Research Article Dual Function of Wnt Signaling during ...downloads.hindawi.com/journals/sci/2015/459301.pdf · Research Article Dual Function of Wnt Signaling during Neuronal Differentiation

Research ArticleDual Function of Wnt Signaling during NeuronalDifferentiation of Mouse Embryonic Stem Cells

Hanjun Kim12 Sewoon Kim1 Yonghee Song1 Wantae Kim1

Qi-Long Ying2 and Eek-hoon Jho1

1Department of Life Science University of Seoul 163 Seoulsiripdaero Dongdaemun-gu Seoul 130-743 Republic of Korea2Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research at USC Keck School of MedicineUniversity of Southern California Los Angeles CA 90033 USA

Correspondence should be addressed to Qi-Long Ying qyinguscedu and Eek-hoon Jho ej70uosackr

Received 19 February 2015 Accepted 24 March 2015

Academic Editor Laura Lasagni

Copyright copy 2015 Hanjun Kim et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Activation ofWnt signaling enhances self-renewal of mouse embryonic and neural stemprogenitor cells In contrast undifferenti-ated ES cells show a very low level of endogenous Wnt signaling and ectopic activation of Wnt signaling has been shown to blockneuronal differentiation Therefore it remains unclear whether or not endogenous Wnt120573-catenin signaling is necessary for self-renewal or neuronal differentiation of ES cells To investigate this we examined the expression profiles ofWnt signaling componentsExpression levels of Wnts known to induce 120573-catenin were very low in undifferentiated ES cells Stable ES cell lines which canmonitor endogenous activity of Wnt120573-catenin signaling suggest that Wnt signaling was very low in undifferentiated ES cellswhereas it increased during embryonic body formation or neuronal differentiation Interestingly application of small moleculeswhich can positively (BIO GSK3120573 inhibitor) or negatively (IWR-1-endo Axin stabilizer) control Wnt120573-catenin signaling suggeststhat activation of that signaling at different time periods had differential effects on neuronal differentiation of 46C ES cells FurtherChIP analysis suggested that 120573-cateninTCF1 complex directly regulated the expression of Sox1 during neuronal differentiationOverall our data suggest that Wnt120573-catenin signaling plays differential roles at different time points of neuronal differentiation

1 Introduction

Embryonic stem (ES) cells are pluripotent and self-renewingcells derived from the inner cell mass of preimplantationblastocysts These cells can differentiate into the followingthree germ layers the ectoderm mesoderm and endodermDue to these characteristics ES cells are considered usefultools in both research and regenerativemedicineTherefore itis important to understand how stem cellsmaintain their self-renewal capacity and differentiate into each specific lineage[1ndash3] However the molecular mechanisms of ES cell self-renewal and differentiation remain poorly understood

ES cells can be differentiated into neuronal cell lineage invitro Generally for differentiation into neuronal cells ES cellsare aggregated to form embryoid bodies (EBs) in suspensionculture for 4 days and treated with retinoic acid (RA) [4]However it is difficult to manipulate a specific lineage asRA treatment has been shown to induce other cell lineages

To avoid these problems a chemically defined mediumfor neuronal differentiation was developed [5 6] UsingN2B27 medium 46C cells (mouse ES cells containing Sox1promoter-driven EGFP) can be reproducibly differentiatedinto a neuronal lineage without EB formation or treatmentwith RA and thereby it has been used to examine the effectsof gene manipulation such as depletion or ectopic expressionon neuronal differentiation [5 6]

Wnts are secreted signaling proteins that regulate a varietyof developmental processes including cell growth motilityand differentiation [7] Wnt120573-catenin signaling is conservedin worms flies fish frogs mice and humans [8] In theabsence of Wnt 120573-catenin forms a ldquodestruction complexrdquoalong with the scaffold protein Axin the tumor suppressorgene product APC and GSK-3120573 that is phosphorylated bycasein kinases (CK1) and glycogen synthase kinase 3120573 (GSK-3120573) Phosphorylation of 120573-catenin triggers its ubiquitinationby 120573-TrCP followed by proteasomal degradation [9 10]

Hindawi Publishing CorporationStem Cells InternationalVolume 2015 Article ID 459301 10 pageshttpdxdoiorg1011552015459301

2 Stem Cells International

However when Wnt binds to its receptor Frizzled andcoreceptor LRP56 the cytoplasmic component dishevelledblocks 120573-catenin degradation leading to the accumulation of120573-catenin in the cytoplasm Stabilized 120573-catenin then entersthe nucleus where it interacts with TCFLEF [11ndash13] andactivates the expression of target genes such as C-Myc CyclinD1 Brachury and Twin

Wnt120573-catenin signaling has been shown to play impor-tant roles in ES cell self-renewal and differentiation [14ndash17]Activation of Wnt120573-catenin signaling prevents differentia-tion of human and mouse ES cells For example activationof Wnt signaling by GSK3120573 inhibitor maintains self-renewalof human and mouse ES cells and Wnt120573-catenin signalingupregulates Stat3 activity and prevents differentiation ofmouse ES cells [18 19] Furthermore TCF3 a terminaltranscription factor in Wnt signaling represses Nanog anddelays differentiation of ES cells [20] However other evi-dence has shown that activation of Wnt120573-catenin signalingby Wnt3a recombinants induces cell differentiation as well asproliferation [21] Wnt120573-catenin signaling is also involved inregulating the neuronal differentiation of ES cells [22] On theother hand it has been shown that theWnt antagonist sFRP2induces neuronal differentiation [23] Therefore the rolesof endogenous Wnt120573-catenin signaling and its componentsin the self-renewal and neuronal differentiation of ES cellsremain controversial

In this study we examined the endogenous level ofWnt120573-catenin signaling components during neuronal differ-entiation The data using reporter ES cell lines representingendogenous Wnt120573-catenin signaling activity showed thatWnt120573-catenin signaling increased during neuronal differ-entiation Interestingly Wnt120573-catenin signaling increaseduntil formation of neuronal precursor cells however it wasreduced during later periods of neuronal differentiationDifferential regulation of neuronal differentiation by thetreatment of activator or inhibitor ofWnt120573-catenin signalingsuggested thatWnt signaling plays dual roles during neuronaldifferentiation

2 Materials and Methods

21 Cell Culture E14 and 46C ES cells (ES cell line inwhich EGFP is substituted into the ORF of the Sox1 gene areporter for differentiation into neural precursor cells) werecultured in mouse ES (mES) cell medium (DMEM (Gibco)supplemented with 15 FBS 2mM GlutaMAX (Gibco)MEMnonessential amino acids 120573-mercaptoethanol (Gibco)tylosin and 1 PenStrep (Gibco)) supplemented with LIF(ESGRO Chemicon) on 02 gelatin-coated dishes Cellswere maintained at 37∘C in a humidified atmosphere of 5CO2

22 Neuronal Differentiation To induce neuronal differ-entiation undifferentiated 46C ES cells were trypsinizedand neutralized with serum-containing medium ES cellswere dissociated in N2B27 medium (DMEMF12 (Gibco)Neurobasal medium (Gibco) N2 supplement (Invitrogen)B27 supplement (Invitrogen) 1mM GlutaMAX (Gibco)

01M120573-mercaptoethanol (Gibco) and 1PenStrep (Gibco))and then plated on a 02 gelatin-coated tissue culturedish (Falcon) containing DMSO (SIGMA) 075 120583M MeBIO(Calbiochem) 075120583MBIO (Calbiochem) [24] and 075 120583MIWR-1-endo (kindly provided by Dr Lawrence Lum Uni-versity of Texas Southwestern Medical Center) [25] N2B27mediumwas changed every other day On the 6th day EGFP-positive cells (Sox1 positive cells) were observed by using afluorescent microscope (Leica DMIRB Leica Microsystems)For production of neurons Sox1-positive cells were replatedon PDL-laminin-coated wells in N2B27 medium containingEGF and FGF-2 N2B27 mediumwas changed every 3-4 days[6]

23 FACS Analysis To analyze 46C ES cell-derived Sox1-GFP-positive neural precursors 46C cells were differentiatedfor 6 days in N2B27mediumwithMeBIO or BIO and DMSOor IWR The cells were then trypsinized and neutralizedwith serum containing medium After centrifugation cellswere washed two times with PBS and resuspended in PBSResuspended cells were analyzed using a FACScan For dataanalysis FACSDiva software was used

24 Transfection and Luciferase Assay Oct4 promoter-EGFP ES cells were stably transfected with TOP-mCherryand Axin2-promoter-mCherry using Amaxa nucleofectoraccording to the manufacturerrsquos instructions Stably trans-fected clones were selected in media containing 800120583gmLof G418 (Gibco)

To measure Wnt signaling activity pTOP-Flash plasmidand pRL-TK plasmid were transfected into mES cells Trans-fected ES cells were cultured in the presence or absenceof LIF for 48 h To measure Sox1 promoter activity undernondifferentiation and neuronal differentiation conditionspGL3-mSox1 promoter plasmid and pRL-TK plasmid weretransfected into 46C cells which were cultured in mESmedium or N2B27 medium with or without 075120583M BIO(Calbiochem) Luciferase activity was measured by Dual-Luciferase reporter assay (Promega)

25 Reverse Transcriptase-PCR Examination of gene expres-sion during neuronal differentiation was carried out byRT-PCR Total RNA was isolated using TRIzol reagent(Invitrogen) according to the manufacturerrsquos protocol cDNAwas synthesized from total RNA (1 120583g) in a total reactionvolume of 20 120583L using ImProm-II Reverse Transcriptase(Promega) with random primers cDNAwas amplified underthe following conditions 94∘C for 2min followed by 20 25or 30 cycles of 94∘C for 45 s 58∘C for 45 s and 72∘C for 50 s

26 Immunofluorescence Analysis Neuronally differentiated46C ES cells for 15 days were fixedwith 4 paraformaldehydeat room temperature for 20min and then permeabilized for20min with permeabilization solution Cells were incubatedwith anti-MapII (Chemicon) primary antibody for 1 h Afterwashing 10 times with PBST (005 Tween 20 10mMNa2HPO4 2mM KH

2PO4 27mM KCl and 137mM NaCl)

cells were incubated with Alexa Fluor 488 (Invitrogen) and

Stem Cells International 3

DAPI (KPL Inc) for 1 h in the dark to stain the nucleusfollowed by washing again five times with PBST Wells weremounted in a drop of Mount Medium (KPL Inc) Signal wasvisualized by fluorescence using amicroscope (LeicaDMIRBLeica Microsystems)

27 Western Blot Analysis Mouse ES cells were seeded in6-well plates and differentiated in ES medium or N2B27mediumwithout LIFmES cells were washedwith phosphate-buffered saline (PBS) and then lysed in RIPA buffer (25mMTris-HCl at pH 80 150mM NaCl 10 glycerol 1 IgepalCA-630 025 deoxycholic acid 2mM EDTA 1mM NaFand 50mM glycerophosphate) on ice for 30min Lysateswere cleared by centrifugation at 12000 rpm for 10minafter which supernatants were collected Bradford (Bio-Rad)assay was used to determine the protein concentrationEqual amounts of protein were boiled and separated bySDS-polyacrylamide electrophoresis gels and transferred to aPVDF membrane (Pall Corporation) Blots were then incu-bated with antiactive-120573-catenin (Millipore) or anti120573-actin(Santa Cruz Biotechnology) antibody Immunocomplexeswere visualized using an enhanced Chemiluminescence kit(Elpis Biotech)

28 Chromatin Immunoprecipitation (ChIP) Assay Cellswere cross-linked with 1 formaldehyde (Sigma) at roomtemperature for 10min and then incubated with 0125Mglycine for 5min with gentle shaking Cells were then washedtwice with PBS before harvesting Cells resuspended withhypotonic buffer (10mM Hepes-KOH pH 78 10mM KCland 15mM MgCl

2) were swollen on ice for 10min and

then passed through a 265 gauge needle six times Aftercentrifugation at top speed for 5min at 4∘C pellets wereincubated with nuclei lysis buffer (1 SDS 50mM Tris-HCl pH 80 and 10mM EDTA) for 10min on ice withoccasional vortexing Chromatin was sheared to an averagelength size of 02sim1 kb by sonication on ice Supernatantwas collected by centrifugation (13000 rpm 10min 4∘C)and the supernatant concentration was determined by aspectrophotometer The appropriate volume of chromatinwas diluted 1 10 in ChIP dilution buffer (001 SDS 20mMTris-HCl pH 80 167mM NaCl 12mM EDTA and 11Triton X-100) after which preclearing was performed at4∘C for 2 h with 10 120583L of protein AG plus-agarose beads(Santa Cruz Biotechnology) For immunoprecipitation goatrabbit-IgG (Bethyl) anti120573-catenin (BD biosciences) anti-TCF3 (Santa Cruz) and anti-TCF1 (cell signaling) antibodieswere administered at 4∘C overnight Immunoprecipitatedchromatins were eluted after which reverse cross-linking wascarried out by addition of 03M NaCl at 65∘C overnightFollowing phenol-chloroform extraction and ethanol pre-cipitation DNA was dissolved in 50120583L of TE buffer(10mM Tris-HCl pH 80 and 1mM EDTA) Sox1-1 primers(Forward 51015840-AGTTCAGCCCTGAGTGAC-31015840 and Reverse51015840-TGGGTGCCTAGCGGAGAG-31015840) Sox1-2 primers (For-ward 51015840-TGGTCTGATCCCAAGTAG-31015840 and Reverse 51015840-TTTCTGAAGCGATTCTCC-31015840) and Axin primers (For-ward 51015840-TAACGCGGGAGCTGAGTGTG-31015840 and Reverse51015840-AAATCCATCGCGAACGGCTG-31015840) were used for PCR

3 Results

31 Increase in Wnt120573-Catenin Signaling Activity duringNeural Differentiation To study the role of Wnt signalingduring neural differentiation we used 46Cmouse embryonicstem (ES) cells (EGFP was substituted into the ORF ofthe Sox1 gene) with the monolayer neural differentiationmethod Upon being cultured in N2B27 medium 46C EScells started to express EGFP from day 4 and outgrowthof neurites was detected from day 8 after replating (Fig-ures 1(a)ndash1(e)) [5] To examine the expression patterns ofWnt components during differentiation we performed RT-PCR analysis using specific primers RT-PCR data showedreduced expression of stemness markers (Oct4 and Nanog)and increased expression of an ectoderm marker (Sox1)during neuronal differentiation Expression levels of manyWntsWnt receptorsWnt coreceptors andWnts componentsalso changed during differentiation (Figure 1(f)) These datasuggest that Wnt signaling might play important roles inself-renewal or neuronal differentiation although its exactfunctions are unknown

Next we asked whether Wnt signaling activity can beregulated under self-renewal and differentiation conditionsTo this end we measured endogenousWnt120573-catenin signal-ing activity under self-renewal and differentiation conditionsusing TOP and Wnt target genes Axin2 promoters (hereaftercalled Ax2P)-driven reporter construct that contain respon-sive TCF binding elements [26 27] Notably reporter activ-ities were low in undifferentiated mouse ES cells whereas itwas increased under absence of LIF conditions (Figure 2(a))To monitor the change status of Wnt signaling during neuraldifferentiation we established reporter ES cell lines (Oct4-GipTOP or Ax2P-mCherry) As expected we observed onlyGFP expression but not mCherry expression in self-renewalcondition due to low Wnt activity (Figure 2(d)) As shownin Figure 2(e) addition of BIO GSK3120573 inhibitor enhancedexpression of mCherry indicating that this cell line reflectsWnt120573-catenin signaling After stable cells were culturedin N2B27 medium for neural differentiation expression ofmCherry was significantly increased in neural precursorcells whereas it was decreased in fully differentiated neurons(Figures 2(f)-2(g)) Consistently the active 120573-catenin wasmore abundant during differentiation induced by the removalof LIF or N2B27 medium which induces embryonic stemcells differentiation toward neuronal lineage reaching amaximum on day 4 (Figure 2(b)) LRP6 phosphorylationon S1490 used an indicator for initial activation of Wnt120573-catenin signaling was increased during neural differentiation(Figure 2(c)) Taken together these data suggest that Wnt120573-catenin signaling is regulated during ES cell differentiationand neural precursor differentiation

32 Treatment of BIO from Days 4 to 6 Enhances NeuralDifferentiation Based on data described above (Figures 1 and2) we asked whether time window of Wnt120573-catenin sig-naling affects neural differentiation For this we sequentiallytreated with BIO (GSK3120573 inhibitor) for indicated duration asdescribed in Figure 3 [24] Addition of BIO in 46C ES cellsfrom day 0 to day 6 completely diminished EGFP expression


(a) (b) (c)

(d) (e)

Oct4

Nanog

Gata4

Brachyury

Sox1

Dvl2

Axin1

Axin2

Undiff 1 3 6 8 11 17 (day)

FACSsorting

Undiff 1 3 6 8 11 17 (day)

FACSsorting

GAPDH

Wnt4

Wnt5a

Wnt7a

Wnt8a

Wnt3a

Frizzled1

Frizzled2

LRP6

120573-cat

(f)

Figure 1 Neuronal differentiation of mouse embryonic stem cells and expression of Wnt and Wnt component genes (a)ndash(e) Using N2B27medium 46C ES cells (Sox1-promoter-GFP) were differentiated to neuronal cells for 14 days GFP in 46C ES cells was expressed duringneuronal differentiation for 6 days ((a) phase contrast (b) Sox1-GFP) and neurite outgrowth increased during neuronal differentiation for8 days (c) and 14 days ((d) phase contrast (e) MAPII (red) DAPI (blue)) (f) RT-PCR results of stem cell marker Wnt and Wnt signalingcomponents during neuronal differentiation During neuronal differentiation RNA levels of manyWnt andWnt signaling components werechanged Scale bars 50120583m


0

02

04

06

08

1

12

14

16

Luci

fera

se ac

tivity

(rel

ativ

e uni

t)

LIF + +

Top-LucAxin2-Luc

minusminus

lowastlowastlowast lowastlowast

(a)

(ABC)

0 2-LIF 4 6 8 (day)

(day)0 2 4 6N2B27

(ABC)

Active 120573-catenin


120573-actin

120573-actin

(b)

(day)0 2 4 6N2B27

p-LRP

120573-actin

(c)

Axin2-mCherry

+MeBIO

(d)

Axin2-mCherry

+BIO

(e)

Top-mCherry

(f)

Axin2-mCherry

(g)

Figure 2 Increase in canonical Wnt signaling during neural differentiation (a) Both TOP and Axin2 promoter luciferase activities in E14 EScells were induced at 48 h after LIF removal (b) Western blot analysis using ABC (active 120573-catenin) and 120573-actin antibodies Active 120573-cateninlevel was the highest on day 4 of neural differentiation (c) p-LRP level was the highest on day 4 of neural differentiation (d)-(e) In Oct4-GipAx2P-mCherry cells only GFP expression was detected mCherry expression was increased after 24 h addition of BIO (075120583M) (f)-(g)mCherry expression increased in the neural precursor region of TOP-mCherry ((d) and (e)) andAx2p-mCherry ((f) and (g)) stable cell linesduring neuronal differentiation Stable cell lines were cultured in N2B27 medium for 14 days Scale bars 100120583m

compared to MeBIO treatment a control analog of BIOwhich displays minimal activity against GSK3120573 (Figures 3(a)and 3(b)) These results were corroborated by FACS analysis(Figures 3(g) and 3(h)) Similar to report that activationof canonical Wnt signaling by GSK3120573 inhibition maintainspluripotency of ES cell we found that BIO treatment could

enhance the expression of stemness marker gene such asNanog under differentiation conditions (Figure 3(i)) [18ndash20 28] On the other hand treatment with BIO from day 4to day 6 surprisingly increased EGFP intensity and EGFP-positive cells (Figures 3(f)ndash3(h)) To test whether or notthe increase in EGFP expression by BIO can be attributed


MeBIO (days 0ndash6)

(a)

BIO (days 0ndash6)

(b)

BIO (days 1ndash6)

(c)

BIO (days 2ndash6)

(d)

BIO (days 3ndash6)

(e)

BIO (days 4ndash6)

(f)

EGFP

0

58

115

173

230

Cou

nt

101 102 103 104 105

MeBIO (days 0ndash6)BIO (days 0ndash6)BIO (days 1ndash6)

BIO (days 2ndash6)BIO (days 3ndash6)BIO (days 4ndash6)

(g)

SOX1

-EG

FP p

ositi

ve ce

lls (

)

01020304050607080

Me (

0ndash6

)

0ndash6

1ndash6

2ndash6

3ndash6

4ndash6BIO (075120583M)

(h)

Oct4

Nanog

Sox1

Tcf1

N2B27+

(days)

LIF ++2ndash4BIO (075120583M)

120573-actin

0ndash2 0ndash20ndash2

0ndash4 0ndash4 0ndash60ndash64ndash6minusminusminusminusminusminus

minusminusminusminus

minus minus

(i)

Figure 3 Increase in neural differentiation of precursor cells by GSK3120573 inhibitor (BIO) during days 4 to 6 in N2B27 medium (a)ndash(f) 46CES cells were cultured in N2B27 medium for 6 days GFP expression was elevated by BIO treatment (075120583M) from days 4 to 6 whereasGFP expression was reduced by BIO treatment (075 120583M) from days 0 to 3 (g) FACS analysis of Sox1-GFP expression during monolayerdifferentiation in N2B27 medium (h) Proportions of Sox1-GFP expressing cells as determined by FACS (i) RT-PCR analysis showed thatNanog and Sox1 expression were elevated by BIO treatment (075 120583M) under nondifferentiation and neural differentiation conditionsrespectively Scale bars 100120583m


4 6 7 9

Neural precursor cell differentiation Neuronal differentiation

+EGFbFGF

+bFGFMeBIO or BIO MeBIO or BIO

15(day)

0

(a)

MeBIO (4sim15 days)

(b)

BIO (4sim7 days)MeBIO (7sim15 days)

(c)

BIO (7sim15 days)

(d)


(e)

Figure 4 Inhibition of neurite formation during late neuronal differentiation by GSK3120573 inhibitor (a) Diagram of MeBIO (075 120583M) orBIO (075 120583M) treatment during late neuronal differentiation of 46C ES cells (b)ndash(e) Cells cultured according to the diagram for 15 days werestained withMAPII antibody Neurite formation was elevated by BIO treatment from days 4 to 7 (c) whereas it was reduced by BIO treatmentfrom days 4 to 15 (d) (e) Cells were cultured with BIO from days 7 to 10 and cultured with MeBIO for 7 days after replating followed bystaining with MAPII antibody Scale bars 100 120583m

to actual neural differentiation we compared the mRNAexpression levels of marker genes in cells treated or untreatedwith BIO We observed that the expression Sox1 mRNAwas increased in ES cells after BIO treatment from day 4compared to untreated cells (Figure 3(i)) These results implythat activation of Wnt120573-catenin signaling for different timeperiods has differential effects on neural differentiation ofmES cells

33 Transient Activation ofWnt120573-Catenin Signaling EnhancesNeuronal Differentiation The finding that the activation ofWnt signaling differentially regulates neuronal differentiationof ES cells promotes us to define the optimal condition forneuronal differentiation To this end we treated with Me-BIO or BIO from 4 to 7 days during neural differentiationfollowed by treatment again with Me-BIO or BIO from8 to 15 days after replating on PDL-laminin-coated plates(Figure 4(a)) Neurite formation induced by BIO from days 4to 7was remarkably enhanced compared toMeBIO treatment(Figures 4(b) and 4(c)) In contrast neurite formation was

completely abolished by BIO treatment from days 7 to 15(Figure 4(d)) Next we checked whether or not neuriteformation derived from precursor cells could be recoveredby withdrawing of BIO during neuronal differentiation Wetreatedwith BIO into precursor cells for 3 days after replatingand then cells were cultured in the absence of BIO for 7 daysmore during neuronal differentiation Interestingly neuriteformation of Map2-positive cells was restored under BIO-deficient conditions (Figure 4(e)) These data indicate thattransient activation ofWnt120573-catenin signaling during neuralprecursor formation may be beneficial for optimal neuronaldifferentiation

34 IWR-1-Endo Treatment from Days 4 to 6 SuppressesDifferentiation of Neural Precursor Cells As GSK3120573 has beenknown to be involved in multiple signaling pathways inaddition to Wnt120573-catenin signaling we elucidated neuraldifferentiation efficiency by using a specific inhibitor IWR-1-endo (Axin stabilizer) [25 29] Similar to data shown inFigure 3 neural differentiation of ES cells was reduced by


Control

(a)


(b)

BIO (days 0ndash2)

(c)

BIO (days 4ndash6)

(d)

IWR (days 0ndash2)

(e)

IWR (days 4ndash6)

(f)

EGFP

0

63

125

188

250

Cou

nt

ControlIWR (days 0ndash2)IWR (days 4ndash6)

101 102 103 104

(g)

(day)DMSO

0

10

20

30

40

50

60

70

4ndash60ndash2IWR (075120583M)

SOX1

-EG

FP p

ositi

ve ce

lls (

)

(h)

Figure 5 Decrease in neural differentiation of precursor cells by an Axin stabilizer (IWR-1-endo) from days 4 to 6 in N2B27medium (a)ndash(f)GFP expression of 46C cells was elevated by BIO treatment (075120583M) from days 4 to 6 whereas it was reduced by IWR-1-endo (075120583M)treatment from days 4 to 6 Cells were cultured in N2B27 medium for 6 days (g) FACS analysis of Sox1-GFP expression during monolayerdifferentiation in N2B27 medium (h) Proportions of Sox1-GFP expressing cells as determined by FACS Scale bars 100120583m

treatment with BIO fromdays 0 to 2 whereas it was increasedfrom days 4 to 6 (Figures 5(a)ndash5(d)) On the contrarytreatment of 46C ES cells with IWR-1-endo from day 4 to day6 resulted in a significant reduction in neural differentiationwhereas Wnt signaling inhibition by IWR-1-endo from days0 to 2 caused a slight increase in differentiation (Figures 5(e)5(f) and 5(h)) These results were further corroborated byFACS analysis of EGFP expression (Figures 5(g) and 5(h))Based on these data we conclude that activation of Wnt120573-catenin signaling on days 4 to 6 is essential for the properformation of neural precursor cells

35 The Expression of Sox1 Is Enhanced by Direct Binding of120573-Catenin-TCF1 to the Promoter during Neural Differentia-tion We found that BIO enhances Sox1 mRNA expression

during neural differentiation (Figure 3(i))Therefore we thenexamined whether 120573-catenin directly could be occupied atpromoter of Sox1 gene during neural differentiation Interest-ingly several conserved putative TCFLEF-binding sites werepresent in a 3 kb promoter region of both human and mouseSox1 gene (Figure 6(a)) In order to verify this hypothesisthat the complex of 120573-catenin and TCFLEF transcriptionfactors could bind to these conserved binding sites we carriedout chromatin immunoprecipitation (ChIP) analysis usingantibodies specific for 120573-catenin TCF1 and TCF3 Notably120573-catenin andTCF1 did not access to Sox1-1 and Sox1-2 regionunder nondifferentiation and early differentiation conditionswhereas they associated with all three potential binding sitesfrom day 4 during monolayer differentiation In additionwe found weak binding of 120573-catenin and TCF1 to Sox1-3


Sox1 mRNA

Sox1-2 Sox1-1

TCFLEFCAAAAA

TT

Sox1-3

300bp

(a)

Sox1-1

Sox1-2

Input TCF1N2B27 0 2 4 0 2 40 2 4

TCF30 2 4

Sox1-3

IgG4

120573-catenin

(b)

Figure 6 Increased Wnt120573-catenin signaling directly regulates Sox1 promoter activity during neural differentiation (a) Schematicrepresentation of binding sites for TCFLEF transcription factors in a 3 kb promoter region of the Sox1 gene Three pairs of PCR primerswere designed based on potential TCFLEF binding sites (b) ChIP analysis of the mouse Sox1 promoter with 120573-catenin TCF1 or TCF3antibodies 120573-catenin and TCF1 bound to the Sox1 promoter from day 4 during neural differentiation

region in self-renewal stem cells This weak 120573-catenin-TCF1binding on Sox1-3 at day 0 may have a rheostatic role intranscriptionally controlling expression of Sox1 gene duringdifferentiation Sox1-3 region is a distal control elementlocated around 3 kb from the transcription initiation siteTherefore we expect that weak interaction of 120573-catenin-TCF1 keeps a ready state to easily activate genes On theother hand TCF3 occupancy a known repressor proteinfor Wnt signaling was not changed during differentiation(Figure 6(b)) These data indicate that 120573-cateninTCF1 com-plex activates Sox1 promoter through direct binding to theconserved TCF binding sites during the late stage of neuralprecursor differentiation

4 Discussion

Here we showed that the expression of various Wnt sig-naling components is dynamically changed during neuronaldifferentiation (Figure 1) but the biological meaning ofthese changes and regulation of the expression of thesegenes are largely unknown However overall Wnt120573-cateninsignaling activity in undifferentiated ES cells seems to below compared to differentiated stage (Figure 2) Although ithas been shown that activation of Wnt120573-catenin signalingby the treatment of BIO or Wnt3a enhances stemness of EScells [18 19 30] these findings are not contradictory to ourfindings Our data suggest that an increase of Wnt120573-cateninsignaling is necessary (Figure 5) but not sufficient to induceneuronal differentiation of ES cells It may be possible thatincrease of Wnt120573-catenin signaling than endogenous levelin undifferentiated state is sufficient to enhance stemnessbut other unknown changes during differentiation alongwithhigh level of Wnt120573-catenin signaling may be required forproper neuronal differentiation of ES cells

Tsao et al [31] showed that Sox1 acts as a tumor sup-pressor by inhibiting TCF-responsive transcriptional activityin hepatocellular carcinoma Interestingly our ChIP analysisshowed that 120573-catenin-TCF complex could regulate theexpression of Sox1 in the middle of neuronal differentia-tion periods (Figure 6) Although further study would benecessary to test the possibility increase of Wnt120573-cateninsignaling induced Sox1 until generation of neural precursorsand then increased level of Sox1 suppresses Wnt120573-cateninsignaling during late neuronal differentiation period

We identified that treatment of BIO at specific time pointsduring differentiation can enhance neuronal differentiation(Figure 3) Treatment of BIO from the beginning of differ-entiation as well as continuous activation of Wnt120573-cateninsignaling until late neuronal differentiation stage blockedneuronal differentiation which might be due to enhancingself-renewal of ES cells and inhibitory effect ofWnt120573-cateninsignaling on late neuronal differentiation period respectivelyas shown before [18 19 23] Our findings could be usefulinformation in therapeutic point of view using stem cellssince the treatment of Wnt120573-catenin signaling activatoralong with small molecules regulating other signaling path-ways will further enhance neuronal differentiation

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by the grants from the NationalResearch Foundation to E Jho (2011-0019353 and2012M3A9C6050109) and the California Institute for Regen-erative Medicine (CIRM) New Faculty Award II (RN2-00938-1) and the CIRM Scientific Excellence throughExploration and Development (SEED) Grant (RS1-00327-1)to Q-L Ying

References

[1] G Keller ldquoEmbryonic stem cell differentiation emergence of anew era in biology and medicinerdquo Genes and Development vol19 no 10 pp 1129ndash1155 2005

[2] A G Smith ldquoEmbryo-derived stem cellsof mice and menrdquoAnnual Review of Cell and Developmental Biology vol 17 pp435ndash462 2001

[3] P P L Tam M Kanai-Azuma and Y Kanai ldquoEarly endo-derm development in vertebrates lineage differentiation andmorphogenetic functionrdquo Current Opinion in Genetics andDevelopment vol 13 no 4 pp 393ndash400 2003

[4] G Bain D Kitchens M Yao J E Huettner and D I GottliebldquoEmbryonic stem cells express neuronal properties in vitrordquoDevelopmental Biology vol 168 no 2 pp 342ndash357 1995


[5] Q-L Ying and A G Smith ldquoDefined conditions for neuralcommitment and differentiationrdquo Methods in Enzymology vol365 pp 327ndash341 2003

[6] Q L Ying M Stavridis D Griffiths M Li and A SmithldquoConversion of embryonic stem cells into neuroectodermalprecursors in adherentmonoculturerdquoNature Biotechnology vol21 no 2 pp 183ndash186 2003

[7] W Kim M Kim and E-H Jho ldquoWntbeta-catenin signallingfrom plasma membrane to nucleusrdquo Biochemical Journal vol450 no 1 pp 9ndash21 2013

[8] A Wodarz and R Nusse ldquoMechanisms of Wnt signalingin developmentrdquo Annual Review of Cell and DevelopmentalBiology vol 14 pp 59ndash88 1998

[9] J RMiller AMHocking J D Brown andR TMoon ldquoMech-anism and function of signal transduction by the Wntbeta-catenin and WntCa2+ pathwaysrdquo Oncogene vol 18 no 55 pp7860ndash7872 1999

[10] J H van Es N Barker and H Clevers ldquoYou Wnt some youlose some oncogenes in the Wnt signaling pathwayrdquo CurrentOpinion in Genetics and Development vol 13 no 1 pp 28ndash332003

[11] J Behrens J P vonKriesM Kuhl et al ldquoFunctional interactionof 120573-catenin with the transcription factor LEF-1rdquo Nature vol382 no 6592 pp 638ndash642 1996

[12] M Molenaar M van de Wetering M Oosterwegel et alldquoXTcf-3 transcription factor mediates 120573-catenin-induced axisformation in xenopus embryosrdquo Cell vol 86 no 3 pp 391ndash3991996

[13] C Mosimann G Hausmann and K Basler ldquoBeta-catenin hitschromatin regulation of Wnt target gene activationrdquo NatureReviewsMolecular Cell Biology vol 10 no 4 pp 276ndash286 2009

[14] M Kleber and L Sommer ldquoWnt signaling and the regulation ofstem cell functionrdquo Current Opinion in Cell Biology vol 16 no6 pp 681ndash687 2004

[15] H Niwa ldquoWnt whatrsquos needed to maintain pluripotencyrdquoNature Cell Biology vol 13 no 9 pp 1024ndash1026 2011

[16] R Nusse ldquoWnt signaling and stem cell controlrdquo Cell Researchvol 18 no 5 pp 523ndash527 2008

[17] S Y Sokol ldquoMaintaining embryonic stem cell pluripotencywithWnt signalingrdquo Development vol 138 no 20 pp 4341ndash43502011

[18] J Hao T-G Li X Qi D-F Zhao and G-Q Zhao ldquoWNT120573-catenin pathway up-regulates Stat3 and converges on LIF toprevent differentiation of mouse embryonic stem cellsrdquo Devel-opmental Biology vol 290 no 1 pp 81ndash91 2006

[19] N Sato L Meijer L Skaltsounis P Greengard and A HBrivanlou ldquoMaintenance of pluripotency in human and mouseembryonic stem cells through activation of Wnt signaling bya pharmacological GSK-3-specific inhibitorrdquo Nature Medicinevol 10 no 1 pp 55ndash63 2004

[20] L Pereira F Yi and B J Merrill ldquoRepression of Nanog genetranscription by Tcf3 limits embryonic stem cell self-renewalrdquoMolecular and Cellular Biology vol 26 no 20 pp 7479ndash74912006

[21] G Dravid Z Ye H Hammond et al ldquoDefining the role ofWnt120573-catenin signaling in the survival proliferation and self-renewal of human embryonic stem cellsrdquo Stem Cells vol 23 no10 pp 1489ndash1501 2005

[22] J J Otero W Fu L Kan A E Cuadra and J A Kesslerldquo120573-catenin signaling is required for neural differentiation ofembryonic stem cellsrdquo Development vol 131 no 15 pp 3545ndash3557 2004

[23] J Aubert H Dunstan I Chambers and A Smith ldquoFunctionalgene screening in embryonic stem cells implicates Wnt antag-onism in neural differentiationrdquo Nature Biotechnology vol 20no 12 pp 1240ndash1245 2002

[24] L Meijer A L Skaltsounis P Magiatis et al ldquoGSK-3-selectiveinhibitors derived from Tyrian purple indirubinsrdquo Chemistry ampBiology vol 10 no 12 pp 1255ndash1266 2003

[25] B ChenM E DodgeW Tang et al ldquoSmall molecule-mediateddisruption of Wnt-dependent signaling in tissue regenerationand cancerrdquo Nature Chemical Biology vol 5 no 2 pp 100ndash1072009

[26] E-H Jho T Zhang C Domon C-K Joo J-N Freundand F Costantini ldquoWnt120573-cateninTcf signaling induces thetranscription of Axin2 a negative regulator of the signalingpathwayrdquoMolecular andCellular Biology vol 22 no 4 pp 1172ndash1183 2002

[27] M van de Wetering R Cavallo D Dooijes et al ldquoArmadillocoactivates transcription driven by the product of theDrosophila segment polarity gene dTCFrdquo Cell vol 88 no6 pp 789ndash799 1997

[28] K F Kelly D YNg G Jayakumaran G AWoodH Koide andB W Doble ldquo120573-catenin enhances Oct-4 activity and reinforcespluripotency through a TCF-independent mechanismrdquo CellStem Cell vol 8 no 2 pp 214ndash227 2011

[29] BW Doble and J RWoodgett ldquoGSK-3 tricks of the trade for amulti-tasking kinaserdquo Journal of Cell Science vol 116 no 7 pp1175ndash1186 2003

[30] Q-L Ying J Wray J Nichols et al ldquoThe ground state ofembryonic stem cell self-renewalrdquoNature vol 453 no 7194 pp519ndash523 2008

[31] C-M Tsao M-D Yan Y-L Shih et al ldquoSOX1 functionsas a tumor suppressor by antagonizing the WNT120573-cateninsignaling pathway in hepatocellular carcinomardquo Hepatologyvol 56 no 6 pp 2277ndash2287 2012

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


However when Wnt binds to its receptor Frizzled andcoreceptor LRP56 the cytoplasmic component dishevelledblocks 120573-catenin degradation leading to the accumulation of120573-catenin in the cytoplasm Stabilized 120573-catenin then entersthe nucleus where it interacts with TCFLEF [11ndash13] andactivates the expression of target genes such as C-Myc CyclinD1 Brachury and Twin

Wnt120573-catenin signaling has been shown to play impor-tant roles in ES cell self-renewal and differentiation [14ndash17]Activation of Wnt120573-catenin signaling prevents differentia-tion of human and mouse ES cells For example activationof Wnt signaling by GSK3120573 inhibitor maintains self-renewalof human and mouse ES cells and Wnt120573-catenin signalingupregulates Stat3 activity and prevents differentiation ofmouse ES cells [18 19] Furthermore TCF3 a terminaltranscription factor in Wnt signaling represses Nanog anddelays differentiation of ES cells [20] However other evi-dence has shown that activation of Wnt120573-catenin signalingby Wnt3a recombinants induces cell differentiation as well asproliferation [21] Wnt120573-catenin signaling is also involved inregulating the neuronal differentiation of ES cells [22] On theother hand it has been shown that theWnt antagonist sFRP2induces neuronal differentiation [23] Therefore the rolesof endogenous Wnt120573-catenin signaling and its componentsin the self-renewal and neuronal differentiation of ES cellsremain controversial

In this study we examined the endogenous level ofWnt120573-catenin signaling components during neuronal differ-entiation The data using reporter ES cell lines representingendogenous Wnt120573-catenin signaling activity showed thatWnt120573-catenin signaling increased during neuronal differ-entiation Interestingly Wnt120573-catenin signaling increaseduntil formation of neuronal precursor cells however it wasreduced during later periods of neuronal differentiationDifferential regulation of neuronal differentiation by thetreatment of activator or inhibitor ofWnt120573-catenin signalingsuggested thatWnt signaling plays dual roles during neuronaldifferentiation

2 Materials and Methods

21 Cell Culture E14 and 46C ES cells (ES cell line inwhich EGFP is substituted into the ORF of the Sox1 gene areporter for differentiation into neural precursor cells) werecultured in mouse ES (mES) cell medium (DMEM (Gibco)supplemented with 15 FBS 2mM GlutaMAX (Gibco)MEMnonessential amino acids 120573-mercaptoethanol (Gibco)tylosin and 1 PenStrep (Gibco)) supplemented with LIF(ESGRO Chemicon) on 02 gelatin-coated dishes Cellswere maintained at 37∘C in a humidified atmosphere of 5CO2

22 Neuronal Differentiation To induce neuronal differ-entiation undifferentiated 46C ES cells were trypsinizedand neutralized with serum-containing medium ES cellswere dissociated in N2B27 medium (DMEMF12 (Gibco)Neurobasal medium (Gibco) N2 supplement (Invitrogen)B27 supplement (Invitrogen) 1mM GlutaMAX (Gibco)

01M120573-mercaptoethanol (Gibco) and 1PenStrep (Gibco))and then plated on a 02 gelatin-coated tissue culturedish (Falcon) containing DMSO (SIGMA) 075 120583M MeBIO(Calbiochem) 075120583MBIO (Calbiochem) [24] and 075 120583MIWR-1-endo (kindly provided by Dr Lawrence Lum Uni-versity of Texas Southwestern Medical Center) [25] N2B27mediumwas changed every other day On the 6th day EGFP-positive cells (Sox1 positive cells) were observed by using afluorescent microscope (Leica DMIRB Leica Microsystems)For production of neurons Sox1-positive cells were replatedon PDL-laminin-coated wells in N2B27 medium containingEGF and FGF-2 N2B27 mediumwas changed every 3-4 days[6]

23 FACS Analysis To analyze 46C ES cell-derived Sox1-GFP-positive neural precursors 46C cells were differentiatedfor 6 days in N2B27mediumwithMeBIO or BIO and DMSOor IWR The cells were then trypsinized and neutralizedwith serum containing medium After centrifugation cellswere washed two times with PBS and resuspended in PBSResuspended cells were analyzed using a FACScan For dataanalysis FACSDiva software was used

24 Transfection and Luciferase Assay Oct4 promoter-EGFP ES cells were stably transfected with TOP-mCherryand Axin2-promoter-mCherry using Amaxa nucleofectoraccording to the manufacturerrsquos instructions Stably trans-fected clones were selected in media containing 800120583gmLof G418 (Gibco)

To measure Wnt signaling activity pTOP-Flash plasmidand pRL-TK plasmid were transfected into mES cells Trans-fected ES cells were cultured in the presence or absenceof LIF for 48 h To measure Sox1 promoter activity undernondifferentiation and neuronal differentiation conditionspGL3-mSox1 promoter plasmid and pRL-TK plasmid weretransfected into 46C cells which were cultured in mESmedium or N2B27 medium with or without 075120583M BIO(Calbiochem) Luciferase activity was measured by Dual-Luciferase reporter assay (Promega)

25 Reverse Transcriptase-PCR Examination of gene expres-sion during neuronal differentiation was carried out byRT-PCR Total RNA was isolated using TRIzol reagent(Invitrogen) according to the manufacturerrsquos protocol cDNAwas synthesized from total RNA (1 120583g) in a total reactionvolume of 20 120583L using ImProm-II Reverse Transcriptase(Promega) with random primers cDNAwas amplified underthe following conditions 94∘C for 2min followed by 20 25or 30 cycles of 94∘C for 45 s 58∘C for 45 s and 72∘C for 50 s

26 Immunofluorescence Analysis Neuronally differentiated46C ES cells for 15 days were fixedwith 4 paraformaldehydeat room temperature for 20min and then permeabilized for20min with permeabilization solution Cells were incubatedwith anti-MapII (Chemicon) primary antibody for 1 h Afterwashing 10 times with PBST (005 Tween 20 10mMNa2HPO4 2mM KH

2PO4 27mM KCl and 137mM NaCl)

cells were incubated with Alexa Fluor 488 (Invitrogen) and







3 Results





(a) (b) (c)

(d) (e)

Oct4

Nanog

Gata4

Brachyury

Sox1

Dvl2

Axin1

Axin2

Undiff 1 3 6 8 11 17 (day)

FACSsorting

Undiff 1 3 6 8 11 17 (day)

FACSsorting

GAPDH

Wnt4

Wnt5a

Wnt7a

Wnt8a

Wnt3a

Frizzled1

Frizzled2

LRP6

120573-cat

(f)



0

02

04

06

08

1

12

14

16

Luci

fera

se ac

tivity

(rel

ativ

e uni

t)

LIF + +

Top-LucAxin2-Luc

minusminus


(a)

(ABC)

0 2-LIF 4 6 8 (day)

(day)0 2 4 6N2B27

(ABC)



120573-actin

120573-actin

(b)

(day)0 2 4 6N2B27

p-LRP

120573-actin

(c)

Axin2-mCherry

+MeBIO

(d)

Axin2-mCherry

+BIO

(e)

Top-mCherry

(f)

Axin2-mCherry

(g)






(a)

BIO (days 0ndash6)

(b)

BIO (days 1ndash6)

(c)

BIO (days 2ndash6)

(d)

BIO (days 3ndash6)

(e)

BIO (days 4ndash6)

(f)

EGFP

0

58

115

173

230

Cou

nt

101 102 103 104 105



(g)

SOX1

-EG

FP p

ositi

ve ce

lls (

)

01020304050607080

Me (

0ndash6

)

0ndash6

1ndash6

2ndash6

3ndash6

4ndash6BIO (075120583M)

(h)

Oct4

Nanog

Sox1

Tcf1

N2B27+

(days)


120573-actin




minus minus

(i)



4 6 7 9


+EGFbFGF


15(day)

0

(a)

MeBIO (4sim15 days)

(b)


(c)

BIO (7sim15 days)

(d)


(e)







Control

(a)


(b)

BIO (days 0ndash2)

(c)

BIO (days 4ndash6)

(d)

IWR (days 0ndash2)

(e)

IWR (days 4ndash6)

(f)

EGFP

0

63

125

188

250

Cou

nt


101 102 103 104

(g)

(day)DMSO

0

10

20

30

40

50

60

70


SOX1

-EG

FP p

ositi

ve ce

lls (

)

(h)






Sox1 mRNA

Sox1-2 Sox1-1

TCFLEFCAAAAA

TT

Sox1-3

300bp

(a)

Sox1-1

Sox1-2

Input TCF1N2B27 0 2 4 0 2 40 2 4

TCF30 2 4

Sox1-3

IgG4

120573-catenin

(b)



4 Discussion






Acknowledgments


References








































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







3 Results





(a) (b) (c)

(d) (e)

Oct4

Nanog

Gata4

Brachyury

Sox1

Dvl2

Axin1

Axin2

Undiff 1 3 6 8 11 17 (day)

FACSsorting

Undiff 1 3 6 8 11 17 (day)

FACSsorting

GAPDH

Wnt4

Wnt5a

Wnt7a

Wnt8a

Wnt3a

Frizzled1

Frizzled2

LRP6

120573-cat

(f)



0

02

04

06

08

1

12

14

16

Luci

fera

se ac

tivity

(rel

ativ

e uni

t)

LIF + +

Top-LucAxin2-Luc

minusminus


(a)

(ABC)

0 2-LIF 4 6 8 (day)

(day)0 2 4 6N2B27

(ABC)



120573-actin

120573-actin

(b)

(day)0 2 4 6N2B27

p-LRP

120573-actin

(c)

Axin2-mCherry

+MeBIO

(d)

Axin2-mCherry

+BIO

(e)

Top-mCherry

(f)

Axin2-mCherry

(g)






(a)

BIO (days 0ndash6)

(b)

BIO (days 1ndash6)

(c)

BIO (days 2ndash6)

(d)

BIO (days 3ndash6)

(e)

BIO (days 4ndash6)

(f)

EGFP

0

58

115

173

230

Cou

nt

101 102 103 104 105



(g)

SOX1

-EG

FP p

ositi

ve ce

lls (

)

01020304050607080

Me (

0ndash6

)

0ndash6

1ndash6

2ndash6

3ndash6

4ndash6BIO (075120583M)

(h)

Oct4

Nanog

Sox1

Tcf1

N2B27+

(days)


120573-actin




minus minus

(i)



4 6 7 9


+EGFbFGF


15(day)

0

(a)

MeBIO (4sim15 days)

(b)


(c)

BIO (7sim15 days)

(d)


(e)







Control

(a)


(b)

BIO (days 0ndash2)

(c)

BIO (days 4ndash6)

(d)

IWR (days 0ndash2)

(e)

IWR (days 4ndash6)

(f)

EGFP

0

63

125

188

250

Cou

nt


101 102 103 104

(g)

(day)DMSO

0

10

20

30

40

50

60

70


SOX1

-EG

FP p

ositi

ve ce

lls (

)

(h)






Sox1 mRNA

Sox1-2 Sox1-1

TCFLEFCAAAAA

TT

Sox1-3

300bp

(a)

Sox1-1

Sox1-2

Input TCF1N2B27 0 2 4 0 2 40 2 4

TCF30 2 4

Sox1-3

IgG4

120573-catenin

(b)



4 Discussion






Acknowledgments


References








































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a) (b) (c)

(d) (e)

Oct4

Nanog

Gata4

Brachyury

Sox1

Dvl2

Axin1

Axin2

Undiff 1 3 6 8 11 17 (day)

FACSsorting

Undiff 1 3 6 8 11 17 (day)

FACSsorting

GAPDH

Wnt4

Wnt5a

Wnt7a

Wnt8a

Wnt3a

Frizzled1

Frizzled2

LRP6

120573-cat

(f)



0

02

04

06

08

1

12

14

16

Luci

fera

se ac

tivity

(rel

ativ

e uni

t)

LIF + +

Top-LucAxin2-Luc

minusminus


(a)

(ABC)

0 2-LIF 4 6 8 (day)

(day)0 2 4 6N2B27

(ABC)



120573-actin

120573-actin

(b)

(day)0 2 4 6N2B27

p-LRP

120573-actin

(c)

Axin2-mCherry

+MeBIO

(d)

Axin2-mCherry

+BIO

(e)

Top-mCherry

(f)

Axin2-mCherry

(g)






(a)

BIO (days 0ndash6)

(b)

BIO (days 1ndash6)

(c)

BIO (days 2ndash6)

(d)

BIO (days 3ndash6)

(e)

BIO (days 4ndash6)

(f)

EGFP

0

58

115

173

230

Cou

nt

101 102 103 104 105



(g)

SOX1

-EG

FP p

ositi

ve ce

lls (

)

01020304050607080

Me (

0ndash6

)

0ndash6

1ndash6

2ndash6

3ndash6

4ndash6BIO (075120583M)

(h)

Oct4

Nanog

Sox1

Tcf1

N2B27+

(days)


120573-actin




minus minus

(i)



4 6 7 9


+EGFbFGF


15(day)

0

(a)

MeBIO (4sim15 days)

(b)


(c)

BIO (7sim15 days)

(d)


(e)







Control

(a)


(b)

BIO (days 0ndash2)

(c)

BIO (days 4ndash6)

(d)

IWR (days 0ndash2)

(e)

IWR (days 4ndash6)

(f)

EGFP

0

63

125

188

250

Cou

nt


101 102 103 104

(g)

(day)DMSO

0

10

20

30

40

50

60

70


SOX1

-EG

FP p

ositi

ve ce

lls (

)

(h)






Sox1 mRNA

Sox1-2 Sox1-1

TCFLEFCAAAAA

TT

Sox1-3

300bp

(a)

Sox1-1

Sox1-2

Input TCF1N2B27 0 2 4 0 2 40 2 4

TCF30 2 4

Sox1-3

IgG4

120573-catenin

(b)



4 Discussion






Acknowledgments


References








































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

02

04

06

08

1

12

14

16

Luci

fera

se ac

tivity

(rel

ativ

e uni

t)

LIF + +

Top-LucAxin2-Luc

minusminus


(a)

(ABC)

0 2-LIF 4 6 8 (day)

(day)0 2 4 6N2B27

(ABC)



120573-actin

120573-actin

(b)

(day)0 2 4 6N2B27

p-LRP

120573-actin

(c)

Axin2-mCherry

+MeBIO

(d)

Axin2-mCherry

+BIO

(e)

Top-mCherry

(f)

Axin2-mCherry

(g)






(a)

BIO (days 0ndash6)

(b)

BIO (days 1ndash6)

(c)

BIO (days 2ndash6)

(d)

BIO (days 3ndash6)

(e)

BIO (days 4ndash6)

(f)

EGFP

0

58

115

173

230

Cou

nt

101 102 103 104 105



(g)

SOX1

-EG

FP p

ositi

ve ce

lls (

)

01020304050607080

Me (

0ndash6

)

0ndash6

1ndash6

2ndash6

3ndash6

4ndash6BIO (075120583M)

(h)

Oct4

Nanog

Sox1

Tcf1

N2B27+

(days)


120573-actin




minus minus

(i)



4 6 7 9


+EGFbFGF


15(day)

0

(a)

MeBIO (4sim15 days)

(b)


(c)

BIO (7sim15 days)

(d)


(e)







Control

(a)


(b)

BIO (days 0ndash2)

(c)

BIO (days 4ndash6)

(d)

IWR (days 0ndash2)

(e)

IWR (days 4ndash6)

(f)

EGFP

0

63

125

188

250

Cou

nt


101 102 103 104

(g)

(day)DMSO

0

10

20

30

40

50

60

70


SOX1

-EG

FP p

ositi

ve ce

lls (

)

(h)






Sox1 mRNA

Sox1-2 Sox1-1

TCFLEFCAAAAA

TT

Sox1-3

300bp

(a)

Sox1-1

Sox1-2

Input TCF1N2B27 0 2 4 0 2 40 2 4

TCF30 2 4

Sox1-3

IgG4

120573-catenin

(b)



4 Discussion






Acknowledgments


References








































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



(a)

BIO (days 0ndash6)

(b)

BIO (days 1ndash6)

(c)

BIO (days 2ndash6)

(d)

BIO (days 3ndash6)

(e)

BIO (days 4ndash6)

(f)

EGFP

0

58

115

173

230

Cou

nt

101 102 103 104 105



(g)

SOX1

-EG

FP p

ositi

ve ce

lls (

)

01020304050607080

Me (

0ndash6

)

0ndash6

1ndash6

2ndash6

3ndash6

4ndash6BIO (075120583M)

(h)

Oct4

Nanog

Sox1

Tcf1

N2B27+

(days)


120573-actin




minus minus

(i)



4 6 7 9


+EGFbFGF


15(day)

0

(a)

MeBIO (4sim15 days)

(b)


(c)

BIO (7sim15 days)

(d)


(e)







Control

(a)


(b)

BIO (days 0ndash2)

(c)

BIO (days 4ndash6)

(d)

IWR (days 0ndash2)

(e)

IWR (days 4ndash6)

(f)

EGFP

0

63

125

188

250

Cou

nt


101 102 103 104

(g)

(day)DMSO

0

10

20

30

40

50

60

70


SOX1

-EG

FP p

ositi

ve ce

lls (

)

(h)






Sox1 mRNA

Sox1-2 Sox1-1

TCFLEFCAAAAA

TT

Sox1-3

300bp

(a)

Sox1-1

Sox1-2

Input TCF1N2B27 0 2 4 0 2 40 2 4

TCF30 2 4

Sox1-3

IgG4

120573-catenin

(b)



4 Discussion






Acknowledgments


References








































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


4 6 7 9


+EGFbFGF


15(day)

0

(a)

MeBIO (4sim15 days)

(b)


(c)

BIO (7sim15 days)

(d)


(e)







Control

(a)


(b)

BIO (days 0ndash2)

(c)

BIO (days 4ndash6)

(d)

IWR (days 0ndash2)

(e)

IWR (days 4ndash6)

(f)

EGFP

0

63

125

188

250

Cou

nt


101 102 103 104

(g)

(day)DMSO

0

10

20

30

40

50

60

70


SOX1

-EG

FP p

ositi

ve ce

lls (

)

(h)






Sox1 mRNA

Sox1-2 Sox1-1

TCFLEFCAAAAA

TT

Sox1-3

300bp

(a)

Sox1-1

Sox1-2

Input TCF1N2B27 0 2 4 0 2 40 2 4

TCF30 2 4

Sox1-3

IgG4

120573-catenin

(b)



4 Discussion






Acknowledgments


References








































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Control

(a)


(b)

BIO (days 0ndash2)

(c)

BIO (days 4ndash6)

(d)

IWR (days 0ndash2)

(e)

IWR (days 4ndash6)

(f)

EGFP

0

63

125

188

250

Cou

nt


101 102 103 104

(g)

(day)DMSO

0

10

20

30

40

50

60

70


SOX1

-EG

FP p

ositi

ve ce

lls (

)

(h)






Sox1 mRNA

Sox1-2 Sox1-1

TCFLEFCAAAAA

TT

Sox1-3

300bp

(a)

Sox1-1

Sox1-2

Input TCF1N2B27 0 2 4 0 2 40 2 4

TCF30 2 4

Sox1-3

IgG4

120573-catenin

(b)



4 Discussion






Acknowledgments


References








































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Sox1 mRNA

Sox1-2 Sox1-1

TCFLEFCAAAAA

TT

Sox1-3

300bp

(a)

Sox1-1

Sox1-2

Input TCF1N2B27 0 2 4 0 2 40 2 4

TCF30 2 4

Sox1-3

IgG4

120573-catenin

(b)



4 Discussion






Acknowledgments


References








































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

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