ORIGINAL ARTICLE

Yehudit Birger1 . Janine Davis1 . Takashi Furusawa .

Eyal Rand . Joram Piatigorsky . Michael Bustin

A role for chromosomal protein HMGN1 in corneal maturation

Received October 18, 2005; accepted in revised from October 19, 2005

Abstract Corneal differentiation and maturation areassociated with major changes in the expression levels ofnumerous genes, including those coding for the chromatin-binding high-mobility group (HMG) proteins. Here wereport that HMGN1, a nucleosome-binding protein thatalters the structure and activity of chromatin, affects thedevelopment of the corneal epithelium in mice. The cor-neal epithelium of Hmgn1� /� mice is thin, has a reducednumber of cells, is poorly stratified, is depleted of supra-basal wing cells, and its most superficial cell layer blisters.In matureHmgn1� /�mice, the basal cells retain the ovoidshape of immature cells, and rest directly on the basalmembrane which is disorganized. Gene expression wasmodified in Hmgn1� /� corneas: glutathione-S-transferase(GST)a 4and GST o 1, epithelial layer-specific markers,were selectively reduced while E-cadherin and a-, b-, and g-catenin, components of adherens junctions, were increased.Immunofluorescence analysis reveals a complete co-local-ization of HMGN1 and p63 in small clusters of basalcorneal epithelial cells of wild-type mice, and an absence ofp63 expressing cells in the central region of theHmgn1� /�

cornea. We suggest that interaction of HMGN1 withchromatin modulates the fidelity of gene expression andaffects corneal development and maturation.

Key words HMG proteins � eye differentiation �corneal epithelium � chromatin

Introduction

Cellular differentiation pathways, including the gener-ation of the mature cornea, involve multiple changes ingene expression, a process known to involve structuraland chemical changes in the chromatin fiber (Felsenfeldand Groudine, 2003; Jaskelioff and Peterson, 2003; Ha-takeyama et al., 2004; Sarmento et al., 2004; Margueronet al., 2005). Proper development of the transparentcornea is essential to normal vision, as together, thecornea and lens refract light onto the retina (Albert andJakobiec, 1994). Cornea development commences earlyin mouse embryos and continues after birth. At birth,the mouse corneal epithelium, derived from head sur-face ectoderm (Pei and Rhodin, 1970), is one to two celllayers thick (Maurice, 1969), and is separated by abasement membrane from the underlying stromal andendothelial cell layers, derived in part from the neuralcrest (Haustein, 1983; Trainor and Tam, 1995).

A tightly regulated interplay between proliferative,differentiative, and cell death processes is required tomaintain the steady rate of corneal epithelial cell re-newal during adult life (Cotsarelis et al., 1989; Lavkeret al., 1991; Kruse, 1994). Undifferentiated stem cells,with a high proliferative potential (capacity to divide),move from the limbus towards the central part of thecornea, into the cuboidal basal cell layer (Nagasaki andZhao, 2003), and transiently become amplifying cells.From this layer, cells committed to terminal differenti-ation migrate outwards into the wing-shaped, supraba-sal cell layer, which gives rise to several layers offlattened, squamous cells that eventually are sloughed(Klyce and Beuerman, 1988; Chung et al., 1992; Sunet al., 2000). By 3 weeks after birth, the epithelium is sixto seven cell layers thick and by 8 weeks of age it1Y. B. and J. D. made equal contributions.

Yehudit Birger1 � Takashi Furusawa � Eyal Rand �Michael BustinProtein Section, Laboratory of MetabolismNational Cancer InstituteBethesda, MD 20892, USATel: 11-301-496-5234E-mail: bustinm@mail.nih.gov

Janine Davis1 � Joram Piatigorsky ( .*)Laboratory of Molecular and Developmental BiologyNational Eye InstituteNational Institutes of HealthBethesda, MD 20892, USATel: 11-301-402-4343E-mail: joramp@nei.nih.gov

U.S. Copyright Clearance Center Code Statement: 0301–4681/2006/7401–19 $ 15.00/0

Differentiation (2006) 74:19–29 DOI:10.1111/j.1432-0436.2006.00054.xr 2006, Copyright the AuthorsJournal compilationr 2006, International Society of Differentiation

reaches its mature size of eight to 10 cell layers (Hay,1979; Chung et al., 1992; Zieske and Wasson, 1993;Collinson et al., 2002).

The proliferative expansion and differentiation of thecorneal epithelium is accompanied by numerous chang-es in gene expression (Stepp et al., 1995; Francesconiet al., 2000; Sun et al., 2000; Yoshida et al., 2000; Ar-gueso and Gipson, 2001; Wang et al., 2001; Fukushimaet al., 2003; West-Mays et al., 2003; Norman et al.,2004). In some cases the expression of specific genes islocalized, or highly enriched, in specific layers. For ex-ample, p63, a member of the p53 transcription factorfamily involved in proliferative and differentiative proc-esses in epithelia (Dai and Segre, 2004; Koster et al.,2004; Westfall and Pietenpol, 2004), is expressed exclu-sively in the basal cell layer (Dua and Azuara-Blanco,2000; Pellegrini et al., 2001; Moore et al., 2002; Hsuehet al., 2004), glutathione-S-transferase (GST) a 4(GSTa4) expression is observed mainly in the supraba-sal region, while another GST isoform, GST o1(GSTo1), is enriched in the superficial cells of the ep-ithelium (Norman et al., 2004).

It is well documented that differentiation-relatedchanges in transcription levels of specific genes are as-sociated with changes in their chromatin structure (Fe-lsenfeld and Groudine, 2003; Jaskelioff and Peterson,2003; Hatakeyama et al., 2004; Sarmento et al., 2004;Margueron et al., 2005). Recent SAGE analysis re-vealed massive changes in gene expression during post-natal maturation of the mouse cornea (Norman et al.,2004). Some of the most affected genes code for chro-matin modifying proteins, including members of thehigh-mobility group (HMG) protein superfamily.HMGs are ubiquitous and relatively abundant nuclearproteins, devoid of enzymatic activity, that bind to thechromatin fiber and induce changes in both the localand the higher-order chromatin structure (Bustin, 1999,2001; Reeves, 2001; Agresti and Bianchi, 2003). HMG-mediated changes in chromatin affect a wide range ofDNA-dependent activities, including transcription;therefore, alterations in their expression levels may leadto global changes in transcription profile, such as seenduring corneal development.

Here we present evidence that the high-mobilitygroup protein N1 (HMGN1) affects the development ofthe corneal epithelium. HMGN1 is known to bind spe-cifically to nucleosome cores, the building block of thechromatin fiber, to reduce the compaction of the chro-matin fiber, to modulate the levels of posttranslationalmodifications in histones (Lim et al., 2004), and to af-fect the rate of transcription (Bustin, 2001). We findthat mice lacking HMGN1 have multiple abnormalitiesin their corneas, some of which were not previouslydescribed. In the corneas of Hmgn1� /� mice, loss ofHMGN1 alters the expression levels of cellular adhe-sion molecules, GSTs, and of p63, a major regulator ofepithelial cell differentiation (Koster et al., 2004; Koster

and Roop, 2004a, b). In the absence of HMGN1, thedevelopment and maintenance of the corneal epitheliumis abnormal and characterized by loss of suprabasalcells, a reduction in the epithelial thickness, and ten-dency to blister. Our studies identify HMGN1 as a newregulator of corneal development.

Materials and methods

RNA blot analysis

A mouse RNA master dot blot (CLONTECH, Palo Alto, CA) wasprobed with 32P-labeled 30UTR of the Hmgn1 cDNA as recom-mended by the manufacturer. The autoradiograms were scannedusing a Molecular Dynamics densitometer and analyzed with Im-ageQuant software (Molecular Dynamics).Hmgn1� /� mice were previously described (Birger et al., 2003).

In situ hybridization

For hybridization of embryo sections, riboprobes were synthesizedfrom a linearized templates containing a 1.2kb cDNA of mouseHmgn1 using T3 and T7 RNA polymerases (Stratagene, La Jolla,CA) and digoxigenin-11-UTP (Roche Diagnostics, Indianapolis, IN).Embryos were fixed in 4% paraformaldehyde (PFA), embedded inparaffin, and 5mm sections were collected. Sections were dewaxed,rinsed with phosphate-buffered saline (PBS), and then subjected toprotease digestion (1mgproteinaseK/ml PBS). Sections were refixed(4% PFA, 0.2% glutalaldehyde/PBS) and acetylated (0.25% aceticanhydrate, 0.1M triethanolamine, 0.1% HCl). Hybridization wasperformed overnight in 50% formamide:5� SSC:1% SDS at 701Cand washed with 5� and 1� SSC. Detection of digoxigenin-labeledhybridization was achieved using an alkaline phosphate (AP)-conju-gated anti-digoxigenin Ab (Roche Diagnostics) at a 1:500 dilutionfollowed by the addition of AP substrates, nitroblue tetrazolium(NBT) and bromo-4-chloro-3-indolylphosphate toluidinium (BCIP)(Roche Diagnostics).Fresh, frozen, 10mm eye sections from 25-day old mice were

fixed, treated with proteinase K (0.2 mg/ml PBS) for 8min, andprocessed for in situ hybridization as described previously (Davis etal., 2003). Riboprobes were synthesized using a DIG RNA Labe-ling Kit (Sp6/T7) (Roche Molecular Biochemicals, Indianapolis,IN) with linearized, proteinase K-treated, plasmid cDNA templatesencoding GST a4 (NM_010357; bases 22–437) and GSTo1(U80819; bases 62–548). Hybridizations were carried out at 551Cusing 200 ng sense or antisense riboprobe/ml hybridization buffer.Hybridization was visualized as described for embryonic sections.The reaction was allowed to proceed until purple color was visible(approximately 90min), at which time reactions for both the senseand antisense riboprobes were terminated.

Real-time PCR

RNA was isolated from mouse corneas using TriZol Reagent (In-vitrogen, Carlsbad, CA) according to the manufacturer’s instruc-tions. Six hundred nanograms of total RNA was reversetranscribed using 1.25U/ml MultiScribe Reverse Transcriptase in30ml of 1� TaqMan RT buffer containing 5.5mMMgCl2, 500 mMof each dNTP, 2.5 mM random hexamers, and 0.4U/ml RNase in-hibitor (Applied Biosystems, Foster City, CA). After incubation at251C for 10min, mixtures were reverse transcribed at 481C for30min followed by heat inactivation at 951C for 5min. Transcriptswere quantified by real-time PCR using reverse transcribed, first-strand cDNA, and SYBR Green PCRMix (Applied Biosystems). Atotal of 12.5ml of 2� SYBR Green PCR mix was mixed with 0.5 mlof reverse transcription product and specific primers (final concen-

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tration: 0.5mM) and analyzed using an ABI PRISM 7900HT. Theprimer sets used in the quantitative real-time PCR were: GSTa4: 50-aaaacccgttacttcccagtgtt-30/50-ggatgtctgcccaactgagc-30, 50-ctcctccagatg-gcccctat-30/50-ggtgacactgcaattggaacc-30, 50-actgtcaagagctaaaacccg-ttact-30/50-gtctgcccaactgagctggt-30, 50-gacatccagctcctagaagcca-30/50-ttgtcttaaatgcctgcagcag-30, and 50-tgaagttctagtgcagcgtgct-3 0/50-ct-ttgcttctggaatgctctg-30; p63: 50-ttgatgccctctctccatcc-3 0/50-gtgcttgac-tgctggaaggac-30, and 50-aggtcgtgagacgtacgagatgt-30/50-gcctgtacgt-ttcgatcgtgt-30; a-actin: 50-aaatcagtgcgtgacatcaaa-3 0/50-tctccagggag-gaagaggat-30, and 50-tcctcctgagcgcaagtactct-3 0/50-gctgatccacatctgct-ggaa-30; E-cadherin: 50-ctgtggacgtggtagacg tg-30/50-cctgacccacac-caaagtct-30, and 50-agactttggtgtgggtcagg-30/50-tgtccctccaaatccgatac-30.

Histology and DAPI staining

Whole eyes were immersion-fixed in 4% PFA in PBS overnight at41C, washed in PBS and then in saline for 30min each, dehydratedthrough a series of ETOH washes, cleared in xylene, and embeddedin paraffin. Prior to staining, 10mm sections were deparaffinized,rehydrated using a graded series of ETOH washes, and rinsed indistilled water for 15min. Eye sections were stained in Gill’s hem-atoxylin and eosin as described (Luna, 1968). Slides were counter-stained with DAPI as described (Davis and Reed, 1996),coverslipped using Aqua-Poly/Mount (Polysciences Inc., Warring-ton, PA), and photographed using a Zeiss Axioplan2 microscopewith Spot camera.

Electron microscopy

Whole eyes were removed and fixed 24 h at room temperature in asolution of 2.5% glutaraldehyde and 6% sucrose buffered to pH 7.2with 50mM sodium cacodylate. Small (0.5� 1mm) portions of thecentral cornea were processed for electron microscopy by dehydra-tion in an ethanol series and embedding in epoxy resin. Ultra thinsections were stained with uranyl acetate and lead citrate and pho-tographed using a JEM-100CX electron microscope (JEOL, Pea-body, MA).

BrdU and TUNEL labeling

Four hours prior to sacrifice, Hmgn1� /� and Hmgn11/1 siblingmice were given an intra-peritoneal injection of BrdU (10mM stocksolution used at 1ml/100 g body weight). Fresh, frozen eye sectionswere prepared, fixed, and washed in PBS. The sections (10 mm) wereincubated in 2N HCl in 0.5% Triton X-100 for 10min followed bya second incubation of fresh 2N HCl in 0.5% Triton X-100 con-taining 1mg/ml pepsin (Sigma P7012, St. Louis, MO) for 30 s. Theslides were washed with PBS three times for 10min each, blocked in10% normal rabbit serum in a humidified chamber, and then in-cubated with a rat monoclonal anti-BrdU antibody (1:100; Accu-rate Chemical and Scientific Corp., Westbury, NY) overnight at41C. Complete processing of the sections was achieved using a ratVectastain Elite ABC kit (Vector Laboratories, Burlingame, CA)and diaminobenzidine. TUNEL labeling on parrafin-fixed sectionwas done with the ApopTag kit obtained from Chemicon, accord-ing to the manufacturer’s instructions.

Immunohistochemistry and immunofluorescence

The following antibodies were used: anti-Hmgn1 peptide 6, anti-N-terminal keratin 12 (K12) (1mg/ml; a gift from W. Kao, University ofCincinnati, Cincinnati, OH), anti-aldehyde dehydrogenase 3 (ALDH3)(gift from R. Lindahl), anti-transketolase (TKT) Ab (Sax et al., 1996),anti-p63 (4A4): sc-8431, mouse monoclonal IgG2a, 1–205 a.a. ofDNp63 of human origin (Santa Cruz Biotechnology, Santa Cruz, CA).

Fresh frozen eye sections (10 mm) were collected on Superfrost/Plus slides (Fisher, Pittsburgh, PA) and stored at � 801C until fur-ther use. Skin analyses were performed with PFA-fixed, paraffin-embedded whole embryos. Immunohistochemistry was performedby VectorStain Elite ABC kit (Vector Laboratories Inc., Burlin-game, CA) with modifications. Briefly, slides were de-paraffinizedby immersing in CitriSolv solution (Fisher Scientific). De-paraf-finized slides were hydrated by incubating 10min for serial dilutedethanol solutions (100%–495%–470%–450%). Slides werewashed with 1�PBS for 10min, permeabilized by 1�PBS with0.1% Triton X-100 for 10min, and then washed with 1� PBS for10min. Slides were immersed in 10mM sodium phosphate (pH6.0)solution and heated by microwave. Sections were blocked with 10%goat normal serum and 3% BSA in PBS at 371C for 1 h and in-cubated with primary antibodies. Slides were washed with 1�PBScontaining 0.1% Tween 20 for 5min and incubated with secondaryantibodies at 371C for 1 h. After incubation, slides were treated asrecommended by the manufacture, and then mounted with AquaPoly/Mount (Polysciences Inc.). For fluorescent microscopy, thesections were incubated with secondary antibodies (1:500 goat anti-mouse 594-Alexa fluor red and goat anti-rabbit 488 Alexa fluorgreen) at RT for 45min then with 0.5 mg/ml Hoechst 33258 in PBSfor 10min. The sections were washed twice with PBS and mountedwith Aqua Poly/Mount (Polysciences Inc.). The location of Hmgn1and p63 proteins was visualized under a fluorescent microscope.

Western blot analysis

Mice (five Hmgn1� /� and five Hmgn11/1) were euthanized andwhole eyes were enucleated and placed in chilled PBS on ice. Usinga dissection microscope, corneas (total 20 corneas) were isolatedfrom the rest of the eye by introducing a small opening at thelimbus with fine forceps, and then separating the cornea from theconjunctiva by pulling on either side of the opening with fine for-ceps. The corneas were trimmed free of any remaining non-cornealtissues with a scalpel blade, frozen on dry ice, and stored at � 801Cuntil further use. Tissues were solubilized in 150mM NaCl, 50mMTris, pH 7.4%, 0.5% NP40, 0.5% Na deoxycholate, 5mM EDTA,0.25% SDS, pepstatin, leupeptin, PMSF, and aprotinin. Proteinconcentration was determined using the Bio-Rad Protein assay(Bio-Rad Laboratories, Hercules, CA). Polyacrylamide gel elect-rophoresis was performed using pre-cast 10% Bis-Tris gels followedby transfer to PVDF membrane in NuPage transfer buffer accord-ing to the manufacturer’s recommendation (Novex: Invitrogen).The membrane was blocked with 5% non-fat dry milk in Tris-buffered saline (Blotto) for 1 h at room temperature and incubatedwith either anti-E-cadherin (Zymed Laboratories Inc., San Fran-cisco, CA), anti a-,b-, or g-catenin (Transduction Laboratories,Lexington, KY) or anti-actin (Santa Cruz Biotechnology) in Blottoat 41C overnight. The membrane was washed and incubated withan anti-goat horseradish peroxidase-conjugated secondary anti-body (1:10,000 in Blotto, Amersham Pharmacia Biotech, Piscat-away, NJ) for 30min. After washing, the immunoreactive complexwas visualized using ECL Plus (Amersham Pharmacia Biotech).

Results

Hmgn1 expression in the embryonic and adult mouseeye

A survey of the Hmgn1 expression in various tissuesrevealed that the level of Hmgn1 mRNA was especiallyhigh in the eye, as compared with the other tissues ex-amined (Fig. 1A). In the dot blots used, the amounts ofRNA in each spot is normalized to the transcription

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levels of eight housekeeping genes; therefore, the inten-sity of the spot is indicative of the relative mRNAabundance in a tissue. The relative abundance ofHmgn1 mRNA in the adult eye was more than twicethat of any other tissue tested, including the kidney anduterus, which also had a relatively high expression level.Hmgn1 mRNA was also detected in lesser amounts inthe epididymus, ovary, prostate gland, and liver.

Several experiments indicated that the expression ofHmgn1 mRNA is related to differentiation (Begumet al., 1990; Crippa et al., 1991; Lehtonen and Leh-tonen, 2001; Korner et al., 2003; Hatakeyama et al.,2004); therefore, we first examined its expression pat-tern in the eye of 14.5-day-old embryos (E14.5), a de-velopmental stage in which the major structures of theeye have been formed. By in situ hybridization, Hmgn1transcripts were detected in the neural retina, lens, andcornea of eyes (Fig. 1B). In a magnified view of thecornea, Hmgn1 mRNA was detected specifically in theepithelial, but not the stromal or endothelial regions ofthe cornea (Fig. 1B), suggesting lineage-dependentspecificity in the timing of Hmgn1 expression.

Consistent with the in situ results for E14.5 eyes,immunohistological analysis revealed that HMGN1protein was localized specifically to the nuclei of theepithelial, but not stromal or endothelial cornea, cells ofthe 25-day-old Hmgn11/1 mice (Fig. 1C). Higher mag-nifications revealed that the HMGN1 protein is presentexclusively in the basal cell layer of the stratified corneaepithelium. The presence of HMGN1 in the basal layerof the epithelium is not unique to the eye; the samepattern was observed in analysis of skin from E16.5 dayor older embryos: HMGN1 is preferentially expressedin the basal skin layer (Fig. 1D).

Interestingly, not all of the cells in the basal layer ofthe corneal epithelium were immunoreactive forHMGN1. Instead, a subset of basal cells, appearing asclusters across the central and peripheral cornea, ex-pressed relatively high levels of the protein (Fig. 1C). Thebasal layer of the corneal epithelium is poised for furtherdifferentiation and is the progenitor of the corneal ep-ithelial cells necessary for the constant renewal of thecornea during adult life (Cotsarelis et al., 1989; Lavkeret al., 1991; Kruse, 1994). The confinement of HMGN1protein to the progenitor cells raises the possibility for arole of this protein in cornea epithelium maturation.

Abnormal corneal epithelium morphology inHmgn1� /� mice

To test the possible involvement of HMGN1 in corneamaturation we first examined the corneal epithelium ofHmgn1� /� and Hmgn11/1 mice that were 25-day old,an age at which the cornea is not fully mature. Histo-logical analysis, by hematoxylin and eosin (H&E)staining, showed a reduction in the thickness of the

Fig. 1 Hmgn1 expression in the eye. (A) Quantitative analysis ofRNA master dot blot (CLONTECH) demonstrating high levels ofHmgn1 expression in the eye. (B) In situ hybridization of Hmgn11/1

embryonic eyes. The arrow in the enlarged box points to the specificexpression detected in the corneal epithelium (EPI). C, cornea; R,retina; L, lens. (C) Immunohistochemistry of 25-day-old Hmgn11/1

and Hmgn1� /� mice corneas using a mouse high-mobility groupprotein N1 (HMGN1) specific antibody. Note the expression of theprotein in a subset of basal cells in the corneal epithelium ofHmgn11/1 (left panel) mice. (D) High HMGN1 expression in thebasal layer of the epidermis, shown are immunofluorescence andcorresponding DAPI stain. For orientation, the basal and outerlayers of the epidermis are outlined.

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epithelium in Hmgn1� /� compared with Hmgn11/1

mice (Fig. 2A). The thinner epithelium corresponded toa 25% reduction in cell number in Hmgn1� /� versusHmgn11/1 mice as determined by counting DAPI-labe-led nuclei (Figs. 2B, 2C). Detailed analysis of the ep-ithelial layers suggested a specific reduction in the

number of wing-shaped cells occupying the suprabasallayer of the epithelium in the Hmgn1� /� corneas (Fig.2A—Sb).

The development of the stratified corneal epitheliumis not completed until 8 weeks after birth (Collinsonet al., 2002). To determine if the lack of HMGN1 alsoaffected the maintenance of the mature corneal epithe-lium, we examined corneal histology at 4 months ofage. Like the 25-day-old corneas, the 4-month-oldHmgn1� /� epithelium was thinner than age-matchedHmgn11/1 littermates (Fig. 2D) with a few, if any, sup-rabasal wing cells (Fig. 2D, magnifications). Moreover,the basal cells of the Hmgn1� /� corneal epithelium donot acquire the cuboidal-shape characteristic of themature basal cells in the corneal epithelium of wild-typemice (Fig. 2D). In 4-month-old Hmgn1� /� mice thebasal cells still have an ovoid shape, characteristic ofimmature basal cells, which in wild-type mice can beseen prior to, or just immediately after, eyelid opening(Zieske, 2004). Additionally, patches of basal cells in theHmgn1� /� cornea appeared to reside directly on thestroma, rather than on the basement membrane thatnormally separates the epithelium from the extracellularstroma (Fig. 2D, arrows). In the control Hmgn11/1

corneas, the basal cells resided on the basement mem-brane, as expected. These results indicate that loss ofHMGN1 disrupts the maturation of the corneal epi-thelium.

We examined the possibility that the decrease in cor-neal cell number in the Hmgn1� /� mice is because ofeither an increase in cells undergoing apoptosis or adecrease in cell proliferation rate. In the TUNEL assay,a small number (0–6) of TUNEL-positive cells weredetected per corneal section from either 25-day-oldHmgn11/1 or Hmgn1� /� mice, indicating that loss ofHMGN1 does not lead to a significant change in therate of apoptosis (not shown). To examine whetherchanges in cell proliferation account for the reduction incorneal cell number in the Hmgn1� /� mice, 25-day-oldmice were sacrificed 4 h after an injection of BrdUand the eyes were processed for immunohistochemistry

b————————————————————————————

Fig. 2 Altered properties of Hmgn1� /� cornea. (A) Thinning ofHmgn1� /� cornea epithelium. Hematoxylin and eosin (H&E)stained sections from the eye of 25-day-old Hmgn11/1 andHmgn1� /� mice reveal a reduced second layer of large nucleatedcells (suprabasal cells) in the Hmgn1� /� samples. E, epithelium; B,basal layer; Sb, suprabasal cells; Se, surface epithelium; Sc, stroma;En, endothelium. (B) Reduced number of epithelial cells inHmgn1� /� corneas. DAPI staining of 25-day-old Hmgn11/1 andHmgn1� /� eye corneas. (C) Decreased cell number in 25-day-oldmice Hmgn1� /� cornea epithelium. The bar graph represents theaverage number of cells counted in six animals. (D) Thinning andstratification changes in H&E-stained 4-month-old Hmgn11/1 andHmgn1� /� mice. Note that in the Hmgn1� /� corneal epitheliumthere are fewer suprabasal wing cells (Sb), the basal membrane(arrows) is disorganized, and the basal cells are oval rather thancolumnar (arrowheads).

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using an anti-BrdU antibody. Surprisingly, there weremore cells incorporating BrdU in S phase in the corneasof Hmgn1� /� compared with Hmgn11/1 mice. On av-erage, there were 58, as compared with 46, BrdU-labe-led cells per corneal section from HMGN1� /� (n5 12)and HMGN11/1 (n5 10) mice, respectively.By t-test analysis the differences were significantwith a po0.02. Thus, loss of HMGN1 altered cornealmorphology and increased the frequency of cells inS phase.

Altered cellular adhesion in the Hmgn1� /� cornea

Abnormal corneal morphogenesis, including a reduc-tion in the number of epithelial cell layers have beenpreviously observed in Sey1/� mice, and could be in-dicative of abnormalities in the adhesion properties ofthe corneal cells (Davis et al., 2003). Electron micros-copy studies revealed that the size and number of des-mosomes in the corneal epithelium were indis-tinguishable between Hmgn11/1 and Hmgn1� /� mice(data not shown). However, the superficial cell layer ofthe epithelium was separated from the underlying layersin the Hmgn1� /� mice as compared with Hmgn11/1

corneas (Fig. 3). The blistering of the epitheliumin Hmgn1� /� mice is reminiscent of bullae formationwhich is characteristic of various ocular diseases(O’Connor, 1983), and suggests adhesion abnormalitiesin these corneas.

To determine whether adherens junctions, a secondtype of adhesive complex present in the epithelium,might be altered in theHmgn1� /� cornea, we examinedthe expression levels of the principal cadherin of epi-thelial tissue, E-cadherin. Quantitative real time PCR

using RNA from 25-day-old corneas showed a 30%increase in the amount of E-cadherin mRNA in corneasfrom Hmgn1� /� mice relative to Hmgn11/1 mice(Fig. 4A). Analysis by Western blot showed a twofoldincrease in the amount of E-cadherin protein in cornealextracts from 25-day-old Hmgn1� /� versus Hmgn11/1

mice (Fig. 4B). In addition, western blots indicate thatthree other molecular components of the adherens junc-tion complex, a-, b-, and g-catenin, were 2.1-fold, 1.7-fold, and 2.3-fold greater, respectively, in corneal ex-tracts from Hmgn1� /� than in extracts from Hmgn11/

1 mice (Fig. 4B).The distribution of E-cadherin protein, visualized by

immunofluorescence, was localized to the plasma mem-brane of epithelial cells. The pattern of fluorescence wassimilar in the two mouse backgrounds with highest ex-pression in the most superficial cells in both Hmgn11/1

and Hmgn1� /� corneal epithelium (Fig. 4C) suggestingthat distribution of E-cadherin protein is not changed inthe Hmgn1 knockout mouse.

Fig. 3 Blistering of the epithelial surface in Hmgn1� /� cornea.Electron micrographs of corneal epithelium demonstrating bullousformation in the superficial epithelial layer of corneas ofHmgn1� /�

mice , but not in their Hmgn11/1 littermates.

Fig. 4 Expression of adhesion molecules in the 25-day-oldHmgn1� /� and Hmgn11/1 corneal epithelium. (A) IncreasedmRNA expression level of E-cadherin (E-cad) in Hmgn1� /� cor-nea. Bars represent quantitative real time PCR of E-cad using RNAisolated from Hmgn1� /� and Hmgn11/1 corneas. Two sets ofprimers were used for the detection of the mRNA product in twoseparate experiments. The results presented in the graph are anaverage of two RT-PCRs for each experiment. The level of expres-sion is normalized to b actin. (B) Altered expression of E-cad, andof a-, b-, g-catenin (-Cat) in Hmgn1� /� cornea. Shown are westernblots of protein extracts from Hmgn1� /� and Hmgn11/1 corneaprobed with the antibodies indicated to the right of each panel.Protein loading normalized to actin. (C) Immunofluorescencestaining of E-cad in Hmgn1� /� (� /� ) and Hmgn11/1 (1/1)cornea indicates a similar distribution of this protein, to the plasmamembrane.

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Altered differentiation in Hmgn1� /� cornealepithelium

The change in cellular morphology and the decreasednumber of cells in the suprabasal layer of the epithelium

suggested that the differentiation pathway may be al-tered in the Hmgn1� /� cornea. To test this possibilitywe examined the levels of GSTa4 and GSTo1 tran-scripts, whose expression is restricted to different layersof the epithelium and correlates with distinct stages ofcorneal epithelial cell differentiation (Norman et al.,2004). In situ hybridization using riboprobes for GSTa4or GSTo1 shows strong hybridization of the antisense(Figs. 5A, 5D), but not the sense (Figs. 5B, 5E) probes,in the middle and superficial epithelial cell layers, re-spectively, in the Hmgn11/1 cornea. In contrast, noGSTa4 is detectable in theHmgn1� /� cornea (Fig. 5C),while the GSTo1 signal is clearly reduced in the super-ficial epithelial layer in the absence of Hmgn1 (Fig. 5F).Quantitative RT-PCR using RNA isolated from 25-day-old corneas confirmed that GSTa4 RNA was de-creased fourfold in the Hmgn1� /� cornea relative towild-type cornea (Fig. 5G), a finding compatible withthe decreased number of cells in the suprabasal layer ofthe epithelium (see Fig. 2). We have previously demon-strated that GSTa4 is most prominently expressed inthese cells (Norman et al., 2004).

The expression of keratin 12 (K12), aldehyde de-hydrogenase 3 (ALDH3), and transketolase (TKT),which constitute abundant, corneal enriched proteins(Liu et al., 1993; Sax et al., 1996; Kays and Piatigorsky,1997), was examined by immunohistochemistry usingspecific antibodies for each protein. As expected, K12,ALDH3, and TKT, were detected in all layers of theepithelium in the Hmgn11/1 cornea (Fig. 5H). The ex-pression levels and localization of K12, ALDH3, andTKT were similar in the Hmgn1� /� 25-day-old micecorneas (Fig. 5H) when compared with their Hmgn11/1

counterparts. We conclude that loss of HMGN1 alteredselectively the levels of proteins in the cornea affectingmostly differentiation related proteins such as E-cad-herin, catenins, GSTa4, and GSTo1.

Altered p63 localization and expression in the corneaepithelium of Hmgn1� /� mice

We noted that the pattern of HMGN1 expression in thecorneal epithelium (Fig. 1) is similar to that of thetranscription factor p63, a major regulator of epidermalmorphogenesis, which may play an analogous role inthe corneal epithelium (Pellegrini et al., 2001). BothHMGN1 (Fig. 1) and p63 (Collinson et al., 2002; Duaet al., 2003; Hsueh et al., 2004) are expressed in inter-mittent clusters of cells in the basal layer of the corneaepithelium. Given this similar pattern of expression andthe role of p63 in epidermal morphogenesis we exam-ined the possibility that the phenotypic changes ob-served in the corneal epithelium of Hmgn1� /� mice islinked to altered p63 expression.

Double-immunofluorescence on 25-day-old wild-typecornea revealed that HMGN1 and p63 colocalize within

Fig. 5 Reduced expression of glutathione-S-transferase a 4(GSTa4) and GST o 1 (GSTo1) in Hmgn1� /� cornea epitheli-um. In situ hybridization with riboprobes for GSTa4 (A–C) andGSTo1 (D–E) genes on Hmgn11/1 and Hmgn1� /� cornea sections(B, E, controls with sense riboprobes). Note the strong hybridiza-tion of the antisense, but not the sense, only in the wild-type (A, D)samples. Note loss of GSTa4 (C) and of GSTo1 (F), in Hmgn1� /�

corneas. (G) Real time RT-PCR for GSTa4 gene using RNA iso-lated from Hmgn1� /� and Hmgn11/1 corneas. Five sets of primerswere used for the detection of the RNA product in two separateexperiments. The results presented in the graph are an average oftwo RT-PCRs for each experiment. The level of expression is rel-ative to actin. (H) Similar expression of aldehyde dehydrogenase 3,transketolase, and keratin 12, in Hmgn11/1 and Hmgn1� /� cor-neas. Immunohistochemistry of cornea from 25-day-old mice.

25

clusters of basal epithelial cells (Fig. 6A). HMGN1protein, visualized using goat anti-mouse alexa fluorred, shows nuclear expression in the small clusters ofbasal cells. A similar staining pattern is observed forp63, visualized using goat anti-rabbit alexa fluor green(Fig. 6A). An overlay of the green and red imagesshowed a perfect correspondence of the expression pat-tern, 100% of the time, indicating that HMGN1 and

p63 are expressed in exactly the same subset of basalcells in the corneal epithelium (Fig. 6A). The overlap inexpression pattern of the two proteins is appreciatedfurther by comparing the Hoechst staining of all nucleiin the same section (Fig. 6A). While most of the cells inthe corneal epithelium were negative for both protein,invariably, cells that were positive for HMGN1 werealso positive for p63.

Immunohistochemical analysis of the cornea of 25-day-old mice revealed that the presence of HMGN1affects the distribution of p63 along the corneal epithe-lium. Whereas in Hmgn11/1 cornea p63 positive clus-ters of basal cells can be detected across both theperipheral and central cornea (Fig. 6B, 1/1), in thecorneal epithelium of their Hmgn1� /� littermates clus-ters of p63-positive basal cells are confined to the pe-ripheral region of the corneal epithelium. No p63protein is observed in the central region of theHmgn1� /� epithelium (Fig. 6B, � /� ). Interestingly,the intensity of the p63 immuno stain in the peripheralregion of the Hmgn1� /� corneas seems to be higherthan in the same region of the Hmgn11/1corneas, sug-gesting that the lack of p63-postive cells in the centralregion is not necessarily because of the absence of suf-ficient p63 protein. Indeed, by quantitative PCR anal-ysis of the levels of p63 transcripts in the corneaof the Hmgn1

� /�mice is higher than in the cornea of

their Hmgn11/1littermates (Fig. 6C). Given the knownfunction of p63 in epithelial development, it is possiblethat changes in p63 levels and in the distribution of p63-positive cells leads to developmental abnormalities inthe corneal epithelium of Hmgn1� /� mice.

Discussion

Our main finding is that HMGN1, a chromatin-bindingprotein that interacts with nucleosomes, plays a role in

b————————————————————————————

Fig. 6 p63 expression in the 25-day-old Hmgn1� /� and Hmgn11/1

corneal epithelium. (A) Double immunofluorescence of high-mo-bility group protein N1 (HMGN1) and p63. The white boxes ineach panel mark a specific area that was enlarged (bottom left).Panels from left to right: Hoechst DNA staining, HMGN1 stainingwith goat anti mouse 594-Alexa fluor red, p63 staining with goatanti rabbit 488 Alexa fluor green, double staining of HMGN1 andp63, the double expressed cells have yellow staining. Note thatHMGN1 and p63 colocalize to the same clusters of basal epithelialcells. (B). Altered distribution pattern of p63-containing cells in thebasal layer of Hmgn1� /� cornea. Immunohistochemistry of 25-day-old wild-type (1/1) and Hmgn1� /� (� /� ) cornea using p63antibody indicates depletion of the p63-positive cells in the centralregion of the Hmgn1� /� corneal epithelium (red circle). (C) In-creased p63 transcript in the corneal Hmgn1� /� epithelium. Quan-titative real time PCR of p63 using RNA isolated from Hmgn1� /�

and Hmgn11/1 corneas. Two sets of primers specific for the com-mon exons of all p63 isoforms were used for the detection of theRNA product in two separate experiments. The results presented inthe bar graph are an average of two PCRs for each experiment.

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the development and maintenance of the corneal epi-thelium. The corneal epithelium of Hmgn1� /� miceis thinner and blisters more readily than that ofHmgn11/1 mice, an indication that loss of HMGN1disturbs the normal stratification program in the epi-thelium. These phenotypic changes are linked to chang-es in the expression levels of GSTs, of structuralcomponents of adherent junctions, and to alterationsin the pattern of expression of p63, a key regulator ofepidermal development and maintenance (Koster et al.,2004; Koster and Roop, 2004a, b; Westfall and Pieten-pol, 2004).

The thinning of the corneal epithelium and increasedtendency to blister in Hmgn1� /� mice may be becauseof a change in its adhesive properties. This finding isconsistent with our earlier studies using Pax 6 mutantmice, where a reduction in the thickness of the cornealepithelium was correlated with changes in adhesion(Davis et al., 2003). In Hmgn1� /� mice, the superficialepithelial layers appeared loosely adhered to the rest ofthe cornea, reminiscent of bullae or blister formation, acondition present in various bullous oculocutaneousdiseases (O’Connor, 1983; Kaufman, 2000). Blistersforming because of a loss in adhesion (Smolin andThoft, 1994; Allen et al., 1996; Koch et al., 1997) mayrepresent a premature sloughing of the superficial cellsand account for, wholly or in part, the observed reduc-tion in the thickness of the epithelium. Interestingly,while in most pathologies associated with blistering theexpression of adhesion molecules is decreased, in theHmgn1� /� corneal epithelium the blistering is associ-ated with an increase in the levels of the adhesion mol-ecules. The changes in adhesion within the Hmgn1� /�

corneal epithelium may be because of misexpression ofE-cadherin, and a-, b-, and g-catenin, all structuralcomponents of adherens junctions, one of several typesof adhesion complexes (Angst et al., 2001). We alsoobserved altered expression of N-cadherin, and changesin cell adhesion and motility, in embryo fibroblastsfrom Hmgn1� /� mice (Rubinstein et al., in press).

We find that the absence of HMGN1 impacts thedifferentiation program of the corneal epithelium. Inthe corneal epithelium of mature Hmgn1� /� mice thebasal cells are ovoid rather than cuboid or columnar.Ovoid shaped basal cells are characteristic of cornealepithelium of very young mice; within 1 week of eyelidopening these cells first become cuboidal and subse-quently acquire the columnar-shape characteristic ofbasal cells of mature corneas (Zieske, 2004). The basalmembrane layer of Hmgn1� /� corneal epithelium isdisorganized and the basal cells penetrate into thestroma (Fig. 2). The suprabasal cell layer is mostly ab-sent in Hmgn1� /� mice, and the up-regulation ofGSTa4 and GSTo1, in the middle and superficialcorneal layers, respectively, is not detected in theHmgn1� /� epithelium; a finding consistent with theabsence of the cells expressing these proteins. The re-

sults suggest that loss of HMGN1 impacts the move-ment of cells from the mitotic, basal layer to the non-mitotic, suprabasal layers. In contrast to the GSTs, theexpression of several other corneal-enriched proteins,such as K12, ALDH3, and TKT, is not affected by theloss of HMGN1. These proteins are expressed through-out the corneal epithelium (Liu et al., 1993; Sax et al.,1996; Kays and Piatigorsky, 1997) while GST expres-sion is confined to distinct epithelial layers (Davis et al.,2003). The results link HMGN1 expression with specificstages of corneal epithelial cell stratification and differ-entiation.

Two key findings of this study are that HMGN1 isexpressed in the same subset of corneal basal cells asp63, and that the distribution of p63-expressing cells isaltered in the Hmgn1� /� cornea. The importance ofp63 during development of ectodermally derived tissueis well documented. p63 null mice have defects in severalepithelial tissues (Mills et al., 1999; Yang et al., 1999),while mutations in p63 are associated with a largenumber of human syndromes including ectodermaldysplasia (ED) (Brunner et al., 2002). Patients withED syndromes often display ocular anomalies includingrecurrent corneal epithelial defects from birth andblindness (Apple, 1998; Donahue et al., 1999; Ca-scallana et al., 2003). Interestingly, p63 is expressedthroughout the basal layer of corneal epithelium in mice(Moore et al., 2002; Dua et al., 2003; Ramaesh et al.,2005) and rats (Hsueh et al., 2004) under the age ofthree months, but is absent in the central portion of thebasal epithelial layer of the cornea by 6 months of age,similar to what we observe in the 25 day old Hmgn1� /�

cornea. The distribution pattern of p63 is altered in theHmgn1� /� cornea and is similar to that of older an-imals. However the overall levels of p63 are increasedrather than decreased. In view of recent findings of un-even distribution of p63 isoforms in limbal keratin-ocytes (Wang et al., 2005) it is conceivable thatHMGN1 protein affects p63 isotype synthesis. Thesedata warrant further experiments to test whether thealtered localization of p63 positive cells in the cornea ofHmgn1� /� mice is causally connected to the abnor-malities in the stratification and differentiation of thecorneal epithelium reported here. We find that the ex-pression of Hmgn1, as well as that of the closely relatedHmgn2 and Hmgn3 genes, is down regulated duringcorneal maturation (Norman et al., 2004). Our presentstudies indicate that HMGN1 protein plays a regulato-ry role during corneal maturation. Our finding may berelevant to regulatory mechanisms in other tissues as wefind that in the skin the expression of HMGN1 is alsolocalized to the basal cell layers (Fig. 1) and very similarto that of p63 (T. Furusawa and M. Bustin, unpub-lished). Misexpression of HMGN proteins in C2C12myoblasts (Pash et al., 1993), in one cell mouse embryos(Mohamed et al., 2001), and in Xenopus zygotes (Ko-rner et al., 2003) leads to developmental abnormalities.

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These, and additional studies (Bustin, 2001) suggestthat proper execution of predetermined differentiationprograms requires regulated expression of HMGN pro-teins. Loss of HMGN1 does not result in majorphenotypic alterations perhaps because of homeostaticmechanisms involving other HMGNs or/and additionalchromatin-binding structural proteins (Agresti andBianchi, 2003; Catez et al., 2004; Bustin et al., 2005).Indeed, the expression of HMGB1, a highly abundantchromatin structural protein whose function partiallyoverlaps with that of HMGNs is also down-regulatedduring corneal maturation (Norman et al., 2004). Theemerging picture suggests that structural proteins thataffect the conformation and accessibility of the chro-matin fiber play regulatory roles in developmental proc-esses including corneal maturation.

Acknowledgments This research was supported by the IntramuralResearch Program of the NIH, Center for Cancer Research, Na-tional Cancer Institute, and National Eye Institute.

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