the effects of natural
genetic variation on placental growth, development, and
physiology as well as an additional comparative model for
these processes as well as disease. We have previously shown
altered expression and epigenetic state of a number of
imprinted loci implicated in human diseases that correlate with
the Peromyscus hybrid phenotypes (e.g., reductions in Cdkn1c
and Phlda2 expression) [41].
We have focused primarily on the dramatic placental defects
in female PO 3 male BW dysgenesis and how they may
underlie fetal mortality. Female PO 3 male BW placental
efficiency (as defined by fetal:placental weight ratio) is reduced
FIG. 7. Quantitation of BrdU (A) and TUNEL (B) assays. Average number
of positive cells per section is shown for each genotype (indicated in key);
bars indicate standard error. Significant differences (P , 0.001) in A are
female bw 3 male po (from the other two classes) at E10 and both hybrid
types (from PO/BW) at E13 and E16. All classes significantly differ from
each other in B except female bw 3 male po from PO/BW at E13 and the
two hybrid types from each other at E16 (all ages are Mus equivalent).
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positive cells, albeit at different points in development: at E10,
female bw 3 male po placentas displayed both greatly
increased numbers of proliferating and dying cells relative to
the parental strains. By E13, however, proliferation had been
reduced to wild-type levels, and cell death was virtually absent.
TUNEL-positive cells increased at E16 but remained below
wild-type levels.
In contrast, female PO 3 male BW cell death remained well
below that of the parental strains at all three time points
examined. While BrdU incorporation did rise to levels well in
excess of those exhibited by the parental strains, this increase
was not apparent until E13. The number of female PO 3 male
BW proliferative cells decreased somewhat from E13 to E16,
reaching roughly equivalent numbers as that of apoptotic cells
at the latter time point.
The localization of cell death and proliferation in PO/BW
and female PO 3 male BW placentas were similar to those
observed in Mus (e.g., late gestation apoptosis in the decidua
and glycogen cells) [36, 37]. The abnormally high levels of
proliferation in the female bw 3 male po placentation were
centered within the spongiotrophoblast layer and maternal
decidua, with apoptotic cell located throughout the placenta
(data not shown).
HYBRID PLACENTAL GROWTH AND PATTERN FORMATION
ACKNOWLEDGMENTS
The Tpbpa probe was courtesy of Dr. Steven Lipkin of Weill Cornell
Medical College. We thank Dr. Grant MacGregor (University of
California, Irvine) and Dr. Steven Kistler (University of South Carolina)
for assistance in microscopy/digital photography.
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The in situ hybridization experiments suggest that both
labyrinth and junctional zone patterning are affected. Because
of our previous demonstration of a reduction in Phlda2
expression [14], we predicted an increase in the number of
female PO 3 male BW Tpbpa staining cells by comparison
with the Mus knockout of this gene [42]. However, this was not
the case; we hypothesize that female PO 3 male BW
overproliferation (and simultaneous reduction in apoptosis)
results in an elevated proportion of improperly differentiated
cells. For example, we frequently observed cells that may be
immature/improperly differentiated TGCs (Fig. 5). Concurrent
experiments assessing TGC and stem-cell markers support this
hypothesis. We suggest that the maternal effect that results in
female PO 3 male BW genomic imprinting perturbations [43]
also results in epigenetic misregulation of lineage allocation
and/or differentiation.
While the maternal effect locus appears to be the major
determinant of the female PO 3 male BW phenotypes, Esx1 is
tightly linked to a locus influencing placental overgrowth in
that cross. Similarities to the Mus targeted mutation strengthen
the case of Esx1 as a candidate for this locus. While there is no
apparent reduction in female PO 3 male BW Esx1 expression,
its spatial patterning is clearly perturbed. As the analyses
clearly indicate, the female PO 3 male BW defects genesis
occurs prior to E10, necessitating earlier analyses.
Preliminary analyses also suggest a role for the X
chromosome in the genetics of the female bw 3 male po
growth retardation. While it is not clear that this potential
linkage will include Esx1, its transient overexpression and
subsequent sharp decline are intriguing. While the female bw 3
male po E16 expression is wild type, the earlier misexpression
and differences in spatial expression may be sufficient to
contribute to the aberrant placental patterning.
The junctional zone marker Tpbpa also appears overexpressed in E10 female bw 3 male po placentas as judged by in
situ hybridization. However, this region is reduced relative to
the parental strains by E13, corroborating our observation that
the higher rate of cell death particularly targets this region.
Concurrent analyses also indicate a premature reduction in
TGC numbers.
It is as yet unclear whether the female bw 3 male po fetal
and postnatal growth retardation is dependent on the placental
defects. These and many other questions make this unique
system ripe for additional study. More detailed analyses are
now feasible because of recent increases in Peromyscus
resources, including BW genome sequencing in progress
(www.ncbi.nlm.nih.gov/Traces/home), more than 90 000 ESTs
in public databases, a nascent genetic map [44], and improved
reproductive technology (unpublished data).
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