a RESEARCH ARTICLE

Ectopic Expression of Fgf3 Leads to AberrantLineage Segregation in the Mouse ParthenotePreimplantation EmbryosYi-Hui Chen1 and John Yu2,3*

Background: Parthenogenetic mammalian embryos were reported to die in utero no later than the 25-somite 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 cellsand less into epiblast and ES cells. Hence we are interested in studying the molecular mechanisms under-lying lineage defects of parthenotes. Results: We found that parthenote inner cell masses (ICMs) containeddecreased numbers of Sox21/Nanog1 epiblast cells but increased numbers of Gata41 primitive endodermcells, indicating an unusual lineage segregation. We demonstrate for the first time that the increasedGata4 level in parthenotes may be explained by the strong up-regulation of Fgf3 and Fgfr2 phosphoryla-tion. Inhibition of Fgfr2 activation by SU5402 in parthenotes restored normal Nanog and Gata4 levelswithout 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 mecha-nisms of the developmental defects of parthenogenetic embryos in both the trophectoderm and ICM.Developmental Dynamics 241:16511664, 2012. VC 2012 Wiley Periodicals, Inc.

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 theparthenogenetic inner cell mass.

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

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 asexualreproduction where the offspring isderived entirely from an unfertilizedfemale gamete. It is a normal process

used by some reptiles and birds to

reproduce. In mammals, partheno-

genesis usually refers to embryonic

development from an artificially acti-

vated 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

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Additional Supporting Information may be found in the online version of this article.1Graduate Institute of Aerospace and Undersea Medicine, National Defense Medical Center, Taipei, Taiwan2Stem Cell Program, Genomics Research Center, Taipei, Taiwan3Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, TaiwanGrant sponsor: Genomics Research Center of Academia Sinica; Grant number: 94M011-1; Grant sponsor: NSC; Grant number: 98-3111-B-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.23851Published online 4 September 2012 in Wiley Online Library (wileyonlinelibrary.com).

DEVELOPMENTAL DYNAMICS 241:16511664, 2012

VC 2012 Wiley Periodicals, Inc.

organisms, is less disputable (Sturm

et al., 1994; Kono et al., 1996; Mog-

netti and Sakkas, 1996). In fact, many

recent studies have demonstrated that

parthenogenesis is an efficient way to

generate histocompatible human em-

bryonic stem (ES) cells for transplan-

tation-based stem cell therapies (Reva-

zova et al., 2008; Hao et al., 2009; Lu

et al., 2010). Histocompatible parthe-

note 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, thefirst lineage segregation event is thesegregation between the trophecto-derm and inner cell mass (ICM) line-ages, which occurs between the mor-ula and blastocyst stages, and thesecond lineage segregation event is thesegregation between the primitiveendoderm and epiblast lineages withinthe ICM (Reik et al., 2003; Morganet al., 2005; Boyer et al., 2006; Dietrichand Hiiragi, 2007). A previous studydemonstrated that the ICM culturesfrom parthenogenetic embryos con-tained mostly differentiated parietalendoderm cells and only a few undif-ferentiated stem cells, suggesting line-age segregation defects in parthenotes(Newman-Smith and Werb, 1995). Inorder to distinguish between the nega-tive effects of in vitro cultures andthat of parthenogenesis per se, ourstudy compared gene expression inparthenotes with that in fertilizedembryos that developed in vitroinstead of in a maternal uterus.In this study, we analyzed expres-

sion of many development-relatedgenes in parthenote morulae andblastocysts, including the trophecto-derm markers Cdx2, Elf5, andTbr2(Eomes), the ICM marker Oct4,the epiblast markers Sox2 and Nanog,the primitive endoderm markerGata4, and the signal transductionfactors Fgf3, Fgf4, and their receptorFgfr2. We found normal levels ofCdx2, Oct4, and Fgf4, but perturbedlevels of Elf5, Tbr2, Sox2, Nanog,Gata4, and Fgf3, as well as increasedFgfr2 phosphorylation in parthenoteembryos. Our study also revealsgreatly increased Gata4 and Fgf3 but

reduced Tbr2, Sox2, and Nanogexpression in parthenotes comparedto controls, indicating cell fate defectsand impaired differentiation capacityof parthenote blastomeres. Hence wesuggest that the increased Fgf3-Fgfr2signaling, the downstream elevationof Gata4 expression and concomitantsuppression of Nanog expression inparthenogenetic preimplantationembryos constitute the major molecu-lar mechanism leading to lineage seg-regation defects of parthenotes.

RESULTS

Reduction of Cell Numbers at

the Early and Late Blastocyst

Stages

To exclude the possibility that the invitro cultures instead of parthenogene-sis per se cause delayed developmentof parthenotes, we used embryos devel-oped from in vitro cultured fertilizedoocytes as controls, instead of thosedirectly from a maternal uterus. Asshown in Figure S1A (which is avail-able online), preimplantation embryosdeveloped separately from a total of 78parthenogenetic and fertilized oocyteswere analyzed on consecutive days af-ter oocyte collection (which wascounted as E0.5). In order to comparethe efficiencies of embryonic develop-ment between controls and parthe-notes during in vitro culturing, weassessed the percentages of embryosreaching each developmental stage ev-ery day. We counted total cell numbersin parthenote and control embryos atboth the morula and blastocyst stages.Morulae obtained at E3.0 usually had

1220 cells and were grouped as earlymorulae, whereas morulae obtainedat E3.5 usually had 2533 cells andwere grouped as late morulae. Blasto-cysts obtained at E3.5, which stillexpressed Oct4 and Nanog in the tro-phectoderm, were grouped as earlyblastocysts (Supp. Fig. S2 and datanot shown). On the other hand, blasto-cysts obtained at E4.5, which have re-stricted Oct4 and Nanog expression inthe ICM, were grouped as late blasto-cysts (Supp. Fig. S2).As shown in Table S1, percentages

of embryos reaching different preim-plantation stages from E1.5 (the cleav-age stage) to E5.5 (the hatched stage)were similar between controls and par-thenotes. Representative charts of sta-tistical analyses at E3.5, E4.5, andE5.5 are shown in Supp. Figure S1C.Taken together, these results indicatethat the developmental timing of par-thenote mouse embryos appears nor-mal from E1.5 to E5.5. We also ana-lyzed the total cell numbers in eachembryo by enumerating the DAPI-stained nuclei on 3D projections con-structed from serial optical sections ofembryos. It was found that total num-bers of cells in parthenote embryoswere significantly decreased at theblastocyst 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 ear-liest transcription factors initiating

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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 (eachrepresenting an individual cell) were counted by MetaMorph. The Students t-test was used forstatistical analyses. Asterisks indicate significantly decreased total cell numbers in parthenoteblastocysts. n 20 for early morulae, n 18 for late morulae, n 19 for early blastocysts, n 21for late blastocysts. P 0.1 between controls and parthenotes at both the early and late morulastages, P < 0.01 between controls and parthenotes at both the early and late blastocyst stages.

1652 CHEN AND YU

trophectoderm differentiation (Russet 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 Elf5directly activates Cdx2 and Tbr2expression (Choi and Sinha, 2006; Nget al., 2008). Hence we assessed thelevels of Cdx2, Elf5, and Tbr2 in par-thenote and control embryos bycounting the nuclei stained with spe-cific markers on 3D projections ofeach embryo (Fig. 2). It was foundthat normal Cdx2 expression in cellslocated on the periphery of the moru-lae (Fig. 2A) and in the trophectodermof blastocysts (Fig. 1B, C). On theother hand, we failed to detect anyspecific Elf5 and Tbr2 signals at themorula stage in either controls or par-thenotes (Fig. 2A, D). The lack of Elf5and Tbr2 expression in normal moru-lae was also described previously(Kwon and Hadjantonakis, 2007; Nget 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 ofCdx2 cells in controls and parthe-notes (Fig. 2AC), percentages ofCdx2 cells in whole embryos werealso comparable between controls andparthenotes (Fig. 2G and Table S2).On the other hand, both Elf5 andTbr2 cells were dramaticallyreduced in parthenotes compared tocontrols at the blastocyst stage (Fig.2B, C, E, F, and Table S3). The per-centages of Elf5 cells are higherthan those of Tbr2 cells, which werelower than 50% of those in the controltrophectoderm, both at the early andlate blastocyst stages (Fig. 2B, C, E,F, and Table S2). We found that, while90% of Elf5 cells are also Tbr2-posi-tive, the remaining 10% of Elf5 cellsdo not express Tbr2. Taken together,our results indicate a normal percent-age of Cdx2 cells but reduced percen-tages of Elf5 and Tbr2 cells in theparthenote trophectoderm.

Decrease of Nanog1 and

Sox21 cells in Parthenotes

Since there was a great decrease intotal cell numbers of parthenote blas-tocysts (Fig. 1), we were interested inassessing the numbers of ICM cells in

parthenote blastocysts. We performeddouble immunostaining for Cdx2 andOct4, and found normal levels of Oct4expression but significantly decreasednumbers of ICM cells in parthenotescompared to controls at both the earlyand late blastocyst stages (Supp. Fig.S2C). This reduction was proportionalto the decrease in total cell numbersin parthenote blastocysts (Fig. 1). Onthe other hand, the percentages ofICM cells in parthenotes were compa-rable with those in controls (Supp.Fig. S2D), concurrent with the compa-rable percentages of trophectodermcells (counted as Cdx2 cells) betweenparthenotes and controls (Fig. 2G andTable S2). The percentages of Oct4

cells in whole embryos were compara-ble between controls and parthenotesat both the morula and blastocyststages (Supp. Fig. S2, Fig. 3, andSupp. Table S2). At the late blastocyststage, when Oct4 expression was com-pletely restricted to the ICM, Oct4

cells constituted 100% of ICM cells inboth control and parthenote embryos(n 20) (Supp. Figs. S2B and S3D).We also examined the expression of

two other pluripotency genes, Sox2and Nanog, in normal fertilized andparthenote embryos. Sox2 isexpressed in the epiblast at the blas-tocyst stage and is required for peri-implantation lineage specification(Hayashi et al., 2002; Avilion et al.,2003; Boyer et al., 2006). Compared tocontrol embryos, we found dramati-cally decreased numbers of Sox2

cells in parthenote embryos at boththe morula and blastocyst stages (Fig.3). Percentages of Sox2 cells inwhole embryos are given in Table S2.As Sox2 cells constitute the epiblastat the blastocyst stage (Hayashi et al.,2002; Avilion et al., 2003), our resultsdemonstrate greatly diminished epi-blast populations in parthenote ICMs.Our analyses of the expression of

another pluripotency gene and epi-blast marker, Nanog (Chamberset al., 2003; Mitsui et al., 2003), fur-ther confirmed our results of Sox2 im-munostaining. We observed colocali-zation of Nanog and Sox2 cells inboth control and parthenote embryosat both the morula and blastocyststages (Supp. Fig. S3), indicatingoverlapping expression of Nanog andSox2 in the same cell lineage duringpreimplantation development. Simi-

lar to Sox2 cells, the numbers andpercentages of Nanog cells in parthe-notes were significantly lower thanthose in controls (Fig. 4 and Supp. Ta-ble S2).

Increase of Gata41 Cells in

Parthenotes

In addition to the epiblast, the othercell lineage in the ICM is the primi-tive endoderm (Rossant et al., 2003;Ralston and Rossant, 2005; Chazaudet al., 2006; Yamanaka et al., 2006).We then assessed expression of theprimitive endoderm marker, Gata4,in parthenotes. Our immunostainingresults showed only scattered Gata4

cells in control morulae at E3.0,whereas almost all cells in parthenotemorulae expressed Gata4 (Fig. 4A, B,and Table S2). Between E4.0 andE4.5, we found a dramaticallyexpanded Gata4 expression domain inparthenote blastocysts, covering notonly the majority of ICM cells but alsomost trophectoderm cells (Fig. 4C).Therefore, almost 90% of total cells inparthenote blastocysts expressedGata4, in contrast to restricted Gata4expression in control primitive endo-derm (Table S2). Percentages ofGata4 cells in ICMs were also signif-icantly higher in parthenotes than incontrols (Fig. 4D). In conclusion, greatdecreases in Sox2 and Nanog epi-blast cells and progenitors in parthe-note embryos were concomitant withdramatic increases of Gata4 primi-tive endoderm cells and progenitors.

Increased Fgf3 Expression

and Fgfr2 Phosphorylation in

Parthenotes

The balance between the expressionlevels of Gata4 and Nanog determinesthe cell fate of becoming, respectively,primitive endoderm or epiblast (Mit-sui et al., 2003; Rossant et al., 2003;Boyer et al., 2006; Chazaud et al.,2006; Yamanaka et al., 2006). Fibro-blast growth factor 4 (Fgf4) signalsthrough its receptor Fgf receptor 2(Fgfr2) and the adaptor protein Grb2to activate Gata4/6 expression andrepress Nanog expression, thus pro-moting primitive endoderm formation(Rappolee et al., 1994; Cheng et al.,1998; Goldin and Papaioannou, 2003;Chazaud et al., 2006; Nichols et al.,

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ABERRANT GENE EXPRESSION IN PARTHENOTE EMBRYOS 1653

2009; Guo et al., 2010; Yamanakaet al., 2010; Frankenberg et al., 2011).Because we observed an increase inGata4 and a decrease in Nanog levelsin parthenote embryos, we examinedthe levels of Fgf4 and Fgfr2 proteinsin parthenotes. At both the morulaand blastocyst stages, it was foundthat immunostaining patterns andintensities of Fgf4 and Fgfr2 werecomparable between controls and par-thenotes (Supp. Fig. S4). It is note-worthy that Fgf4 signals were presentin plasma membranes of all blasto-meres, but were detected in the peri-nuclear regions of Cdx2 blastomeres

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Fig. 2.

Fig. 3.

1654 CHEN AND YU

only (white arrows in Supp. Fig. S4A,B). This indicates that Cdx2 and peri-nuclear Fgf4 expression is mutuallyexclusive among different cells, andthe expression of Fgf4 may be gener-ated only by cells destined to becomethe ICM.Furthermore, in contrast to

unchanged levels of total Fgfr2 in par-thenotes, levels of phosphorylatedFgfr2 in parthenote morulae and blas-tocysts were significantly higher thanthose in controls, as revealed by theincreased intensities of immunostain-ing for Tyr653/654-phosphorylatedFgfr2 (Fig. 5). As Fgfr2 receptors areactivated upon phosphorylation,higher levels of Fgfr2 phosphorylationin parthenotes indicates increasedFgf signaling.Because Fgf signaling is elevated in

parthenotes without an increase ofthe Fgf4 protein level (Supp. Fig.S4A, B), we assessed other Fgfligands to identify which one maycause the increased phosphorylationof Fgfr2 in parthenotes. It was shownthat Fgf3 and Gata4/6 wereco-expressed in the parietal endodermof post-implantation embryos (Shimo-sato et al., 2007; Cai et al., 2008; Pilonet al., 2008). Hence we analyzed Fgf3immunostaining and found that, incontrast to control embryos, whichrevealed only background levels ofFgf3, parthenotes showed strongFgf3-positive signals throughout all

plasma membranes of both morulaeand blastocysts (Fig. 6). Unlike Fgf4,which was expressed in only Cdx2

blastomeres (Supp. Fig. S4A, B), Fgf3was co-expressed with Cdx2 in somenuclei of parthenote blastomeres (Fig.6A, B). Furthermore, Fgf3 was co-expressed with Gata4 in all nuclei ofparthenote blastomeres, both at themorula and blastocyst stages (Fig. 6C,D). We also performed the in situhybridization using an Fgf3 cDNAprobe as described previously (Tanna-hill et al., 1992; Wahl et al., 2007).The levels of Fgf3 mRNA were dra-matically increased in parthenotemorulae and blastocysts comparedwith controls, consistent with our im-munostaining data (Fig. 7). The dra-matic up-regulation of Fgf3 level inparthenotes may contribute to themarked increase and ectopic expres-sion 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 linkagebetween Fgfr2 phosphorylation andGata4/Nanog levels, we treated E3.0parthenogenetic morulae with the

Fgfr2 inhibitor SU5402 and incubatedfor 20 hr, followed by immunostainingto analyze the expression levels ofGata4, Nanog, and Fgf3. SU5402 hasbeen shown to be a potent inhibitor ofFgf signaling in the developing mouseembryos (Zuniga et al., 2004; Calmontet al., 2006; Miura et al., 2006; Di-Gregorio et al., 2007). These previousstudies indicated that a 50% inhibi-tion of Fgfr phosphorylation wasachieved at 1020 mM of SU5402(Mohammadi et al., 1997). Otherpotent inhibitors of Fgf signalinginclude PD173074 and PD184352(Nichols et al., 2009; Yamanaka et al.,2010). Nonetheless, because it wasdemonstrated that SU5402 has a mildeffect on mouse embryonic develop-ment (Miura et al., 2006), we choseSU5402 for our study and tested theeffects of SU5402 at a concentrationbetween 10 and 20 mM.After treatment with 10 mM of

SU5402 for 20 hr, parthenote morulaedisplayed no significant change ofGata4 and Nanog expression,whereas parthenogenetic blastocystsshowed significantly decreasedGata4 cells and increased Nanog

cells, despite that Gata4 was stillexpressed ectopically in many tro-phectoderm cells (compare Fig. 8Awith C). At 15 mM of SU5402, Gata4was detected in less than four tro-phectoderm cells of parthenote blasto-cysts, indicating a dramatic decreasein ectopic Gata4 expression (Fig. 8B);in addition, the numbers of Nanog

cells in parthenotes treated with 15mM of SU5402 were comparable withuntreated control (zygotic) embryos(Fig. 8B, D). At 20 mM of SU5402,Gata4 expression was completely re-stricted to the ICM of parthenotes,and the percentages of Gata4 andNanog cells in whole embryos arecomparable between parthenotes andcontrols (Fig. 8D). Thus, these resultsindicate that 20 mM of SU5402 com-pletely restored the balance betweenGata4 and Nanog levels.It has been shown that treating

normal (fertilized) embryos with 10mM SU5402 significantly down-regu-lates most primitive endoderm-spe-cific markers, including Gata4, andupregulates many epiblast-specificmarkers, including Nanog and Sox2(Guo et al., 2010). Hence we also ana-lyzed the effects of SU5402 on Gata4

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Fig. 3. Reduction in Sox2 levels in parthenote morulae and ICMs. A, C: Immunofluorescenceimages 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 blasto-cyst have dramatically reduced numbers of Sox2 cells (green and yellow colors) (arrows in Aand C). ICM, inner cell mass; TE, trophectoderm. Scale bars in A and C 20 mm. B: Averagepercentages of Oct4 and Sox2 cells in whole embryos at the morula stage. Note the signifi-cantly 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 blastocyststage. Note the significantly reduced percentage of Sox2 cells in parthenote ICMs (red asterisk)(n 20, P < 0.01, Students t-test).

Fig. 2. Decrease in Tbr2 and Elf5 levels in the parthenote trophectoderm. AF: Immunofluores-cence 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 cellsin parthenote trophectoderm. Scale bars in AF 20 mm. G: Comparable percentages ofCdx2 cells (i.e., trophectoderm cells) in whole embryos between controls and parthenotes atthe morula, early blastocyst, and late blastocyst stages (n 27). H: Significantly reduced per-centages of Tbr2 cells in the parthenote trophectoderm (TE) (asterisks) compared to control TEat both the early and late blastocyst stages (n 55, P < 0.01, Students t-test). I: Significantlyreduced percentages of Elf5cells in the parthenote trophectoderm (TE) (asterisks) compared tocontrol TE at both the early and late blastocyst stages (n 28, P < 0.01, Students t-test). Notethat 90% of Elf5cells are also Tbr2-positive, whereas the remaining 10% of Elf5cells do notexpress Tbr2.

ABERRANT GENE EXPRESSION IN PARTHENOTE EMBRYOS 1655

and Nanog expression in control blas-tocysts. We found that SU5402 treat-ment decreased the total numbers ofICM cells, with the numbers ofGata4 cells decreased to a muchhigher extent than the numbers ofNanog cells, leading to the reductionin the percentages of both Nanog

and Gata4 cells in whole blastocysts(Fig. 8AC, E). Therefore, in spite ofthe decreased percentages of Nanog

cells in whole embryos, the percen-tages of Nanog cells in control ICMswere significantly increased from 50.2to 82.6% and 92.3% (12-foldincrease) by 10 and 15 mM SU5402,respectively (Fig. 8E). On the otherhand, the percentages of Gata4 cellsin control ICMs were significantlydecreased from 49.8 to 17.4% and7.8% (310-fold decrease) by 10 and15 mM SU5402, respectively (Fig. 8E).Thus the Nanog/Gata4 gene expres-sion levels in the ICM were inverselycorrelated after treatment withSU5402.Although Gata4 and Nanog expres-

sion was rescued by SU5402

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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 theinner 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 ofNanog nuclei in the parthenote ICM, and arrowheads point to ectopic expression of Gata4 inthe parthenote trophectoderm (TE). Scale bars in A and C &equal; 20 mm. B: Average percen-tages of Nanog and Gata4 cells in whole embryos at the morula stage. Asterisks indicate thesignificantly reduced percentage of Nanog cells and the significantly elevated percentage ofGata4 cells in parthenote morulae (n 27, P < 0.01, Students t-test). D: Average percentagesof Nanog and Gata4 cells in ICMs at the blastocyst stage. Asterisks indicate the significantlydecreased percentage of Nanog cells and the significantly increased percentage of Gata4

cells in parthenote ICMs (n 20, P < 0.01, Students t-test).

Fig. 5. Elevated levels of Fgfr2 phosphorylation in parthenote morulae and blastocysts. AD: Immunofluorescence images of control and parthe-note 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 thecell membranes of parthenote morulae compared to controls. In B and D, arrows point to signals of phosphorylated Fgfr2 on the plasma mem-branes of primitive endoderm cells (i.e., Gata4 cells) in the control blastocyst, while arrowheads indicate significantly higher levels of Fgfr2 phos-phorylation in both the trophectoderm (TE) and inner cell mass (ICM) of the parthenote blastocysts. Scale bars in AD 20 mm.

1656 CHEN AND YU

treatment, we found that the total cellnumbers of parthenogenetic blasto-cysts were still significantly fewer

than controls, even when treated with20 mM of SU5402 (Fig. 8F). ThusSU5402 is capable of restoring normalGata4 and Nanog levels in parthe-notes, but fails to increase total cellnumbers of parthenotes. The decreasein total cell numbers may be attrib-uted to a decrease in cell proliferationor an increase in apoptosis (Hardyand Handyside, 1996; Uranga andArechaga, 1997). Because parthenotesdisplayed increased levels of Fgf3,especially in the nuclei (Fig. 6), andthe nuclear isoform of Fgf3 wasshown to inhibit cell proliferation(Kiefer and Dickson, 1995; Antoineet al., 2005), it was of interest toassess whether Fgf3 expression inparthenotes was affected by SU5402.Surprisingly, even when ectopicGata4 expression was completely sup-pressed by 20 mM of SU5402, the im-munostaining intensity of Fgf3 sig-nals was very strong in most nuclei ofparthenotes (throughout the ICM andtrophectoderm) (Supp. Fig. S5C).Supp. Figure S5D shows that the per-

centages of Gata4 nuclei in parthe-notes decreased as the concentrationof SU5402 increased, whereas thepercentages of Fgf3 nuclei were notaffected by the concentration ofSU5402. Therefore, we conclude thatthe Fgfr2 antagonist SU5402 does notinhibit Fgf3 expression. Given thatthe nuclear isoform of Fgf3 inhibitscell proliferation (Kiefer and Dickson,1995; Antoine et al., 2005), the highlevel of Fgf3 in the nuclei of partheno-genetic embryos may suppress cellproliferation in parthenotes.

DISCUSSION

Our findings in this study are sum-marized in Figure 9. In this study, weanalyzed expression of lineage-spe-cific genes and found lineage segrega-tion defects in parthenogenetic preim-plantation embryos. We found thatthe number of Gata4 primitive endo-derm cells was dramatically increasedwhile the number of Nanog/Sox2

epiblast cells was significantly

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Fig. 6. Increase in Fgf3 levels in parthenote morulae and blastocysts. AD: Immunofluorescence images of control and parthenote embryosstained 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 apparentlybright Fgf3 signals throughout parthenote embryos. White arrows in A indicate co-expression of Fgf3 and Cdx2 in some nuclei of parthenoteblastomeres. White arrows in C indicate co-expression of Fgf3 and Gata4 in all nuclei of parthenote blastomeres. The results demonstrate thatincreased Fgf3 expression in parthenotes is concomitant with increased Gata4 expression. ICM, inner cell mass; TE, trophectoderm. Scale barsin AD 20 mm.

Fig. 7. Increase in Fgf3 expression in par-thenote morulae and blastocysts. Control andparthenote morulae (n 12) and blastocysts(n 10) were hybridized with Fgf3 cDNAusing the in situ hybridization kit. Purple col-ors indicate positive Fgf3 mRNA signals. Notethere was only weak background staining inthe control morula and blastocyst. Fgf3 mRNAsignals are stronger in the cytoplasm andweaker in the nuclei of parthenote embryos,as normally seen in in situ hybridizationresults. Scale bars 30 mm.

ABERRANT GENE EXPRESSION IN PARTHENOTE EMBRYOS 1657

decreased. Although with decreasednumbers, these Nanog/Sox2 epi-blast cells still exist in the parthenoteICM and can be used to derivepatient-specific ES cells for stem celltherapies. On the other hand, thereare other drawbacks of parthenoge-netic embryos, which had been previ-ously reported, including aberrantexpression of imprinted and develop-ment-related genes that disrupt fulldevelopment of organisms (Humph-erys 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 abnor-mal gene expression in the preimplan-tation parthenogenetic embryo, which

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Fig. 8.

Fig. 9.

1658 CHEN AND YU

may affect the derivation and qualityof parthenogenetic ES cells.Consistent with previous studies

(Hardy and Handyside, 1996; Urangaand Ar&eacaute;chaga, 1997), wefound significantly reduced total cellnumbers in parthenote embryos com-pared to control embryos from theearly blastocyst stage, which waslikely due to decreased cell prolifera-tion in parthenotes between the mor-ula and blastocyst stages (Hardy andHandyside, 1996; Uranga andArechaga, 1997). Impaired prolifera-tion and differentiation were observedin both extraembryonic and embry-onic lineages of parthenote embryos,especially in the extraembryonic andembryonic mesoderm as well as prim-itive endoderm lineages (Bartonet al., 1985; Sturm et al., 1994; Mog-netti and Sakkas, 1996). Gata4 andGata6 are two essential marker genesof the primitive endoderm (Boyeret al., 2006; Yamanaka et al., 2006;Cai et al., 2008; Kuijk et al., 2008),which forms yolk sac and is a majoraffected lineage in parthenoteembryos (Sturm et al., 1994; New-man-Smith and Werb, 1995; Chazaudet al., 2006; Yamanaka et al., 2006).Our observation of the overexpressionof the parietal endoderm markergenes, Gata4 and Fgf3, in parthenoteICMs is consistent with the previ-ously reported tendency of parthenoteICM outgrowths to differentiate intoparietal endoderm cells, which are de-scendants of the primitive endoderm

(Newman-Smith and Werb, 1995;Boyer et al., 2006; Cai et al., 2008).Interestingly, we detected Gata4expression as early as the morulastage, while previous studies failed todetect Gata4 expression till the mid-blastocyst stage (Plusa et al., 2008;Silva et al., 2009; Yamanaka et al.,2010). While the same anti-Gata4antibody was used (from Santa CruzBiotechnology, Santa Cruz, CA), differ-ences in experimental procedures maycontribute to the different results. Forexample, while Plusa et al. (2008)fixed embryos in 4% PFA overnight,we fixed for only 15 min after treat-ment with acid Tyrodes solution. TheGata4 staining in morulae should notresult from unspecific signals, as ournegative controls using hepatocyte cul-tures did not show any positive Gata4signals (data not shown).During lineage allocation and specifi-

cation in the ICM, Sox2 and Nanog aretwo key transcription factors requiredfor development of the epiblast, whichforms the embryo body (Avilion et al.,2003; Rossant et al., 2003; Boyer et al.,2006; Chazaud et al., 2006; Yamanakaet al., 2006; Silva et al., 2009; Mes-serschmidt and Kemler, 2010; Franken-berg et al., 2011). In addition, dimin-ished Nanog expression, which wasshown to cause excessive differentia-tion of ICM cells into parietal endodermcells (Mitsui et al., 2003), was observedin parthenote embryos. Therefore, themisregulated balance between theexpression levels of Gata4/6 and Nanog

in parthenote ICMs may lead to exces-sive differentiation into the primitiveendoderm lineage and impaired differ-entiation into the epiblast lineage. It isalso noteworthy that elevated Gata4and reduced Nanog expression in par-thenote embryos was first observed atE3.0, suggesting that aberrant cell dif-ferentiation in parthenotes may occuras early as the morula stage. As Nanogwas demonstrated to maintain self-renewal and the undifferentiated stateof ICM and ES cells (Newman-Smithand Werb, 1995; Mitsui et al., 2003), itis likely that dramatically reducednumbers of Nanog blastomeres in par-thenote morulae indicate a significantlydecreased amount of proliferating cellssince E3.0, thus producing a signifi-cantly lower proliferation rate in par-thenotes compared to controls betweenthe morula and blastocyst stages.It has been reported that Cdx2,

Elf5, and Tbr2(Eomes) are three ofthe earliest genes required for specifi-cation and differentiation of thetrophoblast lineage, with the func-tioning cascade being:Cdx2!Tbr2!Elf5 (Russ et al., 2000;Donnison et al., 2005; Niwa et al.,2005; Strumpf et al., 2005; Hem-berger and Dean, 2007; Jedrusiket al., 2008; Ng et al., 2008; Ralstonand Rossant, 2008). In spite of beingdownstream of Cdx2 and Tbr2, Elf5 isthe key transcription factor creating afeedback loop reinforcing Cdx2 andTbr2 expression in trophectoderm,which is indispensable for the

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Fig. 8. Increased Nanog and decreased Gata4 levels in parthenotes treated with the Fgfr2 inhibitor SU5402. AC: Immunofluorescence imagesfor Nanog (red), Gata4 (green), and DAPI (blue) in control and parthenote blastocysts, which were cultured with the following concentrations ofSU5402 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 numberof 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 blastocyststages, with or without the addition of SU5402. Asterisks indicate significantly increased percentages of Nanog cells and significantly decreasedpercentages of Gata4 cells in SU5402-treated parthenotes compared to untreated ones (n 20, P < 0.01, Students t-test). Note the comparablepercentages of Nanog and Gata4 cells between untreated controls and 20 mM SU5402-treated parthenotes. E: In the ICM of SU5402-treatedcontrols, the percentages of Nanog cells are significantly increased whereas those of Gata4 cells are dramatically decreased after treatmentwith SU5402. Note the extent of change is greater for Gata4 cells (37-fold decrease) than for Nanog cells (less than 2-fold increase). In wholecontrol embryos, both the percentages of Nanog and Gata4 cells are significantly decreased after treatment with SU5402. Note the extent ofdecrease is greater for Gata4 cells (310-fold decrease) than for Nanog cells (less than 2-fold decrease). F: Total cell numbers in untreated con-trols and SU5402-treated or untreated parthenotes at various preimplantation stages. Asterisks indicate significantly decreased total cell numbersin both untreated and SU5402-treated parthenotes compared to controls at the early and late blastocyst stages (n 60, P < 0.01, Students t-test). 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 parthe-nogenetic embryos. In parthenotes, aberrant expression of Fgf3 and lineage-specific genes, including Nanog and Gata4, were observed at boththe morula and blastocyst stages. While the secreted isoform of Fgf3 binds to Fgfr2 and stimulates Fgfr2 phosphorylation and signaling, the nu-clear isoform of Fgf3 inhibits cell proliferation and decreases total cell numbers in parthenotes. The elevated Fgfr2 phosphorylation in parthenotescaused increased Gata4 and decreased Nanog expression, indicating expanded primitive endoderm cells and diminished epiblast cells. Inhibitionof Fgfr2 phosphorylation by SU5402 suppressed Gata4 and enhanced Nanog expression but did not affect Fgf3 expression in parthenotes, indi-cating that Fgfr2, Gata4, and Nanog are all downstream of Fgf3.

ABERRANT GENE EXPRESSION IN PARTHENOTE EMBRYOS 1659

differentiation of the trophoblast line-age (Donnison et al., 2005; Ng et al.,2008). As shown previously, hemizy-gous expression of Elf5 was sufficientto cause a marked decrease in Cdx2protein and Cdx2 and Tbr2 mRNAlevels (Ng et al., 2008).Interestingly, it has been reported

that parthenogenetic embryos show aremarkably higher global DNA meth-ylation level compared to normal (fer-tilized) embryos, especially from the4-cell to the blastocyst stage (Bartonet al., 2001; Li et al., 2009). Previousstudies have demonstrated that theearly demethylation event in embryo-genesis requires the presence of thepaternal genome (Barton et al., 2001;Santos and Dean, 2004; Morganet al., 2005). It is likely that the lackof the paternal genome causes deme-thylation failure in early parthenoge-netic embryos, leading to an elevatedmethylation level of the Elf5 promoterin many of the trophectoderm cells.Reduced Elf5 expression in these tro-phectoderm cells then leads todecreased Tbr2 expression. However,it is interesting that Cdx2 expressionwas not affected by reduced Elf5expression in parthenotes. One possi-bility is that the remaining Elf5expression in some cells is sufficientto maintain non-cell-autonomousCdx2 expression in the neighboringcells. On the other hand, Tbr2 expres-sion may be cell-autonomously regu-lated by Elf5; thus, Tbr2 is expressedonly in Elf5-expressing cells in par-thenote trophectoderm.All of the aforementioned genes

are pivotal factors regulating lineagesegregation and cell fate determina-tion (Russ et al., 2000; Strumpfet al., 2005; Shimosato et al., 2007;Cai et al., 2008). Similar toTbr2(Eomes) homozygous mutants,most parthenote embryos demon-strated a lack of diploid dividingtrophoblast cells, impaired or re-tarded development of the neuroecto-derm and brain, and multiple meso-dermal defects (Sturm et al., 1994;Bulfone et al., 1999; Russ et al.,2000; Kwon and Hadjantonakis,2007). Previous studies using chi-meric mouse embryos have demon-strated that parthenogeneticallyderived cells do contribute to the tro-phectoderm at the blastocyst stage,but not at the post-implantation

stages (Clarke et al., 1988; Thomsonand Solter, 1988, 1989). Therefore,there is an early post-implantationdefect in the parthenogenetic tropho-blast lineage. In spite of the tropho-blast defect, parthenote embryos arecapable of post-implantation develop-ment until the 25-somite stage (ataround E10.0) (Sturm et al., 1994;Penkov et al., 1995; Kono et al.,1996; Mognetti and Sakkas, 1996),and trophoblast giant cells weredetected in parthenote placentae(Barton et al., 1985; Sturm et al.,1994; Mognetti and Sakkas, 1996).On the other hand, Tbr2 homozygousmutants arrested at the peri-implan-tation stage (E6.0E7.5) with differ-entiation defects of the trophecto-derm and incompetence to formtrophoblast stem cells (Russ et al.,2000). This apparent difference isprobably due to the finding thatTbr2 expression is reduced but notcompletely absent in the parthenotetrophectoderm, as observed in thisstudy.Our observation that Cdx2 expres-

sion is normal in the parthenote tro-phectoderm and both Elf5 and Tbr2are expressed in over 40% of parthe-note trophectoderm cells is consistentwith the partially if not completelyspecified trophectoderm lineage inparthenotes (Sturm et al., 1994; Mog-netti and Sakkas, 1996). Interest-ingly, it has been shown that Fgf sig-naling is indispensible fortrophectoderm development andmaintenance of Cdx2 and Tbr2expression (Tanaka et al., 1998; Nich-ols et al., 2009). On the other hand,ectopic Gata4 expression is correlatedwith increased Fgf signaling in theparthenote trophectoderm. Therefore,overexpression of Gata4 may be inde-pendent of reduced Tbr2 expressionin parthenote embryos.It has been shown that Fgf4-Fgfr2

signaling is indispensable for tropho-blast proliferation and the mainte-nance of trophectoderm and ICMidentities between E3.5 and E4.5(Feldman et al., 1995; Chai et al.,1998; Tanaka et al., 1998; Haffner-Krausz et al., 1999; Goldin andPapaioannou, 2003). In this study, wefound elevated Fgfr2 signaling in par-thenotes starting from the 16-cellmorula stage, as indicated byincreased Fgfr2 phosphorylation, in

spite of unchanged Fgf4 expression inparthenotes. We found that Fgf3expression was undetectable orexpressed at a low-to-moderate levelin normal mouse preimplantationembryos (Rappolee et al., 1988, 1994;Zhong et al., 2006), but was stronglyexpressed in parthenote embryos.Our finding that inhibition of Fgfr2suppressed Gata4 but not Fgf3expression in parthenotes (Supp. Fig.S4) indicates that Gata4 is not theonly factor up-regulating Fgf3 expres-sion, and that increased Fgf3 expres-sion is precedent to increased Gata4expression in parthenotes. Our obser-vations indicated that Fgf3 did notup-regulate Gata4 expression whenFgfr2 signaling was inhibited. Itremains to be studied which factor(s)induce(s) Fgf3 overexpression in par-thenotes. Assessing the expressionlevels of Fgf3 in parthenogeneticembryos earlier than the morulastage will unravel the earliest stageat which Fgf3 up-regulation is firstobserved. The factor(s) inducing Fgf3expression in parthenotes should bedetectable at this stage.Interestingly, Fgf3 was reported to

have dual subcellular fates and playdual roles in regulating proliferation,with the secreted isoform promotingbut the nuclear isoform inhibiting cellproliferation (Kiefer et al., 1994; Kie-fer and Dickson, 1995; Antoine et al.,2005). We have shown that Fgf3 im-munostaining signals are apparentlybrighter in the nuclei than on theplasma membranes of parthenotes,indicating more nuclear than secretedFgf3 proteins, thus leading to the in-hibition of cell proliferation. The ex-cessive nuclear Fgf3 proteins maycontribute to the reduction of totalcell numbers in parthenotes. There-fore, increased Fgf3 and ectopicGata4 expression in parthenotetrophectoderm together perturb thebalance between differentiation andproliferation of trophoblast cells.Interestingly, recent studies demon-

strated that inhibition of the FGF/MAPkinase signal caused almost all ICMcells in normal (fertilized) embryos tobecome Nanog epiblast cells, and noor very few Gata4/Gata6 primitiveendoderm cells were detected (Guoet al., 2010; Yamanaka et al., 2010).Consistent with previous findings ofinverse correlation of Nanog/Gata4

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expression levels (Guo et al., 2010;Yamanaka et al., 2010), we foundSU5402 significantly increased the pre-centages of Nanog cells and decreasedthe percentages of Gata4 cells in con-trol ICMs. Interestingly, the extent ofincrease in the percentages of Nanog

cells (less than 2-fold increase) wasmuch lower than the extent of decreasein the percentages of Gata4 cells (upto 67-fold decrease). This is in agree-ment with the previous study showinga 6-fold down-regulation of Gata4expression and 2-fold up-regulation ofNanog expression in embryos treatedwith 10 mM SU5402 from the 16-cellstage for 24 hr (Guo et al., 2010).Our results indicate that the

decrease of total cell numbers in par-thenotes is not associated with aber-rant Nanog or Gata4 expression, asthe restoration of normal Nanog andGata4 expression is not concomitantwith increased cell numbers. Instead,increased Fgf3 expression, which isnot affected by SU5402 treatment,may be associated with decreased cellnumbers in parthenotes, as the exces-sive nuclear Fgf3 proteins may sup-press cell proliferation and thusreduce total cell numbers.

EXPERIMENTAL

PROCEDURES

Parthenogenetic Activation

and In Vitro Culture of

Preimplantation Embryos

Animals used in this study were pur-chased from BioLASCO Taiwan (Tai-pei, Taiwan), and approval wasreceived from the Academia SinicaInstitutional Animal Care and Utili-zation Committee. B6DBA femalemice at 1014 weeks old were supero-vulated by an intraperitoneal (i.p.)injection of 5 IU pregnant mare se-rum gonadotropin (Merck, Darm-stadt, Germany), followed by an i.p.injection of 5 IU human chorionic go-nadotropin (hCG) (Sigma-Aldrich, St.Louis, MO) 4850 hr later. In orderto synchronize the time of fertiliza-tion with the time of parthenogeneticactivation, 12.5 hr after hCG injec-tion, the female mice were separatedinto two groups: the first group wasindividually paired with males of thesame strain for 1.5 hr and thenchecked for copulation plugs, and the

second group was sacrificed while thefirst group was mating. The unfertil-ized oocytes enclosed in cumulusmasses were released from theampullae, and cumulus cells wereremoved by pipetting using a mouth-controlled pipette (with an inner di-ameter of 200300 mm) after 5 min oftreatment with EmbryoMaxVR M2medium containing 50100 U/ml hy-aluronidase (Millipore, Billerica,MA). The unfertilized oocytes werethen washed and equilibrated in 35-ml droplets of potassium simplex opti-mized medium (EmbryoMaxVR

KSOM, Millipore, Billerica, MA) at37C in a humidified atmosphere of5% CO2 in air. After 1 hr of equilibra-tion, parthenogenetic activation wasconducted in CZBG medium contain-ing 10 mM strontium chloride (SrCl2)and 5 mg/ml cytochalasin B for 4.5 hrat 37C with 5% CO2 in air, asdescribed before (Gao, 2006). The for-mation of pronuclei was observedbetween 3 and 3.5 hr after incubationin the activation medium, i.e.,between 16.5 and 17 hr after hCGinjection. The timing of pronulcei for-mation in parthenotes was consistentwith previous observations (Abramc-zuk and Sawicki, 1975).At the same time, for the oocytes of

the first group of female mice, fertiliza-tion was reported to take place from2.5 hr following pairing, and the firstpronuclei were found to appear 2 hr af-ter fertilization (Abramczuk andSawicki, 1975; Hardy and Handyside,1996). Thus the first pronuclei in fer-tilized oocytes appeared about 4.5 hrafter pairing, i.e., approximately 17 hrafter hCG injection. The mated femalemice were sacrificed 4.5 hr after pair-ing, with the cumulus cells removed inthe EmbryoMaxV

R

M2 medium contain-ing 50100 U/ml hyaluronidase (Milli-pore). The presence of pronuclei wasconfirmed under microscope. Accord-ing to our observations, the timepoints of the first pronuclear formationare between 16.5 and 17 hr after hCGinjection for both the fertilized controland parthenogenetic oocytes. Alloocytes at the pronuclei stage werethen transferred to KSOM mediumand incubated till they reached the de-velopmental stage at which they wereanalyzed. Embryos normally cleave totwo-cells at E1.01.5, and form moru-

lae at E2.53.0 (4860 hr in culture).Early blastocysts are formed at E3.5(7072 hr in culture), blastocysts areformed at E4.0 (8084 hr in culture),and late expanded blastocysts areformed at E4.5 (9296 hr in culture).According to our observation, cultur-ing fertilized oocytes in SrCl2-contain-ing medium for the same time asunfertilized (parthenogenetic) oocytesdid not affect immunostaining resultsin these control embryos, indicatingthat chemical activation by SrCl2 doesnot contribute to the differential geneexpression between controls and par-thenotes. These observations are con-sistent with many previous reports(Loren and Lacham-Kaplan, 2006;Kyono et al., 2008; Chen et al., 2010).

Immunofluorescence Staining

For immunostaining, embryos werewashed for 510 s in droplets of acidicTyrodes solution (made by Sigma-Aldrich; purchased from Uni-onward,Taipei, Taiwan) to remove the zonapellucida, and then fixed in 4% para-formaldehyde (Sigma-Aldrich) in 1phosphate-buffered saline (PBS) for 15min at room temperature. Embryoswere then permeabilized with 0.25%Triton X-100 for 15 min, followed bywashing and blocking for 1 hr in block-ing solution containing 0.05% Tween-20, 3% bovine serum albumin (BSA),and 5% normal goat serum in 1 PBS.After blocking, embryos were incu-bated at 4C overnight with the follow-ing primary antibodies diluted inblocking solution: Cdx2 (mouse mono-clonal; 1:100 dilution; BioGenex, SanRamon, CA), Elf5 (mouse monoclonal;1:100 dilution; Santa Cruz Biotechnol-ogy, Santa Cruz, CA), Fgf3 (rabbit pol-yclonal; 1:200 dilution; Abcam, Cam-bridge, MA), Fgf4 (rabbit polyclonal;1:200 dilution; Abcam), Fgfr2 (rabbitpolyclonal; 1:50 dilution; Abgent, SanDiego, CA), phosphor-Fgfr2-pY653/654(rabbit polyclonal; 1:50 dilution;Abgent), Gata4 (mouse monoclonal;1:100 dilution; Santa Cruz Biotechnol-ogy), Nanog (rabbit polyclonal; 1:100dilution; 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 sec-ond day, the embryos were washed

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ABERRANT GENE EXPRESSION IN PARTHENOTE EMBRYOS 1661

and blocked for 1 hr in blocking solu-tion, followed by incubation at roomtemperature for 1 hr with the follow-ing secondary antibodies conjugatedwith fluorophores: goat anti-mouseAlexaFluor 488 (green fluorescence)and goat anti-rabbit AlexaFluor 555(red fluorescence) (Invitrogen Taiwan,Taipei, Taiwan). After incubation, theembryos were washed for 10 min inwashing solution containing 0.2% Tri-ton X-100 in 1 PBS, and counter-stained with 0.2 mg/ml DAPI in wash-ing solution for 10 min, followed bymounting in VectaShield (Vector Labo-ratories, Burlingame, CA) on glassslides.

mRNA In Situ Hybridization

Fgf3 mRNA in situ hybridization wasperformed with DIG-AP RembrandtV

R

Universal RISH and Detection Kit(Invitrogen, Carlsbad, CA), followingthe manufacturers instructions. Thesequence of Fgf3 cDNA probe hasbeen described previously (Tannahillet al., 1992; Wahl et al., 2007).

Confocal Microscopy

Series of confocal sections through 3Dpreserved embryo nuclei were col-lected using a Leica TCS SP5 confocalmicroscope (located on the 5th flour ofGenomics Research Center, AcademiaSinica) equipped with a Super Z gal-vanometer stage and Plan Apo 63/1.4 NA oil immersion objectives. Fluo-rochromes were visualized using anargon laser with an excitation wave-length of 488 nm (for AlexaFluor488), a DPSS laser with a laser line of561 nm (for AlexaFluor 555), and adiode laser with a laser line of 405 nm(for DAPI). For each optical section,images were sequentially collectedusing the XYZ mode for two or threefluorochromes. The pinhole was set to11.5 Airy units and the scan zoomwas 1.5. In order to compare the rel-ative intensities of immunostainingbetween control and parthenoteembryos, identical scanning parame-ters including the strength of laseremissions were maintained for con-trols and parthenotes stained withthe same antibodies. Images of opticalsections were then analyzed usingLeica Application Suite, and 3D andmaximum projections were con-

structed from serial stacks of sectionsfor each embryo.

Cell Counting and Statistical

Analyses

The image files of optical sections ofeach embryo were analyzed by theCount Nuclei/Cell Sorting ApplicationModule for MetaMorph (MetaMorphOffline vers. 7.0; Universal ImagingCorporationTM, Buckinghamshire,UK) to count cell numbers for theentire embryo and for each antibodyimmunostained sample. Statisticalsignificances are represented by Pvalues, which were calculated by Stu-dents t-test. The lower the P value,the more significant the differencebetween control and parthenoteembryos. The difference was regardedas non-significant when P 0.05, assignificant when P < 0.05, and ashighly significant when P < 0.01.

Inhibition of Fgfr2 Signaling

Culturing mouse embryos withSU5402 to inhibit Fgf signaling hasbeen described in previous studies(Zuniga et al., 2004; Calmont et al.,2006; Miura et al., 2006; Di-Gregorioet al., 2007). SU5402 was dissolved in100% dimethyl sulfoxide (DMSO) at10 mM (stock solution) and stored at20C until use. Because a 50% inhi-bition of Fgfr phosphorylation wasachieved at 1020 mM of SU5402(Mohammadi et al., 1997), weassessed the effects of SU5402 at 10,15, and 20 mM, respectively, on thedevelopment of both control and par-thenogenetic embryos. Embryos werecultured in the KSOM medium con-taining different concentrations ofSU5402 for 20 hr since the early mor-ula stage (E3.0) and then analyzed byimmunostaining at the mid-blastocyststage (E4.0). For experimental con-trols, we treated embryos for 20 hrwith an equal concentration of DMSOin KSOM (0.10.2 % final).

ACKNOWLEDGMENTSWe thank Chien-Hong Chen for pro-viding the parthenogenetic activationprotocol and Li-Wen Lo of the CoreFacility of the Genomics ResearchCenter for expert assistance with con-focal microscopy. We also thank Drs.Hung-Chih Kuo and Cheng-Fu Kao in

the Institute of Organismic and Cellu-lar Biology for critical comments onthis project and manuscript. Thiswork was supported by grant 94M011-1 from the Genomics Research Centerof Academia Sinica and NSC 98-3111-B-001-003 to John Yu.

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