Epidermal Growth Factor Receptor Expression Regulates...

Epidermal Growth Factor Receptor ExpressionRegulates Proliferation in the Postnatal Rat RetinaJENNIE L. CLOSE,1 JANICE LIU,2 BURAK GUMUSCU,3 AND THOMAS A. REH1*1Department of Biological Structure, Graduate Program in Neurobiology and Behavior, Health Sciences Center,University of Washington School of Medicine, Seattle, Washington2Primate Research Center, Health Sciences Center, University of Washington, Seattle, Washington3Department of Pediatrics, Nebraska Medical Center, University of Nebraska, Omaha, Nebraska

KEY WORDSM€uller; neurogenesis; regeneration

ABSTRACTEpidermal growth factor (EGF) is known to promote prolif-eration of both retinal progenitors and Muller glia in vitro,but several questions remain concerning an in vivo role forthis factor. In this study, we investigated whether the EGFreceptor (EGFR) is necessary for the maintenance of nor-mal levels of progenitor and Muller glial proliferation invivo. Here, we show that (1) mice with homozygous deletionof the Egfr gene have reduced proliferation in late stages ofretinal histogenesis, (2) EGF is mitogenic for M€uller glia invivo during the first two postnatal weeks in the rodent ret-ina, (3) the effectiveness of EGF as a M€uller glial mitogendeclines in parallel with the decline in EGFR expression asthe retina matures, and (4) following damage to the retinafrom continuous light exposure, EGFR expression is up-regulated in M€uller glia to levels close to those in the neo-natal retina, resulting in a renewed mitotic response toEGF. Together with previous results from other studies,these data indicate that the downregulation of a growthfactor receptor is one mechanism by which glial cellsmaintain mitotic quiescence in the mature nervous sys-tem. VVC 2006 Wiley-Liss, Inc.

INTRODUCTION

The proper regulation of retinal proliferation is impor-tant because the balance of cell numbers and types iscritical to mature retinal function. This regulation isachieved through the action of both cytostatic and mi-totic signaling molecules. For example, we have recentlyshown that transforming growth factor b 2 (TGFb2) isexpressed in the postnatal retina, and inhibits progeni-tor and glial proliferation in vitro and in vivo (Closeet al., 2005). Fibroblast growth factor (FGF), epidermalgrowth factor (EGF), transforming growth factor a(TGFa), transforming growth factor b 3 (TGFb3), andsonic hedgehog (Shh) have all previously been shown topromote retinal progenitor and/or glial proliferation invitro (Anchan and Reh, 1995; Anchan et al., 1991; Jen-sen and Wallace, 1997; Levine et al., 1997; Lillien andCepko, 1992; Moshiri and Reh, 2004; Wang et al., 2002).Some of these factors, such as FGF and Shh, have alsoproven to be mitogenic in vivo (Fischer and Reh, 2002;Fischer et al., 2002a,b; Wang et al., 2002; Wang et al.,2005). While previous cell culture studies have shown

that postnatal retinal cells are more responsive to themitogenic effects of EGF and/or TGFa than their embry-onic counterparts, the effects of the EGF signaling path-way on postnatal retinal proliferation have not beeninvestigated in vivo.

Following the normal period of histogenesis, neural pro-genitors and glial cells do not proliferate in most regionsof the central nervous system (CNS), including the retina(see Close et al., 2005 for review). The maintenance of mi-totic quiescence is likely to be under tight regulation inthe mature nervous system, but little is known about thefactors that inhibit glial proliferation. Previous studieshave shown that when Muller glia are dissociated andplaced in tissue culture, they respond to some of the samegrowth factors as retinal progenitors (Milenkovic et al.,2004; Reichelt et al., 1989; Roque et al., 1992; Schererand Schnitzer, 1994). Thus, it is possible that these mito-gens become limiting in their supply as the retinamatures. Alternatively, the receptors for growth factorsmay become down-regulated as progenitors cease prolif-eration. Although M€uller glia do not normally proliferatein adult animals, these cells can be stimulated to re-enterthe mitotic cycle in posthatch chicks, by treatment witheither neurotoxins or co-injections of FGF and insulin(Fischer and Reh, 2001, 2002; Fischer et al., 2002b); sug-gesting that the factors themselves may be limiting. Stu-dies of the rodent retina suggest that mammalian M€ullerglia may be more resistant to cell cycle entry in responseto damage or growth factor treatments. For instance,treatment with neurotoxins, including ouabain or NMDA,results in only a small number of M€uller glia entering thecell cycle in the adult rodent retina (Dyer and Cepko,2000; Ooto et al., 2004). In experiments with posthatchchickens and rats, some of the proliferating M€uller gliagenerate cells that express markers and morphology ofneurons (Fischer and Reh, 2002; Fischer et al., 2002a,b;Ooto et al., 2004), indicating that M€uller glial re-entryinto the cell cycle may initiate a regenerative process.

Grant sponsor: NIH; Grant numbers: RO1 EY13475 and RO1 NS28308; Grantsponsor: NIH Institutional Neurobiology Training; Grant number: T32 GM07108.

*Correspondence to: Thomas A. Reh, Department of Biological Structure, Neuro-biology and Behavior Program, University of Washington, Seattle, Washington98195, USA. E-mail: [email protected]

Received 10 January 2006; Accepted 19 April 2006

DOI 10.1002/glia.20361

Published online 18 May 2006 in Wiley InterScience (www.interscience.wiley.com).

GLIA 54:94–104 (2006)

VVC 2006 Wiley-Liss, Inc.

Thus, studies of the factors that stimulate M€uller glialproliferation in the mammalian retina may contribute tothe understanding of how retinas may be regeneratedafter damage or disease.

In this study, we have examined the role of epidermalgrowth factor receptor (EGFR) in the regulation of prolif-eration in the postnatal retina. Here, we report thatEGFR is required to maintain normal levels of prolifera-tion in the postnatal retina. We also find that EGF is aneffective mitogen for M€uller glia during the first two post-natal weeks. As the retina matures, this mitogenicresponse declines, with a coincident reduction in the ex-pression of EGFR. However, after light-induced damagein the adult rat retina, EGFR expression increases, re-sulting in a return of the mitotic response of M€uller gliato EGF. These data suggest that one mechanism by whichcell proliferation may be regulated in glia is throughchanges in growth factor receptor expression.

MATERIALS AND METHODSAnimals

All animals used in this study were treated accordingto guidelines of the University of Washington IACUC.Rats were purchased from Charles River Laboratories.Rats and mice were killed by CO2 over-anesthesia andcervical dislocation. Eyes were removed and retinas weredissected in Hank’s buffered salt solution. Mice heterozy-gous for Egfr deletion (Egfrtm1Mag) were obtained fromJackson laboratories (Threadgill et al., 1995). They weremaintained on the CD-1 background and bred as hetero-zygotes. Retinas from littermate wild type animals werecompared with Egfrtm1Mag Egfrtm1Mag mouse retinas.

Injections

Intraocular injections were performed as described pre-viously (Close et al., 2005) for rats at postnatal day 10(P10). For injections made in animals from postnatal day14 to adulthood, rats were anesthetized using ketamine/xylazine and proparacaine topical anesthetic was appliedto the eyes prior to injection. A scalpel blade was used tomake a small incision at the nasal corneal/scleral junc-tion, and growth factors were delivered in 5 lL volumesusing a Hamilton Syringe with a 30.5 G needle attached.Growth factor injections were made using a solution com-posed of sterile phosphate-buffered saline (PBS) and 0.2%bovine serum albumin (BSA) for control, or human recom-binant EGF (R&D systems). Following injection, topicalantibiotic eye lubricant was applied to both eyes to pre-vent drying and infection. BrdU injections were deliveredintra-peritoneally at a dose of 50 mg/kg.

Light Treatment

Albino rats over the age of 14 days were subjected toconstant light from a 15 Watt fluorescent, full spectrumbulb positioned directly over the cage for 7 days. Food,

water, and temperature of the environs were monitoreddaily.

Genotyping

Egfr2/2 homozygous mice were genotyped by polymer-ase chain reaction (PCR) with the forward primer 50-CTC CTC TTC TTC CCG CAC TGT G-30, the reverseprimer 50-CAT TGG TTG TGG CAG CAG TCA CTG-30,the nested forward primer 50-GTC TGT CTC GGA TTAATC CCG GAG-30, and the nested reverse primer 50-CTG CTC GGA TGG CTC TGT AAG TCC-30 for thedetermination of the Egfr gene; the forward primer 50-CTG GCG TTA CCC AAC TTA ATC GC-30, the reverseprimer 50-GTA GGT AGT CAC GCA ACT CGC CG-30,the nested forward primer 50-CGA TCG CCC TTC CCAACA GTT GC-30, and the nested reverse primer 50-CAACGA GAC GTC ACG GAA AAT GC-30 for the identifica-tion of the LacZ gene, using mouse brain or mouse tailprepared with TRIZOL (Invitrogen) following the manu-facturer’s protocol.

Immunohistochemistry

Tissues were rinsed in PBS, fixed in 4% paraformalde-hyde, 4% sucrose in PBS for 1 h, and sunk in 30% su-crose prior to cryosectioning. Cryosections were mountedon Superfrost slides (VWR). Slides and cells stained withanti-BrdU were incubated for 10 min in 4N HCL priorto blocking, and then washed twice with PBS. All block-ing steps were performed at room temperature for 1.5 hin 0.3% TritonX-100 (TX-100) and 5% goat serum inPBS except for those treated with the sheep anti-EGFRantibody, in which fetal bovine serum was substitutedfor goat serum in the blocking step. All primary anti-body staining procedures were performed overnight atroom temperature in 0.3% TX-100 in PBS, except foranti-EGFR incubations, which were performed overnightat 4�C. Following primary incubation, 4 3 15 min wash-es in PBS were performed. Secondary antibody incuba-tions were performed for 1 h at room temperature in0.3% TX-100 in PBS, followed by 4 3 15 min washes inPBS. Sections or coverslips were then allowed to dry,and mounted in Fluoromount-G (Southern Biotechnol-ogy Associates) medium for microscopy. Antibodies usedin this study include mouse anti-BrdU (1:150; G3G4 De-velopmental Studies Hybridoma Bank), rat anti-BrdU(1:100; Accurate), mouse anti-Nestin (1:80; Developmen-tal Studies Hybridoma Bank), rabbit antibovine cellularretinaldehyde binding protein (CRALBP) (1:500; UW55,gift of Jack Saari, University of Washington), rabbitanti-Phospho Histone H3 (1:750; Upstate), sheep anti-EGFR (1:50; Upstate), goat antirabbit Alexa 568 (1:500;Molecular Probes), goat antirabbit Alexa 488 (1:500; Mo-lecular Probes), goat antimouse Alexa 488 (1:500; Molec-ular Probes), goat antimouse Alexa 568 (1:500; Molecu-lar Probes), goat antirat 488 (1:500; Molecular Probes),donkey antisheep Alexa 568 (1:500; Molecular Probes),and goat antiguinea pig Cy-3 (1:700; Chemicon).

95EGF RECEPTOR EXPRESSION

GLIA DOI 10.1002/glia

Quantitative RT PCR

Quantitative RT-PCR was performed as previouslydescribed (Kubota et al., 2004) using an Opticon monitorfrom MJ Research and SYBR Green PCR Master Mix(Applied Biosystems). RNA samples were taken fromthree different individuals at the ages indicated. TotalRNA was collected using TRIZOL (Invitrogen) extrac-tion, cleaned, and DNase treated using RNeasy Mini-Kit(Qiagen), and then quantified by spectrophotometry. Onelg of RNA was used for reverse transcription, usingoligo-dTs and Superscript II reverse transcriptase (Stra-tagene). The resulting cDNA samples from each indivi-dual were run in triplicate for each primer set. Forquantification, the cycle at which a given sample/primercombination reached log-phase was noted, and all sam-ples were normalized to GAPDH levels. Primers usedwere designed to amplify 200 bp of the 30 end of each ratgene examined using Primer3 (MIT), and were obtainedfrom Invitrogen. The sequences of each primer (50-30) areas follows:

GAPDH, 50: AAG GTC ATC CCA GAG CTG AAGAPDH, 30: GTC CTC AGT GTA GCC CAG GAEGFR, 50: ACT CTG ACG GGC TTT GTC ACEGFR, 30: CAA GCG CCA TAG GTC TGT TT

Efficiency curves were performed to determine thenumber of cycles difference represented by a two-fold dilu-tion of template. Template cDNA expressing all ligandsand receptors (P10) was diluted 8-, 16-, 32-, and 64-foldand the average difference for all primers between eachtwofold dilution was found to be one. Therefore, a one-cycle difference represents a twofold dilution in theseexperiments. Error bars shown represent standard errorof the mean of three trials.

Western Blotting

Western blots were performed as previously described(Close et al., 2005), using the following antibodies: sheepanti-EGFR (1:1,000; Upstate), mouse anti-p27 (1:1,000;Transduction Labs), mouse anti-b-Actin (1:5,000; Abcam),goat antimouse horseradish peroxidase (HRP) conjugate(1:20,000; BioRad), and rabbit anti-sheep HRP conjugate(1:20,000). Bands were quantified by scanning the blots.The values were compared with and adjusted for thelevels of b-actin protein for loading control, and thenthese adjusted EGFR expression levels were normalizedto P4 protein levels, and averaged over two separatetrials with two separate protein sample series.

RESULTSEGFR-Deficient Mice Demonstrate a Decrease

in Retinal Proliferation

Previous studies have shown that EGF and relatedligands can stimulate proliferation of retinal progenitors

in vitro (Anchan and Reh, 1995; Anchan et al., 1991;Lillien, 1995; Lillien and Cepko, 1992; Lillien and Wan-cio, 1998). To determine whether EGFR is required tomaintain normal levels of proliferation in vivo, we ana-lyzed the retinas of neonatal mice deficient in the recep-tor (Threadgill et al., 1995). These mice are born at nor-mal Mendelian ratios, and there is no evidence for em-bryonic lethality on the CD-1 background. We genotypedeach litter, sacrificed animals at various postnatal ages,and analyzed retinal sections for differences in mitotic ac-tivity when compared with littermate wild type animals.Mitotic activity was assessed using phospho-Histone-H3immunoreactivity (PH3), a marker of M-phase cells.

In both the wild type and Egfr deficient mouse reti-nas, the PH3 cells were largely confined to the outer(scleral) surface of the retina, equivalent to the ventricu-lar surface of the CNS. In the retinas of newborn mice,there was no obvious difference between the wild typeand Egfr deficient mice in either central or peripheralregions of the retina (Fig. 1A). By postnatal day 3, how-ever, the central retina had fewer PH3-labeled cells inthe Egfr2/2 mouse retina (Figs. 1A,B) although the pe-ripheral regions of the retinas from the two genotypeshad similar numbers of PH3 immunoreactive cells. Bypostnatal day 6, the proliferation of progenitors in cen-tral regions of retina is finished; however, in the periph-eral retinal regions, many PH3 cells are still present(Fig. 1A). We found a significant difference in the num-ber of PH3 cells in peripheral retina between the wildtype and Egfr deficient mice at P6 (Figs. 1A,C). We alsoexamined the expression of Chx10 in late stages of post-natal histogenesis (day 7). We also found a decline inthe number of Chx10 expressing progenitor cells in theEgfr2/2 mouse retinas (Figs. 1D,E). These data indicatethat while the early stages of progenitor proliferationare not significantly affected by the loss of the Egfr,there is progressively less proliferation in the retinas ofEgfr2/2 mice at later stages of histogenesis.

EGF is a M€uller Glial Mitogen In Vivo

Previous studies have implicated EGF in M€uller glialdifferentiation and proliferation (Lillien, 1995; Milenko-vic et al., 2004; Reichelt et al., 1989; Roque et al., 1992;Scherer and Schnitzer, 1994). To determine whetherEGF promotes M€uller glial proliferation in vivo, wemade injections of EGF in progressively older animalsand analyzed the response to EGF. The proliferation ofmultipotent retinal progenitor cells is complete in therat retina at postnatal day 10 (Close et. al., 2005), andthe M€uller glia have developed characteristics of theirdifferentiated phenotype. Thus, intraocular injections ofEGF at this age allow us to separate effects of EGF onprogenitor cells from those on M€uller glia. We made in-traocular injections of EGF into rat pups starting at P10for two consecutive days, followed immediately by 24 hof BrdU administration. Injections of vehicle alone didnot result in significant M€uller glial proliferation (Figs.2A–C). By contrast, we found that at this stage, EGF

96 CLOSE ET AL.


was a potent M€uller glial mitogen. Many cells were im-munoreactive for both BrdU and the M€uller glial markerCRALBP in the inner nuclear layer of the central retinafollowing intraocular EGF injections (Figs. 2D–I). Somedouble-labeled cells were observed in the outer nuclear

layer as well (Figs. 2G–I, asterisks). When the numberof CRALBP/BrdU double-labeled cells was quantified atthe different doses of EGF, we found that the effect ofEGF was dose dependent (Fig. 2J). Control injections ofPBS and 0.2% BSA (PBS/BSA) resulted in an average of10.8 (63.8) CRALBP/BrdU double-positive cells persquare millimeter of retina, while injections of 250 ngEGF resulted in 383 (691.9) CRALBP/BrdU double-posi-tive cells per square millimeter of retina (Fig. 2G). Injec-tions of 500 ng and 1 lg EGF resulted in 765 (616.8)and 695 (677) CRALBP/BrdU double positive cells persquare millimeter, respectively (Fig. 2J). These dataindicate that EGF is a potent M€uller glial mitogen atpostnatal day 10, and that its effects depend on dosage.

The cyclin-dependent kinase inhibitor (cdki) p27kip1 isan important negative regulator of both retinal progeni-tor and M€uller glial proliferation at the G- to S-phasetransition of the cell cycle (Dyer and Cepko, 2000, 2001;Levine et al., 2000). To determine the effects of EGFinjection on p27kip1 expression, we injected 1 lg EGF fortwo consecutive days starting at P10. Protein wasextracted from treated retinas 4 h after the final EGFinjection. Following a protein assay, we performed Wes-tern blots to determine the levels of p27kip1 expressionin control and EGF-treated retinas. Although p27kip1

expression is robust in control-treated retinas, we findthat in retinas treated with 1 lg EGF, p27kip1 proteinlevels are greatly reduced (Fig. 2K). Thus, one of themolecular mechanisms through which EGF promotesproliferation in M€uller glia may be via the downregula-tion of p27kip1.

To determine whether the mitogenic response to EGFpersists at later ages, we performed intraocular injec-tions of the factor in postnatal day 14 (P14) rats. Ratswere injected with either PBS/BSA for control or 1 lgEGF for two consecutive days, followed by 24 h of BrdUinjections. In vehicle-injected eyes, few or no M€uller gliaenter the cell cycle; very few CRALBP/BrdU double-labeled cells were present in the retina (Figs. 3A–C). InP14 EGF-treated animals, some CRALBP/BrdU double-positive cells are present in the retina (Figs. 3D–F,white arrows), though significantly fewer than in P10injected animals. We found that on average, there are5.33 (60.3) CRALBP/BrdU double-labeled cells per mm2

in P14 control-injected retinas, and 43.26 (614.8) BrdU-positive M€uller glia in P14 EGF-treated retinas (Fig.3G). Thus, the number of BrdU labeled M€uller glia inanimals injected with EGF at P14 was only about atenth of that of animals treated at P10 (P < 0.05, pair-wise comparison Student’s t test). When we made simi-lar injections at P21, only an occasional cell was labeledwith BrdU. Thus, there is a dramatic decline in the mi-togenic response of M€uller glia to EGF as the retinamatures.

EGF Receptor Expression Declinesas the Retina Matures

A decline in the expression of the EGFR could under-lie the decline in the response of M€uller glia to intraocu-

Fig. 1. EGFR is required for normal levels of retinal progenitor pro-liferation. Egfr deficient mice and wild type littermates were killed onthe day of birth (P0), or at postnatal day 3 (P3) or postnatal day 6 (P6).(A) Micrographs of sections through the central retina at P0 and P3and of peripheral retina at P6 showing PH3 labeling of mitotic cells inthe retinas. (B) Quantitation of PH31 cells in P3 central retina ofEgfr2/2 mice (black bar) and wt littermate control animals (light graybar). Error bar shows SEM (asterisk: P < 0.005, t test; five animals pergroup). (C) Quantitation of PH31 cells in P6 peripheral retina ofEgfr2/2 mice (black bar) and wt littermate control animals (light graybar). Error bar shows SEM (asterisk: P < 0.002, Students’ t test; sixanimals per group). (D, E) Chx10 immunofluorescence of wild type andEgfr2/2 mouse retina at postnatal day 7 (P7) shows both bipolar celllabeling (arrowhead in INL) and progenitor cells in the ONL (arrow,and insets). There are many Chx101 progenitor cells in the ONL of thewild type (D, arrow and inset D0 at higher magnification), but few inthe Egfr2/2 retina (E, arrow, and inset E0 at higher magnification).ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion celllayer; CB, ciliary body. Asterisk indicates the retinal margin, where afew retinal progenitors are concentrated at this age.



lar EGF injections. To determine whether EGFR levelsdecline quantitatively with the age of the animal, weextracted protein and mRNA from rat retinas at variousages. Protein levels were quantified, and 30 lg of proteinfrom each of the timepoints was analyzed for EGFRexpression via Western blot. EGFR protein levels werehighest at postnatal day 4 in the retina, declining gra-dually until little or no EGFR protein could be detectedat P21 and later (Fig. 4A). Quantitation confirms thistrend (Fig. 4B). When compared with P4 levels of EGFRprotein, P10 EGFR levels drop to 57.1% (63.37), andP10 EGFR levels drop to 41.9% (68.36). In retinas ofanimals P21 or older, EGFR levels are just 6.8% (65.0)of P4 EGFR levels. Quantitation of EGFR mRNA tran-scripts via Q-RT-PCR reveals a similar decline in expres-sion. Compared with adult levels of EGFR mRNA, em-bryonic day 18 (E18) retinas contained on average 11.7-(61.24) fold more EGFR transcripts (Fig. 4C, white bar),while P4 retinas contained 17.1- (61.16) fold moreEGFR transcripts (Fig. 4C, light grey bar). P10 retinascontained an average of 5.3- (61.37) fold more EGFRtranscripts than adult retinas (Fig. 4C). These results

show that at both the protein and mRNA levels, retinalEGFR expression declines with the age of the animal.

To determine which cell types express EGFR in thepostnatal retina, we performed immunofluorescence usingantibodies directed against EGFR and the M€uller glialmarker CRALBP. In the P4 retina, EGFR is expressed pri-marily by the nestin1 progenitor cells (data not shown),but by P10, the EGFR immunoreactivity overlaps wellwith CRALBP-positive M€uller glial cell processes and cellbodies (Figs. 5A–F). Consistent with the Western analysis,there is a decline in the level of EGFR expression in theretina at postnatal day 21 (compare with Figs. 6A–E). Thedecline in EGFR expression correlates with the reductionin the numbers of proliferating M€uller glia in older ani-mals after EGF treatment.

Light-Induced Retinal Damage Causesan Increase in EGFR Expression

Treatment of albino rats with light continuously for1 week results in degeneration of the outer nuclear

Fig. 2. EGF is a M€uller glial mitogen.Rat pups treated with EGF or vehicle con-trol for two consecutive days starting atP10. (A–C) Few BrdU-labeled cells (A,green) are also CRALBP-labeled (C, red) incontrol retinas. (D–F) EGF-treated retinascontained many BrdU-labeled (D, green),CRALBP-labeled cells (F, red, arrowsmarkdouble-labeled cells). (G–I) Higher magnifi-cation micrographs of boxed region in (E)showing examples of double-labeled cellsin the ONL (asterisks). (J) Quantitation ofBrdU/CRALBP double-positive cells inthe various EGF treatment conditions; theresponse appears to saturate at 500 ng(*P < 0.02; **P < 0.01 Students’ t test;error bars 5 SEM). (K) Western blot anal-ysis of protein from animals injected withPBS/BSA or 1 lg EGF starting on P10,lower bands indicate b-actin loading con-trol. [Color figure can be viewed in theonline issue, which is available at www.interscience.wiley.com.]

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layer, rendering M€uller glia reactive and more likely tosurvive long-term in culture (Close et al., 2000; Sarthy,1985). The fact that light damage allows M€uller glia toproliferate in vitro suggests that there may be an up-regulation in the levels of the EGFR. To test this possi-bility, we compared the levels of EGFR protein in reti-nas from control and light-treated animals by Westernblot. We found that EGFR expression levels increasedsignificantly after rats were given light treatment (Fig.4A). When this effect is quantified, EGFR expressionlevels appear to be restored to 86.5% (67.4) of P4 levelsin light-treated animals (Fig. 4B). Immunoflourescentlabeling of retinal sections from light-treated rats con-firmed that an increase in EGFR expression occurred inCRALBP1 M€uller glia. Figure 6 shows the EGFR-immunoreactivity in P21 animals treated for 1 weekwith continuous light when compared with the normalP21 retina. The settings for the confocal microscope werekept the same for the EGFR micrographs from both thecontrol and light-treated retinas. EGFR levels were con-sistently higher in light-treated rats (Figs. 6F–J), whencompared with control P21 animals (Figs. 6A–E); the pri-mary cells labeled with anti-EGFR antibody were theCRALBP1 M€uller glia in the light-treated retinas as wellas the control.

Combined EGF and Light-TreatmentStimulates M€uller Glial Proliferation

To determine whether the changes in EGFR expres-sion after light treatment at P21 can affect the M€ullerglial response to EGF, we performed EGF injections onanimals older than postnatal day 21 that had either

been housed in a normal 12 h light cycle, or had beensubjected to 24 h light treatment in the 7 days prior toinjection. Animals were given intraocular injections ofeither PBS/BSA as a control or 1 lg EGF for two consec-utive days starting after P21, followed by 24 h of BrdUinjections. We found that control injections elicited littleor no proliferative response from M€uller glia (Figs. 7A–C,and data not shown). However, animals that weresubjected to light treatment prior to EGF injectionshowed a substantial number of BrdU-CRALBP double-labeled M€uller glia present in the inner nuclear layer(Figs. 7D–F). When this data is quantified, we foundthat animals not treated with light and receiving intra-ocular PBS/BSA injections contained just 3.8 (60.2)CRALBP-BrdU double-labeled cells per mm2 retina,while EGF injected animals contained 12.3 (65) double-labeled cells per mm2 (Fig. 7G). We find that after lighttreatment, control-injected animals contained 5.8 (61.9)CRALBP-BrdU1 cells per mm2, while those animalsthat received EGF injections, as well as light treatment,had on average 238 (671.4) CRALBP-BrdU1 cells permm2 in their retinas (Fig. 7G). These data support thehypothesis that the M€uller glial response to EGF is sen-sitive to the levels of EGFR expression, and further sug-gest that the proliferative potential of M€uller glia canbe restored to nearly the levels of late-staged progenitorcells with an increase in EGFR expression at laterstages.

DISCUSSION

Here, we show that EGFR expression levels are im-portant in the regulation of postnatal retinal progenitor

Fig. 3. The M€uller glial response toEGFdeclines by postnatal day 14. Rat pupswere injected with EGF or vehicle controlfor two consecutive days starting postnatalday 14. (A–C) Few BrdU-labeled cells (A,green) are CRALBP-labeled (C, red) in con-trol retinas. (D–F) EGF-treated retinascontained some BrdU-labeled (D, green),CRALBP-labeled cells (F, red, arrowsmarkdouble-labeled cells, arrowheads marknonspecific labeling). (G) Quantitation ofBrdU/CRALBP double-positive cells showsan increase in EGF-treated retinas overcontrol (*P < 0.05; Students’ t test; errorbars 5 SEM), but far fewer labeled cellswere present when compared with P10 trea-ted animals (compare with Fig. 2J). [Colorfigure can be viewed in the online issue,which is available at www.interscience.wiley.com.]



proliferation; the retinas of postnatal animals lackingEgfr contain fewer mitotic cells. We also show thatEGFR is expressed in M€uller glia and that EGF is amitogen for M€uller glia in vivo. The effects of EGF di-minish with the age of the animal, and correlating witha decline in the expression of EGFR protein and mRNAin the retina. EGFR expression can be restored toM€uller glia by damaging the retina with continuous ex-posure to light, and this increase in EGFR expressionresults in an increase in the mitotic response of M€ullerglia to EGF injection at later ages.

The results of our analysis of the Egfr deficient miceshow that this receptor is required for normal retinal his-togenesis. Several previous studies have shown that EGFand related ligands can stimulate proliferation of retinalprogenitors (Anchan and Reh, 1995; Anchan et al., 1991;Lillien, 1995; Lillien and Cepko, 1992; Lillien and Wancio,1998). Retinal progenitors express EGFR (Anchan et al.,1991; Lillien, 1995;) and the level of receptor expressionand response to EGF increases in late-staged retinal pro-genitors, when compared with progenitors from early em-bryonic stages of development (Anchan and Reh, 1995;Anchan et al., 1991; Lillien, 1995; Lillien and Cepko,1992). Given the high level of mitotic stimulation causedby EGF in these earlier studies, it is somewhat surprisingthat we find the loss of this receptor causes a relativelyminor phenotype. The severity of an Egfr deficiency inmice is known to be very dependent on the background(Threadgill et al., 1995), and therefore it is possible thatanother molecular pathway compensates for the loss ofthis receptor.

Previous over-expression studies of the EGFR haveshown that this factor can regulate cell fate decisions inthe developing retina. For example, over-expression ofEGFR using a retrovirus directs the progenitors to aMuller glial fate (Lillien, 1995). Therefore, we predictedthat the Egfr2/2 mice would show an absence of Mullerglia. We did not find this to be the case, however, possi-bly because of compensation from another signalingpathway. As noted earlier, since we only found differ-ences in proliferation near the end of histogenesis, itmay be that only the final round of proliferation isabsent in the mutants and therefore only a small changein the relative numbers of Muller glia would occur. Ingeneral, it appears that those factors that regulate pro-liferation or differentiation of retinal progenitors earlyin development, such as Shh (Moshiri and Reh, 2004;Dakubo and Wallace, 2004) and GDF11 (Kim et al.,2005), have more dramatic effects on the relative propor-tions of the different retinal cell types, while those fac-tors that affect proliferation relatively late in histogene-sis, such as p27kip (Dyer and Cepko, 2000; Levine et al.,2000), have more subtle, or undetectable, effects on rela-tive cell-type proportions.

It is also possible that the reduction in proliferationwe observed in the Egfr2/2 mice was due to a specificreduction in the proliferation of Muller glia. Similarly,mice deficient in the cell cycle regulator p27kip display aprolongation of proliferation, and most of the late gener-ated cells adopt a Muller glial fate (Dyer and Cepko,

Fig. 4. EGFR expression levels decline with age in the retina. Pro-tein and mRNA for EGFR decline in the postnatal period. (A) Westernblot shows decline in EGFR protein with the age of the animal; EGFRlevels are undetectable in animals over P21. EGFR receptor expressionincreases in P21 animals after 1 week of light treatment. (B) Quantita-tion of Western blot results normalized to P4 EGFR levels (white bar,100%) shows EGFR expression at P10 is down to (57.1 6 3.4)% whencompared with P4 levels, while P14 levels are down to (41.9 6 8.4)%,and in animals older than P21, EGFR levels are just (6.8 6 4.9)% of P4levels. Following 1 week of light treatment, P21 EGFR levels are ele-vated to (86.5 6 7.5)% when compared with P4. (*ManU: P < 0.001).(C) Q-RTPCR results show that EGFR mRNA levels are highest at ear-lier ages, and lowest in the adult retina. Compared with adult EGFRmRNA levels, EGFR transcripts are 11.7- (61.24) fold more abundantin embryonic day 18 (E18) and 17.1- (61.16) fold more abundant in P4retinal tissue. At P10, EGFR mRNA is 5.3- (61.37) fold more highlyexpressed than in adult rat retinal tissue. (*ManU: P < 0.001).

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2000; Levine et al., 2000). Therefore, it is possible thatin the periphery of the retina, there is a specific Mullerglia progenitor that is lost in the Egfr2/2 mice. Althoughwe cannot rule this out, we do not favor this possibilitybecause (1) we find that the number of Chx101 progeni-tor cells is reduced in the mutants, and Chx10 is notexpressed in the Muller glia (see Fig. 1); and (2) retro-viral lineage studies have shown that Muller glia arenormally generated by a common precursor with theother retinal cell types (Turner and Cepko, 1987).

We also report that EGF is a potent mitogen forM€uller glia in vivo, and their response to this factor par-allels the level of expression of the EGFR. It has beenknown for many years that EGF can stimulate prolifera-tion of M€uller glial cells, though all of the evidence forthis has come from in vitro studies (Milenkovic et al.,2004; Reichelt et al., 1989; Roque et al., 1992; Schererand Schnitzer, 1994). Two previous in vivo studies haveprovided evidence that EGF might stimulate prolifera-tion of progenitors and M€uller glia, though neitherdirectly demonstrated this; Henck et al. (2001) adminis-tered large, systemic doses of EGF to rats aged P1-P6,and observed rosette formation and cellular disruptionof the outer nuclear layer of the retina; Sagar et al.(1991) have found that injection of adult rabbits with ei-ther EGF or TGFa results in the upregulation of c-fosexpression by M€uller glia.

We have also found that M€uller glia express theEGFR in vivo, and that the level of expression declinesduring postnatal development. As noted earlier, severalstudies have analyzed EGFR in developing retina and

shown that the response to this factor increases fromearly to late embryonic development. Prior to our study,it was not known whether the level of this receptorremained high throughout life, or whether it was sub-jected to additional developmental regulation. We havefound that the EGFR is significantly down-regulatedduring the second postnatal week, and that this has aprofound effect on the response of the cells to EGF.Although Roque et al. (1992) reported a high level ofEGFR expression on cultured M€uller glia, our resultssuggest that the receptor was upregulated in vitro, sincein vivo, these cells express only very low levels of recep-tor. Since light damage is frequently used as a methodto enable efficient culture of M€uller glia, it is possiblethat the up-regulation of EGFR, triggered by lightdamage, contributes to this phenomenon. A study byPowers and Planck (1997) also reported EGFR expres-sion in M€uller glia in the developing rat retina. How-ever, there was no quantitation in their study, and thelocalization to Muller glia was not demonstrated withco-labeling for M€uller glial-specific markers.

Our results, together with those of previous studies,indicate that the proliferation of retinal progenitors andM€uller glia are both regulated by EGFR. It is knownthat retinal progenitors and M€uller glia have similargene expression profiles (Blackshaw et al., 2004), andthat Muller glia are among the last cells generated inthe developing retina (Turner and Cepko, 1987). Whilerecent evidence indicates that the cessation of prolifera-tion during development of the retina involves a TGFb2mediated signal (Close et al., 2005), the present results

Fig. 5. EGFR is expressed in CRALBPpositive M€uller glia. Rat retinas from P10were co-labeled with CRALBP and EGFRantibodies to localize the expression of thereceptor. (A–C) P10 retinas show EGFRlabeling (A, red) in the cell bodies and pro-cesses of CRALBP-positive M€uller glia (C,green), (B, merge, arrowheads point toexamples of double-positive cell bodies).(D–F) Higher magnification view of boxedarea indicated in C. ONL, outer nuclearlayer; INL, inner nuclear layer; GCL,ganglion cell layer. [Color figure can beviewed in the online issue, which is avail-able at www.interscience.wiley.com.]



support the hypothesis that regulation of the EGFR alsoplays a role in the maintenance of glial mitotic quiescencein the mature retina. The end of retinal histogenesis isapparently mediated by a combination of at least two fac-tors; in the first postnatal week, TGFb2, released by neu-rons within the retina, inhibits the proliferation of theprogenitors (prior to P7) and the M€uller glia (after P7); inthe second postnatal week, and thereafter, M€uller gliaare maintained in a mitotically quiescent state by thedown-regulation of EGFR. Treatment of mature retinawith inhibitors of the TGFb signaling pathway and/orEGFR ligands can stimulate their proliferation in vitro,but in mature rats, damage to the retina that leads to anup-regulation in EGFR is required for EGF treatment tobe mitotically active in vivo.

There are at least two possible ligands for the EGFRthat are known to be expressed in the developing andmature retina. TGFa (Anchan et al., 1991) is expressedthroughout the period of retinal histogenesis, and bothTGFa (Fassio et al., 1989) and heparin binding epider-mal growth factor (HB-EGF) are expressed in themature retina. HB-EGF is mitogenic for M€uller glia(Hollborn et al., 2005; Milenkovic et al., 2004), and isupregulated in patients with proliferative vitreoretino-pathy (Hollborn et al., 2005). In addition, several othermitogenic factors are known to act through the EGFR(Milenkovic et al., 2004), and therefore it may be moreefficient to regulate the expression of this key receptorthan a diversity of ligands to limit histogenesis in the

developing retina and M€uller glial proliferation in themature retina.

We found that in those animals where EGF stimu-lated proliferation of the Muller glia, not all of theBrdU-labeled Muller cells remained in the inner nuclearlayer. Instead, many migrated to either the outer nu-clear layer, or more rarely, into the ganglion cell layer. Asimilar phenomenon has been observed in the avian ret-ina, following neurotoxin treatments or growth factorinjections, and this proliferative response was followedby a limited production of new neurons (see Fischer andReh, 2001). We therefore examined the BrdU-labeledcells in this study for evidence of neuronal gene expres-sion. We failed to find evidence for neuronal productionafter EGF treatment in the rat retina, using antibodiesagainst rhodopsin, recoverin, bIII-tubulin, or Hu (Closeand Reh, unpublished observations). However, it hasbeen previously shown that conditions that promoteMuller glial proliferation past the normal phase of histo-genesis lead to an accumulation of the over-producedMuller glia in the outer nuclear layer (Levine et al.,2000).

The response of M€uller glia to EGF is similar to themitogenic response of astrocytes (Leutz and Schachner,1981) in other regions of the nervous system and pro-genitors of the developing mammalian CNS (Craig et al.,1996; Fallon et al., 2000; Kuhn et al., 1997; Reynoldsand Weiss, 1996; Reynolds et al., 1992). Astrocytes ofthe mature mammalian brain respond to EGF by re-

Fig. 6. Light treated retinal M€ullerglia express EGFR. Retinas from ani-mals over P21 that had either beenhoused in normal conditions or in 24 hof light treatment were fixed and pro-cessed for immunohistochemistry to de-termine EGFR expression patterns. Set-tings for confocal imaging were identicalfor both control and light-damaged reti-nas, so intensity of the EGFR labelingreflects level of EGFR immunofluores-cence. (A–E) P21 retinas show low levelof EGFR labeling (C and E, red), at lowlevels in CRALBP-positive cells (A andD, green). (D, E) Higher magnification ofboxed area in B. (F–J) Light-treated ani-mals show increased EGFR labeling (Hand J, red) in CRALBP-positive cells (Fand I, green). (I, J) Higher magnificationof boxed region in G. [Color figure canbe viewed in the online issue, which isavailable at www.interscience.wiley.com.]

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entering the cell cycle, both in vitro and in vivo (Doetschet al., 2002; Huff et al., 1990; Kornblum et al., 2000;Leutz and Schachner, 1981). EGFR expression alsodeclines with the age of the animal in other parts of theCNS. For example, EGFR is expressed at relatively highlevels until postnatal day 19 in both astrocytes and Pur-kinje cells, with levels of expression declining thereafter(Gomez-Pinilla et al., 1988). By contrast, EGFR persistsinto adulthood in neurogenic regions, such as the SVZ(Doetsch et al., 2002; Seroogy et al., 1995; Weickertet al., 2000). Together with our data, these results fromother brain regions indicate that EGFR expression levelsin postnatal glia and progenitors may be a limiting fac-tor for proliferation throughout the CNS.

ACKNOWLEDGMENTS

The authors thank Branden Nelson, Paige Etter, andOlivia Berminghan-McDonogh for their critical review ofthe manuscript, and members of the Reh laboratory forconstructive comments. They also thank Melissa Phillipsand Paige Etter for their technical assistance.

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