SUMO-specific protease 2 is essential for modulating p53-Mdm2 in development of trophoblast stem cell niches and lineages.

Abstract

SUMO-specific protease 2 (SENP2) modifies proteins by removing SUMO from its substrates. Although SUMO-specific proteases are known to reverse sumoylation in many defined systems, their importance in mammalian development and pathogenesis remains largely elusive. Here we report that SENP2 is highly expressed in trophoblast cells that are required for placentation. Targeted disruption of SENP2 in mice reveals its essential role in development of all three trophoblast layers. The mutation causes a deficiency in cell cycle progression. SENP2 has a specific role in the G-S transition, which is required for mitotic and endoreduplication cell cycles in trophoblast proliferation and differentiation, respectively. SENP2 ablation disturbs the p53-Mdm2 pathway, affecting the expansion of trophoblast progenitors and their maturation. Reintroducing SENP2 into the mutants can reduce the sumoylation of Mdm2, diminish the p53 level and promote trophoblast development. Furthermore, downregulation of p53 alleviates the SENP2-null phenotypes and stimulation of p53 causes abnormalities in trophoblast proliferation and differentiation, resembling those of the SENP2 mutants. Our data reveal a key genetic pathway, SENP2-Mdm2-p53, underlying trophoblast lineage development, suggesting its pivotal role in cell cycle progression of mitosis and endoreduplication.

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SUMO-Specific Protease 2 Is Essential for
Modulating p53-Mdm2 in Development of
Trophoblast Stem Cell Niches and Lineages
Shang-Yi Chiu1, Naoya Asai2,3, Frank Costantini3, Wei Hsu1*
1 Department of Biomedical Genetics, Center for Oral Biology, James P Wilmot Cancer Center, University of Rochester Medical Center, Rochester, New York, United States of
America, 2 Department of Pathology, Nagoya University, Nagoya, Japan, 3 Department of Genetics and Development, Columbia University Medical Center, New York, New
York, United States of America
SUMO-specific protease 2 (SENP2) modifies proteins by removing SUMO from its substrates. Although SUMO-specific
proteases are known to reverse sumoylation in many defined systems, their importance in mammalian development
and pathogenesis remains largely elusive. Here we report that SENP2 is highly expressed in trophoblast cells that are
required for placentation. Targeted disruption of SENP2 in mice reveals its essential role in development of all three
trophoblast layers. The mutation causes a deficiency in cell cycle progression. SENP2 has a specific role in the G–S
transition, which is required for mitotic and endoreduplication cell cycles in trophoblast proliferation and
differentiation, respectively. SENP2 ablation disturbs the p53–Mdm2 pathway, affecting the expansion of trophoblast
progenitors and their maturation. Reintroducing SENP2 into the mutants can reduce the sumoylation of Mdm2,
diminish the p53 level and promote trophoblast development. Furthermore, downregulation of p53 alleviates the
SENP2-null phenotypes and stimulation of p53 causes abnormalities in trophoblast proliferation and differentiation,
resembling those of the SENP2 mutants. Our data reveal a key genetic pathway, SENP2–Mdm2–p53, underlying
trophoblast lineage development, suggesting its pivotal role in cell cycle progression of mitosis and endoreduplication.
Citation: Chiu S-Y, Asai N, Costantini F, Hsu W (2008) SUMO-specific protease 2 is essential for modulating p53-Mdm2 in development of trophoblast stem cell niches and
lineages. PLoS Biol 6(12): e310. doi:10.1371/journal.pbio.0060310
Introduction
The first two distinct lineages to form in the mammalian
embryos are the outer trophectoderm and the inner cell mass
(ICM) of the blastocyst [1]. The trophectoderm initiates
implantation and invasion of the uterus, processes that are
essential for placental development [2]. This process depends
on the differentiation of trophoblasts, the main and most
important cell types in the placenta [3,4]. The trophoblast
stem (TS) cells in the mural trophectoderm, distal to the ICM,
stop dividing but continue to duplicate their genomes, a
mechanism known as endoreduplication. The polyploid
trophoblast giant cells (TGCs) then develop and eventually
surround the entire fetus [5]. As development proceeds, the
trophoblast progenitors give rise to three distinct layers in
rodents—labyrinth, spongiotrophoblast and TGCs—to form
a functional placenta acting as the maternal–fetal interface
[6]. The fetal–placental blood vessels grow in from the
allantois to generate the fetal parts of the placental
vasculature where the chorioallantoic fusion has occurred
[7]. The labyrinth is formed by extensive branching morpho-
genesis of the labyrinth trophoblast and endothelial cells [8].
The maternal blood passes through the small spaces of the
labyrinth, directly contacting the fetal trophoblast cells to
ensure exchange between the two blood systems. The
labyrinth layer is supported structurally by the spongiotro-
phoblast cells, which are mainly derived from the ectopla-
cental cone and which form a layer separating the labyrinth
from the TGC. The simplicity of placental cell lineages makes
the placenta a valuable model system for understanding
general aspects of development, including branching mor-
phogenesis, lineage-specific determination, cell invasion, and
polyploidy, crucial for cancer development and metastasis.
SENP2 belongs to a family of proteases that remove a small
ubiquitin-related modifier (SUMO) from protein substrates.
SUMO (also known as sentrin), which regulates posttransla-
tional modification of proteins, is a member of the ubiquitin-
like modifier family [9]. This covalent conjugation process is
reversible and highly evolutionary conserved from yeasts to
humans [10]. Unlike ubiquitination, which has a well-
established role in targeting protein degradation, SUMO
modification is involved in protein trafficking, cell cycle, cell
survival, and cell death [11]. SUMO conjugation of proteins
can alter their function, activity, or subcellular localization.
Many sumoylated proteins have been shown to accumulate
preferentially in specific complexes such as the nuclear pore
and PML (promyelocytic leukemia) bodies [12]. Similar to
ubiquitination, sumoylation requires processing, conjugation,
Academic Editor: Margaret A. Goodell, Baylor College of Medicine, United States
of America
Received August 29, 2008; Accepted October 31, 2008; Published December 16,
2008
Copyright: ? 2008 Chiu et al. This is an open-access article distributed under the
terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author
and source are credited.
Abbreviations: E, embryonic day; ES, embryonic stem; ICM, inner cell mass; MEF,
mouse embryonic fibroblast; PI, propidium iodide; PML, promyelocytic leukemia;
RNAi, RNA interference; SUMO, small ubiquitin-related modifier; SENP2, SUMO-
specific protease 2; TGC, trophoblast giant cells; TS, trophoblast stem
* To whom correspondence should be addressed. E-mail: Wei_Hsu@urmc.
rochester.edu
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P PL Lo oS S BIOLOGY

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and transfer. The transfer process, which covalently con-
jugates SUMO polypeptides to their targets, is catalyzed by E3
ligases [13]. The reverse desumoylation process is mediated by
SUMO proteases. The hallmark of these proteases is the
highly conserved carboxyl-terminal SENP domain of ;200
amino acids. SENP2, which is found in three different
alternatively spliced forms, has been localized to the nucleus,
cytoplasmic vesicles and PML nuclear bodies [14–16].
Although SENPs have been shown to catalyze SUMO
modification in various physiological systems, their roles in
mammalian development and pathogenesis are mostly
unknown.
We previously discovered an interaction of SENP2 with
Axin [17,18], a key signaling regulator for the canonical Wnt
pathway. To determine the role of SENP2 in cellular signaling
and the importance of SUMO modification in trophoblast
development, we initiated a genetic analysis in mice. A
SENP2-null mouse strain was created by gene targeting in
embryonic stem (ES) cells. We found that the disruption of
SENP2 leads to developmental defects in all three trophoblast
layers. SENP2 is essential for the G–S transition of both the
mitotic and the endoreduplication cell cycles, which control
the expansion of trophoblast precursors and the maturation
of TGCs, respectively. In the mutants, the loss of SENP2
caused a deregulation of Mdm2, resulting in p53 stimulation.
We also present evidence to support an essential role of
SENP2 in modulating the p53–Mdm2 circuit that underlies
genome replication in mitosis and polyploidy during troph-
oblast development.
Results
Expression of SENP2 in Trophoblast Development
To determine the role of SENP2 and the importance of
SUMO modification in trophoblast development, we first
examined its expression pattern. Strong expression of SENP2
was observed in extraembryonic tissues, including extraem-
bryonic ectoderm, chorion and ectoplacental cone, at
embryonic day (E)7 (Figure 1A). In extraembryonic ectoderm,
its expression started diminishing by E7.5 (Figure 1B). In
addition to these stem cell niche sites, we also detected its
transcript in TS cells (Figure 1C). At E8.5, SENP2 maintained
its ubiquitous expression in trophoblast cells located in the
chorion and ectoplacental cone (Figure 1D–1F). By E9.5 and
E10.5, the SENP2 transcript was detected in all three
trophoblast layers: labyrinth, spongiotrophoblast and TGC
(Figure 1H and 1L). SENP2 was expressed in the labyrinth
trophoblast cells, which derive from the extraembryonic
ectoderm and chorion, upon chorioallantoic fusion at E9.5
(Figure 1I). In the E10.5 labyrinth layer, its expression was
specifically localized to cytotrophoblasts (mononuclear
trophoblasts), adjacent to the maternal blood cells (Figure
1M). Syncytiotrophoblasts, as well as endothelial and blood
cells, appeared to be negative for the staining. In contrast, we
found a uniform expression of SENP2 in spongiotrophoblasts
and TGCs (Figure 1J, 1K, 1N, and 1O), which are derivatives of
the ectoplacental cone. TGCs include primary and secondary
cells, derived from mural trophectoderm and ectoplacental
cone (derivatives of polar trophectoderm), respectively. The
SENP2 transcript was detected in both the primary and
secondary TGCs (Figure 1G, 1K, and 1O). These results imply
an important function of SENP2 in trophoblast progenitors
and their development into all three major layers.
SENP2 Is Required for Extraembryonic Development
A SENP2-null allele was created by the targeted insertion of
a lacZ reporter with pgk-neo cassette into exon 2 and the
deletion of exons 3 to 5 to inactivate all different forms of the
SENP2 gene product (see Materials and Methods for details).
The targeted mouse ES cell clones heterozygous for SENP2
(Figure S1A), obtained by homologous recombination, were
then used to obtain the SENP2lacZmouse strain (Figure S1B).
Mice carrying the targeted allele were subsequently bred with
a Zp3-Cre transgenic strain to remove the pgk-neo cassette
(SENP2-null allele), as confirmed by PCR genotyping analysis
(Figure S1C). RT-PCR analyses further showed that the
SENP2-null allele does not express the SENP2 transcript,
but instead expresses the inserted lacZ gene (Figure S1D).
The SENP2-null heterozygous (hereafter referred to as
SENP2þ/–) mice were viable and fertile without any noticeable
abnormalities. However, we were unable to find SENP2-null
homozygous (hereafter referred to as SENP2–/–) newborns,
implying that they died prematurely. These results prompted
us to investigate whether the loss of SENP2 causes embryonic
lethality. The SENP2–/–embryos appeared to be morpholog-
ically indistinguishable from their SENP2þ/þand SENP2þ/–
littermates at E9.5 (Figure 2A and 2B). However, the SENP2–/–
embryos were significantly smaller or underdeveloped com-
pared with the SENP2þ/þand SENP2þ/–littermates at E10.5
(Figure 2C and 2D). We could not recover the SENP2–/–
embryos after E11.5. This phenotype is often associated with
placental deficiencies, as the embryos begin to rely on
maternal supplies upon allantoic fusion at mid gestation.
Indeed, the SENP2–/–placentas were smaller and paler than
the controls (Figure 2E–2H). The average diameter of E10.5
placentas reduced from 5.2 mm in controls to 3.8 mm in
mutants (Figure 2S, p , 0.0001, n ¼ 7). Histological analyses
revealed a reduction of the TGC layer by E9.5 (Figures 2I and
2J). By E10.5, the thickness of all three trophoblast layers
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SENP2 in Trophoblast Development
Author Summary
Genome replication is essential for both expansion of stem cell
numbers through mitosis and their maturation into certain
specialized cell types through endoreduplication, a unique mech-
anism for multiplying chromosomes without dividing the cell. An
important function of p53 as a guardian of the genome ensures that
the genetic information is properly propagated during these
processes. In this study, we discovered that mice with disruption
of SENP2, an enzyme that removes small molecular signals (called
SUMO) that modify a protein’s behavior and stability, are unable to
form a healthy placenta as a result of deficiencies in the formation of
various trophoblast cell types that give rise to the placenta. In the
mutants, SUMO modification of Mdm2, a protein that monitors the
cellular levels of p53, is deregulated. The loss of SENP2 causes
dislocation of Mdm2, leading to aberrant stimulation of p53. The
precursor cells known as trophoblast stem cells rely on p53 to
proliferate and differentiate into specialized polyploid cells, which
contain multiple copies of chromosomes. In SENP2 mutants, all three
trophoblast layers were substantially defective, with the layer
containing mainly the polyploid cells most severely affected and
diminished. This study reveals a key genetic pathway, SENP2–
Mdm2–p53, which is pivotal for the genome replication underlying
trophoblast cell proliferation and differentiation.

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decreased drastically in the SENP2-null mutants (Figures 2K
and 2L). The TGC layer, which is the layer most severely
affected by the SENP2 mutation, is almost completely
missing. The data suggest that SENP2 has a pivotal role in
development of all three trophoblast layers.
Trophoblast Niche Sites Are Defective in SENP2 Nulls
The placental defects caused by SENP2 deficiency sug-
gested that it is critical for trophoblast development. The
stem cells derived from the trophectoderm develop into
progenitors, which reside in the ectoplacental cone, the
extraembryonic ectoderm, and the chorion. We therefore
examined whether the SENP2 deletion interferes with
formation of these niche sites. In situ hybridization of Tpbpa,
a marker for the ectoplacental cone [19], revealed a drastic
reduction of trophoblast progenitors in the mutants (Figure
2M and 2P). The number of trophoblast progenitors, marked
by Cdx2 expression [20] was also decreased in the SENP2–/–
chorion and extraembryonic ectoderm (Figure 2N, 2O, 2Q,
and 2R). The apparent developmental defects of trophoblast
niche sites suggested that SENP2 might have a role in
trophoblast stem cell development.
Effects of SENP2 Deficiency on Labyrinth Layer
A closer examination of the labyrinth layer was performed
by analyzing the expression of Gcm1, a labyrinth trophoblast
marker that is specifically detected in the chorioallantoic
invasion sites and later in the differentiated syncytiotropho-
blasts [21]. No obvious difference between SENP2þ/þand
SENP2–/–was observed at E9.5 (Figure 3A and 3D). Gcm-1-
positive trophoblast progenitors were clearly identified at the
invasion sites. Therefore, fetal vascular invasion was not
affected by the deletion. However, deficiencies in labyrinth
development of SENP2–/–embryos were evident at E10. The
syncytiotrophoblasts positive for Gcm-1 exhibited punctated
staining in the mutant instead of the continuous thin layers
seen in the wild type, suggesting that their differentiation is
defective (Figure 3B and 3E). By E10.5, the number of the
Gcm1-expressing cells was dramatically reduced in the
mutants (data not shown). At this stage, SENP2 expression is
restricted to the cytotrophoblasts (Figure 1M), a subtype of
TGCs [22]. Therefore, we examined whether cytotrophoblast
development was affected by analyzing a cytotrophoblast
marker, Ctsq [23]. Indeed, the Ctsq-positive cytotrophoblasts
identified in the wild-type labyrinth were completely missing
in the mutants (Figure 3C and 3F). Histological analysis
further showed that both maternal and fetal blood spaces
were enlarged without formation of capillary structures in
the SENP2 mutants (Figure 3G and 3J). Immunostaining of
laminin [24], a basement membrane protein expressed by
endothelial cells that highlight fetal blood spaces, further
revealed a failure of branching morphogenesis in the SENP2-
null fetal vasculature (Figure 3H and 3K). This might be
attributed to a deficiency in endothelial proliferation as the
number of cyclin D1-positive cells (proliferation marker only
Figure 1. SENP2 Is Expressed in Trophoblast Lineage Development
(A and B) In situ hybridization reveals that SENP2 is expressed in the trophoblast stem cell niches, including extraembryonic ectoderm (exe) , chorion
(Ch) and ectoplacental cone (epc) at E7.0 (A) and E7.5 (B).
(C) RT-PCR analysis detected the SENP2 transcript in wild-type (þ/þ), but not knockout (–/–) TS cells.
(D–O) Sections of the E8.5 (D–G), E9.5 (H–K) and E10.5 (L–O) placentas were analyzed by in situ hybridization for the expression of SENP2. Expression was
detected in major extraembryonic tissues. Low magnification images display the overall expression pattern in developing placentas (D,H,L). High
magnification images show expression in specific cell types and layers (E–G,I–K,M–O). The chorion, ectoplacental cone, labyrinth (L), spongiotrophoblast
(S), and TGC (G; 18, primary; 28, secondary) layers are defined by orange, pink, blue, red, and green broken lines, respectively. Arrows indicate specific
expression in mononuclear trophoblasts (cytotrophoblasts) of the labyrinth layer (M).
Em, embryo. Scale bars, 1 mm (D,H,L); 100 lm (A,B,E–G,I–K,M–O).
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detected in endothelial cells) was decreased in the mutants
(Figure 3I and 3L). These data demonstrated that SENP2 is
essential for labyrinth trophoblast development in establish-
ment of the maternal and fetal blood spaces. The presence of
SENP2 in early trophoblast precursors might regulate the
differentiation of specialized cell types at later stages.
Alternatively, its function in the cytotrophoblasts could be
crucial for proper development of syncytiotrophoblasts and
endothelial cells. SENP2 is necessary for development of the
labyrinth layer during placentation.
Spongiotrophoblast Development Is Defective in the
Absence of SENP2
We next examined the spongiotrophoblast layer that is
affected by the SENP2 deletion. In situ hybridization analysis
of Tpbpa [19], a marker for the spongiotrophoblast, revealed
that its expressing cells diminished significantly in the
mutants at E9.5–E10.5 (Figure 3M, 3N, 3P, and 3Q). Histology
confirmed that a rapid expansion of this layer, found in the
wild-type placenta, did not occur in the mutants (Figure 3O
and 3R). As a result, the SENP2-null spongiotrophoblast layer
decreased significantly in volume. Based on the expression of
SENP2 in spongiotrophoblasts (Figure 1J and 1N) and earlier
in their precursors at the ectoplacental cone (Figure 1A, 1B,
and 1F), it is most likely that the abnormalities are primarily
due to its deletion in these tissues. Therefore, spongiotro-
phoblast development requires SENP2 and its disruption
induces abnormalities in the spongiotrophoblast layer.
Impaired Development of TGC in the SENP2-Null
Placentas
Consistent with its expression in early trophoblast develop-
ment, histological analyses revealed a severe abnormality in
the TGC layer (Figure 4A–4H). The SENP2-null primary
TGCs were reduced at E8.5 and completely missing at E9.5
(Figure 4A, 4B, 4E, and 4F). Similarly, the number of
secondary TGCs was decreased at E9.5 and almost disap-
peared at E10.5 (Figure 4C, 4D, 4G, and 4H). In addition, the
size of TGCs was significantly smaller in the SENP2 mutants
(Figure 4D and 4H). The analyses of TGC markers [19,25],
including PL-I (Figure 4I–4P), PL-II (unpublished data), and
p450scc (Figure 4Q–4V), confirmed that the TGC cell
numbers were dramatically decreased in the SENP2 mutants
at all stages examined. We next examined the initiation of
Figure 2. Embryonic and Extraembryonic Abnormalities Caused by SENP2 Deficiency
(A–D) Whole mount analysis of the SENP2þ/þ(A,C) and SENP2–/–(B,D) embryos identified growth restriction induced by the deletion of SENP2 at E9.5
(A,B) and E10.5 (C,D).
(E–L) The placentas of SENP2þ/þ(E,G,I,K) and SENP2–/–(F,H,J,L) were examined in whole mounts (E–H) or transverse sections (I–L) at E9.5 (E,F,I,J) and E10.5
(G,H,K,L). Labyrinth (L), spongiotrophoblast (S) and TGC (G) layers are defined by blue, red and green broken lines, respectively. Note that TGC layer is
missing because of the very few cells present at E10.5 (L).
(M–R) Sections of the E7.5–E8.5 extraembryonic tissues were analyzed by in situ hybridization of the ectoplacental cone (epc) marker Tpbpa (M,P) and
immunostaining of the chorion (Ch) marker Cdx2 (N,O,Q,R), and counterstaining with nuclear fast red and hematoxylin, respectively.
(S) The graph shows the average diameter of the control (þ/þ,þ/–) and mutant (–/–) E10.5 placentas (p , 0.0001, n¼7). Scale bars, 1 mm (A–H); 500 lm
(I–L); 300 lm (M,P); 50; lm (N,O,Q,R).
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TGC differentiation by in situ hybridization of Hand1. Hand1
is required for cell fate determination of TGC, as mice
without Hand1 lack TGCs [26]. Hand1 expression was
detected in the SENP2-null TGCs, suggesting that the initial
induction of TGCs was not affected by the loss of SENP2
(Figure 4W–4Z and 4W9–4Z9). However, later developmental
processes of TGC were impaired in the mutants.
The abnormal development of TGC caused by SENP2
deficiency was further tested using an in vitro differentiation
analysis. The SENP2þ/þand SENP2–/–blastocysts were isolated
at E3.5, and cultured to induce TGC differentiation. TS cells
growing out from the trophectoderm soon attached to the
cultured plates, differentiated, and formed a single tropho-
blast layer. No noticeable difference was observed between
the SENP2þ/þand SENP2–/–blastocysts before hatching (Figure
5A and 5B). About equal amounts of ICM and trophoblast
cells developed after 3 d in culture (Figure 5C and 5D).
However, although the differentiated TGCs were evident in
the SENP2þ/þcultures, their number was significantly reduced
in the SENP2–/–cultures after 6 d (Figure 5E–5H). The average
number of TGC dropped from 40 in the SENP2þ/þculture to
15 in the SENP2–/–(Figure 5I, p¼0.005, n¼6). Consistent with
our in vivo findings, these data suggest that TGC differ-
entiation is severely affected by the loss of SENP2. The results
suggest an essential role for SENP2 in TGC development
during early placentation.
Cell Cycle Defects Caused By SENP2 Deficiency
The SENP2 mutation led to abnormalities in trophoblast
progenitors at niche sites and their development into all
three major trophoblast lineages. These findings imply that
SENP2 might have a general role in cellular regulations
important for expansion of precursors and their differ-
entiation. We speculated that decreases in the numbers of
trophoblast progenitors and specialized cell types might be
due to alterations in cell survival. However, we failed to detect
differences in apoptosis caused by the mutation in tropho-
blast stem cell niches and all three major trophoblast layers in
vivo, or in TS cell culture in vitro (Figure S2). We then
examined whether SENP2 has an important function in the
cell cycle. Investigating the expansion of trophoblast progen-
itors at the niches revealed a deficiency in their cell cycle
progression. The expression of Ki67, a marker detected in all
phases of mitotic cells [18], was detected in virtually all
trophoblast progenitors in stem cell niches, including
extraembryonic ectoderm, chorion, and ectoplacental cone
(Figure 6A, 6C, 6E, and 6G), suggesting that they are actively
cycling cells. We next examined the cell cycle progression rate
among actively cycling cells by measuring the DNA synthesis
rate at S phase using BrdU labeling [18] for 1 h (Figure 6B, 6D,
6F, and 6H). BrdU incorporation specifically measures the
rate of cell cycle progression at S phase, whereas Ki67
identifies all phases of mitotic cells. The cell cycle progression
index (% BrdU-positive cells / % of Ki67-positive cells 3102)
among actively cycling cells decreased 18 units in the mutants
(SENP2þ/þand SENP2þ/–, 67; SENP2–/–, 49; p¼0.0001, n¼6) in
the stem cell niches (Figure 6M). These data suggest a delay in
cell cycle progression of trophoblast progenitors caused by
the SENP2 deletion.
Next, we determined whether similar deficiencies also
affect development of the spongiotrophoblast layer. We
found that this layer expanded rapidly in the wild-type
placenta, but not in the mutants, between E9.5 and E10.5
(data not shown). A portion of the SENP2þ/þspongiotropho-
blasts exited the cell cycle at E10.5 (Figure 6I), whereas almost
all of the SENP2–/–spongiotrophoblasts remained Ki67-
positive (Figure 6K). In E10 SENP2þ/þand SENP2–/–placentas,
Figure 3. Developmental Defects of the SENP2-Null Labyrinth and
Spongiotrophoblast Layers
Sections of E9.5–E11.5 placentas were analyzed by in situ hybridization of
Gcm1 (A,B,D,E), Ctsq (C,F), or Tpbpa (M,N,P,Q) and counterstained with
nuclear fast red (A–F,M,N,P,Q), by histology (G,J,O,R) and immunostaining
of laminin (H,K) or cyclin D1 (I,L), and by counterstaining with
hematoxylin (H,I,K,L).
(A,D) The Gcm-1-positive trophoblast precursors localized to the invasion
site were found in both the E9.5 SENP2þ/þand SENP2–/–placentas.
(B,E) At E10, the SENP2 deletion caused an aberrant reduction in the
Gcm-1 expressing cells. The Gcm-1-positive syncytiotrophoblasts failed
to form an elongated multinuclear structure.
(C,F) The Ctsq-positive cytotrophoblasts identified in the E11.5 wild-type
labyrinth were missing in the mutant.
(G,J) Arrows and arrowheads indicate maternal blood spaces surrounded
by trophoblasts and fetal blood spaces surrounded by endothelia,
respectively.
(H,K) Laminin-labeled basement membrane, highlighting fetal blood
spaces.
(I,L) Cyclin D1 identified the proliferating endothelial cells.
(M,N,P,Q) The number of the Tpbpa-expressing spongiotrophoblasts was
drastically reduced by the loss of SENP2.
(O,R) The thickness of the spongiotrophoblast layer, defined by broken
red lines, decreased significantly.
G, TGC; L, labyrinth; M, maternal decidua; S, spongiotrophoblast. Scale
bars, 200 lm (A,D); 100 lm (B,C,E,F); 50 lm (G–L,O,R); 500 lm (M,N,P,Q).
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spongiotrophoblasts were all positive for Ki67, indicating that
they are actively cycling cells (Figure 6J and 6L). However, the
cell cycle progression index, which mainly reflects the BrdU
incorporation rate, was reduced from 72 in the controls to 53
in the SENP2 mutants (p ¼ 0.0005, n ¼ 3) (Figure 6N). To
examine whether cycling of TGC was also affected by SENP2
deficiency, we determined its cell cycle progression index
(Figure 6O). The cell cycle progression index of TGC
Figure 4. Development of TGCs is Impaired in the SENP2 Mutants
(A–H) Histological analysis of the SENP2þ/þ(A–D) and SENP2–/–(E–H) placentas revealed impaired development of both primary (18G; A,B,E,F) and
secondary (28G; C,D,G,H) caused by SENP2 ablation at E8.5 (A,E), E9.5 (B,C,F,G) and E10.5 (D,H).
(I–P,W–Z9) TGC development was examined by in situ hybridization analysis of specific markers PL-I (I–P) and Hand1 (W–Z and W9–Z9) at the stages
shown (E7.5–E10.5). Stained (blue) sections were counterstained with nuclear fast red. In (I, M), enlargements of the left insets are shown on the right
insets.
(Q–V) Immunostaining of p450scc characterized the TGC in SENP2þ/þ(Q–S) and SENP2–/–(T–V) at E8.5 (Q,T), and E9.5 (R,S,U,V). Immunostained (brown)
sections were counterstained (blue) with hematoxylin.
The TGC layers are defined by broken green lines (A–E,G,H,Q–Z,W9–Z9). AN, anterior neural fold; Em, embryo; G, TGC layer; M, maternal decidua; PS,
primitive streak; S, spongiotrophoblast layer; Yc, yolk sac cavity. Scale bars, 500 lm (I–P); 100 lm (A,B,E,F,Q,R,T,U); 50 lm (C,D,G,H,S,V,W–Z,W9–Z9).
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decreased from 60 in SENP2þ/þto 41 in SENP2–/–(p¼0.0034, n
¼ 4). Taken together, these results suggest that cell cycle
progression was defective in all stem cell niches, and the
spongiotrophoblast and TGC layers, of SENP2 mutants. The
SENP2 mutant cells were trapped or arrested in the cell cycle.
SENP2 Is Essential for the G–S Transition in Mitosis and
Endoreduplication
To further examine stem cell expansion and development,
we derived a number of SENP2–/–TS cell lines from
blastocysts. Immunostaining analyses of Oct4 (an ES cell
marker) [27] and Cdx2 (a TS cell marker) [20] confirmed that
we were able to successfully establish the SENP2-null TS cell
lines (Figure S3). The proliferation rate (BrdU labeling for 1 h)
of the SENP2-null TS cells in vitro was also reduced, compared
to that of the wild-type cells (p ¼ 0.013, n ¼ 3) (Figure 7A).
Although the deficiency in cell cycle progression was also
demonstrated using the TS cells in vitro, the degree of severity
was reduced compared with that seen in the in vivo studies. As
we were aware, the in vitro system does not always recapitulate
the dynamic developmental processes that occur in vivo.
Nevertheless, because of the limited materials available from
the early stages of placenta, the TS cell culture does provide a
valuable system to further those of our investigations that are
otherwise impossible to perform in vivo.
To investigate whether a specific phase of the cell cycle was
defective, we then determined the cell cycle profiles of the
SENP2þ/þand SENP2–/–TS cells by flow cytometry analysis of
PI (propidium iodide) stained cells. There was no significant
difference in the cell population of G2–M between SENP2þ/þ
and SENP2–/–cells (Figure 7B). However, in the SENP2 nulls,
the percentage of cells in G0–G1 was increased (p , 0.0001, n
¼ 4) but the percentage in S was decreased (p ¼ 0.0024, n ¼ 4)
(Figure 7B and 7C). This implied that the mutant cells were
affected at the G1–S transition. To test this hypothesis, we
used nocodazole, a microtubule depolymerizing agent, to
block cell division at M phase. Nocodazole was effective in
synchronizing the SENP2þ/þTS cells at G2–M after 6 h (Figure
7D). However, if cells were arrested or trapped in the G1–S
phase and unable to pass through the cell cycle, there would
be a delay in synchronizing cells by the nocodazole treatment.
Indeed, there were still ;7% of the G0–G1 cells in SENP2–/–,
but none in SENP2þ/þ, 3 h after the treatment. After the 6 h
treatment, a significant number (6.16%) of the SENP2–/–TS
cells remained in G0–G1 (Figure 7D). Even after 24 h, this
population arrested in G0–G1 was still present (data not
shown). The results suggest that SENP2 has a pivotal role in
TS cell cycle progression and the G1–S checkpoint might be
affected by the SENP2 ablation.
Immunostaining of nuclear envelopes with lamin B [28]
revealed that nuclei of the SENP2–/–TGCs were significantly
smaller (Figure 8A–8F). In addition, the mutant TGC nuclei
contained smaller and fewer blue dots upon hematoxylin
staining (Figure 8A–8F), suggesting that the DNA content
might be reduced. These abnormalities are likely caused by a
deficiency in endopolyploidy. An important specialized
process for TGC maturation is endoreduplication, whereby
the genome is amplified without a complete mitosis. The
endoreduplication cycle requires only the G and S phases
[29]. To examine the possibility of a defect in endoredupli-
cation, we induced the TS cells to undergo TGC differ-
entiation in vitro by removal of FGF4, heparin, and mouse
embryonic fibroblast (MEF)-conditioned medium (see Mate-
rials and Methods and [30]). Flow cytometric analysis of the
differentiated cells stained with PI showed that the percent-
age of cells with higher DNA contents (.4N) was drastically
reduced in the SENP2 mutants (Figure 8G). The average
percentage of polyploid cells reduced from 25% (SENP2þ/þ) to
7% (SENP2–/–) (p , 0.0001, n ¼ 5) (Figure 8H). Therefore, the
loss of SENP2 induced a severe deficiency in endopolyploidy.
SENP2 apparently has a dual role in regulating the G–S
transition of mitotic division and endoreduplication during
TS cell proliferation and differentiation, respectively.
Disruption of SENP2 Alters the p53–Mdm2 Circuit in
Trophoblast Development
The cell cycle defects led us to investigate potential
downstream targets involved in trophoblast development.
We specifically focused on those regulators shown to be
conjugated by SUMO. Previous reports showed that SENP2
(also known as Axam) modulates the canonical Wnt pathway
by interacting with its signaling molecules [14,31]. Even
though this led us to identify SENP2 through its binding to
Axin initially, we failed to detect any alteration of Wnt
signaling in the SENP2 mutants. Nor were we able to show
other alternative pathways critical for placentation, e.g.,
Figure 5. In Vitro Differentiation of SENP2-Null Blastocysts into Trophoblast Cells Is Defective
(A–H) Isolated SENP2þ/þ(A) and SENP2–/–(B) blastocysts were cultured for trophoblast differentiation in vitro. Images were taken at culturing day 1 (A,B),
day 3 (C,D) and day 6 (E–H). TS cells, outgrowing from the trophectoderm, differentiated into a single trophoblast cell (TC) layer, whereas the ICM
formed aggregates and sat on top of the trophoblast cells (C,D).
Arrows indicated TGCs present in the cultures (E,F). The cultures were then analyzed by immunostaining of a trophoblast specific marker p450scc
(brown) and counterstaining of hematoxylin (blue) on day 6 (G,H).
(I) The graph represents the average number of TGC present in the SENP2þ/þand SENP2–/–cultures (p ¼ 0.005, n ¼ 6).
Scale bars, 200 lm (E–H); 100 lm (C,D); 50 lm (A,B).
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MAPK and SAPK [32–36], to be involved in the SENP2-
dependent developmental processes. However, when p53 was
examined by immunostaining, we detected an aberrant
accumulation in the nuclei of the developing SENP2–/–TGCs
at E8.5–E10.5 (Figure 9D–9F). In contrast, the SENP2þ/þTGCs
showed no detectable, or very low if any, p53 at these stages
(Figure 9A–9C). The results implied that there is a deficiency
in p53 regulation caused by the SENP2 deletion. Degradation
of p53 is mediated by ubiquitination-dependent proteolysis.
Mdm2, a RING finger E3 ubiquitin ligase that binds to p53,
has an essential role in this process [37–40]. We therefore
tested whether the loss of SENP2 had an effect on Mdm2.
Immunostaining of Mdm2 revealed its localization in both the
SENP2þ/þcytoplasm and the nucleus during early stages
(E8.5–E9.5) of TGC development (Figure 9G and 9H).
However, Mdm2 was mainly located to the nuclei of the
terminally differentiated TGCs at E10.5 (Figure 9I). The
differential subcellular distribution of Mdm2 implies that it
might be critical for development of TGCs. In contrast,
Mdm2 accumulated in nuclei throughout TGC development
in the absence of SENP2 (Figure 9J–9L). The prominent
cytoplasmic staining was lost in the mutants at E8.5–E9.5
(Figure 9J and 9K). Furthermore, the loss of SENP2 also
affected Mdm2 localization in the stem cell niche sites, such
as extraembryonic ectoderm and chorion. Mdm2 clearly
accumulated in the nuclei of the SENP2–/–trophoblast
progenitors, but was evenly distributed in the whole cells of
the controls (Figure 9M and 9N). Similar nuclear accumu-
lations of Mdm2, affecting the p53 level, were also detected in
the SENP2–/–labyrinth and spongiotrophoblast layers (data
not shown). Therefore, Mdm2 appeared to be aberrantly
localized in the stem cell niches and all three major layers of
trophoblast during early embryogenesis. The data suggest
that SENP2 is required for proper localization of Mdm2 and
degradation of p53. Disturbance of SUMO modification by
the SENP2 deletion thus causes deregulation of the p53–
Mdm2 pathway, leading to deficiencies in mitotic and
endoreduplication cell cycle progression and abnormal
trophoblast development.
The accumulation of p53 in the nuclei of SENP2-null
placentas implied that SENP2 negatively modulates the p53–
Mdm2 circuit. To determine the role of p53–Mdm2 in
trophoblast development, we investigated whether SENP2
modulates Mdm2 and p53 at the posttranscriptional level. In
addition to altering the subcellular distribution of Mdm2, the
loss of SENP2 had an effect on posttranslational modification
of Mdm2. The loss of SENP2 disturbed desumoylation of
Mdm2. In the SENP2–/–TS cells, Mdm2 accumulated in the
SUMO conjugated state (Figure 9O). The loss of SENP2
disturbed the ratio of Mdm2 and Mdm2–SUMO. The
sumoylated Mdm2 could also be detected by an anti-SUMO-
1 antibody (Figure 9O) as well as immunoprecipitation–
immunoblot analysis using anti-Mdm2 and anti-SUMO-1
antibodies (data not shown). We encountered a technical
problem in determining the actual amount of the sumoylated
Mdm2 by immunoprecipitation–immunoblot analysis. This is
likely because desumoylation occurs rapidly in isolated cell
extracts whereas immunoprecipitation requires proteins in a
native conformation. Therefore, a straight immunoblot assay
appears to be better suited for quantitative measurements.
To determine whether SUMO modification of Mdm2 is
regulated by SENP2, a plasmid expressing a Myc-tagged
SENP2 (MT–SENP2) under the control of a CMV promoter
was transiently transfected into the mutants. The reintro-
duction of SENP2 altered the ratio of Mdm2 and Mdm2–
SUMO and diminished the level of Mdm2–SUMO, suggesting
that its desumoylation is modulated by SENP2 (Figure 9O).
Immunoblot analysis also revealed an elevation of p53 caused
by the SENP2 deletion in TS cells (Figure 9P). Although p53 is
Figure 6. Defects in Trophoblast Cell Cycle Progression Caused by SENP2
Deficiency
(A–H) In sections of the E7.5 and E8.5 SENP2þ/þ, SENP2þ/–and SENP2–/–
extraembryonic structures, Ki67 staining identified trophoblast progen-
itors undergoing cell cycle progression at the trophoblast stem cell
niches (A,C,E,G). BrdU labeling for 1 h, performed on adjacent sections,
detected the progression rate at S phase (B,D,F,H).
(I–L) SENP2þ/þ(I,J) and SENP2–/–(K,L) spongiotrophoblasts were analyzed
by immunostaining of Ki67 at E10.5 (I,K) and E10 (J,L). Asterisks indicated
the Ki67-negative cells in the SENP2þ/þspongiotrophoblast layer (I). The
adjacent section of E10 placentas were stained with anti-BrdU to obtain
the progression rate.
(M) The graph represents cell cycle progression index, which is the
average progression rate among actively cycling cells (% of BrdU divided
by % of Ki67 3 102), at all stem cell niches, extraembryonic ectoderm,
chorion and ectoplacental cone (p ¼ 0.0001, n ¼ 6). The positive and
negative cells were counted to obtain the percentages of BrdU- and
Ki67-positive cells.
(N,O) The graphs represent the cell cycle progression index of the
spongiotrophoblast (N; p¼0.0005, n¼3) and TGC (O; p¼0.0034, n¼4)
layers.
Ch, chorion; epc, ectoplacental cone; exe, extraembryonic ectoderm; G,
TGC; M, maternal decidua; L, labyrinth; S, spongiotrophoblast. Scale bars,
50 lm (A–L).
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known to be sumoylated, we did not detect obvious
accumulations of the SUMO-conjugated form caused by the
SENP2 ablation. We then tested whether SENP2 is required to
mediate the downregulation of p53 by overexpression of MT–
SENP2. Consistent with our hypothesis, p53 levels were
significantly reduced in the SENP2-null cells transiently
transfected by MT–SENP2 (Figure 9P).
To further confirm that the loss of SENP2 was the primary
cause of the trophoblast defects, we reintroduced MT–SENP2
into SENP2–/–cells. To determine the differentiation process
affected by SENP2 at a more quantitative level, we examined
the expression of a TGC marker, p450scc, by immunoblot
analysis. The expression of p450scc was drastically reduced in
SENP2–/–placentas, confirming the TGC developmental
defects (Figure 9Q). The expression of p450scc was not
detectable in SENP2þ/þTS cells but was highly increased in
the differentiated TGCs, suggesting the success of the in vitro
culture system (Figure 9Q). We did not detect a great
Figure 7. SENP2 Is Critical for the G1–S Transition of Mitotic Division in TS Cells
(A) BrdU labeling for 1 h measured the proliferation rate of the SENP2þ/þand SENP2–/–TS cells in vitro. The graph shows the average percentages of the
BrdU-positive cells in three independent experiments (p ¼ 0.0013, n ¼ 3).
(B,C) Flow cytometric analysis of the PI-stained SENP2þ/þand SENP2–/–TS cells to determine their cell cycle profiles. The result shown in (B) is a
representative of four independent experiments, and the graph in (C) shows the average percentage of the G0–G1 and S populations (n ¼ 4). A
consistent increase in the G0–G1 population (p , 0.0001) and decrease in the S population (p ¼ 0.0024) was detected in the SENP2 mutants.
(D) The SENP2þ/þand SENP2–/–TS cells were treated with nocodazole for 0, 3 and 6 h as indicated. Flow cytometric analyses showed that there was a
delay in synchronizing the SENP2–/–cells upon the nocodazole treatment.
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induction of p450scc in the differentiated SENP2–/–cells,
consistent with our in vivo findings (Figure 9Q). The
reintroduction of MT–SENP2 in the SENP2 mutants led to
an induction of p450scc upon TGC differentiation (Figure
9Q). The p450scc induction level did not reach that of the
SENP2þ/þTGCs, most likely due to the transfection efficacy, in
that not all of the mutants were transfected. Nevertheless,
these data demonstrate that reintroducing SENP2 into the
SENP2–/–TS cells can promote their differentiation into
TGCs. This suggests that SENP2 inactivation is the cause of
the trophoblast developmental defects observed in the
mutants. An aberrant stimulation of p53 might be responsible
for the SENP2-null defects in mitotic division and polyploidy.
In the SENP2 mutants, the dislocation of Mdm2 implied
that its distribution is regulated by the SUMO pathway. We
therefore investigated whether Mdm2 localization is affected
by SUMO. First, immunoblot analysis after cell fractionation
showed that sumoylated Mdm2 is found preferentially in the
nuclear fraction of SENP2–/–cells (Figure 9R). Next, we
examined whether SUMO conjugation alters the subcellular
distribution of Mdm2 in live cells. GFP analysis of TS cells
transiently expressing GFP-tagged Mdm2 or Mdm2–SUMO-1,
revealed their preferential localization. We found that Mdm2
mainly accumulated in the cytoplasm (Figure 9S and 9V), with
occasional distribution to the whole cell (Figure 9T).
However, Mdm2–SUMO-1 displayed a clear nuclear accumu-
lation (Figure 9U), with either a punctated (Figure 9W) or a
nucleolar (Figure 9X) staining pattern. Similar results were
also obtained by the use of Mdm2–SUMO-1GG96–97D, a mutant
lacking the last two glycine residues of SUMO-1, which
prevent further conjugation that might affect subcellular
distribution (data not shown). Therefore, the SENP2 medi-
ated SUMO modification of Mdm2 appears to be crucial for
its subcellular trafficking.
The Requirement for p53 in Mediating the Deficiencies of
SENP2-Null Mutants
To address the importance of p53 in mediating the SENP2-
null phenotype, we tested whether p53 activation is necessary
and sufficient to affect trophoblast proliferation and differ-
entiation. We used both gain-of-function and loss-of-function
analyses. Nutlin-3 is a potent small-molecule antagonist of
Mdm2, which binds to the p53-binding pocket of Mdm2 and
prevents its interaction, thereby stabilizing p53. We first
determined that the Nutlin-3 treatment of the SENP2þ/þcells
could elevate p53 in a dosage-dependent manner, but, most
importantly, to reach the level detected in the SENP2–/–TS
cells (Figure 10A). To examine whether the p53 elevation
induced G1–S arrest, TS cells were treated with Nutlin-3. A
cell cycle profiling assay showed that the Nutlin-3 treatment
caused the wild-type TS cells to accumulate in G0–G1 phase,
similar to the SENP2–/–TS cells (Figure 10C). Next, we
examined whether the elevated level of p53 interfered with
the differentiation process. In the SENP2þ/þTS cells induced
for TGC differentiation, Nutlin-3 significantly reduced the
expression of the TGC marker p450scc (Figure 10E), and
prevented TGC differentiation (Figure 10F–10K). The aver-
age number of TGC decreased significantly in the presence of
Nutlin-3 (Figure 10L, p ¼ 0.006, n ¼ 4). These results support
the hypothesis that stimulation of p53 by alteration in Mdm2
activity induces phenotypic defects in trophoblast prolifer-
ation and differentiation, resembling those observed in the
SENP2 mutants.
To determine whether downregulation of p53 was able to
alleviate the trophoblast deficiencies caused by the SENP2
ablation, we knocked down its cellular levels using an RNA
interference (RNAi) approach. First, immunoblot analysis
showed that the p53 RNAi treatment successfully diminished
its levels in the SENP2–/–TS cells (Figure 10B). The p53 RNAi
treatment also promoted the G1–S transition of the SENP2–/–
TS cells arrested in G0–G1 (Figure 10D). Furthermore,
downregulation of p53 enhanced TGC differentiation of the
SENP2–/–cells, as determined by the expression of p450scc
(Figure 10E). These data demonstrated that stimulation of
p53 is not only necessary to mediate the SENP2-null defects,
but is also sufficient to induce deficiencies in expansion of
trophoblast stem cells and their maturation.
Discussion
This study demonstrates an essential role of SENP2 in
trophoblast lineage development during placentation. All
three major trophoblast layers were affected by SENP2
deficiency. Our data provide an important connection
between SENP2 and the p53–Mdm2 pathway in trophoblast
Figure 8. SENP2 Is Required for Trophoblast Maturation
(A–F) Immunostaining of the E8.5 (A,D), E9.5 (B,E) and E10.5 (C,F) SENP2þ/
þ(A–C) and SENP2–/–(D–F) placentas with lamin B, which marks nuclear
envelopes, shows the size of nuclei. The TGC layers are defined by
broken green lines. The stained (brown) sections were counterstained
(blue) with hematoxylin. Note that the SENP2-null TGCs (D–F) contain
smaller nuclei with less dotted staining (representing nucleoli and
heterochromatin) than the controls (A–C).
(G) Endoreduplication is impaired by the loss of SENP2. The SENP2þ/þand
SENP2–/–TS cells were induced for differentiation into TGCs in vitro. Flow
cytometric analysis of the differentiated SENP2þ/þand SENP2–/–cells,
stained with PI, was used to measure their DNA contents (M1, two to
four copies; M2, more than four copies). The diagram in (G) is a
representative of five independent experiments; the average percen-
tages of the SENP2þ/þand SENP2–/–polyploid cells in all five cultures is
presented in (H) (p , 0.0001, n ¼ 5).
G,TGC layer; M, maternal decidua; S, spongiotrophoblast layer. Scale bars,
50 lm.
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development. The loss of SENP2 caused a deficiency in the G–
S transition, which is required for both the mitotic cell cycle
(containing G1, S, G2, and M phases) and the endocycle
(containing only the G and S phases) during trophoblast
proliferation and differentiation, respectively. The cell cycle
regulators p53 and Mdm2 appear to be critical for SENP2-
dependent trophoblast mitosis and polyploidy. We propose
that the SENP2–Mdm2–p53 pathway has a dual role in the G–
S checkpoint of mitotic division and endoreduplication
(Figure 11A). Although high levels of p53 induce a G1 arrest,
a low level may be necessary to go through the rest of mitosis,
such as through the tetraploid checkpoint. Because of the
omission of M phase in endoreduplication, repression of p53
is essential to produce polyploid cells. Our findings further
suggest that SENP2-dependent SUMO modification controls
the subcellular localization of Mdm2 (Figure 11B). Sumoy-
lated Mdm2, which preferentially accumulates in the nucleus,
likely cannot modulate p53, whereas desumoylated Mdm2,
which can move freely to the cytoplasm, is capable of p53
degradation.
Figure 9. SENP2 Regulates the p53–Mdm2 Circuit During Trophoblast
Development
(A–N) Sections of the E7.5 (M,N), E8.5 (A,D,G,J), E9.5 (B,E,H,K) and E10.5
(C,F,I,L) SENP2þ/þor SENP2þ/–(A–C,G–I,M) and SENP2–/–(D–F,J–L,N)
placentas were stained with an anti-p53 (A–F) or anti-Mdm2 antibody
(G–N). The stained (brown) sections were counterstained with hematox-
ylin (blue).
(D–F) Nuclear accumulations of p53 (arrows) were detected in the SENP2
mutants.
(G–I) In the SENP2þ/þTGCs, Mdm2 predominantly accumulated in the
cytoplasm at E8.5 and E9.5 (arrowheads; G,H), but in the nucleus at E10.5
(arrows; I).
(J–L) Nuclear accumulations of Mdm2 were found throughout the
SENP2–/–TGC development at E8.5–E10.5 (arrows; J,K,L). The TGC layers
are defined by broken green lines.
(M,N) Mdm2 showed clear nuclear localizations in the SENP2–/–
trophoblast progenitors at the niche sites (N), whereas it was evenly
distributed in the controls (M). Enlargements of the insets are shown.
(O) SUMO modification of Mdm2 is regulated by SENP2. Immunoblot
analysis with anti-Mdm2 and anti-SUMO-1 antibodies shows that Mdm2
accumulated in its sumoylated state (Mdm2–SUMO) in the SENP2–/–
trophoblast cells. Two different cell lines (#1 and #2) were examined. The
Mdm2–SUMO band could also be detected by immunoprecipitation–
immunoblot with anti-Mdm2 and anti-SUMO-1 antibodies (data not
shown). Reintroduction of SENP2 into the SENP2–/–TS cells diminished
the Mdm2–SUMO level. Actin level also was analyzed as a loading
control. The number indicates the ratio of Mdm2–SUMO and Mdm2.
(P) The p53 protein level is regulated by SENP2. Protein lysates were
isolated from the SENP2þ/þand SENP2–/–TS cells with or without
transfection of MT–SENP2. Immunoblot analysis with an anti-p53
antibody revealed the steady state levels of p53 and actin (loading
control). Inactivation of SENP2 induced an accumulation of p53 in
trophoblasts. Reintroduction of SENP2 down regulated p53 in the SENP2-
null mutants. The number represents the expression level of p53 in
SENP2–/–relative to that in SENP2þ/þ.
(Q) SENP2 is necessary and sufficient to induce trophoblast differ-
entiation. Protein lysates were isolated from the SENP2þ/þand SENP2–/–
placentas at E10.5, and the SENP2þ/þand SENP2–/–TS cells with or
without transfection of MT–SENP2. The TS cells were cultured in
differentiation media for 6 d to obtain the differentiated TGCs.
Immunoblot analysis with an antibody that recognizes either p450scc
or MT revealed the steady state protein level. The levels of ER protein
calnexin and actin were analyzed as loading controls. The number shows
the quantitative difference in p450scc expression.
(R) Preferential accumulations of Mdm2 (arrow) and Mdm2–SUMO
(arrowheads) in TS cells. Nuclear (N) and cytoplasmic (C) extracts of
SENP2–/–were analyzed by immunoblot with anti-Mdm2 and anti-SUMO-
1 antibodies. Asterisk indicates non specific reaction detected after cell
fractionation.
(S–X) Mdm2 and Mdm2–SUMO are differentially localized in the cell. TS
cells transfected by the GFP-tagged Mdm2 (S,T,V) or Mdm2–SUMO-1
fusion (U,W,X) under control of a CMV promoter were analyzed by GFP
analysis with either phase contrast (S–U) or by immunofluorescence
microscopy (blue, DAPI) (V–X).
G, TGC layer; M, maternal decidua; S, spongiotrophoblast layer; Yc, yolk
sac cavity. Scale bars, 50 lm (A–N,S–U); 20 lm (V–X).
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This study provides evidence to support an important
function of p53, as a guardian of the genome to control
polyploidy. An endoreduplication deficiency was previously
observed in embryos lacking cyclin E proteins [41]. In
contrast to the SENP2-null deficiencies, the loss of cyclin E
proteins did not affect TGC differentiation. It is conceivable
that cyclin E, which functions in late G1 phase to promote S-
phase entry, acts further downstream of the SENP2–Mdm2–
p53 pathway. In the SENP2 mutants, we detected alterations
of this regulatory pathway not only in the stem cell niche site,
but also in the differentiated trophoblast layer. A recent
report found that an increased number of TGCs were
detected in the p53-null placentas [42], further supporting
our hypothesis. SENP2 might also be involved in a crucial step
of p53-dependent aneuploidy, genome instability and tu-
morigenesis [43]. Polyploid cells have several different fates.
They can arrest in the cell cycle mediated by the tetraploidy
checkpoint, which then triggers apoptosis. However, the lack
Figure 10. Repression of p53 Is Necessary and Sufficient to Promote Trophoblast Proliferation and Differentiation
(A,B) Nutlin-3 stimulates p53 in a dosage-dependent manner (A) and accumulation of p53 in the SENP2-nulls can be knocked down by RNAi (B). Protein
lysates, isolated from SENP2þ/þand SENP2–/–TS cells treated with Nutlin-3 (A) or transfected by p53 RNAi (B), were analyzed for the p53 expression by
immunoblot. Calnexin was used as a loading control.
(C) Activation of p53 by Nutlin-3 caused a delay in the G1–S transition. Flow cytometric analysis of PI-stained SENP2þ/þTS cells determined the cell cycle
profiles without Nutlin-3 or affected by Nutlin-3 treatment for 24 or 48 h. The Nutlin-3 (8 lM) treatment induced a cell cycle arrest at G1–S.
(D) The p53 RNAi treatment alleviates the cell cycle defects caused by the SENP2 deletion. The SENP2–/–TS cells with or without p53 RNAi (100 nM) were
treated with nocodazole for 30 h. Flow cytometric analyses revealed that the cell population arrested in G0–G1 of SENP2–/–TS cells was reduced by the
p53 knockdown. (C,D) are representatives of two independent experiments.
(E) Stimulation of p53 is necessary and sufficient to inhibit trophoblast maturation. Protein lysates, isolated from SENP2þ/þand SENP2–/–cells with or
without the Nutlin-3 (8 lM) treatment and the transfection of p53 RNAi, were analyzed by immunoblot analysis for the expression of a TGC marker
p450scc. Calnexin was used as a loading control.
(F–K) Nutlin-3 inhibits differentiation of blastocysts into TGCs. Isolated blastocysts were cultured for trophoblast differentiation in the absence (F–H) and
presence (I–K) of Nutlin-3 (8 lM) in vitro. Images were taken at culturing day 1 (F,I), day 3 (G,J) and day 6 (H,K). The cultures were then analyzed by
immunostaining of a TGC-specific marker p450scc (brown) and counterstaining by hematoxylin (blue) on day 6 (H,K). Asterisks indicate TGCs.
(L) The graph shows the average number of TGC present in the cultures (p ¼ 0.006, n ¼ 4).
Scale bars, 100 lm (F–K).
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SENP2 in Trophoblast Development

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of p53 allows these cells, as they escape from the arrest to
undergo multipolar mitosis, to become aneuploid [44–46].
The nature of trophoblast development provides a system to
elucidate the regulatory mechanism underlying polyploidy.
Because of the biochemical activity of SENP2, the SENP2-null
model offers a unique opportunity to further investigate the
modulation of the p53–Mdm2 circuit by SUMO in normal
developmental programming of polyploidy. The knowledge
obtained here might be applicable to malignant trans-
formation processes associated with polyploidy.
SENP2 is also known as Axam, which has been shown to
modulate Wnt signaling by interacting with Axin, a scaffold
protein involved in targeting b-catenin for degradation
[14,17]. Although biochemical studies suggested that SENP2
could regulate the canonical Wnt pathway by SUMO
modulation of a LEF/TCF transcription factor [31], there
was no in vivo evidence to support this idea. We failed to
detect alterations in Wnt–b-catenin signaling in the SENP2
mutant placentas (SC and WH, unpublished data) although
this might occur in other tissues. SUMO modification of Axin
has been shown to modulate its effects on JNK signaling [36].
Neither JNK, nor the related p38 and Erk1/2 factors that are
important for placental function [32–35], seem to be involved
in the SENP2-mediated trophoblast development (SC and
WH, unpublished data). However, we identified the p53–
Mdm2 pathway as a downstream target of SENP2. Our data
imply that SUMO modification mediated by SENP2 is
required for proper localization and function of Mdm2,
which in turn controls p53 stability during trophoblast
development. Not only does stimulation of p53 induce
phenotypic defects resembling those of the SENP2 inactiva-
tion, but downregulation of p53 alleviates the trophoblast
deficiencies caused by SENP2 deficiency. It is conceivable that
Wnt or JNK/SAPK signaling regulated by SENP2 is critical for
another cell type and lineage development. The generation of
mouse models permitting conditional inactivation of SENP2
will aid these studies and determine its essential role in other
developmental processes.
The loss of SENP2 disturbs the balance of SUMO
modification. Although sumoylation of Mdm2 has been
described [47], it was not clear whether this modification
dictates subcellular distribution. Our data provide evidence
that cellular distribution of Mdm2 is regulated by the SUMO
pathway. Disruption of SENP2, leading to an accumulation of
Mdm2 ina hyper-sumoylated state, induces its mislocalization.
Many sumoylated proteins, including PML, preferentially
accumulate in specific complexes called PML nuclear bodies
[12]. Sumoylation of PML is essential not only for these
nuclear bodies to form but also for other sumoylated proteins
to concentrate there. Although the biological function of PML
nuclear bodies remains largely elusive, subsequent recruit-
ment of proteins can modulate transcription activity. It has
been shown that sumoylation of PML directs p53 to nuclear
bodies, leading to a stimulation of its transcriptional and pro-
apoptotic activities [48,49]. These effects can be regulated by
sumoylation of p53 [11,50,51]. Because of technical limitations
and, more importantly, SUMO regulation of a number of p53
regulators (Mdm2, MdmX, and PML), the functional con-
sequences of sumoylation have been difficult to elucidate. As
SUMO modification of PML and p53 is a key determinant for
maintaining genome integrity [12], our data imply that SENP2
might mediate this maintenance.
Using a mouse model with disruption of SENP2, this study
suggests a novel role of SUMO modification in cell cycle
progression and induction of polyploidy. Sumoylation, which
dictates Mdm2 trafficking, is crucial for modulation of the
p53–Mdm2 circuit. Further studies focusing on the detailed
mechanistic switch of the SENP2–Mdm2–p53 pathway and its
implications in other developmental and pathogenic pro-
cesses promise important insights into the role of SUMO
modification in mammalian development and disease.
Materials and Methods
Mouse strains. Genomic DNA fragments containing the SENP2
gene (Accession number NC_000082) were isolated by PCR and
cloned into the pGEM vector. The 59 arm contained sequences from
the first coding exon to the beginning of the second coding exon,
which encodes the first 49 amino acids of SENP2. The 39 arm included
parts of the fifth intron and the sixth coding exon. A b-galactosidase
cDNA was fused in-frame to the second coding exon of SENP2. The
Figure 11. Model for the SENP2–Mdm2–p53 Pathway in Trophoblast
Development
(A) Diagram illustrating the p53–Mdm2 circuit regulated by SENP2 in the
trophoblast cell cycle. Stimulation of Mdm2 by SENP2 leads to
degradation of p53. Cellular levels of p53 control the G–S transition
that has a dual role in TGC development. The G–S phase is required for
both mitotic division (cell cycle: G1, S, G2, and M) and endoreduplication
(endocycle: G and S only) during expansion of trophoblast stem cells and
maturation of trophoblasts, respectively. Although a low p53 level is
essential for stem cell proliferation, inhibition of p53 is required upon
differentiation.
(B) Schematic representation for the mechanism underlying the
regulation of p53 and Mdm2 by the SUMO pathway. SENP2 activates
Mdm2 by removing SUMO that permits the modulation of p53 by Mdm2
in the nucleus. The ubiquitin-conjugated p53 is then degraded in the
cytoplasm.
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SENP2lacZ /þmutant ES cell lines were generated by electroporation of
the targeting vector into CSL3 ES cells [52]. Correct homologous
recombination at the SENP2 locus was confirmed by Southern
blotting (Figure S1A). ES cell clones were injected into blastocysts to
generate chimeras that were bred to obtain mice carrying the
targeted allele. Mice were genotyped by PCR analysis using primers
(G1: 59-ctgttttctactgcagtggacac-39, G3: 59-gatacttgtagaaaggcctagtat-39
and K1: 59-taaccgtgcatctgccagtttga-39) to identify the wild-type and
mutant SENP2 locus (Figure S1B). To delete the neo cassette flanked
by two loxP sites, the SENP2lacZ/þstrain was crossed with the Zp3-Cre
strain as described [53]. PCR genotyping was performed to confirm
the removal of neo and the presence of lacZ as described (Figure S1C)
[52]. Care and use of experimental animals described in this work
comply with guidelines and policies of the University Committee on
Animal Resources at the University of Rochester.
DNA and RNA. The pCS2-SENP2 clone, containing the Myc-tagged
SENP2 cDNA, was generated by inserting a blunt-ended 1.7 kb Not1–
Spe1 fragment into the blunt-ended Xho1–Xba1 sites of pCS2 vector
[54]. The GFP-tagged Mdm2 expression vector (pGFP-Mdm2) was
generated by ligation of a full length Mdm2 [55] and GFP (BD
bioscience) cDNA fragments. The GFP-tagged Mdm2–SUMO expres-
sion vector was created by insertion of a SUMO-1 fragment [51] into
the pGFP-Mdm2 plasmid. To generate the pBS-SENP2 clone for
making the RNA probes, a 400 bp BamH1–EcoR1 fragment of the
pCS2-SENP2 clone was cloned into the same restriction sites in pBS
vector (Stratagene). To generate RNA probes for in situ hybridization,
DNAplasmidspBS-Gcm1,pBS-Hand1, pCR4-PL-I, pCR4-Tpbpa, pBS-
Ctsq, and pBS-SENP2 [19,21,23,56] were linearized and transcribed in
vitrousingRNApolymerasesT3,T7,andSP6(Promega).PlasmidDNA
transfection was performed by Lipofectamine 2000 (Invitrogen)-
mediated transfer with 4 lg pCS2-MT–SENP2, 1 lg pGFP-Mdm2, 1 lg
pGFP-Mdm2–SUMO-1, or 10–100 nM p53 siRNA (Santa Cruz). Cells
were plated (1.53105cells in a 30 mm dish for protein extraction, 23
104cells in a 24-well dish for GFP analysis, and 53105cells in a 60 mm
dish for flow cytometry) 24 h prior to the transfection procedure. The
transfected cells were harvested after 48 or 72 h for further analyses.
Total RNA, isolated using Trizol (Invitrogen), was used to produce
cDNA according to the manufacturer’s instructions (SuperScript III,
Invitrogen). The reverse transcription products were subject to PCR
amplifications of the SENP2-lacZ fusion transcript using primers 59-
cagtctctacaatgctgcc-39 and 59-ctgtcactctgatctttgg-39 (exons 3–5), pri-
mers 59-gtgagctgatgagttctgg-39 and 59-gtcgctccaataactttcg-39 (exons 4–
6), primers 59-ggaggagcagaatcatgg-39 and 59-ctcaaaatctcatctggtgg-39
(exons 8–11) and primers 59-cattaccagttggtctggtg-39 and 59-gctgcaa-
taaacaagttccg-39 (lacZ). The PCR reaction was performed by denatu-
ration at 94 8C for 5 min and 30 cycles of amplification (94 8C for 30 s,
53 8C for 30 s, and 72 8C for 45 s), followed by a 7-min extension at 72
8C.
Embryo and cell cultures. Mouse blastocysts were recovered and
cultured in DMEM medium containing 15% FBS, 100 lM b-
mercaptoethanol, 100 lM non-essential amino acid, and 100 lg/ml
penicillin-streptomycin, in a humidified 5% CO2incubator at 37 8C.
Cultured embryos were hatched and attached to dishes after 24–36 h.
The differentiated trophoblasts became identifiable in a few days. For
genotyping, cultured cells were incubated in 10 ll buffer containing
25 mM NaOH and 0.2 mM EDTA, pH 12 for 1 h at 95 8C, followed by
the addition of 10 ll buffer containing 40 mM Tris-HCl, pH 5.0.
Lysates were subject to PCR analysis. The SENP2 wild-type allele was
detected by a nested PCR assay. Primers 59- ctgttttctactgcagtggacac-39
and 59-gctgcctggagtttatctactgtag-39 were used for the first PCR
reaction, performed with 35 cycles of amplification (94 8C for 30 s,
60 8C for 30 s, and 72 8C for 2 min 30 s), followed by a 7-min extension
at 72 8C. Subsequently, the first PCR products were subject to a
second PCR reaction using the method described for genotyping the
SENP2 wild-type mouse strain. For genotyping the SENP2 mutant
culture, the same method for the SENP2 mutant mouse strain was
used.
To establish the TS cell lines [30], blastocysts were recovered in TS
medium (RPMI-1640 medium containing 20% fetal bovine serum, 1
mM sodium pyruvate, 100 lM b-mercaptoethanol, 100 lg/ml
penicillin–streptomycin), plus 25 ng/ml FGF4 and 1 ng/ml heparin.
Briefly, each blastocyst was placed in a culture dish with mitomycin C-
treated MEF feeders and cultured in a humidified 5% CO2incubator
at 37 8C. The blastocysts were hatched and attached to the dishes in
24–36 h. After 48 h, a small outgrowth from a blastocyst was formed
and cultured in TS medium containing 25 ng/ml FGF4 and 1 ng/ml
heparin. After 72–96 h, the outgrowths were ready to be disaggre-
gated by the addition of 0.25% trypsin/EDTA and incubation for 3
min at 37 8C. The disaggregated cells were continuously cultured in
TS medium with the presence of FGF4 and heparin. The TS cell
colonies began to appear after days 6 to 10, and continued to be
cultured until they were about 50% confluent. After expanding the
cultures on the feeders for one or two passages, MEF-free TS cells
were obtained and maintained in media containing 70% MEF-
conditioned medium, 30% TS medium, 37.5 ng/ml FGF4, and 1.5 ng/
ml heparin. To differentiate TS cells into TGC, cells were cultured in
TS medium with no additions [30]. For BrdU labeling of the cultured
cells, 30 lg/ml BrdU (Sigma) was added in the media for 1 h. The
labeled cells were then fixed with methanol/acetone (1:1), followed by
immunostaining analysis. For cell cycle analysis by flow cytometry, 83
105(for mitotic cell cycle) or 105(for endoreduplication cycle) TS
cells were cultured in 6 cm dishes in TS media plus FGF4, heparin,
and MEF-conditioned medium (undifferentiated medium) for 2 d,
and TS media only (differentiated medium) for 6 d, respectively. Cells
were then harvested by trypsinization and fixed in 70% ethanol at 4
8C for at least 24 h. Cells were then treated with RNase (1 mg/ml) for
30 min, followed by PI staining (20 lg/ml) for 10 min at room
temperature. Samples were analyzed by an Epics Elite ESP (Coulter
Electronics) set to collect 10,000 events. The percentage of cells in
G0–G1, S, G2–M or with polyploidy were determined using ModFit
LT software. For synchronizing cells in M phase, 3 lM nocodazole was
added to the media. Nuclear and cytoplasmic fractionations of TS
cells were extracted using an NE-PER extraction kit according to the
manufacturer’s protocol (PIERCE).
In situ hybridization. Paraffin sections were treated with buffer
containing 0.1 M Tris-HCl and 0.1 M EDTA (pH 8.0) plus 1 lg/ml
proteinase K for 30 min, and washed with the same buffer without
proteinase K for 5 min at 37 8C. Samples were then incubated with
buffer containing 0.2 M Tris-HCl (pH 8.0) and 0.1 M glycine for 10
min at room temp, followed by post-fixing with 4% paraformalde-
hyde in PBS buffer for 20 min and a 20-min wash in PBS buffer at
room temperature. The sections were incubated in buffer containing
0.1 M triethanolamine (pH 8.0) for 10 min, followed by 0.25% (v/v)
acetic anhydride in 0.1 M triethanolamine (pH 8.0) buffer for 10 min
and by 23 SSC (13 SSC: 0.15 M sodium chloride and 15 mM sodium
citrate, pH 5.5) buffer for 10 min. After dehydration through ethanol
gradients and air drying for 2 h, sections were incubated with
digoxygenin-labeled probes (1 lg/ml) in 53 SSC buffer containing
50% formamide, 50 lg/ml yeast tRNA and 1% SDS overnight at 70 8C.
Samples were then washed three times with 53SSC buffer for 15 min
at 70 8C and 23SSC buffer containing 50% formamide for 10 min at
45 8C before incubating with buffer containing 20 lg/ml RNase A, 5
U/ml RNase T1, 0.5 M sodium chloride, 10 mM Tris (pH 8.0) and 1
mM EDTA (pH 8.0) for 30 min at 37 8C. After washing with 23SSC for
10 min at 37 8C and 0.13 SSC for 10 min at 45 8C, samples were
incubated in MBST buffer containing 60 mM maleic acid, 0.15 M
sodium chloride, and 0.1% Tween-20, pH 7.5 for 10 min and blocked
with 10% goat serum in MBST for 2 h at room temperature. After
incubating with anti-digoxygenin antibody (Roche) in the blocking
buffer for overnight at 4 8C, sections were washed with NTMT buffer
(100 mM sodium chloride, 100 mM Tris, pH 9.5, 50 mM magnesium
chloride and 0.1 % Tween 20) and incubated in NTMT plus 2 mM
levamisole overnight at 4 8C. To visualize the bound signals, samples
were incubated with BM-purple (Roche) for 2 h to several days. The
reaction was stopped by incubating in PBS buffer, followed by
counterstaining with nuclear fast red.
Histology, immunostaining and immunoblotting. Samples were
fixed, paraffin embedded, sectioned, and stained with hematoxylin/
eosin for histological evaluation as described [57]. Tissue sections
were subject to immunological staining with avidin:biotinylated
enzyme complex as described [18,58]. Proteins were extracted from
TS cells using M-PER reagent (PIERCE) with the addition of protease
inhibitor cocktail (Sigma-Aldrich), 1 mM sodium molybdate, 1 mM
sodium vanadate, and 10 mM N-ethylmaleimide, or SDS lysis buffer
(2% SDS, 10% glycerol, and 50 mM Tris, pH 6.8). Protein extracts
were subject to immunoblotting as described [54]. Bound primary
antibodies were detected with horseradish peroxidase-conjugated
secondary antibodies (Vector Lab), followed by ECL-mediated
visualization (GE HealthCare) and autoradiography. Mouse mono-
clonal antibodies anti-actin (Thermo Fisher; 1:1,000), anti-BrdU
(Thermo Fisher; 1:300), anti-Cdx2 (BioGenex; 1:1), anti-MDM2 (Santa
Cruz; 1:100), and anti-SUMO-1 (Zymed; 1:2,000); rabbit polyclonal
antibodies anti-calnexin (Stressgene; 1:2,000), anti-cyclin D1 (Neo-
marker; 1:100), anti-Ki67 (Neomarker; 1:400), anti-laminin (Sigma-
Aldrich; 1:25), anti-Myc tag (CalBioChem; 1:400), anti-Oct4 (Santa
Cruz; 1:200), anti-p53 (Santa Cruz; 1:50), and anti-p450scc (Chemicon;
1:200); and goat polyclonal antibody anti-lamin B (Santa Cruz; 1:100)
were used as primary antibodies. BrdU incorporation analysis was
performed by intraperitoneal injection of BrdU (250 lg/g of body
weight) into pregnant females for 1 h. Placentas were recovered,
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SENP2 in Trophoblast Development

Page 15

fixed, embedded, sectioned, and subject to immunostaining as
described [18,57].
Supporting Information
Figure S1. Creation of Mice Carrying a SENP2-Null Allele
(A–C) The targeted locus contains an in-frame insertion of lacZ into
the second exon of SENP2 and a pgk-neo gene, flanked by loxP sites,
for positive selection. Diphtheria toxin (DTA) was used for negative
selection. The pgk-neo was removed by Cre-mediated recombination
to generate the null allele as described in Materials and Methods.
Southern (A) and PCR analyses (B,C) examined the targeted and null
alleles. (A) Using a 59 external probe, the EcoRV-digested wild-type
(WT, 10.3 kb) and knock-in (KI, 7.9 kb) bands were detected in the
targeted ES cells by Southern blotting. Mice carrying either the
targeted (B) or the null (C) allele were analyzed by PCR for the WT
and KI alleles and the neo and lacZ genes as indicated.
(D) RT-PCR analyses detected the transcripts of SENP2 and lacZ in
the control (þ/þ) and homozygous (–/–) E10.5 embryos, respectively.
Found at doi:10.1371/journal.pbio.0060310.sg001 (692 KB TIF).
Figure S2. Programmed Cell Death Is Not Affected by the SENP2
Deletion
Sections of the SENP2þ/þ(A, C, E, G) and SENP2–/–(B, D, F, H)
extraembryonic structures were analyzed for terminal deoxynucleo-
tidyl transferase-mediated dUTP–biotin nick end labeling (TUNEL)
staining using fluorescent (green) or immunohistochemical (brown)
assays at E7.5 (A,B) and E9.5 (C–H). No significant differences
between the SENP2þ/þand SENP2–/–were found at the trophoblast
stem cell niches (A,B), labyrinth (C,D), spongiotrophoblast (E,F), or
TGC layers (n ¼ 2).
(I,J) TUNEL staining identifies cell death in the SENP2þ/þ(I) and
SENP2–/–(J) TS cell cultures.
(K,L) TS (K) and mesenchymal (L) cell cultures induced for apoptosis
with 50 lM dexamethasone for 24 h were also analyzed as positive
controls. Fluorescently and immunohistochemically labeled samples
were counterstained with DAPI and hematoxylin, respectively.
(M) The graph represents the average percentage of apoptotic cells in
the SENP2þ/þand SENP2–/–samples (n ¼ 2), and the percentage of
apoptotic cells in TS and mesenchymal (MSC) controls.
Found at doi:10.1371/journal.pbio.0060310.sg002 (3.15 MB TIF).
Figure S3. Development of the SENP2þ/þand SENP2–/–TS Cell Lines
The TS cell lines were derived from blastocysts isolated at E3.5.
Immunostaining analyses of Oct4 (first row) and Cdx2 (second row)
were performed on SENP2þ/þES (first column), SENP2þ/þTS (second
column), and SENP2–/–TS (third column) cells. Cells were counter-
stained by DAPI (third row). Scale bar, 50 lm.
Found at doi:10.1371/journal.pbio.0060310.sg003 (1.73 MB TIF).
Acknowledgments
We thank James C. Cross, Toshio Harigaya, Ron Hay, Hiroaki
Kataoka, and Arnold Levine for reagents; Peter Keng for technical
advice; James C. Cross for discussion; and Anthony Mirando for
critical reading of the manuscript.
Author contributions. SC and WH conceived and designed the
experiments. SC, NA, and WH performed the experiments. SC, NA,
and WH analyzed the data. FC contributed reagents/materials/analysis
tools. SC, FC, and WH wrote the paper.
Funding. WH is supported by National Institutes of Health (NIH)
grant CA106308. FC is supported by NIH grant HD044265.
Competing interests. The authors have declared that no competing
interests exist.
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