temporal expression changes during differentiation of neural stem cells derived from mouse embryonic...

Journal of Cellular Biochemistry 93:563–578 (2004)

Temporal Expression Changes During Differentiationof Neural Stem Cells Derived From Mouse EmbryonicStem Cell

Joon-Ik Ahn,1 Ki-Hwan Lee,1 Dong-Mi Shin,1 Jae-Won Shim,1 Chang-mee Kim,2 Hyun Kim,2

Sang-Hun Lee,1 and Yong-Sung Lee1*1Department of Biochemistry, College of Medicine, Hanyang University, Seoul 133-791, South Korea2Department of Anatomy, College of Medicine, Korea University, Seoul 136-705, South Korea

Abstract Temporal analysis in gene expression during differentiation of neural stem cells (NSCs) was performed byusing in-housemicroarrays composed of 10,368 genes. The changes inmRNA level weremeasured during differentiationday 1, 2, 3, 6, 12, and 15. Out of 10,368 genes analyzed, 259 genes were up-regulated or down-regulated by 2-fold ormore at least at one time-point during differentiation, and were classified into six clusters based on their expressionpatterns by K-means clustering. Clusters characterized by gradual increase have large numbers of genes involved intransport and cell adhesion; those which showed gradual decrease have much of genes in nucleic acid metabolism, cellcycle, transcription factor, and RNA processing. In situ hybridization (ISH) validated microarray data and it also showedthat Fox M1, cyclin D2, and CDK4 were highly expressed in CNS germinal zones and ectonucleotide pyrophosphatase/phosphodiesterase 2 (Enpp2) was highly expressed in choroid plexus where stem/progenitor cells are possibly located.Together, this clustering analysis of expressionpatterns of functionally classified genesmaygive insight intounderstandingof CNS development and mechanisms of NSCs proliferation and differentiation. J. Cell. Biochem. 93: 563–578, 2004.� 2004 Wiley-Liss, Inc.

Key words: microarray; proliferation; in situ hybridization; germinal zone; choroid plexus

Neural stem cells (NSCs) are multipotentand self-renewing cell population able to gene-rate the three major central nervous system(CNS) lineages neurons, astrocytes, and oligo-dendrocytes. Therefore, NSCs is thought of as areservoirwith thepotential to becomeanyone ofthe thousands of cell types within the CNS[McKay, 1997; Ross et al., 2003].Analyses of a variety of mammalian model

systems have identified helix-loop-helix (HLH)transcription factors as a regulator of earlydifferentiation of NSCs. The different classes

of HLH factors cross-regulate one another tomediate the effect of several signaling systemson a large number of target genes. HLH tran-scription factors participate at different stagesof NSC differentiation, such as formation ofprogenitor cells, initiation of differentiation,cell fate determination, neurite outgrowth,and synaptogenesis, and the timing of differ-entiation is regulated by the balance of thesefactors [Morrison, 2001; Ross et al., 2003].

The signaling molecules that regulate thetransition of NSCs from proliferation to differ-entiation are beginning to be identified, withseveral growth factors, including basic fibro-blast growth factor, epidermal growth factor,brain-derived neurotrophic factor, and notchligands [Vescovi et al., 1993; Zigova et al., 1998;Benraiss et al., 2001; Caldwell et al., 2001].In addition to these factors on the stem cellproliferation and differentiation, identificationof other factors will advance the understandingof CNS development and the potential for theuse of stem cells as therapeutic agents [Karstenet al., 2003].

� 2004 Wiley-Liss, Inc.

Grant sponsor: Korean Ministry of Health & Welfare;Grant number: 01-PJ10-PG8-01EC01-0023.

*Correspondence to: Dr. Yong-Sung Lee, Department ofBiochemistry, College of Medicine, Hanyang University,Seoul 133-791, South Korea.E-mail: [email protected]

Received 10 February 2004; Accepted 20 April 2004

DOI 10.1002/jcb.20168

To identify regulatory genes that are involvedin NSCs proliferation and differentiation, wehave performed in-house-made cDNA micro-array, which allows simultaneous assessmentof the expression of potentially thousands ofgenes. Recently, some microarray analyseswere performed to investigate the molecularmechanism underlying NSC proliferation andpluripotency [Geschwind et al., 2001; Luo et al.,2002]. However, most genes identified in thesemicroarrays do not overlap with each other.Elucidation of common genetic factor in variousstem cell populations is hampered by differentexperimental design and analysis. To highlightconserved genes expressed during differen-tiation in NSCs, we compared list of genesidentified in this embryonic stem cell (ES)-derived NSCs population with genes identifiedin other stem cell populations, such as primaryneural progenitor cells [Karsten et al., 2003].

MATERIALS AND METHODS

ES Cell-Derived Neural Stem Cellsand Differentiation

The NSCs were generated through five-stagemethod as described [Lee et al., 2000]. Nestin-positive cells (NSCs, stage 4) were expanded for4 days. The medium was changed every 2 days.Differentiation (stage 5) was induced byremoval of bFGF. The cells were incubatedunder differentiation conditions for 1, 2, 3, 6, 12,and 15 days. The total RNA of each time-pointwas extracted using Tri-Reagent (MRC, Inc.,Cincinnati, OH) according to the manufac-turer’s instructions.

Microarray Fabrication and Hybridization

A total of 10,368 cDNA clones were spottedonto SuperAldehyde slides (Telechem, Sunnyvale,CA) using a MicroGrid TAS arrayer (Genomicsolutions, Huntingdon, United Kingdom) aspreviously described [Lee et al., 2002]. Themicroarray consisted of 6,531 clones from theNational Institute of Aging (NIA), 1,243 clonesfrom the Brain Molecular Anatomy Project(BMAP), 2,060 clones from Incyte, and hous-keeping genes and yeast DNA as negativecontrols.

Probes were made from 2 mg of total RNAfromNSCs (stage 4) for control and 2 mg of RNAfrom each differentiated cells RNA (stages 5),using a dendrimer labeling kit (3DNA 350RP;Genisphere, Hatfield, PA). For each experiment,

at least four replicates were performed, and twoof these were repeated with the fluorophoresreversed to eliminate false-positive results.

Scanning and Data Analysis

The two fluorescent images from each slidewere scanned separately using a GMS 418scanner (Affymetrix, Santa Clara, CA). Theimages were analyzed using ImaGene 4.2(Biodiscovery, El Segundo, CA) and MAAS(Gaiagene, Seoul, Korea) software. Cy3:Cy5intensity ratios from each gene were calculatedand subsequently normalized with nonlinearlowess normalization. To filter out unreliabledata, spots with signal-to-noise ratios below 2were flagged. The non-flagged genes in at leastthree out of four experiments were used forfurther analysis. We took the median valuefrom the gene expression ratio of each of fourindependent experiments and selected geneswhose expression level differed from theirmeanexpression level by at least 2-fold at one ormoredifferentiation time. K-means clustering wasapplied to genesusing theEuclidean distance asa similarity measurement, as implemented inthe software program Genesis (http://genome.tugraz.at, Sturn et al. [2002]). Genes showingmore than a 2-fold difference in expression levelwere divided into categories according to theirmolecular function using the classificationscheme in Gene OntologyTM. To examine sta-tistical significance for frequencies of genes ofeach functional group in each cluster, two-sidedFisher’s exact test was performed.

Immunocytochemistry

The cells were grown on polyornithine (15 mg/ml, Sigma, St. Louis, MO)/fibronectin (1 mg/ml,Sigma) coated coverslips and fixed with 4%paraformaldehyde/0.15% picric acid in 0.1%bovine serum albumin (BSA)/phosphate-buf-fered saline (PBS). Subsequently, the cells werewashed three-times with BSA/PBS and per-meabilized with Triton X-100/PBS. The cellswere incubated for 12 h at 48C with polyclonalnestin antibodies (1:50) (Matha Marvin andRon McKay, National Institute of Health,Bethesda,MD), polyclonal neuron-specific classIII b-tubulin (Tuj1) antibodies (1:2,000) (Babco,Richmond, CA) and polyclonal glial fibrillaryacidic protein (GFAP)antibodies (1:400) (DAKO,Glostrup, Denmark). For detection of primaryantibodies, fluorescence labeled (FITC or Cy3)secondary antibodies (Jackson Immunore-

564 Ahn et al.

search Laboratories, West Grove, PA) were usedaccording to the specifications of the manufac-turer. Cells were mounted in Vectashield con-taining 40, 6-diamidino-2-phenylindole (DAPI;Vector Laboratories, Burlingame, CA) andanalyzed under an epifluorescence microscope(Nikon, Tokyo, Japan).

Semi-Quantitative andReal-Time Quantitative RT-PCR

For semi-quantitative and real-time quanti-tative RT-PCR, beta-actin was used as aninternal control with the following primer sets;50-ctt tat ggt gtggtc gcaga-30 and50-tca ggg tag tcagcc atg tg-30. For each sample from each time-point, the same total RNAused for cDNAmicro-array hybridization was reverse-transcribedusing 1 mM oligo (dT) primer with SuperscriptII reverse transcriptase (Invitrogen, Carlsbad,CA). The thermal profiles consisted of 958C for5 min for initial denaturing, followed by 25–30 cycles of 958C for 30 s, 588C for 30 s, and 728Cfor 30 s. All RT-PCR reactions were repeatedat least three-times. Triplicate real-time RT-PCR experiments were performed using iCycleriQ system (Bio-Rad, Hercules, CA) and theSYBR Green I dye (Molecular probes, Eugene,OR). Amelting curvewas obtained for eachPCRproduct after each run to confirm that the signalcorresponded to a unique amplicon of theproduct size. The following primers were used:50-ggagtgtcgcttagaggtgc-30 and 50-tccagaaagc-caagagaagc-30 for nestin; 50-tgtcagaggagccc-gaggtc-30 and 50-ccaagagcagcccatcaaag-30 fortyrosine hydroxylase (TH); 50-aaggtagccgtgtgt-gacatc-30 and 50-accaggtcattcatgttgctc-30 for Tuj1;50-ccaaactggctgatgtctacc-30 and 50-gcttcatgtgc-ctcctgtcta-30 for GFAP; 50-tccctacataacgggagc-ag-30 and 50-aactcaggccaagcgataga-30 for insu-lin-like growth factor 2 (Igf2); 50-ttttcatcttgg-cagctgtg-30 and 50-acactccactgccattctcc-30 forpleiotrophin; 50-ttccccagaagcgaaatatg-30 and50-tgaccccattcctttctgac-30 for ectonucleotide pyr-ophosphatase/phosphodiesterase 2 (Enpp2); 50-gatgatttccgagggagaca-30 and 50-catgaggaatgt-cagccaga-30 for N-myc downstream regulated2 (Ndr2); 50-gaggaaacagcaccttcagc-30and 50-ag-gcaatgtctccttgatgg-30 for forkhead box M1(FoxM1); 50-ggaagagactgttggaagagga-30and 50-ctgataagcccaggctagaaga-30 for thymopoietin.

In Situ Hybridization

ISH were performed on E13.5, E18.5, P0, P7,and P14 BL6 mouse brains and embryos sec-

tioned at 12 mm. Sections were thaw-mountedonto gelatin-coated slides, fixed in 4% parafor-maldehyde, treated with 0.25% acetic anhy-dride in 0.1 M triethanolamine/0.9% NaCl(pH 8.0), dehydrated and defatted in ethanoland chloroform, and finally air-dried. Tran-scription of antisense probes was carried outusing the Riboprobe (Promega, Madison, WI) inthe presence of a-35S-UTP (Amersham, Buck-inghamshire, England). Sections were hybri-dized overnight at 538C with 5� 105 cpm oflabeled probe per slide, treated with RNase A(20 mg/ml, Boehringer-Mannheim, Mannheim,Germany) for 30 min at 258C, washed sequen-tially for 60 min in 2� SSC (1� SSC is 0.15 Msodium chloride, 0.015 M sodium citrate, pH7.2) at 508C, 60 min in 0.2� SSC at 558C, and60 min in 0.2� SSC at 60� 8C, briefly rinsed ina graded series of ethanol containing 0.3 Mammonium acetate and dried. Hybridizedradioactivity was visualized after 6 days’ expo-sure using-max film (Amersham, ArlingtonHeights, IL).

RESULTS

Generation and Differentiation ofNeural Stem Cells

To generate NSCs, we adopted lineage selec-tion (five-stage) method that leads to theefficient selection and proliferation of NSCsfrom ES cell. We performed immunocytochem-istry to evaluate generation and differentiationof NSCs. As shown in Figure 1A, a majority ofcells in the stage 4 were labeled with nestin,which is commonly used marker for NSCs.After 6 day of bFGF removal, differentiatedcells were immunostained with Tuj1 and GFAPwhich is specific marker for neurons andastrocytes, respectively. Furthermore, morpho-logical changes were observed. TheNSCs beganextension of neurite-like structures at day 2after removal bFGF. The number and length ofneurite-like structures were increased as thedifferentiation time of cultures evolves (Fig. 1B).We also performed semiquantitative RT-PCRanalysis for neuronal and glial phenotypicgenes (Fig. 1C). Nestin showed little expres-sion since differentiation day 12. Tuj1 andGFAP were gradually increased during differ-entiation. Additionally, TH was also graduallyexpressed, because our culture method gene-rates dopaminergic neurons in high yield [Leeet al., 2000].

Gene Expression in Differentiating Neural Stem Cells 565

Identification of Significantly Expressed Genes

To examine the temporal changes in geneexpression during differentiation comprehen-sively, we performed gene expression profiling.Total RNA derived from differentiated cell at 1,2, 3, 6, 12, or 15 days and the control (NSCs)were subjected to cDNA synthesis. Both cDNAswere subsequentlymixed and hybridizedwith amicroarray. After hybridization, specific Cy3and Cy5 dye labeling for each cDNA (NSCs vs.differentiating cells) were carried out in chips.The flagged genes at more than one time-pointwere not subjected to further analysis (K-meansclustering). The data set of differentially ex-pressed genes including missing value is avail-able at; http://neurogenomics.hanyang.ac.kr.Out of the 10,368 genes represented on the chip,259 genes were up- or down-regulated 2-foldor more at least at one time-point during thedifferentiation (Table I).

To determine the validity of results obtain-ed by the microarray analysis, six randomlyselected genes (Igf2,pleiotrophin, thymopoietin,

Enpp2, FoxM1, Ndr2) were subjected to real-time quantitative PCR analysis. These expres-sion pattern observed by PCR showed a goodconcordance with the pattern assayed bymicro-array (Fig. 2).

Temporal Cluster Analysis

To characterize patterns of transcriptsexpressionduring the time course of differentia-tion, K-means clustering was performed on the259 genes, and these genes were classified intosix clusters (Fig. 3). The temporal clusteranalysis revealed that four clusters of tran-scripts whose the expression was either up-regulated (cluster A, 26 genes and B, 75 genes)or down-regulated (clusterD, 71genes andE, 39genes). Cluster F (45 genes) showed gradualincrease by day 6 and slight decrease on day 15.Cluster C represented only three genes (RIKENcDNA 1700011I03 gene, exportin 4, BRF2) thatwere markedly decreased by day 3, but return-ed to undifferentiated level on day 12 andincreased at day 15.

Fig. 1. Differentiation of neural stem cells (NSCs). A: Immuno-cytochemistry for NSCs, neurons, and astrocytes. Most cells(DAPI nuclear staining, red) at stage 4 were immunostained withthe intermediate filament nestin (green)which ismarker of NSCs.Differentiation of NSCs was induced by withdrawal of bFGF.After 6 day of bFGF removal, differentiated cells wereimmunostained with Tuj1 (green) and GFAP (red). Scale bar,20 mm. B: Phase-contrast images illustrate the morphological

changes during differentiation of NSCs. The neurite-like struc-tures were first detected at differentiation day 2. Arrow indicatesthe neurite-like structures at day 2. C: The generation anddifferentiationofNSCswere further validatedbyRT-PCRanalysisfor phenotypic genes (Nestin, Tuj1, GFAP, and TH). [Color figurecan be viewed in the online issue, which is available atwww.interscience.wiley.com.]

566 Ahn et al.

TABLE

I.Signifi

cantlyExpressedGenesDurin

gDifferentiationofNSCs

No.

Cluster

Foldch

ange(log

arithm,base

2)

Gen

Bank

Title

Resultof

Karsten

etal.[2003]

1day

2day

3day

6day

12day

15day

1A

�0.029

0.040

�0.089

0.461

1.691

1.325

BG073613

Insu

lin-likegrowth

factor

22

A�0.031

0.047

0.521

0.875

2.129

2.065

AA444490

Tissu

einhibitor

ofmetallop

roteinase

2Up-reg

ulatedin

DC

3A

�0.146

0.105

0.148

0.331

1.877

1.874

BG069465

Cd63antigen

4A

�0.196

0.008

0.199

0.494

1.767

1.603

BG086970

GlutathioneS-transferase,mu1

5A

0.070

0.117

0.207

0.804

2.186

2.005

BG074103

H19fetalliver

mRNA

6A

0.295

0.070

0.285

0.973

1.365

1.376

BG079167

Zincfinger

protein

42

7A

0.354

0.604

0.853

0.604

1.190

1.461

BG080229

Aldolase

3,C

isoform

Up-reg

ulatedin

DC

8A

0.101

0.299

0.640

0.821

2.478

2.179

AA063753

ATP-bindingcassette,su

b-familyA

(ABC1),mem

ber

19

A�0.228

0.410

0.851

1.013

2.459

2.203

BG070737

Solute

carrierfamily1,mem

ber

310

A�0.158

�0.135

�0.116

0.125

1.600

1.664

BG087410

CD9antigen

11

A�0.059

0.238

0.190

0.595

1.789

1.610

BG072683

Prion

protein

Up-reg

ulatedin

DC

12

A�0.130

0.041

0.359

0.591

1.782

1.786

W83974

Carbox

ypep

tidase

EUp-reg

ulatedin

DC

13

A0.038

0.330

0.569

1.044

1.397

1.182

AW060338

Gamma-aminob

utyricacidreceptor,

subunit

beta3

14

A0.040

0.228

0.433

1.087

1.839

1.587

BG077733

ATPase,Naþ/K

þtransp

orting,beta1polypep

tide

15

A0.063

�0.108

0.431

0.494

1.410

1.416

AA518639

Aldolase

1,A

isoform

16

A�0.132

�0.035

0.107

0.662

1.624

1.401

BG085991

Integralmem

braneprotein

2B

17

A0.242

0.045

0.274

0.546

2.186

1.956

AI854310

Cocaineandamphetamineregulatedtranscript

18

A0.499

0.279

0.140

1.021

1.549

1.609

BG076160

Mem

brane-sp

anning4-dom

ains,

subfamilyA,mem

ber

6D

19

A�0.018

0.052

�0.153

0.264

1.637

1.776

W83447

Serine(orcysteine)

proteinase

inhibitor,cladeA,mem

ber

3G

20

A0.138

�0.275

0.042

0.262

2.151

2.305

W78651

CystatinC

Up-reg

ulatedin

DC

21

A�0.007

0.626

0.109

0.864

1.670

1.591

AI853802

Phosphofru

ctok

inase,platelet

22

A�0.066

0.508

0.917

1.295

1.788

1.838

AI844824

Endothelin

receptortypeB

23

A�0.037

�0.040

0.088

0.809

1.565

1.535

BG070501

GlutathioneS-transferase,mu2

24

A�0.451

0.145

0.272

0.996

2.550

2.420

BG081834

ATPase,Naþ/K

þtransp

orting,beta2polypep

tide

25

A�0.257

�0.057

�0.133

0.622

1.486

1.786

BG075757

CD

81antigen

26

A�0.005

�0.477

�0.091

1.475

1.102

1.513

BG077818

Metallothionein1

27

B�0.256

�0.143

�0.029

0.109

0.767

1.156

AA002439

Annex

inA5

28

B�0.134

0.014

0.075

�0.388

1.678

1.591

BG064768

RIK

EN

cDNA

1810020C02gen

e29

B0.334

0.568

�0.140

0.611

0.967

1.004

AU022809

Deleted

inpolyposis

130

B�0.367

�0.215

�0.175

0.024

0.464

1.137

W89518

Annex

inA2

31

B�0.268

0.204

0.601

0.536

0.983

1.242

W75931

Secretogranin

III

Up-reg

ulatedin

DC

32

B�0.079

�0.162

0.127

�0.041

0.489

1.106

AA038826

Keratincomplex2,basic,

gen

e4

33

B�0.071

0.140

�0.002

0.280

0.765

1.082

BG084347

Dev

elop

men

talpluripoten

cyassociated5

34

B0.016

0.212

�0.003

0.350

1.247

1.628

BG074398

SPARC-like1(m

ast9,hev

in)

35

B0.105

0.057

0.227

0.371

1.093

1.041

AI854816

Bon

emorphog

enetic

protein

receptor,

type1B

36

B�0.235

0.162

0.350

0.331

1.153

1.010

BG083549

Protein

tyrosinephosphatase

4a3

37

B�0.139

�0.095

0.106

0.679

1.342

1.161

BG086357

Procollagen

,typeIII,alpha1

38

B�0.246

0.136

0.119

0.551

0.776

1.191

BG088450

Low

den

sity

lipop

rotein

receptor-relatedprotein

associated

protein

139

B�0.054

�0.070

0.095

0.123

1.058

1.648

AA108928

RIK

EN

cDNA

9530006B08gen

e40

B�0.043

0.009

0.349

0.171

0.871

1.730

BG069468

F-box

only

protein

15

41

B�0.068

0.256

0.497

0.077

1.085

0.740

BG083306

Interleu

kin

6signaltransd

ucer

42

B�0.449

�0.255

�0.025

�0.041

0.776

1.078

BG086434

Phospholipase

A2,groupVII

Up-reg

ulatedin

DC

43

B0.164

0.117

0.334

0.562

0.929

1.152

AA198668

Acidbetaglucosidase

44

B0.270

�0.066

0.054

0.373

0.291

1.101

AU017674

Musmusculustranscribed

sequen

ce45

B�0.247

�0.154

�0.097

0.017

0.624

1.082

BG072874

Secretedacidic

cysteinerich

glycoprotein

46

B�0.017

0.002

0.202

0.678

1.027

0.948

BG087506

Glutamate

deh

ydrogen

ase

47

B�0.060

�0.106

0.186

0.347

1.520

1.328

W16221

Procollagen

,typeVI,alpha1

(Con

tinued

)


TABLE

I.(C

ontinued

)

No.

Cluster

Foldch

ange(log

arithm,base

2)

Gen

Bank

Title

Resultof

Karsten

etal.[2003]

1day

2day

3day

6day

12day

15day

48

B�0.382

�0.309

0.132

�0.330

0.818

1.269

AA030447

Peripherin

149

B0.099

0.041

0.157

0.346

1.009

1.098

BG085219

RIK

EN

cDNA

1810009M01gen

e50

B�0.341

�0.151

�0.154

�0.143

1.374

1.945

W29607

Enoy

lcoen

zymeA

hydratase

1,perox

isom

al

51

B�0.319

�0.800

�0.186

�1.116

0.316

0.892

AA122925

Carbon

icanhydrase

2Up-reg

ulatedin

NS

52

B�0.240

0.136

0.015

�0.341

1.420

1.518

AA105295

BCL2/aden

ovirusE1B

19kDa-interactingprotein

1,NIP

3a

Up-reg

ulatedin

NS

53

B0.218

0.141

0.136

0.675

1.011

1.376

BG079209

RIK

EN

cDNA

1700029I01gen

e54

B0.021

0.030

0.306

0.341

1.302

1.253

BG065432

ATP-bindingcassette,su

b-familyF(G

CN20),mem

ber

355

B�0.009

0.139

0.162

0.616

1.218

0.876

BG065450

Unknow

nEST

56

B0.049

0.146

0.219

0.686

0.717

1.152

BG071897

Fattyacidbindingprotein

3,muscle

andhea

rt57

B�0.170

0.043

0.239

0.219

0.789

1.110

AI836517

Pleiotrop

hin

aUp-reg

ulatedin

NS

58

B0.007

0.092

0.071

0.083

1.159

1.274

BG064176

Lysosomal-associatedprotein

transm

embrane5

59

B�0.162

�0.204

�0.197

0.260

1.046

1.135

BG064802

Secretedacidic

cysteinerich

glycoprotein

60

B0.188

0.273

0.281

0.489

0.719

1.003

BG079424

Orn

ithineaminotransferase

61

B0.050

�0.196

�0.102

0.305

1.328

1.816

BG073601

Diazepam

bindinginhibitor

62

B�0.224

�0.611

�0.236

0.211

0.780

1.317

AA276440

Selen

oprotein

P,plasm

a,1

Up-reg

ulatedin

DC

63

B�0.039

�0.147

�0.134

0.442

1.050

1.112

BG070071

Hea

tsh

ockprotein

164

B�0.045

�0.022

�0.027

0.024

0.973

1.306

AI835702

GM2gangliosideactivatorprotein

65

B0.150

0.308

0.265

0.752

1.118

1.164

BG084031

Sim

ilarto

hypotheticalprotein

FLJ90036

66

B0.087

�0.001

�0.379

0.206

0.775

1.093

BG087985

Laminin

B1su

bunit

167

B�0.065

0.354

�0.137

0.562

1.187

0.954

AA023786

Polycystic

kidney

disea

se2

68

B0.122

�0.059

�0.111

0.620

1.086

1.577

BG085352

Procollagen

,typeIV

,alpha1

69

B0.038

�0.142

�0.149

0.242

0.942

1.205

BG084610

Microrchidia

70

B0.029

�0.155

�0.244

�0.017

0.898

1.315

BG066605

Laminin,gamma1

71

B0.084

0.096

�0.424

�0.239

1.214

1.023

BG073394

Adducin3(gamma)

Up-reg

ulatedin

DC

72

B�0.012

�0.203

�0.044

0.721

0.999

1.344

BG087341

Procollagen

,typeIV

,alpha2

73

B�0.125

�0.779

�0.827

�0.350

1.282

1.289

AW557873

Inhibitor

ofDNA

binding3

74

B0.026

�0.204

�0.191

0.366

1.516

1.231

AA034564

Procollagen

,typeV,alpha2

75

B0.131

�0.018

�0.389

0.203

0.424

1.085

BG067246

PCTAIR

E-m

otifprotein

kinase

376

B0.125

0.207

0.158

0.320

1.325

1.466

BG070449

RIK

EN

cDNA

4933436C10gen

e77

B0.060

0.082

0.018

0.555

0.814

1.071

AA230924

Myosin

lightch

ain,alkali,non

muscle

78

B0.114

0.270

0.328

0.623

0.632

1.199

BG067269

Aden

ylate

kinase

279

B0.166

0.213

0.090

0.518

1.055

0.963

BG087365

Strom

alcellderived

factor

receptor1

80

B0.027

�0.031

�0.130

0.326

1.125

1.800

BG082965

Cyclin-dep

enden

tkinase

881

B�0.019

0.077

�0.052

0.060

0.805

1.038

BG069516

Solute

carrierfamily6,mem

ber

882

B�0.012

�0.010

0.153

0.182

0.976

1.046

BG071424

Integralmem

braneprotein

2C

83

B0.055

0.148

0.176

0.856

0.882

1.201

BG083801

RIK

EN

cDNA

2610036A22gen

e84

B�0.131

�0.297

�0.573

�0.134

0.985

1.115

BG085415

Gapjunctionmem

branech

annel

protein

alpha1

85

B0.229

�0.133

�0.029

0.221

1.267

0.901

AA030540

Aden

inephosphoribosyltransferase

86

B0.049

�0.516

�1.004

�0.146

1.213

1.598

AA109951

Beta-2

microglobulin

87

B0.327

�0.225

�0.216

�0.020

1.170

0.917

BG084290

Inhibitor

ofDNA

binding2

88

B0.198

0.058

0.056

0.449

0.762

1.028

BG086136

Zincfinger

protein

51

89

B�0.158

0.250

�0.119

�0.561

1.312

1.199

BG086605

Thioredox

ininteractingprotein

Up-reg

ulatedin

DC

90

B�0.391

�0.714

�0.780

�0.182

0.586

1.467

BG078028

Lectin,galactosebinding,soluble

191

B0.081

�0.041

0.014

0.493

1.046

1.307

BG079624

Serine(orcysteine)

proteinase

inhibitor,cladeE,mem

ber

292

B�0.009

�0.023

�0.118

0.365

1.235

1.137

AW491453

Calpain

693

B�0.211

�0.023

0.095

0.397

0.787

1.051

BG084582

Lectin,galactosebinding,soluble

894

B�0.072

0.193

�0.043

0.663

1.308

0.974

AI849826

Catenin

delta

295

B�0.230

�0.490

0.178

�0.523

0.975

1.451

AI835385

Lactate

deh

ydrogen

ase

1,A

chain

568 Ahn et al.

96

B0.285

0.147

�0.350

�0.269

1.052

0.837

BG070902

Nuclea

rprotein

197

B�0.069

0.048

0.053

1.008

1.033

1.045

AI385510

Cadherin

13a

Up-reg

ulatedin

NS

98

B�0.006

�0.025

�0.309

0.199

0.372

1.108

BG067443


sequen

ces

99

B0.249

0.146

�0.083

0.178

0.837

1.188

BG085167

Lysosomalmem

braneglycoprotein

2100

B0.130

�0.103

�0.207

0.222

0.755

1.048

BG080373

Expressed

sequen

ceAW743433

101

B�0.199

0.108

0.252

0.452

1.024

0.728

BG088310

Prosa

posin

102

C0.986

0.843

�1.324

0.272

�0.373

1.051

AU022767

Exportin4

103

C1.308

0.777

�0.947

0.436

0.132

1.006

BG068317

RIK

EN

cDNA

1700011I03gen

e104

C1.220

1.120

�1.131

0.431

0.061

0.862

BG068328

BRF2,su

bunit

ofRNA

polymerase

IIItranscription

initiation

factor

105

D�0.143

�0.264

�0.492

�1.193

�1.136

�0.817

AA466087

H2A

histonefamily,mem

ber

ZUp-reg

ulatedin

NS

106

D�0.011

�0.267

�0.509

�1.489

�1.217

�0.661

AW490674

ect2

oncogen

eUp-reg

ulatedin

NS

107

D�0.135

�0.082

�0.352

�1.026

�1.364

�0.517

BG066442

Karyop

herin

(importin)alpha2

108

D�0.243

�0.074

�0.454

�1.107

�0.991

�0.770

BG068146

Calm

odulinbindingprotein

1109

D�0.104

�0.085

�0.705

�1.486

�1.542

�0.603

BG079172

Top

oisomerase

(DNA)II

alpha

110

D�0.061

�0.364

�0.437

�0.677

�1.351

�1.155

BG081202

Sperminesynthase

111

D�0.546

�0.431

�0.280

�1.048

�0.397

�0.286

BG065190

Aden

osinekinase

112

D�0.224

0.229

�0.295

�1.108

�1.153

�1.078

BG080962

cDNA

sequen

ceBC034753

113

D�0.072

0.045

�0.416

�0.832

�1.054

�0.571

BG082035

Activatorof

Sphase

kinase

114

D�0.121

�0.095

�0.493

�0.844

�1.366

�1.286

BG075630

RIK

EN

cDNA

2310035M22gen

e115

D�0.072

�0.286

�0.425

�1.268

�0.885

�0.698

BG068666

Kinesin

familymem

ber

23

116

D�0.110

�0.345

�0.390

�0.679

�1.223

�0.735

AI836129

Highmob

ilitygroupnucleosomalbindingdom

ain

2117

D0.005

�0.308

�0.638

�1.614

�1.650

�0.744

BG082403

Histone1,H4m

118

D�0.110

�0.266

�0.579

�1.116

�0.594

�0.432

AA060205

Zincfinger

protein

36,C3H

type-like1

119

D�0.085

�0.280

�0.363

�0.925

�1.088

�0.607

BG074248

Kinesin

familymem

ber

22,pseudog

ene

120

D0.089

�0.277

�0.530

�1.057

�1.324

�0.933

BG087310

DEK

oncogen

e(D

NA

binding)

121

D0.011

�0.254

�0.649

�1.327

�1.055

�0.868

BG072056

Cen

trom

ereautoantigen

A122

D�0.035

�0.259

�0.729

�1.376

�1.192

�0.568

AA396324

Cyclin

B1

Up-reg

ulatedin

NS

123

D0.124

�0.263

�0.507

�0.517

�1.131

�0.695

BG066359

Ubiquitin

specificprotease

1124

D0.045

�0.416

�0.819

�1.311

�1.594

�1.525

BG066232

Highmob

ilitygroupAT-hook2

125

D0.073

�0.375

�0.657

�0.791

�1.198

�0.753

BG072545

CDC28protein

kinase

1Up-reg

ulatedin

NS

126

D�0.085

�0.199

�0.356

�0.566

�1.096

�1.082

BG063923

H3histone,

family3B

127

D�0.081

�0.157

�0.549

�0.708

�0.780

�1.071

BG085335

RIK

EN

cDNA

2600016F06gen

e128

D�0.018

�0.317

�0.613

�0.996

�0.803

�1.020

W81792

Cytoch

romeP450,family2,su

bfamilyd,polypep

tide10

129

D�0.201

�0.278

�0.862

�2.048

�1.827

�1.504

AI836520

Histone3,H2bb

130

D0.032

�0.205

�0.357

�1.512

�0.889

�0.841

BG066499

Gen

erich

cluster,C8gen

e131

D0.131

�0.293

�0.562

�0.809

�1.286

�1.028

BG076805

Nuclea

rautoantigen

icsp

erm

protein

(histone-binding)

132

D�0.174

�0.154

�0.779

�1.380

�1.249

�0.897

BG082508

DNA

segmen

t,Chr2,ERATO

Doi

750,ex

pressed

133

D0.061

�0.152

�0.500

�1.600

�1.410

�1.026

BG085939

RIK

EN

cDNA

A730011O11gen

e134

D�0.151

�0.148

�0.512

�0.509

�1.042

�1.074

AW547625

Cyclin

D2

Up-reg

ulatedin

NS

135

D0.122

�0.354

�0.177

�0.838

�1.342

�0.860

AA030433

Bloom

syndromehom

olog

(human)

136

D0.194

�0.193

�0.506

�0.941

�1.430

�1.274

AI835559

Thymop

oietin

137

D0.010

�0.382

�0.667

�1.081

�1.213

�0.908

BG078138

Ribon

ucleotidereductase

M2

138

D0.006

�0.185

�0.700

�1.199

�1.125

�0.338

BG072979

RIK

EN

cDNA

2610201A12gen

e139

D0.227

�0.308

�0.373

�0.819

�1.110

�0.804

AW553739

Ttk

protein

kinase

140

D0.066

�0.534

�0.336

�0.376

�1.822

�1.176

AA034857

RNA

bindingmotifprotein

3141

D0.011

0.521

�0.420

�1.184

�0.666

�0.957

AI844331

Pyrroline-5-carbox

ylate

synthetase

Up-reg

ulatedin

NS

142

D0.144

�0.112

�0.650

�1.201

�1.606

�1.029

BG069688

Cyclin

A2

143

D�0.158

�0.327

�0.686

�1.241

�1.209

�0.476

BG069168

Pituitary

tumor-transforming1

Up-reg

ulatedin

NS

144

D0.151

�0.197

�0.531

�0.953

�0.839

�1.212

BG087468

Forkhea

dbox

M1

Up-reg

ulatedin

NS

145

D0.221

�0.352

�0.417

�0.770

�1.041

�0.904

AA544948

Minichromosom

emaintenance

defi

cien

t7(S

.cerevisiae)

Up-reg

ulatedin

NS

146

D0.131

�0.350

�0.745

�1.335

�1.026

�1.123

BG068759

Transforming,acidic

coiled

-coilcontainingprotein

3Up-reg

ulatedin

NS

147

D0.263

�0.425

�0.669

�0.755

�1.024

�0.945

AA153012

Chromatinassem

bly

factor

1,su

bunit

A(p150)

Up-reg

ulatedin

NS

148

D�0.062

�0.205

�0.618

�1.865

�1.370

�1.157

AA049416

Histone1,H1c

149

D0.077

�0.152

�0.860

�1.304

�1.240

�0.596

BG079289

RIK

EN

cDNA

2700099C18gen

e150

D�0.204

�0.404

�0.778

�0.454

�1.014

�0.097

AA120637

RIK

EN

cDNA

5430410E06gen

e(C

ontinued

)


TABLE

I.(C

ontinued

)

No.

Cluster

Foldch

ange(log

arithm,base

2)

Gen

Bank

Title

Resultof

Karsten

etal.[2003]

1day

2day

3day

6day

12day

15day

151

D0.251

�0.407

�0.596

�1.133

�1.395

�1.128

AA276998

Serine/threon

inekinase

18

152

D�0.123

�0.307

�1.091

�1.907

�2.380

�1.825

AI847223

Sem

adom

ain,transm

embranedom

ain

andcytoplasm

icdom

ain,6C

153

D�0.046

�0.412

�0.951

�1.524

�1.290

�0.442

BG071965

Cyclin

B1,relatedsequen

ce1

154

D0.257

�0.418

�0.526

�0.958

�1.103

�0.905

BG071804

Buddinguninhibited

byben

zimidazoles1hom

olog

,beta(S.

cerevisiae)

155

D0.175

�0.264

�0.675

�0.751

�1.060

�0.942

BG088398

Glucocorticoidinducedtranscript1

156

D�0.332

0.146

�0.361

�1.075

�0.272

�0.418

BG076333

Methylenetetrahydrofolate

deh

ydrogen

ase

(NADþ

dep

enden

t)Up-reg

ulatedin

NS

157

D�0.015

�0.247

�0.558

�0.920

�1.013

�0.877

AI853169

Hypotheticalprotein

C530022J18

158

D�0.024

�0.492

�0.617

�1.103

�0.967

�0.868

BG064686

Chorionic

somatomammotropin

hormon

e1

159

D�0.062

�0.546

�0.510

�0.396

�1.054

�0.799

BG069969

Tcraen

hancer-bindingfactor

interactingprotein

1160

D�0.029

�0.395

�0.715

�0.753

�1.204

�0.528

BG085427

Highmob

ilitygroupbox

2Up-reg

ulatedin

NS

161

D�0.041

�0.225

�0.114

�0.597

�1.301

�0.890

BG086151

CDK2(cyclin-dep

enden

tkinase

2)-associatedprotein

1162

D0.072

�0.538

�0.407

�0.575

�1.536

�1.068

BG077092

RIK

EN

cDNA

2600016C11gen

e163

D0.330

�0.033

�0.860

�1.061

�1.044

�0.345

BG081629

SMC4stru

cturalmaintenance

ofch

romosom

es4-like1(yea

st)

164

D0.252

�0.360

�0.909

�1.108

�1.386

�0.901

BG067860

MAD2(m

itotic

arrestdefi

cien

t,hom

olog

)-like1(yea

st)

Up-reg

ulatedin

NS

165

D0.229

�0.130

�0.606

�1.106

�1.167

�1.118

BG083621

Poliovirusreceptor-related3

166

D�0.156

�0.417

�0.606

�0.529

�1.111

�1.137

AA498281

Lamin

B1

167

D0.010

�0.619

�0.818

�1.223

�1.052

�0.478

BG064846

Celldivisioncycle2hom

olog

A(S

.pom

be)

Up-reg

ulatedin

NS

168

D�0.182

�0.376

�0.321

�0.529

�1.024

�0.380

BG085860

Smallnuclea

rribon

ucleoprotein

polypep

tideG

169

D0.267

�0.271

�0.807

�1.600

�0.993

�0.546

BG086805

Buddinguninhibited

byben

zimidazoles1hom

olog

(S.cerevisiae)

170

D�0.042

�0.479

�0.462

�0.647

�1.732

�0.994

BG064598

Proliferatingcellnuclea

rantigen

Up-reg

ulatedin

NS

171

D�0.208

�0.420

�0.605

�1.280

�1.106

�0.636

BG080690

ShcSH2-dom

ain

bindingprotein

1Up-reg

ulatedin

NS

172

D�0.182

�0.480

�0.654

�1.732

�1.498

�1.048

BG069986

Polymyositis/sclerod

ermaautoantigen

1173

D�0.009

�0.215

�0.286

�0.671

�1.014

�0.011

BG083522

CDC28protein

kinase

regulatory

subunit

2174

D0.140

�0.240

�0.547

�1.140

�0.559

�0.620

AI893902

Sperm

associatedantigen

5Up-reg

ulatedin

NS

175

D0.040

�0.362

�0.723

�1.134

�0.867

�0.313

BG077844

SMC2stru

cturalmaintenance

ofch

romosom

es2-like1(yea

st)

176

E�0.436

0.290

0.372

0.005

�0.940

�1.309

BG071846

Transd

ucin-likeen

hancerof

split4,E(spl)hom

olog

(Drosophila)

177

E�0.031

0.170

0.209

0.090

�0.809

�1.303

BG085705

RIK

EN

cDNA

4933432H23gen

e178

E�0.020

0.207

0.378

0.188

�1.180

�0.786

AW554258

Midnolin

179

E�0.012

0.106

0.036

�0.307

�1.151

�1.029

BG074931

Enhancerof

zestehom

olog

2(D

rosophila)

180

E�0.049

0.083

0.016

�0.656

�1.280

�1.464

AA498495

Erythrocyte

protein

band4.1

181

E0.280

�0.065

0.290

�0.096

�0.963

�1.018

BG079889

Isop

entenyl-diphosphate

delta

isom

erase

Up-reg

ulatedin

NS

182

E�0.053

�0.151

�0.165

�0.521

�1.303

�1.740

AA242226

Cadherin

2183

E0.045

0.370

0.729

0.558

�0.777

�1.451

AI846983

Microtubule-associatedprotein

1B

184

E0.027

0.594

�0.009

0.061

�1.021

�0.683

BG081111

Top

oisomerase

(DNA)I

185

E�0.032

0.295

0.323

�0.302

�1.005

�1.080

BG086306

Polyhom

eotic-like2(D

rosophila)

186

E0.091

�0.298

�0.104

0.285

�1.271

�1.421

AI836508

Splicingfactor,arginine/serine-rich

2(SC-35)

187

E0.225

0.069

0.066

0.053

�1.166

�1.550

BG086100

RIK

EN

cDNA

1110055B05gen

e188

E0.062

�0.016

�0.070

�0.463

�1.083

�0.643

BG072534

Heterog

eneousnuclea

rribon

ucleoprotein

A1

189

E0.074

�0.207

�0.444

�0.388

�1.182

�0.971

BG064278

Nucleosomeassem

bly

protein

1-like1

190

E0.014

0.427

0.101

�0.368

�0.708

�1.304

BG071313

Cyclin-dep

enden

tkinase

inhibitor

1C

(P57)

191

E0.097

�0.029

�0.268

�0.035

�1.433

�1.308

BG074524

Heterog

eneousnuclea

rribon

ucleoprotein

A/B

192

E0.054

�0.088

�0.235

�0.516

�1.289

�1.285

BG085009

Cyclin-dep

enden

tkinase

4193

E0.016

�0.058

�0.085

�0.192

�1.071

�1.247

BG087161

Latrop

hilin

2194

E0.109

�0.027

�0.013

�0.315

�0.715

�1.011

AA048831

Highmob

ilitygroupbox

3195

E0.051

�0.011

�0.248

�0.374

�1.054

�0.845

AI848377

cDNA

sequen

ceBC010348

196

E0.272

�0.116

0.182

�0.525

�0.929

�1.070

AI323209

Makorin,ringfinger

protein,3

197

E0.435

�0.230

�0.405

�0.298

�0.870

�1.021

BG078653

Heterog

eneousnuclea

rribon

ucleoprotein

D

570 Ahn et al.

198

E0.058

�0.030

�0.182

�0.025

�0.677

�1.093

AA212717

C-terminalbindingprotein

1199

E0.111

0.438

0.447

0.243

�0.618

�1.013

AU045483

Ubiquilin

2200

E0.120

�0.138

�0.123

�0.568

�0.729

�1.009

AA059891

Upstream

regulatory

elem

entbindingprotein

1201

E�0.079

0.136

0.273

0.133

�0.308

�1.263

BG070656

SRY-box

containinggen

e4

202

E0.052

�0.288

�0.369

�0.166

�1.011

�1.410

BG087241

Mesod

erm

specifictranscript

203

E0.080

0.155

�0.173

�0.272

�1.006

�0.790

BG085781

Chromob

oxhom

olog

1(D

rosophilaHP1beta)

204

E�0.001

�0.219

�0.155

0.273

�1.171

�0.744

BG078157

Splicingfactor,arginine/serine-rich

3(SRp20)

Up-reg

ulatedin

NS

205

E0.250

0.155

0.216

0.425

�1.351

�1.165

AA125197

Actin

dep

enden

tregulatorof

chromatin,su

bfamilye,

mem

ber

1206

E0.076

�0.036

�0.057

�0.508

�1.051

�0.706

BG079001

Gelon

gation

factor

207

E�0.027

�0.174

0.242

0.296

�0.447

�1.084

AA120351

Ubiquitin-con

jugatingen

zymeE2I

208

E�0.197

�0.304

�0.420

�0.365

�1.211

�1.397

AA032448

Zincfinger

protein

162

209

E0.010

0.328

0.546

0.491

�0.688

�1.425

AI840073

Neu

ronatin

210

E0.112

0.273

0.091

0.364

�1.320

�1.393

BG072739

SRY-box

containinggen

e11

211

E�0.070

0.163

0.565

0.400

�0.645

�1.102

AI838805

Kru

ppel-likefactor

15

212

E�0.026

0.201

0.139

�0.293

�1.071

�1.026

BG087208

H1histonefamily,mem

ber

0213

E0.047

0.062

�0.227

�0.111

�0.918

�1.161

BG087103

KH

dom

ain

containing,RNA

binding,signaltransd

uction

associated1

214

E0.024

�0.232

�0.084

�0.276

�0.904

�1.002

AA209061

Thioredox

inreductase

1215

F�0.058

0.668

1.329

1.175

0.847

0.281

W18828

Dihydropyrimidinase-like3

216

F�0.098

0.607

1.024

0.657

1.248

0.638

AI842333

Dou

ble

cortin

andcalcium/calm

odulin-dep

enden

tprotein

kinase-like1

217

F0.145

0.474

0.150

1.121

0.298

0.223

BG064500

Hea

tsh

ockprotein

218

F0.007

0.567

0.788

1.177

1.157

0.868

BG086292

Amyloid

beta(A

4)precu

rsor

protein

219

F�0.048

0.386

1.080

1.200

0.371

0.145

AA275865

ALL1-fusedgen

efrom

chromosom

e1q

220

F�0.014

0.612

1.017

0.803

0.712

0.402

BG083182

Guaninedea

minase

221

F�0.077

0.070

0.345

0.697

1.200

0.821

AA547428

Protein

kinase,cA

MPdep

enden

t,catalytic,

beta

222

F0.142

0.081

0.788

0.989

1.057

0.799

AI854566

Synaptotagmin

7223

F0.169

0.552

1.067

1.295

0.280

0.169

BG087115

Myeloidecotropic

viralintegration

site-relatedgen

e1

224

F�0.071

0.479

0.757

0.768

1.112

0.578

AI323062

Guaninenucleotidebindingprotein,alphao

225

F0.108

0.488

0.820

1.165

0.244

�0.349

AA231471

Sialyltransferase

8(alpha-2,8-sialyltransferase)B

226

F�0.122

0.310

0.754

0.714

1.011

0.556

AA269845

Glycoprotein

m6a

227

F�0.100

0.719

0.791

0.834

1.032

0.597

AI835125

Pleck

strinhom

olog

y,Sec7andcoiled

-coildom

ains2

228

F0.123

0.591

1.023

0.636

0.439

0.423

BG087300

p21(C

DKN1A)-activatedkinase

4229

F0.138

0.381

0.648

1.167

1.327

1.008

BG086955

Brain

protein

44-like

230

F0.085

0.474

0.837

0.983

1.133

0.836

BG082280

RIK

EN

cDNA

9430072K23gen

e231

F0.063

0.302

0.557

0.725

0.595

1.049

BG070401

Cyclin

M2

232

F�0.050

0.514

1.065

1.061

1.121

0.806

W42241

Stathmin-like4

233

F�0.235

0.192

0.867

0.480

1.556

0.745

AI840345

Gamma-aminob

utyricacid(G

ABA-A

)transp

orter1

234

F0.078

0.695

1.327

2.075

2.036

1.150

AA028410


tau

Up-uregulatedin

DC

235

F0.134

�0.129

�0.065

1.758

0.592

0.927

BG063925

Metallothionein2

236

F0.077

0.557

0.863

1.160

1.252

0.838

AI853686

Glutamate

receptor,

ionotropic,AMPA3(alpha3)

237

F0.203

0.366

0.635

1.303

1.353

0.884

AA521764

Recep

tor(calciton

in)activitymod

ifyingprotein

2Up-reg

ulatedin

DC

238

F0.040

0.679

0.725

1.311

1.026

0.242

BG075834

RIK

EN

cDNA

3110039L19gen

e239

F�0.190

0.429

0.338

1.144

0.838

0.685

BG076172

Calm

odulin1

240

F0.362

0.540

0.697

1.015

0.488

0.314

AI844249

RIK

EN

cDNA

1110003P13gen

e241

F0.073

0.321

0.736

1.082

0.310

�0.015

W66622

TEA

dom

ain

familymem

ber

2242

F�0.274

�0.166

0.059

0.751

1.094

0.395

AI837858

Protein

kinase

C,beta

243

F0.234

0.237

0.709

1.273

1.139

0.729

AI835523

Protein

kinase

inhibitor,alpha

244

F0.113

0.320

0.456

1.306

0.551

0.275

BG081562

Reg

ulatorof

G-protein

signaling2

245

F0.009

0.227

0.611

1.039

1.068

0.972

BG067399

Tropom

odulin2

246

F0.239

0.578

0.620

0.829

1.190

0.887

BG084093

N-m

ycdow

nstream

regulated2

247

F0.023

0.505

0.660

1.141

0.685

0.380

BG086006

EphrinB3

248

F0.400

0.573

0.490

1.058

0.822

0.624

BG080627

Nuclea

rresp

iratory

factor

1249

F0.206

0.266

1.176

1.538

0.905

0.215

AI849758


2250

F0.287

0.355

0.323

0.797

0.876

1.012

BG069134


sequen

ces

251

F0.435

0.548

0.575

1.193

0.685

0.733

BG066404


sequen

ces

(Con

tinued

)


We also categorized the 259 genes based ontheir functions, referring toGeneOntologyTMandclassified them into 14 groups (Fig. 4). Frequen-cies of these functionally classified genes in eachcluster are shown in Figure 4, with statisticalsignificance evaluated by Fisher’s exact test forhigh and low frequencies. High frequencieswere observed as follows: ‘‘Transport’’-relatedgenes in the cluster A; ‘‘cell adhesion’’-relatedgenes in the cluster B; both ‘‘nucleic acidmetabolism’’- and ‘‘cell cycle’’-related genes inthe cluster D; both ‘‘transcription’’- and ‘‘RNAprocessing’’-related genes in the cluster E.

Transport-related genes. Amonggenes inthe clusterAwith properties of gradual increaseduring differentiation, significantly frequentwere members of the category ‘‘transport’’-related genes. These genes are a ATP-bindingcassette, sub-familyA,member1(ABCA1); solutecarrier family 1, member 3; gamma-aminobu-tyric acid receptor, subunit beta 3; ATPase,Naþ/Kþ transporting, beta 1 and cocaine andamphetamine regulated transcript (CART). Ofthem, ABCA1 gene was reported to be highlyexpressed in CA1 and CA3 pyramidal neurons[Koldamova et al., 2003] and CART gene, ex-pressed in hypothalamus neuron [Larsen et al.,2003] and may selectively regulate certain cen-tral dopamine neuronal activities [Shieh, 2003].

Cell adhesion-related genes. Genes of‘‘cell adhesion’’ were significantly enriched incluster B. These included procollagen, type VI,alpha 1; procollagen, type IV, alpha 1; procolla-gen, type IV, alpha 2; laminin B1 subunit 1;laminin, gamma 1; catenin delta 2; cadherin 13.Specially, mRNA level of procollagen, type IV,alpha 1 was up-regulated after differentiationday 12 (2.8-fold) and this gene previouslyreported as a marker of neural differentiation[Clifford et al., 1996] and a promoting factorfor the differentiation of neuronal progenitors[Ali et al., 1998].

Cell cycle-related genes. The majorityof ‘‘cell cycle’’ related genes was included incluster D and was down-regulated duringdifferentiation. With regard to cell cycle check-point transition, the expression of cyclin A2, B1,D2, and cell division cycle 2 homolog A (cdc2A)were repressed. In addition, this cluster in-cluded activator of S phase kinase; CDC28protein kinase 1; transforming, acidic coiled-coil containing protein 3; budding uninhibitedby benzimidazoles 1 homolog beta (Mm.29133);budding uninhibited by benzimidazoles 1 homo-

TABLE

I.(C

ontinued

)

No.

Cluster

Foldch

ange(log

arithm,base

2)

Gen

Bank

Title

Resultof

Karsten

etal.[2003]

1day

2day

3day

6day

12day

15day

252

F0.249

0.465

0.723

1.001

0.505

0.534

BG069707

CD1d1antigen

253

F�0.026

0.091

0.134

0.961

1.272

0.836

BG086024

Activatedleukocyte

celladhesionmolecule

254

F�0.174

0.396

0.376

0.607

1.004

0.383

BG073000

Protocadherin

gammasu

bfamilyB,4

255

F�0.254

0.022

0.328

1.259

0.915

0.726

AI852463

Protein

tyrosinephosphatase,receptortype,

T256

F0.015

0.063

0.088

1.094

0.721

0.924

AI323177

Neu

rofibromatosis2

257

F0.165

0.388

0.738

1.039

0.531

0.250

AA463173

Solute

carrierfamily11,mem

ber

2258

F0.134

0.162

0.531

1.005

�0.094

�0.262

BG077853

Coa

tomer

protein

complex,su

bunit

gamma2

259

F0.173

0.067

0.056

1.048

�0.156

�0.358

BG067712

RIK

EN

cDNA

6720463E02gen

e

aGen

eswithop

positeex

pressionpatternin

overlapped

gen

esof

bothsystem

s.

572 Ahn et al.

log (Mm.2185); MAD2 (mitotic arrest deficient,homolog)-like 1 and CDC28 protein kinaseregulatory subunit 2.Nucleic acid metabolism-related genes.

Among genes in the cluster D with properties ofdecrease during differentiation, significantlyfrequent were also members of the category‘‘nucleic acid metabolism’’-related genes. Therewas a decrease in the expression of genesinvolved in DNA packaging, replication, repair,nucleosome assembly, and chromosome segre-gation, such as H2A histone family, member Z;topoisomerase II alpha; high mobility groupnucleosomal binding domain 2; centromereautoantigen A; Bloom syndrome homolog;ribonucleotide reductase M2; minichromosomemaintenance deficient 7; chromatin assemblyfactor 1; proliferating cell nuclear antigen(PCNA); high mobility group box 2 and SMC2structural maintenance of chromosomes 2-like

1. Altered expressions of these functional geneswere concomitant with the decrease in expres-sion of G1/S cell cycle regulatory genes.

Transcription regulators. Transcriptionregulators which may be involved in NSCsproliferation and maintenance showed highfrequency in cluster E and included transdu-cin-like enhancer of split 4 (TLE 4); enhancer ofzeste homolog 2 (EZL2); ring finger protein 3;SRY-box containing gene 4, 11 (Sox 4, 11); actindependent regulator of chromatin, subfamily e,member 1 andKruppel-like factor 15. BothSox4and 11 were previously reported to be highlyexpressed in ventricular zones and down-regulated in neurons as they become mature[Cheung et al., 2000].

In Vivo Expression Patterns

We performed ISH for four genes. Of them,three genes (FoxM1, cyclinD2, andCDK4) were

Fig. 2. Verification of microarray data. Real-time RT-PCRwas used to verify the data from the microarray.Each value is expressed as fold change (logarithm, base 2) in mRNA levels at each differentiation time-pointcompared to the control NSCs, and represents the mean� SEM of three independent experiments.Microarray data of each gene was displayed as a horizontal strip. [Color figure can be viewed in the onlineissue, which is available at www.interscience.wiley.com.]


highly expressed in the NSCs, while Enpp2showed high expression level in differentiatedcells as shown by RT-PCR. In ISH, Fox M1,cyclin D2, and CDK4 were highly expressed inthe germinal zone during embryonic develop-ment. Enpp2 showed high and specific expres-sion in the choroid plexus of brain fromE13.5 toP14 (Fig. 5A,B). The choroid plexus has recentlybeen shown to be essential organizing centersfor inducing dorsal neuron fates and sustainingneuron function [Awatramani et al., 2003] andwas identified as a possible location of stem/progenitor cells [Li et al., 2002]. Moreover,lysophosphatidic acid (LPA), catalyzed by Enpp2,was shown to increase terminal mitosis ofneural precursor cells and promote their cellcycle exit [Kingsbury et al., 2003].

DISCUSSION

Comparision of Two Expression Profiles

Our experiment used a comprehensivemolec-ular approach to identify temporal changes ingene expression that occur during differentia-tion ofNSCs.A total of 259geneswere regulated

during differentiation. The 259 gene expressionprofiles provided us with an opportunity tocompare or contrast our analysis with those ofother investigators who have used similarmethodologies. Karsten et al. [2003] reported adata set consisting of 318 significantly changedexpression profiles in proliferating NSCs (NS)and its 24 h differentiated cells (DC) in vitro.There were 36 overlapped genes (defined asUnigene IDs) represented in both data sets(Table I). In addition, 33 of 36 overlapped geneswere found to have very similar expressionpatterns in both systems, which suggest a goodoverall agreement (91.6%) between the resultsof these two groups. However, these genesaccounted for very small proportion in totaloverlapped genes of our 10K chip and UCLA 9Karray which had been used in Karsten et al.experiment (36/2,312 genes). There could beseveral reasons to explain these consequences.One major possibility is difference in thedetermination of significantly expressed genesin microarray data set (stringent 2-fold changecutoff method vs. false discovery rate method).Other reasons are differences in NSC population,

Fig. 3. K-means clustering of genes with expression levels that changed during NSCs differentiation. K-means clusteringwasused to group the259 identifiedgenes into clusters basedon similar expressionover thesix time-point. These 259 genes were classified into 6 clusters (A–F).

574 Ahn et al.

differentiation protocol, and microarray label-ing method. Although we used NSCs derivedfrom ES cells, Karsten et al. used neural pro-genitor cells whichwere obtained fromP0mouse.

Cell Cycle Regulation and Inhibition ofProneuronal Genes in NSC Proliferation

The clustering analysis of expression patternsof 259 genes and following statistical analysisbased on functional classification revealed re-

markable features of changes in gene expres-sionduringNSCdifferentiation. Themajority ofcell cycle, nucleic acid metabolism, transcrip-tion, and RNA processing related genes wereoverexpressed inNSCs. Notably, the up-regula-tion of G1-S cell cycle regulation, nucleic acidsynthesis, DNA replication, packaging, andrepair genes inNSCswere consistent with theirself-renewal properties. These changes alsoindicate a progressive switch from biosynthetic

Fig. 4. Distribution of functionally categorized genes in each cluster. Frequencies of genes of eachfunctional category are shown. Significantly high (**, P<0.01; *, P<0.05) and low ({, P<0.05) frequencieswere evaluated by Fisher’s exact test.


activity towards more functional activity in thedifferentiating NSCs.

Anumber of transcription factors required forNSCsproliferation through cell cycle regulationwere suppressed as cells differentiated. A goodexample is Fox M1 mRNA, which activatescyclin B1 [Leung et al., 2001]. Cyclin B1 is a keyregulatory component of the G2/M phase tran-sition in the cell cycle. Germinal zone expres-sion and cell cycle regulator activation of FoxM1 support its important role in NSCs prolif-eration. Another transcription factor is EZL2.Concordance with the hypothesis that retino-blastoma protein complex (pRb) may play acentral role inNSC proliferation [Karsten et al.,2003], we observed a down-regulation of EZL2

at 12 and 15 day. Previous study reportedthat EZL2 is a downstream target of pRb-E2Fpathway and essential for proliferating cells[Bracken et al., 2003]. Additionally, there wereincreases in mRNA of inhibitor of DNA binding(Id) 3 at differentiation day 12 and 15. Idspromote cell cycle progression by interactingwith components of the cell cyclemachinery andinhibit the precocious differentiation of neuralprogenitors into neurons and oligodendrocytes.Though Id3 is necessary for proliferating NSCs,previous study showed that Id3 may play animportant role in the regulation of astrocyteproliferation [Tzeng and de Vellis, 1997]. Inter-estingly, only one member of cyclin family,cyclin M2, exhibited an alteration in differen-

Fig. 5. In situ hybridization of selected transcripts that showed altered expression level during NSCsdifferentiation. A: Sagittal and coronal sections of each age were hybridized with 35S labeled antisensecRNAs. The arrows indicate germinal zone and arrowheads indicates choroid plexus at each age. B: Highermagnification of germinal zone (cdk4) and choroid plexus expression (Enpp2).

576 Ahn et al.

tiated cells and an opposite expression patternagainst other cyclins. The precise role of thisgene during differentiation remains to be con-firmed through function-based assays.In addition to cell cycle regulation, our

analysis showed that inhibition of proneuronalgenes is continued during proliferation (beforeterminal differentiation). TLE 4, a mammalianhomologue of Drosophila groucho, was signifi-cantly down-regulated at differentiation day 15.Notch stimulation leads to activation of the ex-pression of Enhancer of split complex [E (spl)-C]ranscripts. Their protein products thenmediatethe negative regulation of the expression ofproneural genes. The transcriptional repres-sor function of E (spl)-C proteins requiresthe activity of the product of groucho [Grbavecet al., 1998]. Taken together, these alteredexpression levels strongly suggest that cell cycleregulation and negative regulation of prone-uronal genes have high influences on NSCproliferation.

Up-Regulation of Neurite Outgrowth and AxonGuidance Related Genes in Differentiated Cells

A variety of extracellular cues, consisting ofsecreted molecules, cell–cell contact and cell–extracellular matrix (ECM) interaction, areresponsible for neurite outgrowth and axonguidance [Wei et al., 2002]. Genes encodingmembers of a number of these signaling path-ways are up-regulated. Pleiotrophin, a cytokinethat induce neurite outgrowth [Deuel et al.,2002], is overexpressed during differentiationand also reported to exhibit a trophic effect onsurvival of dopaminergic neurons [Hida et al.,2003]. Another secreted molecule, annexin A2,induce neuritogenesis and differentiation of thecell line PC12, suggesting involvement in NSCdifferentiation [Jacovina et al., 2001]. EphrinB3, guidance cue molecule, was also expressed.Ephrin-Eph receptor system has been shown tomediate contact-dependent repulsion involvedin axon guidance [Cook et al., 1998].Many molecules have been isolated from the

ECM and found to be very good at supportingneurite out growth. ECM proteins, such aslaminins and various forms of collagens in ourchip, are also up-regulated. Most growth cones,especially those in the CNS, grow along thesurfaces of other cells and axons. In thisfunction, they are supported largely by class ofgrowth-promotingmolecules that are expressed

on the cell surfaces and known as cell adhesionmolecules (CAM) [Walsh and Doherty, 1997].mRNA for activated leukocyte CAM (Alcam)in the cluster F was also up-regulated. Neuriteformation requires extensive cytoskeletonremodeling. Microtubules provide structuralsupport and act as substrate for the fast axonaltransport of vesicles [Signor and Scholey, 2000].Microtubule proteins including microtubuleassociated protein (MAP) 2 and MAP tau, wereoverexpressed.

Cellular signaling pathways thatmake use ofCAMs, repulsive factors, attractive factors, andgrowth factors are received by receptors on thesurface of the growth cone. Receptors that havetyrosine phosphatase activity have been foundin abundance on growth cones [Holland et al.,1998]. Tyrosine phosphatase 4a3 in the clusterB was up-regulated at differentiation day 12and 15 and tyrosine phosphatase, receptor typeT in the cluster F was up-regulated at day 6.

In conclusion, we used microarray analysisduring differentiation of NSCs to identify re-gulatory molecules. We identified 259 modu-lated genes, which could be divided into 14functional groups. The further statistical ana-lysis showed that cell cycle regulation, nucleicacid metabolism, and negative regulation ofproneuronal genes play crucial roles in NSCsproliferation and its self-renewal. Also, in dif-ferentiated cells, a variety of neurite outgrowthand axon guidance related genes were detectedas significantly expressed genes. Genes thatwe identified in this study will advance theunderstanding of mechanisms underlying pro-liferation/differentiation of NSCs and thepotential for the use of stem cells as therapeuticagents.

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