DEVELOPMENTAL DYNAMICS 241:16511664, 2012

RESEARCH ARTICLE

Ectopic Expression of Fgf3 Leads to Aberrant

Lineage Segregation in the Mouse Parthenote
Preimplantation Embryos

Developmental Dynamics

Yi-Hui Chen1 and John Yu2,3*

Background: Parthenogenetic mammalian embryos were reported to die in utero no later than the 25somite stage due to abnormal development of both embryonic and extraembryonic lineages. Interestingly,
it has been shown that parthenogenetic ICM cells tend to differentiate more into primitive endoderm cells
and less into epiblast and ES cells. Hence we are interested in studying the molecular mechanisms underlying lineage defects of parthenotes. Results: We found that parthenote inner cell masses (ICMs) contained
decreased numbers of Sox21/Nanog1 epiblast cells but increased numbers of Gata41 primitive endoderm
cells, indicating an unusual lineage segregation. We demonstrate for the first time that the increased
Gata4 level in parthenotes may be explained by the strong up-regulation of Fgf3 and Fgfr2 phosphorylation. Inhibition of Fgfr2 activation by SU5402 in parthenotes restored normal Nanog and Gata4 levels
without affecting Fgf3, indicating that Fgf3 is upstream of Fgfr2 activation. In parthenote trophectoderm,
we detected normal Cdx2 but ectopic Gata4 expression and reduced Elf5 and Tbr2(Eomes) levels.
Conclusions: Taken together, our work provides for the first time the insight into the molecular mechanisms of the developmental defects of parthenogenetic embryos in both the trophectoderm and ICM.
Developmental Dynamics 241:16511664, 2012. V 2012 Wiley Periodicals, Inc.
C

Key words: epiblast; inner cell mass; lineage segregation; mouse embryo; parthenogenetic; primitive endoderm;
trophectoderm
Key findings

Molecular mechanisms of the developmental defects of parthenogenetic embryos were unraveled.

Decreased Sox21 and Nanog1 epiblast cells but increased Gata41 primitive endoderm cells were observed in the
parthenogenetic inner cell mass.

Ectopic Gata4 expression and reduced Elf5 and Tbr2(Eomes) expression were observed in the parthenogenetic
trophectoderm.

Up-regulation of Fgfr2 phosphorylation leads to increased Gata4 and decreased Nanog expression in parthenotes.
Accepted 5 August 2012

INTRODUCTION
Parthenogenesis is a form of asexual
reproduction where the offspring is
derived entirely from an unfertilized
female gamete. It is a normal process

used by some reptiles and birds to

reproduce. In mammals, parthenogenesis usually refers to embryonic
development from an artificially activated oocyte without fertilization by a

sperm. While using normal human

embryos to derive ES cells is ethically
disputable, using parthenogenetic
embryos (called parthenotes), which
are incapable of developing into full

Additional Supporting Information may be found in the online version of this article.
1
Graduate Institute of Aerospace and Undersea Medicine, National Defense Medical Center, Taipei, Taiwan
2
Stem Cell Program, Genomics Research Center, Taipei, Taiwan
3
Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan
Grant sponsor: Genomics Research Center of Academia Sinica; Grant number: 94M011-1; Grant sponsor: NSC; Grant number: 98-3111B-001-003.
*Correspondence to: John Yu, Genomics Research Center, Academia Sinica, Nankang, Taipei 11529, Taiwan.
E-mail: johnyu@gate.sinica.edu.tw
DOI 10.1002/dvdy.23851
Published online 4 September 2012 in Wiley Online Library (wileyonlinelibrary.com).

C 2012 Wiley Periodicals, Inc.

V

Developmental Dynamics

1652 CHEN AND YU

organisms, is less disputable (Sturm

et al., 1994; Kono et al., 1996; Mognetti and Sakkas, 1996). In fact, many
recent studies have demonstrated that
parthenogenesis is an efficient way to
generate histocompatible human embryonic stem (ES) cells for transplantation-based stem cell therapies (Revazova et al., 2008; Hao et al., 2009; Lu
et al., 2010). Histocompatible parthenote ES cells represent an important
milestone in stem cell therapies, as
they allow partial MHC matching to a
substantial population of unrelated
transplant recipients (Cheng, 2008;
Revazova et al., 2008).
In mammalian embryogenesis, the
first lineage segregation event is the
segregation between the trophectoderm and inner cell mass (ICM) lineages, which occurs between the morula and blastocyst stages, and the
second lineage segregation event is the
segregation between the primitive
endoderm and epiblast lineages within
the ICM (Reik et al., 2003; Morgan
et al., 2005; Boyer et al., 2006; Dietrich
and Hiiragi, 2007). A previous study
demonstrated that the ICM cultures
from parthenogenetic embryos contained mostly differentiated parietal
endoderm cells and only a few undifferentiated stem cells, suggesting lineage segregation defects in parthenotes
(Newman-Smith and Werb, 1995). In
order to distinguish between the negative effects of in vitro cultures and
that of parthenogenesis per se, our
study compared gene expression in
parthenotes with that in fertilized
embryos that developed in vitro
instead of in a maternal uterus.
In this study, we analyzed expression of many development-related
genes in parthenote morulae and
blastocysts, including the trophectoderm markers Cdx2, Elf5, and
Tbr2(Eomes), the ICM marker Oct4,
the epiblast markers Sox2 and Nanog,
the primitive endoderm marker
Gata4, and the signal transduction
factors Fgf3, Fgf4, and their receptor
Fgfr2. We found normal levels of
Cdx2, Oct4, and Fgf4, but perturbed
levels of Elf5, Tbr2, Sox2, Nanog,
Gata4, and Fgf3, as well as increased
Fgfr2 phosphorylation in parthenote
embryos. Our study also reveals
greatly increased Gata4 and Fgf3 but

Fig. 1. Total cell numbers in control and parthenote embryos at various preimplantation stages.
All nuclei of all cells in each embryo were stained with DAPI and the total numbers of nuclei (each
representing an individual cell) were counted by MetaMorph. The Students t-test was used for
statistical analyses. Asterisks indicate significantly decreased total cell numbers in parthenote
blastocysts. n 20 for early morulae, n 18 for late morulae, n 19 for early blastocysts, n 21
for late blastocysts. P 0.1 between controls and parthenotes at both the early and late morula
stages, P < 0.01 between controls and parthenotes at both the early and late blastocyst stages.

reduced Tbr2, Sox2, and Nanog

expression in parthenotes compared
to controls, indicating cell fate defects
and impaired differentiation capacity
of parthenote blastomeres. Hence we
suggest that the increased Fgf3-Fgfr2
signaling, the downstream elevation
of Gata4 expression and concomitant
suppression of Nanog expression in
parthenogenetic
preimplantation
embryos constitute the major molecular mechanism leading to lineage segregation defects of parthenotes.

RESULTS
Reduction of Cell Numbers at
the Early and Late Blastocyst
Stages
To exclude the possibility that the in
vitro cultures instead of parthenogenesis per se cause delayed development
of parthenotes, we used embryos developed from in vitro cultured fertilized
oocytes as controls, instead of those
directly from a maternal uterus. As
shown in Figure S1A (which is available online), preimplantation embryos
developed separately from a total of 78
parthenogenetic and fertilized oocytes
were analyzed on consecutive days after oocyte collection (which was
counted as E0.5). In order to compare
the efficiencies of embryonic development between controls and parthenotes during in vitro culturing, we
assessed the percentages of embryos
reaching each developmental stage every day. We counted total cell numbers
in parthenote and control embryos at
both the morula and blastocyst stages.
Morulae obtained at E3.0 usually had

1220 cells and were grouped as early

morulae, whereas morulae obtained
at E3.5 usually had 2533 cells and
were grouped as late morulae. Blastocysts obtained at E3.5, which still
expressed Oct4 and Nanog in the trophectoderm, were grouped as early
blastocysts (Supp. Fig. S2 and data
not shown). On the other hand, blastocysts obtained at E4.5, which have restricted Oct4 and Nanog expression in
the ICM, were grouped as late blastocysts (Supp. Fig. S2).
As shown in Table S1, percentages
of embryos reaching different preimplantation stages from E1.5 (the cleavage stage) to E5.5 (the hatched stage)
were similar between controls and parthenotes. Representative charts of statistical analyses at E3.5, E4.5, and
E5.5 are shown in Supp. Figure S1C.
Taken together, these results indicate
that the developmental timing of parthenote mouse embryos appears normal from E1.5 to E5.5. We also analyzed the total cell numbers in each
embryo by enumerating the DAPIstained nuclei on 3D projections constructed from serial optical sections of
embryos. It was found that total numbers of cells in parthenote embryos
were significantly decreased at the
blastocyst stage (Fig. 1).

Normal Expression of Cdx2

and Reduced Elf51 and Tbr21
Cells in the Trophectoderm of
Parthenotes
It has been reported that Cdx2, Elf5,
and Tbr2(Eomes) are three of the earliest transcription factors initiating

Developmental Dynamics

ABERRANT GENE EXPRESSION IN PARTHENOTE EMBRYOS 1653

trophectoderm differentiation (Russ

et al., 2000; Donnison et al., 2005;
Niwa et al., 2005; Strumpf et al.,
2005; Hemberger and Dean, 2007;
Jedrusik et al., 2008; Ng et al., 2008;
Ralston and Rossant, 2008), and Elf5
directly activates Cdx2 and Tbr2
expression (Choi and Sinha, 2006; Ng
et al., 2008). Hence we assessed the
levels of Cdx2, Elf5, and Tbr2 in parthenote and control embryos by
counting the nuclei stained with specific markers on 3D projections of
each embryo (Fig. 2). It was found
that normal Cdx2 expression in cells
located on the periphery of the morulae (Fig. 2A) and in the trophectoderm
of blastocysts (Fig. 1B, C). On the
other hand, we failed to detect any
specific Elf5 and Tbr2 signals at the
morula stage in either controls or parthenotes (Fig. 2A, D). The lack of Elf5
and Tbr2 expression in normal morulae was also described previously
(Kwon and Hadjantonakis, 2007; Ng
et al., 2008; Ralston and Rossant,
2008; Hemberger et al., 2009).
The distribution of specific markers
in whole embryos is shown in Supp.
Table S2. With similar numbers of
Cdx2 cells in controls and parthenotes (Fig. 2AC), percentages of
Cdx2 cells in whole embryos were
also comparable between controls and
parthenotes (Fig. 2G and Table S2).
On the other hand, both Elf5 and
Tbr2
cells
were
dramatically
reduced in parthenotes compared to
controls at the blastocyst stage (Fig.
2B, C, E, F, and Table S3). The percentages of Elf5 cells are higher
than those of Tbr2 cells, which were
lower than 50% of those in the control
trophectoderm, both at the early and
late blastocyst stages (Fig. 2B, C, E,
F, and Table S2). We found that, while
90% of Elf5 cells are also Tbr2-positive, the remaining 10% of Elf5 cells
do not express Tbr2. Taken together,
our results indicate a normal percentage of Cdx2 cells but reduced percentages of Elf5 and Tbr2 cells in the
parthenote trophectoderm.

Decrease of Nanog1 and

Sox21 cells in Parthenotes
Since there was a great decrease in
total cell numbers of parthenote blastocysts (Fig. 1), we were interested in
assessing the numbers of ICM cells in

parthenote blastocysts. We performed

double immunostaining for Cdx2 and
Oct4, and found normal levels of Oct4
expression but significantly decreased
numbers of ICM cells in parthenotes
compared to controls at both the early
and late blastocyst stages (Supp. Fig.
S2C). This reduction was proportional
to the decrease in total cell numbers
in parthenote blastocysts (Fig. 1). On
the other hand, the percentages of
ICM cells in parthenotes were comparable with those in controls (Supp.
Fig. S2D), concurrent with the comparable percentages of trophectoderm
cells (counted as Cdx2 cells) between
parthenotes and controls (Fig. 2G and
Table S2). The percentages of Oct4
cells in whole embryos were comparable between controls and parthenotes
at both the morula and blastocyst
stages (Supp. Fig. S2, Fig. 3, and
Supp. Table S2). At the late blastocyst
stage, when Oct4 expression was completely restricted to the ICM, Oct4
cells constituted 100% of ICM cells in
both control and parthenote embryos
(n 20) (Supp. Figs. S2B and S3D).
We also examined the expression of
two other pluripotency genes, Sox2
and Nanog, in normal fertilized and
parthenote
embryos.
Sox2
is
expressed in the epiblast at the blastocyst stage and is required for periimplantation lineage specification
(Hayashi et al., 2002; Avilion et al.,
2003; Boyer et al., 2006). Compared to
control embryos, we found dramatically decreased numbers of Sox2
cells in parthenote embryos at both
the morula and blastocyst stages (Fig.
3). Percentages of Sox2 cells in
whole embryos are given in Table S2.
As Sox2 cells constitute the epiblast
at the blastocyst stage (Hayashi et al.,
2002; Avilion et al., 2003), our results
demonstrate greatly diminished epiblast populations in parthenote ICMs.
Our analyses of the expression of
another pluripotency gene and epiblast marker, Nanog (Chambers
et al., 2003; Mitsui et al., 2003), further confirmed our results of Sox2 immunostaining. We observed colocalization of Nanog and Sox2 cells in
both control and parthenote embryos
at both the morula and blastocyst
stages (Supp. Fig. S3), indicating
overlapping expression of Nanog and
Sox2 in the same cell lineage during
preimplantation development. Simi-

lar to Sox2 cells, the numbers and

percentages of Nanog cells in parthenotes were significantly lower than
those in controls (Fig. 4 and Supp. Table S2).

Increase of Gata41 Cells in

Parthenotes
In addition to the epiblast, the other
cell lineage in the ICM is the primitive endoderm (Rossant et al., 2003;
Ralston and Rossant, 2005; Chazaud
et al., 2006; Yamanaka et al., 2006).
We then assessed expression of the
primitive endoderm marker, Gata4,
in parthenotes. Our immunostaining
results showed only scattered Gata4
cells in control morulae at E3.0,
whereas almost all cells in parthenote
morulae expressed Gata4 (Fig. 4A, B,
and Table S2). Between E4.0 and
E4.5, we found a dramatically
expanded Gata4 expression domain in
parthenote blastocysts, covering not
only the majority of ICM cells but also
most trophectoderm cells (Fig. 4C).
Therefore, almost 90% of total cells in
parthenote blastocysts expressed
Gata4, in contrast to restricted Gata4
expression in control primitive endoderm (Table S2). Percentages of
Gata4 cells in ICMs were also significantly higher in parthenotes than in
controls (Fig. 4D). In conclusion, great
decreases in Sox2 and Nanog epiblast cells and progenitors in parthenote embryos were concomitant with
dramatic increases of Gata4 primitive endoderm cells and progenitors.

Increased Fgf3 Expression

and Fgfr2 Phosphorylation in
Parthenotes
The balance between the expression
levels of Gata4 and Nanog determines
the cell fate of becoming, respectively,
primitive endoderm or epiblast (Mitsui et al., 2003; Rossant et al., 2003;
Boyer et al., 2006; Chazaud et al.,
2006; Yamanaka et al., 2006). Fibroblast growth factor 4 (Fgf4) signals
through its receptor Fgf receptor 2
(Fgfr2) and the adaptor protein Grb2
to activate Gata4/6 expression and
repress Nanog expression, thus promoting primitive endoderm formation
(Rappolee et al., 1994; Cheng et al.,
1998; Goldin and Papaioannou, 2003;
Chazaud et al., 2006; Nichols et al.,

Developmental Dynamics

1654 CHEN AND YU

Fig. 2.

2009; Guo et al., 2010; Yamanaka

et al., 2010; Frankenberg et al., 2011).
Because we observed an increase in
Gata4 and a decrease in Nanog levels
in parthenote embryos, we examined
the levels of Fgf4 and Fgfr2 proteins
in parthenotes. At both the morula
and blastocyst stages, it was found
that immunostaining patterns and
intensities of Fgf4 and Fgfr2 were
comparable between controls and parthenotes (Supp. Fig. S4). It is noteworthy that Fgf4 signals were present
in plasma membranes of all blastomeres, but were detected in the perinuclear regions of Cdx2 blastomeres

Fig. 3.

Developmental Dynamics

ABERRANT GENE EXPRESSION IN PARTHENOTE EMBRYOS 1655

only (white arrows in Supp. Fig. S4A,

B). This indicates that Cdx2 and perinuclear Fgf4 expression is mutually
exclusive among different cells, and
the expression of Fgf4 may be generated only by cells destined to become
the ICM.
Furthermore,
in
contrast
to
unchanged levels of total Fgfr2 in parthenotes, levels of phosphorylated
Fgfr2 in parthenote morulae and blastocysts were significantly higher than
those in controls, as revealed by the
increased intensities of immunostaining for Tyr653/654-phosphorylated
Fgfr2 (Fig. 5). As Fgfr2 receptors are
activated
upon
phosphorylation,
higher levels of Fgfr2 phosphorylation
in parthenotes indicates increased
Fgf signaling.
Because Fgf signaling is elevated in
parthenotes without an increase of
the Fgf4 protein level (Supp. Fig.
S4A, B), we assessed other Fgf
ligands to identify which one may
cause the increased phosphorylation
of Fgfr2 in parthenotes. It was shown
that Fgf3 and Gata4/6 were
co-expressed in the parietal endoderm
of post-implantation embryos (Shimosato et al., 2007; Cai et al., 2008; Pilon
et al., 2008). Hence we analyzed Fgf3
immunostaining and found that, in
contrast to control embryos, which
revealed only background levels of
Fgf3, parthenotes showed strong
Fgf3-positive signals throughout all

plasma membranes of both morulae

and blastocysts (Fig. 6). Unlike Fgf4,
which was expressed in only Cdx2
blastomeres (Supp. Fig. S4A, B), Fgf3
was co-expressed with Cdx2 in some
nuclei of parthenote blastomeres (Fig.
6A, B). Furthermore, Fgf3 was coexpressed with Gata4 in all nuclei of
parthenote blastomeres, both at the
morula and blastocyst stages (Fig. 6C,
D). We also performed the in situ
hybridization using an Fgf3 cDNA
probe as described previously (Tannahill et al., 1992; Wahl et al., 2007).
The levels of Fgf3 mRNA were dramatically increased in parthenote
morulae and blastocysts compared
with controls, consistent with our immunostaining data (Fig. 7). The dramatic up-regulation of Fgf3 level in
parthenotes may contribute to the
marked increase and ectopic expression of Gata4 (Figs. 4, 6).

Inhibition of Fgfr2
Phosphorylation Restores
Normal Nanog and Gata4
Expression in Parthenotes
But Does Not Increase Total
Cell Numbers of Parthenote
Embryos
To further verify the causal linkage
between Fgfr2 phosphorylation and
Gata4/Nanog levels, we treated E3.0
parthenogenetic morulae with the

Fig. 2. Decrease in Tbr2 and Elf5 levels in the parthenote trophectoderm. AF: Immunofluorescence images of control and parthenote embryos stained for Cdx2 (green), Tbr2 (red), and DAPI
(blue) at the morula stage (E3.0) (A), early blastocyst stage (E3.5) (B), and late blastocyst stage
(E4.5) (C), and stained for Elf5 (green), Tbr2 (red), and DAPI (blue) at the morula stage (E3.0) (D),
early blastocyst stage (E3.5) (E), and late blastocyst stage (E4.5) (F). Arrows point to Tbr2 cells
in parthenote trophectoderm. Scale bars in AF 20 mm. G: Comparable percentages of
Cdx2 cells (i.e., trophectoderm cells) in whole embryos between controls and parthenotes at
the morula, early blastocyst, and late blastocyst stages (n 27). H: Significantly reduced percentages of Tbr2 cells in the parthenote trophectoderm (TE) (asterisks) compared to control TE
at both the early and late blastocyst stages (n 55, P < 0.01, Students t-test). I: Significantly
reduced percentages of Elf5cells in the parthenote trophectoderm (TE) (asterisks) compared to
control TE at both the early and late blastocyst stages (n 28, P < 0.01, Students t-test). Note
that 90% of Elf5cells are also Tbr2-positive, whereas the remaining 10% of Elf5cells do not
express Tbr2.
Fig. 3. Reduction in Sox2 levels in parthenote morulae and ICMs. A, C: Immunofluorescence
images of control and parthenote embryos stained for Oct4 (red), Sox2 (green), and DAPI (blue)
at the morula (E3.0) (A) and late blastocyst (E4.5) (C) stages. The parthenote morula and blastocyst have dramatically reduced numbers of Sox2 cells (green and yellow colors) (arrows in A
and C). ICM, inner cell mass; TE, trophectoderm. Scale bars in A and C 20 mm. B: Average
percentages of Oct4 and Sox2 cells in whole embryos at the morula stage. Note the significantly reduced percentage of Sox2 cells in parthenote morulae (asterisk) (n 26, P < 0.01,
Students t-test). D: Average percentages of Oct4 and Sox2 cells in ICMs at the blastocyst
stage. Note the significantly reduced percentage of Sox2 cells in parthenote ICMs (red asterisk)
(n 20, P < 0.01, Students t-test).

Fgfr2 inhibitor SU5402 and incubated

for 20 hr, followed by immunostaining
to analyze the expression levels of
Gata4, Nanog, and Fgf3. SU5402 has
been shown to be a potent inhibitor of
Fgf signaling in the developing mouse
embryos (Zuniga et al., 2004; Calmont
et al., 2006; Miura et al., 2006; DiGregorio et al., 2007). These previous
studies indicated that a 50% inhibition of Fgfr phosphorylation was
achieved at 1020 mM of SU5402
(Mohammadi et al., 1997). Other
potent inhibitors of Fgf signaling
include PD173074 and PD184352
(Nichols et al., 2009; Yamanaka et al.,
2010). Nonetheless, because it was
demonstrated that SU5402 has a mild
effect on mouse embryonic development (Miura et al., 2006), we chose
SU5402 for our study and tested the
effects of SU5402 at a concentration
between 10 and 20 mM.
After treatment with 10 mM of
SU5402 for 20 hr, parthenote morulae
displayed no significant change of
Gata4
and
Nanog
expression,
whereas parthenogenetic blastocysts
showed
significantly
decreased
Gata4 cells and increased Nanog
cells, despite that Gata4 was still
expressed ectopically in many trophectoderm cells (compare Fig. 8A
with C). At 15 mM of SU5402, Gata4
was detected in less than four trophectoderm cells of parthenote blastocysts, indicating a dramatic decrease
in ectopic Gata4 expression (Fig. 8B);
in addition, the numbers of Nanog
cells in parthenotes treated with 15
mM of SU5402 were comparable with
untreated control (zygotic) embryos
(Fig. 8B, D). At 20 mM of SU5402,
Gata4 expression was completely restricted to the ICM of parthenotes,
and the percentages of Gata4 and
Nanog cells in whole embryos are
comparable between parthenotes and
controls (Fig. 8D). Thus, these results
indicate that 20 mM of SU5402 completely restored the balance between
Gata4 and Nanog levels.
It has been shown that treating
normal (fertilized) embryos with 10
mM SU5402 significantly down-regulates most primitive endoderm-specific markers, including Gata4, and
upregulates many epiblast-specific
markers, including Nanog and Sox2
(Guo et al., 2010). Hence we also analyzed the effects of SU5402 on Gata4

Developmental Dynamics

1656 CHEN AND YU

Fig. 4. Decrease in Nanog and increase in Gata4 levels in parthenote morulae and blastocysts.
A, C: Immunofluorescence images of control and parthenote embryos stained for Nanog (red),
Gata4 (green), and DAPI (blue) at the morula (E3.0) (A) and late blastocyst (E4.5) (C) stages.
Note the expression of both Nanog and Gata4 in late blastocysts is completely restricted to the
inner cell mass (ICM). In A, arrows point to Gata4 nuclei in the control morula and Nanog
nuclei in the parthenote morula. In C, arrows indicate the dramatically reduced number of
Nanog nuclei in the parthenote ICM, and arrowheads point to ectopic expression of Gata4 in
the parthenote trophectoderm (TE). Scale bars in A and C &equal; 20 mm. B: Average percentages of Nanog and Gata4 cells in whole embryos at the morula stage. Asterisks indicate the
significantly reduced percentage of Nanog cells and the significantly elevated percentage of
Gata4 cells in parthenote morulae (n 27, P < 0.01, Students t-test). D: Average percentages
of Nanog and Gata4 cells in ICMs at the blastocyst stage. Asterisks indicate the significantly
decreased percentage of Nanog cells and the significantly increased percentage of Gata4
cells in parthenote ICMs (n 20, P < 0.01, Students t-test).

and Nanog expression in control blastocysts. We found that SU5402 treatment decreased the total numbers of
ICM cells, with the numbers of
Gata4 cells decreased to a much
higher extent than the numbers of
Nanog cells, leading to the reduction
in the percentages of both Nanog
and Gata4 cells in whole blastocysts
(Fig. 8AC, E). Therefore, in spite of
the decreased percentages of Nanog
cells in whole embryos, the percentages of Nanog cells in control ICMs
were significantly increased from 50.2
to 82.6% and 92.3% (12-fold
increase) by 10 and 15 mM SU5402,
respectively (Fig. 8E). On the other
hand, the percentages of Gata4 cells
in control ICMs were significantly
decreased from 49.8 to 17.4% and
7.8% (310-fold decrease) by 10 and
15 mM SU5402, respectively (Fig. 8E).
Thus the Nanog/Gata4 gene expression levels in the ICM were inversely
correlated after treatment with
SU5402.
Although Gata4 and Nanog expression was rescued by SU5402

Fig. 5. Elevated levels of Fgfr2 phosphorylation in parthenote morulae and blastocysts. AD: Immunofluorescence images of control and parthenote embryos stained for phosphorylated Fgfr2 (red), DAPI (blue), and Cdx2 (green) (A, B) or Gata4 (green) (C, D) at the morula (E3.0) (n 26) (A,
C) and late blastocyst (E4.5) (n 22) (B, D) stages. Arrowheads in A and C demonstrate greatly increased signals of Fgfr2 phosphorylation on the
cell membranes of parthenote morulae compared to controls. In B and D, arrows point to signals of phosphorylated Fgfr2 on the plasma membranes of primitive endoderm cells (i.e., Gata4 cells) in the control blastocyst, while arrowheads indicate significantly higher levels of Fgfr2 phosphorylation in both the trophectoderm (TE) and inner cell mass (ICM) of the parthenote blastocysts. Scale bars in AD 20 mm.

Developmental Dynamics

ABERRANT GENE EXPRESSION IN PARTHENOTE EMBRYOS 1657

Fig. 6. Increase in Fgf3 levels in parthenote morulae and blastocysts. AD: Immunofluorescence images of control and parthenote embryos
stained for Fgf3 (red), DAPI (blue), and Cdx2 (green) (A, B) or Gata4 (green) (C, D) at the morula (E3.0) (n 28) (A, C) and late blastocyst (E4.5)
(n 20) (B, D) stages. Note the background levels of Fgf3 immunostaining in control morulae and blastocysts, in contrast with the apparently
bright Fgf3 signals throughout parthenote embryos. White arrows in A indicate co-expression of Fgf3 and Cdx2 in some nuclei of parthenote
blastomeres. White arrows in C indicate co-expression of Fgf3 and Gata4 in all nuclei of parthenote blastomeres. The results demonstrate that
increased Fgf3 expression in parthenotes is concomitant with increased Gata4 expression. ICM, inner cell mass; TE, trophectoderm. Scale bars
in AD 20 mm.

Fig. 7. Increase in Fgf3 expression in parthenote morulae and blastocysts. Control and
parthenote morulae (n 12) and blastocysts
(n 10) were hybridized with Fgf3 cDNA
using the in situ hybridization kit. Purple colors indicate positive Fgf3 mRNA signals. Note
there was only weak background staining in
the control morula and blastocyst. Fgf3 mRNA
signals are stronger in the cytoplasm and
weaker in the nuclei of parthenote embryos,
as normally seen in in situ hybridization
results. Scale bars 30 mm.

treatment, we found that the total cell

numbers of parthenogenetic blastocysts were still significantly fewer

than controls, even when treated with

20 mM of SU5402 (Fig. 8F). Thus
SU5402 is capable of restoring normal
Gata4 and Nanog levels in parthenotes, but fails to increase total cell
numbers of parthenotes. The decrease
in total cell numbers may be attributed to a decrease in cell proliferation
or an increase in apoptosis (Hardy
and Handyside, 1996; Uranga and
Arechaga, 1997). Because parthenotes
displayed increased levels of Fgf3,
especially in the nuclei (Fig. 6), and
the nuclear isoform of Fgf3 was
shown to inhibit cell proliferation
(Kiefer and Dickson, 1995; Antoine
et al., 2005), it was of interest to
assess whether Fgf3 expression in
parthenotes was affected by SU5402.
Surprisingly, even when ectopic
Gata4 expression was completely suppressed by 20 mM of SU5402, the immunostaining intensity of Fgf3 signals was very strong in most nuclei of
parthenotes (throughout the ICM and
trophectoderm) (Supp. Fig. S5C).
Supp. Figure S5D shows that the per-

centages of Gata4 nuclei in parthenotes decreased as the concentration

of SU5402 increased, whereas the
percentages of Fgf3 nuclei were not
affected by the concentration of
SU5402. Therefore, we conclude that
the Fgfr2 antagonist SU5402 does not
inhibit Fgf3 expression. Given that
the nuclear isoform of Fgf3 inhibits
cell proliferation (Kiefer and Dickson,
1995; Antoine et al., 2005), the high
level of Fgf3 in the nuclei of parthenogenetic embryos may suppress cell
proliferation in parthenotes.

DISCUSSION
Our findings in this study are summarized in Figure 9. In this study, we
analyzed expression of lineage-specific genes and found lineage segregation defects in parthenogenetic preimplantation embryos. We found that
the number of Gata4 primitive endoderm cells was dramatically increased
while the number of Nanog/Sox2
epiblast cells was significantly

Developmental Dynamics

1658 CHEN AND YU

Fig. 8.

decreased. Although with decreased

numbers, these Nanog/Sox2 epiblast cells still exist in the parthenote
ICM and can be used to derive
patient-specific ES cells for stem cell
therapies. On the other hand, there
are other drawbacks of parthenogenetic embryos, which had been previously reported, including aberrant
expression of imprinted and development-related genes that disrupt full
development of organisms (Humpherys et al., 2001; Zvetkova et al., 2005;
Bonk et al., 2007; Jiang et al., 2007;
Mitalipov et al., 2007; Horii et al.,
2008). Our study showed the abnormal gene expression in the preimplantation parthenogenetic embryo, which
Fig. 9.

Developmental Dynamics

ABERRANT GENE EXPRESSION IN PARTHENOTE EMBRYOS 1659

may affect the derivation and quality

of parthenogenetic ES cells.
Consistent with previous studies
(Hardy and Handyside, 1996; Uranga
and Ar&eacaute;chaga, 1997), we
found significantly reduced total cell
numbers in parthenote embryos compared to control embryos from the
early blastocyst stage, which was
likely due to decreased cell proliferation in parthenotes between the morula and blastocyst stages (Hardy and
Handyside,
1996;
Uranga
and
Arechaga, 1997). Impaired proliferation and differentiation were observed
in both extraembryonic and embryonic lineages of parthenote embryos,
especially in the extraembryonic and
embryonic mesoderm as well as primitive endoderm lineages (Barton
et al., 1985; Sturm et al., 1994; Mognetti and Sakkas, 1996). Gata4 and
Gata6 are two essential marker genes
of the primitive endoderm (Boyer
et al., 2006; Yamanaka et al., 2006;
Cai et al., 2008; Kuijk et al., 2008),
which forms yolk sac and is a major
affected
lineage
in
parthenote
embryos (Sturm et al., 1994; Newman-Smith and Werb, 1995; Chazaud
et al., 2006; Yamanaka et al., 2006).
Our observation of the overexpression
of the parietal endoderm marker
genes, Gata4 and Fgf3, in parthenote
ICMs is consistent with the previously reported tendency of parthenote
ICM outgrowths to differentiate into
parietal endoderm cells, which are descendants of the primitive endoderm

(Newman-Smith and Werb, 1995;

Boyer et al., 2006; Cai et al., 2008).
Interestingly, we detected Gata4
expression as early as the morula
stage, while previous studies failed to
detect Gata4 expression till the midblastocyst stage (Plusa et al., 2008;
Silva et al., 2009; Yamanaka et al.,
2010). While the same anti-Gata4
antibody was used (from Santa Cruz
Biotechnology, Santa Cruz, CA), differences in experimental procedures may
contribute to the different results. For
example, while Plusa et al. (2008)
fixed embryos in 4% PFA overnight,
we fixed for only 15 min after treatment with acid Tyrodes solution. The
Gata4 staining in morulae should not
result from unspecific signals, as our
negative controls using hepatocyte cultures did not show any positive Gata4
signals (data not shown).
During lineage allocation and specification in the ICM, Sox2 and Nanog are
two key transcription factors required
for development of the epiblast, which
forms the embryo body (Avilion et al.,
2003; Rossant et al., 2003; Boyer et al.,
2006; Chazaud et al., 2006; Yamanaka
et al., 2006; Silva et al., 2009; Messerschmidt and Kemler, 2010; Frankenberg et al., 2011). In addition, diminished Nanog expression, which was
shown to cause excessive differentiation of ICM cells into parietal endoderm
cells (Mitsui et al., 2003), was observed
in parthenote embryos. Therefore, the
misregulated balance between the
expression levels of Gata4/6 and Nanog

in parthenote ICMs may lead to excessive differentiation into the primitive

endoderm lineage and impaired differentiation into the epiblast lineage. It is
also noteworthy that elevated Gata4
and reduced Nanog expression in parthenote embryos was first observed at
E3.0, suggesting that aberrant cell differentiation in parthenotes may occur
as early as the morula stage. As Nanog
was demonstrated to maintain selfrenewal and the undifferentiated state
of ICM and ES cells (Newman-Smith
and Werb, 1995; Mitsui et al., 2003), it
is likely that dramatically reduced
numbers of Nanog blastomeres in parthenote morulae indicate a significantly
decreased amount of proliferating cells
since E3.0, thus producing a significantly lower proliferation rate in parthenotes compared to controls between
the morula and blastocyst stages.
It has been reported that Cdx2,
Elf5, and Tbr2(Eomes) are three of
the earliest genes required for specification and differentiation of the
trophoblast lineage, with the functioning
cascade
being:
Cdx2!Tbr2!Elf5 (Russ et al., 2000;
Donnison et al., 2005; Niwa et al.,
2005; Strumpf et al., 2005; Hemberger and Dean, 2007; Jedrusik
et al., 2008; Ng et al., 2008; Ralston
and Rossant, 2008). In spite of being
downstream of Cdx2 and Tbr2, Elf5 is
the key transcription factor creating a
feedback loop reinforcing Cdx2 and
Tbr2 expression in trophectoderm,
which is indispensable for the

Fig. 8. Increased Nanog and decreased Gata4 levels in parthenotes treated with the Fgfr2 inhibitor SU5402. AC: Immunofluorescence images
for Nanog (red), Gata4 (green), and DAPI (blue) in control and parthenote blastocysts, which were cultured with the following concentrations of
SU5402 for 20 hr since the early morula stage (E3.0): 10 mM (A), 15 mM (B), and 20 mM (C) (n 20 for each concentration). Note that the number
of green-colored Gata4 nuclei decreases in both controls and parthenotes as the concentration of SU5402 increases. Arrows point to Gata4
cells in the trophectoderm. D: Average percentages of Nanog and Gata4 cells in whole parthenote embryos at the morula and blastocyst
stages, with or without the addition of SU5402. Asterisks indicate significantly increased percentages of Nanog cells and significantly decreased
percentages of Gata4 cells in SU5402-treated parthenotes compared to untreated ones (n 20, P < 0.01, Students t-test). Note the comparable
percentages of Nanog and Gata4 cells between untreated controls and 20 mM SU5402-treated parthenotes. E: In the ICM of SU5402-treated
controls, the percentages of Nanog cells are significantly increased whereas those of Gata4 cells are dramatically decreased after treatment
with SU5402. Note the extent of change is greater for Gata4 cells (37-fold decrease) than for Nanog cells (less than 2-fold increase). In whole
control embryos, both the percentages of Nanog and Gata4 cells are significantly decreased after treatment with SU5402. Note the extent of
decrease is greater for Gata4 cells (310-fold decrease) than for Nanog cells (less than 2-fold decrease). F: Total cell numbers in untreated controls and SU5402-treated or untreated parthenotes at various preimplantation stages. Asterisks indicate significantly decreased total cell numbers
in both untreated and SU5402-treated parthenotes compared to controls at the early and late blastocyst stages (n 60, P < 0.01, Students ttest). ICM, inner cell mass; TE, trophectoderm. Scale bars in AC 20 mm.
Fig. 9. Model of molecular mechanisms underlying decreased total cell numbers, increased Gata4 and decreased Nanog expression in parthenogenetic embryos. In parthenotes, aberrant expression of Fgf3 and lineage-specific genes, including Nanog and Gata4, were observed at both
the morula and blastocyst stages. While the secreted isoform of Fgf3 binds to Fgfr2 and stimulates Fgfr2 phosphorylation and signaling, the nuclear isoform of Fgf3 inhibits cell proliferation and decreases total cell numbers in parthenotes. The elevated Fgfr2 phosphorylation in parthenotes
caused increased Gata4 and decreased Nanog expression, indicating expanded primitive endoderm cells and diminished epiblast cells. Inhibition
of Fgfr2 phosphorylation by SU5402 suppressed Gata4 and enhanced Nanog expression but did not affect Fgf3 expression in parthenotes, indicating that Fgfr2, Gata4, and Nanog are all downstream of Fgf3.

Developmental Dynamics

1660 CHEN AND YU

differentiation of the trophoblast lineage (Donnison et al., 2005; Ng et al.,

2008). As shown previously, hemizygous expression of Elf5 was sufficient
to cause a marked decrease in Cdx2
protein and Cdx2 and Tbr2 mRNA
levels (Ng et al., 2008).
Interestingly, it has been reported
that parthenogenetic embryos show a
remarkably higher global DNA methylation level compared to normal (fertilized) embryos, especially from the
4-cell to the blastocyst stage (Barton
et al., 2001; Li et al., 2009). Previous
studies have demonstrated that the
early demethylation event in embryogenesis requires the presence of the
paternal genome (Barton et al., 2001;
Santos and Dean, 2004; Morgan
et al., 2005). It is likely that the lack
of the paternal genome causes demethylation failure in early parthenogenetic embryos, leading to an elevated
methylation level of the Elf5 promoter
in many of the trophectoderm cells.
Reduced Elf5 expression in these trophectoderm cells then leads to
decreased Tbr2 expression. However,
it is interesting that Cdx2 expression
was not affected by reduced Elf5
expression in parthenotes. One possibility is that the remaining Elf5
expression in some cells is sufficient
to maintain non-cell-autonomous
Cdx2 expression in the neighboring
cells. On the other hand, Tbr2 expression may be cell-autonomously regulated by Elf5; thus, Tbr2 is expressed
only in Elf5-expressing cells in parthenote trophectoderm.
All of the aforementioned genes
are pivotal factors regulating lineage
segregation and cell fate determination (Russ et al., 2000; Strumpf
et al., 2005; Shimosato et al., 2007;
Cai et al., 2008). Similar to
Tbr2(Eomes) homozygous mutants,
most parthenote embryos demonstrated a lack of diploid dividing
trophoblast cells, impaired or retarded development of the neuroectoderm and brain, and multiple mesodermal defects (Sturm et al., 1994;
Bulfone et al., 1999; Russ et al.,
2000; Kwon and Hadjantonakis,
2007). Previous studies using chimeric mouse embryos have demonstrated
that
parthenogenetically
derived cells do contribute to the trophectoderm at the blastocyst stage,
but not at the post-implantation

stages (Clarke et al., 1988; Thomson

and Solter, 1988, 1989). Therefore,
there is an early post-implantation
defect in the parthenogenetic trophoblast lineage. In spite of the trophoblast defect, parthenote embryos are
capable of post-implantation development until the 25-somite stage (at
around E10.0) (Sturm et al., 1994;
Penkov et al., 1995; Kono et al.,
1996; Mognetti and Sakkas, 1996),
and trophoblast giant cells were
detected in parthenote placentae
(Barton et al., 1985; Sturm et al.,
1994; Mognetti and Sakkas, 1996).
On the other hand, Tbr2 homozygous
mutants arrested at the peri-implantation stage (E6.0E7.5) with differentiation defects of the trophectoderm and incompetence to form
trophoblast stem cells (Russ et al.,
2000). This apparent difference is
probably due to the finding that
Tbr2 expression is reduced but not
completely absent in the parthenote
trophectoderm, as observed in this
study.
Our observation that Cdx2 expression is normal in the parthenote trophectoderm and both Elf5 and Tbr2
are expressed in over 40% of parthenote trophectoderm cells is consistent
with the partially if not completely
specified trophectoderm lineage in
parthenotes (Sturm et al., 1994; Mognetti and Sakkas, 1996). Interestingly, it has been shown that Fgf signaling
is
indispensible
for
trophectoderm
development
and
maintenance of Cdx2 and Tbr2
expression (Tanaka et al., 1998; Nichols et al., 2009). On the other hand,
ectopic Gata4 expression is correlated
with increased Fgf signaling in the
parthenote trophectoderm. Therefore,
overexpression of Gata4 may be independent of reduced Tbr2 expression
in parthenote embryos.
It has been shown that Fgf4-Fgfr2
signaling is indispensable for trophoblast proliferation and the maintenance of trophectoderm and ICM
identities between E3.5 and E4.5
(Feldman et al., 1995; Chai et al.,
1998; Tanaka et al., 1998; HaffnerKrausz et al., 1999; Goldin and
Papaioannou, 2003). In this study, we
found elevated Fgfr2 signaling in parthenotes starting from the 16-cell
morula stage, as indicated by
increased Fgfr2 phosphorylation, in

spite of unchanged Fgf4 expression in

parthenotes. We found that Fgf3
expression was undetectable or
expressed at a low-to-moderate level
in normal mouse preimplantation
embryos (Rappolee et al., 1988, 1994;
Zhong et al., 2006), but was strongly
expressed in parthenote embryos.
Our finding that inhibition of Fgfr2
suppressed Gata4 but not Fgf3
expression in parthenotes (Supp. Fig.
S4) indicates that Gata4 is not the
only factor up-regulating Fgf3 expression, and that increased Fgf3 expression is precedent to increased Gata4
expression in parthenotes. Our observations indicated that Fgf3 did not
up-regulate Gata4 expression when
Fgfr2 signaling was inhibited. It
remains to be studied which factor(s)
induce(s) Fgf3 overexpression in parthenotes. Assessing the expression
levels of Fgf3 in parthenogenetic
embryos earlier than the morula
stage will unravel the earliest stage
at which Fgf3 up-regulation is first
observed. The factor(s) inducing Fgf3
expression in parthenotes should be
detectable at this stage.
Interestingly, Fgf3 was reported to
have dual subcellular fates and play
dual roles in regulating proliferation,
with the secreted isoform promoting
but the nuclear isoform inhibiting cell
proliferation (Kiefer et al., 1994; Kiefer and Dickson, 1995; Antoine et al.,
2005). We have shown that Fgf3 immunostaining signals are apparently
brighter in the nuclei than on the
plasma membranes of parthenotes,
indicating more nuclear than secreted
Fgf3 proteins, thus leading to the inhibition of cell proliferation. The excessive nuclear Fgf3 proteins may
contribute to the reduction of total
cell numbers in parthenotes. Therefore, increased Fgf3 and ectopic
Gata4 expression in parthenote
trophectoderm together perturb the
balance between differentiation and
proliferation of trophoblast cells.
Interestingly, recent studies demonstrated that inhibition of the FGF/MAP
kinase signal caused almost all ICM
cells in normal (fertilized) embryos to
become Nanog epiblast cells, and no
or very few Gata4/Gata6 primitive
endoderm cells were detected (Guo
et al., 2010; Yamanaka et al., 2010).
Consistent with previous findings of
inverse correlation of Nanog/Gata4

Developmental Dynamics

ABERRANT GENE EXPRESSION IN PARTHENOTE EMBRYOS 1661

expression levels (Guo et al., 2010;

Yamanaka et al., 2010), we found
SU5402 significantly increased the precentages of Nanog cells and decreased
the percentages of Gata4 cells in control ICMs. Interestingly, the extent of
increase in the percentages of Nanog
cells (less than 2-fold increase) was
much lower than the extent of decrease
in the percentages of Gata4 cells (up
to 67-fold decrease). This is in agreement with the previous study showing
a 6-fold down-regulation of Gata4
expression and 2-fold up-regulation of
Nanog expression in embryos treated
with 10 mM SU5402 from the 16-cell
stage for 24 hr (Guo et al., 2010).
Our results indicate that the
decrease of total cell numbers in parthenotes is not associated with aberrant Nanog or Gata4 expression, as
the restoration of normal Nanog and
Gata4 expression is not concomitant
with increased cell numbers. Instead,
increased Fgf3 expression, which is
not affected by SU5402 treatment,
may be associated with decreased cell
numbers in parthenotes, as the excessive nuclear Fgf3 proteins may suppress cell proliferation and thus
reduce total cell numbers.

EXPERIMENTAL
PROCEDURES
Parthenogenetic Activation
and In Vitro Culture of
Preimplantation Embryos
Animals used in this study were purchased from BioLASCO Taiwan (Taipei, Taiwan), and approval was
received from the Academia Sinica
Institutional Animal Care and Utilization Committee. B6DBA female
mice at 1014 weeks old were superovulated by an intraperitoneal (i.p.)
injection of 5 IU pregnant mare serum gonadotropin (Merck, Darmstadt, Germany), followed by an i.p.
injection of 5 IU human chorionic gonadotropin (hCG) (Sigma-Aldrich, St.
Louis, MO) 4850 hr later. In order
to synchronize the time of fertilization with the time of parthenogenetic
activation, 12.5 hr after hCG injection, the female mice were separated
into two groups: the first group was
individually paired with males of the
same strain for 1.5 hr and then
checked for copulation plugs, and the

second group was sacrificed while the

first group was mating. The unfertilized oocytes enclosed in cumulus
masses were released from the
ampullae, and cumulus cells were
removed by pipetting using a mouthcontrolled pipette (with an inner diameter of 200300 mm) after 5 min of
R
treatment with EmbryoMaxV
M2
medium containing 50100 U/ml hyaluronidase (Millipore, Billerica,
MA). The unfertilized oocytes were
then washed and equilibrated in 35ml droplets of potassium simplex optiR
mized
medium
(EmbryoMaxV
KSOM, Millipore, Billerica, MA) at
37 C in a humidified atmosphere of
5% CO2 in air. After 1 hr of equilibration, parthenogenetic activation was
conducted in CZBG medium containing 10 mM strontium chloride (SrCl2)
and 5 mg/ml cytochalasin B for 4.5 hr
at 37 C with 5% CO2 in air, as
described before (Gao, 2006). The formation of pronuclei was observed
between 3 and 3.5 hr after incubation
in the activation medium, i.e.,
between 16.5 and 17 hr after hCG
injection. The timing of pronulcei formation in parthenotes was consistent
with previous observations (Abramczuk and Sawicki, 1975).
At the same time, for the oocytes of
the first group of female mice, fertilization was reported to take place from
2.5 hr following pairing, and the first
pronuclei were found to appear 2 hr after fertilization (Abramczuk and
Sawicki, 1975; Hardy and Handyside,
1996). Thus the first pronuclei in fertilized oocytes appeared about 4.5 hr
after pairing, i.e., approximately 17 hr
after hCG injection. The mated female
mice were sacrificed 4.5 hr after pairing, with the cumulus cells removed in
the EmbryoMaxV M2 medium containing 50100 U/ml hyaluronidase (Millipore). The presence of pronuclei was
confirmed under microscope. According to our observations, the time
points of the first pronuclear formation
are between 16.5 and 17 hr after hCG
injection for both the fertilized control
and parthenogenetic oocytes. All
oocytes at the pronuclei stage were
then transferred to KSOM medium
and incubated till they reached the developmental stage at which they were
analyzed. Embryos normally cleave to
two-cells at E1.01.5, and form moruR

lae at E2.53.0 (4860 hr in culture).

Early blastocysts are formed at E3.5
(7072 hr in culture), blastocysts are
formed at E4.0 (8084 hr in culture),
and late expanded blastocysts are
formed at E4.5 (9296 hr in culture).
According to our observation, culturing fertilized oocytes in SrCl2-containing medium for the same time as
unfertilized (parthenogenetic) oocytes
did not affect immunostaining results
in these control embryos, indicating
that chemical activation by SrCl2 does
not contribute to the differential gene
expression between controls and parthenotes. These observations are consistent with many previous reports
(Loren and Lacham-Kaplan, 2006;
Kyono et al., 2008; Chen et al., 2010).

Immunofluorescence Staining
For immunostaining, embryos were
washed for 510 s in droplets of acidic
Tyrodes solution (made by SigmaAldrich; purchased from Uni-onward,
Taipei, Taiwan) to remove the zona
pellucida, and then fixed in 4% paraformaldehyde (Sigma-Aldrich) in 1
phosphate-buffered saline (PBS) for 15
min at room temperature. Embryos
were then permeabilized with 0.25%
Triton X-100 for 15 min, followed by
washing and blocking for 1 hr in blocking solution containing 0.05% Tween20, 3% bovine serum albumin (BSA),
and 5% normal goat serum in 1 PBS.
After blocking, embryos were incubated at 4 C overnight with the following primary antibodies diluted in
blocking solution: Cdx2 (mouse monoclonal; 1:100 dilution; BioGenex, San
Ramon, CA), Elf5 (mouse monoclonal;
1:100 dilution; Santa Cruz Biotechnology, Santa Cruz, CA), Fgf3 (rabbit polyclonal; 1:200 dilution; Abcam, Cambridge, MA), Fgf4 (rabbit polyclonal;
1:200 dilution; Abcam), Fgfr2 (rabbit
polyclonal; 1:50 dilution; Abgent, San
Diego, CA), phosphor-Fgfr2-pY653/654
(rabbit polyclonal; 1:50 dilution;
Abgent), Gata4 (mouse monoclonal;
1:100 dilution; Santa Cruz Biotechnology), Nanog (rabbit polyclonal; 1:100
dilution; ReproCELL, Tokyo, Japan),
Oct4 (rabbit polyclonal; 1:200 dilution;
Santa Cruz Biotechnology), Sox2
(mouse monoclonal; 1:100 dilution;
Millipore), and Tbr2 (rabbit polyclonal;
1:100 dilution; Millipore). On the second day, the embryos were washed

1662 CHEN AND YU

and blocked for 1 hr in blocking solution, followed by incubation at room

temperature for 1 hr with the following secondary antibodies conjugated
with fluorophores: goat anti-mouse
AlexaFluor 488 (green fluorescence)
and goat anti-rabbit AlexaFluor 555
(red fluorescence) (Invitrogen Taiwan,
Taipei, Taiwan). After incubation, the
embryos were washed for 10 min in
washing solution containing 0.2% Triton X-100 in 1 PBS, and counterstained with 0.2 mg/ml DAPI in washing solution for 10 min, followed by
mounting in VectaShield (Vector Laboratories, Burlingame, CA) on glass
slides.

mRNA In Situ Hybridization

Fgf3 mRNA in situ hybridization was
performed with DIG-AP RembrandtV
Universal RISH and Detection Kit
(Invitrogen, Carlsbad, CA), following
the manufacturers instructions. The
sequence of Fgf3 cDNA probe has
been described previously (Tannahill
et al., 1992; Wahl et al., 2007).

Developmental Dynamics

Confocal Microscopy
Series of confocal sections through 3D
preserved embryo nuclei were collected using a Leica TCS SP5 confocal
microscope (located on the 5th flour of
Genomics Research Center, Academia
Sinica) equipped with a Super Z galvanometer stage and Plan Apo 63/
1.4 NA oil immersion objectives. Fluorochromes were visualized using an
argon laser with an excitation wavelength of 488 nm (for AlexaFluor
488), a DPSS laser with a laser line of
561 nm (for AlexaFluor 555), and a
diode laser with a laser line of 405 nm
(for DAPI). For each optical section,
images were sequentially collected
using the XYZ mode for two or three
fluorochromes. The pinhole was set to
11.5 Airy units and the scan zoom
was 1.5. In order to compare the relative intensities of immunostaining
between control and parthenote
embryos, identical scanning parameters including the strength of laser
emissions were maintained for controls and parthenotes stained with
the same antibodies. Images of optical
sections were then analyzed using
Leica Application Suite, and 3D and
maximum projections were con-

structed from serial stacks of sections

for each embryo.

Cell Counting and Statistical

Analyses
The image files of optical sections of
each embryo were analyzed by the
Count Nuclei/Cell Sorting Application
Module for MetaMorph (MetaMorph
Offline vers. 7.0; Universal Imaging
CorporationTM,
Buckinghamshire,
UK) to count cell numbers for the
entire embryo and for each antibody
immunostained sample. Statistical
significances are represented by P
values, which were calculated by Students t-test. The lower the P value,
the more significant the difference
between control and parthenote
embryos. The difference was regarded
as non-significant when P  0.05, as
significant when P < 0.05, and as
highly significant when P < 0.01.

Inhibition of Fgfr2 Signaling

Culturing mouse embryos with
SU5402 to inhibit Fgf signaling has
been described in previous studies
(Zuniga et al., 2004; Calmont et al.,
2006; Miura et al., 2006; Di-Gregorio
et al., 2007). SU5402 was dissolved in
100% dimethyl sulfoxide (DMSO) at
10 mM (stock solution) and stored at
20 C until use. Because a 50% inhibition of Fgfr phosphorylation was
achieved at 1020 mM of SU5402
(Mohammadi et al., 1997), we
assessed the effects of SU5402 at 10,
15, and 20 mM, respectively, on the
development of both control and parthenogenetic embryos. Embryos were
cultured in the KSOM medium containing different concentrations of
SU5402 for 20 hr since the early morula stage (E3.0) and then analyzed by
immunostaining at the mid-blastocyst
stage (E4.0). For experimental controls, we treated embryos for 20 hr
with an equal concentration of DMSO
in KSOM (0.10.2 % final).

ACKNOWLEDGMENTS
We thank Chien-Hong Chen for providing the parthenogenetic activation
protocol and Li-Wen Lo of the Core
Facility of the Genomics Research
Center for expert assistance with confocal microscopy. We also thank Drs.
Hung-Chih Kuo and Cheng-Fu Kao in

the Institute of Organismic and Cellular Biology for critical comments on

this project and manuscript. This
work was supported by grant 94M0111 from the Genomics Research Center
of Academia Sinica and NSC 98-3111B-001-003 to John Yu.

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