Microarray analysis of retinal gene expression in Egr-1 knockoutmice

Ruth Schippert, Frank Schaeffel, Marita Pauline Feldkaemper

Institute for Ophthalmic Research, Section of Neurobiology of the Eye, University Eye Hospital Tuebingen, Tuebingen, Germany

Purpose: We found earlier that 42 day-old Egr-1 knockout mice had longer eyes and a more myopic refractive errorcompared to their wild-types. To identify genes that could be responsible for the temporarily enhanced axial eye growth,a microarray analysis was performed in knockout and wild-type mice at the postnatal ages of 30 and 42 days.Methods: The retinas of homozygous and wild-type Egr-1 knockout mice (Taconic, Ry, Denmark) were prepared forRNA isolation (RNeasy Mini Kit, Qiagen) at the age of 30 or 42 days, respectively (n=12 each). Three retinas were pooledand labeled cRNA was made. The samples were hybridized to Affymetrix GeneChip Mouse Genome 430 2.0 Arrays.Hybridization signals were calculated using GC-RMA normalization. Genes were identified as differentially expressed ifthey showed a fold-change (FC) of at least 1.5 and a p-value <0.05. A false-discovery rate of 5% was applied. Ten geneswith potential biologic relevance were examined further with semiquantitative real-time RT–PCR.Results: Comparing mRNA expression levels between wild-type and homozygous Egr-1 knockout mice, we found 73differentially expressed genes at the age of 30 days and 135 genes at the age of 42 days. Testing for differences in geneexpression between the two ages (30 versus 42 days), 54 genes were differently expressed in wild-type mice and 215genes in homozygous animals. Based on three networks proposed by Ingenuity pathway analysis software, nine differentlyexpressed genes in the homozygous Egr-1 knockout mice were chosen for further validation by real-time RT–PCR, threegenes in each network. In addition, the gene that was most prominently regulated in the knockout mice, compared to wild-type, at both 30 days and 42 days of age (protocadherin beta-9 [Pcdhb9]), was tested with real-time RT–PCR. Changesin four of the ten genes could be confirmed by real-time RT–PCR: nuclear prelamin A recognition factor (Narf),oxoglutarate dehydrogenase (Ogdh), selenium binding protein 1 (Selenbp1), and Pcdhb9. Except for Pcdhb9, the geneswhose mRNA expression levels were validated were listed in one of the networks proposed by Ingenuity pathway analysissoftware. In addition to these genes, the software proposed several key-regulators which did not change in our study:retinoic acid, vascular endothelial growth factor A (VEGF-A), FBJ murine osteosarcoma viral oncogene homolog(cFos), and others.Conclusions: Identification of genes that are differentially regulated during the development period between postnatalday 30 (when both homozygous and wild-type mice still have the same axial length) and day 42 (where the difference ineye length is apparent) could improve the understanding of mechanisms for the control of axial eye growth and may leadto potential targets for pharmacological intervention. With the aid of pathway-analysis software, a coarse picture ofpossible biochemical pathways could be generated. Although the mRNA expression levels of proteins proposed by thesoftware, like VEGF, FOS, retinoic acid (RA) receptors, or cellular RA binding protein, did not show any changes in ourexperiment, these molecules have previously been implicated in the signaling cascades controlling axial eye growth.According to the pathway-analysis software, they represent links between several proteins whose mRNA expression waschanged in our study.

Myopia is becoming an increasing problem, especially inindustrial nations. It is widely believed that both hereditaryand environmental factors contribute to the development ofmyopia. Several molecules have already been identified in theretina that appear to be involved in the visual control of axialelongation of the eye (e.g., dopamine [1-3], retinoic acid[4-6], nitric oxide [7-9], vasoactive intestinal polypeptide[10-12]). Another factor that was found to be involved was

Correspondence to: Marita Pauline Feldkaemper, Institute forOphthalmic Research, Section of Neurobiology of the Eye,University Eye Hospital Tuebingen, Calwerstrasse 7/1, 72076Tuebingen, Germany; Phone: +49 7071/29-87424; FAX: +497071/29-5196; email: marita.feldkaemper@uni-tuebingen.de

the transcription factor Egr-1 (early growth responseprotein-1), the mammalian ortholog to the avian proteinZENK (also called Tis8, Ngfi-A, Kro×−24, Zif268 in otherspecies). By means of immunocytochemistry it was initiallyfound that the number of ZENK-immunoreactive amacrinecells in the retina of chicks is increased under conditions thatlead to a reduction in eye growth (myopic defocus, recoveryof myopia) and decreased under conditions that enhanceocular growth (hyperopic defocus, form-deprivation). Thesechanges were most prominent and distinct in a specific subsetof amacrine cells, the glucagon-containing amacrine cells[13,14]. Recently, this bi-directional response was detectedby means of immunohistochemistry in another glucagon-containing cell type of the chicken retina as well, the so-called

Molecular Vision 2009; 15:2720-2739 <http://www.molvis.org/molvis/v15/a288>Received 9 June 2009 | Accepted 4 December 2009 | Published 10 December 2009

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bullwhip-cells [15]. Moreover, a downregulation of Egr-1mRNA in total retinal tissue was found in mice after shortperiods of form-deprivation [16]. All of these experimentssuggested that Egr-1 (ZENK) is an important factor incontrolling eye growth, at least in some animal models formyopia. However, it should be noticed that mRNA levels ofEgr-1 in the total retina of chicks do not seem to show thisbidirectional response consistently. Although ZENK mRNAlevels are upregulated in total retinal samples within one hourafter diffuser removal of former form-deprived chicks(recovery of myopia) [17], treatment with both minus lensesand plus lenses for one day reduced the amount of ZENKmRNA in the total retina of chicks in both cases, suggestingthat the role of Egr-1 is complex and may vary among specialcell types [18,19]. Unfortunately, no study is available thatinvestigated the time course of Egr-1 mRNA changes indetail. Therefore, current knowledge about the regulation ofEgr-1 during increased or decreased eye growth is stilllimited.

Studies on Egr-1 knockout mice were in line with thehypothesis that Egr-1 has a function in the regulation of eyegrowth. Homozygous knockout mice, lacking functionalEgr-1 protein, developed relative axial myopia at the age of42 and 56 days (compared to heterozygous and wild-typeEgr-1 knockout mice [20]). The difference in axial lengthdeclined with increasing age, but the differences in therefractive state persisted. Paraxial schematic eye modelingsuggested that other optical elements, possibly the lens, hadalso changed in the Egr-1 knockout mice. This is notsurprising, given that Egr-1 was absent not only from retinalamacrine cells but from all cells of the body. The effect oflacking Egr-1 protein should have long-ranging effects onother cells in the retina, eye, and the autonomic nervoussystem or the endocrine system.

Egr-1 is known to have a function in a variety of biologicprocesses (e.g., cell proliferation [21], brain plasticity andlearning [22], apoptosis [23]) and several target genes of Egr-1have already been identified. Egr-1-overexpression insynovial fibroblasts leads to an increased expression ofcollagen type 1 and of tissue inhibitor of metalloproteinasestype 1 and 3 (TIMP1 and TIMP3) [24]. Since the induction ofmyopia is associated with scleral thinning through reducedaccumulation of collagen and increased degradation of scleraltissue [25-27], the reduction of Egr-1-stimulated collagenexpression and the reduced inhibition of degrading enzymes(such as the matrix-metalloproteinases that are repressed byTIMPs) that could take place in animals without functionalEgr-1 protein, could explain the myopic phenotype of thesemice. Other genes that are already known to be influenced byEgr-1 are for instance platelet-derived growth factor-A and -B (PDGF-A and -B) [28,29], basic fibroblast growth factor(bFGF) [30] and transforming growth factor-beta (TGF-β)[31].

Because of the complex role of Egr-1 in the regulation ofvarious other proteins, and the differences in axial eye lengthbetween the Egr-1 knockout mice and the wild-type mice, wehave studied the role of Egr-1 in the retina in more detail.Retinal samples of Egr-1 knockout and wild-type mice at theage of 30 days (no difference in axial eye length yet) and 42days (already a difference in axial eye length of 59 µm) werecompared regarding their mRNA expression changes, bothbetween the two genotypes and within the same genotypebetween the two age groups.

METHODSAnimals: Experiments were conducted in accordance with theARVO Statement for the Use of Animals in Ophthalmic andVision Research and were approved by the UniversityCommission for Animal Welfare. Egr-1 knockout mice,generated on C57/BL6 background, were purchased fromTaconic (Ry, Denmark) and bred in the animal facilities of theinstitute after a breeding permission was obtained from thecompany. Since female homozygous knockout mice aresterile because of a deficiency of luteinizing hormone-beta(which is due to the lack of Egr-1 [32]), only heterozygousfemales were bred. Animals were housed in standard cageswith their littermates under a 12 h light/dark cycle withunrestricted access to water and food pellets. Illumination wasprovided by standard fluorescent lamps and wasapproximately 200 lx. Standard PCR was performed todetermine genotype (specific primer sequences provided byTaconic) and gender (with primers designed for the geneencoding the sex-determining region Y represents as SRY).

Male mice were killed by an overdose of diethyl ether atthe mean age of 30 days (p29-p31) or 42 days (p41-p43). Eyeswere enucleated and retinas were prepared carefully to ensurethat the samples were not contaminated with retinal pigmentepithelium. Tissue was snap-frozen in liquid nitrogen. Theretinas of 12 homozygous Egr-1 knockout mice (hm) and 12wild-type mice (wt) of the same strain were prepared for bothtime points (48 animals in total). Three single retinas fromdifferent mice were then pooled to obtain four samples pergroup (wt/30, wt/42, hm/30, hm/42), and RNA was isolatedusing the RNeasy Mini Kit (Qiagen, Hilden, Germany)according to the manufacturer's instructions.Microarray: Quality check of RNA, cDNA synthesis andlabeling and the actual microarray analysis was performed bythe Affymetrix Resource Facility at the University ofTuebingen. The quality of total RNA was monitored byAgilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto,CA) following the manufacturer's instructions. Generation ofdouble-stranded cDNA, preparation and labeling of cRNA,hybridization to 430 2.0 Mouse Genome Arrays (Affymetrix,Santa Clara, CA), washing, and scanning were performedaccording to the standard Affymetrix protocol. Scanning andanalysis were performed using the Affymetrix Microarray

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Suite Software (version 5.0) and the signal intensities wereanalyzed using ArrayAssist 5.5.1 (Stratagene, La Jolla, CA).

Data were normalized using the GC-RMA normalizationmethod which uses the GC content of the probes innormalization with RMA (Robust Multi-Array). To correctfor multiple testing, a false-discovery rate of 5% was applied.All comparisons of mRNA expression levels between thegroups were performed using un-paired t-tests. Genes wereidentified as differentially expressed if they showed a fold-change (FC) of at least 1.5 with a p value lower than 0.05. Foldchange was calculated by dividing the experimental value(lens-treated, t) by the control value (untreated control, c). Ifthe relative signal intensity of the control was higher than theintensity of the treated samples, the negative reciprocal wascalculated (-c/t). A fold change of 1 or −1 therefore indicatesno change, while a fold change of 2 equals a doubling inproduct, and a fold change of −2 equals a halving in transcriptabundance.

The data discussed in this publication have beendeposited in the National Center for BiotechnologyInformation (NCBI's) Gene Expression Omnibus (GEO) andare accessible through GEO Series accession numberGSE16974.Pathway analysis: The list of differently expressed genes wassubjected to a subsequent post-analysis task to find the mainbiologic processes associated with the experimental system.The “Ingenuity Pathways Analysis” Software 5.0 (IPA,Ingenuity Systems) was applied to elucidate putativepathways associated with the gene expression changes in theretinas of the Egr-1 knockout mice between the age of 30 daysand 42 days. For this purpose, 215 genes which were classedas “differently expressed,” e.g., those whose retinal mRNAexpression in the knockout mice at p30 was significantlydifferent from the expression at p42, were analyzed andtheoretical networks and pathways were computed. The IPAis a manually curated database of functional interactions andcontains previously published findings from peer-reviewedpublications. Interactions between proteins and molecules inthe proposed networks are therefore supported by publishedinformation which is associated by the program with knownbiologic pathways. It should be noted here that the interactionspresented in the networks are not specific for the retina orbrain tissue, as the database contains literature from manydifferent research areas. If the mRNA expression levels ofmany proteins present in one proposed network have actuallybeen found to be changed, it is likely that they are connectedwith each other and that their changes may represent aresponse to the lack of Egr-1.Real-time RT–PCR: Based on three networks found in thehomozygous Egr-1 knockout mice computed by IngenuityPathways Analysis Software (hm/30 versus hm/42, see Figure1A-C), nine genes were chosen for further validation of theirexpression changes by real-time RT–PCR (three genes per

network). For network A we chose: A kinase anchor protein9 (Akap9), SET domain, bifurcated 1 (Setdb1) andstaphylococcal nuclease domain containing 1 (Snd1). Thethree genes representing network B were: corticotropinreleasing hormone (Crh), insulin-like growth factor bindingprotein 3 (Igfbp3), and LIM and SH3 protein 1 (Lasp1).Finally, from network C we chose: nuclear prelamin Arecognition factor (Narf), oxoglutarate dehydrogenase(Ogdh), and selenium binding protein 1 (Selenbp1). Inaddition, protocadherin-beta 9 (Pcdhb9), the gene that showedthe highest fold-change in mRNA expression levels in acomparison between wild-type and knockout mice, was testedwith real-time PCR.

The primer sequences, product lengths, NCBI accessionnumbers and network classifications of the genes tested areshown in Table 1. From each sample, 1 µg of RNA was reversetranscribed using M-MLV reverse transcriptase (Promega,Mannheim, Germany), 500 ng oligo (dT)15 primer and 50 ngof a random primer mixture (Invitrogen, Solingen, Germany).Semiquantitative real-time RT–PCR was performed with theaid of QuantiTect SYBR Green master mix kit of Qiagen onthe iCycler iQ Multicolor Real-Time PCR Detection Systemfrom Bio-Rad (Hercules, CA). All samples were analyzed intriplicate with a template amount corresponding to 2 ng ofRNA. Hypoxanthine-phosphoribosyl-transferase (HPRT)was used as a housekeeping gene and all PCR products weresubjected to automated sequencing to ensure amplification ofthe correct sequences.Statistics and data analysis: Data were analyzed using thesoftware JMP 5.1 (SAS Institute, Cary, NC) and Excel(Microsoft Corporation, Redmond, WA). The mean cyclethreshold (Ct) value of each triplet was taken and the meannormalized expression (MNE) was computed as previouslydescribed [33]. To test for differences between the four groups(wt/30, wt/42, hm/30, hm/42), ANOVA’s (ANOVA) wereapplied for each gene. In case the ANOVA was significant(p<0.05), a paired Student's t-test was applied as a post-hoctest to test for differences between wt/30 versus wt/42 and hm/30 versus hm/42.

RESULTSMicroarray:

Age-related comparisons (wt/30 versus wt/42) in wild-type mice—Comparing mRNA expression levels between the30 days old and the 42 days old wild-type mice, 54 genes wereclassified as differentially expressed (with a minimum FC of±1.5 and a p-value lower than 0.05). The corresponding genesare listed in Appendix 1 together with the fold-changes and p-values that were determined in homozygous Egr-1 knockoutmice at the two ages. Seventeen genes showed reduced mRNAexpression in the 42 days old wild-type mice compared to the30 days old mice. Thirty-seven genes were higher expressed.The maximum fold-changes were −2.40 and 2.62,respectively.

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Figure 1. Networks predicted by Ingenuity Pathway Analysis in the homozygous Egr-1 knockout mice. Networks proposed by IngenuityPathways Analysis Software. All genes whose mRNA expression levels were found to be differentially regulated in the knockout mice betweenthe age of 30 days and 42 days are highlighted in gray. Encircled are those genes that were chosen for validation by real-time RT–PCR. Adetailed legend describing the symbols used in this scheme is enclosed in the figure. Asterisks denote changes in gene expression that werevalidated using real-time PCR.

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Age-related comparisons (hm/30 versus hm/42) in Egr-1knockout mice: Two hundred fifteen genes had changed theirexpression levels in the homozygous Egr-1 knockout micebetween the age of 30 and 42 days (see Appendix 2 for a listof those genes). Higher mRNA expression was found in 176genes at 42 days, compared to 30 days, while 39 genes showedreduced mRNA expression. Age-dependent changes in geneexpression ranged here between 2.49 fold and −4.01 fold. Apathway analysis was performed based on this list ofdifferentially expressed genes. Genes that were further studiedusing semiquantitative real-time RT–PCR are shown in boldin Appendix 2.

Eight genes were differently expressed at the two ages inboth wild-type and homozygous Egr-1 knockout mice (shownin italics and underlined in Appendix 1 and Appendix 2). Thedirections of their changes were the same in wild-type- andhomozygous Egr-1 knockout mice.Egr-1-related comparisons (wt/30 versus hm/30 and wt/42versus hm/42): In the 30 days old mice, the lack of Egr-1 wasassociated with different mRNA expression levels of 73genes, with 39 upregulated and 34 downregulated (wt/30versus hm/30, see Table 2 for a list of those genes). In the 42days old mice, 135 genes were differently expressedcompared to the wild-type. One hundred and thirteen geneswere upregulated, and 22 genes were downregulated (wt/42versus hm/42, see Table 3 for a list of those genes).

Thirteen genes showed up in both lists (shown in italicsand underlined in both Table 2 and Table 3) and except forone gene (dystrophin (DMD)), the regulation of mRNAexpression levels was in the same direction at both 30 daysand 42 days. The gene that showed the highest difference inmRNA expression levels (Pcdhb9, shown in bold in Table 2and Table 3), was further studied using semiquantitative real-time RT–PCR.Pathway analyses: Data obtained by comparison of themRNA expression patterns in the homozygous Egr-1knockout mice between the age of 30 days and 42 days (hm/

30 versus hm/42, see Appendix 2, 215 genes) were analyzedusing the software “Ingenuity Pathway Analysis.” Severalnetworks were identified that could be involved in retinalsignaling. We chose three networks that appeared different inthe homozygous Egr-1 knockout mice (see Figure 1A-C) tofind candidates for validation by real-time RT–PCR.Networks proposed by the software were common signalingpathways. All genes that were found to be differentlyexpressed in the knockout mice are indicated by filled graysymbols in Figure 1. Genes labeled as open symbols representintermediate metabolic steps, determined from the currentliterature by the software. Molecules represented in networkA (Figure 1A) are regulated by mitogen-activated proteinkinases (MAPK), which respond to a variety of extracellularstimuli and regulate various cellular activities, such as geneexpression, mitosis, differentiation, and cell survival/apoptosis [34]. These kinases seem to be effector proteins innetwork B (Figure 1B) as well, together with insulin, the earlyresponse transcription factor activator-protein 1 (Ap1) andanother ubiquitous transcription factor, nuclear factor-kappaB (NfkB). The central molecules in network C (Figure 1C) areretinoic acid, vascular endothelial growth factor A (Vegf-A),V-fos FBJ murine osteosarcoma viral oncogene homolog(Fos) and beta-estradiol.

Furthermore, several pathways were identified in both thewild-type and the homozygous knockout mice (wt/30 versuswt/42 and hm/30 versus hm/42, shown in Table 4). The lackof Egr-1 seems to affect a variety of pathways and manyfundamental pathways are part of this list (e.g., synaptic long-term depression and potentiation, PDGF- and chemokinesignaling, and others).Real-time RT–PCR: Based on the three networks identifiedby Ingenuity Pathways Analysis Software (hm/30 versus hm/42, Figure 1A-C), nine genes were chosen for validation oftheir changes in mRNA expression by real-time RT–PCR,with three genes present in each network. In addition,protocadherin-beta 9 (Pcdhb9), the gene that showed thehighest fold-change in comparison between the wild-type and

TABLE 1. DESCRIPTION OF PRIMERS.

GeneNCBI accession

number Forward primer (5′-3′) Reverse primer (5′-3′) Amplicon NetworkAkap9 NM_194462.2 GTCTTCAGATGGTAGAAAAGGA GTTAGCTGTGAGCTGAGTTATG 173 bp ASetdb1 NM_018877.2 GATAGCTCCTGCCGAGACTTC CTGCCATCCACCTCTTCAAC 143 bp ASnd1 NM_019776.2 ACGCTGATGAGTTTGGCTACA CCACGACAGAGGAGGTTTC 171 bp ACrh NM_205769.1 CATTCTTGAGGGGTGGCTA CTCTTACACAACCAAATTGACC 116 bp B

Igfbp3 NM_008343.2 TCTTGGGGTCCTTCTCAAA CCTCCAGACACAGGCTCC 194 bp BLasp1 NM_010688.3 ATACCATGAGGAGTTTGAGAAG ACCATAGGACGAGGTCATCT 196 bp BNarf NM_026272.2 GATAGCATCCCTTCAGCCCT TTCATCAAACCCCTTTATCTCC 156 bp COgdh NM_010956.3 GCTAGTCTCTTCCTTGACTG AACTTACTCATGCCATTGTC 184 bp C

Selenbp1 NM_009150.3 GTGCAACGTGAGCAGTTT CTGCATCCCCAGGCTTCT 161 bp CPcdhb9 NM_053134.3 TTTAGGAGAAACTACCTTGTGC TGAGCATTAAAGTCACTTGAGG 195 bp noneHRPT NM_013556.2 CCAGTAAAATTAGCAGGTGTTC GATAAGCGACAATCTACCAGAG 179 bp none

Shown are the NCBI accession numbers, primer sequences, product sizes and the assignment to the networks of the genes thatwere further tested with real-time RT–PCR.

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30 v

shm

/p30

)p-

valu

e (w

t/p3

0 vs

hm

/p3

0)

FC (w

t/p42

vs

hm/p

42)

p-va

lue

(wt/

p42

vs h

m/

p42)

1454

713_

s_at

Hdc

hist

idin

e de

carb

oxyl

ase

1.80

0.01

501.

210.

0278

1449

106_

atG

px3

glut

athi

one

pero

xida

se 3

1.93

0.04

72−1

.80

0.24

4114

5297

5_at

Agx

t2l1

alan

ine-

glyo

xyla

te a

min

otra

nsfe

rase

2-li

ke 1

2.29

0.00

72−1

.24

0.16

0614

2432

5_at

Esco

1es

tabl

ishm

ent o

f coh

esio

n 1

hom

olog

1 (S

. cer

evis

iae)

−1.5

30.

0239

−1.2

20.

1796

1430

996_

atEt

nk1

etha

nola

min

e ki

nase

1−1

.53

0.00

76−1

.13

0.46

6814

4148

6_at

Fkbp

15FK

506

bind

ing

prot

ein

15−1

.54

0.00

40−1

.38

0.10

8514

3473

4_at

E130

016E

03R

ikR

IKEN

cD

NA

E13

0016

E03

gene

−1.5

70.

0446

−1.0

00.

9955

1458

363_

atZd

hhc1

7zi

nc fi

nger

, DH

HC

dom

ain

cont

aini

ng 1

7−1

.58

0.00

12−1

.25

0.08

1414

4055

3_at

Mec

rm

itoch

ondr

ial t

rans

-2-e

noyl

-CoA

redu

ctas

e−1

.58

0.02

62−1

.16

0.17

6914

4563

2_at

Ogd

hox

oglu

tara

te d

ehyd

roge

nase

(lip

oam

ide)

−1.5

80.

0050

1.09

0.63

2214

4035

1_at

Birc

4B

acul

ovira

l IA

P re

peat

-con

tain

ing

4−1

.75

0.02

831.

020.

8804

1457

732_

atPc

mtd

2Pr

otei

n-L-

isoa

spar

tate

O-m

ethy

ltran

sfer

ase

dom

ain

cont

aini

ng 2

−2.0

00.

0016

−1.0

10.

9495

1439

843_

atC

amk4

calc

ium

/cal

mod

ulin

-dep

ende

nt p

rote

in k

inas

e IV

−2.

190.

0000

−1.

950.

0006

Bin

ding

(24)

1438

295_

atG

lcci

1G

luco

corti

coid

indu

ced

trans

crip

t 11.

500.

0044

1.21

0.22

3914

4131

7_x_

atJa

kmip

1ja

nus k

inas

e an

d m

icro

tubu

le in

tera

ctin

g pr

otei

n 1

1.50

0.02

561.

040.

5407

1434

203_

atFa

m10

7aFa

m10

7a fa

mily

with

sequ

ence

sim

ilarit

y 10

7, m

embe

r A1.

520.

0244

−1.0

70.

4713

1451

602_

atSn

x6so

rting

nex

in 6

1.55

0.04

561.

060.

6713

1428

942_

atM

t2m

etal

loth

ione

in 2

1.57

0.00

08−1

.30

0.00

7814

3538

6_at

Vw

fV

on W

illeb

rand

fact

or h

omol

og1.

600.

0158

1.31

0.01

6614

5221

7_at

Ahn

akA

HN

AK

nuc

leop

rote

in (d

esm

oyok

in)

1.61

0.00

73−1

.01

0.93

4814

2266

0_at

LOC

6712

37si

mila

r to

Puta

tive

RN

A-b

indi

ng p

rote

in 3

1.62

0.01

21−1

.04

0.66

0314

5635

1_at

Brd8

brom

odom

ain

cont

aini

ng 8

1.67

0.00

031.

660.

0005

1429

239_

a_at

Star

d4St

AR-r

elat

ed li

pid

tran

sfer

(STA

RT) d

omai

n co

ntai

ning

42.

180.

0020

1.79

0.02

9514

1758

0_s_

atSe

lenb

p1se

leni

um b

indi

ng p

rote

in 1

2.34

0.01

021.

230.

0676

1451

692_

atTm

co6

tran

smem

bran

e an

d co

iled-

coil

dom

ains

64.

100.

0000

2.73

0.00

0214

2287

7_at

Pcdh

b12

prot

ocad

herin

bet

a 12

−1.5

20.

0070

−1.4

10.

0010

1437

694_

atB

B11

4266

Expr

esse

d se

quen

ce B

B11

4266

−1.5

30.

0318

−1.1

30.

1606

1421

132_

atPv

rl3po

liovi

rus r

ecep

tor-

rela

ted

3−1

.56

0.01

531.

020.

7130

1436

981_

a_at

Yw

haz

tyro

sine

3-m

onoo

xyge

nase

act

ivat

ion

prot

ein,

zet

a−1

.56

0.00

24−1

.04

0.57

1214

4063

2_at

Pcdh

b4pr

otoc

adhe

rin b

eta

4−1

.57

0.04

46−1

.58

0.08

3314

4952

7_at

Pcdh

b7pr

otoc

adhe

rin

beta

7−

1.58

0.01

55−

1.64

0.00

1614

3056

9_at

Ttc9

cte

tratri

cope

ptid

e re

peat

dom

ain

9C−1

.58

0.00

89−1

.02

0.84

7814

4395

0_at

A63

0042

L21R

ikR

IKEN

cD

NA

A63

0042

L21

gene

−1.6

40.

0345

1.33

0.06

0014

2195

3_at

Crk

lv-

crk

sarc

oma

viru

s CT1

0 on

coge

ne h

omol

og (a

vian

)-lik

e−1

.66

0.02

161.

010.

9614

Molecular Vision 2009; 15:2720-2739 <http://www.molvis.org/molvis/v15/a288> © 2009 Molecular Vision

2726

TAB

LE 2

. CO

NTI

NU

ED.

Aff

ymet

rix

IDG

ene

sym

bol

Gen

e tit

leFC

(wt/p

30 v

shm

/p30

)p-

valu

e (w

t/p3

0 vs

hm

/p3

0)

FC (w

t/p42

vs

hm/p

42)

p-va

lue

(wt/

p42

vs h

m/

p42)

1426

458_

atSl

map

sarc

olem

ma

asso

ciat

ed p

rote

in−1

.68

0.01

64−1

.39

0.19

3914

4331

5_at

Dm

dD

ystr

ophi

n−

2.45

0.04

921.

660.

0103

1422

640_

atPc

dhb9

prot

ocad

heri

n be

ta 9

−14.

340.

0000

−17.

480.

0000

Unk

now

n (8

)14

1746

0_at

Ifitm

2in

terf

eron

indu

ced

trans

mem

bran

e pr

otei

n 2

1.53

0.04

78−1

.03

0.70

1514

1727

5_at

Mal

mye

lin a

nd ly

mph

ocyt

e pr

otei

n, T

-cel

l diff

eren

tiatio

n pr

otei

n1.

590.

0059

1.16

0.13

1614

5363

2_at

4930

538K

18R

ikR

IKEN

cD

NA

493

0538

K18

gen

e1.

610.

0215

1.07

0.50

3214

3481

7_s_

atR

prd2

Reg

ulat

ion

of n

ucle

ar p

re-m

RN

A d

omai

n co

ntai

ning

21.

750.

0387

1.13

0.22

2214

6004

9_s_

at15

0001

5O10

Rik

RIK

EN c

DN

A 1

5000

15O

10 g

ene

1.77

0.01

631.

340.

3090

1447

553_

x_at

Ric

3R

esis

tanc

e to

inhi

bito

rs o

f cho

lines

tera

se 3

hom

olog

(C. e

lega

ns)

−1.5

60.

0314

1.33

0.16

5314

5163

4_at

2810

051F

02R

ikR

IKEN

cD

NA

281

0051

F02

gene

−1.6

30.

0438

1.10

70.

5203

1428

909_

atA1

3004

0M12

Rik

RIK

EN c

DN

A A1

3004

0M12

gen

e−

1.90

0.00

01−

1.62

0.03

68N

ot A

nnot

ated

(2)

1441

430_

at1.

560.

0106

−1.1

30.

2617

1442

733_

at−1

.55

0.02

44−1

.05

0.73

78

Show

n ar

e A

ffym

etrix

ID, g

ene

sym

bol,

gene

title

, fol

d ch

ange

(FC

) and

p-v

alue

s tha

t ind

icat

e th

e di

ffer

ence

s in

mR

NA

exp

ress

ion

leve

ls b

etw

een

the

wild

-type

and

the

hom

ozyg

ous E

gr-1

kno

ckou

t mic

e bo

th a

t the

age

of 3

0 da

ys a

nd 4

2 da

ys. G

enes

wer

e so

rted

afte

r GO

ann

otat

ions

. Gen

es th

at w

ere

sign

ifica

ntly

cha

nged

(with

a F

C >

1.5

and

p-v

alue

< 0

.05)

in b

oth

the

30 d

ays o

ld m

ice

and

the

42 d

ays o

ld m

ice

are

show

n in

ital

ics a

nd a

re u

nder

lined

. The

gen

e th

at sh

owed

the

high

est

diff

eren

ces i

n m

RN

A e

xpre

ssio

n le

vels

bet

wee

n th

e ho

moz

ygou

s and

the

wild

-type

mic

e (P

cdhb

9 (sh

own

in b

old)

) was

furth

er in

vest

igat

ed u

sing

real

-tim

e PC

R.

Molecular Vision 2009; 15:2720-2739 <http://www.molvis.org/molvis/v15/a288> © 2009 Molecular Vision

2727

TAB

LE 3

. LIS

T O

F GEN

ES T

HA

T W

ERE

DIF

FER

ENTI

ALL

Y E

XPR

ESSE

D B

ETW

EEN

WIL

D-T

YPE

AN

D H

OM

OZY

GO

US E

GR-

1 K

NO

CK

OU

T M

ICE

AT

THE

AG

E O

F 42

DA

YS (

WT/

42 V

ERSU

S HM

/42)

.

Aff

ymet

rix

IDG

ene

sym

bol

Gen

e tit

leFC

(wt/p

42 v

shm

/p42

)

p-va

lue

(wt/

p42

vs h

m/

p42)

FC (w

t/p30

vs

hm/p

30)

p-va

lue

(wt/

p30

vs h

m/

p30)

Stru

ctur

al M

olec

ule

Act

ivity

(1)

1421

811_

atTh

bs1

thro

mbo

spon

din

1−2

.09

0.01

951.

060.

7630

Tra

nspo

rter

Act

ivity

(4)

1458

916_

atSl

c12a

6So

lute

car

rier f

amily

12,

mem

ber 6

1.61

0.00

76−1

.08

0.66

9714

5749

7_at

Syt1

Syna

ptot

agm

in I

1.64

0.00

52−1

.16

0.62

1314

3604

4_at

Scn7

aso

dium

cha

nnel

, vol

tage

-gat

ed, t

ype

VII,

alph

a−

1.89

0.00

03−

1.28

0.03

8314

1584

4_at

Syt4

syna

ptot

agm

in IV

−2.

260.

0000

−2.

550.

0000

Enz

yme

Reg

ulat

or A

ctiv

ity (7

)14

4467

1_at

Ras

al2

RA

S pr

otei

n ac

tivat

or li

ke 2

1.53

0.03

63−1

.30

0.29

6214

4659

5_at

Itsn2

inte

rsec

tin 2

1.53

0.01

82−1

.26

0.47

8414

4034

7_at

Arh

gap1

0R

ho G

TPas

e ac

tivat

ing

prot

ein

101.

570.

0115

1.02

0.93

2414

4138

6_at

Rap

gef1

Rap

gua

nine

nuc

leot

ide

exch

ange

fact

or 1

1.61

0.01

94−1

.12

0.15

1514

4530

7_at

Aut

s2A

utis

m su

scep

tibili

ty c

andi

date

21.

860.

0009

−1.1

30.

7187

1442

897_

at26

1002

4E20

Rik

RIK

EN c

DN

A 2

6100

24E2

0 ge

ne−1

.50

0.02

19−1

.39

0.00

9514

1618

8_at

Gm

2aG

M2

gang

liosi

de a

ctiv

ator

pro

tein

−1.7

90.

0475

−1.0

20.

8384

Mol

ecul

ar T

rans

duce

r A

ctiv

ity (8

)14

5846

9_at

Cbl

bC

asita

s B-li

neag

e ly

mph

oma

b1.

610.

0089

−1.1

30.

6019

1455

967_

atSo

rbs1

sorb

in a

nd S

H3

dom

ain

cont

aini

ng 1

1.58

0.01

111.

100.

7148

1445

555_

atTr

pm3

Tran

sien

t rec

epto

r pot

entia

l cat

ion

chan

nel,

subf

amily

M, m

embe

r3

1.64

0.04

56−1

.13

0.57

03

1443

279_

atN

lkN

emo

like

kina

se1.

550.

0307

−1.0

90.

7229

1441

498_

atPt

prd

Prot

ein

tyro

sine

pho

spha

tase

, rec

epto

r typ

e, D

1.57

0.04

991.

120.

8303

1441

220_

atM

agi2

Mem

bran

e as

soci

ated

gua

nyla

te k

inas

e, W

W a

nd P

DZ

dom

ain

cont

aini

ng 2

1.93

0.03

161.

100.

8184

1422

723_

atSt

ra6

stim

ulat

ed b

y re

tinoi

c ac

id g

ene

6−1

.59

0.02

051.

220.

3171

1417

205_

atK

delr2

KD

EL (L

ys-A

sp-G

lu-L

eu) e

ndop

lasm

ic re

ticul

um p

rote

in re

tent

ion

rece

ptor

2−1

.53

0.00

271.

140.

3870

Tra

nscr

iptio

n R

egul

ator

Act

ivity

(13)

1441

615_

atC

bfa2

t2co

re-b

indi

ng fa

ctor

, run

t dom

ain,

alp

ha su

buni

t 2, t

rans

loca

ted

to, 2

(hum

an)

1.53

0.02

80−1

.02

0.92

091

1445

914_

atN

rf1

Nuc

lear

resp

irato

ry fa

ctor

11.

543

0.00

161.

310.

5977

1441

140_

atR

ere

Arg

inin

e gl

utam

ic a

cid

dipe

ptid

e (R

E) re

peat

s1.

550.

0021

1.27

0.35

2414

5668

6_at

Zfhx

1aZi

nc fi

nger

hom

eobo

x 1a

1.56

0.01

591.

040.

7871

1439

946_

atM

ef2c

Myo

cyte

enh

ance

r fac

tor 2

C1.

620.

0041

−1.1

40.

5653

1458

661_

atLc

orLi

gand

dep

ende

nt n

ucle

ar re

cept

or c

orep

ress

or1.

630.

0265

−1.0

40.

9105

1446

953_

atTc

f4Tr

ansc

riptio

n fa

ctor

41.

630.

0097

1.03

0.95

6714

4569

5_at

Atx

n1A

taxi

n 1

1.73

0.01

10−1

.40

0.36

9214

1706

5_at

Egr1

earl

y gr

owth

resp

onse

11.

790.

0004

1.99

0.00

0014

4351

1_at

Ror

aR

AR

-rel

ated

orp

han

rece

ptor

alp

ha1.

960.

0294

−1.1

80.

5929

1436

329_

atEg

r3ea

rly g

row

th re

spon

se 3

−1.5

10.

0326

−1.3

00.

1831

1443

897_

atD

dit3

DN

A-d

amag

e in

duci

ble

trans

crip

t 3−1

.55

0.02

971.

110.

6698

1417

930_

atN

ab2

Ngf

i-A b

indi

ng p

rote

in 2

−2.

880.

0001

−2.

400.

0035

Molecular Vision 2009; 15:2720-2739 <http://www.molvis.org/molvis/v15/a288> © 2009 Molecular Vision

2728

TAB

LE 3

. CO

NTI

NU

ED.

Aff

ymet

rix

IDG

ene

sym

bol

Gen

e tit

leFC

(wt/p

42 v

shm

/p42

)

p-va

lue

(wt/

p42

vs h

m/

p42)

FC (w

t/p30

vs

hm/p

30)

p-va

lue

(wt/

p30

vs h

m/

p30)

Cat

alyt

ic A

ctiv

ity(1

7)14

5866

3_at

Larg

eLi

ke-g

lyco

syltr

ansf

eras

e1.

500.

0298

1.37

0.24

7414

4281

3_at

Dgk

iD

iacy

lgly

cero

l kin

ase,

iota

1.53

0.04

48−1.00

0.99

8214

3527

3_at

War

s2try

ptop

hany

l tR

NA

synt

heta

se 2

(mito

chon

dria

l)1.

530.

0008

1.10

0.60

1114

4344

5_at

Dia

p3D

iaph

anou

s hom

olog

3 (D

roso

phila

)1.

610.

0162

−1.32

0.06

8214

4216

3_at

Hac

e1H

ECT

dom

ain

and

anky

rin re

peat

con

tain

ing,

E3

ubiq

uitin

pro

tein

ligas

e 1

1.64

0.00

43−1.11

0.70

30

1417

024_

atH

ars

hist

idyl

-tRN

A sy

nthe

tase

1.66

0.00

001.

660.

0000

1440

066_

atSm

arca

d1SW

I/SN

F-re

late

d, m

atrix

-ass

ocia

ted

actin

-dep

ende

nt re

gula

tor o

fch

rom

atin

, sub

fam

ily a

1.69

0.04

201.

160.

1397

1445

395_

atPr

kca

Prot

ein

kina

se C

, alp

ha1.

690.

0030

1.04

0.93

6914

3858

3_at

Ern1

Endo

plas

mic

retic

ulum

(ER

) to

nucl

eus s

igna

ling

11.

710.

0264

−1.50

0.09

6914

4641

2_at

Ww

oxW

W d

omai

n-co

ntai

ning

oxi

dore

duct

ase

1.76

0.01

521.

104

0.65

5014

4518

8_at

Gph

nG

ephy

rin1.

800.

0010

−1.13

0.72

2714

5590

5_at

2610

507B

11R

ikR

IKEN

cD

NA

261

0507

B11

gen

e−1.50

0.04

35−1.15

0.34

2314

3017

7_at

Ube

2bub

iqui

tin-c

onju

gatin

g en

zym

e E2B

, RA

D6

hom

olog

y (S

. cer

evis

iae)

−1.52

0.00

99−1.33

0.20

7114

3954

0_at

Mar

ch2

mem

bran

e-as

soci

ated

ring

fing

er (C

3HC

4) 2

−1.52

0.01

15−1.40

0.04

9314

1661

3_at

Cyp

1b1

cyto

chro

me

P450

, fam

ily 1

, sub

fam

ily b

, pol

ypep

tide

1−1.54

0.04

02−1.19

0.37

1714

2102

4_at

Agp

at1

1-ac

ylgl

ycer

ol-3

-pho

spha

te O

-acy

ltran

sfer

ase

1−1.55

0.00

481.

020.

8328

1439

843_

atC

amk4

calc

ium

/cal

mod

ulin

-dep

ende

nt p

rote

in k

inas

e IV

−1.

950.

0006

−2.

190.

0000

Bin

ding

(30)

1441

567_

atM

yo9a

Myo

sin

IXa

1.50

0.02

491.

070.

8016

1447

381_

atC

psf6

Cle

avag

e an

d po

lyad

enyl

atio

n sp

ecifi

c fa

ctor

61.

510.

0298

1.12

0.54

6614

5677

3_at

Nup

l2nu

cleo

porin

like

21.

510.

0362

−1.07

0.68

1414

4137

3_at

Msi

2M

usas

hi h

omol

og 2

(Dro

soph

ila)

1.53

0.00

461.

140.

6634

1436

382_

atZb

tb12

zinc

fing

er a

nd B

TB d

omai

n co

ntai

ning

12

1.54

0.03

59−1.18

0.12

6214

5630

3_at

Phf1

4PH

D fi

nger

pro

tein

14

1.54

0.01

781.

070.

8732

1443

199_

atLr

ch3

Leuc

ine-

rich

repe

ats a

nd c

alpo

nin

hom

olog

y (C

H) d

omai

nco

ntai

ning

31.

550.

0347

−1.29

0.29

64

1443

337_

atG

rip1

Glu

tam

ate

rece

ptor

inte

ract

ing

prot

ein

11.

550.

0332

−1.30

0.51

4714

4761

5_at

Fmn1

Form

in 1

1.55

0.00

82−1.19

0.64

7014

5589

3_at

Rsp

o2R

-spo

ndin

2 h

omol

og (X

enop

us la

evis

)1.

550.

0419

1.09

0.54

6414

4627

9_at

Neg

r1N

euro

nal g

row

th re

gula

tor 1

1.58

0.03

141.

020.

9288

1445

329_

atD

tnb

dyst

robr

evin

, bet

a1.

580.

0214

−1.07

0.79

4414

4438

4_at

Jazf

1JA

ZF z

inc

finge

r 11.

580.

0358

−1.34

0.29

8014

3912

3_at

Phf2

1aPH

D fi

nger

pro

tein

21A

1.60

0.01

02−1.14

0.67

5614

4176

9_at

Arl1

5A

DP-

ribos

ylat

ion

fact

or-li

ke 1

51.

610.

0200

−1.03

0.93

5114

4241

1_at

Glc

ci1

Glu

coco

rtico

id in

duce

d tra

nscr

ipt 1

1.63

0.00

68−1.16

0.62

0614

4055

1_at

Dna

jc1

Dna

J (H

sp40

) hom

olog

, sub

fam

ily C

, mem

ber 1

1.66

0.02

801.

210.

5996

1440

543_

atD

9300

36F2

2Rik

RIK

EN c

DN

A D

9300

36F2

2 ge

ne1.

660.

0428

−1.02

0.94

8214

4331

5_at

Dm

dD

ystr

ophi

n1.

660.

0103

−2.

450.

0492

1456

351_

atBr

d8br

omod

omai

n co

ntai

ning

81.

660.

0005

1.67

0.00

0314

4648

1_at

Apb

b2A

myl

oid

beta

(A4)

pre

curs

or p

rote

in-b

indi

ng, f

amily

B, m

embe

r 21.

680.

0279

1.06

0.83

85

Molecular Vision 2009; 15:2720-2739 <http://www.molvis.org/molvis/v15/a288> © 2009 Molecular Vision

2729

TAB

LE 3

. CO

NTI

NU

ED.

Aff

ymet

rix

IDG

ene

sym

bol

Gen

e tit

leFC

(wt/p

42 v

shm

/p42

)

p-va

lue

(wt/

p42

vs h

m/

p42)

FC (w

t/p30

vs

hm/p

30)

p-va

lue

(wt/

p30

vs h

m/

p30)

1458

263_

atC

ugbp

2C

UG

trip

let r

epea

t, R

NA

bin

ding

pro

tein

21.

740.

0041

−1.10

0.81

7614

4006

7_at

Nca

m1

Neu

ral c

ell a

dhes

ion

mol

ecul

e 1

1.75

0.01

711.

030.

9102

1444

488_

atC

adps

Ca<

2+>d

epen

dent

act

ivat

or p

rote

in fo

r sec

retio

n1.

760.

0054

1.01

0.98

0114

4133

0_at

Crb

1cr

umbs

hom

olog

1 (D

roso

phila

)1.

990.

0214

−1.12

0.41

6314

2923

9_a_

atSt

ard4

StAR

-rel

ated

lipi

d tr

ansf

er (S

TART

) dom

ain

cont

aini

ng 4

2.18

0.00

1991

1.79

0.02

9514

5169

2_at

Tmco

6tr

ansm

embr

ane

and

coile

d-co

il do

mai

ns 6

2.73

0.00

024.

100.

000

1449

548_

atEf

nb2

ephr

in B

2−1.58

0.04

36−1.05

0.82

9114

4952

7_at

Pcdh

b7pr

otoc

adhe

rin

beta

7−

1.64

0.00

16−

1.58

0.01

5514

2264

0_at

Pcdh

b9pr

otoc

adhe

rin

beta

9−1

7.48

0.00

00−1

4.34

0.00

00U

nkno

wn

(43)

1454

397_

at46

3241

8H02

Rik

RIK

EN c

DN

A 4

6324

18H

02 g

ene

1.50

0.00

66−1.05

0.87

2514

3271

3_at

6430

709C

05R

ikR

IKEN

cD

NA

643

0709

C05

gen

e1.

510.

0031

−1.05

0.89

2314

4250

9_at

Evi5

Ecot

ropi

c vi

ral i

nteg

ratio

n si

te 5

1.52

0.01

56−1.03

0.90

0514

2990

0_at

5330

406M

23R

ikR

IKEN

cD

NA

533

0406

M23

gen

e1.

520.

0119

1.12

0.80

0214

4465

1_at

LOC

5530

89hy

poth

etic

al L

OC

5530

891.

530.

0230

−1.42

0.37

1014

5940

9_at

Ccd

c109

aco

iled-

coil

dom

ain

cont

aini

ng 1

09A

1.53

0.03

371.

010.

9616

1440

570_

atLh

fpl3

Lipo

ma

HM

GIC

fusi

on p

artn

er-li

ke 3

1.53

0.01

0572

1.29

0.44

4014

3019

5_at

2810

043O

03R

ikR

IKEN

cD

NA

281

0043

O03

gen

e1.

540.

0153

−1.16

0.75

6414

3878

8_at

D5W

su15

2eD

NA

segm

ent,

Chr

5, W

ayne

Sta

te U

nive

rsity

152

1.54

0.00

601.

150.

7128

1444

137_

atA

4301

08G

06R

ikR

IKEN

cD

NA

A43

0108

G06

gen

e1.

540.

0017

1.03

0.86

7114

5778

1_at

Kcn

q1ot

1K

CN

Q1

over

lapp

ing

trans

crip

t 11.

540.

0013

−1.00

0.99

9314

3009

6_at

2900

017F

05R

ikR

IKEN

cD

NA

290

0017

F05

gene

1.56

0.01

601.

140.

5974

1443

088_

at99

3003

1P18

Rik

RIK

EN c

DN

A 9

9300

31P1

8 ge

ne1.

560.

0026

1.16

0.73

2614

5870

6_at

2610

035D

17R

ikR

IKEN

cD

NA

261

0035

D17

gen

e1.

560.

0048

−1.08

0.59

9214

5370

6_at

2900

042A

17R

ikR

IKEN

cD

NA

290

0042

A17

gen

e1.

570.

0272

1.02

0.93

7314

4348

9_at

Vps

13b

Vac

uola

r pro

tein

sorti

ng 1

3B (y

east

)1.

570.

0012

1.01

0.95

7514

4060

4_at

8030

494B

02R

ikR

iken

cD

NA

803

0494

B02

gen

e1.

570.

0255

−1.35

0.25

0714

4444

5_at

C77

648

expr

esse

d se

quen

ce C

7764

81.

570.

0475

−1.10

0.63

4314

3383

7_at

8430

408G

22R

ikR

IKEN

cD

NA

843

0408

G22

gen

e1.

570.

0226

−1.00

0.99

4314

5750

8_at

C43

0003

N24

Rik

RIK

EN c

DN

A C

4300

03N

24 g

ene

1.60

0.00

22−1.34

0.39

7414

5455

8_at

5430

416B

10R

ikR

IKEN

cD

NA

543

0416

B10

gen

e1.

600.

0256

−1.07

0.86

6514

4089

2_at

BC

0176

47C

DN

A se

quen

ce B

C01

7647

1.63

0.04

03−1.06

0.85

7214

4146

7_at

Tspa

n5Te

trasp

anin

51.

640.

0438

−1.10

0.66

5814

6010

1_at

NR

XN

3N

eure

xin

31.

670.

0353

−1.05

0.91

4414

4410

9_at

C13

0009

A20

Rik

RIK

EN c

DN

A C

1300

09A

20 g

ene

1.67

0.01

091.

010.

9683

1454

424_

at26

1004

0L17

Rik

RIK

EN c

DN

A 2

6100

40L1

7 ge

ne1.

680.

0058

1.06

0.86

9214

3326

6_at

2810

416A

17R

ikR

IKEN

cD

NA

281

0416

A17

gen

e1.

660.

0005

−1.14

0.76

6014

4256

1_at

Mam

dc1

MA

M d

omai

n co

ntai

ning

11.

690.

0202

−1.06

0.90

4014

5390

6_at

Med

13l

med

iato

r com

plex

subu

nit 1

3-lik

e1.

690.

0010

1.02

0.94

1514

4320

1_at

Gpc

6G

lypi

can

61.

700.

0288

−1.20

0.65

94

Molecular Vision 2009; 15:2720-2739 <http://www.molvis.org/molvis/v15/a288> © 2009 Molecular Vision

2730

TAB

LE 3

. CO

NTI

NU

ED.

Aff

ymet

rix

IDG

ene

sym

bol

Gen

e tit

leFC

(wt/p

42 v

shm

/p42

)

p-va

lue

(wt/

p42

vs h

m/

p42)

FC (w

t/p30

vs

hm/p

30)

p-va

lue

(wt/

p30

vs h

m/

p30)

1458

309_

atD

ip2c

disc

o-in

tera

ctin

g pr

otei

n 2

hom

olog

C1.

730.

0250

−1.04

0.87

9414

5850

5_at

LOC

5529

01hy

poth

etic

al L

OC

5529

011.

750.

0206

−1.20

0.56

9314

2997

7_at

9030

425L

15R

ikR

IKEN

cD

NA

903

0425

L15

gene

1.76

0.00

68−1.07

0.92

0214

4051

3_at

C80

258

expr

esse

d se

quen

ce C

8025

81.

790.

0100

−1.20

0.63

8414

2987

0_at

C63

0040

K21

Rik

RIK

EN c

DN

A C

6300

40K

21 g

ene

1.79

0.00

29−1.01

0.97

9714

4610

2_at

D9E

rtd29

2eD

NA

segm

ent,

Chr

9, E

RA

TO D

oi 2

92, e

xpre

ssed

1.81

0.00

28−1.23

0.27

1114

5877

9_at

8030

445P

17R

ikR

IKEN

cD

NA

803

0445

P17

gene

1.83

0.01

54−1.17

0.76

3599

1453

897_

atC

0300

14A

21R

ikR

IKEN

cD

NA

C03

0014

A21

gen

e1.

850.

0025

−1.46

0.26

5201

1446

606_

atLO

C62

5175

hypo

thet

ical

pro

tein

A63

0054

D14

1.86

0.00

621.

300.

1872

2914

3275

7_at

2900

011L

18R

ikR

IKEN

cD

NA

290

0011

L18

gene

1.90

0.02

321.

300.

6230

9614

4123

1_at

LOC

1000

4201

6hy

poth

etic

al p

rote

in L

OC

1000

4201

61.

920.

0002

−1.06

0.84

0764

1439

086_

atA

9300

09L0

7Rik

RIK

EN c

DN

A A

9300

09L0

7 ge

ne−1.58

0.02

941.

190.

3401

514

2890

9_at

A130

040M

12Ri

kRI

KEN

cD

NA

A130

040M

12 g

ene

−1.

620.

0368

−1.

900.

0001

Not

Ann

otat

ed (1

2)14

4679

9_at

1.50

0.00

30−1.10

0.82

2414

4327

1_at

1.51

0.03

84−1.46

0.31

0114

4174

0_at

1.52

0.02

84−1.22

0.55

9314

4352

6_at

1.52

0.00

621.

030.

9444

1458

077_

at1.

550.

0148

−1.01

0.97

5814

4574

0_at

1.56

0.04

56−1.00

0.99

9814

3540

9_at

1.61

0.02

201.

590.

0878

1439

999_

at1.

630.

0223

−1.11

0.71

3314

4374

4_at

1.72

0.01

28−1.14

0.71

4814

4462

2_at

1.82

0.02

24−1.02

0.95

3914

5747

9_at

1.95

0.00

60−1.00

0.98

1514

5959

5_at

2.18

0.00

191.

070.

8169

Show

n ar

e A

ffym

etrix

ID, g

ene

sym

bol,

gene

title

, fol

d ch

ange

(FC

) and

p-v

alue

s tha

t ind

icat

e th

e di

ffer

ence

s in

gene

exp

ress

ion

leve

ls b

etw

een

the

wild

-type

and

the

hom

ozyg

ous

Egr-

1 k

nock

out m

ice

both

at t

he a

ge o

f 42

days

and

30

days

. Gen

es w

ere

sorte

d af

ter G

O a

nnot

atio

ns. G

enes

that

wer

e si

gnifi

cant

ly c

hang

ed (w

itha

FC >

1.5

and

p-v

alue

< 0

.05)

in b

oth

the

42 d

ays

old

mic

e an

d th

e 30

day

s ol

d m

ice

are

show

n in

ital

ics

and

are

unde

rline

d. T

he g

ene

that

sho

wed

the

high

est

diff

eren

ces i

n m

RN

A e

xpre

ssio

n le

vels

bet

wee

n th

e ho

moz

ygou

s and

the

wild

-type

mic

e (P

cdhb

9 [s

how

n in

bol

d]) w

as fu

rther

inve

stig

ated

usi

ng re

al-ti

me

PCR

.

Molecular Vision 2009; 15:2720-2739 <http://www.molvis.org/molvis/v15/a288> © 2009 Molecular Vision

2731

the knockout mice (wt/30 versus hm/30: 14-fold lowerexpressed in the homozygous mice and wt/42 versus hm/42:17-fold lower expressed) was tested with real-time RT–PCR.The changes in mRNA expression levels of four of the genescould be validated (Figure 2). Three of them were chosenbased on network C (Narf, Ogdh, and Selenbp1) and the genewhich showed the biggest difference in mRNA expressionlevels between wild-type and knockout mice, Pcdhb9, wasalso confirmed.

The relative signal intensities obtained by microarrayanalysis (GC-RMA based, shown are the log transformedmean relative signal intensities ±SD, n=4 each) of all fourgroups tested are shown in Figure 2, upper graphs. The lowergraphs shows the mean MNE-values ±SD obtained by real-time RT–PCR (n=4 each). The results of the ANOVA isindicated in the header of the figures for each gene and thefold-changes (FC) and p values (p) of the un-paired Student'st-tests as post-hoc tests for the comparisons between the 30days old mice and the 42 days old mice are shown within thegraphs.

Note that the comparison of the Pcdhb9 expression is notbetween the two age groups, but between homozygousknockout mice and wild-type mice at both time points.

Nuclear prelamin A recognition factor (Narf) was lowerexpressed in the 42 days old homozygous mice, compared tothe 30 days old mice in both the microarray analysis (hm/42versus hm/30: −1.65 fold, p<0.001) and the real-time PCRexperiment (−1.53 fold, p<0.05). The mRNA expression didnot differ in the wild-type mice over time in either experiment.

Also Oxoglutarate dehydrogenase (Ogdh) was differentonly in the homozygous Egr-1 knockout mice and showed nochanges in mRNA expression levels over time in the wild-type. Post-hoc analysis confirmed that mRNA expressionlevels in the 30 day-old homozygous mice was significantlylower, compared to 42 day-old animals (hm/30 versus hm/42:1.69-fold, with p<0.01 in the MA experiment and 1.51 foldwith p<0.01 in the PCR experiment).

The mRNA expression levels of selenium binding protein1 (Selenbp1) were higher in the 30 days old knockout mice,compared to the 42 days old homozygous mice (hm/42 versushm/30: −2.52 fold with p<0.001 in the MA experiment and

TABLE 4. PATHWAYS IDENTIFIED IN THE WILD-TYPE AND THE HOMOZYGOUS EGR-1 KNOCKOUT MICE BY INGENUITY PATHWAY ANALYSIS SOFTWARE(WT/30 VERSUS WT/42 AND HM/30 VERSUS HM/42).

Pathways detected in wild-type mice (30 days versus 42 days, 54 differentially expressed genes):

Pathway MoleculesArachidonic Acid Metabolism Gpx3, Cyp2d6, PtgdsAcute Phase Response Signaling Ttr, Tf, A2mFXR/RXR Activation Pon1, Cyp27a1Pathways detected in Egr-1 knockout-mice (30 days versus 42 days, 215 differentially expressed genes):

Pathway MoleculesSynaptic Long-term Depression Plcb4, Itpr2, Igf1r, Gna13, Prkca, Prkcb1Synaptic Long-term Potentiation Plcb4, Itpr2, Atf2, Prkca, Prkcb1Huntington's Disease Signaling Gnb1, Plcb4, Igf1r, Zdhhc17, Atf2, Prkca, Prkcb1PDGF Signaling Crkl, Abl1, Prkca, Prkcb1Chemokine Signaling Plcb4, Ppp1r12b, Prkca, Prkcb1VDR/RXR Activation Igfbp3, Ncoa1, Prkca, Prkcb1FGF Signaling Crkl, Fgf11, Atf2, PrkcaEphrin Receptor Signaling Gnb1, Crkl, Abl1, Gna13, Atf2, Rapgef1Axonal Guidance Signaling Gnb1, Plcb4, Pfn1, Crkl, Abl1, Gna13, Prkca, Prkcb1Lysine Degradation Setdb1, Nsd1, OgdhActin Cytoskeleton Signaling Pfn1, Diaph3, Crkl, Ppp1r12b, Fgf11, Gna13Circadian Rhythm Signaling Arntl, Atf2G-Protein Coupled Receptor Signaling Pde8a, Plcb4, Atf2, Prkca, Prkcb1Integrin Signaling Tspan5, Crkl, Ppp1r12b, Abl1, Rapgef1Neuregulin Signaling Crkl, Prkca, Prkcb1SAPK/JNK Signaling Gnb1, Crkl, Gna13, Atf2ERK/MAPK Signaling Crkl, Atf2, Rapgef1, Prkca, Prkcb1

Shown are the descriptions of the pathways and the molecules involved in both the wild-type and the homozygous Egr-1 knockoutmice that changed their mRNA expression patterns over the investigated period of time (p30 versus p42) based on IngenuityPathway Analysis Software.

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−2.23 fold with p<0.001 in the PCR experiment) and again,no such changes were observed in the wild-type.

In case of protocadherin-beta 9 (Pcdhb9), mRNAexpression levels did not change with age, but were muchlower in the knockout mice. Real-time PCR detected afivefold decline in the expression of Pcdhb9 in knockout mice,both at p30 and at p42, whereas the microarray analysisdetermined an even larger decline (hm/30 versus wt/30: −14fold and hm/42 versus wt/42: −17 fold, with p<0.001 each).

DISCUSSIONEarlier studies suggested that Egr-1 might play a role in eyegrowth regulation in chicks [13] and mice [20,35]. The currentstudy attempted to determine which genes were differentiallyregulated in Egr-1 knockout mice at two differentdevelopmental stages. Since Egr-1 knockout mice had longereyes at age 42 days, compared to wild-type mice, but not atday 30, it was hoped that a correlation could be found between

the expression of certain genes and changes in eye growth.Furthermore, it was hoped that some of the changes mightoccur that relate to genes or factors that are already known tobe involved in the regulation of axial eye growth in animalmodels.

The analysis of retinal gene expression in homozygousEgr-1 knockout mice and wild-type mice at different ages(p30 and p42) provided a huge amount of data. Depending onthe comparisons (wild-type versus homozygous, 30 daysversus 42 days), different information about gene expressionwere obtained and different conclusions could be drawn. Thefocus in this paper was on changes that occur in homozygousEgr-1 knockout mice and wild-type mice between 30 and 42days (wt/30 versus wt/42 and hm/30 versus hm/42). Someconsistent changes in mRNA expression patterns were foundin the Egr-1 knockout mice compared to wild-type mice.Whether these genes are directly involved in the temporarily

Figure 2. Genes whose mRNAexpression levels could be validated byreal-time RT–PCR. Mean relativeexpression values ±SD (n=4) obtainedby microarray analysis and meannormalized expression values obtainedby real-time PCR (n=4). The p-values ofthe ANOVA's can be seen in the headingof the figures. The fold-changes and p-values (determined by un-pairedStudent's t-test as post-hoc analysis)were computed for the comparisonbetween 30 days and 42 days oldknockout- and wild-type mice (wt/30versus wt/42 and hm/30 versus. hm/42)and are shown within the figures. Pleasenote that in the case of Pcdhb9, thecomparison is between the knockoutand the wild-type mice at both ages (wt/30 versus. hm/30 and wt42 versus hm/42).

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enhanced axial eye growth, or represent just epiphenomena,remains undefined and needs to be determined by furtherstudies.Microarray:

Gene expression changes over time (p30 versus p42)—It was not surprising that in wild-type mice only 54 genesshowed differential mRNA expression over time (wt/30versus wt/42, see Appendix 1), since the mouse retina isgenerally considered to be mature at the age of about p21[36]. In the homozygous Egr-1 knockout mice, 215 genes(roughly four times as many) were differently expressed (hm/30 versus hm/42, Appendix 2). Eight genes showed similarchanges in mRNA expression in both the homozygous and thewild-type Egr-1 knockout mice (shown in italics andunderlined in Appendix 1 and Appendix 2). They likelyencode for proteins that are involved in normal retinal functionand are not related to the abnormal ocular growth in theknockout mice.

The average mean fold-changes in this study were1.48±0.41, which is in line with findings from othermicroarray studies [16,19,37,38]. It has to be kept in mind thatglobal gene expression measurements in a heterogeneoustissue like the retina are difficult to interpret since largechanges in mRNA expression in a subset of cells might beobscured by changes in the opposite direction in a different,perhaps even more abundant population of cells. For the samereason, large changes in mRNA expression in a rare cell typemight go unnoticed because they generate only a smallfraction of the total mRNA. In the future, quantitativeimmunohistochemistry might help to detect changes in geneproducts which are localized to certain cells types.

Differences in gene expression in Egr-1 knockout miceand their wild-type: Thirteen genes were differentiallyexpressed in knockout mice, compared to wild-type, at bothages tested (Table 2 and Table 3: genes are shown in italicsand underlined). Except for dystrophin (DMD), the regulationof those genes was in the same direction at both ages. Thesegenes are therefore most probably target genes of Egr-1 andare not directly related to the developmental changes observedin these mice. Nevertheless, these thirteen genes are of interestbecause they were not previously described as possible targetsfor the Egr-1 protein. Still, whether the interaction with theEgr-1 protein is direct or indirect, has yet to be experimentallyvalidated using other techniques like chromatinimmunoprecipitation. The Egr-1 knockout mice used in ourstudy contain several in-frame stop codons in the Egr-1 codingsequence, upstream of the zinc finger DNA binding domain.Parts of the mRNA sequence of Egr-1 can therefore still betranscribed but the stop-codons lead to the functionalelimination of the protein Egr1. The truncated Egr-1 mRNAscan still bind to the microarray. Upregulation of Egr-1 mRNAitself in the knockout mice can be explained by the fact thatEgr-1 can suppress its own expression, as has been shown intissue culture [39]. A negative feedback mechanism has

already been described elsewhere and can be seen for Nab2(Ngfi-A-binding protein-2) [40]. This protein was massivelydownregulated in Egr-1 knockout mice and is assumed to bea major regulator of Egr-1 function, since it is induced by thesame stimuli that induce Egr-1. Dystrophin (DMD) mRNAexpression was lower at p30 in the homozygous mice,compared to the wild-type mice, and higher at p42. Anenhanced DMD content may therefore be correlated withenhanced axial eye growth. DMD is a plasma membrane-associated cytoskeletal protein of the spectrin superfamily andits absence or functional deficiency is the cause of severaltypes of muscular dystrophies in humans. In some of thesepatients, retinal function is affected as well, as reflected in areduced b-wave in the electroretinograms (ERG). DMDisoforms have been localized to Müller cells andphotoreceptor terminals, so the abnormality in the ERGs ismost likely due to a disturbance of neurotransmission betweenphotoreceptors and ON-bipolar cells [41]. Interestingly, thelack of the dystrophin isoform Dp71 leads to impairedclustering of two Müller glia cell proteins in mice, namely theinwardly rectifying potassium channel Kir4.1 and the waterpore aquaporin 4 (AQP4) [42]. Both Kir4.1 and AQP4 havealready been implicated in the development of myopia andtheir role as a conduit for movement of retinal fluid into thevitreous was suggested [43,44].

Protocadherin beta 9 (Pcdhb9) was the most heavilyregulated gene in this study (wt/30 versus hm/30: 14-folddownregulated and wt/42 versus hm/42: 17-folddownregulated in the homozygous Egr-1 knockout mice).Protocadherins are calcium-dependent cell–cell adhesionmolecules. Their specific functions are unknown, but theymost likely play a critical role in the establishment andfunction of specific cell-cell neural connections [45]. Themassive downregulation of this gene suggests a tight control,either directly by Egr-1, or indirectly by other Egr-1-regulatedgenes.

Ingenuity pathway analysis: The list of genes that weredifferentially expressed in the Egr-1 knockout mice betweenthe age of 30 and 42 days (hm/30 versus hm/42) was analyzedusing Ingenuity Pathway Analysis Software. Many ubiquitoussignaling pathways in the retina seem to be involved.Therefore, it is difficult to define those genes that areresponsible for the development of the relative myopia in the42-day-old Egr-1 knockout mice. As can be seen in Figures1A-C, genes that showed altered mRNA expression in thisstudy were not the key regulators in the functional networksproposed by the software. As already mentioned above, thesepathway schemes are based on known interactions betweenmolecules. They provide only suggestions for possibleinteractions. Involvement of the intermediate proteins and co-factors in these pathways is, therefore, possible but notproven. Furthermore, since the changes in mRNA expressionlevels of any gene chosen from network A and B could not be

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validated in all cases, it is difficult to speculate about theinvolvement of these networks as illustrated in Figure 1.

The only data available on visual function in Egr-1knockout mice are from optomotor experiments. They did notshow any differences in contrast sensitivity or spatialresolution to the wild-type [20]. Interestingly, the lack ofEgr-1 does not seem to affect other sensory systems in themouse, like the auditory system. Auditory function wasstudied by evoked brainstem responses (ABR) andotoacoustic emissions (DPOAE), but no differences werefound between wild-types and knockout mice (Dr. LukasRüttiger, Tuebingen Hearing Research Centre [THRC],Tuebingen, Germany, personal communication 2008).Therefore the function of Egr-1 in the regulation of axial eyegrowth seems to be quite specific despite the fact that commonsignaling pathways were affected.Real-time RT–PCR:

Validation of genes—The mRNA expression levels offour out of the ten tested genes could be validated (Figure 2).To be able to compare the relative expression levels asdetermined by microarray analysis (MA) and real-time PCR(PCR) easily, Figure 2 shows the results of MA and PCRexperiments separately for each gene. Non-validated genesare not represented in this figure. The fold-changesdetermined by PCR and MA of Narf, Ogdh, and Selenbp1were very similar whereas for Pcdhb9, the magnitude ofchanges was severely overestimated by MA. Considering thisfact, the involvement of network A and B is morequestionable, since none of the genes tested with real-timePCR could be validated. On the other hand, only three geneswere tested in each network, and others which would havebeen validated upon testing might have been overlooked. Ourprimers were designed to bind to the same part of the sequencethat was detected by the oligonucleotide probes on theAffymetrix chip (to avoid detection of different isoforms) andshould have been appropriate to validate the microarrayresults. The applied false-discovery-rate of 5% thus does notseem to reflect the true errors that are unavoidable in thistechnique. These observations confirm that validation of theresults of microarray analyses by other techniques (in this casesemiquantitative real-time RT–PCR) should be mandatory.

Genes whose transcription could be validated: Thefunctions of the three genes which could be validated withreal-time PCR are described in more detail below:

Narf—The expression of Nuclear prelamin Arecognition factor was found to be lowest in the 42 days oldknockout mice. Prenylation and methylation occurs at the C-terminal end of proteins and was initially believed to beimportant only for membrane attachment. However, anotherrole for prenylation appears to be the mediation of protein–protein interactions [46]. The only nuclear proteins known tobe prenylated in mammalian cells are prelamin A- and B-typelamins. Lamins are fibrous proteins providing structural

function and transcriptional regulation in the cell nucleus, butthe cellular role of both the prenylated prelamin A precursorand Narf, which is known to bind to the farnesylated prelaminA, is unknown and its role in the retina is unclear.

Ogdh—Oxoglutarate dehydrogenase mRNA expressionwas significantly enhanced in 42-day-old knockout mice.Ogdh is a mitochondrial enzyme complex, comprising of threedifferent subunits (Ogdh, Dld, Dlst) that converts 2-oxoglutatate into succinyl-CoA and carbon dioxide in theKrebs cycle. Its reduction in patients with Alzheimer diseasesuggests an altered metabolism of the nervous tissue [47].Unfortunately, no information is available yet for the role ofthis enzyme in the retina, but it could well be that the changesin mRNA expression of this gene reflects changes in themetabolic rate of retinal cells in the homozygous Egr-1knockout mice.

Selenbp1—Egr-1 knockout mice showed a highexpression of Selenbp1 at the age of 30 days, with asubsequent decline. Selenium binding protein has been shownin several studies to be downregulated in cancer [48,49]. Aprotective effect of selenium in preventing maculardegeneration has also been shown [50] and another seleniumtransporter (selenoprotein P) has been found to be upregulatedin the retina of chicks that were treated with either positive ornegative lenses [51]. Selenbp1 expression can be blocked byTGF-β in smooth muscle cells [52] and this protein (TGF-β)has already been implicated in myopia [53,54] and in theregulation of programmed cell death in the retina [55]. Therole of selenium and its associated binding proteins in theretina of Egr-1 knockout mice merits further investigation.

Involvement of network C: Although the genes for whichexpression changes could be validated belong to this network,the involvement of the “key molecules” presented in Figure1C remains speculative. No mRNA expression changes ofe.g., VEGF, cFos, or EGF were found in this study althougholigonucleotides representing mRNAs of VEGF, cFos,EGF, and retinoic acid receptors are present on the Affymetrixchip used in this study. Obviously, the lack of functional Egr-1protein did not affect mRNA expression levels of these genes.

Nevertheless, some of those molecules have already beenproposed to play a role in the regulation of eye growth, or wereshown to change in the retina in response to various opticalstimuli [4-6,13,16,56-62]. The fact that they seem to linkseveral factors that were found to have changed mRNAexpression levels in Egr-1 knockout mice is in line with theidea that these molecules are involved in the regulation of eyegrowth.

Outlook: This study provided a list of genes that appearassociated with Egr-1 signaling and/or altered eye growth inEgr-1 knockout mice. It is difficult to define specific roles tothose genes in eye growth in the Egr-1 knockout mice.Analysis of possibly involved networks show that the lack ofEgr-1 affects common pathways in the retina. Furthermore,

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other important factors (like cFos, VEGF, NfkB) may play arole as well, perhaps partially taking over the role of Egr-1 inthe retina of Egr-1 knockout mice. We believe that themicroarray analysis is a powerful tool to detect NEWcandidates, rather than to look at the “usual suspects.” It is,however, difficult to use the microarray technology inheterogeneous tissue like retina. There are now advancedmethods available to focus on certain cell types (e.g., lasercapture microdissection of fluorescent-activated cell sorting).Nevertheless, some interesting genes were found in this study.In addition, several direct or indirect target genes of Egr-1could be identified, including the most prominently regulatedgene protocadherin-beta 9. The list of differentially expressedgenes is accessible online in the Gene Expression Omnibus(GEO) Database and might be useful not only to researchersin the field of myopia.

ACKNOWLEDGMENTSThis study was supported by the German ResearchFoundation, DFG Sch518/13–1. We thank Prof. Dr.Wissinger of the Molecular Genetics Department of theOpthalmic Research Institute in Tuebingen for sequencing ofour samples.

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Appendix 1. List of differentially expressed genes in the wild-type micebetween p30 and p42 days (wt/30 versus wt/42).

To access the data, click or select the words “Appendix1.” This will initiate the download of a (pdf) archive thatcontains the file. Affymetrix ID, gene symbol, gene title, foldchange (FC) and p-values of both the wild-type mice and thehomozygous Egr-1 knockout mice are shown. Genes were

sorted after GO annotations. Genes that were significantlychanged (with a FC > 1.5 and p-value < 0.05) in both the wild-type mice and the homozygous Egr-1 knockout mice areshown in italics and are underlined.

Appendix 2. List of genes that were differentially expressed in thehomozygous Egr-1 knockout mice between p30 and p42 (hm/30 versus hm/42).

To access the data, click or select the words “Appendix2.” This will initiate the download of an Excel (.xls) file thatcontains the file.Affymetrix ID, gene symbol, gene title, foldchange (FC), and p-values of both the homozygous Egr-1knockout mice and the wild-type mice are shown. Genes were

sorted after GO annotations. Genes that were significantlychanged (with a FC >1.5 and p-value <0.05) in both thehomozygous wild-type mice and the Egr-1 knockout mice areshown in italics and are underlined. Genes that were chosenfor real-time RT–PCR validation are shown in bold.

Molecular Vision 2009; 15:2720-2739 <http://www.molvis.org/molvis/v15/a288> © 2009 Molecular Vision

The print version of this article was created on 7 December 2009. This reflects all typographical corrections and errata to thearticle through that date. Details of any changes may be found in the online version of the article.

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